ADIOLOGY 1,.11 NCOLOGY June 2002 Vol. 36 No. 2 Ljubljana ISSN 1318-2099 ADIOLOGY ANO NCOLOGY 1 year we observed response in 4 cases including 1 CR (RR 57 %). In TFI =1 year subgroup we observed response in 6 cases also with 1 CR (RR 67 %). TFI was not statistically significant for response (p < 0,4349). Median TTP is 6 month with 4 patients remaining progres­sion free. Patients with TFI > 1 year tend to have longer TTP (p = 0,0201). Median OS has not been reached with 10 patients surviving. We administered 599 cycles including 473 cycles of trastuzumab and paclitaxel with no dose adjustment. One patient developed hypersensitivity reaction on the first trastuzumab infusion and was excluded from study. The most common toxicity was trastuzumab infusion related pyretic reaction observed in 6 patients. Dose limiting adverse effect which led to the treatment discontinuation was cardio-toxicity. Ejection fraction decline grade 2 occurred in 1 patient and grade 3 also in 1 patient. Six patients ex­perienced grade 3 neuropathy. There were observed 1 episode of grade 4 neutropenia and grade 3 anemia. We noted 4 episodes of grade 3 infection without neutropenia. Grade 3 elevation of liver function tests oc­curred in 6 patients with no need of dose reduction. There were observed 1 episode of grade 3 hyperglyce­mia and 1 episode of grade 3 weight gain. Other grade 3 or 4 toxicity was not detected. Conclusions. Trastuzumab and paclitaxel have shown activity and good tolerability in HER-2/neu overex-pressing metastatic breast cancer patients. Tumor response in 10 responding taxanes pretreated patients was independent on TFI, but patients with longer TFI tend to be longer progression free. Key words: breast neoplasms – drug therapy; paclitaxel; treatment outcome; trastuzumab Introduction The HER-2/neu (c-erbB-2) oncogene encodes a 185 kD transmembrane protein with tyrosi­ne kinase activity. This glycoprotein belongs to the family of epidermal growth factor re­ceptors.1,2 Overexpression of HER-2/neu was found in several solid tumors including bre­ast cancer, lung cancer, prostate cancer, ova­rian, gastric and pancreatic cancer.3,4 In bre­ast cancer overexpression of HER-2/neu has important biological consequences. In precli­nical studies HER-2/neu was associated with higher tumorigenicity and metastatic potenti-al.5,6 HER-2/neu overexpression is conside­red as a negative prognostic factor in women with breast cancer and in several studies was confirmed the correlation with shorter disea­se free survival, overall survival and more ag­gressive tumors.7,8 HER-2/neu receptor crea­tes homodimers or heterodimers with other members of epidermal growth factor receptor family (EGFR, HER-3, HER-4) on the cell sur­face which leads to triggering a cascade of growth signals. Studies performed with viral ligands suggest that HER-2/neu evolved as li­gandless receptor9 or this ligand has not been identified so far. The most common and most potent association occurs between HER-2/neu and HER-3. Interestingly, HER-3 has no intrinsic tyrosine kinase activity and can­not respond to ligand binding unless associa­tes with another receptor, such as HER-2, which provides the intracellular signaling.10 Some level of HER-2/neu overexpression is found in 25 %-30 % breast cancers with HER-2/neu gene amplification detected in 95 % of the specimens.7, 11, 12 There are many possible ways to test for HER-2/neu overex- Received 17 May 2002 Accepted 31 May 2002 Correspondence to:Filip Janku, Comprehensive Can­cer Center, Charles University Prague, Onkologicka klinika VFN a 1. LF UK, U Nemocnice 2, 128 08 Praha 2, Czech Republic; janku@vfn.cz pression in tumor cells. Immunohistochemi­stry measuring protein expression on the cell surface is widely practiced by pathologists around the world and is fast and relatively cheap. Fluorescence in situ hybridization (FISH) measuring gene amplification is easily reproducible but expensive and not widely available. In general, there is a good agree­ment between the testing strategies. Howe­ver, there are cases of immunohistochemical “positive” tests with no evidence of gene am­plification. There are conversely cases of ge­netic amplification without increased surface expression.13 Currently used scoring system for immunohistochemistry has the scale from 0 to 3+. Results 0 and 1+ are understood as negative and 2+ and 3+ as positive.14 Nowa­days, there is trend to find only 3+ results as positive because in this subgroup is a concor­dance with FISH over 75 %15, 16. In 2+ subgroup the level of agreement rea­ches only 24%-39% with significantly lower response rates when treated with monoclonal antibody directed against this protein.15-17 The definition and standardization of optimal HER-2/neu assay is still in a process. Using the specific humanized anti-HER-2/neu monoclonal antibody trastuzumab (Herceptin; Roche ®) we can block the acti­vity of HER-2/neu protein and stimulate anti­body dependent cellular cytotoxicity.18 Tra­stuzumab demonstrated activity in clinical trials in women with HER-2/neu overexpres-sing metastatic breast cancer as a single agent achieving response rates ranged from 12 % to 27 %.19-21 Clinical trials based on preclinical evidence of synergy with many chemothera­peutic agents have been conducted. Trastuzu­mab was combined with cisplatin,22 or pacli-taxel,16 or vinorelbine23 in phase II studies with higher response rates than expected for chemotherapy alone. The pivotal phase III tri­al compared trastuzumab + chemotherapy to chemotherapy alone (either doxorubicin, cyclophosphamide or paclitaxel). Data indica­ted that trastuzumab in combination with chemotherapy produced significantly increa­sed time to progression, response rate and overall survival. Of particular note is that ad­dition of trastuzumab to paclitaxel therapy more than doubled median time to progressi­on and almost doubled the response rate.24 Regimens with combination of chemotherapy and trastuzumab were generally well tolera­ted. Pyretic reaction following first trastuzu­mab infusion occurred in 40 % of patients. So­me degree of cardiac dysfunction was observed in 27 % of patients treated with tra­stuzumab plus doxorubicin and cyclophos­phamide which excluded this combination from further clinical use. In trastuzumab plus paclitaxel arm such events were observed in 12 % of patients and only in 2 % were serio­us.24 However, trastuzumab plus paclitaxel seems to be a very potent combination, the vast majority of patients in our study were treated with another taxane, docetaxel, in the first or second line treatment. It needs to be clarified whether docetaxel pretreatment and time from the last docetaxel administration have any significant impact on treatment out­comes. Patients and methods Eligibility Women with histologically confirmed meta­static breast cancer overexpressing HER-2/neu were eligible for the purpose of this study. Patients had to be from 18 to 75 years old, with performance status at least 60 % ac­cording to the Karnofsky scale. All patients signed written informed consent. Patients were pretreated with two or more regimens for metastatic disease. In case of early recur­rence after adjuvant chemotherapy (less than 12 month) also patients pretreated only with one regimen for metastatic disease were eligi­ble. All patients were previously treated with antracyclines (mainly in the adjuvant setting) and all except one with taxanes. Any hormo­nal treatment except LHRH analogs had to be discontinued before study entry. Laboratory criteria included absolute neutrophile count (ANC) > 1 000/ul, hemoglobin > 80 g/l, plate­lets > 100 000/ul, adequate hepatic and renal function. Left ventricular ejection fraction had to exceed 50 % with exclusion of patients with history of serious cardiac disease. Pati­ents with clinically unstable metatastases to the brain were not allowed to enter the study. Patients were ineligible if they had a history of other malignancy (except carcinoma in situ of the cervix or nonmelanoma skin carcino­ma). Women with childbearing potential had to use reliable contraception while on study and had a negative pregnancy test before en­tering the study. Baseline evaluation included a complete physical examination, history, complete blood count with differential and platelet count, chemistry, echocardiogram, le­sion measurement as appropriate for disease assessment. Her-2/neu status was determi­ned using rabbit 4D5 antihuman HER-2/neu polyclonal antibody (HercepTest). Treatment Trastuzumab was administered 4 mg/kg in­travenously over 90 minutes as a loading do­se with subsequent weekly doses 2 mg/kg over 30 minutes. Paclitaxel 80 mg/m2 was administered intravenously over 60 minutes the day after trastuzumab loading dose and subsequently the same day after trastuzumab infusion. The treatment was delivered in the outpatient clinic of our department. Trea­tment was administered every week until di­sease progression or unacceptable toxicity. Paclitaxel could have been discontinued due to toxicity with further administration of tra­stuzumab alone until disease progression. Routine premedication before paclitaxel infu­sion consisted of 8 mg of dexamethasone in­travenously (IV), 100 mg cimetidine or 20 mg famotidine IV and 1 mg clemastine IV. Pacli-taxel was omitted or discontinued for hema­tologic toxicity (ANC < 1000/ul, platelets < 100000/ul), peripheral neuropathy grade 3 and higher. Response and toxicity evaluation Complete blood count was obtained every week and every other week when paclitaxel was discontinued. Serum biochemistry was repeated every four weeks. Echocardiography was performed at least every 16 weeks and at any other time if necessary. Toxicity was gra­ded according to Common Toxicity Criteria National Cancer Institute Version 2.0. The response was evaluated every three month. The same method as at baseline was used throughout the study. Complete respon­se was defined as a complete disappearance of all signs of tumor confirmed after 4 weeks or later. A partial response was defined as a more than 50 % reduction in the sum of pro­ducts. Progressive disease was defined as 25 % or bigger increase in the sum of pro­ducts. All other cases were evaluated as a sta­ble disease. Immunohistochemical analyses All specimens of either primary or metastatic tumor were tested for overexpression of HER-2/neu with polyclonal rabbit antihuman anti­body (HercepTest DAKO®). We used widely accepted scale when score 3+ is strongly posi­tive, score 2+ is moderately positive, score 1+ means weak positivity, and score 0 is negati­ve. Only patients with 3+ and 2+ results were eligible for protocol. No FISH analyses were performed. Statistical methods The primary endpoint of this trial was the overall response to the regimen combining trastuzumab and paclitaxel. The time to pro­gression (TTP) was defined as a time from study entry to progression. Overall survival (OS) was defined as a time from study entry to death. The median time to progression and median overall survival was estimated by the Kaplan-Meier method. TTP was censored in the following circumstances: patient was still receiving treatment without evidence of pro­gression, patient died of unknown cause wi­thout evidence of clinical deterioration due to breast cancer and patient discontinued trea­tment for any reason without evidence of cli­nical deterioration due to breast cancer befo­re discontinuation. The same criteria was applied for OS. All patients treated with me­tastatic breast cancer documented at study entry and treated were included in the effi­cacy intent-to-treat population. Safety analy­sis included all patients received at least one dose of study drug. Results Efficacy data Between July 1999 and January 2001, 17 eligi­ble patients were enrolled in our institution onto this study. The characteristics are listed in table 1. Only two patients had 2+ Hercep-test, all other results were 3+. All patients ex­cept one were pretreated with taxanes for me­tastatic disease (14 with docetaxel and 2 with paclitaxel administered every 3 weeks). Pati­ents were stratified according to the taxane-free interval to two groups: TFI > 1 year; TFI = 1 year. Altogether 599 cycles of treatment inclu­ding 473 cycles of trastuzumab plus paclita­xel were delivered. The median number of treatment cycles per patient was 33 (1-78). In one case only first dose of trastuzumab was given with subsequent severe hypersensitive reaction. This patient could not have been evaluated for response. There were no princi­pal protocol deviation. Paclitaxel was discon­tinued or omitted due to toxicity in 11 pati- Table 1. Patient characteristics (n = 17) Patients Characteristics No. % Age Median 50 Range 36-66 Prior chemotherapy 2 prior regimens = 3 prior regimens No. of metastatic sites 1 2 = 3 Visceral metastases Taxane free interval > 1 year = 1 year not pretreated with taxanes IHC HER2/neu (Herceptest) 3+ 2+ IHC ER ER + ER ­Unknown 6 11 7 4 6 11 7 9 1 15 2 4 11 2 35 65 41 24 35 64 41 53 6 88 12 24 64 12 Table 2. Response to therapy Patients Response No. % Overall response Complete response Partial response Stable disease Disease progression Nonassessable Response in patients with TFI = 1 year Response in patients with TFI > 1 year Response in patients with 3+ IHC 10 2 8 2 4 1 6 4 10 59 12 47 12 23 6 67 57 67 ents with permanent discontinuation in 6 pa­tients. Response data are listed in Table 2. There was 2 CRs and 8 PRs with an objective res­ponse rate 59 % in the intent-to-treat popula­tion. Two patients had stable disease for at le­ast 24 weeks and 4 patients progressed on therapy. The first CR occurred in 53 years old woman with infiltration of soft tissues of chest wall, and the second CR occurred in 36 years old woman with liver involvement. The first CR was maintained for 47 weeks and the second CR was still maintained at the time of analysis (45 weeks from the first documenta­tion). One patient with PR after 16 cycles was referred to surgery for removal of residual di­sease in contralateral breast. She remains di­sease free at the time of analysis. In the sub­group with TFI > 1 year was observed 3 PRs and 1 CR. In the subgroup with TFI = 1 year were achieved 5 PRs and 1 CR. TFI does not have any impact on the overall response (p < 0,4349). The median time to tumor progression in the intent-to-treat population was 6 month (range: 1-23,5) (Figure 1). At the time of analy­sis 4 patients (3 with TFI > 1 year 1 with TFI = 1 year) remained free of progression. Pati­ents with TFI > 1 year tended to stay longer progression free (p = 0,0201). The median survival was not reached at the time of analyses when 10 patients were alive. The 1-year survival in the intent-to-treat popu­lation was 53 % and the 2-year survival 12 %. Safety and toxicity data All 17 patients were evaluated for toxicity (Table 3). In total there were delivered 599 cy­cles including 473 cycles of paclitaxel plus trastuzumab with no dose adjustments to 17 patients. The median number of cycles per patient was 33 (range: 1-78). Paclitaxel was omitted or discontinued in 11 patients. Pacli­taxel toxicity, mainly neurotoxicity, led to permanent discontinuation of this drug in 6 patients. The most common adverse event was infusion related pyretic reaction after the first trastuzumab infusion in 6 patients (35 %). One patient experienced serious hypersensitivity reaction with dyspnea, shor­tness of breath and hypertension when recei­ving first trastuzumab infusion. This event led to treatment discontinuation. The dose li­miting adverse effect was also cardiotoxicity. Ejection fraction decline grade 2 occurred in 1 and grade 3 in 1 patient. The treatment was discontinued in both cases when one patient was in CR and one in PR with no further tu­mor regression. Hematological toxicity was very modest. We noted only 1 episode of gra­de 4 neutropenia and 1 episode of grade 3 anemia. No growth factors were administered and only 3 units of blood transfusion were gi- Table 3. Toxicity profile of weekly trastuzumab and paclitaxel NCI Grade (% of patients) Toxicity 2 3 4 Neutropenia Leucopenia Anemia Neuropathy Cardiotoxicity Infection Hypacusis Edema Nausea/Vomiting Heartburns Onycholysis Weight gain Transaminitis High glucose Infusion related reaction: 6 (35%) - 3 (18%) 1 (6%) 2 (12%) 1 (6%) 5 (29%) 2 (12%) 4 (24%) 1 (6%) 1 (6%) 1 (6%) 6 (35%) 3 (18%) - --1 (6%) 6 (35%) 1 (6%) 4 (24%) -----1 (6%) 6 (35 %) 1 (6%) 1 (6 %) ------------- ven. There were observed 3 episodes of grade 3 infection without neutropenia treated with antibiotics with no further complications. Grade 3 elevation of liver function tests oc­curred in 6 patients with no need of dose re­duction. Six patients experienced grade 3 ne­uropathy, which led to paclitaxel discontinu­ation in 5 patients. Other serious toxicity was very rare. We observed grade 3 weight gain in 1 patient, grade 2 weight gain in 6 patients and 1 episode of grade 3 hyperglycemia. It re­mains to be answered whether weight gain is related either to dexamethasone used as a premedication or to study drugs. Other toxi­city was only marginal (Table 3). Discussion At the time we started to accrue patients to this trial there were no phase II or III data pu­blished about the efficacy of trastuzumab plus weekly paclitaxel. We assumed sufficient effi­cacy based on results of pivotal trial combining trastuzumab with chemotherapy.24 This pivotal, multicentre phase III trial ran­domized 469 HER2 positive (2+, 3+) previo­usly untreated metastatic breast cancer pati­ents either to receive chemotherapy alone or chemotherapy plus trastuzumab. Four arms were designed as follows: AC every 3 weeks alone, AC plus trastuzumab weekly, paclita­xel every 3 weeks alone, or paclitaxel plus tra­stuzumab. The addition of trastuzumab to chemotherapy almost doubled response rate and prolonged overall survival. When we compare paclitaxel alone to paclitaxel plus trastuzumab the response rate was even mo­re than doubled however the response rate in paclitaxel alone was lower than expected. Most potent has been the combination of AC and trastuzumab but high proportion of car­diac events excluded this combination from further investigation. Nevertheless some gra­de of cardiac dysfunction occurred in both trastuzumab arms. In another pivotal phase II trial trastuzu­mab was administered as a single agent to 222 metastatic breast cancer patients who had failed on previous 1st or 2nd line chemo­therapy.20 The overall response rate in pretre­ated population was 15 % and 18 % in 3+ po­pulation. In the subset with positive FISH response rate was even 20 % and furthermore no FISH negative patient responded to the­rapy. The evaluation of HER2 expression was further investigated. There was found 75-89 % concordance between FISH positivity and 3+ result of immunohystochemistry16,17 but only 24-39 % of patients who were 2+ positive by immunohistochemistry had also positive FISH result. The relative lack of benefit in 2+ population implicated the suggestion that all 2+ results should be confirmed by FISH befo­re the treatment initiation. The results from preclinical studies sho­wed that trastuzumab has synergic or additi­ve effect with some other drugs including vi-norelbine, carboplatin, cisplatin, docetaxel, gemcitabin etc. Interesting data has come up from the trial with weekly trastuzumab and vinrolebine as the 1st, 2nd and 3rd line therapy of metastatic breast cancer with overall res­ponse rate exceeding 70 %.23 Just recently the­re were published data from trastuzumab and gemcitabine phase II trial.25 In several ongo­ing trials is trastuzumab combined with we­ekly docetaxel or carboplatin plus minus do­cetaxel.26 Because of promising safety and efficacy profile of trastuzumab combinations in the metastatic setting, this novel biologic agent now entered adjuvant breast cancer trials in the United States and Europe. Breast Cancer International Research Group conducts a cli­nical trial (BCIRG 006) for node positive early breast cancer patients.27 Other examples of this approach include the National Surgical Breast and Bowel Project clinical trial B-31, the North Central Cancer Treatment Group adjuvant trial 9831 and the Eastern Coopera- Figure 2. Trastuzumab adjuvant trials. PTX = paclitaxel; T = trastuzumab; AC = doxorubicin and cyclophosphamide; DTX = docetaxel, CBDCA = carboplatin q3w = every 3 weeks; w = weekly; S = surgery; RT = radiotherapy; CT chemotherapy 1) ECOG 2198 (N+) PTX q3w × 4 + T .AC × 4 .No T .T × 52 w 2) Intergroup NCCTG N 9831 (N+) AC q3w x 4 .PTX w × 12 .PTX w × 12 .T x 52 weeks . PTX w × 12 + T w . T × 40 weeks 3) NSABP B-31 (N+) AC q3w × 4 .PTX × 4 .PTX × 4 + T × 52 weeks 4) BCIRG (N+) number of adverse event. As the first group we assessed the effect of this combination with respect to the interval from the last taxa­ne administration – taxane free interval. We did not found any statistically significant cor­relation between taxane free interval and ove­rall response rate. Nevertheless, patients with longer taxane free interval tended to stay lon­ger progression free. References. 1. Hynes NE, Stern DF. The biology of erbB2­/neu/HER-2 and its role in cancer. Biochem Biophys Acta 1994; 1198: 165-84. 2. Akiyama T, Sudo C, Ogawara H, Toyoshima K, Ya-mamoto T. The product of the human c-erbB-2 ge­ne: A 185-kilodalton glycoprotein with tyrosine ki­nase activity. Science 1986; 232: 1644-6. ´ a AC × 4 .DTX × 4 3. Hung MC, Lau YK. Basic science of HER-2/neu: a ´ a AC × 4 .DTX × 4 + T × 52 weeks review. Semin Oncol 1999; 26: 51-9. ´ a DTX + CBDCA × 6 + T × 52 weeks 5) BIG HERA 4. Novotny J, Vedralova J, Kleibel Z, et al. C-erb-B-2 expression and k-ras mutations and pancreatic S + CT + RT .T q3w 1 year .T q3w 2nd year .observation .observation tive Oncology Group trial 2198 (Figure 2).26 The Breast International Group conducts an adjuvant trial called HERA which slightly dif­fers from other trials mentioned before beca­use trastuzumab is planned to be administe­red every 3 weeks (Figure 2).26,28 Completion of these ongoing trials through collaborative efforts between patients and scientific com­munity will allow us to obtain the results we so eagerly await. Conclusions In this small single institution prospective open labeled clinical trial we showed that tra­stuzumab and paclitaxel is active in the trea­tment of HER-2/neu overexpressing metasta­tic breast cancer patients with only limited cancer. Correlation with clinical course and patho­logical characteristic. Proc Am Soc Clin Oncol 1999; 19: 294a, [Abstract 1150]. 5. Pegram MD, Finn RS, Arzoo K, Beryt M, Pietras RJ, Slamon DJ. The effect of HER-2/neu overex­pression on chemotherapeutic drug sensitivity in human breast and ovarian cells. Oncogene 1997; 15(5): 537-47 6. Tan M, Yao J, Yu D. Overexpression of the c-erbB­2 gene enhanced intrinsic metastatic potential in human breast cancer cells without increasing the­ir transformation abilities. Cancer Res 1997; 57: 1199-205. 7. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ul­lrich A, McGuire WL. Human breast cancer: Cor­relation of relaps and survival with amplification of the HER-2/neu oncogene. Science 1987; 235: 177-82. 8. Gusterson BA, Gelber RD, Goldhirsch A, Price KN, Save-Soderborgh J, Anbazhagan R, et al. Pro­gnostic importance of c-erbB-2 expression in bre­ast cancer. J Clin Oncol 1992; 10(7): 1049-56. 9. Wallasch C, Weiss FU, Niederfellner G, Jallal B, Is-sing W, Ullrich A. Heregulin-dependent regulati­on of HER2/neu oncogenic signaling by heterdi­merization with HER3. EMBO J 1995; 14: 4267-75. 10. Wang SC, Hung MC. HER2 overexpression and cancer targeting. Sem Oncol 2001; 28(suppl 16): 115-24. 11. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE,et al. Studies of the HER-2/neu proto-oncogene in human breast and ovari­an cancer. Science 1989; 244: 707-12. 12. Berger MS, Locher GW, Saurer S, Gullick WJ, Wa­terfield MD, Groner B, et al. Correlation of c-erbB­2 gene amplification and protein expression in hu­man breast carcinoma with nodal status and nuclear grading. Cancer Res 1988; 48:1238-43. 13. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Comparison of fluorescence in situ hybridization and immunohistochemistry for the evaluation of HER-2/neu in breast cancer. J Clin Oncol 1999; 17: 1974-82. 14. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Specificity of HerceptTest in determi­ning HER-2/neu status of breast cancer using the United States Food and Drug Administration ap­proved scoring system. J Clin Oncol 1999; 17: 1983­87. 15. Seidman AD, Fornier MN, Esteva FJ, et al. Final re­port: weekly (W) Herceptin (H) and taxol (T) for metastatic breast cancer (MBC): analyses for effi­cacy by HER2 immunophenotype [immunohisto-chemistry (IHC)] and gene amplification [fluore­scent in situ hybridization (FISH)]. Proc Am Soc Clin Oncol 2000; 19: 83a, [Abstract 319]. 16. Seidman AD, Fornier MN, Esteva FJ, Tan L, Kapta-in S, Bach A, et al. Weekly trastuzumab and pacli­taxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification. J Clin Oncol 2000; 19: 2587-95. 17. Mass RD, Sanders C, Charlene K, et al. The con­cordance between clinical trials assay (CTA) and fluorescence in situ hybridization (FISH) in Her-ceptin pivotal trials. Proc Am Soc Clin Oncol 2000; 19: 75a, [Abstract 291]. 18. Harwerth IM, Wels W, Schlegel J, Muller M, Hynes NE. Monoclonal antibodies directed to the erbB-2 receptor inhibt in vivo tumor cell growth. Br J Cancer 1993; 68: 1140-5. 19. Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L, et al. Phase II study of of we­ekly intravenous recombinant humanized anti­p185HER2 monoclonal antibody in patients with HER2/neu overexpressing metastatic breast can­cer. J Clin Oncol 1996; 14: 737-44. 20. Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, et al. Multinational study of the efficacy and safety of humanized an­ti-HER2 monoclonal antibody in women who have HER2 overexpressing metastatic breast cancer that has progressed after chemotherapy for meta­static disease. J Clin Oncol 1999; 17: 2639-48. 21. Vogel C, Cobleigh M, Tripathy D, et al. First-line, nonhormonal treatment of women with HER2 overexpressing metastatic breast cancer with Her-ceptin (trastuzumab, humanized anti-HER2 anti­body). Proc Am Soc Clin Oncol 2000; 19: 71a, [Ab­stract 275]. 22. Pegram MD, Lipton A, Hayes DF, Weber BL, Ba-selga JM, Tripathy D, et al.. Phase II study of re-ceptor-enhanced chemosensitivity using recombi­nant humanized antibody-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998; 16: 2659-71. 23. Burstein HJ, Kuter I, Campos SM, Gelman RS, Tri­bou L, Parker LM, et al. Clinical activity of trastu­zumab and vinorelbine in women with HER2-ove­rexpressing metastatic breast cancer. J Clin Oncol 2001; 19(10): 2722-30 24. Slamon D, Leyland-Jones B, Shak S, et al.. Additi­on of Herceptin (humanized anti-HER2 antibody ) to first line chemotherapy for HER2 overexpres-sing metastatic breast cancer (HER2+/MBC) mar­kedly increases anticancer activity: A randomized multinational controlled phase III trial. Proc Am Soc Clin Oncol 1998; 17: 98a [Abstract 377]. 25. O’Shaughnessy JA, Vukelja S, Marsland T. Gemci­tabine and trastuzamab for HER-2 positive meta­static breast cancer: preliminary results of a phase II study. Proc San Antonio Breast Cancer Symp 2001; [Abstract 523]. 26. Winer EP, Burstein HJ. New combinations with Herceptin in metastatic breast cancer. Oncology 2001; 61 (Suppl. 2): 50-7. 27. Hortobagyi GN, Perez EA. Integration of trastuzu­mab into adjuvant systemic therapy of breast can­cer:ongoing and planned clinical trials. Sem Oncol 2001; 28 (Suppl 16): 41-6. 28. Hortobagyi GN. Overview of treatment results with trastuzumab (Herceptin) in metastatic breast cancer. Sem Oncol 2001; 28 (Suppl 18): 43-7. Radiol Oncol 2002; 36(2): 131-43. review Natural inhibitors of tumor-associated proteases Ulla Magdolen1, Janna Krol1, Sumito Sato1,3, Markus M. Mueller4, Stefan Sperl5, Achim Kr.ger2, Manfred Schmitt1, and Viktor Magdolen1, * 1Klinische Forschergruppe der Frauenklinik and 2Institut f.r Experimentelle Onkologie und Therapieforschung der Technischen Universit-t M.nchen, Germany; 3School of Biological and Molecular Sciences, Oxford Brookes University, Great Britain; 4Max-Planck-Institut f.r Biochemie, Martinsried, Germany; 5Wilex AG, M.nchen, Germany The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases in­volved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagena­ses, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the ac­tivity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or control­led inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease in­hibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tu­mor tissue is not balanced. Key words: cystein protease; cystatin; matrix metalloproteinase; serine protease; serpin; tissue inhibitor of matrix matalloproteinase Received 11 March 2002 Accepted 10 April 2002 Correspondence to: Viktor Magdolen, Ph.D., Klini­sche Forschergruppe der Frauenklinik der TU M.nchen, Klinikum rechts der Isar, Ismaninger Str. 22, This paper was presented at the Ň2nd Conference on D-81675 M.nchen; Germany. Tel.: ++49-89-4140-2493; Experimental Tumour BiologyÓ, Bovec, Slovenia, Fax: ++49-89-4140-7410; E-mail: viktor@magdolen.de March 14-17, 2002. PAI-1 and PAI-2, natural inhibitors of the urokinase-type plasminogen activator (uPA) The tumor cell surface-associated urokinase-type plasminogen activator system consists of the serine protease uPA, its receptor uPAR, and the inhibitors PAI-1 and PAI-2. Various normal and cancer cells produce uPA as a sin-gle-chain pro-enzyme (pro-uPA) that is prote­olytically converted to an active two-chain form, e.g. by plasmin or cysteine proteases such as cathepsins B and L.1 Binding of uPA to uPAR (CD87) focuses the proteolytic acti­vity on the tumor cell surface. In addition to uPAR, tumor cells also express binding sites for plasmin(ogen). uPAR-bound uPA effi­ciently converts tumor cell-associated plasmi­nogen into plasmin, an active serine protease with broad substrate specificity. Plasmin de­grades a variety of components of the extra­cellular matrix (e.g. fibrin, fibronectin, or la-minin) and activates several matrix metalloproteases that additionally break down certain macromolecules of the extracel­lular matrix such as fiber-forming collagens and/or the basement membrane protein col­lagen IV.2,3 The uPA/uPAR system is under the con­trol of the plasminogen activator inhibitors type 1 (PAI-1) and type 2 (PAI-2) both belon­ging to the serine protease inhibitor super-fa­mily (serpins) and sharing 55 % sequence ho­mology (amino acid identity: 33 %). Their structural similarity was shown by X-ray cry­stal structures of active mutants of PAI-1 and PAI-2 (Figure 1A, B).4,5 These two inhibitors interact with uPA and the other plasminogen activator, tPA (tissue-type plasminogen acti­vator), forming 1:1 stoichiometric complexes with the respective target protease. tPA, in contrast to uPA, does not bind to a high-affi­nity receptor on tumor cell surfaces and, the­refore, does not promote tumor cell-associa­ted pericellular proteolysis. Whereas PAI-1 recognizes and inhibits all active forms of the proteases (two-chain uPA as well as single-and two-chain tPA), PAI-2 only acts as an in­hibitor for the two-chain forms of uPA and tPA.6 For inhibition, the surface-exposed re­active center loop (RCL) of PAI-1 or PAI-2 in­teracts with the reactive site of the target pro­tease. Initially, the P1-P1Ő-bond of the inhibitor (R346-M347 in the case of PAI-1; R380-T381 in the case of PAI-2) is cleaved and a covalent bond between the hydroxyl-group of the catalytic serine residue of the protease and the carboxyl-group of the P1-residue of the RCL of the serpin is formed (acyl-enzyme intermediate). Upon cleavage of the P1-P1Ő­bond, the RCL is rapidly inserted into the central b-sheet A as additional b-strand 4A, which leads to the translocation (> 70 Ĺ) of the protease across the plane of b-sheet A of the serpin and the formation of a stable enz-yme/inhibitor complex. The structure of PAI­1 or PAI-2 in complex with a target protease has not been solved yet, however, the X-ray crystal structure of another serpin-protease complex (trypsin/antitrypsin) has recently been published.7 The experimental data con­firm the theoretical model for the inhibition of serine proteases by serpins.8,9 In the try­psin/antitrypsin complex, the structure of the hyperstable serpin is hardly changed, where­as the active site of the protease is massively disordered, which prevents the release of the protease from the complex.7 PAI-1 is synthesized as a 402 aa-protein (inclusive an N-terminal signal peptide) and secreted by the cell in a glycosylated form. The mature protein (379 aa; M: » 50,000) r contains no cysteines and, therefore, no di­sulfide bridges. Active PAI-1 is meta-stable and spontaneously converts to a latent form (which does not inhibit its target serine prote­ases) by inserting a major part of its RCL into the central b-sheet A (Figure 1C). The biologi­cally active conformation of PAI-1 (half-life: » two hours) is stabilized by vitronectin (Vn), an extracellular matrix (ECM) and plasma protein (half life of PAI-1 bound to Vn: » four hours).10 Upon binding of Vn to active PAI-1 Figure 1. The serpins PAI-1 and PAI-2 Ribbon representation of the tertiary structure of PAI-1 and PAI-2: (A) active conformation of PAI-1 (pdb-code: 1B3K), (B) active conformation of PAI-2 (pdb-code: 1BY7), (C) latent conformation of PAI-1(pdb-code: 1DVN), and (D) substrate cleaved form of PAI-1 (pdb-code: 9PAI). The central b-sheet is colored green, the reactive center loop (RCL) is yellow and the localization of the P1 and P1Ő residues are blue. In the active conformation of PAI-1 (A), the RCL represents a very flexible loop which is interlaced as an additional b-strand in the latent conformation (C), while in the substrate cleaved form the essential residues P1 and P1Ő of the RCL are pulled far apart (D). Because of its flexibility, the localisation of the RCL of PAI-2 was not clearly defined in the X-ray structure and is therefore not depicted in (B). This is also true for several other parts of the molecule. The figures were drawn on the coordinates from the Brookhaven Protein Database using the computer program INSIGHT II (Molecular Simulations Inc.). structural changes in PAI-1 are induced, pro­viding it with the inhibitory properties aga­inst the serine proteases other than uPA and tPA, namely thrombin and activated protein C.11,12 Recently, alpha(1)-acid glycoprotein has been shown to interact with PAI-1 as well and to stabilize the active form of this inhibi­tor.13 In addition to Vn and alpha(1)-acid glycoprotein, PAI-1 interacts with heparin, fi­brin, and Đ when present as a PAI-1/uPA complex Đ with members of the lipoprotein receptor-related protein (LRP) receptor fa­mily.6 Under certain conditions, when the distortion of the active site of the protease in complex with the serpin cannot keep pace with ester bond hydrolysis, PAI-1 can also be cleaved in a substrate-like manner, and the inhibitor is released from the active protease as the so-called RCL-cleaved form of PAI-1 (Figure D).14,15 PAI-2 lacks a cleavable signal peptide and is mainly present intracellularly in a non-glycosylated form (415 aa; M: » 47,000). Only r a small amount of PAI-2 (» 20%) is glycosyla-ted and secreted (M: » 55,000). PAI-2 sponta- r neously forms polymers, very likely by a loop-sheet polymerisation mechanism, in which the RCL of one molecule inserts as an additional b-strand into the central b-sheet of another molecule.16 The mainly intracellular location of PAI-2 developed a hypothesis that this serpin has some other functions in addi­tion to the inhibition of plasmin generation (which occurs extracellularly). In fact, PAI-2 has been shown to inhibit apoptosis.17 Fur­thermore, PAI-2 may function as an antiviral agent and be of relevance in AlzheimerŐs dise­ase and in some inflammatory reactions.16,18 The inhibitory effect of PAI-2 depends on both the active site and interhelical loop be­tween helices C and D. This loop has been im­plicated in transglutaminase-catalyzed cross-linking of PAI-2 to cell membranes.17 Besides the target proteases uPA and tPA (and trans-glutaminases), no further intra- or extracellu­lar interaction partners of PAI-2 have been identified so far. In addition to PAI-1 and PAI-2, there are other serpins, e.g. proteinase nexin-1, protein C inhibitor, and maspin (a serpin with tumor suppressive activity), which are also capable to inhibit uPA (and tPA) under physiological conditions.3,19 PAI-1 and PAI-2 in cancer In a variety of malignancies such as breast, ovarian, esophageal, gastric, colorectal or he-patocellular cancer, a strong clinical progno­stic impact has been attributed to compo­nents of the uPA-system, especially PAI-1 and uPA that are statistically independent factors with the capacity to predict the proba­bility of disease-free and/or overall survi­val.1,20-23 In general, elevated tumor antigen levels of PAI-1 and/or uPA are associated with poor disease outcome and are conduc­tive to tumor cell spread and metastases. The clinical finding that the uPA inhibitor PAI-1 does not exert a protective function but is an indicator of bad prognosis for cancer patients appears rather striking at first sight. How­ever, additional functions of PAI-1 have been described, which strongly suggest an involve­ment of PAI-1 in the tumor promoting proces­ses, especially in the modulation of tumor cell attachment or migration and in angiogene- sis.24,25 As the binding sites of PAI-1 and uPAR on the ECM protein Vn overlap, PAI-1 is able to regulate uPAR-mediated cell adhesi­on by competing with uPAR for binding to Vn. Furthermore, PAI-1/Vn-interaction also affects integrin-mediated cell adhesion to Vn by sterically blocking integrin binding to the RGD (Arg-Gly-Asp) sequence which is imme­diately adjacent to the PAI-1 binding site on Vn.26,27 Malignant murine keratinocytes, transplanted into PAI-1-deficient mice, did not invade the surrounding tissue (local inva­sion). Additionally, the PAI-1-deficient hosts failed to vascularize the implanted tumor cells. Upon intravenous injection of an ade-noviral vector expressing human PAI-1 in these tumor-bearing mice, tumor cell invasi­on and associated angiogenesis were resto­red.28 Some recently published data report of plasmin involvement in the assembly of new tumor vessels and indicate that PAI-1 is es­sential for controlling excessive plasmin pro­teolysis which would otherwise prevent the formation of these vessels.25,29 The PAI-1/Vn-interaction may also play a part in tumor ne­ovascularization.29 In contrast to the consistent association of high tumor tissue concentrations of PAI-1 (and uPA and uPAR) with poor prognosis, various studies analyzing the prognostic impact of PAI-2 have shown different associations betwe­en the PAI-2 levels in tumor tissue and patient survival. On the one hand, high antigen levels of PAI-2 in tumor tissue have been associated with good prognosis in patients with breast cancer, small-cell lung cancer and ovarian can­cer, but on the other, with a poor prognosis in colorectal and endometrial cancer.30,31 Biochemical properties of the TIMPs, the inhibitors of matrix metalloproteases In addition to the uPA/plasmin system, there is compelling evidence that matrix metallo-proteases (MMPs) also act as key players in the events that underlie tumor disseminati­on.32 Tumor and stromal cells produce solu­ble and cell-surface anchored MMPs, which mediate ECM degradation, release of seque­stered latent growth and angiogenic factors, and activation of latent growth factors. The proteolytic activity of MMPs is controlled by the so-called TIMPs. Currently four members of the TIMP-fa­mily (TIMP-1, -2, -3 and -4) are known and characterized.33 These are small proteins with a molecular weight between 21 kDa and 28 kDa and are secreted by many different cell types. They share common structural features including an N-terminal and a C-terminal subdomain, each stabilized by 3 disulfide bonds. Each TIMP has 12 conserved cysteine residues, contributing to the secondary struc­ture and their ability to inhibit MMPs.34,35 TIMP-1 is glycosylated (8-9 kDa), TIMP-3 can contain sugar components up to 7 kDa;36 whereas TIMP-2 and TIMP-4 are non-glyco­sylated. The N-terminal domain of the molecules harbors the inhibitory activity which forms a tight 1:1 non-covalent complex with the ca­talytic center of active MMPs (Figure 2). Bin­ding to latent MMPs occurs in a 1:1 stoichio­metry at the C-terminal region of individual MMPs.37 Recombinant truncated TIMPs con­taining only the N-terminal domain retain most of their inhibitory activity towards Figure 2. The TIMP-1/MMP-3 complex. Ribbon representation of the TIMP-1/MMP-3 complex structure (pdb-code: 1UEA): the N-terminal domain of TIMP-1 (residues 2-126) is colored yellow, the C-termi­nal domain in magenta; MMP-3 is green, catalytic zinc is red, calcium is blue and selenium orange. Only the N-terminal domain of the TIMP-1 molecule performs interactions to the MMP-3 protein in the complex. The figures were drawn on the coordinates from the Brookhaven Protein Database using the computer pro­gram INSIGHT II (Molecular Simulations Inc.) MMPs.38 The four different TIMPs bind to most subtypes of latent and active forms of MMPs with only minor differences in their in­hibitory potential. Variable affinities were elucidated for TIMP-1 and TIMP-2, the former binding to the latent form of MMP-9 with a higher affinity than the latter, while the con­verse relationship was found for binding to latent MMP-2.39 High levels of TIMP-2 or -3, but not of TIMP-1 inhibit the activity of MT1­MMP, thereby preventing the latent MMP-2 activation.40 The C-terminal domain of the TIMPs is more variable and is involved in the interacti­on with pro-MMPs.41,42 Furthermore, this do­main might be responsible for the additional biological functions of the TIMPs in prolifera­tion, angiogenesis, and apoptosis.43,44 These effects are independent of the inhibitory function of TIMPs; however, the mechanisms of these actions are not understood yet. TIMP-3 is unique because it is unsoluble and tightly bound to the extracellular matrix by its C-terminal domain. Furthermore, TIMP-3 is correlated with the hereditary dis­ease Sorsby`s fundus dystrophy that is cau­sed by a single base pair exchange in the se­quence coding for the C-terminal domain.45 Modulation of tumor growth and metastasis by TIMP expression in the tumor environment Recent clinical studies have stated TIMP-1 and TIMP-2 to be rather tumor-promoting mo­lecules, as they were found to be significantly overexpressed in patients with poor progno-sis.46,47 However, it is important to note that, in these studies, the MMP/TIMP ratio was not determined, while the evaluation of either TIMP or MMP expression alone is likely not sufficient for prognostication of malignancies. It is generally accepted that the net proteolytic activity in the tissue is responsible for tumor cell invasion-promoting ECM turnover. Con­sequently, (gene-) therapeutic intervention by overexpression of TIMPs in the tumor micro-environment is supposed to inhibit ECM de­gradation and metastasis.48 The MMPs, the target molecules for the TIMPs, are proteolyti­cally active in the extracellular space. Therefo­re, in a therapeutic approach, it is not neces­sary that all tumor cells are transduced by gene therapy vehicles to express elevated le­vels of TIMPs. It is sufficient that host cells, located at the interface surrounding the pri­mary tumor or the invading cells in the target organ of metastasis, overexpress and secrete TIMPs. The proteolytic balance in the tumor-host environment could thus be shifted in fa­vor of blocking proteolysis, resulting in the in­hibition of tumor invasion and metastasis. TIMP-1 transgenic mice have been used to elucidate the feasibility of such an approach and to assess the protective potential of host TIMP-1 on primary tumor growth and meta­stasis. In the first of these studies, the cross­ing of transgenic mice that constitutively ove­rexpress TIMP-1 in the liver with transgenics expressing SV40 T antigen, which develop he-patocellular carcinoma, resulted in inhibiting the tumor initiation, growth, and angiogene­sis.49 In another approach, two transgenic mo­use lines were used, one overexpressing TIMP-1 (TIMP-1high), and the other expressing the antisense TIMP-1 RNA, leading to TIMP-1 reduction in the tissue (TIMP-1low).50 TIMP-1 overexpression (TIMP-1high) inhibited tumor growth and spontaneous metastasis of an aggressive T-cell lymphoma, thereby prolon­ging the survival of mice. Opposite effects oc­curred in TIMP-1low mice: experimental metastasis assays demonstrated that TIMP-1­compromised livers in TIMP-1low mice sho­wed at least a doubling of metastatic foci and numerous additional micrometastases, indi­cative of increased host susceptibility.51 In another experimental setting, experimental metastasis of a fibrosarcoma in the brain could significantly be inhibited in transgenic mice overexpressing TIMP-1.50 Similar studi-es with transgenics for TIMP-2, -3, and -4 ha­ve not been reported so far. The encouraging results with TIMP transgenic mice have sti­mulated preclinical gene therapy experiments in mice. Employing adenoviral vectors for TIMP-2 gene transfer to the liver of mice pre­vented the growth of colorectal metastasis in this organ.52 Recently, the protection of the host environment was provided by the drama­tic overexpression of TIMP-1 due to adenovi­ral gene transfer, inhibiting the growth of ex­perimental liver metastasis of the T-cell lymphoma (employed in the studies with the transgenic mice mentioned above) and colo-rectal carcinoma.53 The systemic increase of TIMP-4 due to intramuscular gene delivery re­sulted in the inhibition of WilmŐs tumor growth,54 but induced mammary tumorigene-sis, most likely due to the anti-apoptotic fea­tures of TIMP-4.55 Gene therapy with vectors encoding TIMP-1 or TIMP-3 to protect the host environment from metastasis has not yet been documented. The vast literature on the genetic alteration of tumor cells themselves with TIMPs has re­vealed conflicting data on the usefulness of natural TIMPs in the direct genetic modifica­tion of tumor cells. However, these studies in­dicate that genetic engineering of TIMPs de­void of their additional biological functions (e.g. growth factor activity) to increase anti-tu­mor specificity, might be a useful therapeutic approach.33 Cystatins, the natural inhibitors of cysteine proteases Cystatins comprise single-chain inhibitory proteins that reversibly inhibit the proteolytic activity of cysteine proteases, which are wi­dely distributed in the human body.56-59 Three types of cystatins are present in verte­brates: type-1 cystatins that are synthesized without a signal peptide and generally pre­sent in the cell (cystatin A and B, also named stefin A and B); the secretory type-2 single-domain cystatins (C, D, M/E, F, S, SN, SA) and type-3 multi-domain cystatins (high and low molecular weight kininogens). The type-1 cystatins (approx. 100 aa; M: » 11- 12,000) r lack both disulfide bridges and carbohydrate groups. Type-2 cystatins (e.g. chicken cystatin and human cystatin C) are molecules of about 120 aa (M: » 13-14,000) and are characterized r by two intrachain disulfide bonds located to­wards the C-terminus. With the exception of the rat cystatin C, type-2 cystatins are non­glycosylated.60 Type-3 cystatins encompass three type-2 cystatin-like domains that most probably arose by gene duplications.61 They contain additional disulfide bonds and are glycosylated. The secondary structures of chicken cysta-tin, human cystatin C, and the type-1 cystatins are very similar. A cystatin molecule consists of an N-terminal straight five-turn a-helix ((1) and a five-stranded antiparallel b-pleated she­et (b1 N-terminal and b2-b5 C-terminal), twi­sted and wrapped around the a-helix.62 In the case of human cystatin C, no evidence was fo­und for an a-helical conformation of the regi­on unique to the type-2 cystatins (aa T71-L91) that was found in chicken cystatin.63 As further demonstrated by crystallogra­phic studies, human cystatin C forms very tight symmetric dimers by a mechanism cal­led three-dimensional domain swapping.64 Furthermore, higher aggregates may arise through this mechanism in an open-ended way, in which partially unfolded molecules are linked into infinite chains, also found in the brain arteries of elderly people with amy­loid angiopathy. An even more severe disease is associated with the L68Q mutant of human cystatin C that destabilizes the monomers and increases the stability of the partially un­folded intermediate causing massive amyloi­dosis, cerebral hemorrhage, and death in yo­ung adults.63 Cystatins form equimolar, tight and rever­sible complexes with papain-like cysteine Figure 3. Chicken cystatin. Ribbon representation of the structure of chicken cystatin (pdb-code: 1CEW). The residues essential for the inhibition of cysteine proteases are distributed on three different adjacent loops and are colored blue. The figures were drawn on the coordinates from the Brookhaven Protein Database using the computer pro­gram INSIGHT II (Molecular Simulations Inc.) proteases.65 There are three well-conserved regions in the cystatin superfamily that have been implicated in cysteine protease inhibiti­on (Figure 3). These regions are (i) a region near the N-terminus, (ii) a first hairpin loop containing the highly conserved sequence Gln-Xaa-Val-Xaa-Gly, and (iii) a second hair­pin loop containing a Pro-Trp pair.62,66 All three regions contain several hydrophobic re­sidues, indicating that hydrophobic interacti­ons play an important role in the interaction of cystatins with target molecules. Based on structural theory, these three regions penetra­te the active site of the enzyme in such a way that the papain active site Cys25 residue is blocked.62 An additional reactive site in the loop between the a-helix and the first strand of the main b-pleated sheet with its Asn39 re­sidue was detected and shown to be responsi­ble for the inhibition of mammalian legumin by some cystatins.67 The role of cystatins in cancer progression In mammals, cystatins are found in relatively high concentration in biological fluids such as seminal plasma, cerebrospinal fluid, plasma, saliva, and urine. Cystatins A, B, and C are present in high molar concentration in vario­us cells and tissues,66,68 whereas cystatins D, S, SN, and SA are almost limited to saliva, te­ars, and seminal plasma.59,69 Kininogens are major plasma proteins, involved in the tonus regulation of blood vessels and coagulation in addition to their function as cysteine endo-peptidase inhibitors.65 A large number of normal and patho-physiological processes are controlled by ba­lancing cysteine proteases and their inhibi­tors. Uncontrolled proteolysis by human cysteine proteases can cause irreversible da­mage such as inflammatory diseases, neuro­logical disorders, infection, and tumor meta­stases.70-74 Cathepsin B, H, and L, primarily lysosomal cysteine proteases, are also impor­tant matrix proteases that are involved, toge­ther with plasminogen activator and metallo-proteases, in cancer invasion by degrading extracellular matrix components.75-78 Cysta-tin C is the strongest inhibitor of cysteine proteases, it was therefore most frequently investigated in tumor invasion and metasta­sis. Tumor-associated expression of cystatin C was at first detected in the ascitic fluid from patients with ovarian cancer.79 Cystatin C is also increased in the blood of patients with breast cancer,80 fibrosarcoma,81 melano­ma,71 colorectal carcinoma,73 and lung can­cer.82 In these studies, it was reported that the cystatin C content in melanoma, colorec­tal, and lung cancer patients is associated with the progression of the malignant disea­se. Furthermore, cathepsin B/cystatin C com­plexes were found to be less abundant in the blood of patients with malignant tumors than in healthy controls indicating an imbalance between cysteine proteases and cystatin C in cancer cells.83 In comparison to normal tissue, a significant decrease of cystatin acti­vity in breast carcinomas compared to nor­mal tissue was noted80 also in the cerebrospi­nal fluid and blood from patients with brain tumors.84 An elevated concentration of the latent (inactive) fraction of cystatins was de­termined in the blood of patients with head and neck cancer and in the urine of patients with colorectal cancer.85,86 The significant de­crease of the inhibitory activity of cystatins in biological fluids in cancer patients may be taken as a further support to the assumption of an involvement of cysteine proteases in tu­mor progression and metastasis.75 Cystatin C activity is, in fact, correlated to the malignant phenotype as shown by in vitro and in vivo tumor model systems. Transfecti-on of cystatin C cDNA into B16 melanoma cells led to an inhibition of tumor cell invasi­on through an artificial matrix barrier in vitro and to a significant reduction of the number of lung metastases after the injection into the tail vein of nude mice.87,88 The inhibitory ef­fect of cystatin C on tumor cell invasion was also demonstrated in in vitro Matrigel assays using transfected murine SCC-VII squamous carcinoma cells,89 ras-transformed breast epi­thelial cells,90 human fibrosarcoma, and co­lon carcinoma cell lines.81 It is worth to men­tion that, in cystatin C-deficient mice, a reduction of lung colonization of mouse mela­noma cells (expressing cystatin C) was obser­ved.91 This indicates that an excessively high local cysteine protease activity may inhibit metastatic spread to some tissues. 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Cysteine proteinase inhibitors stefin A and stefin B in operable carcinoma of the head and neck Primož Strojan1, Marjan Budihna1, Lojze Šmid2, Branka Svetic3, Ivan Vrhovec3, Janko Kos4,5, Janez Škrk6 1Department of Radiotherapy, 3Department of Biochemistry, 6Department of Tumor Biology, Institute of Oncology, Ljubljana, Slovenia, 2Department of Otorhinolaryngology and Cervicofacial Surgery, University Medical Centre, Ljubljana, Slovenia 4Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Ljubljana Slovenia 5Department of Biochemical Research and Drug Design, Research and Development Division, KRKA, d.d., Ljubljana, Slovenia Purpose. To evaluate the significance of cysteine proteinase inhibitors stefins (Stefs) A and B for a trea­tment decision and prognosis in operable squamous cell carcinoma of the head and neck (SCCHN). Patients and methods. Stefs A and B concentrations were determined immunobiochemically using ELISAs in cytosols prepared from the tumor and adjacent normal mucosa from 91 patients with operable SCCHN. The median follow-up period of patients alive at the close-out date was 5.8 years (range, 5-9.3 years). Results. Stef A concentrations were significantly higher in tumor compared to normal mucosa (P=0.05). When a subgroup with clinically palpable node(s) at presentation was taken into consideration (n = 57), a significant difference in Stef A (P = 0.03) and Stef B (P = 0.02) concentrations between those with negative and positive necks, as determined on histopathological examination, was observed. On the univariate survi­val analysis, higher Stefs’ concentrations turned to be prognostically advantageous. Stef A proved its inde­pendent prognostic significance also on multivariate setting. Conclusions. With the capability to differentiate between the pN0- and pN+-stages of the disease in the pa­tients originally presented as node-positive, Stefs A and B could be useful markers when deciding on the ex­tent of neck surgery. In addition, both Stefs proved to be reliable prognosticators for survival in patients with operable SCCHN. Key words: head and neck neoplasms; carcinoma, squamous cell; cysteine proteinases inhibitors Received 22 March 2002 Accepted 29 April 2002 Correspondence to: Assist. Prof. Primož Strojan, M.D., Ph.D., Department of Radiation Oncology, In­stitute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slo-Acknowledgement: This work was supported by the venia; Phone: +386 1 232 3063; Fax: +386 1 431 41 80; Ministry of Science and Technology of the Republic of E-mail: pstrojan@onko-i.si Slovenia Grant J3-3010-0302-01. Introduction Alterations in the balance between cysteine proteinases and their endogenous inhibitors have been postulated to contribute to mali­gnant progression. A large body of literature has been accumulating evidence to suggest that they could be used as prognostic markers in a large spectrum of benign and malignant diseases.1 However, their prognostic signifi­cance in the squamous cell carcinoma of the head and neck (SCCHN) was much less inve­stigated. Our research team conducted the most extensive research in this field and arri­ved at the conclusion that the cytosolic con­centrations of the inhibitors stefins (Stefs) A and B were strongly related to the survival probability.2 The results on their serum con­centrations as well as those related to protei­nases were negative in this respect.2,3 The present study is a re-analysis of the da­ta on Stefs A and B in patients with exclusively operable disease, exploiting the advantage of longer follow-up. In addition to the prognostic value of Stefs A and B, their significance for a treatment decision was analysed. Patients and methods Patients Ninety-one previously untreated patients with primary operable squamous cell carcino­ma of the head and neck entered the study. The routine diagnostic work-up comprised a clinical examination and endoscopy of the ae­rodigestive tract, chest X-ray, and standard haematological and biochemical tests. In all patients, therapeutic surgery of the primary tumour related to the lesion extension, and neck node dissection were performed. Posto­perative radiotherapy was applied in all but nine patients with low-risk disease. The radi­otherapy doses were adapted to the disease extent and ranged between 50-66 Gy (medi­an, 56 Gy). Patients were irradiated on a Co­balt-60 unit or a 5-MV linear accelerator, with a daily dose of 1.8-2 Gy, 5 days per week. Tu­mors were staged after the histopathological examination of surgical specimens (patholo­gical stage) according to UICC TNM classifi­cation4, clinical N-stage before surgery was also recorded. The histopathological grade was defined according to WHO criteria.5 Cli­nical features of the patients and their tumors are shown in Table 1. As of October 31, 2001 (close-out date), 53 patients died: 22 due to the disease recurren­ce and/or dissemination and 31 due to causes other than the treated malignant disease. Thirty-three patients were alive with no signs of the disease and five patients were lost from follow-up; they were considered to be ineligi­ble for the analysis of survival. The median follow-up period of all eligible patients as cal­culated from the date of surgery was 4.3 years Table 1. Patient and tumor characteristics (n = 91) Patients Age: Median, 58 years (range, 37-72 years) Sex: Female, 6; Male, 85 Tumors Site: Oral cavity, 16 Oropharynx, 21 Hypopharyngis, 11 Larynx, 43 pTNM-stage: pN0 pN1 pN2 pN3 total pT1 3 2 2 0 7 pT2 13 4 13 1 31 pT3 15 2 11 0 28 pT4 10 2 13 0 25 total 41 10 39 1 91 Degree of differentiation: Grade1, 1 Grade2, 71 Grade3, 12 Grade, 7 x Extracapsular extension: 34a Tumor emboli in lymph node vessels: 7a a pN+ patient only, n = 50 . (range, 0.1-9.3 years), and those alive at the last follow up examination was 5.8 years (ran­ge, 5-9.3 years). Biochemical analysis of stefins For biochemical analysis of Stef A and Stef B, tissue specimens from the primary tumor and adjacent normal mucosa (matched pairs) we­re collected during surgery. The tissue cyto-sols were prepared as described in details el­sewhere.6 For quantitative analysis of Stefs A and B in tissue cytosols, commercially availa­ble ELISAs (KRKA d.d., Novo mesto, Slove­nia) were used, as developed at Jožef Stefan Institute, Ljubljana, Slovenia.6 The concentra­tions of Stefs in tissue cytosols were expres­sed as ng/mg of total protein (ng/mgp). Statistical analysis The differences between the median concen­trations of each of the Stefs in match pairs and various prognostic groups were determi­ned using nonparametric Wilcoxon signed rank test and Mann-Whitney U-test. In the analysis of the disease-free survival (DFS, lo­cal and/or regional recurrence and/or syste­mic dissemination considered as event) and the disease-specific survival (DSS, deaths from disease-unrelated causes censored), Ka­plan-Meier product-limit method7 was used, and the differences between the groups were tested by the log-rank test.8 The patients we­re grouped according to the cut-off concentra­tions of Stef A and Stef B, at which maximal differences in the survival rates were determi­ned. All tests were two-sided and the results were considered statistically significant at the probability level of 0.05. Results The distribution of Stefs A and B concentrati­ons in matched pairs is represented in Figure 1. Stef A concentrations were significantly higher in tumors compared to normal muco­sa (467 vs. 346 ng/mgp, P = 0.05); however, the difference in Stef B concentrations was not significant (285 vs. 269 ng/mgp, P > 0.05). At presentation, 34 patients were staged as node-negative (clinical stage, cN0) and 57 as node positive (cN). When a subgroup with + clinically positive neck nodes was taken into consideration, a significant difference in Stef A (536 vs. 380 ng/mgp, P = 0.03) and Stef B (382 vs. 240 ng/mgp, P = 0.02) concentrations was observed between those with negative and those with positive necks, as determined by histopathological examination after sur­gery (Figure 2). In node-negative subgroup, however, this difference didn’t reach the level of statistical significance. No statistically im­portant relationship was observed between Figure 2. Distribution of tumor concentrations of stefins between patients with histopathologically determined negative and positive necks, as measured in a group with clinically palpable nodes at presentation. The top and the bottom of the box represent the 25th and 75th percentiles, respectively, and the ends of the bars represent the rang. The line in the box is the median value. n, number of samples. Figure 3. Disease-free survival (DFS) and disease-specific survival (DSS) of patients with respect to the optimal cut­off concentrations of stefin A and stefin B. The numbers in parentheses indicate the number of events/total in each Table 2. Univariate and multivariate analysis of survival (n = 86) group. Stef concentrations and other established 0.0008, PDSS < 0.0001) (Figure 3). In ad­(PDFS = prognostic factors. dition, the pN0- and lower overall stage of the On univariate analysis, longer DFS and disease, the absence of extracapsular extensi- DSS correlated with higher concentrations of on and tumor emboli in lymph node vessels Stef A (PDFS = 0.0002, PDSS = 0.0003) and Stef B were also harbingers of favourable outcome Disease-free survival Disease-specific survival Variable Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis n %, 5-yr P-value P-value RR %, 5-yr P-value P-value RR Stefin A = 212 ng/mgp 22 40 0.0002 0.02 0.31 42 0.0003 0.04 0.35 > 212 ng/mgp 64 79 83 Stefin B = 112.6 ng/mgp 10 30 0.0008 NS 30 <0.0001 0.06 0.33 > 112.6 ng/mgp 76 75 79 Age =58 yrs 43 73 NS – 75 NS – > 58 yrs 43 65 68 pT-stage T1-3 62 73 NS – 79 NS – T4 24 60 66 pN-stage N0 37 80 0.08 NS 88 0.01 NS N1-3 49 61 60 pTNM StageI-III 36 85 0.01 NS 91 0.001 0.07 3.84 StageIV 50 57 59 Tumor site Larynx 38 77 NS – 80 NS – Non-larynxa 48 64 66 Tumor differentiation Grade1-2 70 70 NS – 74 NS – Grade3 12 73 73 Extracapsular spread Negative 52 81 0.003 0.05 4.71 87 0.0003 0.03 6.13 Positive 34 50 47 Tumor emboli Negative 76 71 0.03 NS 75 0.03 NS Positive 7 50 33 aOral cavity, oropharynx and hypopharynx. n, Number of patients; RR, Risk ratio; NS, Not significant. (Table 2). Radiation dose and other classical prognostic factors didn’t come out as progno­stically important. In Cox multivariate regres­sion analysis for DFS and DSS, only Stef A tu­mor concentrations (PDFS = 0.02, PDSS = 0.04) and extracapsular extension were retained in the final model (Table 2). Discussion In the present study we showed that Stef A and Stef B concentrations could be useful mar­kers when deciding on treatment intensity, and reliable prognosticators in patients with SCCHN. With the analysis restricted exclusi­vely to the patients with operable disease and the maturity of follow-up data, places a higher emphasis on the reliability of present results. As in our previous study on more hetero­geneous group of patients,2 only Stef A, but not Stef B, concentrations differed between tumor and normal mucosa. However, in nu­merous other reports on their levels and/or activity in malignant tissue compared with normal tissue the results are inconclusive, too.1 It seems that a significant elevation of inhibitor production is not the only option when proteolytic activity in tumor tissue is in­creased: it is more likely that the ratio betwe­en active/non-active or functioning/malfunc­tioning inhibitor molecules determines the net proteolytic potential in the cells. The new and the most important finding in the present study is that, in patients with clinically positive neck nodes at presentation (cN), Stef A and Stef B concentrations emer- + ged as reliable predictors of lymph-node in­volvement with tumor cells. From clinical po­int of view, this differentiation is of utmost importance because, in a considerable pro­portion of patients with SCCHN (in the pre­sent series, 26 %), the nodes are enlarged due to inflammation rather than tumor infiltrati­on. Those patients could be spared of more aggressive therapy, i.e. extensive neck sur­gery and/or high-dose radiotherapy, and trea­tment related side-effects. In this context, even though there is an overlap of individual values of inhibitor concentrations between those with pathologically positive and negati­ve necks, Stefs alone or in combination with other biological or clinical markers that wo­uld increase their diagnostic accuracy war­rant further evaluation. In patients with clinically undetectable no­des at diagnosis (cN0), Stefs had no potential to predict pN-stage of the disease, which is not of critical importance from clinical pers­pective. In this group, 76% of patients were without tumor cells at histopathological exa­mination after neck dissection. If surgery is technically correctly performed, postoperati­ve radiotherapy is not indicated and its side effects could be avoided. Only a minority of patients (24 %) were found to have a micro­scopic tumor deposits in the neck nodes which are highly curable with a moderate-do­se radiotherapy, i.e. = 95 % cure rate with 50 Gy.9 As the pattern of spread of neoplastic cells from the primary tumor to regional lymph nodes is predictable,10 the risk of geo­graphic miss could be neglected. In addition, those neck regions with the highest risk for bearing micrometastases are usually in imme­diate vicinity of the primary. The most welco­me consequence is that when there is an indi­cation for primary to be irradiated postopera­tively, the majority if not all nodal basins at risk are also included in the high-dose irradi­ation volume. Following these propositions, neck surgery is not always necessary prior to irradiation and these patients can be spared of its morbidity. The results of univariate analysis of the survival showed that the patients with Stef A or Stef B concentrations above the calculated cut-off concentrations do significantly better than those with lower concentrations of either inhibitor. In addition, Stef A tumor concentra­tion turned to be independent prognosticator for the risk of relapse and death in our group of patients. These results are consistent with our earlier observation2 and further support the concept of protective role of Stefs A and B, previously raised in the studies on carcinoma of the breast11 and lung.12 The studies that contradict this assumption are those by Kuo­pio et al.13 on breast cancer and by Kos et al.14 on colorectal cancer. However, in the first, the Stefs’ content was determined immunohisto-chemically, while in the other, their extracel­lular, i.e. serum concentrations were measu­red, which may reflect the involvement of Stefs in mechanisms other than the control of extracellular matrix degradation and invasion of tumor cells. Because the origin of inhibitor molecules in different sample types and/or modes of quantification of their content diffe-red substantially from the type of analysis used in our and related reports,11,12 a simple comparison would be inadmissible. Accor­ding to our experience, the prognostic poten­tial of immunohistochemically determined Stef expression and their serum concentrati­ons in SCCHN is yet to be defined.3,15 On the basis of the presented results our conclusions would be as follows: (1) With the capability to differentiate between pathologi­cally positive and negative necks in patients originally presented as node-positive, Stef A and Stef B could be useful markers when de­ciding on the extent of neck surgery; (2) Stefs A and B proved to be reliable prognosticators for the survival of patients with operable SC­CHN. References 1. Kos J, Lah TT. Cysteine proteinases and their en­dogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer. [Review]. Oncol Rep 1998; 5: 1349-61. 2. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Kos J, et al. Prognostic significance of cysteine pro-teinases cathepsins B and L and their endogenous inhibitors stefins A and B in patients with squa­mous cell carcinoma of the head and neck. Clin Cancer Res 2000; 6: 1052-62. 3. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I,Škrk J. Cathepsin B and L and stefin A and B levels as serum tumour markers in squamous cell carci­noma of the head and neck. Neoplasma 2001; 48: 66-71. 4. Sobin LH, Wittekind Ch. TNM classification of malignant tumours. International Union Against Cancer (UICC). 5th ed. New York: Wiley-Liss; 1997. p. 20-37. 5. Azzopardi JG, Chepizk OF, Hartman WH. Interna­tional histological classification of tumours no.2: histo­logical typing of breast tumours. 2nd ed. Geneva: World Health Organisation; 1981. 6. Kos J, Šmid A, Krašovec M, Svetic B, Lenarcic B, Vrhovec I, et al. Lysosomal proteases cathepsins D, B, H, L and their inhibitors stefins A and B in head and neck cancer. Biol Chem Hoppe-Seyler 1995; 376: 401-5. 7. Kaplan EL, Meier P. Nonparametric estimation from incomplete observation. J Am Stat Assoc 1958; 53: 457-81. 8. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, et al. Design and analysis of ran-domised clinical trials requiring prolonged obser­vation of each patients. II. Analysis and examples. Br J Cancer 1977; 35: 1-39. 9. Withers HR, Peters LJ, Taylor JMGP. Dose-respon­se relationship for radiation therapy of subclinical disease. Int J Radiat Oncol Biol Phys 1995; 31: 353­9. 10. Grégoir V, Coche E, Cosnard G, Hamoir M, Rey-chler. Selection and delineation of lymph node tar­get volumes in head and neck conformal radiothe­rapy. Proposal for standardizing terminology and procedure based on the surgical experience. Radi-other Oncol 2000; 56: 135-50. 11. Lah TT, Kos J, Blejec A, Frkovic-Georgijo S, Golo-uh R, Vrhovec I, et al. The expression of lysosomal proteinases and their inhibitors in breast cancer: possible relationship to prognosis of the disease. Pathol Oncol Res 1997; 3: 89-99. 12. Ebert E, Werle B, Jülke B, Kopitar-Jerala N, Kos J, Lah T, et al. Expression of cysteine proteinase in­hibitors stefin A, stefin B and cystatin C in human lung tumors. Adv Exp Med Biol 1997; 421: 259-65. 13. Kuopio T, Kankaanranta A, Jalava P, Kronqvist P, Kotkansalo T, Weber E, et al. Cysteine proteinase inhibitor cystatin A in breast cancer. Cancer Res 1998; 58: 432-6. 14. Kos J, Krašovec M, Cimerman N, Nielsen HJ, Chri­stensen IJ, Brünner N. Cysteine proteinase inhibi­tors stefin A, stefin B, and cystatin C in sera from patients with colorectal cancer: relation to progno­sis. Clin Cancer Res 2001; 6: 505-11. 15. Strojan P, Šmid L, Budihna M, Gale N, Svetic B, Vrhovec I, et al. The expression of stefins A and B in supraglottic carcinoma: immunobiochemical and immunohistochemical study. In: Bosatra A, Gale N, Michaels L, Pavelic K, Vizjak A, Zidar N, et al, editors. Epithelial tumours of the head and neck. Proceeding of the XXXIst memorial meeting for profes­sor Janez Plecnik, Ljubljana 2000. Ljubljana: Institu­te of Pathology, Faculty of Medicine University of Ljubljana; 2000. p. 38-42. Radiol Oncol 2002; 36(2): 153. 1st Workshop on Experimental Tumour Biology March 16 – 18, 2000 Bovec, Slovenia Organised by Tamara Lah (National Institute of Biology) and Janko Kos (Jožef Stefan Institute & Krka, d.d., Ljubljana) Application of modern experimental methods in cancer biology generates new knowledge to understand the mechanisms of development and progression of malignant disease. With the rapid development of molecu­lar biology, new tools are becoming available to study tumour progression – from carcinogenesis to tumour cell invasion and metastasis. The aim of the 1st Workshop in Experimental Tumour Biology was to select some topics, covered by the scientists from some research laboratories in Slovenia, working in the field of tumour biology and by their collaborators from abroad. It has become essential, especially in a small scien­tific community, such as Slovenia, that the researches in related areas are well and promptly informed with the latest data of their work. The exchange of information in this field is especially important for young in­vestigators, starting their careers, and this Workshop is giving them the opportunity to get a new knowled­ge and to discuss the research and experimental problems with their young colleagues and senior researches. The workshop was divided into four major sections. Carcinogenesis starts from the initial DNA damage, which is caused by external agents, present in food and beverages, by radiation and other environmental pollutants. To study their effects at molecular level, several new tests were introduced in vitro at cellular le­vel and in vivo in animal models. This is covered in the first section on Detection of genetic alterations in cancer, where mutagenicity assays, including the newly developed Comet assays, and the tumorigenicity as­says in animal models were applied to test the effects of heavy metals and bacterial toxins, possibly present in drinking water. Next, in vitro and in vivo studies of tumour progression were discussed, where recent data on breast and brain tumour cells invasion were presented. Diagnosis of malignant progression and prognosis of the dise­ase are clearly related and can be improved by detecting new biological markers of malignancy, such as pro-teolytical enzymes, especially cathepsins and their endogenous inhibitors. This was covered in the section of Immunoassays in cancer biology. The growth of malignant tumours is associated with angiogenesis, new vessels formation, induced by the tu­mour cells, and understanding of the process is essential for prognosis as well as for the development of new therapeutic anti-angiogenesis agents, as discussed the in last section on Tumour angiogenesis. We strongly believe that the success of the first workshop will lead to next meetings of this kind and we ho­pe that they will become traditional in the future. Radiol Oncol 2002; 36(2): 154-5. Genetic toxicology: from exposure detection to cancer prevention Metka Filipic Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenija There is a general concern that many environmental chemicals to which humans are exposed are genotoxic and may cause cancer. The main problem is not only to identify the environmental genotoxic pollutants, but also to characterise their mode of action at cellular and molecular levels. To identify genotoxic environmen­tal pollutants we are using selected in vitro short-term assays. For fast screening for the presence of geno-toxic substances in wastewater and fresh wa­ters samples we use modifed SOS/umu test and Salmonella/microsomal assay. The modi­fied assays are sensitive enough to detect low concentrations of genotoxic pollutants in non-concentrated water samples. We have demonstrated by using bacterial strains with elevated nitroreductase and/or O-acetyltrans­ferase activity, that nitro polyaromatic hydro­carbons are important genotoxic contami­nantns in water. We suggest to use the modifed SOS/umu for rutine controling the efficiency of wastewater treatment plants. For detection of mutations, which are the consequence of DNA damage and the proces­sing of the damaged DNA, we are using the Comet assay or single-cell gel electrophoresis (SCGE). This is a sensitive method for detec­ting DNA strand breaks at the level of indivi­dual cells. Cells embedded in agarose are lysed, subjected to alkaline unvinding, electro-phoresed and stained. DNA, broken and rela­xed in the electric field, migrates toward the anode, resembling a shape of a comet with bright fluorescent head and a tail region, which increases, as the DNA damage gets mo­re severe. The “comets” are measured and analysed using video image analysis: DNA da­mage is quantified by tail length, tail moment and percentage of DNA in the tail. In our hands, the comet assay proved to be a valuable tool to study the biochemical and physicoche­mical mechanisms of DNA damage and repa­ir. Based on the Comet assay we are develo­ping a biomarker for detection differences in DNA repair capacity in populations exposed to heavy metals (Cd and Pb) compared to non-exposed. As heavy metals inhibit DNA repair, we supose that in populations exposed to he­avy metals cellular DNA repair capacity is af­fected, leading to enhanced risk for cancer. For identification of cancer preventive agents and potential antimutagens, we found, that the extract of Salvia officinalis inhibited UV induced SOS response and mutagenesis in bacteria. The results indicate that Salvia of-ficinalis extract acts as an inhibitor of SOS functions as well as a promoter of DNA exci­sion repair processes. For screening large number of samples we use SOS/umu test, mo-difed for detection of potential antimutagens. The samples that show antimuatgenic poten­tial in SOS/umu test are than tested for anti-mutagenic activity in bacterial reverse mutati­on assay using different strains to allow insight in to the mode of action. Using this approach, we tested 121 mushroom extracts for antigenotoxic activity. Seven of them inhi­bited UV induced SOS response by more than 50 % and three of them were effective al­so against oxidative damage. Radiol Oncol 2002; 36(2): 156-8. Cadmium induced DNA damage in human hepatoma (Hep G2) and Chinese hamster ovary (CHO) cells Tanja Fatur and Metka Filipic Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000, Ljubljana, Slovenia Introduction Cadmuim (Cd) is one of the most important heavy metal environmental toxicants. It accu­mulates in human tissues, particularly in ki­dney and liver. Cd is classified as probable hu­man carcinogen by IARC.1 The genotoxic potential of Cd is rather weak and restricted to high cytotoxic concentrations. However, at low concentrations Cd enhances genotoxicity of other DNA damaging agents.2 It was shown that Cd interferes with nucleotide excision re­pair (NER) by inhibiting DNA damage recogni­tion and incision step of NER.3 The aim of the study was explore the DNA damaging potenti­al of Cd and its interference with the repair of UV induced DNA damage in vitro, using Co­met assay. We studied the induction of DNA single strand breaks (ssb) by low nanomolar concentrations of CdCl2 after different durati­on of exposure on human hepatoma cell line (Hep G2). The effect of CdCl2 on the repair of UV induced DNA damage was studied in Chi­nese hamster ovary cells (CHO). The results of the formation and disappearance of DNA ssb after different periods of recovery after the UV irradiation, reflecting the nucleotide excision repair (NER) kinetics, are presented. Materials and methods Cell lines Human hepatoma cells (HepG2) cells were cultured in Williams Medium containing 10 % FBS at 37 °C in humidified atmosphere with 5% CO2. Chinese hamster ovarian (CHO) cells were cultured in F-12 medium contai­ning 10% FBS at 37°C in humidified atmos­phere with 5 % CO2. Treatment of cells HepG2 cells were incubated with low, non-to­xic concentrations of CdCl2 (10 nM, 100 nM and 1000 nM) in complete growth medium for 3, 6, 9, 12, 24 and 72 h. After the incubati­on period the cells were harvested and su­bjected to alkaline single cell electrophoresis (Comet assay). CHO cells were seeded in 25 cm2 tissue culture flasks one day prior to tre­atment with Cd. Five hours prior to the UV ir­radiation the cells were treated with 1 µM and 10 µM CdCl2 in complete growth medium. Af­ter the treatment the cells were washed with PBS, harvested, suspended in PBS and UV ir­radiated (20’’, 50-cm distance). The irradiated cells were subjected to Comet assay after the recovery period of 0, 10, 20, 30 and 60 minu­tes at 37 °C. Comet assay The cells (HepG2 or CHO) were embedded in 1 % LMP agarose on pre-coated microscope slide and lysed at 4 °C for 1 h (2.5M NaCl, 100mM EDTA, 10 mM Tris, 1 % Triton X-100, pH10). The slides were then placed in the electrophoresis solution (300 mM NaOH, 1 mM EDTANa2, pH13) for 20 min at 4 °C to allow DNA unwinding and electrophoresed for 20 min at 25V (300 mA). After electropho­resis slides were neutralized (0,4 M Tris, pH 7,5) and stained with EtBr. 50-100 cell nuclei per each experimental point were examined at 400 magnification using a fluorescence mi­croscope (Olympus) and analyzed with the software VisCOMET. The DNA damage is ex­pressed as comet tail moment, which is defi­ned as product of the comet length and per­centage of DNA in tail. Results and discussion Figure 1 shows the effects of low non-toxic concentrations of CdCl2 on DNA damage in HepG2 cells. Level of DNA damage was as­sessed after different periods of incubation with CdCl2 by measuring the increase of co­met tail moments. Incubation with 10 nM, 100 nM and 1000 nM CdCl2 caused a dose-dependent increase of DNA damage. The DNA damage increased also with the increa­sing time of exposure to CdCl2 up to 12 h. However, when the cells were incubated for 24 h the DNA damage was lower than after 12 h incubation. After 72 h incubation DNA damage was detected only in cells treated with 1000 nM CdCl2. This time dependent decrease of DNA damage can be due to the Cd mediated induction of the synthesis of metallothioneins, which are known to play role in cellular defence mechanism against Cd toxicity.4 UV irradiation induces pyrimidine dimers and 6-4 photoproducts that are repaired pre­dominantly by NER. With the Comet assay ssb are detected, which reflect the incision step of the NER. In CdCl2 pretreated cells the tail moment was lower than in control cells (Figure 2) indicating that CdCl2 prevented the incision step of NER. After 60 minutes of re­covery the residual ssb were higher in CdCl2 treated cells compared to the control, which might reflect either slower NER or inhibition of ligation step of NER. This result confirms the interference of Cd with NER. Figure 2. DNA repair kinetic of UV induced DNA da­mage in CHO cells. Non-treated or CdCl2 pre-treated cells were exposed to UV irradiation and than incuba­ted at 37oC. At different intervals samples were taken for comet assay. 100 comets per experimental point were analysed by image analysis system. Conclusion In conclusion, our results in Hep2G cells sho­wed that ssb were induced at 10 nM of CdCl2, which is the concentration that corresponds to the concentrations of Cd found in blood of environmentally exposed population.5 This damage was detected only after short time of exposure. In CHO cells, we demonstrated, that when cells are exposed to CdCl2 for a short time, the repair of UV induced DNA da­mage was inhibited. Further experiments are in progress to explore the role of metallothio­neins in protection against genotoxic and co-genotoxic effects of Cd. References 4. Waalkers MP and Goering PL. Metallothionein and other Cd-binding proteins: Recent develo­pments. Chem Res Toxicol 1990; 3: 281-8. 1. IARC. Monographs on the Evaluation of Carcino­genic Risks to Humans: Beryllium, Cd, Mercury 5. Skerfving S, Bencko V, Vachter M, Schütz A, Ger-and Exposures in Glass Manufacturing Indu­hardsson L. Environmental health in the Baltic re­stry,Vol 58, IARC, Lyon, 1993. gion-toxic metals. Scand J Work Env Hea 1999; 25 (Suppl 3): 40-64. 2. Hartwig A. Current aspects in metal genotoxicity. Biometals 1995; 8: 3-11. 3. Hartwig A, Schlepegrell R, Dally H. Interaction of carcinogenic metal compounds with deoxyribonu­cleic acid repair processes. Ann Clin Lab Sci 1996; 26: 31-8. Radiol Oncol 2002; 36(2): 159-61. In vitro genotoxicity of microcystin-RR on primary cultured rat hepatocites and Hep G2 cell line detected by Comet assay Bojana Žegura1, Metka Filipic1, Dušan Šuput2, Tamara Lah1 and Bojan Sedmak1 1National Institute of Biology, Vecna pot 111, 1001 Ljubljana, Slovenia; 2Medical Faculty, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia Introduction Microcystins are hepatotoxic cyclic heptapep-tides produced by different species of bloom forming cyanobacteria (Microcystis, Anabaena, Nostoc Oscillatoria).1 The primary target of the toxin is the liver. The uptake of microcystins into the hepatocites occurs via carrier-media­ted transport system. Microcystins cause cytoskeletal damage, necrosis and pooling of blood in the liver, with a consequent increase in the liver weight. The cause of death is a massive hepatic haemorrhage.2 Microcystins are inhibitors of serine/threo-nine protein phosphatases 1 and 2A and act as tumor-promoters.3 Ito and coworkers4 de­monstrated that microcystins induced neo­plastic nodules in the liver after repeated injections without an initiator, which indica­tes that they might act also as tumor initia­tors. The aim of our studies was to elucidate possible genotoxic effects of microcystin-RR (MCYST-RR) at molecular level using Comet assay. The Comet assay is a sensitive method for detection of DNA strand breaks at the le­vel of a single cell.5 DNA single-strand breaks can lead to mutations, which are the first step in carcinogensis. Materials and methods Primary cultured rat hepatocytes and the cell line Hep G2 Primary rat hepatocytes were isolated from rat liver by a three-step collagenase perfusion method described by Doyle.6 The isolated he­patocytes and Hep G2 (human hepatoma cell line) were cultured in Williams Medium con­taining 10 % FBS for four h at 37 °C in humi­dified atmosphere with 5 % CO2. Cell treatment The primary cultured rat hepatocytes were rin­sed with phosphate buffer saline (PBS) (Mg2+ and Ca2+ free) and then incubated with diffe­rent concentrations (0.01, 0.1 and 1 µg/ml) of microcystin-RR (MCYST-RR) in Williams Me­dium containing 10 % FBS for 3 and 13 h. Con­trol cultures were treated with solvent (metha­nol) only. After the treatment hepatocytes were rinsed with PBS, trypsinised and centrifuged at 1000 rpm for 10 min. Similarly, the Hep G2 cells were incubated with different concentrati­ons of MCYST-RR in Williams Medium contai­ning 10 % FBS for 13 h, rinsed with PBS, trypsi­nised and centrifuged at 1000 rpm for 10 min Comet assay 30 µl of a freshly prepared suspension of cells (3,5x105) and 70 µl low melting point agarose were added to a fully frosted microscope slide, precoated with 80 µl of 1 % normal melting po­int agarose, covered with the top slide. The top slide was removed after solidifying and the cells were lysed at 4 °C for 1 h (2.5M NaCl, 100mM EDTA, 10 mM Tris, 1 % Triton X-100, pH10). The slides were then placed in the elec­trophoresis solution (300mM NaOH, 1mM EDTANa2, pH13) for about 20 min at 4 °C to allow DNA unwinding before electrophoresis. Samples were electrophoresed for 20 min at 25 V (300 mA). After electrophoresis the slides were placed in 0.4 M Tris buffer (pH7.5) for 15 minutes in order to neutralise and then stained with ethidium bromide (5 µg/ml). The 100 cell nuclei were examined at 400 magnification us­ing a fluorescence microscope (Olympus) and analyzed with the software VisCOMET. MTT cytotoxicity test was performed as described previously by Immamura.7 Results Significant increase in DNA damage of the primary cultured rat hepatocytes treated with MCYST-RR when compared to the (solvent) control was observed only after prolonged treatment for 13 h. The MTT test showed that MCYST-RR was not toxic at the tested con­centrations (results not shown). Hep G2 cells were treated with the same concentrations of MC-RR and for the same ti­me as primary cultured rat hepatocytes. The results showed a dose-response similar to that of isolated cells. MC-RR was not toxic to Hep G2 cells at the tested concentrations. Discussion The genotoxic potential of microcystin RR was evaluated since it is the most frequent and abundant hepatotoxin in Slovene surface water bodies.8 The results demonstrated that MCYST-RR induced dose dependent DNA damage in both, primary cultured rat hepa­tocytes and in Hep G2 cell line at the nonto­xic concentrations. With the comet assay DNA damage has already been demonstrated in primary cultured rat hepatocytes after the Figure 1. Olive tail moment of primary cultured rat hepatocytes (A) and the cell line Hep G2 (B) treated with dif­ferent concentrations of microcystin-RR. The cells were treated for 13 h. Mean values are presented. Methanol (sol­vent) was used as negative and B(a)P was used as a positive control. treatment with MCYST-LR (1 µg/ml).9 In the­ir experiments the DNA damage was induced after 4 h treatment, while in our experiment MCYST-RR was effective only after treatment for 13 h. MCYST-RR is known to be 10 times less biologically active in comparison to MCYST-LR.10 This could explain the differen­ce in exposure time, needed to induce DNA damage. The fact, that the effects are time de­pendent corroborates the importance of chro­nic in vivo experiments for the extrapolation of health risks. References 1. Carmichael WW. Cyanobacteria secondary meta­bolites – the cyanotoxins. A review. J Appl Bact 1992; 72: 445-59. 2. Dawson RM. The toxicology of microcystins. Toxi-con 1997; 36: 953-62. 3. Holmes CFB, Boland MP. Inhibitors of protein phosphatase-1 and -2A; two of the major seri­ne/threonine protein phosphatases involved in cellular regulation. Curr Opin Struc Biol 1993; 3: 934-43. 4. Ito E, Kondo F, Terao K, Harada K. Neoplastic no­dular formation in mouse liver induced by repea­ted intraperitoneal injection of microcystin-LR. To-xicon 1997; 35: 1353-457. 5. Fairbairn DW, Olive PL and O’Neill KL. The co­met assay: a comprehensive review. Mutat Res 1995; 339: 37-59. 6. Doyle A, Griffiths JB and Newell DG. Cell & Tis­sue Culture: Laboratory Procedures. Morule 12B: 14, 16, 17, 1996. 7. Immamura H, Takao S, Aikou T. A modified inva­sion-3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenylte­trazolium bromide assay for quantitating tumor cell invasion. Cancer Res 1994; 54: 3620-24. 8. Sedmak B, Kosi G. Microcystins in Slovene fresh­waters (Central Europe) – first report. Nat toxins 1997; 5: 64-73. 9. Ding W, Shen H, Zhu H, Lee B, Ohg C. Genotoxi-city of microcistic cyanobacteria extract of water source in China. Mutat Res 1999; 442: 69-77. 10. Rinehart KL, Namikoshi M and Choi BW. Structu­re and biosynthesis of toxins from blue-green al­gae (cyanobacteria). J Appl Physiol 1994; 6: 159-76. Radiol Oncol 2002; 36(2): 162-4. Co-operative effects in tumorigenicity. The microcystin example. Bojan Sedmak1 and Dušan Šuput2 1Department of Ecotoxinology and Ecotoxicology, National Institute of Biology, Vecna pot 111, 1001 Ljubljana, Slovenia; 2Department of Pathophysiology, Medical Faculty, University of Ljubljana, Zaloška 4, 1001 Ljubljana, Slovenia. Introduction Cyanobacteria have been implicated in many deaths of livestock, wildlife and human thro­ughout the world.1 They produce a broad ran­ge of biologically active substances including proteinase inhibitors2, endotoxins (LPS), which are generally present in gram-negative bacteria3, and a variety of other toxic compo­unds.4 These substances are released in the water environment during the senescence of the bloom and can penetrate in the water sup­ply system. Little attention has been paid to possible synergistic interactions between the­se biologically active substances in tumor promotion and tumor initiation. With few exceptions5, the vast majority of experiments used for the human risk asses­sment of cyanobacteria have been performed using purified microcystins.6 To evaluate li­ver injuries such as cirrhosis and hepatocellu­lar carcinoma high doses of pure microc­ystins have been used.7 The present paper presents an attempt to verify the possible synergistic effects of different biologically ac­tive substances we have tested the toxic ef­fects of lyophilized hepatotoxic cyanobacteria in comparison to the effects produced by the same amounts of purified microcystins. Materials and methods The toxicity was estimated by a mouse bioas-say and the microcystins isolated and deter­mined as described elsewhere.8 Acute and sub chronic experiments were performed on Wistar rats and New Zealand rabbits, as de­scribed previously.9 The toxic material was injected intraperitoneallly (i.p.). Results and discussion In our experiments we have been able to de­tect precancerosis after application of much lower amount of microcystins in lyophilized cyanobacteria (2 LD50 injected during the 5 weeks period). This amount is on average 4 fold lower, than the amount used by other au­thors in order to produce similar injuries by i.p. application of purified microcystins. Since endotoxins (LPS) present in cyanobacteria have a profound effect on detoxification of microcystins by the suppression of glutathio­ne S- transferase activity10 they may enable the accumulation of microcystins in the liver of exposed organisms (Figure 1). The persistence of microcystins in liver cells may be responsible for the more pronounced changes in the liver of animals treated with whole cyanobacteria in comparison to the ani­mals treated with pure microcystins (Figure 2). Experimental data imply that microcystins can no longer be treated merely as tumor pro­moters since it has been demonstrated that prolonged exposure to microcystins can indu­ce neoplastic nodular formation.7 Additio­nally, DNA damages have been observed as a result of exposure to microcystins.11 Figure 1. Inhibition of microcystin detoxification in the presence of endotoxin (LPS) as suggested by Pflugmaher and coworkers. Figure 2. Prolonged action of microcystis on liver cells due to the inhibition of detoxification. We must be aware that several other toxic or carcinogenic substances found in environ­mental waters can be dissolved, adsorbed or trapped on the surface of cyanobacteria. The­refore, in our opinion the health risk of cya­nobacteria can not be established only on the basis of microcystin contents, meaning that the risk is underestimated. Conclusion New experimental data on cyanobacterial to­xicity suggest that the extrapolation of the he­alth risk of microcystins from acute and sub chronic experiments performed mainly with purified toxins is not adequate. Other biologi­cally active substances normally present in cyanobacterial blooms can amplify their toxic effects. There are also evidences, that microc­ystins are able to damage the genetic materi­al of exposed animals and can even induce formation of neoplastic nodular formation. Therefore, at least in our opinion, the health risk of natural cyanobacterial blooms is unde­restimated. References 1. Gorham PR, Carmichael WW. Hazards of fresh­water blue-green algae (cyanobacteria). In: Lembi CA, Waaland JR, editors: Algae and human affairs. New York: Cambridge University Press; 1988. p. 404-31. 2. Banker R, Carmeli S. Inhibitors of serine proteases from a waterbloom of the cyanobacterium Microc­ystis sp. Tetrahedron 1999; 55: 10835-44. 3. Keleti G, Sykora JL, Libby EC, Shapiro MA. Com­position and biological properties of lipopolysac­charides isolated from Schizothrix calcicola (Ag.) Gomont (Cyanobacteria). Appl Environ Microb 1979; 38: 471-7. 4. Carmichael WW. Freshwater cyanobacteria (blue-green algae) toxins. In: Ownby CL, Odell GV, edi­tors: Natural toxins, characterisation, pharmacology and therapeutics. Oxford: Pergamon Press, 1989. p. 3-16. 5. Falconer IA, Burch MD, Steffensen DA, Choice M, Coverdale RO. Toxicity of the blue-green alga (cya-nobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal model for hu­man injury risk assessment. Environ Toxicol Wat Quality 1994; 9: 131-9. 6. Fawell JK, Mitchell RE, Everett DJ, Hill RE. The to­xicity of cyanobacterial toxins in the mouse: I mi-crocystin-LR. Hum Exp Toxicol 1998; 18: 162-7. 7. Ito E, Kondo F, Terao K, Harada K-I. Short commu­nications. Neoplastic nodular formation in mouse liver induced by repeated intraperitoneal injecti­ons of microcystin-LR. Toxicon 1997; 35: 1453-7. 8. Sedmak B, Kosi G. Microcystins in Slovene fresh­waters (Central Europe) – first report. Nat Toxins 1997; 5: 445-59. 9. Frangež R, Kosec M, Beravs K, Demšar F, SedmakB, Šuput D. MRI revealed chronic liver injuries ca­used by microcystins. Proc Intl Soc Magn Reson Med 1999; 7: 1139. 10. Pflugmacher S, Best JH, Wiegand C, Codd GA. In­hibition of human recombinant glutathione S-transferase activity by cyanobacterial lipopolysac­charides. Supporting the hypothesis of the influ­ence of lipopolysaccharide on toxicity of microc-ystin-LR. 9th Int. Conf. HAB 2000, 7 – 11 February 2000, Hobart, Tasmania, p200. 11. Rao PVL, Bhattacharya R, Parida MM, Jana AM, Bhaskar ASB. Freshwater cyanobacterium Microc­ystis aeruginosa (UTEX 2385) induced DNA dama­ge in vivo and in vitro. Environ Toxicol Phar 1998; 5: 1-6. Radiol Oncol 2002; 36(2): 165-7. Chronic exposure to cyanobacterial lyophilisate reveals stronger effects than exposure to purified microcystins – a MRI study Dušan Šuput1, Aleksandra Milutinovic1, Igor Serša2 and Bojan Sedmak3 1School of Medicine, Institute of Pathophysiology, 1001 Ljubljana, Slovenia; 2Inštitut Jožef Stefan, Jamova 39, 1000 Ljubljana, Slovenija; 3Department of Ecotoxinology and Ecotoxicology, National Institute of Biology, Vecna pot, 1000 Ljubljana, Slovenia Introduction Microcystins are potent hepatotoxins, tumor promoters, and carcinogens. Although they are present in surface water bodies worldwi­de little has been done to assess the effects of chronic exposure to these substances in hu­man population. This may partly be due to the fact that acute intoxication by these sub­stances is rare in man. Recent experience from Brazil where a number of dialysis pati­ents died due to the presence of microcystins in the water used for dialysis is a serious war­ning. Chronic exposure to microcystins is less dramatic, but more widespread. Therefore the aim of our study was to test magnetic re­sonance imaging (MRI) as a noninvasive and harmless method for the early detection of changes in liver tissue after chronic exposure to microcystins. It should be emphasized that living organism are in most cases chronically exposed to toxic cyanobacteria and/or to the­ir dissolved contents, which consist of a bro­ad range of biologically active substances and not only microcystins.1-4 Therefore the se­cond aim of our study was to assess the pos­sible synergistic interactions between these substances in chronic intoxication by using either purified microcystins or cyanobacterial lyophilisate (CL). Methods Male albino rats (Wistar) weighing 165-190 g were used in all experiments. The animals were kept at standard room condition (room temperature 24 °C, daily-night interval at 12 hours, exposed to 60 lux artificial light). The number of animals was kept at minimum. The experiments were performed with the gu­idelines of IST. Either cyanobacterial lyophili-sate or purified microcystins were injected i.p. in the intervals of 3 days for 2 months, the total cumulative dose of microcystins (either in the lyophilisate or purified substance) was 2 LD50. Magnetic resonance imaging of the rat’s li­ver was performed on a Bruker Biospec sys­tem with a 2.35 T horizontal bore magnet on animals anesthetized with i.p. injection of Xylazin (15 mg/kg), Ketanest (100 mg/kg) and atropine (0.3 mg/kg). In this study, standard T1 weighted spin-echo magnetic resonance imaging method with echo time 18 ms and re­petition rate 400 ms was used. Signal avera­ging of 10 signal acquisitions was used to im­prove the signal to noise ratio. In order to extract the liver volume, rats were imaged in seven consecutive slices in two experiments, first in transverse and then in coronal slice orientation. Imaging filed of view and slice thickness was at each experiment adjusted so that imaging volume covered the whole liver region. Thus, field of view was in the range 8­10 cm and slice thickens 3-4 mm. T1 weig­hted MR images had good contrast between liver and surrounding tissues that allowed precise liver region selection and its area cal­culation for each slice. Liver volume was later calculated as a sum of liver areas in all slices multiplied by the slice thickness. Results and discussion In acute experiments MRI revealed enlarge­ment of liver in both groups of experimental animals, and there was no difference whether they received only purified microcystins or the same amount of microcystins present in the CL. Significant differences have been ob­served between the group that received CL or purified microcystins only in the chronic ex­periments. Liver from the animals injected with the microcystin- LR showed only minor changes on the signal intensity on MRI ima­ges, and patho -morphological examination of abdominal organs on sacrificed animals sho­wed degenerative changes in one animal on­ly. The MR images of the liver from all ani­mals injected with CL showed irregular changes in the signal intensity and nodular formations have been observed. Patho -mor­phological examination of sacrificed animals showed granular structure of liver edges, and enlargement of kidneys. However, degenera­tive changes characterised as periportal fibro­sis and fatty infiltrations were observed in both groups of chronically treated animals. Changes were more pronounced in the case of the animals treated with CL. No tumours were detected in any of the treated animals, which is in agreement with the data from oth­er authors5 that showed that longer exposu­res and higher dosages are necessary to pro­duce carcinogenic effects. Extracts from toxic cyanobacteria induce DNA damage in vitro as well as in vivo.7 This implies that microcystins can no longer be treated merely as tumour promoters. The en­largement of kidney shown in our experi­ments suggests that exposure to toxic cyano-bacteria also affects other organs, which has already been proposed.8 Microcystins could be responsible for these effects, but the com­bined action of the constituents of cyanobac­teria is evidently important as kidney enlarge­ment has been observed only in the group of animals treated with CL. The data show that not only microcystins but also other compo­nents of the cyanobacterial bloom can affect the health of population.6 Therefore the he­alth risk of hepatotoxic cyanobacteria estima­ted from the microcystin content is underesti­mated. Figure 1. MR images of the liver exposed to cyanobacterial lyophilisate or to purified microcystins alone. A. Con­trol liver is of a dark gray and homogeneous colour. B: Chronic exposure to cyanobacterial lyophilisate (CL) results in liver degeneration shown as non-homogeneous signal from the liver seen as white patches. C) Chronic exposu­re to microcystins gives rise to only slight changes in the liver structure as seen by MRI. References 1. Banker R, Carmeli S. Inhibitors of serine proteases from a waterbloom of the cyanobacterium Microc­ystis sp. Tetrahedron 1999; 55: 10835-44. 2. Keleti G, Sykora JL, Libby EC, Shapiro MA. Com­position and biological properties of lipopolysac­charides isolated from Schizothrix calcicola (Ag.) Gomont (Cyanobacteria). Appl Environ Microb 1979; 38: 471-7. 3. Carmichael WW. Freshwater cyanobacteria (blue-green algae) toxins. In: Ownby CL, Odell GV, edi­tors: Natural toxins, characterisation, pharmacology and therapeutics. Oxford: Pergamon Press; 1989. p. 3-16. 4. Namikoshi M, Rinehart KL. Bioactive compouds produced by cyanobacteria. J Ind Microbiol 1996; 17: 373-84. 5. Ito E, Kondo F, Terao K, Harada K-I. Short commu­nications. Neoplastic nodular formation in mouse liver induced by repeated intraperitoneal injecti­ons of microcystin-LR. Toxicon 1997; 35: 1453-7. 6. Pflugmacher S, Best JH, Wiegand C, Codd GA. In­hibition of human recombinant glutathione S-transferase activity by cyanobacterial lipopolysac­charides. Supporting the hypothesis of the influ­ence of lipopolysaccharide on toxicity of microc-ystin-LR. 9th Int. Conf. HAB 2000, 7 – 11 February 2000, Hobart, Tasmania, p200. 7. Rao PVL, Bhattacharya R, Parida MM, Jana AM, Bhaskar ASB. Freshwater cyanobacterium Microc­ystis aeruginosa (UTEX 2385) induced DNA dama­ge in vivo and in vitro. Environ Toxicol Phar 1998; 5: 1-6. 8. Bhattacharya R, Sugendran K, Dangi RS, Rao PVL. Toxicity evaluation of freshwater cyanobacterium Microcystis aeruginosa PCC 7806: II Nephrotoxi-city in rats. Biomed. Environ Sci 1997; 10: 93-101. Radiol Oncol 2002; 36(2): 168. Tumor progression and invasion Tamara T. Lah Department of Genetic Toxicology and Cancer Biology, National Institute of Biology Vecna pot 111, Ljubljana, Slovenija Cancer is the disease of the gene. The very first event that damages nuclear DNA is initiated by an initiator (substance, radiation), which may cause different effects, such as mutations, DNA breaks, etc, which may or may not cause cancer. Additional hits, which do lead to the development of cancer, are caused by tumor pro­moters. At present, detailed knowledge of all the events leading to a malignant disease, are not yet understo­od. Molecular description of tumor progression envisages that each type of cancer will progress in stages: the step-wise genetic changes accompanying this progression, are unique to each type of cancer. To study these events, various in vitro and in vivo approaches were developed. The first part of the workshop will be dedi­cated to the methodology used to identify DNA damage, while the second part will be dedicated to the expe­rimental models used to identify biological markers, associated with tumor and/or endothelial cell invasion. Metastasis of primary tumors is comprised of biologically distinct steps and is rather inef­ficient process. Local tumor cell invasion is a common denominator in many of these steps and was first described by Liotta1 as a three-step process, where, first specific attachment (I) is followed by the induction and/or release or hydrolytic – proteolytic – enzymes, which degrade (II) extracellular matrix components, thereby facilitating tumor cell locomotion (III) and penetratioan into the host tissues. Several experimental models are used to study the mo­lecular mechanisms of invasion. Most in vitro invasion assays are using natural tissues, such as amnion membrane, eye lens and fragments of various tissues, such as chicken heart, mou­se liver, lung, etc. Organotypic co-culture mo­dels were mostly used for studying brain tu­mor invasion. The most simple are invasion assays using modified Boyden chambers2,3 and Matrigel or other isolated proteins of extracel­lular matrix. However, the results may not be reproducible even when using the same sys­tem, due to several methodological problems, comprising variability in composition, concen­tration and preparation of Matrigel (or other proteins), quality of filters, treatment of cells (pH, trypsinization, organic solvents), chemoa­tractants, time of the assays and methods used for the quantitation of the invasion. The data in the literature confirmed that various proteina­se inhibitors were effective in partial inhibition of invasion, indicating that one or several pro-teinases are involved in this process. The re­sults on inhibition of invasion of breast tumo­ur cells, using synthetic and natural (peptide) cysteine proteinase inhibitors are not yet con­clusive, due to variability of methodological ap­proaches, described in the literature. References 1. Liota LA. Tumor invasion and metastasis. Role of extracellular matrix. Cancer Res 1989; 46: 1-7. 2. Albini A, Iwamoto, Y, Kleinman HK, Martin GR, AAronson SA, Kozlowksi JM, McEwan RN. A ra­pid in vitro assay for quantiting the invasive poten­tial of tumor cells. Cancer Res 1987; 47: 3239-45. Radiol Oncol 2002; 36(2): 169-71. In vitro invasion of transfected human breast epithelial cells MCF10A-neoT Nataša Sever, Nataša Levicar, Irena Zajc, Aleš Bervar and Tamara T. Lah Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia Introduction Tumor invasion and metastasis are responsi­ble for the progression of malignant disease. These processes are facilitated by the upregu­lation of various types of proteinases. In re­cent years, experimental and clinical studies have been carried out using inhibitors of MMPs,1,2 plasmin and plasminogen activa-tors,3 as well as cysteine proteinases.4,5,6,7 They inhibit either growth,7 motility8 or the invasive potential4,5 of various types of tumo­urs. The aim of the study was to evaluate the effectiveness of synthetic and peptide protei­nase inhibitors in reducing in vitro invasion of MCF10A cells, transfected with ras oncogene. Material and methods MCF10A-neoT cell line was established by transfection of MCF10A cells with T24 c-Ha­ras oncogene9 and has an acquired ability to grow as xenograft in nude mice, forming small nodules, which progress sporadically into invasive carcinomas of different histolo­gical types. Cells were grown to 80 % conflu-ency and 24 h before harvesting, the medium was replaced with SFM (serum free medium). Invasion assay The method described by Albini and co-wor­kers10 was used with minor modifications. Poly­carbonate filters (Costar, USA) with 12 µm po­rosity were coated with fibronectin (25 ng/mm, Sigma, Germany) on the lower surface and with Matrigel (0.9 µg/mm, Becton Dickinson, USA) on the upper surface, dried overnight and reconstituted with 200 µl SFM for 1h at 37 °C. Maximum inhibition of invasion was ob­served using SFM containing proteinase inhibi­tors (Bachem, Switzerland) at the following non-cytotoxic concentrations: 10 µM E64, 100 µM E64-d, 20 µM Ca-074, 0.5 µM Ca-074Me, 20 µM Z-FA-FMK, 0.5 µM Clik 148, 50 µM Z­FF-CHN2, 1 µM pepstatin A, 100 µg/ml aproti­nin and 10 µM BB94. Cytotoxicity was determi­ned using MTT (1-(4,5 dimethyltiazol-2-yl)-2,5 diphenyl tetrazolium bromide) viability assay. Cells were harvested and seeded (200,000 cells in 0.5 ml SFM) to the upper chamber. After 21 h incubation, MTT (Sigma, Germany) at the final concentration of 0.5 mg/ml was added.3 The cells were further incubated for 3 h at 37 °C to allow the formation of formazan crystals. The crystals from upper and lower chambers were separated and pelleted by centrifugation at 15000 rpm for 5 min and dissolved in 1 ml of DMSO. Optical density (OD) was measured at 570 nm (reference filter 690 nm). Invasiveness of the cells was calculated as the ratio of the OD in the lower chamber to the sum of ODs in the lower and upper chambers. Results The ability of inhibitors of cysteine, aspartic, serine and metalloproteinases to reduce inva­ Figure 1. Inhibition of MCF10A-neoT cells by synthetic inhibitors of invasion. sion of MCF10A-neoT cells was determined (Figure 1). Membrane-permeant cysteine pro-teinase inhibitors, E64-d (28 %) and Z-FF­CHN2 (40 %), were the most effective, follo­wed by the selective CatB inhibitor, Ca-074Me (about 20 %). Inhibition of invasion by Z-FA-FMK (40 %) and the selective CatL in­hibitor, Clik 148 (14 %) was not statistically si­gnificant. Among the inhibitors of other types of proteinases, only pepstatin significantly in­hibited the invasion (28 %). Aprotinin redu­ced the invasiveness to the similar extent. The broad spectrum matrix metalloproteina­se inhibitor BB94 did not inhibit invasiveness. Data is expressed as percentage of the con­trol. Error bars depict standard error of the mean values of three independent experi­ments. Statistical significance (*) was deter­mined by two tailed t-test with assumed equ­al variance, and p < 0.05 was considered significant. Discussion The MCF10A-neoT cell line was used to deter­mine the effect of proteinase inhibitors on in­vasion. Cysteine proteinase inhibitors were found to be more effective than the inhibitors of other classes of proteinases. Therefore, we conclude that cysteine proteinases contribute significantly to the process of invasion. CatB inhibitors proved more effective than CatL in­hibitors, suggesting that CatB plays a more important role than CatL. Since the derivati­ves of CatB inhibitors, which can enter the cells, were found to be most effective, the cells probably degrade collagen also intracel­lularly, as reported previously. The aspartic proteinase inhibitor pepstatin A also reduced the invasion, so CatD may also be involved in breast tumor cell invasion.12 Similar to re­ports in human esophageal and ovarian carci­noma cells in vivo13, we did not observe inhi­bition of invasion by the broad spectrum inhibitor of MMPs, BB94. This is in contrast to the previous reports of its inhibitory effect if invasion in other cell types.4,5 Such discre­pancies may reflect differences in the expres­sion and activation of MMPs in various cells. Our results support the hypothesis that the cysteine proteinase CatB plays an active role in invasion of transformed human breast cell lines. These findings could have an impact on the search for new anti-invasive and anti-me­tastatic agents Acknowledgements The authors thank Dr.B.F. Sloane (Wayne State University, USA) for the cells, Dr. P. Brown (British Biotech Pharmaceutical,UK) for the inhibitor BB94, Dr. N. Katunuma (To­kushima Bunri University, Japan) for Clik 148 and Dr. C.J. Van Noorden (University of Am­sterdam, The Netherlands) for the inhibitor Z-FA-FMK. This work was supported by the Ministry of Education, Science and Sport, programme #105-509). References 1. Kohn CE, Liotta LA. Molecular insights into can­cer invasion: Strategies for prevention and inter­vention. Cancer Res 1995; 55: 1856-62. 2. Botos I, Scapozza L, Zhang D, Liotta LA, Meyer EF. Batimastat, a potent matrix metalloproteinase inhibitor, exhibits an unexpected mode of bin­ding. Proc Natl Acad Sci USA 1996; 93: 2749-54. 3. Holst-Hansen C, Johannessen B, Hoyer-Hansen G, Romer J, Ellis V, Brünner N. Urokinase-type pla­sminogen activation in three human breast cancer cell lines correlates with their in vitro invasiveness. Clin Exp Metastas 1996; 14: 279-307. 4. Kolkhorst V, Stürzenbecher J, Wiederanders B. In­hibition of tumor cell invasion by proteinase inhi­bitors: correlation with the proteinase profile. J Cancer Res Clin Oncol 1998; 124: 598-606. 5. Stonelake PS, Jones CE, Neoptolomos JP, Baker PR. Proteinase inhibitors reduce basement mem­brane degradation by human breast cancer cell li­ne. Br J Cancer 1997; 75: 951-9. 6. Bjornland K, Buo L, Kjoniksen I, Larsen M, Fod­stad O, Johansen HT, Aasen AO. Cysteine protei­nase inhibitors reduce malignant melanoma cell invasion in vitro. Anticancer Res 1996; 16: 1627-32. 7. Van Noorden CJF, Jonges TGN, Meade Tollin LC, Smith RE, Koehler A. In vivo inhbition of cysteine proteinases delays the onset of growth of human pancreatic cancer explants. Br J Cancer 2000; 82: 931-6. 8. Boike G, Lah T, Sloane BF, Rozhin J, Honn K, Gu­irguis R, Strache ML, LiottaLA, Schiffmann E A possible role for cysteine proteinase and its inhibi­tors in motility of malignant melanoma and other tumour cells. Melanoma Res 1991; 1: 333-40. 9. Basolo F, Elliot J, Tait L, Chen XQ, Maloney T, Rus­so IH, Pauley R, Momiki S, Kaamano J, Klein-Szanto AJP, Koszalka M, Rosso J. Transformation of human breast epithelial cells by c-Ha-ras onco­gene. Mol Carcinogen 1991; 4: 25-35. 10. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN. A ra­pid in vitro assay for quantitation of the invasive potential of tumor cells. Cancer Res 1987; 47: 3239­45. 11. Sameni M, Moin K, Sloane BF. Imaging proteolysis by living human breast cancer cells. Neoplasia 2000; 2: 496-504. 12. Rochefort H, Garcia M, Glondu M, Laurent V, Li-audet E, Rey JM, Roger P. Cathepsin D in breast cancer: Mechanisms and clinical application, a 1999 overview. Clin Chem Acta 2000; 291: 157-70. 13. Della Porta PD, Soeltl R, Krell WH, Collins K, Odonoghue M, Schmitt M, Krueger A. Combined treatment with serine proteinase inhibitor aproti­nin and matrix metalloproteinase inhibitor bati­mastat (BB94) does not prevent invasion of human esophageal and ovarian carcinoma cells in vivo. Anticancer Res 1999; 19: 3809-16. Radiol Oncol 2002; 36(2): 172-3. The effect of E-64 and monoclonal antibodies on proliferative and invasive activity of ras transformed human breast epithelial cell line MCF10A neoT tested in in vitro assays Aleš Premzl1 and Janko Kos2 1Jožef Stefan Institute, Department of Biochemistry and Molecular Biology, Ljubljana, Slovenia; 2Krka, d.d., Research and Development Division, Department of Biochemical Research and Drug Design, Ljubljana, Slovenia Introduction The characteristic of malignant tumours is their potential to invade normal host tissue and to metastasize to distant organs.1,2 Tumo­ur cell invasion of the extracellular matrix (ECM) is described3 as a multi-step process. First, tumour cells need to be attached to the components of the ECM. Next step is the lo­cal degradation of ECM components, follo­wed by the migration of the tumour cell thro­ugh locally modified matrix. Invasive behavi-our of tumour cells requires proteolytic acti­vity associated with the concerted action of various intra and/or extracellular proteinases, including a cysteine proteinase cathepsin B. Cathepsin B was found localized on the surfa­ce of different brain and breast cancer cells, including MCF10A neoT.4 Another prerequi­site for tumour cells to form distant metasta­ses is tumour cell proliferation.2 The effect of E-64, an irreversible cysteine proteinase inhibitor and various monoclonal antibodies raised to human cathepsin B, was tested on ras transformed human breast ep­ithelial cell line MCF10A neoT, using in vitro proliferation and invasion assays. Materials and methods Hybridoma technique, first demonstrated by Köhler and Millstein in 19755, was used to pre­pare mouse anti-cathepsin B monoclonal anti­bodies. Recombinant cathepsin B was used for immunization of Balb/c mice. For in vitro pro­liferation and invasion assays we used MAbs, derived from two clones of hybridoma cells. The MCF10A neo T, ras transformed hu­man breast epithelial cell line was used in the in vitro assays. The cells were cultured in DMEM/F12 (1:1), with 5 % foetal calf serum (FCS) and supplemented with insulin, epider­mal growth factor, hydrocortisone and antibi­otics at 37 °C and 5 % CO2 in humidified atmosphere, to about 70-80 % confluency. The assays were performed using the same DMEM/F12 medium, however, FCS used was purified on the affinity chromatography co­lumn using immobilized papain. The MTT-cell proliferation assay6 was used to assess possible influence of inhibitor and antibodies on tumour cell proliferation. It is a quantitative colorimetric assay based on clea­vage of the tetrazolium salt MTT (3-(4,5-di­methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into water insoluble, dark-blue for-mazan crystals by the mitochondrial enzyme succinate-dehydrogenase.7 Assays were per­formed in quadruplicates. To study the effects of MAbs and E-64 on tumour cell invasion, Costar Transwells with 12 mm polycarbonate filters and 12 µm pore size were used. Filters were coated with Ma-trigel (Becton Dickinson).8 Assays were per­formed in triplicates. Results and conclusions In the proliferation assay, MAbs derived from clone A and E-64 didn’t show any effect in the concentration range tested (2-0,01 µM and 100-0,1 µM). MAbs derived from clone B ex­pressed a slight anti-proliferative effect in a dose-dependent manner (2-0,01 µM). Results of in vitro invasion assay show a strong inhibitory effect expressed on the in­vasion of MCF10A neoT cells by E-64. MAbs derived from clone B had no effect, whereas MAbs from clone A expressed a 20 % inhibiti­on compared to control at 1 µM concentrati­on. References 1. Liotta LA. Cancer cell invasion and metastasis. Sci Am 1992; 266: 34-41. 2. Liotta LA, Kohn E. Cancer invasion and metasta­ses. JAMA 1990; 263: 1123-6. 3. Liotta LA. Tumor invasion and metastases-role of the extracellular matrix: Rhoads memorial award lecture. Cancer Res 1986; 46: 1-7. 4. Kos J, Lah TT. Cysteine proteinases and their en­dogenous inhibitors: Target proteins for progno­sis, diagnosis and therapy in cancer (Review). On-col Rep 1998; 5: 1349-61. 5. Goding JW. Antibody production by hybridomas (Review). J Immunol Meth 1980; 39: 285-308. 6. Holst-Hansen C, Brünner N. MTT-Cell proliferati­on assay. Cell Biology: A laboratory handbook. Acad Press, 16-18, 1998. 7. Slater TF, Sawyer B, Strauli U. Studies on succina-te-tetrazolium reductase systems. III. Points of co­upling of four different tetrazolium salts. Biochim Biophys Acta 1963; 77: 383-93. 8. Holst-Hansen C, Johannessen B, Hoyer-Hansen G, Romer J, Ellis V, Brünner N. Urokinase-type pla­sminogen activation in three human breast cancer cell lines correlates with their in vitro invasive­ness. Clin Exp Metastas 1996; 14: 297-307. Radiol Oncol 2002; 36(2): 174-5. Brain tumour migration and invasion: The role of in vitro model systems Geoffrey J Pilkington Experimental Neuro-oncology Group, Department of Neuropathology, Institute of Psychiatry, King’s College, London, De Crespigny Park, Denmark Hill, London SE5 8AF UK Human primary (intrinsic) brain tumours rarely metastasis to distant organs, however they do show a mar­ked propensity for diffuse infiltrative invasion of the contiguous, normal brain tissue. This is arguably the most important biological feature of this group of – predominantly glial – neoplasms. Single neoplastic glia may migrate several millimetres, or even centimetres from the major tumour mass and there is increasing evidence that during the migratory phase these cells transiently arrest from the cell cycle therefore rendering them refractory to therapeutic radiation. Moreover, they are protected from the action of the majority of cytotoxic drugs by virtue of their investment within areas of intact blood-brain barrier. These migratory, so-called “guerrilla” cells later return to the division phase, under hitherto unknown microenvironmental cues, to form local recurrences of the primary tumour. The terms “migration” and “invasion” in the context of oncology frequently tend to be used interchangeably however, strictly speaking mi­gration refers to the simple movement of cells within a tissue without causing specific dama­ge to the host tissue while invasion infers the movement of cells with consequent detriment to the normal cellular elements. Ideally, in or­der to elucidate the underlying mechanisms and patterns of invasion this phenomenon should be studied in in vivo model systems. Animal models of brain tumour, however, do not generally fulfil the criteria required for such studies. Indeed, while intracranial tumo­urs induced by chemical carcinogens such as ethyl nitrosourea (ENU) do invade the brain, they occur at inconsistent locations and at differing latency (which is long) and inciden­ce. Transplantable glial tumours tend to have rapid growth rates, growing by expansion ra­ther than diffuse invasion and\ showing only limited dissemination, generally along the va­scular basal laminae. To date the only convin­cing demonstration of invasion in an animal model comes from the direct xenotransplan­tation of human biopsy tissue into immuno­deficient rats. This model, however, suffers from lack of reproducibility and therefore ineffective statistical accountability. In order that invasion of glial tumours can be studied in the laboratory it has been neces­sary to develop a variety of in vitro approa­ches. Although each of these approaches suf­fers from intrinsic flaws they have collecti­vely provided considerable information re­garding the cellular mechanisms and molecu­lar pathways which underlie the process of brain tumour invasiveness. The design of such models is of great im­portance since the culture microsystem ex­erts an influence on the invasive and motile properties of neoplastic cells. Although a simple scratch across a conflu­ent monolayer facilitates monitoring of mi­gration, more sophisticated techniques have been developed. For example, cells may be se­eded into cloning rings on extracellular ma­trix protein coated substrates and incubated in culture for a 12 hour period, then the ring may be removed and migration away from the cell colony assessed by sequential photo­micrography. It is also possible to examine the move­ment of tumour cells across the substrate of a culture dish by time-lapse video photomicro­scopy. Here, the substrate can be varied and putative chemorepellant or chemoattractant factors, as well as agents that may promote or retard invasion, may be added to the growth medium and their effects studied over a peri­od of many hours. Using this approach the trajectories of cell movement may be tracked and the intervals between cell divisions log­ged. Another method employs modified Boy-den chambers. In essence, commercially ava­ilable “Transwell” units, incorporating inserts with polycarbonate membrane filters (porosi­ties of 8 to 12 microns are generally suitable for the study of human neoplastic glia), are set up with a chemoattractant containing me­dium (eg platelet-derived growth factor or tu­mour cell conditioned medium) in the lower chamber and cells for assay are seeded onto the top of the filter in the upper chamber (the two chambers are separated by the filter alo­ne). After a pre-determined period of incuba­tion, filters are removed and cells which have migrated to the lower side of the filter may be stained with simple haematological stains such as modified Papanicolau (Diff-Quik) or by immunocytochemical methods: electron microscopy may also be carried out. Cells on the non-migratory (upper) side of the filter are either ignored, as unfocussed cells, or may be removed by scraping, counts can then be made of migratory cell populations. By co­ating the polycarbonate filter with a thin layer of extracellular matrix (ECM) components such as “Matrigel” or with growth inhibited, viable non-neoplastic cells, the migration as­say can be converted to an assay for invasive­ness. Such assays not only monitor the moti­le propensity of cells but also require that the cells adhere to and subsequently degrade the ECM in order to permit invasion. Three-dimensional confrontational models where normal tissue is maintained in the pre­sence of neoplastic glial cells has enabled as­sessment of invasive potential and has helped to elucidate the interaction between normal and neoplastic cells during invasion. In early studies, cell lines derived from ENU-induced brain tumours in the rat were confronted with embryonic chick heart fragments and this system has proved to be of value in asses­sing invasion of human brain tumours. Inde­ed, the degree of invasiveness in this system has been shown to correlate well with mali­gnancy and clinical evolution of the neo­plasms. For example, malignant gliomas inva­de into the normal tissue and destroy it while meningiomas surround but do not invade the “host” tissue. In other three-dimensional sys­tems re-aggregated foetal brain has formed the target for in vitro invasion by both human and experimental animal brain tumour cell li­nes or short-term, early passage cultures ma­intained as either monolayer cultures or as multicellular tumour spheroids. Optic nerve, too, has provided a neural target with which to study invasion and has yielded informati­on concerning the phagocytic activity of hu­man and animal gliomas during invasion, as well as providing morphological evidence of interaction with the ECM. Radiol Oncol 2002; 36(2): 176-9. Cathepsins and cystatins in extracellular fluids – useful biological markers in cancer Janko Kos1,2 and Ana Schweiger1 Jožef Stefan Institute, Department of Biochemistry and Molecular Biology and Krka, d.d., R&D Division, Department of Biochemical Research and Drug Design, 1000 Ljubljana, Slovenia Proteases of all four classes have been shown to participate in processes of tumor growth, vascularisation, invasion and metastasis. Their levels in tumor tissue extracts can provide useful clinical information to pre­dict disease free and overall survival in various types of cancer. Recently we found that cysteine proteina­ses cathepsins B and H and their endogenous protein inhibitors stefins A, B and cystatin C can also predict prognosis when measured extracellularly. In melanoma and colorectal cancer patients high serum levels of cathepsins as well as high levels of stefins A and B and cystatin C correlated with shorter survival. On the other hand, cathepsin B/cystatin C complex was found to be less abundant in sera of patients with mali­gnant tumors than in those with benign diseases or in healthy controls. Introduction Alterations in expression, processing and lo­calization of cysteine proteinases (CPs) in tu­mor tissues have been observed at various le­vels when compared to their normal and benign tissue counterparts.1 CPs can be tran­slocated to the plasma membrane or secreted from tumor cells where they presumably par­ticipate in the degradation of components of the extracellular matrix and basement mem­brane.1,2 The mechanism of secretion is not fully understood3, however, it is known that cathepsins can be secreted from normal and tumor cells as precursors or active enzymes. CPs may be involved also in the formation of new blood vessels, which enable feeding of the growing tumor.4,5 Relation of mRNA, ac­tivity or protein levels of CPs in tumors with clinical characteristics of cancer patients has shown that these molecules are highly predic­tive for the length of survival and may be used for assessment of risk of relapse or de­ath for cancer patients.4 Detection of these proteins in extracellular fluids may extend their application to primary diagnosis6-8, to the assessment of response to selected che­motherapy9 and to the monitoring of mali­gnant disease. Quantization of cathepsins and their inhibi­tors in extracellular fluids Cathepsins B, H and L have been determined in extracellular fluids of cancer patients by measuring enzymatic activity or immunoche­mically by ELISA. In first case various chro­mogenic and fluorogenic substrates and synthetic inhibitors have been used in experi­mental procedures, resulting in more or less specific signal for individual enzyme. In ELI-SAs specific monoclonal and polyclonal anti­bodies, raised to individual human antigens have been used, providing reliable informati­on of the protein level of each enzyme in ex­tracellular fluids.10,11 However, for most of cathepsins only the total protein level can be assessed by ELISA, excluding the informati­on on active, pro or complexed enzyme forms. Only for detection of cathepsinB/cystatin C complex a specific ELISA has been desi­gned.11 Extracellular levels of the inhibitors of CP have been defined by measuring total cystei­ne proteinase inhibitor (CPI) activity or by specific ELISAs.13 Automated particle-enhan­ced immunoturbodimetric or immunonephe­lometric assays have been designed for detec­ting of cystatin C in blood.12,13 Extracellular cathepsins and their inhibitors as diagnostic or prognostic indicators The activity and protein levels of cathepsins and inhibitors have been determined in fluids surrounding tumors, such as bronchoalveolar lavage fluid of lung cancer patients and asci­tes fluid of ovarian carcinoma patients and in blood and urine.11 High levels of cathepsin B have been repor­ted in sera of patients with breast, ovarian, uterine, liver, pancreatic, melanoma, colorec­tal and lung cancer.4,11 In patients with colo-rectal and uterine carcinoma cathepsin B pro­tein or activity levels correlated with tumor stage.8,14,15 Additionally, in this type of mali­gnancy cathepsin B protein concentration was found to correlate with different modes of pa­tient treatment.16 Increased levels of cathe­psin B were found in urine of patients with ga­stric cancer17 but not in urine from breast 18 or bladder carcinoma.19 Cathepsin B was fo­und as a significant prognostic marker in sera of patients with melanoma9 and colorectal cancer.14 Patients with high levels of serum cathepsin B experienced high risk of death. Cathepsin H was increased in sera of pati­ents with melanoma, colorectal, lung and he­ad and neck cancer.11 In melanoma, its prote­in level was significantly increased within the group of patients who did not respond to the combined chemoimmunotherapy, compared with the group of responders, indicating the potential of this enzyme in predicting the ef­fectiveness of the therapy.9 In patients with head and neck cancer cathepsin H serum le­vels correlated with histological grade20 and in melanoma and lung cancer its high levels correlated with shorter overall survival.9,21 Cathepsin L activity levels were found in­creased in sera of breast, pancreatic, liver and colorectal cancer.11 Its protein level was fo­und to be increased in sera of patients with ovarian carcinoma and suggested in combina­tion with CA 125 and CA 72-4 as better mar­ker for detection of ovarian cancer than the methods currently used in clinical practice.7 Stefin A and stefin B have been detected in ascitic fluid from ovarian carcinoma22 and in bronchoalveolar fluid of lung cancer pati­ents.23 Increased serum levels of stefin A in patients with hepatocellular carcinoma and li­ver cirrhosis correlated with tumor size and with the number of neoplastic lesions.6 Stefin A, but not stefin B levels were moderately in­creased also in sera of patients with colorec­tal and lung cancer.24 Cystatin C was also in­creased in sera of cancer patients.11 In melanoma, colorectal and lung cancer its le­vels correlated with the progression of the malignant disease. Since cystatin C has also been proposed as an accurate marker of glo­merular filtration rate (GFR), its levels in can­cer patients should be very carefully evalua­ted before clinical application of this new GFR marker. Stefin A, stefin B and cystatin C have been reported as significant prognostic markers in sera of patients with colorectal cancer.24 High levels of all three inhibitors correlated with shorter overall survival altho­ugh for stefin A the difference between high and low risk patients was not statistically si­gnificant. Stefin B was the strongest progno­sticator of all three inhibitors and the combi-nation with cathepsin B or CEA further strati­fied the risk of death. In sera of patients with colorectal and lung cancer the level of cathepsin B/cystatin C complex was also determined.11 The complex was significantly less abundant in sera of pa­tients bearing malignant lung tumors than in those with benign lung diseases or in healthy controls. Similarly, in colorectal cancer sera, its level was lower in Dukes’ stages C and D than in early stages A and B. The inverse cor­relation found in this study between mali­gnant progression and stability of the com­plex, supports the hypothesis of hindered in­hibitory capability during cancer progression. Conclusions Quantization of cysteine cathepsins and their inhibitors in extracellular fluids as com­pared with tumor tissue cytosols has many advantages. Besides prognostic information their levels can be used also for primary dia­gnosis, for the assessment of response to se­lected chemotherapy and for the monitoring of malignant disease. Additionally, the need for careful histological examination of tumor tissue, inherent problems with tissue hetero­geneity and problems with the choice of ex­traction buffer do not apply to extracellular samples. On the other hand, the levels of ca-thepsins and their inhibitors in blood and other bodily fluids are much lower than in tis­sue extracts and their assessment requires more sensitive assays. Future activities sho­uld be focused on standardization and quality assurance of assays and on definition of sub­populations of cancer patients who would be­nefit most from the information provided by these new extracellular biological markers. References 1. Sloane BF, Moin K, Lah TT. Lysosomal enzymes and their endogenous inhibitors in neoplasia. In: Pretlow TG and Pretlow TP, editors: Biochemical and molecular aspects of selected cancers. New York: Academic Press; 1994. p 411-66. 2. Keppler D, Sloane BF. Cathepsin B: Multiple enz­yme forms from a single gene and their relation to cancer. Enzyme Protein 1996; 49: 94-105. 3. Kornfeld S. Lysosomal enzyme targeting. Biochem Soc T 1990; 18: 367-74. 4. Kos J, Lah T. Cysteine proteinases and their endo­genous inhibitors: Target proteins for prognosis, diagnosis and therapy in cancer (Review). Oncol Rep 1998; 5: 1349-61. 5. Strojnik T, Kos J, Židanik B, Golouh R, Lah T. Ca-thepsin B immunostaining in tumor and endothe­lial cells is a new prognostic factor for survival in patients with brain tumors. Clin Cancer Res 1999; 5: 559-67. 6. Leto G, Tuminello, FM, Pizzolanti G, Montallo G, Soresi M and Gebbia N. Lysosomal cathepsins B and L and stefin A blood levels in patients with he-paticellular carcinoma and/or liver cirrhosis: po­tential clinical implications. Oncol 1997; 54: 79-83. 7. Nishida Y, Kohno K, Kawamata T, Morimitsu K, Kuwano M and Miyakawa I: Increased cathepsin L levels in serum in some patients with ovarian can­cer: comparison with CA125 and CA72-4. Gynecol Oncol 1995; 56: 357-61. 8. Warwas M, Haczynska H, Gerber J and Nowak M. Cathepsin B-like activity as a serum tumor marker in ovarian carcinoma. Eur J Clin Chem Clin Biochem 1997; 35: 301-4. 9. Kos J, Štabuc B, Schweiger A, Krašovec M, Cimer-man N, Kopitar-Jerala N and Vrhovec I. Cathe-psins B, H, L, and their inhibitors stefin A and cystatin C in sera of melanoma patients. Clin Can­cer Res 1997; 3: 1815-22. 10. Kos J, Šmid A, Krašovec, M, Svetic B, Lenarcic B, Vrhovec I, Škrk J and Turk V. Lysosomal proteases Cathepsins D, B, H, L and their inhibitors stefins A and B in head and neck cancer. Biol Chem 1995; 376: 401-5. 11. Kos J, Werle B, Lah T, Brunner N. Cysteine prote­inases and their inhibitors in extracellular fluids: Markers for diagnosis and prognosis in cancer. Int J Biol Marker 2000; 15: 84-9. 12. Kyhse-Andersen J, Schmidt C, Nordin G, Anders-son B, Nilsson-Ehle P, Lindstr(m V and Grubb A. Serum cystatin C, determined by a rapid, automa­ted particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomeru­lar filtration rate. Clin Chem 1994; 40: 1921-6. 13. Finney H, Newman DJ, Gruber W, Merle P, Price CP. Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems. Clin Chem 1997; 43: 1016-22. 14. Kos J, Nielsen HJ, Krašovec M, Christensen IJ, Ci-merman N, Stephens RW and Brunner N. Progno­stic values of cathepsin B and carcinoembryonic antigen in sera of patients with colorectal cancer. Clin Cancer Res 1998; 4: 1511-6. 15. Makarewicz R, Drewa G, Szymanski W and Sko­nieczna Makarewicz I. Cathepsin B in predicting the extend of cervix carcinoma. Neoplasma 1995; 42: 21-4. 16. Bhuvarahamurthy V, Govindasamy S. Extracellu­lar matrix components and proteolytic enzymes in uterine cervical carcinoma. Mol Cell Biochem 1995; 144: 35-43. 17. Hirano T, Manabe T, Takeuchi S. Serum cathepsin B levels and urinary excretion of cathepsin B in the cancer patients with remote metastasis. Cancer Lett 1993; 70: 41-4. 18. Dengler R, Lah T, Gabrijelcic D, Turk V, Fritz H, Emmerich B. Detection of cathepsin B in tumor cytosol and urine of breast cancer patients. Biomed Biochim Acta 1991; 50: 555-60. 19. Sier CFM, Casetta G, Verheijen JH, Tizziani A, Agape V, Kos J, Blasi F, Hanemaaijer R. Enhanced urinary gelatinase activities (MMP-2 and MMP-9) are associated with early stage bladder carcinoma: a comparison with clinically used tumor markers. Clin Cancer Res 2000; 6: 2333-40. 20. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I,Kos J, Škrk J. Cathepsin H in squamous cell carci­noma of the head and neck. Radiol Oncol 1999; 33: 143-51. 21. Schweiger A, Staib A, Werle B, Krašovec M, Lah TT, Ebert W, Turk V, Kos J. Cysteine proteinase cathepsin H in tumors and sera of lung cancer pa­tients: relation to prognosis and cigarette smo­king. Brit J Cancer 2000; 82: 782-8. 22. Lah TT, Kokalj-Kunovar M, Kastelic L, Babnik J, Stolfa A, Rainer S, Turk V. Cystatins and stefins in ascites fluid from ovarian carcinoma. Cancer Lett 1991; 61: 243-53. 23. Luethgens K, Gabrijelcic D, Turk V, Ebert W, Trefz G, Lah T. Cathepsin B and cysteine proteina­se inhibitors in bronchoalveolar lavage fluid of lung cancer patients. Cancer Detect Prev 1993; 17: 387-97. 24. Kos J, Krašovec M, Cimerman N, Jorgen-Nielsen H, Christensen IJ, Brunner N. Cysteine proteinase inhibitors stefin A, stefin B and cystatin C in sera of patients with colorectal cancer: relation to pro­gnosis. Clin Cancer Res 2000; 6: 505-11. Radiol Oncol 2002; 36(2): 180-2. Circadian rhythms of cysteine proteinases and cystatins, potential tumour markers, in normal sera Nina Cimerman1, Pika Meško-Brguljan2, Marta Krašovec1, Stanislav Šuškovic2 and Janko Kos1,3 1Krka, d.d., Research and Development Division, Department of Biochemical Research and Drug Design, Ljubljana, Slovenia; 2University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia; 3Jožef Stefan Institute, Department of Biochemistry and Molecular Biology, Ljubljana, Slovenia Introduction Circadian day/night variations have been evi­denced in all major groups of organisms and at all levels of organisation of the organism. Circadian intra-individual variations are known for a number of analyses in serum in­cluding tumour-associated markers.1-4 It was suggested that the serum levels of cysteine proteinases and their inhibitors may be of cli­nical importance for prognosis and diagnosis in cancer.5 Since known circadian rhythms are important for choosing the best sampling time, interpretation of the results of a diagno­stic test, patient monitoring, and timing of a therapy, our objective was to establish 24-h variations of cysteine proteinases, cathepsins B, H, L, and their low molecular weight inhi­bitors, stefin A, stefin B, and cystatin C, in se­ra from healthy subjects. Materials and methods This study, which included eight clinically symptom-free adults (median age, 31 years; range, 22-64 years; 5 females, 3 men), was performed as reported previously.6,7 Before entering the study the volunteers had given informed written consent, and procedures were approved and performed in accordance with the guidelines of the regional medical ethics committee. Meals were served at 08:00, 12:30 and 18:00 h. The lights were switched off from 22:00 to 07:00 h. Blood was taken by venipuncture in up­right position according to National Commit­tee for Clinical Laboratory Standards appro­ved standard H3-A3. Seven samples were collected at 4-hour intervals beginning at 08:00 h. Blood was clotted at room temperatu­re and centrifuged subsequently at 3000 rpm. Serum was separated, aliquoted and frozen at -20 °C until analysis. Measurements of all proteins were done by specific ELISAs (Krka, d.d., Novo mesto, Slovenia) as described previously.8,9 Mean values and SE were computed at fi­xed hours for each subject during the 24-hour monitoring. All data were analysed by one-way ANOVA and by cosinor analysis invol­ving the fit of a 24-hour cosine curve by the method of least squares10,11 as reported previ­ously.6,7 The correlation between the parame­ters examined was assessed by the nonpara­metric Spearman rank correlation test. Two-sided P values < 0.05 were considered si­gnificant. Results and discussion The 24-h patterns of cathepsins B, H, L, cysta-tin C, stefins A and B were investigated in se­ra of clinically healthy subjects. To minimise the factors such as posture, activity, food in­gestion, stress, sleep or wakefulness, which could contribute to the variations, the su­bjects were required to maintain the same re­gime during the study and 2 days before the beginning. All tested proteins in normal sera were in the nM concentration range and among them cystatin C was the most abun­dant since only cystatin C is localised extra­cellularly (Table 1). Common feature of 24­hour patterns was that cystatins and cathepsins reached their minimal values in the resting period, except for cathepsin B and stefin B, which were close to the daily mean throughout the day. Comparing all patterns of cathepsins and cystatins a significant rela­tionship between the variations was observed only for cathepsins H and L, indicating possi­ble similar regulation of expression. To eliminate between individual variabi­lity, each individual’s data were transformed to deviation from that individual’s 24-hour mean. All data were subjected to ANOVA, which validated any apparent differences by comparing different time points. Since ANO­VA can fail to obtain the actual high point of the rhythm, data were analysed also individu­ally and as a group for circadian rhythm by single and population mean cosinor analysis, which involves the fit of a 24-hour cosine cur­ve by the method of least squares. The me­thod provides both the probability of rejecti­on of the zero amplitude hypothesis for a chosen period (24 h in our case) and rhythm characteristics: the mesor (24-hour adjusted means), the amplitude (half the difference between the maximum and the minimum fit­ted cosine function), and the acrophase (time of maximum in fitted cosine function, with midnight as the phase reference). ANOVA re­vealed no significant time effect except for transformed data of cathepsins H and L and both stefins (Table 1). Using single cosinor analysis no significant rhythm was revealed, except for cystatin C, stefin A and cathepsin H, where only one subject demonstrated a circadian variation (P = 0.04). On the group level using population mean cosinor analysis, the patterns of all investigated serum prote- Table 1. Circadian characteristics for serum cathepsin B (CB), cathepsin H (CH), cathepsin L (CL), cystatin C (CC), stefin A (SA) and stefin B (SB), measured every 4 hours for 24 hours in healthy subjects. 24-h means with range between subjects are presented. Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. ANOVA Least-squares fit of 24-h cosine Serum Units 24-h mean Range F P P Mesor Amplitude Acrophase protein ±2 SE ±SE ±SE ±SE (h) CB ng/ml 4.5 ±0.5 3.5 – 7.5 0.02 1.0 0.7 4.5 ±0.5 0.1 08:40 % of mean 19 ±2 11 – 26 0.4 0.9 0.8 100 ±0.2 1 06:55 CH ng/ml 15.0 ±2.2 7.8 – 24.4 0.4 0.9 0.1 14.8 ±2.2 1.6 11:51 % of mean 112 ±51 18 – 440 2.8 0.02 0.2 99 ±1 15 11:13 CL ng/ml 18.1 ±2.7 7.5 – 32.3 0.5 0.8 0.02 18.0 ±2.7 2.1 ±0.5 11:38 ±00:41 % of mean 66 ±17 7 – 113 6.6 <0.001 0.03 99 ±0.3 15 ±4 12:01 ±00:47 CC ng/ml 681.9 ±39.4 438.2 – 1156.0 0.4 0.9 0.2 675.5 ±76.2 40.2 07:19 % of mean 60 ±9 27 – 90 1.9 0.1 0.2 99 ±0.3 5 07:23 SA ng/ml 6.1 ±0.4 4.2 – 8.2 1.0 0.4 0.2 6.1 ±0.4 0.3 14:26 % of mean 85 ±7 32 – 77 3.1 0.01 0.2 100 ±1 6 14:34 SB ng/ml 3.0 ±1.2 0.5 – 8.8 0.05 1.0 0.1 3.0 ±1.2 0.1 06:52 % of mean 49 ±7 19 – 86 4.3 0.002 0.2 100 ±0.3 5 03:46 ins showed no significant circadian rhythm with the exception of cathepsin L where the rhythm exhibited a small amplitude, ranging from 5-24 % of the 24-hour mean, and an acro-phase localised at around 12 h (Table 1). Conclusion We conclude that the time of sampling in the course of day has a minor influence on mea­surements of cathepsin L, and none on cathe­psins B and H, stefins A and B, and cystatin C in normal sera which underlines their use­fulness as potential clinical markers. The pos­sible changes in their circadian structure with different types of cancer will be of considera­ble interest. References 1. Emile C, Fermand JP, Danon F. Interleukin-6 se­rum levels in patients with multiple myeloma. Brit J Haematol 1994; 86: 439-40. 2. Micke O, Schafer U, Wormann B, Hiddemann W, Willich N. Circadian variations of interleukin-2 re­ceptors, serum thymidine kinase and beta-2-mi­croglobulin in non-Hodgkin’s lymphoma and nor­mal controls. Anticancer Res 1997; 17: 3007-10. 3. Hallek A, Touitou Y, Levi F, Mechkouri M, Bogdan A, Bailleul F, Senekowitsch R and Emmerich B. Se­rum thymidine kinase levels are elevated and ex­hibited diurnal variations in patients with advan­ced ovarian cancer. Clin Chim Acta 1997; 267: 155­66. 4. Touitou Y, Bogdan A, Levi F, Benavides M, Auzeby A. Disruption of circadian patterns of serum corti-sol in breast and ovarian cancer patients: relation­ships with tumour marker antigens. Brit J Cancer 1996; 74: 1248-52. 5. Kos J, Lah T. Cysteine proteinases and their endo­genous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer. Oncol Rep 1998; 5: 1349-61. 6. Cimerman N, Meško Brguljan P, Krašovec M, Šu­škovic S, Kos J. Circadian characteristics of cathe­psins B, H, L, and stefins A and B, potential mar­kers for disease, in normal sera. Clin Chim Acta 1999; 282: 211-8. 7. Cimerman N, Meško Brguljan P, Krašovec M, Šu­škovic S, Kos J. Twenty-four hour variations of cystatin C and total cysteine proteinase inhibitory activity in sera from healthy subjects. Clin Chim Acta 2000; 291: 89-95. 8. Kos J, Šmid A, Krašovec M, Svetic B, Lenarcic B, Vrhovec I, et al. Lysosomal proteases cathepsins D, B, H, L and their inhibitors stefins A and B in head and neck cancer. Biol Chem Hoppe-Seyler 1995; 376: 401-5. 9. Kos J, Krašovec M, Cimerman N, Nielsen HJ, Chri­stensen, Brünner N. Cysteine proteinase inhibi­tors stefin A, stefin B, and cystatin C in sera from patients with colorectal cancer: Relation to pro­gnosis. Clin Cancer Res 2000; 6: 505-11. 10. Nelson W, Tong YL, Lee JK, Halberg F. Methods for cosinor rhythmometry. Chronobiologia 1997; 6: 305-23. 11. Mojon A, Fernandez JR, Hermida RC. Chronolab: an interactive software package for chronobiologic time series analysis written for the Macintosh computer. Chronobiol Int 1992; 9: 403-12. Radiol Oncol 2002; 36(2): 183-4. Immunohistochemical analysis of cathepsin B and cathepsin S in tumors, parenchyma and regional lymph nodes of the lung Bernd Werle Ruperta-Carola-University Heidelberg, Medizinische Klinik und Poliklinik, 69115 Heidelberg, Germany Introduction The detection of antigens on tissue sections by using specific antibodies (immunohisto-chemistry (IHC)) is an important approach in identifying cell types, which express the spe­cific antigens. Although the immunohistoche­mical analysis (IHA) is a relatively well esta­blished and simple method, the major problem remains to detect low concentrati­ons of specific antigens. In this respect, the efforts are mainly focused on improved de­tection systems after binding of the specific primary antibody. Cathepsin (cath) S is present at rather low levels in lung tissue extracts1, therefore low expression of cath S at the cellular level is ex­pected. In fact, cath S was hardly detected by using standard procedures described in the li­terature. To solve this problem we establi­shed a highly sensitive staining protocol ba­sed on the CSA detection system from DAKO (Hamburg, Germany) in combination with antigen retrieval approach. Material and methods In order to identify cell populations produ­cing cath S, we performed IHC on tissue sec­tions deriving from formalin-fixed, paraffin-embedded tumors (n = 36), non-tumors pa-renchyma (n = 6) and regional lymph nodes of the lung (n = 10). In addition, the expression of cath S in lung cell populations was compa­red with those of cath B.2 Furthermore, the expression of both cysteine-cathepsins was correlated with clinical-pathological parame­ters of lung cancer patients. Results Cath S is expressed in alveolar type II cells, macrophages, bronchial epithelial cells and lymphocytes. The enzyme showed a lysoso­mal distribution over the cytoplasm. In addi­tion, we observed differences in the staining intensity of cath S in macrophages, alveolar type II cells and lymphocytes, as well. In so­me bronchial epithelial cells a more restricted localization in the basal proliferating zone of the epithelium was observed. Immune reacti­ve cath S was not found in alveolar type I cells. However, cath S could also be detected in lung tumors, independently of their origin. We found a positive reaction in adenocarci­nomas, squamous cell carcinomas and small cell carcinomas. Remarkably, a weak positive reaction at sides of interaction between tu­mor cells of the small cell carcinoma and the extracellular matrix of the alveoli could be ob­served. It should be noted that also in alveo­lar duct cells a focal positive reaction was vi­sible. Cath B could easily be detected using a standard protocol in combination with the ABC-system (Vector Laboratories, Serva Hei­delberg), but without the need of an additio­nal enhancing step. This clearly indicates that cath B is expressed at much higher levels in various cell populations compared with cath S. Cath B could also be detected in alveolar type II cells, macrophages, bronchial epitheli­al cells and various tumors. In contrast to cath S, we were not able to localize cath B in lymphocytes. The expression of cath S did not correlate with clinical-pathological para­meters of lung cancer patients, while for cath B SpN1/PnP/pN3 primary tumors were more frequently labelled than PnP tumors. Conclusion We established a highly sensitive IHC proto­col for the detection of cath S in tissue secti­ons deriving from tumors, parenchyma and regional lymph nodes of the lung. Our results show that of the two cysteine cathepsins (B and S), only cath S seems to be produced by lymphocytes, This indicates that cath S may be involved in regulatory mechanisms of the immune response. References 1. Werle B, Staib A, Jülke B, Ebert W, Zladoidsky, P, Sekirnik A, Kos J, Spiess E. Fluorometric microas-says for the determination of cathepsin L and ca-thepsin S activities in tissue extracts. Biol Chem 1999; 380: 1109-16. 2. Werle B, Lötterle H, Schanzenbšcher U, Lah TT, Kalman E, Kayser K, Bülzebruck H, Schirren J, Krasovec M, Kos J, Spiess E.. Immunochemical analysis of cathepsin B in lung tumors: an inde­pendent prognostic factor for squamous cell carci­noma patients. Brit J Cancer 1999; 81: 510-9. Radiol Oncol 2002; 36(2): 185-8. Characterization of monoclonal antibodies against MHC class II-associated p41 invariant chain fragment Valentina Zavašnik Bergant1, Nataša Kopitar-Jerala1, Tadeja Bevec1 and Janko Kos1,2 1Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; 2Krka, d.d., R & D Division, Department of Biochemical Research and Drug Design, 1000 Ljubljana, Slovenia Mouse monoclonal antibodies directed against human MHC class II-associated p41 invariant chain fra­gment have been generated. Mice were immunized with human recombinant Ii-isoform p26. For hybridoma production mouse splenocytes and myeloma cells were fused. Hybridoma cells were screened using ELISA and immunoblotting. Three cell lines (42B10, 42G11 and 43C8) were used for production of specific anti­bodies, which reacted with p41 fragment and did not bind to cathepsins L or S or their proenyzmes. As pri­mary antibody for immunofluorescence staining of lymph node tissue sections clone 2C12 MAb was selec­ted. Specific localization of p41 fragment in certain cells in lymph nodes was observed. Introduction MHC class II molecules display antigenic pep­tides on cell surface of APC (dendritic cells, B cells, macrophages, thymic epithelial cells) for recognition by CD4+ T lymphocytes.1,2,3 The MHC class II-associated Ii is a transmembrane protein that complexes with newly synthesi­zed MHC class II heterodimers and directs their trafficking through the endosomal com­partments of APC. The luminal domain of Ii organizes MHC class II dimers into nonome­ric complexes and prevents premature associ­ation of MHC class II molecules with endoge­nous polypeptides. Within endosomal/lysoso­mal compartments, Ii undergoes stepwise pro­teolytic degradation to yield progressively smaller fragments that remain associated with the peptide-binding groove of MHC class II dimmers.4 Dissociation of CLIP (a set of 3 kDa peptides) from the peptide-binding groove al­lows loading and subsequent surface expres­sion of MHC class II molecules with antigenic peptides generated from endocytosed or pha­gocytosed protein. The key enzymes that de­grade Ii are the cysteine proteases (cathepsins S5, L6 and/or V7). In human Ii exists in two al­ternatively spliced forms, p31 in p413, the lat­ter containing an additional 64-amino acid se­quence at the C-terminal end (hereafter called the p41 fragment). The discovery of p41 fra­gment-cathepsin L complex isolated from hu­man liver8 and its known crystal structure9, led to the suggestion that the p41 fragment (Ii respectively) may enhance antigen presentati­on by providing a mechanism to inhibit other­wise destructive cathepsin L but not cathe­psin S activity.10 The aim of the present study was to generate specific mouse monoclonal antibodies directed against MHC class II-asso­ciated p41 invariant chain. Furthermore these antibodies were used in immunohistochemi-cal studies of p41 fragment in lymph-node tis­sue. Materials and methods Preparing and purification of antibodies Human recombinant Ii-isoform p26, com­prising only the luminal domain11 (together with p41 fragment) was purified by Ni-chela­te chromatography (a gift from Dr. Klaus Dor­nmair, Max Planck Institute, Martinsried) and was subsequently used as an antigen for immunization of mice. BALB/c mice were injected subcutaneously with p26 (50 µg/mo­use) emulsified in complete Freund’s adju­vant, followed by intraperitoneal injections of the same amount of antigen in incomplete Freund’s adjuvant. Test bleeds were taken and titer of specific antibodies determined using antigen immobilized ELISA in which recombinant p26 and p41 fragment-cathepsin L complex, respectively, were used as anti­gens. The mouse with the highest titer was boosted intraperitoneally with p26 (50 µg/mouse). For hybridoma production sple­nocytes and myeloma cells (NS1/1-Ag4-1) were fused by a modification of the method of Kohler and Milstein.12 The screening of the positive wells was performed by antigen im­mobilised ELISA as described above for test bleeds. Hybridomas producing antibodies against p41 fragment were cloned twice by means of the limiting dilution method13 and expanded into large volumes. The cell culture supernatants were concentrated by ultrafil­tration and MAbs purified by affinity chro­matography on Protein A-Sepharose (Phar­macia, Sweden). Antibody containing fractions were pooled and dialysed against PBS, pH 7.2. Small aliquots of purified anti­bodies were stored at – 20 °C. Immunoblotting Samples were first separated by SDS-PAGE on 8 – 25 % polyacrylamide gels using Phast-System (Pharmacia, Sweden). After the elec­trophoresis the proteins were transferred on­to PVDF membrane (Millipore, USA) by pas­sive diffusion accelerated with higher tempe­rature. Non-specific binding sites were bloc­ked with 0.4 % Tween 20 in PBS, pH 7.2. After this and all subsequent steps the membrane was washed with PBS, pH 7.2 containing 0.5 % Tween. Primary anti-p41 fragment anti­bodies were incubated with the membrane, followed by secondary goat anti-mouse IgG conjugated to HRP (Dianova, Germany). De­tection was performed using 0.05 % DAB (Si­gma, USA) and 0.01 % H2O2 in 50 mM Tris-HCl buffer, pH 7.5. Immunofluorescence tissue staining Sections from formalin fixed, paraffin em­bedded lymph nodes were used for IHA. Tis­sue sections on micro cover glasses were de­paraffinised in xylene and rehydrated through ethanol series. They were placed in 10 mM sodium citrate buffer, pH 6.0 and put into a microwave oven (5 min, 400 W) for an­tigen retrieval. Non-specific binding sites we­re blocked with 3 % BSA in PBS, pH 7.4. After this and all subsequent steps tissue sections were rinsed in PBS, pH 7.4. Primary anti-p41 fragment antibody was added (clone 2C12, 25 µg/ml, for 2 h at 37 °C), followed by Alexa FluorTM 488-labeled goat anti-mouse IgG se­condary antibody. Molecular Probes, USA). Tissue sections were mounted on slides with ProLongTM AntiFade Kit (Molecular Probes, USA). Fluorescence microscopy and optical slicing were performed by confocal laser scanning microscope LSM 510 (Carl Zeiss Inc., USA). Results Purified monoclonal antibodies were tested for specificity using immunoblotting. As shown in Figure 1, 2C12 MAb (derived from clone 42B10) reacted with p41 fragment in complex with cathepsin L (32 kDa, lane 5), with p41 fragment detached from the com­plex (14 kDa, lane 1 and 2), as well as with re­combinant p26 (lane 3). There was no cross-reactivity observed with recombinant proca­thepsin L (lane 4), cathepsin L (31 kDa, lane 1) nor its heavy chain (25 kDa, lane 1) and light chain (below 14 kDa, lane 1), respectively. Also, antibody specificity and cross-reac­tvity towards different cathepsins was tested by ELISA. Recombinant (pro)cathepsins L and S were added to the wells instead of p26 Figure 1. Demonstration of the specificity of anti-p41 fragment Mab. (A) SDS-PAGE silver staining. (B) Im­munoblot of the equivalent gel stained with 2C12 MAb. Samples: (lane 1) p41 fragment-cathepsin L complex reduced (with 5 % 2-mercaptoethanol) and exposed to 100 °C in the presence of SDS for 5 minu­tes; (lane 2) p41 fragment detached from the native complex by HPLC; (lane 3) p26 reduced (with 5 % 2­mercaptoethanol) and exposed to 100 °C in the pre­sence of SDS for 5 minutes; (lane 4) nonreduced re­combinant procathepsin L; (lane 5) nonreduced p41 fragment-cathepsin L complex; (lane 6) LMW stan­dards. or p41 fragment-cathepsin L complex. With all three selected cell lines negligible reacti­vity was observed. For immunohistochemical localization of p41 fragment in lymph node tissue sections 2C12 MAb was selected. Re­sults are shown in Figure 2. Figure 2. Immunohistochemical staining of lymph-no­de tissue sections for p41 fragment. Conclusions Specific monoclonal antibodies recognizing MHC class II-associated p41 fragment were successfully produced. They do not cross re­act with cathepsins L nor S or their proenz­ymes. We have shown specific localization of Ii fragments in certain cells in lymph nodes. These antibodies provide new tools for inve­stigating subcellular colocalization of Ii toge­ther with cathepsins S and L. Acknowledgements This work was supported by the Ministry of Science and Technology of the Republic of Slovenia. The authors thank Professor Robert Zorec and Dr. Marko Kreft for their assistan­ce with confocal laser scanning microscope. References 1. Nakagawa TY, Brissette WH, Lira PD, Griffiths RJ, Petrushova N, Stock J, McNeish J D, Eastman S, Howard ED, Clarke SRM, Rosloniec EF, Elliot EA, Rudensky AY. Impaired invariant chain degradati­on and antigen presentation and diminished colla­gen-induced arthritis in cathepsin S null mice. Im­munity 1999; 10: 207-17. 2. Riese RJ, Mitchell RN, Villadangos JA, Shi, G-P, Palmer JT, Karp ER, De Sanctis GT, Ploegh HL, Chapman HA. Cathepsin S activity regulates anti­gen presentation and immunity. J Clin Invest 1998; 101: 2351-63. 3. Chapman HA. Endosomal proteolysis and MHC class II. function. Curr Opin Immunol 1998; 10: 93­102. 4. Shi G-P, Villadangos JA, Dranoff G, Small C, Gu L, Haley KJ, Riese R, Ploegh HL, Chapman HA. Cathepsin S required for normal MHC Class II peptide loading and center development. Immu­nity 1999; 10: 197-206. 5. Cresswell, P. Proteases, processing and thymic se­lection. Science 1998; 280: 394-5. 6. Nakagawa T, Roth W, Wong P, Nelson A, Farr A, Deussing J, Villadangos JA, Ploegh H, PetersC, Ru-densky AY. Cathepsin L: Critical role in Ii degra­dation and CD4 T cell selection in the thymus. Sci­ence 1998; 280: 450-3. 7. Brömme D, Li Z, Barnes M, Mehler E. Human ca-thepsin V functional expression, tissue distributi­on, electrostatic surface potential, enzymatic cha­racterization and chromosomal localization. Biochemistry 1999; 38: 2377- 85. 8. Bevec T, Stoka V, Pungercic G, Dolenc I, Turk V. Major histocompatibility complex class II-associa­ted p41 invariant chain fragment is a strong inhi­bitor of lysosomal cathepsin L. J Exp Med 1996; 183: 1331-8. 9. Guncar G, Pungercic G, Klemencic I, Turk V, Turk D. Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathe­psins L and S. EMBO J 1999; 18: 793-803. 10. Turk D, Guncar G, Turk V. The p41 fragment sto­ry. IUBMB Life 1999; 48: 7-12. 11. Strubin M, Mach B, Long E O, The complete sequ­ence of the mRNA for the-HLA-DR-associated in­variant chain reveals a polypeptide with an unusu­al transembrane polarity. EMBO J 1984; 3: 869-72. 12. Kohler G, Milstein C. Continuous cultures of fu­sed cells secreting antibody of predefined specifi­city. Nature 1975; 265: 495-7. 13. Robb A. J. Microcloning and replica plating of mammalian cells. Science 1970; 170: 857-8. Radiol Oncol 2002; 36(2): 189. In vitro and in vivo angiogenic assays Nils Brünner Finsen Laboratory, Strandboulevarden 49, DK-2100, Copenhagen, Denmark The formation of new vessels in a tumour requires a number of steps including chemotaxis, migration, pro­liferation and tubular formation of the endothelial cells. Thus, in vitro assays, which dissect out each of the­se steps or a combination of these, can be designed. For example, tubular formation assays can be conduc­ted using collagen gels as matrix and angiogenic factors such as VEGF and bFGF as stimuli. In addition to studying these functions of the endothelial cells under various conditions, these assays can also be used to test the importance of different molecules including molecules with a potential inhibitory effect on tumour angiogenesis. The next question that arises is which endothelial cells to use. HUVEC (human umbilical ve­in endothelial cells) can be obtained from commercial sources and these cells can grow for approximately 15 to 20 passages. Another possibility is to establish primary cultures of endothelial cells. We have used this technique to establish primary lung endothelial cell cultures from wild-type mice and from knock-out mice. The resulting cell lines can then be compared using some of the above-mentioned in vitro assays. A large number of in vivo angiogenesis assays have been described. The more common ones are implantation of bFGF or VEGF pellets either just subcutaneously, in a dorsal air sack or embedded in Matrigel. Other mo­dels include wound healing, retina damage etc. However, caution should be taken regarding extrapolating re­sults from assays including non-malignant conditions to tumour angiogenesis. The mediators of tumour an-giogenesis may very well be different from those mediating angiogenesis in non-tumour conditions. Radio/ 011col 2002; 36(2): 189. In vitro and in vivo angiogenic assays Nils Briinner Finsen Laboratory, Strandboulevarden 49, DK-2100, Copenhagen, Denmark The formation oj new vessels in a tumour requires a number of steps including chemotaxis, migration, pro­liferation and tubular fonnation of the endothe/ial cells. Thus, in vitro assays, which dissect out each of the­se steps or a combination of these, can be designed. For example, tubular formation assays can be conduc­ted using collagen gels as nzatrix and angiogenic factors such as VEGF and bFGF as stimuli. In addition to studying these functions oj the endothelial cel/s under various conditions, these assays can also be used to test the illlportance oj different Jnolecules including molecules with a potential inhibitory effect on tumour angiogenesis. The next question that arises is which endothelial cel/s to use. HLlVEC (human umbilical ve­in endothelial cells) can be obtained from commercial sources and thcse cel/s can grmu far approximately 15 to 20 passages. Another possibility is to establish primary cultures oj endothe/ial cel/s. We have used this technique to estab/ish primary lung endothelial celi cultures from wild-type mice and from knoc/c-out mice. The resulting celi lines can then be compared using some oj the above-mentioned in vitro assays. A large number oj in vivo angiogenesis assays have been described. The more common ones are implantation oj bFGF or VEGF pellets either Just subcutaneously, in a dorsal air sack or embedded in Matrigel. Other mo­de/s include wound healing, retina damage etc. However, caution should be ta/cen regarding extrapolating re­sults frorn assays including non-malignant conditions to tumour angiogenesis. The mediators oj lumour an­giogenesis may very well bc diffcrent from thosc mediating angiogcnesis in 11011-tumour conditions. Slove11ia11 abslracl Radio/ 011col 2002 36(2): 87-90. Nepopolna spontana raztrganina uretra. Prikaz primera Bor kovic Z, Srdoc D, Bedalov G Izhodišca. Avtorji so v prispevku predstavili primer spontane delne raztrganine uretra med na­padom ledvicnih kolik, ki ga je povzrocil ledvicni kamen v proksimalnem delu uretra v višini processus transversusa L3. Prikaz primera. Urografsko slikanje je pokazalo razširitev ureteralnega kanala pa tudi ledvicne­ga meha. V višini pielo-ureternega prehoda je slika pokazala iztekanje kontrastnega sredstva. Kontrastno sredstvo je bilo razlito ob musculusu psoas. Precne slike, narejene z racunalniško to­mografijo, so pokazale razlito kontrastno sredstvo vzdolž medialnega in dorzalnega dela perire­nalnega prostora in distalno ob musculus psoas. Ureter je bil na sliki prikazan od raztrganine na pielo-ureternem prehodu do mesta ledvicnega kamna. Na uretru ni bilo znamenj o poškodbah parenhima ali o krvavitvah v perirenalnem prostoru. Med operacijo smo našli mesto raztrgani­ne, ki je bilo obdano z veliko kolicino perirenalne in periureterne tekocine. Po odstranitvi kamna smo raztrganino kirurško zašili. Pooperativna urografija in slika uretra z racunalniško tomogra­fijo sta bili normalni. Zakljucki. Pri delni spontani raztrganini uretra še vedno vidimo v uretru kontrastno sredstvo di­stalno od mesta raztrganine in iztekajoce vzdož musculusa psoas. Radio/ 011col 2002; 36(2): 91-4. Gastropareza pri mladi bolnici s sladkorno boleznijo. Prikaz primera Kovacic P, Jamar B Izhodišca. Gastropareza je upocasnjeno praznjenje želodca in se pojavlja pri razlicnih bolezen­skih stanjih, na primer po vagotomiji ali pri sistemskih boleznih kot so sladkorna bolezen, skle­rodermija in amiloidoza. Namen tega clanka je predstaviti rentgensko preiskavo, ki je enostav­na, zanesljiva in neinvazivna, kot alternativno metodo drugim metodam za oceno praznjenja želodca. Prikaz primera. Enaindvajsetletna ženska je bila sprejeta zaradi suma na avtonomno nevropati­jo. Zadnjih deset let je imela od insulina odvisno sladkorno bolezen tipa I. Ob sprejemu je opi­sovala nauzejo, bruhanje, oslabelost in obcasne vrtoglavice. Pri rentgenski preiskavi z barijem je bil viden dilatiran, aperistalticen požiralnik, v aperistalticnem želodcu so bili ostanki hrane, in nobene strukturne spremembe, ki bi povzrocala oviro praznjenja želodca, ni bilo videti. Zakljucki. Scintigrafija trenutno velja za standardno preiskavno metodo za oceno praznjenja že­lodca. Želodcno motoriko pregledujejo še z ultrazvokom, elekrogastrografijo in antroduodenal­no manometrijo, praznjenje želodca pa ocenjujejo tudi z magnetno rezonanco in radiopacnimi markerji.Vloga rentgenske preiskave z barijem še ni dokoncno razjasnjena. Radio/ 011cu/ 2002; 36(2): 95-102. Magnetna resonancna spektroskopija. Pregled metode in njena uporaba v klinicni neuroradiologiji KorenA Izhodišca. Magnetno resonancna spektroskopija (MRS) je sorazmerno nova diagnosticna meto­da. Možganovina je zelo primerno tkivo za te vrste analizo. V praksi pa je z MRS mogoce anali­zirati le majhno število sestavin, ki se so v možganskem tkivu. Uporabnost metode v neuroradi­ologiji in pri klinicnem delu narašca, saj pomaga tako pri diferencialni diagnostiki patoloških procesov kot pri opredelitvi progresije bolezni oz. uspeha terapije. Pri analizi rezultatov preiska­ve je potrebno upoštevati številne dejavnike, ki lahko vplivajo na objektivnost izvida. Magnetno resonancni tomograf na klinicnem inštitutu za radiologijo v KC v Ljubljani omogoca sodobne MRS protokole, ki jih uporabljamo v diagnostiki nevroloških in drugih bolezni. Zakljucki. MRS omogoca spektralno analizo snovi v izbranem volumnu tkiva in s tem vpogled v njegovo metabolno stanje. Radio/ 011col 2002; 36(2): 103-8. Napredki v s kontrastom ojacani MR-angiografiji: Indikacije in omejitve Aschauer MA, Stollberger R, Ebner F Izhodišca. Z gadoliniurn(Gd)-ojacana tri-dimenzionalna (3D) magnetno resonancna angiografija (MRA) je novejša tehnika, s katero hitro dobimo podatke visoke locljivosti tako za prikaz arterij kot ven v celem telesu. Zakljucki. S kontrastom ojacana 3D MRA predstavlja mejnik za neinvaziven slikovni prikaz ži­lja. Metem, ko je bila klinicna uporabnost 3D MRA že dokazana v številnih podrocjih žilne dia­gnostike, bo stalen razvoj strojne in programske opreme, kakor tudi novih kontrastnih sredstev pripeljal do nadaljnega širjenja indikacij. Rutinsko lahko opravljamo 3D prikaz ledvic, ureterjev in mehurja, s cimer lahko prikažemo in ocenimo zaporo, zapoznelo funkcijo, polnitvene defek­te in mase v predelu ledvic. Bolniki z vzpodbujevalniki srca, niso primerni za preiskavo z MRA, nekatere vrste endoprotez in imobilizacijskih sredstev pa povzrocajo pomembne artefakte, ki skrijejo pomembne. Locljivost CE-3D-MRA je nižja v primerjavi z konvencionalno angiografijo, omejena pa je tudi locljivost majhnih perifernih arterij. Slove11ia11 abstract Radio/ Onco/ 2002; 36(2): 109-19. Poškodbe normalnega tkiva zaradi radioterapije in kemotearpije: vloga citokinov in adhezijskih molekul Plevova P Ozadje. Ionizirajoce sevanje in citostatiki, ki jih uporabljamo za zdravljenje raka, poškodujejo normalno tkivo in sprožijo celovit odgovor tako na celularni kot molekularni ravni, vanj pa so vpleteni tudi citokini in adhezijske molekule. Metode. Zbrali in pregledali smo že objavljene podatke. Rezultai in zakljucki. Razlicni citokini in adhezjske molekule, kot so nekrotizirajoci faktor alfa interleukini 1, 2, 4 in 6, intereferon gama, granulocite in makrofage stimulirajoci faktor, trans­formirajoci rastni faktor beta, faktor aktiviranja krvnih plošcic, intercelicna adhezijka molekula 1, vaskularno-celicna adhezijska molekula-1, in selektini E in P so vpleteni v odgovor normal­nega tkiva na zdravljenje z ionozirajocim sevanjem in kemoterapijo in so odgovorni za poškod­be tkiva zaradi zdravljenja ter nezaželenih ucinkov teh nacinov zdravljenja, na primer vrocine, anoreksije, utrujenosit, zaviranja hematopoeze ter akutnega in poznega lokalnega odziva na zdravljenje. Radio/ 011co/ 2002; 36(2): 121-9. Ucinkovitost trastuzumaba in paclitaxela pri zdravljenju bolnic z metastatskim rakom dojke in izraženim HER-2/neu onkogenom Janku F, Petruzelka L, Pribylova O, Vedralova J, Honova H, Pecen L, Zimovjanova M, Pazdrova G, Safanda M, Konopasek B, Zemanova M Izhodišce. Trastuzumab je poznan kot ucinkovito zdravilo pri bolnicah z rakom dojke in izraže­nem HER2/neu onkogenom. V prospektivni študiji smo proucevali ucinkovitost, varnost in to­ksicnost trastuzumaba in paclitaxela pri metastatskem raku dojke, ki je bil v progresu po pred­hodnem zdravljenju. Bolniki in metode. Vkljucili smo 17 bolnic z histološko potrjenim rakom dojke, s stanjem zmo­gljivosti po Karnofskem vsaj 60 %, srednjo starostjo 50 let (36-66), ki so bile prej zdravljene z vsaj dvema shemama kemoterapije. Izražanje HER-2/neu smo preverjali z Herceptest ® (DAKO) pri vseh 17 bolnicah. Petnajst vzorcev je bilo 3+ positivnih, dva vzorca pa 2+ positivna. Vse bolnice razen ene so bile prej zdravljene s taksani. Interval brez taksanov (TFI) smo definirali kot cas med zadnjo aplikacijo taksanov in pricetkom študije za vsako bolnico posebej. TFI je bil daljši od enega leta pri 7 bolnicah, TFI krajši od enega leta pa pri 9. Zacetna doza Trastuzumaba je bi­la 4 mg/kg i.v., nato pa 2 mg/kg i.v. vsak teden. Paclitaxel smo aplicirali v dozi 80 mg/m2 i.v. vsak teden do napredovanja bolezni ali pa nesprejemljive toksicnosti. Ocenjevali smo stopnjo odgo­vora (RR), cas do napredovanja bolezni (TTP), preživetje (OS) in toksicnost. Rezultati. V populaciji z namenom zdravljenja smo ugotovili objektiven odgovor pri 10 bolnicah (59 %), vkljucno z dvema popolnima odgovoroma (CR). V podskupini z TFI > 1 leto smo ugoto­vili odgovor v štirih primerih, vkljucno z enim CR (RR 57 %). V podskupini z TFI < 1 leto pa smo ugotovili odgovor v 6 primerih vkljucno z enim CR (RR 67%). TFI ni bil statisticno pomemben za odgovor (p<0,4349). Srednji TTP znaša 6 mesecev , 4 bolnice pa so še vedno brez progresa. Bolnice z TFI > 1 leto imajo daljši TTP (p 0,0201). Pri 10 živih bolnicah nismo dosegli srednje­ga OS. Aplicirali smo 599 krogov terapije vkljucno z 473 krogi trastuzumaba in paclitaxela brez prilagajanja odmerka. Ena bolnica je pri prvi aplikaciji trastuzumaba razvila preobcutljivostno reakcijo in smo jo izkljucili iz študije. Najpogostejši toksicni ucinek je bila z infuzijo trastuzu­maba povezana pireticna reakcija, ki smo jo opazili pri šestih bolnicah. Edini stranski ucinek, ki je pripeljal do prekinitve zdravljenja je bila kardiotoksicnost. Zmanjšanje iztisne frakcije druge stopnje se je pojavilo pri eni bolnici, ravno tako pa tudi tretje stopnje. Šest bolnic je izkusilo nev­ropatijo tretje stopnje. Pri eni bolnici se je pojavila nevtropenija cetrte stopnje in anemija tretje stopnje. Ugotovili smo štiri primere infekcije tretje stopnje brez nevtropenije. Poslabšanje testov jetrne funkcije tretje stopnje smo ugotovili pri šestih bolnicah, niso pa zahtevali prilagoditve odmerka. Ugotovili smo še en primer hiperglikemije tretje stopnje in en primer povecanja tele­sne teže tretje stopnje. Zakljucki. Trastuzumab in paclitaxel sta pokazala ucinkovitost in dobro toleranco pri bolnicah z metastatski mrakom dojke z prekomernim izražanje HER-2/neu. Odgovor tumorja pri desetih bolnicah, ki so so bile prej zdravljene s taksani in so odgovorile na zdravljenje ni bil odvisen od TFI, so pa bolnice z daljšim TFI imele daljši TTP. Radio/ 011col 2002; 36(2): 131-43. Naravni inhibitorji proteaz v tumorju Magdolen U, Krol J, Sato S, Mueller MM, Speri S, Kriiger A, Schmitt M, Magdolen V Preoblikovanje zunaj celicnega matriksa je osnova mnogih normalnih bioloških procesov, na pri­mer razvoja, morfogeneze in celjenja rane. Preoblikovanje zunajcelicnega matriksa opazimo tu­di pri zelo resnih patoloških okvarah, na primer pri aterosklerozi, fibrozi, invazivnosti tumorja in razvoju metastaz. V takšno preoblikovanje so najpogosteje vpletene serinske proteaze (še zla­sti plazminogenski aktivator-urokinaza / plazminski sistem), metaloproteaze matriksa (družina približno 20 od Zn odvisnih endopeptidaz, vkljucno s kolagenazami, želatinizami, stromelizini in metaloproteazami membranskega tipa) ter cisteinske proteaze. Dejavnost teh proteaz in vivo v zunajcelicnem prostoru uravnavata aktiviranje zimogena in nadzorovana inhibicija. Vpregle­dnem clanku predstavljamo zgradbo in biokemicne lastnosti pomembnih inhibitorjev proteaz, na primer inhibitor plazminogenskega aktivatorja tipa 1 in 2 (PAI-1 in PAI-2,) tkivnih inhibitor­jev metaloproteinaz (TIMP-1, -2, -3 in -4) in inhibitorjev cisteinskih proteaz cistatin C, ki so po­vezani z razvojem tumorja. Zanimivo je, da nekateri od teh inhibitorjev tumorskih proteaz opravljajo hkrati vec nalog, ki pravzaprav bolj pospešujejo kot zavirajo napredovanje tumorja, ce prisotnost inhibitorjev v tumorskem tkivu ni uravnoteženea. Radio/ 011col 2002; 36(2): 145-51. Inhibitorja cisteinskih proteinaz stefin A in stefin B pri operabilnem karcinomu glave in vratu Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Kos J, Škrk J Namen. Ovrednotiti vlogo inhibitorjev cisteinskih proteinaz stefinov A in B v procesu odlocanja o zdravljenju in njihov napovedni pomen pri operabilnem plošcatocelicnem karcinomu glave in vratu. Bolniki in metode. Koncentracije stefinov A in B so bile izmerjene imunobiokemicno z uporabo ELISA testov v citosolih, pripravljenih iz tkiva tumorja in okolne zdrave sluznice 91 bolnikov z operabilnim plošcatocelicnim karcinomom glave in vratu. Ob zakljucku opazovanega obdobja je znašal srednji cas spremljanja preživelih bolnikov 5,8 let (razpon 5-9,3 leta). Rezultati. Koncentracija stefina A je bila statisticno pomembno višja v vzorcih tumorja kot v vzorcih zdrave sluznice (P = 0.05). V skupini bolnikov s klinicno tipnimi bezgavkami pred zdrav­ljenjem (11 = 57) je bila ugotovljena signifikantna razlika v koncentracijah stefina A (P= 0.03) in stefina B (P= 0.02) med tistimi s histopatološko potrjeno prizadetostjo bezgavk in tistimi z nepri­zadetostjo vratnih bezgavk. V univariatni analizi preživetja so se kot prognosticno ugodnejše iz­kazale visoke koncentracije stefinov. Stefin A je potrdil svoj neodvisen napovedni pomen tudi v multivariatni analizi. Zakljucki. Kot dejavnika, zmožna razlikovati med patološkima stadijema bolezni pN0 in pN+ pri bolnikih s klinicno ugotovljeno prizadetostjo bezgavk, bi stefina A in B lahko vplivala na odlo­citev o obsegu operacije na vratu. Oba stefina sta se izkazala kot zanesljiva kazalca za napoved preživetja bolnikov z operabilnim plošcatocelicnim karcinomom glave in vratu. Notices Notices submitted for publication should contain a mailing address, phone and/or fax number and/or e-mail oj a Contact person or department. CT scanning ]11/le 13-16, 2002 "7th The meeting Annual Computed Body Tomography for the Technologist" will take place in Las Vegas, Nevada, USA. Contact Conference Co-ordinator, Office of Conti­nuing Medica! Education, Johns Hopkins University School of Medicine, Turner 20/720 Rutland Avenue, Baltimore, Maryland 21205-2195, USA; or call +1 410 955 2959; or fax +1 410 955 0807; or e-mail cmenet@ljh­mi.edu; or see http://www.med.jhu.edu/cme Bronchology and bronchoesophagology }lll!C 16-19, 2002 The "12th World Congress for Bronchology" and the "12th World Congress for Bronchoesophagology" will be offered in Boston, USA. Contact Congress Secretariat. Tufts University School of Medicine. Office of Continuing Education, 136 Harrison Avenue, Boston, MA 02111, USA, or call +1 617 636 6509; or fax +1 617 636 0472; or see http://www.aabronchology.org Brachytherapy ]ulle 16-20, 2002 The ESTRO teaching course "Modem Brachy­therapy Techniques" will take place in Lisbon, Portugal. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@estro.be; or see http://www.estro.be Clinical oncology June 21-22, 2002 The "3rd International Anglo-Croatian Symposium on Clinical Oncology" in collaboration with "51 Radiotherapy Club" (UK) meeting will be offered in Dubrovnik Cavtat, Croatia. Contact Dr. Fedor Šantek, Executive Secretary; Medica! school, Clinic of Oncology and Radiotherapy, University Hospital Centre Rebro, Kišpaticeva 12, Zagreb, Croatia; or call +385 1 4552 333. Radiotherapy June 23-27, 2002 The ESTRO teaching course "IMRT and Other Con­formal Techniques in Practice" will take place in Amsterdam, The Netherlands. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@estro.be; or see http://www.estro.be Radiotherapy ]1111e 23-27, 2002 The ESTRO teaching course "Imaging for Target Volu­me Determination in Radiotherapy" will take place in Coimbra, Portugal. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail infoa,lestro. be; or see http://www.estro.be Oncology ]une 30 -July 5, 2002 The "18th UICC International Cancer Congress" will be offered in Oslo, Norway. Contact Norwegian Cancer Society, P.O. Box 5327 Majorstua, N-0304 Oslo, Norway, or call +47 22 59 30 00; or fax +47 22 60 69 80; or e-mail cancer@loslo2002.org Radiology ]uly 1-5, 2002 The "22nc1 lnternational Congress of Radiology (ICR 2002)" will take place in Cancun, Mcxico. Contact B.P. Servimed, S.A. de C.V., at Insergentes Sur No. 1188 50 piso, Col. Del Valle, 03210 Mexico DF, Belgium; or call +525 575 9931; or fax +525 559 9407; or e-mail fmricr« 1>servimed.com.mx Oncology july 3-5, 2002 The ESO course "Cancer Economics and Evidence­Based Medicine" will take place in Sapporo, Japan. Contact ESO Office, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 02 43359611; or fax +39 02 43359640; or e-mail esomi«Dtin.it; or see http://www.cancerworld.org Biomedical spectroscopy July 7-10, 2002 The "First International Conference on Biomedical Spectroscopy: Fram Molecules to Men" will take place in Cardiff, Wales, United Kingdom. Contact Dr Parvez l. Haris, Department of Biological Sciences, De Montfort University, The Gateway, Leicester, LEl 9BH, United Kingdom; or call +44 116 2506306; or fax +44 116 2577287; or e-mail: pharis«ildmu.ac.uk; or see http://www.dmu.ac. uk/in/biospectra/ CT scanning ]11/y 25-28, 2002 The "10111 Annual Advanced Topics in CT Scanning: The 2002 Edition" will take place at Lake Tahoe, NV, USA Contact Conference Co-ordinator, Office of Conti­nuing Medica! Education, Johns Hopkins University School of Medicine, Turner 20/720 Rutland Avenue, Baltimore, Maryland 21205-2195, USA; or call +1 410 955 2959; or fax +1 410 955 0807; or e-rnail cmenel«''ih­mi.edu; or see http://www.med.jhu.edu/cme Clinical Oncology Augusl 4-9, 2002 The "Masterclass in Clinical Oncology" will take place in Montecatini Terme, Jtaly. Contact Dr. Wolfgang Gatzermeier, ESO Office, Viale Beatrice d'Este 37, 20122 Milan, Italy; or call +39 0258317850; or fax +39 02 433 59640; or e-mail es­oweb@tin.it Radiation physics August 25-29, 2002 The ESTRO tcaching course "Physics far Clinical Radiotherapy" will take place in Leuven, Belgium. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info«11estro.be; or see http://www.estro.be Radiobiology A11g11st 25-29, 2002 The ESTRO teaching course "Basic Clinical Ra­diobiology" will take place in St. Petersburg, Russia. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info«iiestro.be; or see http://www.estro.be Oncohaematology A11g11st 29-30, 2002 The ESO course will take place in Buenos Aires, Argentina. Contact G. Farante, ESO Headquarters, ESO Latin Arnerica Office, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 02 583] 73]8; or fax +39 02 5832'1266; or e-rnail esolatin«Dtin.it; or see http://www.cancer­world.org; or Argentina Office, A. Ranca ti, Florida 833 (lo), "J 005 Buenos Aires; Phone +54 11 45118078; Fax +54 1] 45118079. Oncohaematology August 31 -Seple111ber 1, 2002 The ESO course will take place in Bahia, Brazil. Contact G. Farante, ESO Headquarters, ESO Latin Arnerica Office, Viale Beatrice d'Estc 37, 20122 Milan, ltaly; or call +39 02 583] 7318; or fax +39 02 58321266; or c-mail esolatin«Dtin.it; or see http://www.cancer­world.org; or c-mail afrasson«llhotrnail.corn Prostate cancer Seplc111ber 1-3, 2002 The ESTRO teaching course "Brachytherapy for Prostate Cancer" will take place in Utrecht, the Nethcrlands. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgiurn; or call +32 7759340; or fax +32 2 7795494; or e-rnail info(alestro.be; or see http://www.estro.be 198 Notices Lung cancer September 1-4, 2002 "81h The Central European Lung Cancer Confe­ rence" will be offered in Vienna, Austria. Contact Conference Secretariat, Mondial Congress, Faulmanngasse 4, A-1040 Vienna, Austria; or call +43 1 588 04 O; or fax +43 1 586 91 85; or e-mail con­gress@mondial.at Lung cancer September5-7, 2002 The "2nd lnternational Conference on New Perspec­tives in the Treatment of Small Celi Lung Cancer" will be offered in Lausanne, Switzerland. Contact Imedex, 70 Technology Drive, Alpharetta, GA, 30005 3969 USA; or call +1 770 751 7332; or fax +1 770 751 7334; or e-mail meetings@imedex.com; or see http://www.imedex.com Lung cancer September 8-12, 2002 The "IASLC Workshop on Progress and Guidelines in the Management of Non Small Cell Cancer" will be offered in Bruges, Belgium. Contact Secretariat, P. van Houtte, Dept. Ra­diotherapy, Institute Jules Bordet, Rue Heger-Bordet 1, B-1000 Brussels, Belgium; or call +32 2 541 3830; or fax +32 2 538 7542; or e-mail paul.vanhoutteaobordet.be Medica! physics September 9-13, 2002 The "lOth lnternational Congress on Boron Neutron Capture Therapy" will take place in Essen, Germany. Contact Dr. Ray Moss with e-mail mosstiJljrc.nl Radiation therapy September 17-21, 2002 The 21st Annual ESTRO Meeting will take place in Prague, Czech Republic. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call + 32 7759340; or fax + 32 2 7795494; or e-mail info@estro.be; or see http://www.es­tro.be Oncology September 19-21, 2002 The ESO course "The Challenge of Cancer. A Cen­ tral Role for General Practice" will take place in Dublin, lreland. Contact ESO Headquarters, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 02 43359611; or fax +39 02 43359640; or e-mail esomi@tin.it; or see http://www.cancerworld.org Neuroradiology September 16-28, 2002 The course "Johns Hopkins Neuroradiology Re­ view" will take place in Baltimore, Maryland, USA. Contact Conference Co-ordinator, Office of Conti­nuing Medica! Education, Johns Hopkins University School of Medicine, Turner 20/720 Rutland Avenue, Baltimore, Maryland 21205-2195, USA; or call +1 410 955 2959; or fax +1 410 955 0807; or e-mail cmenet@jh­mi.edu; or see http://www.med.jhu.edu/cme Ultrasound September 20-22, 2002 The course "31 st Annual Diagnostic Ultrasound in Gynecology and Obstetrics and Abdomen" will take place in Baltimore, Maryland, USA. Contact Conference Co-ordinator, Office of Conti­nuing Medica! Education, Johns Hopkins University School of Medicine, Turner 20/720 Rutland Avenue, Baltimore, Maryland 21205-2195, USA; or call +1 410 955 2959; or fax +1 410 955 0807; or e-mail cmenet@jh­mi.edu; or see http://www.med.jhu.edu/cme Oncology September 29 -October 3, 2002 The "2nd World Assembly on Tobacco Counters Health" will be offered in New Delhi, India. Contact Convenor, WATCH 2002, 509-B, Sarita Vihar, New Delhi 110 044, lndia; or call +91 11 694 4551; or fax +91 11 694 4472; or e-mail cancerak@del6.vsnl.net.in; or see http://www.watch-2000.org Radiation therapy October 6-9, 2002 ASTRO Annual meeting will be held in New Orleans, Louisiana, USA. Contact American Society for Therapeutic Radio­logy and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or see http://www.astro.org Notices 199 Cancer imaging October 7-9, 2002 The 3rd Annual Teaching Course will be organised by International Cancer lmaging Society (IC!S 2002) and it will take place in Paris, France. Contact ICIS Secretariat, BIR Conference Office, 36 Portland Place, London, w·1 B 1A T, U .K.; or call +44 20 7307 1416; or fax +44 20 7307 1414; or e-mail rebec­ca.gladdishQobir.org.uk Salivary glands October 7-12, 2002 The master course about cancer in salivary glands will take place at European Institute of Oncology in Milan, ltaly. Call P. Lonati, +39 02 5748 9490; or fax +39 02 5748 9491; or e-mail head&neck@ieo.it Colorectal cancer October 24-25, 2002 The "2nd Colorectal Cancer Conference" will take place in Rome, ltaly. Contact ESO Office, Viale Beatricc d'Este 37, 20122 Milan, ltaly; or call +39 02 43359611; or fax +39 02 43359640; or e-mail esomi<1Dtin.it; or see http://www.cancerworld.org Radiation oncology November 10-16, 2002 The ESTRO teaching course "Evidence-Based Ra­diation Oncology: Methodological Basis and Clinical Application" will take place in Tenerife, Spain. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info<1,'estro.be; or see http://www.es­tro.be Breast cancer November 12-73, 2002 The ESO course will take place in New York, USA. Contact ESO Headquarters, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 02 43359611; or fax +39 02 43359640; or e-mail esomW.otin.it; or see http://www.cancerworld.org; or R. Boschi-Belgin, ESO US Office, American-ltalian Cancer Foundation, 112 East 71st Street -2B, New York NY 10021, USA; Phone +1 212 6289090; Fax +1 212 5176089; e-mail aicf<1,uaicfonline.org; http://www.aicfonline.org Breast cancer Novenzber21-23, 2002 The ESO course "Current Breast Cancer Manage­ment" will take place in Johannesburg, South Africa. Contact ESO Headquarters, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 02 43359611; or fax +39 02 43359640; or e-mail esomi<1Jltin.it; or see http://www.cancerworld.org Radiation oncology Marc/z 15-19, 2003 The "2nd lnternational Conference on Translation Research and Pre-Clinical Strategies in Radiation On­cology, ICTR 2003" will be offered in Lugano, Switzerland. Fax +41 91 820 9044, or c-mail jbernier<11'pop.cunet.ch, or see http://www.osg.ch/ictr2003.htm1 Biomedicine April 2-4, 2003 The "5th International Conference on Simulations in Biomedicine" will be offered in Ljubljana, Slovenia. Contact Ms. Gabriella Cossutta, Conference Secreta­riat, Biomedicine 2003, Wessex Institute of Technolo­gy, Ashurst Lodge, Ashurst, Southampton, SO40 7 AA, UK; or call +44 238 029 3232; or fax +44 238 029 2853; or e-mail gcossutta<1Dwessex.ac.uk; or see http://www.wessex.ac.uk/conferences/2003/biomed03 Allergology and clinical immunology Jzme 7-1 J, 2003 The "2211d Congress of the European Academy of Allergology and Clinical Immunology" take place in Paris, France. Contact Congrex Sweden AB, Attn: EAACI 2003, Linnegatan 89A, P.O. Box 5619, SE-l14 86 Stockholm, Sweden, or call +46 8 459 66 00; or fax +46 8 661 91 25; or e-mail eaaci2003«1'congrex.se; or see http://www.eaaci.org Lung cancer August 10-14, 2003 The "lOth World Conference of the lnternational Association for the Study of Lung Cancer" will be of­ fercd in Vancouver, Canada. Contact 10th World Conference of Lung Cancer, c/o lnternational Conference Services, 604-850 West Hastings, Vancouver BC Canada V6C lEl, or call +1 604 681 2153; or fax +l 604 681 1049; or e-mail con­ ference«,12003worldlungcancer.org Radio/ Ollcol 2002; 36(2): 196-200. 200 Notices Radiation therapy September 21-25, 2003 The ESTRO 22 / ECCO 12 Meeting will take place in Copenhagen, Denmark. Contact FECS office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@estro.be; or see http://www.fecs.be Radiation therapy October 19-23, 2003 ASTRO Annual meeting will be held in Salt Lake City, Utah, USA. Contact American Society for Therapeutic Radiology and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or see http://www.astro.org Radiation therapy September 12-16, 2004 The 23rd Annual ESTRO Meeting will be held. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@lestro.be; or see http://www.es­tro.be Radiation therapy October 3-7, 2004 ASTRO Annual meeting will be held in Atlanta, USA. Contact American Society for Therapeutic Radiology and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or see http:// www.astro.org As a service to our readers, notices of meetings or courses will be inserted free of charge. Please sent information to the Editorial office, Radiology and Oncology, Zaloška 2, Sl-1000 Ljubljana, Slovenia. - - kapsule v svetu najvec predpisovani sistemski(; antimikotik edini peroralni sistemski antimikotik za zdravljenje vaginalne kandidoze, ki ga je odobril FDA Skrajšano navodilo Flukonazol je sistemski antimikotik iz skupine triazolov. Odmerjanje pri razlicnih indikacijah: vaginalna kandidoza 150 mg v enkratnem odmerku mukozna kandidoza 50 do 100 mg na dan dermatomikoze 50 mg na dan ali 150 mg na teden sistemska kandidoza prvi dan 400 mg, nato od 200 do 40 · Najvecji dnevni odmerek je 80 preprecevanje kandidoze 50 do 400 mg na dan kriptokokni meningitis prvi dan 400 mg, · . nato od 200 do 400 mg na dtm .·• ; i? > . vzdrževalno zdravljenje 200mg na dan ;':?, '( '.,,: ''.: Kontraindikacije: Preobcutljivost za zdravilo ali sestavine zd.avil.! ln:..f.kgije,: .;; " · odmerku flukonazola za zdravljenje vaginalne kandldoze. klinicno Pri veckratnih in vecjih odmerkih so možne interakcije s terfe varfarinom, derivati sulfonilureje, hidroklorotiazidom, feriitol teofilinom, indinavirom in midazolamom, Nosecnost. indoje . le, ce je korist zdravljenja za mater vecja od tveganja za plod; s flukonazolom ne dojijo. Stranski ucinki: Povezani sopre napenjanje, bolecine v trebuhu, driska, zelo redko seJioja anafilaksija in angioedem -v tem primeru takoj preneham glivicnimi obolenji lahko pride do levkopenije in trol}lbo encimov. Oprema in nacin izdajanja: 7 kapsul.po 50 r11\;l:, · · · ·150 mg. Na zdravniški recept. 1/99. . . <' Podrobnejše informacije so na voljo pri proizvajalcu, FONDACIJA "DOCENT DR. J. CHOLEWA" JE NEPROFITNO, NEINSTITUCIONALNO IN NESTRANKARSKO ZDRUŽENJE POSAMEZNIKOV, USTANOV IN ORGANIZACIJ, KI ŽELIJO MATERIALNO SPODBUJATI IN POGLABLJATI RAZISKOVALNO DEJAVNOST V ONKOLOGIJI. MESESNELOVA 9 1000 LJUBLJANA TEL 01 519 12 77 FAKS 01 251 81 13 ŽR: 501 00-620-1 33-05-1 0331 1 5-2 14 779 Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education -A Report f or the Second Quarter of 2002 The new circumstances, difficulties and problems associated with maintaining regular con­tacts with the donors were taken into consideration and seriously discussed on all levels by the members of the "Dr. J. Cholewa Foundation for Cancer Research and Education". 1t is hoped that some of the new approaches considered in contacts and communications with the donors will produce some tangible results in the near future. It is by now commonly under­stood that it would be naive to expect the same leve] of generosity by the donors in tirne of economic downturn following the unfortunate and violent events on September 11 th, 2001, in New York and later also in the other parts of the world. The decision was taken to increase the amount of the "Dr. J Cholewa Foundation for Cancer Research and Education" annual prize in order to give further incentive to young researchers in all parts of Slovenia. It is a long-time held position of the Foundation that high quality re­search work in oncology and related scientific fields is taking place and should be further en­couraged in all parts of Slovenia where the interest to promote such research exists. It is thus perceived that the quality of research will improve and that the results of cancer rescarch may find its way to the practical application in hospital wards a lot easier, and that in this way the attempts to publish and present the research results in respectable and influential international oncology journals, international meetings and conferences and other events of scientific importance, may gain another impetus. The Foundation therefore also continues to support the regular publication of "Radiology and Oncology" international scientific journal that is edited, published and printed in Ljubljana, Slovenia. With this in mind, a nmnber of grants was thus also awarded to experts from various parts of Slovenia in order to attend var­ious conferences and meetings in the field of oncology in Slovenia and around the world. The Foundation is sad to announce that Mr. Metod Rotar, one of its founding members, passed away in May 2002. Mr. Metod Rotar had a successful career in government and in banking during his lifetime, and his rich experience and knowlcdge were instrumenta! in bringing about the idea of the "Dr. J. Cholewa Foundation for Cancer Research and Education", in maintaining the high spirits and zeal for activity among its members during the initial and most important passes of the fledgling Foundation, as well as later, when it had to adapt to the new circumstances. Mr. Metod Rotar will be greatly missed by the re­maining members of the Foundation. Tomaž Benulic, MD Andret Plesnicar, MD Borut Stabuc, MD, PhD Sanolabor epoetin alfa limafne uredno6 pri bolnikih z rakom Dodatne informacije o zdravilu lahko dobite pri imetniku dovoljenja za promet: JANSSEN-CILAG JOHNSON & JOHNSON S, E. Podružnica Ljubljana, Šmartinska cesta 1-40, 1000 Ljubljana. E-mail:jac_sfo@jpjsl.jnj, PE: Stritarjeva 5, 4000 Kranj, Slovenija tel.: (0)4/ 2015 050, fax: (0)4/ 2015 055 e-mail: kemomed@siol.net KEMOMED Promega IZDELKI ZA MOLEKULARNO BIOLOGIJO PLASTIKA ZA CELICNE KULTURE SANYO UFEeno-NOLOGIES,M CISTA VODA ZA LABORATORIJ SKRINJE CELICNE KULTURE IN HLADILNIKI BIOHJT ELEKTRONSKE IN MEHANSKE AVTOMATSKE PIPETE zastopa naslednja podjetja Kottermann (Nemcija): INTEGRA BIOSCIE.CES (Švica): laboratorijsko pohištvo, laboratorijska oprema za ].ikrobiologijo,varnostne omare za kisline, biologijo celic, molekularno biologijo luge, topila, pline in strupe, I ventilacijska tehnika in digestorji C..:;.:.;.... DAKO (Danska): specialna laboratprijska plastika testi za aplikacijo v imunohistokemiji, za aplikacijo v imupologiji, mikro­patologiji, mikrobiologiji, virologiji, biologiji-virologiji, ipd., fehanske eno­mono-in poliklonalna protitelesa in veckanalne pi9ete in nastavki SVANOVA Biotech (Švedska): EVL (Nizozemska): Elisa testi za diagnostiko v veterini diagnosticni testi za uporabo v veteri arski medicini NOVODIRECT BIOBLOCK (Francija): kompletna oprema in pripomocki HURNER (Nemcija): za delo v laboratoriju ventilrcijska tehnika GFL (Nemcija): CSL -Biosciences: laboratorijski aparati, omare in diagnosticni tksti za uporabo skrinje za globoko zamrzovanje v veteriharski medicini ANGELANTONI SCIENTIFICA (Italija): BIOM.RICA (ZDA): hladilna tehnika in aparati za laboratorije, hitri testi ka diagnostiko, transfuzijo, patologijo in sodno medicino I EIA /R!A testi EHRET (Nemcija): CHARLES ISCHI (Svica): laminar flow tehnika, inkubatorji, specialna oprema za testiranje izdelkov sušilniki, suhi sterilizatorji in oprema v farmacevtski induhriji;aparati za za laboratorijsko vzrejo živali -kletke procesno kontrolo in ko trolo kvalitete ROSYS -ANTHOS (Avstrija): fotometri, avtomatski pralni sistem za mikrotitrine plošce LABORMED d.o.o. 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How7 Through industry­the diagnostic and treatment modalities. That's what we leading technology, increased productivity measures for call Best Practice Oncology Care. Siemens medical Solutions that help OUR COVENANT TO HELP CUSTOMERS ACHIEVE CLINICAL EXCELLENCE WITHOUT COMPROMISE TO BE RELENTLESS IN OUR PURSUIT OF INNOVATION TO NEVER FORGET THAT OUR WORK CAN SAVE, LENGTHEN ANO IM­PROVE LIVES "There is nothing more powerful than an intellect fueled by the passion to help make a difference." Our scientists, designers and engineers at Philips Medica! Systems have embraced your mission as their own. Which is why the world's medica! community can count on us to continue delivering breakthrough technology and superior services. To reach the peo­ple who share your passion for clinical excellence, vis­it us at www.medical.philips.com. PHILIPS Leli!Mk.h« Rodiology ond 011cology Instructions f or authors Editorial policy of the journal Radiology and Oncology is to publish original scientific pa­pers, profcssional papers, review articles, case reports and varia (editorials, reviews, short communications, professional information, book reviews, letters, etc.) pertinent to diag­nostic and interventional radiology, computer­ized tomography, magnetic resonance, ultra­sound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiol­ogy, radiophysics and radiation protection. The Editorial Board requires that the paper has not been published or submitted for publica­tion elsewhere: the authors are responsible for all statements in their papers. Accepted arti­cles become the property of the journal and therefore cannot be published elsewhere with­out written permission from the editorial board. Papers concerning the work on hu­mans, must comply with the principles of the declaration of Helsinki (1964). The approval of the ethical committee must then be stated on the manuscript. Papers with questionable jus­tification will be rejected. Manuscript written in English should be submitted to the Editorial Office in triplicate (the original and two copies), including the il­lustrations: Radiology and Oncology, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia; (Phone: +386 1 432 00 68, Tel./Fax: +386 1 433 74 10, E-mail: gsersa([Donko-i.si). Authors are also asked to submit their manu­scripts on a 3.5" 1.44 Mb formatted diskette. The type of computer and word-processing package should be specified (W ord for Windows is preferred). All articles are subjected to editorial review and review by independent referee selected by the editorial board. Manuscripts which do not comply with the technical requirements stated herein will be returned to the authors for cor­rection before peer-review. Rejected manu­scripts are generally returned to authors, how­ever, the journal cannot be held responsible for their loss. The editorial board reserves the right to ask authors to make appropriate changes in the contents as well as grammatical and stylistic corrections when necessary. The expenses of additional editorial work and re­quests for reprints will be charged to the au­thors. General instructions• Radiology and Onco­logy will consider manuscripts prepared accor­ding to the Vancouver Agreement (N Engl J Med 1991; 324: 424-8, BMJ 1991; 302: 6772; JA­MA 1997; 277: 927-34.). Type the manuscript double spaced on one side with a 4 cm margin at the top and left hand side of the sheet. Write the paper in grammatically and stylisti­cally correct language. Avoid abbreviations unless previously explained. The technical da­ta should conform to the SI system. The man­uscript, including the references may not ex­ceed 15 typewritten pages, and the number of figures and tables is limited to 4. If appropri­ate, organize the text so that it includes: Introduction, Material and methods, Results and Discussion. Exceptionally, the results and discussion can be combined in a single sec­tion. Start each section on a new page, and number each page consecutively with Arabic numerals. Title page should include a concise and in­formative title, followed by the full name(s) of the author(s); the institutional affiliation of each author; the name and address of the cor­responding author (including telephone, fax and e-mail), and an abbreviated title. This should be followed by the abstract page, sum­marising in less than 200 words the reasons for the study, experimental approach, the major findings (with specific data if possible), and the principal conclusions, and providing 3-6 key words for indexing purposes. Structured ab­stracts are preferred. lf possible, the authors are requested to submit also slovenian version of the title and abstract. The text of the report should then proceed as follows: Introduction should state the purpose of the article and summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study. Material and methods should provide enough information to enable experiments to be re­peated. New methods should be described in detail. Reports on human and animal subjects should include a statement that ethical ap­proval of the study was obtained. Results should be presented clearly and concisely without repeating the data in the ta­bles and figures. Emphasis should be on clear and precise presentation of results and their significance in relation to the aim of the inves­tigation. Discussion should explain the results rather than simply repeating them and interpret their significance and draw conclusions. It should review the results of the study in the light of previously published work. Illustrations and tables must be numbered and referred to in the text, with appropriate location indicated in the text margin. Illu­strations must be labelled on the back with the author's name, figure number and orien­tation, and should be accompanied by a de­scriptive legend on a separate page. Line drawings should be supplied in a form suit­able for high-quality reproduction. Photo­graphs should be glossy prints of high quality with as much contrast as the subject allows. They should be cropped as close as possible to the area of interest. In photographs mask the identities of the patients. Tables should be typed double spaced, with descriptive title and, if appropriate, units of numerical meas­urements included in column heading. References must be numbered in the order in which they appear in the text and their cor­responding numbers quoted in the text. Authors are responsible for the accuracy of their references. References to the Abstracts and Letters to the Editor must be identified as such. Citation of papers in preparation, or sub­mitted for publication, unpublished observa­tions, and personal communications should not be included in the reference list. If essen­tial, such material may be incorporated in the appropriate place in the text. References fol­low the style of Index Medicus. Ali authors should be listed when their number does not exceed six; when there are seven or more au­thors, the first six listed are followed by "et al.". The following are some examples of refer­ences from articles, books and book chapters: Dent RAG, Cole P. In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95. Chapman S, Nakielny R. A guide to radiolog­ica/ procedures. London: Bailliere Tindall; 1986. Evans R, Alexander P. Mechanisms of ex­tracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology oj macrophage. New York: Academic Press; 1976. p. 45-74. Page proofs will be faxed to the correspon­ding author whenever possible. It is their re­sponsibility to check the proofs carefully and fax a list of essential corrections to the editori­al office within 48 hours of receipt. If correc­tions are not received by the stated deadline, proof-reading will be carried out by the edi­tors. Reprints: Fifty reprints are free of charge, for more contact editorial board. For reprint infomzation contact: Intematio11al Reprint Corporation, 287 East "H" Street, Benicia, CA 94510, USA. Tei: (707) 746-8740; Fax: (707) 746-1643; E-mail: reprints@intlrepri11ts.com