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ADIOLOGY AND NCOLOGY Edi to rial office RadiologtJ and Oncology December 2003 Instih1te oj OncologiJ Vol. 37 No. 4 Zaloška 2 Pages 213-2 79 SI-1000 Ljubljana ISSN 1318-2099 Slovenia UDC 616-006 Phone: +386 1 5879 369 CODEN: RONCEM Phone/Fax: +386 1 5879 434 E-1nail: gsersa@onko-i.si Aims and scope Radiology and Oncology is a journal devoted to publication oj original contributions in diagnostic and interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinica/ and experimental oncologiJ, radiobiology, radiophysics and radia/ion protection. Editor-in-Chief Editor-in-Chief Emeritus Gregor Serša Tomaž Benulic Ljubljana, Slovenia Ljubljana, Slovenia Executive Editor Editor Viljem Kovac Uroš Smrdel Ljubljana, Slovenia Ljubljana, Slovenia Editorial board Marija Auersperg Valentin Fidler Maja Osmak Ljubljana, Slovenia Ljubljana, Slovenia Zagreb, Croatia Nada Bešenski Be1a Fornet Branko Palcic Zagreb, Croatia Budapest, Hungary Vancouver, Ca nada Karl H. Bohuslavizki Tullio Giraldi ]urica Papa Hamburg, Gennany Trieste, Italy Zagreb, Croatia Haris Boka Andrija Hebrang Dušan Pavcnik Zagreb, Croatia Zagreb, Croatia Portland, USA Nataša V. Budihna Ltiszl6 Horvtith Stojan Plesnicar Ljubljana, Slovenia Pecs, Hungary Ljubljana, Slovenia Marjan Budihna Berta Jereb Ervin B. Podgoršak Ljubljana, Slovenia Ljubljana, Slovenia Montreal, Canada Malte Clausen Vladimir Jevtic Jan C. Roos Hamburg, Germany Ljubljana, Slovenia Amsterdam, Netherlands Christoph Clemm H. Dieter Kogelnik Slavko Šimunic Munchen, Germany Salzburg, Austria Zagreb ,_, Croatia Mario Corsi Jurij Lindtner Lojze Smid Udine, Italy Ljubljana, Slovenia Ljubljan:,a,Slovenia Ljubomir Diankov Ivan Lovasic Borut Stabuc Sofia, Bulgaria Rijeka, Croatia Ljubljana, Slovenia Christian Dittrich Marijan Lovrencic Andrea Veronesi Vienna, Austria Zagreb, Croatia Aviano, Ital y Ivan Drinkovic Luka Milas Živa Zupancic Zagreb, Croatia Houston, USA Ljubljana, Slovenia Gillian Duchesne Metka Milcinski Melbourne, Australia Ljubljana, Slovenia Publisher Association of Radiology and Oncologi; Affiliated with S/ovenian Medica/ Association -Slovenian Association of Radiology, Nuclear Medicine Society, Slovenian Society for Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medica/ Association -Croatian Societi; of Radiologi; Societas Radiologorum Hungarorum Friuli-Venezia Giulia regiona/ groups of S.I.R.M. (Italian Society of Medica/ Radiologi;) Copyright © Radiologi; and Oncologi;. Ali rights reserved. Reader for English Mojca Cakš Key words Eva Klemencic Secretaries Milica Harisch Mira Klemencic Design Monika Fink-Serša Printed by Imprint d.o.o., Ljubljana, Slovenia Published quarterly in 700 copies Bank account number 02010-0090006751 Foreign currency account number 010-7100-900067 /4 NLB d.d., Podružnica Ljubljana Center, Ljubljana S. W.I.F. T. Code LJBASI2X Subscription fee for institutions EUR 100 (16000 SIT), individuals EUR 50 (5000 SIT) The publication of this joumal is subsidized by the Ministry of Education, Science and Sport of the Republic of Slovenia. Indexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EM.BASE/ Excerpta Medica Sci Base This joumal is printed on acid-free paper Radiologi; and Oncologi; is available on the internet at: http://www.onko-i.si/radiolog/rno.html ISSN 1581-3207 COLOGY Ljubljana, Slovenia ISSN 1318-2099 December 2003 UDC 616-006 Vol. 37 No. 4 CODEN: RONCEM CONTENTS CLINICAL ONCOLOGY Brain metastases in lung cancer. Impact of prognostic factors on patient survival Smrdel U, Zwitter M, Kovac V 213 Endobronchial metastasis as first manifestation of renal cell carcinoma Kaneko Y, Haraguchi N, Kodama T, Kagohashi K, Ishii Y, Satoh H, Sekizawa K 217 Pubic bone metastasis as first manifestation of lung cancer Kodama T, Satoh H, Ueno T, Homma S, Sekizawa K 221 EXPERIMENTAL ONCOLOGY Detection of apoptotic cells in tumour paraffin sections Pižem J, Car A 225 Expression of cathepsin B is related to tumorigenicity of breast cancer cell lines Zajc I, Frangež L, Lah TT 233 Cysteine and aspartic proteases cathepsins B and D determine the invasiveness of MCFlOA neoT cells Premzl A, Kos J 241 Experiencing professional strains of nurses, radiation engineers and physicians working at the Institute of Oncology in Ljubljana Škufca Smrdel AC 249 An outline of the history of radiotherapy at the Institute of Oncology in Ljubljana from its beginning till 1980s Okiješa Lukic A, Hiibscher K SLOVENIAN ABSTRACTS 257 267 NOTICES 275 AUTHORS INDEX 2003 SUBJECT INDEX 2003 Radiol Oncol 2003; 37(4): 213-6. Brain metastases in lung cancer. Impact of prognostic factors on patient survival Uroš Smrdel, Matjaž Zwitter, Viljem Kovac Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia Background. Brain metastases are common patterns of dissemination in lung cancer patients. In this paper we would like to assess the pattern of brain metastases in lung cancer patients and the impact of prognos­tic factors on the survival of lung cancer patients with brain metastases. Patients and methods. In the year 1998 there were 974 registered patients with lung cancer in Slovenia, six hundred and fifteen of them were treated at the Institute of Oncology Ljubljana and we analyzed them. Among 615 patients 137 (22.3 %) of them have had brain metastases during a natural course of disease. Results. For 12 patients presenting with solitary brain metastases (most of them were undertaken metasta­sectomy) median survival was 7.6 months, while in patients with multiple brain metastases the median sur­vival was 2.8 months (p = 0.0018). Of the 137 patients 45 (32.8 %) were small cell lung cancer patients, 43 (31.4 %) were adenocarcinoma patients and 19 (13.9 %) were squamous cell carcinoma patients. Patients with performance status (WHO scale) less than 2 had the median survival time 3.7 months while patients with performance status 2 or more had median survival time 2.7 moths (p=0.0448). Conclusions. Patients with solitary brain metastases had better survival comparing with those who had multiple metastases. It is surprisingly that the portion of brain metastases patients with adenocarcinoma is almost equal to those with small-call lung cancer therefore, the prophylactic cranial radiation becomes actu­al for both groups of patients. The performance status of patients with brain metastases remains very im­portant prognostic factor. Key words: lung neoplasms; brain neoplasms secondary; survival analysis Received 20 November 2003 Accepted 2 December 2003 Correspondence to: Uroš Smrdel, M.D., Department of Radiotherapy, Institute of Oncology; Zaloška 2, 1000 Ljubljana, Slovenia; Phone +386 1 5879 622; Fax +386 1 5879 400; E-mail: usmrdel@onko-i.si Introduction Brain metastases are common patterns of dis­semination in lung cancer patients. In the natural course of disease in around 17% of pa­tients with squamous carcinoma, in 24% of patients with anaplastic carcinoma, in 39% of patients with adenocarcinoma and in 42% with small cell carcinoma brain metastases will develop.1 Admittedly non-small cell car­cinoma of the lung patients with single re-sectable metastases will gain from metasta­sectomy followed by the external beam cra­nial irradiation.2-4 In those patients a gain can be objectified as the survival. Unfortunately, those patients represent only about 10% of lung cancer patients with brain metastases. The rest 90% of lung cancer patients are ei­ther small cell lung cancer patients or pa­tients with multiple or irresectable metas­tases which do not benefit from the same treatment. The majority of patients are thus offered the choice of palliative an external beam cra­nial irradiation or a corticosteroid treatment as the best supportive care.3 In last few years there is a tendency towards shorter radiation courses. There is evidence that the size and the number of fractions do not influence the outcome of treatment and duration of re­sponse.5,6 Furthermore, it seems that the only factor influencing the response is the severity of neurological deficit and that those patients presenting with the most severe deficit usual­ly respond better than those with only minor neurological changes. On the other hand those presenting with minor neurological changes fare better in the term of survival, which is expected.7,8 An acceptable approach towards the treat­ment of brain metastases is corticosteroid ther­apy alone, which is particularly appropriate for those elderly in poor physical condition.9 At the Institute of oncology in Ljubljana we are currently using 5 x 4 Gy, 10 x 3 Gy, 12 x 2.5 Gy and 14 x 2.5 Gy. In this paper we would like to assess the pattern of brain metastases in lung cancer patients and the impact of treatment on the outcome of lung cancer patients with brain metastases. Patients and methods The Cancer registry of Slovenia collects data on all cancer patients in Slovenia. In the year 1998 there were registered 974 patients with lung cancer,10 six hundred and fifteen of them were treated at the Institute of Oncology Ljubljana. In the year 2003 we re­viewed all these 615 patients who were at least once examined and managed at our Institute. The median age of lung cancer patients was 62.66 years (min. 36.01; max. 89.54 stan­dard deviation 10.029); male, female ratio was 4:1; histological types were: 155 (25.2 %) adenocarcinoma, 217 (35.3 %) squamous car­cinoma, 32 (5.2 %) anaplastic carcinoma, 132 (21.5 %) small cell carcinoma, 45 (7.3 %) non small cell carcinoma, 10 (1.6 %) mixed carci­noma, 1 (0.2 %) bronchioloalveolar carcino­ma. Twenty-one patients (3.4 %) had no his­tological diagnosis prior to their death. Among 615 patients 137 (22.3 %) have had brain metastases during the natural course of disease. Of these 137 patients 22 (16 %) presented with brain metastases at the time of a diagno­sis and 115 (84 %) underwent a treatment and developed brain metastases later on. Patients were treated for brain metastases either with radiotherapy alone (110 patients (80.3 %)) or in cases with solitary brain metas­tases with metastasectomy followed by radio­therapy (12 patients (8.8 %)). Due to a poor performance status 15 patients (10.9 %) re­ceived no oncological treatment except the best supportive care. Results The median survival for all 615 patients diag­nosed with lung cancer in 1998 was 8.9 months (range: min. 2 days, max. 62.6 months; 95 % confidence interval 7.93 - 9.9 months). For patients with brain metastases the me­dian survival was 3.1 months (range: min 0 days, max: 52.2 months) For 12 patients presenting with solitary brain metastases (the most of them were un­dertaken metastasectomy), median survival was 7.6 months, (range: min 2.4 months, max 52.2 months, 95% confidence interval 2.9 ­ 12.4 months) compared to those with multi­ple brain metastases for whom the median survival was 2.8 months (range: min 0 days, max 50.4 months; 95% confidence interval 2.1- 3.5 months). The difference is statistical­ly significant (log-rank p = 0.0018) (Figure 1). Of the 137 patients with brain metastases 45 or 32.8 % were small cell lung cancer pa­tients, 43 or 31.4 % were adenocarcinoma pa­tients and 19 or 13.9 % were squamous cell carcinoma patients (Figure 2). Predictably an important factor influenc-ing the survival in lung cancer patients with brain metastases is a performance status. In those patients with performance status on WHO scale less than 2 a median survival was 3.7 months while for patients with a perform­ance status on WHO scale 2 or more a medi­an survival was 2.7 moths. This small differ­ence is statistically significant with p=0.0448 (Figure 3). There were 18 patients with adenocarcino-ma confined to chest, later on presented with brain metastases, their median survival was 16.1 months (range: min 4.7 months max: 56.6 month) and their median survival after the diagnosis of brain metastases was 2.3 months (range: min. 0 days max 50.4 months) Discussion As already shown elsewhere1 a fair propor­tion of lung cancer patients will be, during the course of their disease, presented with brain metastases. At our Institute 22.3 % of lung cancer patients reported in 1998 pre­sented with brain metastases in the course of disease. There was still a prevalence of squamous cell carcinoma in all patients, but in those with brain metastases, there is almost an equal proportion of small cell lung cancer and adenocarcinoma (32.8 % and 31.4 % respec­tively). In most historical studies there was a well-established prevalence of squamous cell carcinoma,11 but in last years we are starting to recognize a rise in adenocarcinoma pa­tients.12 In our retrospective of 155 patients with adenocarcinoma 43 or 27.7 % presented with brain metastases which is comparable with 45 of 132 or 34 % for non small cell lung cancer and far above 19 of 217 or 8.8 % for squamous carcinoma. Since of 43 adenocarcinoma patients with brain metastases 18 or 41 % at presentation had a disease confined to chest and either re­ceived surgery or radiotherapy with curative intent, we could assume that with the in­crease in adenocarcinoma incidence there will also be the increase in the number of pa­tients who will present with brain metastases after the radical treatment. This presumption opens space for screening for brain metas­tases in this patients and open question of prophylactic brain irradiation in adenocarci­noma patients.13,14 For patients with solitary brain metastases the effective treatment is metastasectomy fol­lowed by brain irradiation, which in patients with good performance status yield a survival superior to irradiation alone.15 However, sim­ilar results are achieved with the use of cra­nial stereotactic radiosurgery.16 References 1. Line DH, Deeley TJ. The necropsy findings in car­cinoma of the bronchus. Br J Did Chest 1971; 65: 238-42. 2. Taimur S, Edelman MJ. Treatment options for brain metastases in patients with non-small-cell lung cancer. Curr Oncol Rep 2003; 5: 342-6. 3. Taimur S, Edelman MJ. Treatment options for brain metastases in patients with non-small cell lung can­cer. Curr Treat Options Oncol 2003; 4: 89-95. 4. Schuchert MJ, Luketich JD. Solitary sites of metastatic disease in non-small cell lung cancer. Curr Treat Options Oncol 2003; 4: 65-79. 5. Regine WF, Scott C, Murray K, Curran W. Neurocognitive outcome in brain metastases pa­tients treated with accelerated-fractionation vs. ac-celerated-hyperfractionated radiotherapy: an analysis from Radiation Therapy Oncology Group Study 91-04. Int J Radiat Oncol Biol Phys 2001; 51: 711-7. 6. Murray KJ, Scott C, Zachariah B, Michalski JM, Demas W, Vora NL, et al. Importance of the mini-mental status examination in the treatment of pa­tients with brain metastases: a report from the Radiation Therapy Oncology Group protocol 91­ 04. Int J Radiat Oncol Biol Phys 2000; 48: 59-64. 7. Nieder C, Nestle U, Motaref B, Walter K, Niewald M, Schnabel K. Prognostic factors in brain metas­tases: should patients be selected for aggressive treatment according to recursive partitioning analysis (RPA) classes? Int J Radiat Oncol Biol Phys 2000; 46: 297-302. 8. Soffietti R, Ruda R, Mutani R. Management of brain metastases. J Neurol 2002; 249: 1357-69. 9. Oneschuk D, Bruera E. Palliative management of brain metastases. Support Care Cancer 1998; 6: 365­72. 10. Cancer Registry of Slovenia. Cancer incidence in Slovenia 1998. Report No. 40. Ljubljana: Institute of Oncology Ljubljana; 2001. 11. Johnson W. Histologic and cytologic patterns of lung cancer in 2580 men and woman over a 15­year period. Acta Cytol 1988; 32: 163-8. 12. Janssen-Heijnen ML, Coebergh JW. The changing epidemiology of lung cancer in Europe. Lung Cancer 2003; 41: 245-58. 13. Gore EM. Prophylactic cranial irradiation for pa­tients with locally advanced non-small-cell lung cancer. Oncology (Huntingt). 2003; 17: 775-9; dis­cussion 779-80, 784, 787 passim. 14. Komaki R, Cox JD, Stark. Frequency of brain metastasis in adenocarcinoma and larg carcinoma of the lung: correlation with survival. Int J Radiat Oncol Biol Phys 1983; 9: 1467-70. 15. Rodrigus P, de Brouwer P, Raaymakers E. Brain metastases and non-small cell lung cancer. Prognostic factors and correlation with survival af­ter irradiation. Lung Cancer 2001; 32: 129-36. 16. Sheehan JP, Sun MH, Kondziolka D, Flickinger J, Lunsford LD. Radiosurgery for non-small cell lung carcinoma metastatic to the brain: long-term out­comes and prognostic factors influencing patient survival time and local tumor control. J Neurosurg 2002; 97: 1276-81. Radiol Oncol 2003; 37(4): 217-9. Endobronchial metastasis as first manifestation of renal cell carcinoma Yoshiko Kaneko, Norihiro Haraguchi, Takahide Kodama, Katsunori Kagohashi, Yukio Ishii, Hiroaki Satoh, Kiyohisa Sekizawa Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Japan Background. In the majority of cases of endobronchial metastasis, presence of a primary tumour antedat­ed the diagnosis of the metastasis. We showed a case of endobronchial metastasis as first manifestation of renal cell carcinoma. Case report. A 61-year-old man was admitted to our hospital complaining of cough of 3 months duration. Chest CT scan showed a polypoid mass in the right upper lobe bronchus. Biopsy of the lesion was obtained, and microscopic examination showed metastatic renal cell carcinoma of the bronchial wall. Conclusions. When endobronchial lesion occurs in the absence of clinical evidence of a primary tumour, appropriate diagnostic studies should be undertaken to exclude the possibility of an asymptomatic ex-trathoracic tumour. Key words: bronchial neoplasms - secondary; carcinoma, renal cell - diagnosis Introduction Case report The lung is a common site of metastasis in re­nal cell carcinoma.1 However, endobronchial metastasis as the first manifestation of renal cell carcinoma seems to be uncommon.2-7 We report here clinical findings of such a rare case. Received 2 November 2003 Accepted 16 November 2003 Correspondence to: Hiroaki Satoh, MD, Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba-city, Ibaraki, 305­8575, Japan; Phone: +81 29 853 3210; Fax: +81 29 853 3320; E-mail: hirosato@md.tsukuba.ac.jp A 61-year-old man was admitted to our hos­pital complaining of cough of 3 months dura­tion. He began to smoke cigarettes at the age of 20 years and consumed two packages dai­ly thereafter. He had no previous diseases. On physical examination, his blood pressure was 140/70 mmHg and pulse rate 80/min and regular. Enlarged lymph nodes were not de­tected. On percussion and auscultation of the chest, increased dullness and diminished breath sounds were noted in the upper half of the right lung field. Routine blood tests and ECG were normal. The urine was normal. Chest X-ray revealed right upper lobe atelectasis with localized pleural effusion. Chest CT scan showed a polypoid mass in the right upper lobe bronchus (Figure 1). On bronchoscopy, an obstructive, polypoid mass was found in the right upper bronchus. Biopsy of the lesion was obtained, and microscopic examination showed metastatic renal cell carcinoma of the bronchial wall. CT scan of the abdomen re­vealed a 4-cm tumour in the lower pole of the right kidney, but regional lymph node swelling was not observed (Figure 2). No oth­er distant metastatic lesions than lung were found. Thereafter, nephrectomy was per­formed, and the tumour was confirmed as re­nal cell carcinoma pathologically. Discussion Endobronchial metastases are a late manifes­tation in the course of solid tumour.8-13 In the majority of cases clinical manifestations of the presence of a primary extrathoracic tu­mour antedated the diagnosis of endo-bronchial metastasis. 8-13 Occasionally, how­ever, clinical and roentgenographic features of endobronchial metastasis preceded recog­nition of the primary tumour.2-7 In 1975, Braman and Whitcob reported 7 of the 15 re­nal tumours were accompanied by symptoms of a bronchial metastasis three weeks to ten months before discovery of the primary renal cell carcinoma.14 Thereafter, however, there have been few reports of endobronchial metastasis being diagnosed before the detec­tion of the primary extrathoracic tumour.7,15 In the reports from our country, seven among the 37 patients with endobronchial metasta­sis from renal cell carcinoma preceded recog­nition of the primary tumour.15 Recently, Katsimbri et al. reported 8 case of endo-bronchial metastasis from various organs. In all of the 8 patients, however, clinical mani­festations of the presence of a primary ex-trathoracic tumour antedated the diagnosis of the endobronchial metastases, and the medi­an interval of endobronchial metastases diag­nosis from the diagnosis of the primary tu­mour was 41 months.12 Although a few, however, there are reports of endobronchial metastatic lesions being di­agnosed before the detection of the primary tumour.2-7 In this case report, we showed that a cer­tain type of renal cell carcinoma develops en-dobronchial metastasis with no regional lymph node swelling, and such hematoge-nous distant metastasis may not necessarily associated with enlarged size of primary le­sion. When endobronchial neoplastic lesion occurs in the absence of clinical evidence of an extrathoracic primary tumour, the bronchial neoplasma is almost certain to be a primary lung cancer. Nevertheless, if atypical clinical or pathological features as primary lung cancer are present, appropriate diagnos­tic studies should be undertaken to exclude the possibility of an asymptomatic extratho­racic tumour as observed in our case. References 1. Lim DJ, Carter MF. Computerized tomography in the preoperative staging for pulmonary metas­tases in patients with renal cell carcinoma. J Urol 1993; 150: 1112-4. 2. Maytum CK, Vinson PP. Pulmonary metastasis from hypernephroma with ulceration into a bronchus simulating primary bronchial carcino­ma. Arch Otolaryngol 1936; 23: 101-4. 3. Nofsinger CD, Vision PP. Intrabronchial metasta­sis of hypernephroma simulating primary bronchial carcinoma. JAMA 1942; 119: 944-5. 4. Gerle R, Feison B. Metastatic endobronchial hy­pernephroma. Dis Chest 1963; 44: 225-33. 5. Silverberg SG, Evans RH, Koehler AL. Clinical and pathologic features of initial metastatic pre­sentations of renal cell carcinoma. Cancer 1969; 23: 1126-32. 6. Case records of the Massachusetts General Hospital. Case-13-1972. N Engl J Med 1972; 286: 713-9. 7. Carlin BW, Harreell JH, Olson LK, Moser JM. Endobronchial metastases due to colorectal carci­noma. Chest 1989; 96: 1110-4. 8. Baumgartner WA, Mark JBD. Metastatic maling­nancies from distant sites to the tracheobronchial tree. J Thorac Cardiovasc Surg 1980; 79: 499-503. 9. Berg HK, Petrelli NJ, Herrera L, Lopez C, Mittelman A. Endobronchial metastasis from colorectal carci­noma. Dis Colon Rectum 1984; 24: 745-8. 10. Noy S, Michowitz M, Lazebnik N, Baratz M. Endobronchial metastasis of renal cell carcinoma. J Surg Oncol 1986; 31: 268-70. 11. Heitmiller RF, Marasco WJ, Hruban RH, Marsh BR. Endobronchial metastases. J Thorac Cardiovasc Surg 1993; 106: 537-42. 12. Katsimbri PP, Bamias AT, Froudarakis ME, Peponis IA, Constantopolos SH, Pavidis NA. Endobronchial metastases secondary to solid tu­mours: report of eight cases and review of litera­ture. Lung Cancer 2000; 28: 163-70. 13. Sakamoto N, Inafuku S, Shimoji H, Nomura K, Honma K, Kawabata T, et al. Endobronchial metastasis from renal cell carcinoma: report of a case. Surg Today 2000; 30: 744-6. 14. Braman SS, Whitcomb ME. Endobronchial meast­asis. Arch Intern Med 1975; 135: 543-7. 15. Tomita M, Shino Y, Sugaya S, Ikemoto I, Oishi Y. A case of lung collapse caused by endobronchial metastasis from renal cell carcinoma re-inflated with laser and electrosurgical snaring. Acta Urol Jpn 2002; 48: 459-62. Radiol Oncol 2003; 37(4): 221-4. Pubic bone metastasis as first manifestation of lung cancer Takahide Kodama, Hiroaki Satoh, Takahiro Ueno, Shinsuke Homma, Kiyohisa Sekizawa Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Japan Background. Pubic bone metastasis as the initial manifestation of lung cancer is very rare, and it may sim­ulate parasymphyseal insufficiency fracture of the pubic bone, which is observed in postmenopausal women and elderly people. Case report. A 65-year-old woman developed pain in the right flank, which extended to the anterior aspect of the thigh. A pelvic X-ray showed osteolytic lesion in the right pubic ramus. Chest radiograph revealed a nodular mass in the right middle lobe of the lung. Transbronchal biopsy of the mass led to the diagnosis of lung adenocarcinoma. The patient was given radiotherapy of osteolytic lesion in her right pubic ramus and the pain was controlled with a combination of morphine sulfate. Conclusions. When unusual bone metastasis is found in the absence of a primary tumor, investigation must include chest radiographs. Key words: pubic bone; bone neoplasms - secondary; lung neoplasms; adenocarcinoma Introduction Lung cancer is a severe disease often diag­nosed a late stage when surgical resection is no longer possible because of local advance­ment or distant metastasis.1 Bone metastases from lung cancer may occur early in the clin­ical course and are usually discovered with severe pain.2-4 Received 2 November 2003 Accepted 16 November 2003 Correspondence to: Hiroaki Satoh, M.D., Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba-city, Ibaraki, 305­8575, Japan; Phone: +81 298 53 3210; Fax: +81 298 53 3320; E-mail: hirosato@md.tsukuba.ac.jp The spine and ribs are often the earliest sites of bone metastases, whereas the skull, femur, humerus, and scapula are involved lat­er.5 Pubic bone metastasis as the initial man­ifestation of lung cancer is very rare. We re­port a case of particular interest because of unusual bone metastatic site and the relative­ly slow progression of the neoplasm. Case report A 65-year-old woman was referred to her or­thopedist with pain in the right flank, which extended to the anterior aspect of the thigh for three months. A pelvic radiograph showed an osteolytic lesion in her right pubic ramus (Figure 1). Because the pain was not controlled with usual analgesic drugs and ex­tended to the thigh, she was referred to the orthopedic division in our hospital. On ad­mission, plain chest radiograph revealed a nodular mass in the right lung (Figure 2), therefeore she was consulted to Division of Respiratory Medicine. She had no complaints other than the pain in the right flank and thigh. Chest CT scan showed a 6 cm nodule in right middle robe with right hilar lymph node swelling. Bone scan showed hot uptake in pubic bone (Figure 3). Transbronchal biop­sy of the right middle lobe led to the diagno­sis of lung adenocarcinoma. A brain magnet­ic resonance imaging showed a 2.0 cm metastatic lesion in the right temporal lobe for which an osmotherapy and stereotactic radiosurgery were performed. The patient was given radiotherapy for osteolytic lesion in her right pubic ramus and the pain was controlled with a combination of morphine sulfate. The patient was discharged, however, she returned to our hospital because of pathological fracture of the right femur. To control the pain, total hip replacement under general anesthesia was performed. Thereafter she was discharged again. Three months lat­er, right massive pleural effusion was devel­oped and she was admitted to our hospital. Eleven months since the diagnosis of bone metastasis, the patient died of respiratory failure. Discussion Patients may first receive medical attention as the result of skeletal metastasis from an unknown primary tumor. For such individu­als, imaging studies may help to identify the primary lesion.6,7 Some primary tumors tend to result in metastases that are purely lytic in nature, whereas others tend to be associated with varying degrees of sclerosis.8 Pubic bone metastasis may simulate parasymphyseal in­sufficiency fracture of the pubic bone, which is observed in postmenopausal women and elderly people.9-11 The parasymphyseal insuf­ficiency fracture is a commonly regarded form of stress fracture in patients with osteo­porosis. Pathologically, lysis and callus for­mation produce a destructive malignant ap­pearing lesion.9 In our patient, at the time of initial diagnosis the pubic lesion was recog­nized on radiography as an insufficiency frac­ture of the symphysis pubis or a primary bone tumor because of its unusual localiza­tion of bone metastasis. Metastatic site other than pubic bone was not found at the time of initial diagnosis. Among lung cancers, adenocarcinomas are more heterogeneous in progression than oth­er cell types of lung cancer.12 Therefore, some cases of lung adenocarcinoma grow very rap­idly, and others are slowly progressive.12 Our patient had relatively slow progres­sion despite of the first sign of distant metastatic lesion. In patients with advanced lung cancer, the major goal of treatment is re­covery of the performance status of the pa­tient and the relief of pain. In a certain per­cent of cases, however, intensive systemic chemotherapy would be indicated as an adju­vant to local therapy such as radiotherapy and/or surgical procedures. Although the ef­ficacy or duration was limited, radiotherapy at metastatic bone sites and analgesics im­proved quality of life in our patient. When unusual bone metastases are found in the absence of a primary tumor, investiga­tion must include chest radiographs. Chest CT scan occasionally may be helpful in diag­nosing lung cancer, which is not obvious on plain chest radiograph. Therefore, chest CT scan and bronchoscopy would be undertaken when there is a clinical indication. References 1. Satoh H, Ishikawa H, Kamma H, Yamashita YT, Takahashi H, Ohtsuka M, et al. Serum sialyl X-i antigen levels in non-small cell lung cancer: corre­lation with distant metastasis and survival. Clin Cancer Res 1997; 4: 495-9. 2. Gilbert HA, Kagan AR, Nussbaum H, Rao AR, Satzman J, Chan P, et al. Evaluation of radiation therapy for bone metastases: pain relief and qual­ity of life. Am J Roentgenol 1977; 129: 1095-6. 3. Lote K, Walloe A, Bjersand A. Bone metastasis. Prognosis, diagnosis and treatment. Acta Radiol Oncol 1986; 25: 227-32. 4. Nishino T, Yoshida A, Satoh H, Hagiya M, Sakamoto T, Yamashita YT, et al. Atypical presen­tation of vertebral bone metastasis from lung can­cer. Oncol Rep 1999; 6: 781-3. 5. Nielsen OS, Munro AJ, Tannock IF. Bone metas­tases: pathophysiology and management policy. J Clin Oncol 1991; 9: 509-24. 6. Rougraff BT, Kneisl JS, Simon MA. Skeletal metas­tases of unknown origin. A prospective study of a diagnostic strategy. J Bone Joint Surg Am 1993; 75: 1276-81. 7. Katagiri H, Takahashi M, Inagaki J, Sugiura H, Ito S, Iwate H. Determining the site of the primary cancer in patients with skeletal metastasis of un­known origin: a retrospective study. Cancer 1999; 86: 533-7. 8. Aoki J, Yamamoto I, Hino M, Shigeno C, Kiamura N, Itoh H, et al. Sclerotic bone metastasis: radio­logic-pathologic correlation. Radiology 1986; 159: 127-32. 9. Casey D, Mirra J, Staple TW. Parasymphyseal in­sufficiency fractures of the os pubis. Am J Roent­genol 1984; 142: 581-6. 10. McCarthy B, Dorfman HD. Pubic osteolysis. Clin Orthop 1990; 251: 300-7. 11. Resnick D. Diagnosis of bone and joint disorders. 3rd edn. Philadelphia: Saunders; 1995. p. 1783-853. 12. Latief KH, White CS, Protopapas Z, Attar S, Krasna MJ. Search for a primary lung neoplasm in patients with brain metastasis: is the chest radi­ograph sufficient? Am J Roentgenol 1997; 168: 1339­44. Radiol Oncol 2003; 37(4): 225-32. review Detection of apoptotic cells in tumour paraffin sections Jože Pižem1, Andrej C2 1Institute of Pathology and 2Institute for Histology and Embryology, Medical Faculty, Ljubljana, Slovenia Apoptosis is a distinct form of cell death characterised by specific morphological features and regulated by complex molecular mechanisms. Its deregulation is fundamental for tumour growth and progression and, moreover, anticancer therapies suppress tumour growth mainly by induction of apoptosis. Since the extent of apoptosis in a tumour may have prognostic as well as therapeutic implications, much effort has been in­vested in developing specific methods that can be routinely used to detect apoptotic cells in archival forma­lin-fixed paraffin-embedded tissue. Complex molecular pathways are involved in the regulation of apoptosis. Pro-apoptotic signals trigger acti­vation of caspases that specifically cleave target proteins. Cleavage of proteins (caspase substrates) is re­sponsible for morphological changes of apoptotic cells and DNA fragmentation. In the last decade, detection of apoptotic cells in formalin-fixed tumour tissue sections has been based mainly on morphology and char­acteristic DNA fragmentation. Recently, specific antibodies to activated caspases and cleaved target proteins (including cytokeratin 18, actin and PARP) have been produced that enable accurate detection of apoptosis in paraffin sections. Key words: neoplasms; apoptosis; caspases; DNA fragmentation Introduction Apoptosis, or programmed cell death, is a complex, tightly regulated and conserved process, whereby individual cells die without injuring neighbouring cells or provoking any Received 28 November 2003 Accepted 10 December 2003 Correspondence to: Jože Pižem, Institute of Pathology, Medical Faculty, Korytkova 2, 1000 Ljubljana, Slovenia. Phone: 01 543 7161; E-mail: jozepizem@ hotmail.com inflammatory reaction. It is essential for the maintenance of homeostasis in multicellular organisms and is a critical component in the cellular response to injury.1 Apoptosis is im­plicated in the patogenesis of various condi­tions, such as cancer, autoimmune disorders and neurodegenerative diseases. An imbal­ance between cell proliferation and apoptosis is fundamental for tumour growth and pro­gression. Because of its wide ranging implica­tions and possible therapeutic interventions, apoptosis is one of the most investigated ar­eas in biological science.2 Since various proteins implicated in apop­tosis regulation have been reported to be mu­tated in cancer, and it has become evident that different anticancer therapeutic modali­ties suppress tumour growth mainly by in­duction of apoptosis, accurate quantification of the extent of apoptosis in tissue specimens may have therapeutic as well as prognostic implications.3,4 Many different biochemical, cytochemical and in situ methods are available for apopto-sis detection,5-8 but only a limited number of them are applicable on paraffin sections. Because of the preserved cell morphology in archival formalin-fixed paraffin-embedded tissue, there has been considerable interest in developing methods that could specifically demonstrate apoptotic cells in tissue sec­tions. In the last decade, the morphological method and the TUNEL method (terminal transferase mediated d-UTP biotin nick end labelling) have been almost exclusively used for apoptosis detection on paraffin sections. Since great progress in understanding the mechanisms of apoptosis has been made, several new methods for apoptosis detection on paraffin sections have been introduced re­cently. This article deals with apoptosis de­tection techniques applicable to archival paraffin sections with an emphasis on tu­mour tissue. Apoptotic pathway Initially, apoptosis was recognised on mor­phological grounds as a well defined type of cell death.9 During apoptosis, the cell shrinks, it detaches from the surrounding cells, the nucleus becomes condensed and fragmented, and finally, apoptotic bodies are formed. In the last two decades, the molecu­lar basis of apoptosis has been elucidated. It has become apparent that there is a cascade of events from pro-apoptotic signalling to well defined morphological changes. Caspases (cysteine proteases that cleave their substrates following an aspartate residue) have been recognised as key mole­cules in the apoptotic cascade.5,10 At least 14 different caspases have been characterised to date. They are constitutively expressed as in­active pro-enzymes (pro-caspases) in virtually all animal cells. Pro-caspases (32 - 56 kDa) contain a large subunit (17 - 21 kDa), a small subunit (10 - 13 kDa) and an N-terminal pro-domain. Pro-caspase activation requires pro­teolytic cleavage of the pro-caspase chain at two caspase specific cleavage sites, yielding a large and a small subunit. Active caspase is a heterotetramer composed of two large and two small subunits. In mammalian cells, apoptosis can be initi­ated by either intracellular or extracellular pathways that induce initiator caspase activa­tion (caspases 8 and 9). Activated caspase 8 and/or 9, in turn, induce cascade activation of effector caspases (caspases 3, 6 and 7), which cleave different target proteins. Demolition of the target proteins induces DNA fragmenta­tion and is responsible for morphological changes of the apoptotic cells (Figure 1). Figure 1. Apoptotic pathway. Different pro-apoptotic signals converge to induce activation of the caspase cascade. Activation of initiator caspases (8 and 9) is followed by activation of effector caspases which, in turn, specifically cleave different target proteins (cas­pase substrates). Cleavage of the target proteins in­duces DNA fragmentation and provokes morphologi­cal changes of the apoptotic cells. Detection of apoptotic cells based on morphology Apoptotic cells can readily be identified by means of routine histological staining meth­ods, such as haematoxylin and eosin. Detection of apoptotic cells is based on char­acteristic morphological features.11 The cyto­plasm of apoptotic cells is condensed and eosinophilic. Chromatin is condensed, mar-ginated at the nuclear membrane, and nu­clear fragments are later seen (Figure 2a). Finally, the apoptotic cells desintegrate into membrane-bound apoptotic bodies, which are phagocytosed by neighbouring cells and macrophages.12 During apoptosis, the cell membrane retains its integrity and lysosomal enzymes are not released to the surrounding tissue, so no inflammatory reaction is elicit­ed. All morphological features are best viewed by electron microscope, but this is im­practical for screening large tissue areas.13 Visible changes in cell morphology are the final event of the apoptotic process and are estimated to take from two to three hours, compared to 12 to 24 hours that are needed for the entire apoptotic process to be com­pleted.12,14 The basis for changes in the apop­totic cell morphology is caspase mediated cleavage of the target proteins (Figure 1). The asynchronicity of apoptosis, comple­tion of the morphological phase in a few hours and immediate clearing from the tissue usually results in a very low number of apop­totic cells in tissues.11 Because morphological changes may be inconspicuous, the lowest numbers of apoptotic cells are usually detect­ed by morphological criteria only.12 An apoptotic index is used as a measure of the extent of apoptosis, which is defined in tumour tissues as the percentage of apoptotic cells of all tumour cells. Because apoptotic cells frequently appear in clusters, enough fields need to be included in the analysis. To guarantee representativeness, at least 20 fields of 1000x magnification should be ex­amined.15 The identification of apoptotic cells, and thus inter-observer variation, great­ly depends on the magnification, so a higher magnification should be used. Inflammatory cells that are frequently found among tumour cells may substantially influence the apoptot­ic index. They may be difficult to differentiate from apoptotic cells (inflammatory cells are also frequently apoptotic) as well as from the population of tumour cells analysed.12 Generally, assessing apoptosis based solely on its morphology is reasonably reliable and inexpensive, although tedious and fairly inter-observer dependent. It might be accurate for some tissues,15-17 but for more accurate as­sessment, morphology should be used in com­bination with more specific methods, which detect apoptotic cells earlier in the apoptotic process. However, morphology based on rou­tine staining methods remains, even today, a major tool of apoptosis detection and is criti­cal for validation of new techniques. Detection of DNA fragmentation In situ detection of DNA fragmentation has been the most widely used method for detec­tion of apoptosis.12 The orderly internucleoso­mal fragmentation of DNA into 180 to 200 base-pair fragments is the biochemical hall­mark of apoptosis.16 It is induced by caspase mediated cleavage of ICAD (inhibitor of CAD) leading to activation of CAD (caspase-activat­ed deoxyribonuclease), which in turn, cleaves DNA.18 DNA fragments can be detected by enzy­matic labelling of the 3’-hydoxyl ends with modified nucleotides. The most sensitive and specific in situ method for detection of DNA fragmentation is the TUNEL method (terminal deoxynucleotidyl transferase-mediated-dUTP nick end labelling) (Figure 2b). It was intro­duced for use in tissue sections in 1992 by Gavrieli et al.19 As its name implies, the TUNEL method is based on the addition of la-belled nucleotides to free 3’-hydoxyl ends of single or double-strand DNA breaks catalysed by a terminal deoxynucleotidyl transferase (TdT) enzyme. Following in situ enzyme reac­tion, the incorporated labelled nucleotides can be detected by immunohistochemistry. Nuclear counterstains are used to facilitate recognition of apoptotic morphology.11 There are many commercially available apoptosis detection kits that rely on detection of DNA fragments.16 TUNEL labelling greatly depends on tissue fixation, tissue pretreatment and the concen­tration of TdT. Overlong formalin fixation and insufficient pretreatment may give rise to false negative results. On the other hand, excessive pretreatment, necrosis, autolysis or extensive DNA repair may lead to false positive re­sults.12 In some tissues, proteinase pretreat­ment may result in release of endogeneous en-donucleases, which may cause false positive results with TUNEL staining.20 Generally, TUNEL gives higher apoptotic indices than the morphological method, at least partly because DNA fragmentation slightly precedes light mi­croscopic morphological changes.14 TUNEL has been the most widely used method for apoptosis detection on tumour material. However, there are some drawbacks that war­rant caution. TUNEL is an in situ enzymatic method followed by an immunohistochemical reaction and, due to its complexity, the results depend greatly on tissue preparation, pretreat­ment and reaction conditions.12 Optimisation of TUNEL staining therefore requires stan­dardisation of the above mentioned technical factors. Moreover, DNA fragmentation is a late event in apoptosis, it is not an obligatory feature of apoptosis, and may be absent.16 Immunohistochemical detection of cleaved caspase 3 As a result of increased knowledge about the molecular mechanisms of apoptosis, detec­ tion of apoptotic cells based on morphology and detection of DNA fragmentation is now advancing to more specific methods. Proteins selectively activated, or protein fragments generated during the process of apoptosis, have been characterised, and specific anti­bodies against them allow reliable detection of apoptotic cells, distinguishing them from necrotic cells.11 Caspase 3 is activated during most apop­totic processes and is believed to be the main effector caspase. Its activation is directly or indirectly responsible for cleavage of the tar­get proteins, which leads to characteristic DNA fragmentation and morphological changes of apoptotic cells.21 The appearance of the active form of caspase 3 in the cell un­dergoing apoptosis is an early event during apoptosis and precedes the development of classical morphological features. At the time of caspase 3 activation, the cell is fully com­mitted to death and apoptosis is said to run beyond the ‘point of no return’.22 Detection of cleaved caspase 3 enables detection of apoptosis even before the morphological changes of apoptosis appear. Unprocessed caspase 3 is a 32 kDa protein which is cleaved by upstream caspases into 17 kDa and 12 kDa active fragments.5 This cleavage creates or unmasks new epitopes. Specific antibodies are available that recog­nise specifically the large subunit of processed caspase 3, but do not react with un­processed caspase 3 and are therefore specif­ic for the active caspase 3 (Figure 2c). These antibodies have been shown to work on paraffin sections.11,23,24 Processed caspase 3 is a highly specific marker for apoptosis, since no activation of the caspase cascade has been found in necrotic cells.11 The detection of active caspase 3 in cells can therefore be used as a discriminating criterion to distin­guish apoptosis from necrosis. Different studies have confirmed the use­fulness of antibodies to cleaved caspase 3 in detecting apoptotic cells in non-tumour as well in tumour tissues.23-25 In breast tissue, a strong correlation between the apoptotic index as­sessed by morphology in haematoxilin and eosin stained sections and the apoptotic index assessed using antibodies to cleaved caspase 3 has been reported. The immunohistochemical reaction makes recognition of apoptotic cells easier and reduces subjectivity in interpreta­tion, thus reducing inter-observer variability.24 One study investigating apoptosis detec­tion in normal tissues, showed prominent im­munostaining to cleaved caspase 3 in germi­nal centres and in neutrophilic granulo­cytes.23 A prominent granulocyte infiltration can be found in tumour tissues. In our expe­rience, many (but not all) granulocytes are positive to cleaved caspase 3. It is not clear whether this represents specific detection of apoptosis of granulocytes. However, positive granulocytes should not be confused with tu­mour apoptotic cells. In some tumours, no cleaved caspase 3 could be detected. This might reflect a low level of apoptotic activity, but the expression of uncleaved caspase 3 may nevertheless be downregulated in tumours.26 Immunohistochemical detection of caspase-cleaved target proteins It has become evident that caspase mediated cleaveage of target proteins at specific consen­sus sequences represents a unique feature of apoptosis. At least 100 different caspase sub­strates have been identified. Among them are structural proteins, such as actin and cytoker­atins, cell signalling molecules, regulators of cell cycle and DNA repair, such as PARP (polyADP-ribose polymerase), regulators of cell-cell interactions, inhibitors of endonucle­ases, such as ICAD, and others.5,27 Their cleav­age can result in their inactivation or activa­tion, leading to morphological changes of apoptotic cells and DNA fragmentation. Cleavage of target proteins may generate or unmask new epitopes. The generation of antibodies to such epitopes, which recognise cleaved but not uncleaved target proteins, has been reported for several target proteins. Cytokeratins are intermediary filaments of epithelial cells. Different cytokeratins are found in different epithelial cells as well as in epithelial tumours, so they are considered differentiation markers for epithelia and ep­ithelial tumours.28 Cytokeratin 18 is ex­pressed in simple nonstratified, ductular and pseudostratified epithelia (hepatocytes, renal tubular cells, ductular epithelia, mesotheli-um, respiratory epithelium).16,29 Caspase me­diated cleavage of cytokeratin 18 leads to ex­posure of an epitope that can be recognised by the binding of specific antibodies. The M30 antibody recognises cleaved but not un-cleaved cytokeratin 18.5,29 This antibody works on paraffin sections and has been shown to detect apoptotic cells in neoplastic simple epithelia as well in lung and colonic carcinoma. Detection of cleaved cytokeratin 18 precedes TUNEL positivity, indicating that cleavage of cytokeratin represents an early event, but in the late phase of apoptosis, the M30 epitope is lost. Its use is, however, limit­ed to detecting apoptotic cells in epithelial tis­sues or tumours expressing cytokeratin 18. Actin filaments are present in all human cells. During apoptosis, caspase-mediated cleavage of actin generates an actin fragment, which is specifically recognised by a poly-clonal antibody, called fractin.30 Positive fractin immunostaining of apoptotic cells has been shown in several neoplastic and non-neoplastic tissues. Fractin preferentially stains apoptotic bodies, indicating that cas­pase cleavage of actin filaments occurs late in the apoptosis. PARP (poly ADP-ribose polymerase) is a 116 kDa nuclear protein that is implicated in DNA repair. During apoptosis, it is one of the earliest proteins cleaved (and thus inactivat­ed) by caspases.31 PARP cleavage generates an 89 kDa C-terminal fragment and a 24 kDa N-terminal peptide. The 89 kDa fragment can be detected by the use of specific antibodies (Figure 2d).32 PARP degradation may precede DNA cleavage, as evidenced by the presence of the 89 kDa fragment in TUNEL negative cells. Therefore, in addition to late apoptosis (TUNEL positive), antibodies against cleaved PARP can detect early apoptosis before DNA cleavage.33 Conclusions Three decades ago, based on the morphology, apoptosis was recognised as a special type of cell death. Later, fragmentation of nuclear DNA was recognised as a biochemical hall­mark of apoptosis, and methods for detection of DNA fragments were developed. Recently, the apoptotic pathway has been elucidated and caspases have been recognised as key regulatory and executioner molecules of apoptosis. Their activation, followed by the cleavage of target proteins, precedes and is responsible for DNA fragmentation and mor­phological changes of apoptotic cells. Based on this knowledge, new more specific and sensitive in situ immunohistochemical meth­ods for the detection of apoptotic cells have been developed that enable accurate and rou- bd Figure 2. Apoptotic cells in tumour tissues. (a) Numerous apoptotic cells in multiple myeloma, as revealed by con­densed eosinophilic cytoplasm and condensed and fragmented nuclei in haematoxylin and eosin sections. (b) An apoptotic cell in hepatocellular carcinoma labelled by the TUNEL method. (c) Apoptotic cells immunostained against active caspase 3, the same tumour tissue as in (a). (d) Apoptotic cells in squamous cell carcinoma im­munostained against cleaved PARP. Magnification 400x a,c,d and 1000x b. tine detection of apoptosis on formalin-fixed and paraffin-embedded tissue. References 1. Raff M. Cell suicide for beginners. Nature 1998; 396: 119-22. 2. Gastman BR. Apoptosis and its clinical impact. Head Neck 2001; 23: 409-25. 3. Ryungsa K, Kazuaki T, Yoko U, Manabu E, Hideki I, Tetsuya T. Current status of the molecular mech­anisms of anticancer drug-induced apoptosis. Cancer Chemother Pharmacol 2002; 50: 343-52. 4. Dukers DF, Meijer CJ, ten Berge RL, Vos W, Ossenkoppele GJ, Oudejans JJ. High numbers of caspase 3-positive Reed-Sternberg cells in pre­treatment biopsy specimens of patients with Hodgkin disease predict favourable clinical out­come. Blood 2002; 100(1): 36-42. 5. Kler C, Orrenius S, Zhivotovsky B. Evaluation of caspase activity in apoptotic cells. J Immunol Methods 2002; 265: 97-100. 6. Willingham MC. Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 1999; 47(9): 1101-9. 7. Castedo M, Ferri K, Roumier T, Metivier D, Zamzami N, Kroemer G. Quantification of mito­chondrial alterations associated with apoptosis. J Immunol Methods 2002; 265: 39-47. 8. Van Heerde WL, Robert-Offerman S, Dumont E, Hofstra L, Doevendans PA, Smits JF et al. Markers of apoptosis in cardiovascular tissues: focus on Annexin V. Cardiavasc Res 2000; 45: 449-59. 9. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basis biological phenomenon with wide-ranging impli­cations in tissue kinetics. Br J Cancer 1972; 26: 239­57. 10. Pižem J, C A. Kaspaze. Med Razgl 2001; 40: 283­91. 11. Stadelmann C, Lassmann H. Detection of apopto-sis in tissue sections. Cell Tissue Res 2000; 301: 19-31. 12. Soini Y, Paakko P, Lehto V-P. Histopathological evaluation of apoptosis in cancer. Am J Pathol 1998; 153: 1041-53. 13. Cummings MC, Winterford CM, Walker NI. Apoptosis. Am J Surg Pathol 1997; 21(1): 88-101. 14. Elsässer A, Suzuki K, Lorenz-Meywe S, Bode C, Schaper J. The role of apoptosis in myocardial ishemia: a critical appraisal. Basic Res Cardiol 2001; 96: 219-26. 15. Van de Schepop HAM, de Jong JS, van Diest PJ, Baak JPA. Counting of apoptotic cells: a method­ological study in invasive breast cancer. J Clin Pathol Mol Pathol 1996: 49: 214-17. 16. Alison MR. Identifying and quantifying apoptosis: a growth industry in the face of death. J Pathol 1999; 188: 117-8. 17. Pižem J, Ferlan-Marolt V, Luzar B, C A. Proliferative and apoptotic activity in hepatocellu­lar carcinoma and surrounding non-neoplastic liver tissue. Pflers Arch 2000; 422(6) [Suppl1]: 174-6. 18. Zheng TS, Hunot S, Kuida K, Flavell RA. Caspase knockouts: matters of life and death. Cell Death Differ 1999; 6: 1043-53. 19. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identifi­cation of programmed cell death in situ via specif­ic labeling of nuclear DNA fragmentation. J cell Biol 1992; 119(3): 493-501. 20. Stäheliin BJ, Marti U, Solioz M Zimmermann H, Reichen J. False positive staining in the TUNEL as­say to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyocarbonate. J Clin Pathol Mol Pathol 1998; 51: 204-8. 21. Porter AG, Jänicke RU. Emerging roles of caspase­3 in apoptosis. Cell Death Differ 1999; 6: 99-104. 22. Slee EA, Adrain C, Martin SJ. Serial killers: order­ing caspase activation events in apoptosis. Cell Death Differ 1999; 6: 1067-74. 23. Gown AM, Willingham MC. Improved detection of apoptotic cells in archival paraffin sections: im­munohistochemistry using antibodies to cleaved caspase 3. J Histochem Cytochem 2002; 50(4): 449-54. 24. Hadjiloucas I, Gilmore AP, Bundred NJ, Streuli CH. Assessment of apoptosis in human breast tis­sue using an antibody against the active form of caspase 3: relation to tumour histopathological characteristics. Br J Cancer 2001; 85(10): 1522-6. 25. Urase K, Fujita E, Miho Y, Kouroku Y, Mukasa T, Yagi Y et al. Detection of activated caspase-3 (CPP32) in the vertebrate nervous system during development by cleavage site-directed antiserum. Develop Brain Res 1998; 111: 77-87. 26. C A, Pižem J, Gale N. The expression of Bcl-2 and pro-caspase 3 in head and neck squamous cell carcinoma. Zdrav Vest 2002; 71(Suppl III): 39-44. 27. Nicholson DW. Caspase structure, photolytic sub­strates, and function during apoptotic cell death. Cell Death Differ 1999; 6: 1028-42. 28. Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathol 2002; 40: 403­39. 29. Leers MPG, Kgen W, Bjklund V, Bergman T, Tribbick G, Persson B et al. Immunocytochemical detection and mapping of cytokeratin 18 neo-epi­tope exposed during early apoptosis. J Pathol 1999: 187: 567-72. 30. Suurmeijer AJH, van der Wijk J, van Veldhuisen DJ, Yang F, Cole GM. Fractin immunostainig for the detection of apoptotic cells and apoptotic bod­ies in formalin-fixed and paraffin-embedded tis­sue. Lab Invest 1999; 79(5): 619-20. 31. Duriez PJ, Shah GM. Cleavage of poly(ADP-ri­bose) polymerase: a sensitive parameter to study cell death. Biochem Cell Biol 1997; 75(4): 337-49. 32. Dukers DF, Oudejans JJ, Vos W, ten Berge RL, Meijer CJ. Apoptosis in B-cell lymphomas and re­active lymphoid tissues always involves activation of caspase 3 as determined by a new in situ detec­tion method. J Pathol 2002; 196(3): 307-15. 33. Soldani C, Bottone MG, Pellicciari C, Scovassi AI. Two-color fluorescence detection of Poly (ADP-Rybose) Polymerase (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells. Eur J Histochem 2001; 45(4): 389-92. Radiol Oncol 2003; 37(4): 233-40. Expression of cathepsin B is related to tumorigenicity of breast cancer cell lines Irena Zajc, Leonida Frangež, Tamara T. Lah Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia Background. The lysosomal cysteine proteases cathepsins B (CatB) and L (CatL) and their endogenous in­hibitors, stefins A (StA) and B (StB), are widely thought to be involved in the progression of human breast carcinoma. Previously we showed that, in model breast carcinoma cell lines, the reported tumorigenicity was not directly related to their in vitro invasive potential.1 However, CatL expression was positively related to the invasiveness of the cells and inversely related to the levels of StA. Here we challenge the hypothesis that imbalance between CatB and the two stefins is associated either with the invasiveness or the reported tu­morigenicity of the panel of selected breast carcinoma cells. Results. We investigated levels of mRNA, protein and activity for CatB in the panel of human breast car­cinoma cell lines whose tumorigenicity in vivo increased in the order MCF-7 < MDA-MB468 < MDA­MB231 < MDA-MB435, the most invasive being MDA-231. Levels of expression of mRNA, protein and ac­tivity for CatB were highly correlated and increased progressively with cell tumorigenicity. The ratio of CatB to stefins was shifted in favour of CatB in the more tumorigenic cell lines. Conclusions. Since CatL has been shown previously to be associated with invasive potential and, in this study, CatB expression was found positively associated with the tumorigenicity of the same breast carcino­ma cell lines, the two cathepsins in these cells do not appear to be regulated in a coordinated manner. CatB expression and the ratio between CatB and stefins increased progressively with tumorigenicity of the cells and suggests a similar situation in human tumours in vivo. Key words: breast neoplasms; cathepsin B; neoplasm invasiveness; tumor cells, cultured Received 16 November 2003 Accepted 28 November 2003 Correspondence to: Irena Zajc, PhD, Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia; Fax: + 386 1 257 38 47; Phone: + 386 1 423 33 88; E-mail: irena.zajc@nib.si Introduction Lysosomal cathepsins comprise a variety of peptidases of different clans, among which ClanA (CA) includes the cysteine peptidases cathepsins (Cats) B and L, which belong to so-called papain family.2 The association of the two cathepsins with tumour progression is well established in the literature.3,4 In clinical studies of breast carcinoma patients, elevated levels of CatB are associated with bad prog­nosis of patient survival.5-9 The activities of cysteine cathepsins are regulated by their endogenous inhibitors, a large superfamily of cystatins.10 The stefin (St) family comprises intracellular inhibitors, of which levels of StA and StB were found to be altered in tumour cells11 and in clinical samples from cancer patients.3,7,9,12-13 There­fore, the molecular mechanisms responsible for the biological roles of CatB and CatL in tu­mour cells, together with their inhibitors, StA and StB, need to be elucidated at the molecu­lar level. Tumour metastasis is a multi-step process, starting by detachment of tumour cells from the primary tumours, invasion through the extracellular matrix and/or basement mem­branes of vasculature, to reach the blood flow, which carries the tumour cells to distant organs where organ selective invasion and growth of tumour cells into the secondary site takes place. Invasion is therefore the com­mon denominator of many metastatic steps, a process which is associated with tissue re-modelling. Presumably, this is induced by tu­mour cells which are triggered to express, se­crete and/or activate a battery of proteolytic enzymes at their cell surface. Extracellularly and at the plasma membrane, metallopro­teinases and the plasminogen activator/plas-min system may initiate the extracellular ma­trix degradation. However, it has been pro­posed that Cats B and L initiate the proteolyt­ic cascade by specific activation of pro-uroki­nase and/or metalloproteinases.14 Further­more, intracellular degradation of extracellu­lar matrix components occurs during the in­vasion process15-16 which involves activation of lysosomal cathepsins, including CatB.17 Moreover, recent reports show that the inva­sion of tumour cells is significantly impaired when the intracellular activity of cysteine cathepsins is blocked.16-18 Tumorigenicity is a key characteristic of the malignant cancer cell, although the po­tential to form tumours at the secondary site may not directly reflect its invasive potential. In the two models of breast carcinoma cell lines, we have demonstrated, that the tumori­genicity of the cell lines was not strictly relat­ed to their in vitro invasiveness in Matrigel.1,19 Here, we used a model of four selected hu­man breast cancer lines: MCF7, MDA-MB468, MDA-MB231 and MDA-MB435, which have been reported to differ in tumorigenicity and metastasis in vivo.20,21 Their phenotype varies from epithelial (MCF7) to mesenchymal (MDA-MB435), with highly increased expres­sion of vimentin and downregulated expres­sion of estrogen receptors.22 However, not much is known about the expression of lyso­somal proteinases, except our recent report on the positive association of CatL mRNA and protein expression, and an inverse corre­lation of StA expression, with the invasive­ness of the cells in this model.1 In the present study we used the same cell model with the aim (a) to determine mRNA, protein and ac­tivity levels for CatB, (b) to relate protein ex­pression of CatB to those of StA and StB and (c) to relate these to the invasiveness and pro­gressive tumorigenicity of the human breast carcinoma cell lines. Materials and methods Cells and their characteristics: tumorigenic and invasive potentials Human breast carcinoma cell lines were ob­tained from the ATCC cell bank and cultured under conditions recommended by the sup­plier. The cells range from poorly to highly tumorigenic and metastatic in the order, MCF7 < MDA-MB468 < MDA-MB231 < MDA­MB435.20 The MCF7 cell line is poorly tu­morigenic and non-metastatic, MDA-MB468 cells exhibit low tumorigenic and low metastatic activities, while MDA-MB231 and MDA-MB435 cell lines are both highly tu­morigenic and metastatic, the latter produc­ing the highest number of distant metas­tases.21 The invasiveness of this panel of cell lines was determined in vitro, using the Matrigel assay.1 Invasiveness ranged from MCF7, MDA-MB468, MDA-MB435 to MDA­MB231 which was the most invasive cell. Thus invasiveness does not parallel tumori­genicity in the two most tumorigenic cell lines. RNA analysis - Northern analysis and real time PCR Total RNA was extracted from cells by TRIzol Reagent, according to the instructions of the supplier (Gibco, UK). CatB mRNA was deter­mined by Northern analysis and by quantita­tive RT-PCR. For Northern analysis, 15µg of RNA was electrophoresed through agarose/ formaldehyde gel and hybridized with full length CatB cDNA probe that was non-ra­dioactively labelled with digoxygenin, accord­ing to the instructions of the supplier. The cDNA probe was kindly provided by Dr. Boris Turk, Josef Stefan Institute, Ljubljana, Slovenia. The signals were detected by chemi­luminescence, using CDP StarTM System (Boehringer, Germany). A fluorescence-based real-time, quantita­tive RT-PCR method developed by Perkin Elmer ABI (TaqMan), was used to measure CatB RNA levels in cell extracts. 1µg of total RNA was reverse transcribed using High-Capacity cDNA Archive Kit and PCR ampli­fied with TaqMan Universal PCR Master Mix according to the instructions of the supplier (both Applied Biosystems, USA). The se­quence of the CatB forward primer was 5’­CTCTATGAATCCCATGTAGGGTGC-3’, 5’-CCTGTTTGTAGGTCGGGCTG-3’ for the reverse primer and 5’-CCCTGTGAGCAC­CACGTCAACGG-3’ for the TaqMan probe. Amplification of 18S ribosomal RNA was per­formed as an internal control. Protein concentration Cells were homogenised in Tris buffer (50mM Tris, pH 6.9, 0.05% Brij 35, 0.5mM DTT, 5mM EDTA, and 10µM pepstatin A) at 4°C using Tissue TearorTM (Biospec Products Inc., USA). The homogenates were centrifuged at 12,000 rpm for 15 min and supernatants (cell lysates) were stored at -20°C. Total protein concentra­tion was determined by Bio-Rad protein assay (Bio-Rad, USA) using bovine serum albumin as standard. Protein concentration of CatB was measured in cell lysates using a specific ELISA kit (Krka d.d., Slovenia). The ELISAs recognised total CatB protein, i.e. the precur­sor and the active forms as well as CatB com­plexed with the inhibitors. Cathepsin B activity The activity of CatB was determined using the fluorogenic substrate Z-Phe-Arg-AMC (modi­fied from Lah12), which is not selective for CatB, but also measures other cathepsins, such as CatL. First, the total cysteine pepti­dase activity was determined as the differ­ence between the total activity and the back­ground activity of the non-cysteine peptidas­es; this was determined using the general cys­teine peptidase inhibitor, E64c, at a final con­centration of 16µM. CatB activity was meas­ured by adding its selective inhibitor, 10µM CA-074, and determining the residual activi­ty. CatB activity was obtained as the differ­ence between total cysteine protease activity and residual activity. One enzyme unit (EU) is defined as the quantity releasing 1µmol of AMC per min. Specific activity is expressed as mEU/mg of total protein in the cell lysates. Statistical significance between measure­ments was determined by the two tailed t-test and p<0.05 was considered as significant. Figure 1. CatB mRNA expression in MCF7 and MDA­MB cell lines determined by Northern analysis (A) and quantitative RT-PCR (B). (A) For Northern analysis the total RNA was electrophoresed and hybridised with full length CatB DNA probe, as described in Material and methods. Total RNA stained with EtBr served as loading and transfer control (shown underneath). CatB expression is shown typically as two bands, one at 4.1kb and the other by 2.2.bp. (B) For quantitative analysis the total RNA was reverse transcribed and PCR amplified using CatB specific primers, as de­scribed in Material and methods. The results were normalised to 18S RNA. Error bars depict standard er­ror of the mean. CatB mRNA levels were similar in MCF7 and MDA-MB468, but significantly higher in MDA-MB231 and MDA-MB435 cells. Results Expression of cathepsin B in four human breast carcinoma cell lines Expression of CatB was determined in the four breast cancer cell lines that differ in their tumorigenicity and invasive potential. Levels of CatB mRNA, protein and enzyme activity correlated highly (r>0.99). All three levels in- Figure 2. Protein concentration (A) and enzymatic ac­tivity (B) of CatB in MCF7 and MDA-MB cell lines. Cell lysates were prepared as described in Material and methods. (A) CatB protein was measured by ELISA, and expressed as ng/mg total protein. The differences in protein concentration were statistically significant. (B) CatB activity was determined as described in Materials and methods.. CatB enzymatic activities were significantly different, except between MCF7 and MDA-MB468 cells. Error bars depict standard er­ror of the mean. creased with the degree of tumorigenicity of these breast carcinoma cells (Figures 1 and 2). Figure 1 shows CatB mRNA expression in MCF7 and the three MDA-MB cell lines de­termined by Northern analysis (A) and quan­titative RT-PCR (B). The highest CatB mRNA level was detected in the most tumorigenic MDA-MB435 cells, slightly less in the most invasive MDA-MB231 cells, whereas both poorly tumorigenic cell lines MCF7 and MDA-MB468 expressed significantly lower levels of CatB. However, the difference be­tween the latter two cell lines was not statis­tically significant. Figure 3. The molar concentrations of StA and StB (A) and the molar ratio between CatB and StB (B) in MCF7 and MDA-MB cell lines. Protein concentrations of StA, StB and CatB were measured in cell lysates with ELISA (see above) and their molar concentrations were calculated. (A) Both stefins were the highest in MCF7 cells and significally lower in metastatic MDA-MB cell lines. Note the difference in scale. (B) The molar ratio between CatB and StB increased progressively with tumorigenicity of the cells. All the differences be­tween the cell lines were statistically significant. Error bars depict standard error of the mean. Concentrations of CatB protein in MCF7 and the three MDA-MB cell lines are present­ed in Figure 2A. The lowest concentration was observed in the non-metastatic and poor­ly tumorigenic MCF7 cells and increased pro­gressively from MDA-MB468, MDA-MB231 to MDA-MB435, the most tumorigenic cell line. All the differences were statistically sig­nificant. The protein concentration of CatB in cell lysates increased more than 14 fold, from 25 ng/mg (cca 1 nM) in MCF7 cells to 350 ng/mg protein (14 nM) in MDA-MB435 cells. Figure 2B shows CatB proteolytic activities in MCF7 and the three MDA-MB cell lines. As for protein concentration, specific activity of CatB was the lowest in MCF7 cells and in­creased progressively with tumorigenicity of the cells. The difference is statistically signif­icant between the high and low tumorigenic lines, but not between the poorly tumorigenic cell lines MCF7 and MDA-MB468. Correlation between cathepsin B and stefins in four breast carcinoma cell lines Protein concentrations of StA and StB in the same breast cancer cell lines have been shown to be lower in cells with higher tu­morigenicity.1 As shown in Figure 3A, StB in the cell lysates was 2.4 fold lower in MDA­MB435, the most tumorigenic cell line, (59 ng/mg; 5,4 nM) than in MCF7 (about 126 ng/mg; 11 nM), whereas the levels of StA were about 10 fold lower, at 2 ng/mg protein (0.19 nM) and 0.2 ng/mg protein (0.018 nM) respectively. The molar ratio of the protein concentration of CatB to that of StB in the lysates of MCF7 and the three MDA-MB cell lines was determined (Figure 3B). This ratio was lowest in the MCF7 cell line and in­creased with tumorigenicity of the cells, as was observed with CatB levels alone. Discussion Cathepsin B and CatL were initially consid­ered to be products of single copy house­keeping genes, their expression being neces­sary for normal protein turnover in the cells. Surprisingly, homozygous CatB-deficient mice have an apparently normal phenotype, 23 suggesting redundancy of the genes, whereas CatL-deficient mice have periodic shading of fur and abnormal skin, but are otherwise viable,24 suggesting cell-specific functions of this enzyme. Their expression is regulated at the gene level,25-26 by mRNA splicing27-28 and by posttranslational modifi­cation (reviewed by Frosch29). In the present study, we have determined the expression of cathepsin B at the mRNA, protein and activi­ty levels in MCF7, MDA-MB468, MDA­MB231 and MDA-MB435 breast cancer cell lines of increasing tumorigenicity in vivo.20 CatB expression at all three levels is highly correlated. This suggests that the initial regu­lation of CatB occurs at the genetic level, the regulation of CatB transcription during devel­opment of the tumorigenic phenotype being most probably modulated through multiple GC boxes.25 This needs to be elucidated fur­ther. However, in a model comprising cells derived from MCF-10A manipulation to re­sult in distinct invasive and tumorigenic phe­notypes, we observed that CatB was signifi­cantly related to invasiveness but not tumori­genicity,19 although again all three levels of CatB expression correlated well. Presumably, expression of other gene profiles in the two panels of breast carcinoma cell lines is re­sponsible for up regulation of CatB in relation to the invasive and/or tumorigenic potential. There are also differences in the regulation of CatB and CatL. We found previously that, in contrast to CatB, CatL was highly in­creased at mRNA and protein levels, but was lowered at the activity level in the most inva­sive of the four cell lines, MDA-MB231.1 This may be due to selective inhibition of CatL ac­tivity by endogenous inhibitors, or by anoth­er defect in its intralysosomal processing. Similar differences in expression of CatL be­tween different levels were observed in the MCF10A model, suggesting that, in contrast to CatB, CatL regulation of expression also occurs posttranslationally. This supports our previous observation on differential regula­tion of cathepsins in breast cancer cell lines30 and in clinical samples of breast tumours.7,8 The ultimate regulation of CatB and CatL activities in the cells results from binding by the endogenous inhibitors, the stefins.31 Alteration of their levels in tumours, presum­ably downregulation, was reported.3,11 In the cell line model used here, we have shown pre­viously that StA levels decrease significantly with increasing tumorigenicity, in line with the hypothesis that imbalance between cysteine proteinases and their inhibitors facilitates tu­mour progression. StB levels were higher in the MCF-7 cells than in the other three inva­sive and tumorigenic cell lines.1 It is notewor­thy that the molar concentration of stefins was higher in low tumorigenic cells lines, but was lower than that of CatB in the most invasive and tumorigenic cells. This would suggest in­sufficient inhibition of CatB activity, since the complexes between cysteine cathepsins and cystatins are equimolar, as shown by crystal­lography31 and kinetic measurements.32 Although measurements in cell lysates may not completely parallel the situation in living cells, the result is a good indication that the balance between cathepsins and stefins is drastically altered in this panel of cell lines. We conclude that, in this model of breast car­cinoma cells, upregulation of CatB is a char­acteristic of the highly tumorigenic cell phe­notype. Together with our previous studies on this cell model, the results presented here confirm that cathepsins B and L are impor­tant in the processes of tumorigenicity and in­vasiveness of the cells, respectively, but are not regulated in a coordinated manner. Furthermore, the imbalance between prote­olytic enzymes and their inhibitors may facil­itate the development of a malignant pheno­type in breast cancer. If confirmed by further studies, both cathepsins could constitute po­tential targets for anti-invasive therapy. Acknowledgements This study was funded by Ministry of Education, Science and Sport of Republic of Slovenia (Programme 0105-509 to T.L). We are also grateful to Prof. Dr. Roger Pain for critical reading of the manuscript. References 1. Zajc I, Sever N, Bervar A, Lah TT. Expression of cys­teine peptidase cathepsin L and its inhibitors stefins A and B in relation to tumorigenicity of breast can­cer cell lines. Cancer Lett 2002; 187: 185-90. 2. Dickinson DP. Cysteine peptidases of mammals: Their biological roles and potential effects in the oral cavity and other tissues in health and disease. Crit Rev Oral Biol Med 2002; 13(3): 238-75. 3. Kos J, Lah TT. Cysteine proteinases and their en­dogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer. Oncol Rep 1998; 5: 1349-61. 4. Yan S, Sameni M, Sloane BF. Cathepsin B and hu­man tumor progression. Biol Chem 1998; 379: 113­23. 5. Foekens JA, Kos J, Peters HA, Kravec M, Look MP, Cimerman N, et al. Prognostic significance of cathepsins B and L in primary human breast can­cer. J Clin Oncol 1998; 16: 1013-21. 6. Thomssen C, Schmitt M, Goretzki L, Oppelt P, Pache L, Dettmar P, et al. Prognostic value of the cys­teine proteases cathepsin B and cathepsin L in hu­man breast cancer. Clin Cancer Res 1995; 1: 741-46. 7. Lah TT, Kos J, Blejec A, Frkovic-Grazio S, Golouh R, Vrhovec I, Turk V. The expression of lysosomal proteinases and their Inhibitors in breast cancer: Possible relationship to prognosis of the disease. Pathol Oncol Res 1997; 3: 221-33. 8. Lah TT, Cercek M, Blejec A, Kos J, Gorodetsky E, Somers R, et al. Cathepsin B, a prognostic indica­tor in lymph node-negative breast carcinoma pa­tients: comparison with cathepsin D, cathepsin L and other clinical indicators. Clin Cancer Res 2000; 6: 578-84. 9. Levicar N, Kos J, Blejec A, Golouh R, Vrhovec I, Frkovic-Grazio S, Lah TT. Comparison of potential biological markers cathepsin B, cathepsin L, stefin A and stefin B with urokinase and plasminogen activator inhibitor-1 and clinicopathological data of breast carcinoma patients. Cancer Detect Prev 2002; 26: 42-49. 10. Turk V, Bode W. The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 1991; 285: 213-19. 11. Calkins CC, Sloane BF. Mammalian cysteine pro­tease inhibitors: biochemical properties and pos­sible roles in tumor progression. Biol Chem Hoppe Seyler 1995; 376: 71-80. 12. Lah TT, Kokalj-Kunovar M, ätrukelj B, Pungercar J, Barlic-Maganja D, Drobnic-Korok M, et al. Stefins and lysosomal cathepsins B, L and D in hu­man breast carcinoma. Int J Cancer 1992; 50: 36-44. 13. Kuopio T, Kankaanranta A, Jalava P, Kronqvist P, Kotkansalo T, Weber, et al. Cysteine proteinase in­hibitor cystatin A in breast cancer. Cancer Res 1998; 58: 432-36. 14. Levicar N, Nuttall RK, Lah TT. Proteases in human brain tumours. Acta Neurochir 2003; 145: 825-38. 15. Everts V, Creemers LB, Docherty AJP, Beertsen W. Intracellular versus extracellular digestion of colla­gen. In: Davidivich Z and Norton LA, editors. Biological Mechanisms of Tooth Movement and Craniofacial Adaptation. Harvard Society for Advancement of Orthodontics, Boston: Ma Press; 1996. p. 309-16. 16. Premzl A, Zavarik-Bergant V, Turk V, Kos J. Intracellular and extracellular cathepsin B facili­tate invasion of MCF-10A neoT cells through re­constituted extracellular matrix in vitro. Exp Cell Res 2003; 283: 206-14. 17. Szpaderska AM, Frankfater A. An intracellular form of cathepsin B contributes to invasiveness in cancer. Cancer Res 2001; 61: 3493-500. 18. Sameni M, Dosescu J, Sloane BF. Imaging proteol­ysis by living human glioma cells. Biol Chem 2001; 382: 785-88. 19. Bervar A, Zajc I, Sever N, Katunuma N, Sloane BF, Lah TT. Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors. Biol Chem 2003; 384: 447-55. 20. Xin H, Stephans JC, Duan X, Harrowe G, Kim E, Grieshammer U et al. Identification of a novel as-partic-like peptidase differentially expressed in human breast cancer cell lines. Biochim Biophys Acta 2000; 1501: 125-37. 21. Price JE, Polyzos A, Zhang RD, Daniels LM. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 1990; 50: 717-21. 22. Thompson EW, Paik S, Brner N, Sommers CL, Zugmaier G, Clarke R, et al. Association of in­creased basement membrane invasiveness with absence of estrogen receptor and expression of vi-mentin in human breast cancer cell lines. J Cell Physiol 1992; 150: 534-44. 23. Deussing J, Roth W, Saftig P, Peters C, Ploegh HL, Villadangos JA. Cathepsin B and D are dispensa­ble for major histocompatibility complex class II- mediated antigen presentation. Proc Natl Acad Sci USA 1998; 95: 4516-21. 24. Nakagawa T, Roth W, Wong P, Nelson A, Farr A, Deussing J, et al. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thy­mus. Science 1998; 280: 450-3. 25. Yan, S, Berquin IM, Troen, BR, Sloane BF. Transcription of human cathepsin B is mediated by Sp1 and Ets family factors in glioma. DNA Cell Biol 2000; 19: 79-1. 26. Chauhan SS, Popescu NC, Ray D, Fleischmann R, Gottesman MM, Troen BR. Cloning, genomic or-ganisation and chromosomal localisation of hu­man cathepsin L. J Biol Chem 1993; 268(2); 1039­45. 27. Mehtani S, Gong Q, Panella J, Subbiah S, Peffley DM, Frankfater A. In vivo expression of an alter­ natively spliced human tumour message that en­codes a truncated form of cathepsin B. J Biol Chem 1998; 273(21); 13236-44. 28. Abudula A, Rommerskirsch W, Weber E, Gunther D, Wiederanders B. Splice variants of human cathepsin L mRNA show different expression rates. Biol Chem 2001; 382: 1583-91. 29. Frosch BA, Berquin I, Emmert-Buck M, Moin K, Sloane BF. Molecular recognition, membrane as­sociation and secretion of tumour cathepsin B. AMPIS 1999; 107: 28-37. 30. Lah TT, Calaf G, Kalman E, Shinde BG, Somers R, Russo J, et al. Cathepsins D, B, and L in trans­formed human breast epithelial cells. Breast Cancer Res Treat 1996; 39: 221-33. 31. Turk D, Guncar G. Lysosomal cysteine proteases (cathepsins): promising targets. Acta Crystallogr D Biol Crystallogr 2003; 59: 203-13. 32. Lah TT, Moin K, Clifford JL, Helmer KM, Day NA, Crissman JD, et al. Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma. Biochim Biophys Acta 1989; 993: 63-73. Radiol Oncol 2003; 37(4): 241-8. Cysteine and aspartic proteases cathepsins B and D determine the invasiveness of MCF10A neoT cells Aleš Premzl1 and Janko Kos1,2 1Jožef Stefan Institute, Department of Biochemistry and Molecular Biology; 2Krka, d.d., Research and Development Division, Department of Biochemical Research and Drug Design, Ljubljana, Slovenia Background. Lysosomal cathepsins B and D have been reported to play a role in various processes leading to progression of malignant disease. In ras-transformed MCF10A neoT cells both enzymes show similar vesicular distribution in perinuclear and peripheral cytoplasmic regions. Results. The co-localization of cathepsins B and D in some vesicles as defined by confocal microscopy sup­ports their co-ordinate activity in the proteolytic cascade. On the other hand, we showed that stefin A, an endogenous intracellular inhibitor of cysteine proteases, did not co-localize with cathepsin B and is presum­ably not involved in regulation of its enzymatic activity within the vesicles. Intracellular localization of both enzymes was confined to similar vesicles as the fluorescent degradation products of DQ-collagen IV either in individual cells or cell spheroids. The capability of these two enzymes to degrade collagen and other com­ponents of extracellular matrix is further supported by the results of Matrigel invasion assay. We showed that specific intracellular (CA-074 Me) and extracellular (CA-074) inhibitors of cathepsin B and pepstatin A, an inhibitor of cathepsin D, significantly reduced invasion of MCF10A neoT cells. Our results also show that in contrast to some other studies the activation peptide of pro-cathepsin D exhibited no mitogenic ef­fect on MCF10A neoT, MCF-7 or HEK-293 cells. Conclusion. We conclude that lysosomal cysteine proteases cathepsins B and D predominantly participate in degradation of extracellular matrix and facilitate invasion of tumour cells. Key words: tumor cells, cultured; cathepsin B; cathepsin D; neoplasms invasiveness Received 28 November 2003 Accepted 5 December 2003 Correspondence to: Janko Kos, Ph. D., Jožef Stefan Institute, Dept. of Biochemistry and Molecular Biology, Jamova 39, 1000 Ljubljana, Slovenia; Phone: +386 1 423 3832; Fax: +386 1 423 38 33; E-mail: janko.kos@krka.si Introduction Dissemination of malignant cells of primary tumours to distant tissues and the formation of secondary tumours is the primary cause of treatment failure and death of cancer pa­tients. Besides serine and metallo proteases, lysosomal cysteine proteases cathepsins B and L and aspartic protease cathepsin D have been shown to participate in processes of tu­mour progression, including tumour cell growth, invasion and metastasis.1-3 Alte­rations in expression, processing and subcel­lular distribution of these three cathepsins have been linked with more aggressive tu­mour behaviour, increased risk of relapse and shorter survival of cancer patients.1, 4 In numerous studies it has been reported that proteolytic activity of cysteine proteases is involved in degradation and remodelling of extracellular matrix (ECM), a crucial step in tumour cell invasion.5 Degradation of ECM in living tumour cells was localized either extra­cellularly or intracellularly, following endocy­tosis of partially degraded ECM proteins. Recently, we have demonstrated that tumour cells can use both proteolytic pathways si­multaneously.6 Cathepsin B has been sug­gested to facilitate ECM degradation and sub­sequent tumour cell invasion directly by in­tra- or extracellular degradation of ECM com­ponents or indirectly, by activation of other enzymes of the proteolytic cascade mediating this process.7 In contrast to cathepsins B and L, which are believed to be mainly involved in the in­vasion of tumour cells, the importance of cathepsin D in cancer progression is less clear. In breast cancer catalytically inactive cathepsin D pro-peptide was proposed to act as a mitogen, promoting tumour cell prolifer­ation.8 Additionally, we showed that active enzyme can promote tumour invasion in vit­ro.9 This is in line with clinical studies, re­vealing the correlation of the mature form of cathepsin D with poor prognosis of cancer patients.10 In this study, cathepsins B and D have been simultaneously evaluated in MCF10A neoT cells with regard to their impact on cell proliferation, degradation of ECM and tu­mour cell invasion. Additionally, the localiza­tion of these two cathepsins and stefin A was determined in this cell line by immunofluo­rescence labeling and confocal microscopy. Materials and methods Cell cultures Ras-transformed human breast epithelial cell line MCF-10A neoT was obtained from Prof. B. F. Sloane (Wayne State University, Detroit, USA). Human cell lines HEK-293 and MCF-7 were obtained from the American Tissue Culture Collection. MCF-10A neoT cells origi­nate from MCF-10, a human breast epithelial cell line derived from a patient with fibrocystic breast disease that underwent spontaneous immortalization in culture and grows attached as MCF-10A cell line. Co-transfection of MCF­10A cells with plasmid containing the neomycin resistance gene and human T24 mu­tated c-Ha-ras oncogene using the calcium phosphate method resulted in MCF-10A neoT cells.11,12 MCF-10A neoT cells were cultured as monolayers in DMEM/F12 medium supple­mented with Hepes, 5% FCS, antibiotics and growth factors. Human breast cancer cell line MCF-7 and transformed human embryonic kidney cell line HEK-293 were cultured in MEM supplemented with 2 mM L-glutamine, Earle’s BSS and adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 10 % FCS. Cell lines were cultured at 37°C in a hu­midified atmosphere containing 5% CO2. Cysteine protease inhibitors The natural reversible tight-binding protein inhibitor chicken cystatin13 and synthetic ir­reversible epoxysuccinyl inhibitor E-64 (Sigma, St. Louis, USA) were used as general cysteine proteinase inhibitors. Cathepsin B was inhibited with selective membrane im­permeable epoxide derivative CA-074 (Bachem, Bubendorf, Switzerland) and mem­brane-permeable pro-inhibitor CA-074Me (Sigma, St. Louis, USA ). Cathepsin L was in­hibited by CLIK-1489 (a gift from Prof. Nobuhiko Katunuma, Tokushima Bunri University, Japan) and cathepsin D by pep-statin A (Sigma, St. Louis, USA). Cell viability and proliferation assays Cytotoxic and/or proliferative effects of cys­teine protease inhibitors on MCF-10A neoT cells were tested as described.14 Cells were added to a final concentration of 5 x 104 cells/200 µl per well of a 96 well microtiter plate. Appropriate concentrations of in­hibitors or control media were added. Plates were incubated for 24 hours at 37°C and 5% CO2. The medium was carefully removed, 200 µl of 0.5 mg/ml MTT (3-4,5-dimethylthiazol­2,5 diphenyl tetrazolium bromide, Sigma, St. Louis, USA) was added and incubated for three hours at 37°C and 5% CO2. The medi­um was carefully removed and formazan crystals dissolved in 200 µl/well of iso­propanol. Absorbance was measured on an ELISA microplate reader at 570 nm, reference filter 690 nm. All tests were performed in quadruplicate. Cell invasion assays Effects of protease inhibitors on invasion were tested using the modified method de­scribed by Holst-Hansen et al.15 Transwells (Costar Corning, New York, USA) with 12 mm polycarbonate filters, 12 µm pore size, were used. 25 µl of 100 µg/ml fibronectin were applied on the lower side of the filters, which were left for one hour in a laminar hood to dry. The upper side of the filters was coated with 100 µl of 1 mg/ml Matrigel (Becton Dickinson, San Diego, USA) and 100 µlof DMEM/F12 was added. The Matrigel was dried overnight at room temperature in a lam­inar hood and reconstituted with 200 µl of medium for one hour at 37°C. The upper compartments were filled with 0.5 ml of cell suspension, final concentration 4 x 105 cells/ml, containing the appropriate concen­tration of the inhibitor. The lower compart­ments were filled with 1.5 ml of medium con­taining the same concentration of the in­hibitor. The plates were incubated for 24 hours at 37°C and 5% CO2. MTT was added to a final concentration of 0.5 mg/ml to the upper and lower compartments and plates were incubated for an additional 3 hours. Media from both compartments were trans­ferred separately to Eppendorf tubes, and centrifuged at 6200 rpm for 5 minutes. Supernatants were discarded and the for-mazan crystals which remained dissolved in 1 ml of isopropanol. The colour intensity of the dissolved formazan crystals was measured as described above. As controls, cells were incu­bated with medium containing the appropri­ate volumes of distilled water, ethyl acetate and DMSO, the solvents used for preparation of concentrated solutions of the inhibitors and monoclonal antibody. Invasion was recorded as the percentage of cells that pene­trated the Matrigel coated filters compared to controls, and was calculated as ODlower / ODlower+ODupper x 100. All tests were per­formed in triplicate. Mitogenic effect of procathepsin D activation peptide 18 amino acids long cathepsin D activation peptide8 (IAKGPVSKYSQAVPAVTE) as a part of cathepsin D pro-peptide, was synthesised by NeoSystems (Calgary, Canada) at 95% pu­rity. The mitogenic effect of the peptide was tested as described for cytotoxicity and pro­liferation assays, with minor modifications. Cells were added to a final concentration of 5 x 103 cells/200 µl per well of a 96 well mi-crotiter plate. The activation peptide was added in a range of 0.001-10 µM concentra­tions. Plates were incubated for the period of 1-5 days at 37°C and 5% CO2 and analysed as described above by MTT assay. Immunocytochemistry Cathepsins B and D, and stefin A were local­ized in MCF-10A neoT cells fixed in ice-cold methanol and permeabilized in 0.1% saponin in PBS, pH 7.4. Non-specific staining was blocked with 0.2% BSA in PBS, pH 7.4. Primary antibodies used were rabbit anti-hu­man cathepsin B polyclonal antibody, mouse Figure 1. Immunolocalization studies in MCF-10A neoT cells. (A) Co-localization (yellow) of cathepsin B (red fluorescence) and cathepsin D (green fluores­cence). (B) MCF-10A neoT spheroids of living cells. Green fluorescent degradation products are predomi­nantly localised within the cells. (C) Localization of cathepsin B (green fluorescence) and stefin A (red flu­orescence). Bars, 20 ?M. anti-human cathepsin D D101 monoclonal antibody and mouse anti-human stefin A C5/2 monoclonal antibody (all KRKA d.d., Novo mesto, Slovenia). Antibodies recognize precursors, mature forms and enzyme/in­hibitor complexes of all three antigens. Secondary antibodies were goat anti-rabbit labelled with Alexa Fluor 488 and goat anti-mouse labelled with Alexa Fluor 546 (Molecular Probes, Eugene, USA). Controls were run in the absence of primary antibod­ies, but in the presence of secondary antibody and pre-immune goat serum. Pro Long Antifade Kit was used for mounting cover-slips on glass slides. A Zeiss LSM 510 confo­cal microscope was used to observe the cells. Proteolysis assays Pre-cooled glass coverslips were coated with 25 µg/ml of the quenched fluorescent sub­strate DQ-collagen IV (Molecular Probes, Eugene, USA) suspended in Matrigel, 10 mg/ml, for 10 minutes at 4°C. Cells were plat­ed and grown for a period of 72 hours. A Zeiss LSM 310 confocal microscope with 40 X water immersion objective was used to ob­serve the cells or cell spheroids for fluores­cent degradation products of DQ-collagen IV. Results Immunolocalization Cathepsins B and D showed similar vesicular distribution in MCF-10A neoT cells, but the level of their expression varied between cells. They were localized both in the perinuclear and in the peripheral cytoplasmic region (Figure 1A) and both enzymes co-localized in some of the vesicles. Stefin A, an endogenous intracellular inhibitor of cysteine proteases, localized evenly in the cell cytoplasm, but no co-localization with cathepsin B was observed (Figure 1C). Degradation of DQ-collagen IV by living MCF­10A neoT spheroids The ability of MCF-10A neoT cells to degrade extracellular matrix was analysed by the nov­el confocal assay desribed by Sameni et al.16 using quenched fluorescent substrate DQ-collagen IV. MCF-10A neoT cells plated on a DQ-collagen IV/Matrigel matrix formed sphe- Figure 2. Effect of cysteine and aspartic protease in­hibitors on Matrigel invasion by MCF-10A neoT cells. Concentration of chicken cystatin was 2 mM, all oth­er inhibitors were used at 10 mM concentration. Data are represented as mean ( S.D. of two independent de­terminations performed in triplicate. roids after 72 hours. Fluorescent degradation products seen as green fluorescence were ob­served inside the cells forming the body of the spheroid (Figure 1B). They were localised in vesicular structures as observed in individ­ual cells.6 Individual spots of degradation were observed also in the pericellular regions. Effect of protease inhibitors on the viability and proliferation of MCF-10A neoT cells Prior to the invasion assays, the selected pro­tease inhibitors were tested for their possible cytotoxic and proliferative effects. No effect on cell viability and/or proliferation was ob­served within the same concentration range as used in invasion assays (data not shown). Effect of protease inhibitors on Matrigel invasion of MCF-10A neoT cells The roles of cathepsins B and D in the inva­sion of MCF-10A neoT cells were determined with selected natural and synthetic inhibitors of cysteine and aspartic proteases. All in­hibitors decreased cell invasion after 24 h (Figure 2). Inhibition of aspartic protease cathepsin D with 10 µM pepstatin A resulted in 22.1 ± 0.8 % decrease in invasion of MCF­10A neoT cells. The effect of pepstatin A was much lower compared to general cysteine Figure 3. Effect of pro-cathepsin D activation peptide IAKGPVSKYSQAVPAVTE on the growth of cancer cell lines. After five days incubation the number of cells was determined by MTT method and compared to controls protease inhibitors chicken cystatin (70.2 ± 17.8 % at 2 µM) and E-64 (47.5 ±12.7 % at 10 µM). To assess the contribution of cathepsin B, CA-074, a specific inhibitor of extracellular cathepsin B and membrane-permeable ana­logue CA-074 Me were used. CA-074 and CA­074 Me reduced invasion by 24.9 ±1.2 % and 27.0 ± 8.7 % at 10 µM, respectively. For com­parison, 10 µM concentration of CLIK-148, a specific synthetic inhibitor of cathepsin L re­sulted in 27.7 ± 7.1 % decrease in cell inva­sion. Effect of cathepsin D activation peptide on tumour cell proliferation To investigate the mitogenic effect of the acti­vation peptide of pro-cathepsin D, a corre­sponding synthetic peptide was used in MTT proliferation assay. In contrast to results pub­lished by Vetvicka et al.8 and Fusek and Vetvicka17, no significant mitogenic effect was observed on MCF-10A neoT and MCF-7 cell proliferation within the comparable con-cenration range after five days (Figure 3). Additionally, no proliferative activity was ob­served in human embryonic kidney cells HEK-293 used as negative control. Discussion Metastatic process depends on the ability of tumour cells to invade through ECM. This process is facilitated by proteolytic degrada­tion of various ECM proteins, including differ­ent types of collagens, laminin and fibronectin by proteases. The common believe is, that this process takes place extracellularly at the inva­sive front of tumour cells. New techniques, like confocal laser scaning microscopy enable analysis of proteolytic degradation of quenched fluorescent protein substrates, like DQ-collagen IV, in living cells and the results show that cells differ in their sites of matrix remodeling, located either extracellularly or in­tracellularly.5,16 We found that invasive MCF­10A neoT cells accumulate fluorescent prod­ucts of digested DQ-collagen IV inside the cells, in vesicle-like structures.6 This result was confirmed in this study by using 3-dimension­al spheroid cultures of MCF-10A neoT cells. Again, positive vesicular staining was found in most of the cells forming the body of the sphe­roid. In addition, individual spots of pericellu­lar fluorescence observed both in individual cells and in spheroid cultures suggest, that MCF-10A neoT cells can simultaneously use both pathways of ECM degradation. Several studies have implicated cysteine proteases in invasion and tumour progres­sion. Therefore, we tested the impact of gen­eral and specific cysteine protease inhibitors on invasiveness of MCF10A neoT cells in the concentration range not affecting cell viabili­ty and proliferation. Both general inhibitors, i.e. the reversible tight-binding protein in­hibitor chicken cystatin13 and the irreversible inhibitor E-64 were the most effective in Matrigel invasion assay, with 70.2 ± 17.8 % (2 µM concentration) and 47.5 ± 12.7 % (10 µM) inhibition of invasion, respectively. CA­074, an inhibitor of extracellular cathepsin B, decreased invasion by 24.9 ± 1.2 % at 10 µM, a result is comparable to the effect of intra­cellular cathepsin B inhibitor CA-074 Me (27.0 ± 8.7 %). We may expect that the in­hibitor, capable to impair the activity of both, intracellular and extracellular fraction of cathepsin B would be even more effective to decrease tumour cell invasion. Further, we demonstrated that cathepsin L also partici­pates in this process. However, none of the inhibitors completely blocked MCF-10A neoT cell invasion, suggesting the involvement of other, presumably serine and matrix metallo proteases in invasion process. Our finding, that aspartic protease in­hibitor pepstatin A also reduced Matrigel in­vasion of MCF-10A neoT cells, suggests that beside cysteine cathepsins B and L, active as-partic protease cathepsin D is implicated in invasion process as well. This is in contrast with reports of Johnson et al.18 that the irre­versible peptide inhibitor pepstatin A was in­effective in inhibiting MCF-7 tumour cell in­vasion in vitro. In our case treatment of MCF­10A neoT cells with 10 µM pepstatin A result­ed in 22.1 ± 0.8 % decrease in invasion. Reduction of invasion was even higher when SQAPI-like natural protein inhibitor of aspar-tic proteases was used.9 Besides direct degradation of ECM, cathep-sin D was suggested to act as an initiator pro­tease upstream in a proteolytic cascade acti­vating pro-cathepsin B7. Activated cathepsin B can further convert serine protease pro-urokinase type plasminogen activator (uPA) into active enzyme uPA, which, in turn, is able to activate plasmin and matrix metallo proteases. These enzymes can then actively degrade various ECM components. Our re­sults support the involvement of cathepsin D in the proteolytic cascade. Cathepsin D was co-localized with cathepsin B in the same cy­toplasmic vesicles in MCF10A neoT cells and is, therefore, able to activate procathepsin B. However, we have to be aware that antibody used for localization of cathepsin B recognis-es besides precursor also other forms of the enzyme19 and that other assays and experi­ments, including specific fluorogenic sub-strates are needed to determine the ratio be­tween pro and active forms of cathepsin B and to assess the rate of its activation. The effect of endogenous protease in­hibitors on degradation of ECM in MCF10A neoT cells remains to be evaluated. Whereas no endogenous inhibitor of cathepsin D is known so far, the activity of cysteine proteas­es cathepsins B and L are regulated by their endogenous inhibitors, the cystatins.1 In MCF10A neoT cells we localised an intracel­lular inhibitor stefin A, which was difusely distributed throughout cell cytoplasm. It was not co-localised with cathepsin B what indi­cates that at least inside the cells it is not im­portant for regulation of cathepsin B depend­ent degradation of ECM. The results are sim­ilar to that of Calkins and Sloane20 in he-patoma cells, reporting differetial intracellu­lar distribution of cathepsins and stefins. In conclusion, our results show that lyso­somal cathepsins B and D, overexpressed in most of malignant tumours, predominantly participate in degradation of ECM and facili­tate tumour cell invasion. Regulation of their enzymatic activities by exogenous inhibitors represents a new possibility for therapeutic intervention in cancer patients. Aknowledgement This work was supported by Ministry of Education, Science and Sport of the Republic of Slovenia. References 1. 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. 2. Foekens JA, Look MP, de Vries JB, Meijer-van Gelder ME, van Putten WLJ, Klijn JGM. Cathepsin D in primary breast cancer: prognostic evaluation involving 2810 patients. Brit J Cancer 1999; 79: 300-7. 3. Lah TT, Cercek M, Blejec A, Kos J, Gorodetsky E, Somers R, Daskal I. Cathepsin B, a prognostic in­dicator in lymph node-negative breast carcinoma patients: comparison with cathepsin D, cathepsin L, and other clinical indicators. Clin Cancer Res 2000; 6: 578-84. 4. Sloane BF, Moin K, Sameni M, Tait LR, Rozhin J, Ziegler G. Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-ras oncogene. J Cell Sci 1994; 107: 373-84. 5. Sameni M, Moin K, and Sloane BF. Imaging prote­olysis by living human breast cancer cells. Neoplasia 2000; 2: 496-504. 6. Premzl A, Zavašnik-Bergant V, Turk V, Kos J. Intracellular and extracellular cathepsin B facili­tate invasion of MCF-10A neoT cells through re­constituted extracellular matrix in vitro. Exp Cell Res 2003; 283: 206-14. 7. Schmitt M, Jänicke F, and Graeff H. Tumor-asso­ciated proteases. Fibrinolysis 1992; 6: Suppl 4, 3-26. 8. Vetvicka V, Vetvickova J, Fusek M. Anti-human procathepsin D activation peptide antibodies in­hibit breast cancer development. Breast Cancer Res Treat 1999; 57: 261-9. 9. Premzl A, Puizdar V, Zavašnik-Bergant V, Kopitar-Jerala N, Lah TT, Katunuma N, Sloane BF, Turk V, Kos J. Invasion of ras-transformed breast epithe­lial cells depends on the proteolytic activity of cys­teine and aspartic proteinases. Biol Chem 2001; 382: 853-7. 10. Brouillet JP, Spyratos F, Hacene K, Fauque J, Freiss G, Dupont F, Maudelonde T, Rochefort H. Immunoradiometric assay of pro-cathepsin D in breast cancer cytosol: relative prognostic value versus total cathepsin D. Eur J Cancer 1993; 29: 1248-51. 11. Soule HD, Maloney TM, Wolman SR, Peterson Jr. WD, Brenz R, McGrath CM et al. Isolations and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 1990; 50: 6075-86. 12. Basolo F, Elliot J, Tait L, Chen QC, Maloney TM, Russo IH, et al. Transformation of human breast epithelial cells by c-Ha-ras oncogene. Mol Carcinogen 1991; 4: 25-35. 13. Kos J, Dolinar M, Turk V. Isolation and character-isation of chicken L- and H-kininogens and their interaction with chicken cysteine proteinases and papain. Agents Actions (Suppl.) 1992; 38: 331-9. 14. Holst-Hansen C, Brner N. (1998). MTT-Cell pro­liferation assay. In: Celis E, editor. Cell Biology: A laboratory handbook. San Diego: Academic Press; 1998. p. 16-18. 15. Holst-Hansen C, Johannessen B, Hoyer-Hansen G, Romer J, Ellis V, Brner N. Urokinase-type plas­minogen activation in three human breast cancer cell lines correlates with their in vitro invasive­ness. Clin Exp Metastasis 1996; 14: 297-307. 16. Sameni, M, Dosescu J, Sloane BF. Imaging prote­olysis by living human glioma cells. Biol Chem 2001; 382: 785-8. 17. Fusek M, Vetvicka V. Mitogenic function of hu­man procathepsin D: the role of the propeptide. Biochem J 1994; 303: 775-80. 18. Johnson MD, Torri JA, Lippman ME, Dickson RB. The role of cathepsin D in the invasiveness of hu­man breast cancer cells. Cancer Res 1993; 53: 873­7.18. 19. Kos, J, Šmid, A, Krašovec, M, Svetic, B, Lenarcic, B, Vrhovec, I, Škrk, J, Turk, V. Lysosomal pro-teinases cathepsins D, B, H, L and their inhibitors stefins A and B in head and neck cancer. Biol Chem Hoppe-Seyler 1995; 376: 401-5.19. 20. Calkins CC, Sloane BF . Mammalian cysteine pro­tease inhibitors: Biochemical properties and possi­ble roles in tumour progression. Biol Chem 1995; 376: 71-80. Radiol Oncol 2003; 37(4): 249-55. Experiencing professional strains of nurses, radiation engineers and physicians working at the Institute of oncology in Ljubljana Andreja Cirila Škufca Smrdel Department of Psychooncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Background. Since 1974 the term burnout is used in psychology. Burnout describes the end result of stress and has been described by Maslach comprising three basic components: emotional exhaustion, depersonal­ization and low personal accomplishment. In this paper we would like to describe some aspects of burnout experiences of the employees of the Institute of Oncology in Ljubljana. Subjects and methods. We used Questionnaire of professional stress, created by Žunter Nagy and Kocmur. In our research 137 health workers from four professional groups participated: physicians, graduated nurs­es, nurses and radiation engineers, representing 38% of all employees. Results. We found out that in the experience the professional stressof all four professional groups is rela­tively equalized. The most prominent feelings are of fatigue, irritability and work overload. There were no signs of depersonalization - as described by Maslach - reported in our group. In nurses and in radiation en­gineers a distress is significantly more often displayed due to poorer personal income and poorer material status. Nurses reported significantly more often the intention to change work position (51%), institution (57%) or job (47%). Conclusions. Workstress impacts on the experience and on the thought patterns in those participating in the study. We can describe those signs as burnout signs. However, there are more new questions opening in the future as well as the need to a longitudinal approach to the research of this more and more prominent field. Key words: medical oncology; stress, psychological; burnout, professional Received 20 November 2003 Accepted 2 December 2003 Correspondence to: Andreja Cirila Škufca Smrdel, psychologist, Department of Psychooncology, Institute of Oncology; Zaloška 2, 1000 Ljubljana, Slovenia; Phone +386 1 5879 181; Fax +386 1 5879 400; E-mail: askufca@onko-i.si Introduction The term »burnout« was first used by Herbert Freudenberger in 1974 when he described problems in so the called »help professions«. He described problems appearing when one encounters situations that are above his abili­ty, power and energy. A decade later Christine Maslach defined »burnout« with the help of three basic dimensions - emotion­al exhaustion, depersonalisation (non com­passionate way of treating the patients, in­creased emotional distance from the patients) and reduced personal accomplishment.1 The development of the burnout syndrome is influenced by the intertwined influence of many factors. Some personal types are more prone to the burnout development as others.2 Maslach especially described the following personal factors: low level of hardiness, poor self-esteem, an external locus of control and avoidant coping style. She found out that fe­males, younger employees and singles are more prone to the burnout development. Beside the individual factors the burnout is also dependent upon the organization and nature of the job. Among those night work and work in shifts, lack of the personal and poor shift organisation stand out in the health system. The good team serves as an impor­tant source of emotional support and as such the safety factor; on the other hand conflicts at work are important risk factors for the burnout. The problem of burnout is common in health care workers in every speciality. The research finding shows that the burnout prevalence among health care workers varies from 28% (Ramirez et al, 1996)3 to 56% (Whippen and Canellos, 1991).4 In the field of oncology the work with incurable patients was described as the most important risk fac­tor. The therapy often has a limited impact; health care workers are confronted with the difference between real state of the affairs and expectations - theirs as well as patients’ and patient’s relatives. Due to a constant con­tact with dying and the constant loss of pa­tients some thought that the burnout is more pronounced in workers in the field of oncolo­gy than in other medical fields; however, some researches discarded this hypothesis.5,6 Research findings confirmed the Maslach findings that women, younger and single health care workers are more prone to the burnout.4,7 The aim of this paper is to present findings considering health care workers at the Institute of Oncology in Ljubljana and shed light to how physicians-oncologists, nurses and radiation engineers are experiencing the burdens of professional and personal life. Subjects and methods When carrying out this study, there was no standardized questionnaire for measuring burnout in Slovenia. Therefore, we decided to use Questionnaire of professional stress cre­ated by Žunter Nagy and Kocmur, which was fashioned for health care workers in the field of psychiatry.8 Participants ranged items on 5-degree scales, some questions were dichotomized. For a statistical analysis we used chi-squared test with Yates correction and in the interval type of results we used Student t-test. All the physicians-oncologists, nurses, graduated nurses and radiation engineers working at the Institute of Oncology in Ljubljana (which is the only comprehensive cancer centre in Slovenia) received the ques­tionnaire. We got 137 usefully filled ques­tionnaires, representing 38% of those sent, which is comparable with the data from the literature.4 Response rate was the greatest among the nurses (59.3 %), followed by physi­cians (21 %) and radiation engineers (19.3 %). Results Demographics The average age of the participants was 32 years for radiation engineers and 40 years for physicians; the combined average age for all groups was 35 years. Like the age the average work time was the lowest for the group of ra­diation engineers (8.8 years, and for the other Table1. Demographics of medical oncology staff Physicians (n=30) nurses (n=57) graduated nurses (n=26) radiation engineers (n=25) Age under 25 yr 0 36 % 24 % 29 % 26 - 35 yr 37 % 13 % 31 % 37 % 36 - 45 yr 27 % 33 % 20 % 13 % 46 and more yr 37 % 16 % 23 % 21 % Average work time 14.1 yr 13.9 yr 13.6 yr 8.78 yr Gender Male Female 27 % 73 % 11 % 89 % 7 % 93 % 31 % 69 % Marital status Single 23 % 32 % 19 % 60 % Married 73 % 65 % 81 % 36 % divorced/widowed 3 % 3 % 0 4 % Children None 34 % 43 % 24 % 50 % one or more 66 % 57 % 76 % 50 % Direct work with patients 83 % 96 % 100 % 100% three groups between 13 and 14 years, re­spectively). The majority of participants were females (83 %); the proportion of females was the greatest among nurses and graduated nurses (90 %). Three quarters of physicians and graduated nurses were married and had at least one child; in radiation engineer’s two thirds were singles and half of all without children (Table 1). For the most of working time 83 % of the physicians and almost all participants from other professional groups were working directly with patients. Work and professional life Participants from all professional groups are in general satisfied with their profession and their relation to patients (average mark >4). They are less satisfied as regards the profes­sional relations with their colleagues as well as their superiors; their position within the work organization, amount of responsibility they are trusted with; the possibilities of con­tinuous education and possibilities of ad­vancement. The results differ significantly ac­cording to groups in the item of satisfaction with salary (F = 3.519, p = 0.017), where physicians and graduated nurses showing significantly higher mark in comparison to other two groups. What all have in common is the feeling of being overloaded and emo­tionally too involved in their work. There were no statistically significant dif­ferences between groups in describing their relations toward patients. In their description there a positive, almost idealistic view is dom­inant. Participants are »almost all the time« understanding, patient, considerate and »al­most never« impatient, indifferent, they are never rude (Figure 1). Although there are no statistically significant differences between groups, there is an interesting feeling of guilt in the group of physicians, which could be at­tributed to their responsibility for treatment, as well as to their adoption of the responsi­bility for disease outcome. Participants estimated that their attitude towards patients did not change in the past years of professional work and remained »al­most always« the same as their attitude to­wards patients at the beginning of their work. Radiol Oncol 2003; 37(4): 249-55. Results are the same also when comparing their marks in relation to the work time of participants. The failure of treatment was a prominent factor influencing the experience of work stress in all professional groups. Among or­ganization factors there is a pre-eminent lack of labourers and as a consequence too crammed timetable (Figure 2). Differences be­tween average item marks are small; follow­ing the described three items there are pre­tentiousness of work with the patients and close contact with dying. We found statistically significant differ­ences among professional groups in items concerning organization matters. Physicians and both groups of nurses estimate more of­ten as radiation engineers that their distress is connected to the lack of personnel (F = 3.30, p = 0.022). Shift work is more burden­some to nurses as to radiation engineers (F = 5.149, p = 0.002); radiation engineers work in shifts, but they have a shorten work time; ra­diation engineers also most often estimate that there is enough personnel. The other two groups do not have a shift work but have in­stead of this 24 hours duty. Physicians and graduate nurses are on du­ty on average 2-3 days per month. As a ra­tionale for taking duty they are citing in the first place the needs of the institution, while physicians more often cite the material needs (F = 3.073, p = 0.049). Nevertheless, half of graduate nurses and one third of physicians do not want to be on duty less often, which is in both groups linked with bonus allowance. Among the ways of settling the problems in work place the discussion is pre-eminent in all professional groups, with colleagues, superiors and discussions at home. Recreation is important in the same measure. A statistically significant difference between professional groups is evident only in one item. When encountering problems in work place nurses are more likely to enter sick leave even for minor physical strains (F = 2.796, p = 0.043). Statistically significant differences are showing in the item about thinking on work problems at home (F = 4.237; p = 0.007). Transfer of work situation in the home envi­ronment is most pronounced in physicians who could be linked to their responsibility for treatment. This transfer is the least pro­nounced in radiation engineers. On the question about considering the change of profession, specialization or work field/work position within the institution there are statistically significant differences in all items (F = 2.67, p = 0.050; F = 3.71, p = 0.012; F = 6.11, p = 0.001). While physicians and radiation engineers are never or almost never considering this, there is a group of nurses with the marked deviation in the pos­itive direction, while in the meantime their answers to questions on the contentment with profession and work place were no dif­ferent from the others. Family life All professional groups estimated that a part­ner ”almost always” understands the nature of their work (average mark 4.03). While they are satisfied with the emotional relationship they are having with a partner, the emotional Figure 2. The estimated stress sources on work places of four professional groups. satisfaction with children in all groups is esti­mated between ”most of the time” and ”al­ways”. The relationship with a partner is -like in the all complex of questions regarding per­sonal life - presented in the very positive light - and we can assume idealized. The relation with a partner is almost always loving and comprehensive, items with a negative conno­tation such as impatient, indifferent, bur­dened with guilt are estimated as ”almost never”. A statistically significant difference between groups was found for the item indif­ferent (F = 3.245, p = 0.025), which is least ex­pressed in the group of physicians. In estimating which factors of the home environment are connected with stress in par­ticipants the estimations are in all groups cen­tred ”mostly” on ”almost never”, which means that they almost never connect their stress with factors at home. The highest rankings are material problems (between ”almost never” and ”sometimes”) with statistically significant differences between groups (F = 5.425, p = 0.002); material problems are highest ranking by nurses and lowest by physicians. The item comparison according to age failed to show statistically significant differences. Personal life In the last three years more than 90 % of nurs­es and radiation engineers and 73 % of physi­cians completed the physical exam. Ten per­cent of physicians and 15 % of nurses have a chronical disease, most often hypertension, spine problems (nurses), asthma and mi­graines. Among regular smokers there are mostly nurses (20 % of participants), and less physicians (6.7 %); but the differences in the frequency allocations are not statistically sig­nificant. According to gender among regular smokers there are 48 % males and 13% fe­males. Of the psychoactive substances the participants do not use benzodiazepines, an­ti-depressants or anti-psychotics; they also al­most never consummate alcohol. The differ­ences between groups are not statistically sig­nificant. 2-3 % of participants sometimes think of suicide, and the same proportion cites the attempt in the past; there are no dif­ferences between groups. Bad psychical con­dition is in all groups most often expressed as fatigue and irritability (median estimate 2.5­ 3) and less often as anxiety, weakness or loneliness (equal or lower than 2). Consi­dering this there are no significant differ­ences between groups. In all groups there is sometimes felling of overworking. Discussion Burnout is a complex phenomena and is a re­sult of mutual co-influence of work stress (re­lated to type and nature of work combined with its organization) and underlying person­ality; with return negative implications for a person with worse attitude toward a patient and decreased efficiency of work as well as for patients one is in care of and organization. Mutual co-influence of those factors re­quires a complex research, which had quite expanded in last few years; nevertheless there is a lack of longitudinal researches in research settings.2 Despite findings that un­derlying personality could have greater influ­ence on the burnout development than work stress, the research of personality characteris­tics is still a challenge for the further re­search.5 This study has not by-passed those faults (while there the possibility of longitudinal setting is still open). To investigate one - only institution is related with smaller number of participants, and the descriptive nature of ap­proach enables the investigation of specific social and organizational aspects useful in planning future strategies for the prevention of burnout at the level of a particular organi­zation. The comparison of all four professional groups shows that in the experiencing the professional stress all four are relatively equalized. In all included professional groups, the most stressful are those situations where the treatment was unsuccessful, which is related to encounter with mortality. The or­ganizational factors are represented even more - poor labourers covering resulting in stuffed schedule. Professional and personal stress result thus above all in feelings of fa­tigue, irritability and work overload. Beside this nurses and radiation engineers display distress due to poorer personal in­come and poorer material status. Shift work decreases their presence in family life and creates conflicts between their professional role and other life roles. Beside this many young nurses not only more frequently think about changing their profession but also to change their work place fairly quick. Signs of depersonalization as defined by Maslach were not encountered - on the con­trary, there is explicitly positive, even idealis­tic view on e.g. one’s attitude towards pa­tients. We can not therefore rule out a hypo­thesis that this can express phenomena of a denial and no criticism to the state of affairs. There are two so called burnout protection factors pointed to by the results. The first one is the expressed satisfaction at work and the other social support experienced by the em­ployees when the relationships within the team are satisfactory and in situations, when troubles occur and co-workers help to get out of them.9 Due to burnout negative consequences on health professionals, as well as patients and a health organization, it is important to recog­nize early signs of burnout and act preven­tively. The factors we can use for this are in­corporation of appropriate contents in the ed­ucational process, communication skills training, appropriate work organization and unburdening of negative emotions and final­ly raising consciousness on burnout with ap­propriate recording. This study of course is only a static picture of a current situation and warrants further longitudinal approach. References 1. Maslach C, Schaufeli WB, Leiter MP. Job burnout. Annu Rev Psychol 2001; 52: 397-422. 2. Penson RT, Dignan FL, Canellos GP, Picard CL, Lynch TJ Jr. Burnout: caring for the caregivers. Oncologist 2000; 5: 425-434. 3. Whippen D, Canellos G. Burnout sindrome in the practice of oncology: results of a random survey of 1000 oncologists. J Clin Oncol 1991; 9: 1916-20. 4. Ramirez AJ, Graham J, Richards MA, Timothy AR. Stress at work for the clinical oncologist. Clin Oncol 1996; 8: 137-9. 5. Blenkin H, Deary I, Sadler A, Agius R. Stress in NHS consultants. BMJ 1995; 310: 534. 6. Ramirez AJ, Graham J, Richards MA, Cull A, Gregory WM, Leaning MS, et al. Burnout and psy­chiatric disorder among cancer clinicians. Br J Cancer 1995; 71: 1263-9. 7. Ramirez AJ, Graham J, Richards MA, Cull A, Gregory WM, et al. Mental health of hospital con­sultants: The effects of stress and satisfaction at work. Lancet 1996; 347: 724-8. 8. Žunter Nagy A, Kocmur M. Poklicne obremenitve zdravnikov [Physicians Professional Strains]. Zdrav Vest 1998; 67: 67-73. 9. Kash KM, Holland JC, Breitbart W, Berenson S, Dougherty J, Ouellette-Kobasa S, et al. Stress and Burnout in Oncology. Oncology (Huntingt) 2000; 14: 1621-33. Radiol Oncol 2003; 37(4): 257-66. An outline of the history of radiotherapy at the Institute of Oncology in Ljubljana from its beginning till 1980s Aleksandra Oklješa Lukic, Karmen Hscher Department of Radiotherapy, Institute of Oncology, Ljubljana, Slovenia Background. The article presents the milestone events in the history of radiotherapy at the Institute of Oncology since its establishment till 1980s. It reviews the facts deduced from various jubilee publications, seminar reports and staff interviews of the Institute of Oncology. The aim of the article is to present the chronological history of radiotherapy at the Institute of Oncology, and to supplement the fragmented and incomplete records written in the past. Conclusions. Available records are occasionally discrepant, but the most significant events in the history of the Institute of Oncology and its Radiotherapy Ward can nevertheless be ascertained. Key words: radiotherapy - history - trends; medical oncology; Ljubljana Introduction The inception of the Institute of Oncology goes back to October 1917, when Dr Josip Cholewa (Figure 1), Chief Physician of the re­gional hospital in Brežice, used his modest re­sources to found an oncological laboratory. He successfully performed experiments in it, and published results in national and foreign Received 3 September 2003 Accepted 14 September 2003 Correspondence to: Aleksandra Oklješa Lukic, Dipl. Ing. Radiol., Department of Radiotherapy, Institute of Oncology, Zaloška 2, Ljubljana, Slovenia; Phone +386 1 522 3749; Fax: 386 1 4319 108; E mail: aokljesa@onko-i.si scientific literature. Cholewa had studied medicine at the University of Krakow, and later specialized in surgery. While practising the latter, he became interested in oncology. His experimental inducement of cancer was based on the realization that cancer in hu­mans did not differ significantly from cancer in other mammals.1 He maintained contacts with prominent oncologists in the World, and attended many congresses abroad. In September 1921, he lectured on induced blas­toma in a white mouse in Zagreb. His work aroused keen interest abroad. He was the ini­tiator of the »Yugoslav Committee for Cancer Abatement«, and instrumental in the estab­lishment of the Institute for Research and Treatment of Neoplasms of Dravska banovina (an administrative unit of the pre-war Yugoslavia, corresponding largely to the pres-ent-day Slovenia) in1937.2 Other people, besides Cholewa, must be given credit for the establishment of the Institute. One of them was Dr. Gerlovic, Principal of the State Hospital for Mental Disorders in Ljubljana, whose rich experi­ence was always at the disposal of the Institute. Janko Dolžan, Senior Inspector of Dravska banovina, was a regular visitor to the Institute. Ever since the lecture of Professor Blumental in Ljubljana in 1935, the establish­ment of the Institute was strongly supported by Dr. Mayer, while Dr. Pogacnik, Head of the Department of Otology at the General Hospital in Ljubljana, supplied resources for radium treatment as early as 1928. The Institute for Research and Treatment of Neoplasms (IRTN) Prior to the establishment of the Institute for Radiol Oncol 2003; 37(4): 257-66. Research and Treatment of Neoplasms in Dravska banovina (IRTN), oncological patients had been treated in surgical, gynecological, otological, ophtalmological and other wards in hospitals throughout Slovenia, and the treatment consisted of surgical operations for the most part. Surgeons invested great efforts in saving operable patients, but their interest rapidly ebbed in case of recurring disease, metastases, or inoperable tumours. The new institute filled the void in the cancer treat­ment of the time. The IRTN was opened in the southeastern extension of the military hospital in Šem­peter. On the ground floor, a boiler room and facilities for test animals were installed. Four patient rooms with 30 beds, an office, a li­brary, the principal’s office, a tea kitchenette, toilets, two bathrooms, a waiting room, an outpatient surgery, roentgen - and radium-therapy facilities, and an operating theatre were provided on the first floor.3 The radiotherapy equipment of the IRTN consisted of three units.4 The Siemens Stabivolt (Figure 2) was used for deep therapy, and its orthovoltage roentgen tube worked at 15 mA and 200kV. The tube was mounted on a special casing, which functioned as the shield against high voltage and secondary ra­diation. The tube was adjustable along all three axes, so that the rays could reach any part of the body. At the time, the Stabilivolt was a state-of-the-art radiotherapeutical de-vice. Its high quality enabled the Oncological Institute to use it until the year 1977, for no less than four decades. The second unit of the radiotherapy equip­ment was a Schafer-Witte, used for contact in-travaginal irradiation. It worked at the maxi­mum voltage of 100 kV and with maximum amperage of 5 mA. A special x-ray tube irra­diated neoplasms with straight beams or at an angle, so that cervical cancer could be treated without damaging healthy tissue. The third device was a Chaoul, similar to the Schafer-Witte in construction, but working at a lower voltage of 60 kV and maximum am­perage of 5 mA. The apparatus was used for contact irradiation of superficial neoplasms. The roentgen tube was cooled with water. All three pieces of radiotherapy equipment had timers on their respective switch boards, which controlled the duration of irradiation. The control unit was separated from the de­vices with lead-coated partitions and lead-glass windows, shielding the staff from radia­tion. The dosimetry was performed with an electrostatic Hammer dosimeter with two ioni­zation cells. The southeastern extension of former mil­itary barracks in Šempeter, where the IRTN was set up, was short of room, but an operat­ing theatre with adjoining scrubbing facilities was nevertheless installed. It was small, but fully equipped for major surgeries. The attic accommodated separate rooms for patients’ wardrobe, sterilization, chemical, chemobio-logical and histopathological laboratories, pharmacy, filing cabinets and storage, three rooms for physicians, a room for orderlies, three rooms for nurses, a bathroom and toi­lets. Although the IRTN was fully equipped for research and treatment of neoplasms, the shortage of room soon became an impedi­ment, since, in addition to the Institute’s own patients, outpatients were sent there for ra­diotherapy by other medical institutions. Meals were delivered from the nearby hospi­tal for mental diseases, where the laundry was done as well. The IRTN’s resources depended largely on external sponsors. The purchase of 300 mg of radium, for example, was made possible by the Savings Bank of Dravska banovina. The ma­terial lasted for quite some time, since the Schafer-Witte and the Chaoul substantially re­lieved and partly replaced the radium-thera­py.3 The Principal of the IRTN was Dr Cholewa, the Chief Physician was Dr Lev Šavnik, a gynecologist, who had previously been in charge of radiotherapy at the Roentgen Department of the State General Hospital in Ljubljana. The two of them were assisted by three younger physicians. The histopathological laboratory was lead by Professor Alija Košir, the roentgen equip­ment, radium and other physical appliances were taken care of by France Avcin, an elec­troengineer, the scientific chemical laborato­ry was lead by Professor Vladimir Premru. From World War II to the Institute of Oncology During the Italian occupation (1941-1943), the IRTN of Dravska Banovina was replaced, to a limited degree, by the Institute for Neoplasms of the Ljubljana Province (since Dravska banovina no longer existed). After the death of Dr Cholewa in 1943, the manage­ment was taken over by Professor Šavnik. In August 1945, the Institute merged with the Roentgen Institute of the former General Hospital, and became the Institute of Roentgenology and Radiology of the newly es­tablished University Hospitals in Ljubljana.5 It was headed by Dr. Josip Hebein. Already in April 1946, the two institutes were separated again, and the name of the former changed to Institution of Oncology. In December 1948, the Chair of Oncology and Radiotherapy was opened and the same time the name Institution of Oncology changed to Institute of Oncology. The management of the Institute, which was still attached to the University Hospitals, was taken over by Professor Šavnik, who remained its principal until the year 1963. During the first few years after the war, ra­diotherapy was the fastest developing activi­ty, propelled by the newly acquired equip­ment. Until 1949, radiotherapy was per­formed by only one radiologist, who was in charge of x-ray diagnostics as well. The short­age of manpower made it necessary to engage physicians who were trained as radiothera­pists on the job. After the year 1949, the Institute started to educate radiotherapists systematically. In 1955, two physicians (Dr. Danica Žitnik and Dr. Majda Mackovšek­Peršic) successfully passed the specialist ex­am in radiotherapy. This achievement was the beginning of radiotherapy as an inde­pendent branch of medicine in Slovenia. By the year 1963 six other physicians had passed the same exam.4 At that time, radiotherapy was the most important therapeutic activity at the Institute. The equipment was relatively modest in the beginning, but it improved considerably over the first few years. In addition to the Stabili-volt and two other out-dated roentgen devi­ces, the Institute acquired, in 1952, a Siemens Stabilipan (300 kV), suitable for superficial ir­radiation, and allowing 3 to 5 combinations of voltage (Figure 3). It was used primarily in the treatment of superficial and semi-deep seated tumours. The focus of source-skin distance and the size of the field were regulated by a special accessory unit, an applicator, which re­duced the scattering of rays and adjusted the field of irradiation. In those years, the Institute received 600 mg of radium from the United Nations Relief and Rehabilitation Agency (UNRRA). In 1955, the irradiation of patients with a 31 MeV Betatron (Figure 4) started, which was another milestone event. The Betatron was the first circular accelerator and, due to its high energy potential, the first device en­abling supervoltage radiotherapy. It was bought by the Institute of Physics Jožef Stefan, and also installed there. It was used both for research and for treatment. Unfortunately, patients had to be transported all the way through the city, which made the therapy even more complicated. During the irradia­tion, patients were placed on an adapted op­erating table. The position of the patient had to be adjusted to the horizontal emanation of beams - patients with lung cancer were treat­ed in sitting position. In 1958, the idea of complete autonomy of the Institute was reborn. The aspirations re­sulted in the Institute of Oncology becoming and independent institution on August 1, 1961, exactly after 23 years of its existence. The development of radiotherapy at the Institute of Oncology from 1961 till 1980 The period between 1961 and 1970 was marked by the efforts to build new facilities and acquire larger premises, as well as to ex­pand the research activity. The Institute ob­tained some new radiotherapy equipment, but the shortage of room hindered its effi­ciency. Towards the end of the 1960s, the blueprints of a new building for teleradio-therapy were designed. By the year 1968, the bulk of resources necessary for the construc­tion of the new Institute of Oncology had been accumulated in the fund for the construc­tion of the new Institute of Oncology (set up in 1965). The building was to be erected on the right side of the Ljubljanica river. The facili­ties for the teleradiotherapy were the first to be built, but the plans fell through because of the construction of the new University Clinical Centre. The first supervoltage device with sealed radioactive source used at the Institute of Oncology (in 1962) was a Siemens Gamatron (Figure 5), a Co60 unit, with the initial activi­ty of 111 TBq (3000 Ci). The activity of the cobalt source (Co60) was 111 TBq, and its head made of wolfram, which shielded the environment from radiation, weighed more than half a ton. It was used for deep irradia­tion, and soon nick-named the Cobalt Bomb. In addition, two roentgen diagnostic devices were bought. They were used for tracing the localizations of irradiated fields and the posi­tion of brachytherapeutic sources of radium, as well as for diagnostic purposes.5 The body contour, in which the position of the tumour and of other organs was marked while devising the irradiation plan, was traced with the help of a lead wire (Figure 6). It was wrapped around the patient, then care­fully removed and copied to tracing paper. The method was very imprecise, since distor­tions of the wire were inevitable during its re­moval from the patient. In the middle of the 1960s the body con­tours were traced with a special needle contour device (NCD), with radially arranged metal needles (Figure 7). The tips of the needles were pressed against the patient’s body, and then the dots were copied to the tracing paper and connected into a line. In the beginning, the protection of healthy tissue around the irradiated area consisted of standardized lead blocks of different shapes (Figure 8), attached to an acrylic plate (or in­serted between two such plates). This method is still applied with sealed sources of radia­tion (cobalt), but for linear accelerators it was replaced by customized shields in the 1990s. The only exception in the use of standardized shields was the treatment of patients with Hodgkin’s disease. Custom shields were made for them at an early date. Depending on the shape of the part of the body that needed shielding, holes were cut out of a Styrofoam board with a hot wire, filled with protective lead pellets, and sealed with paraffin. It is crucial that the position of the patient remains the same throughout the therapy, es­pecially when the patient’s head and neck with many sensitive organs are within the ir­radiation field. It is the only way to make irra­diation effective, and the protection reliable. In the beginning, patients with tumours in the head and neck were secured in their posi­tion with the help of a special cardboard pro­file (Figure 9), which allowed the resumption of the position during the planning and dur­ing the irradiation process. The profile was taken with the help of a special device made of metal needles. Once the position of the pa­tient was secure, the metal needles were arranged along the patient’s profile and fixed. The contour was copied to the cardboard and the profile was cut out of it. In 1969, another sealed source apparatus (Co60) joined the slightly out-dated and over­loaded Siemens Gamatron. It was a Theratron 80, 222 TBq (6000 Ci), made in Canada (Figure 10). It was provisionally set up in the adapted former garage near the isotopic labo­ratory. Its specialty was the so-called »beam stopper«, which intercepted the exiting radia­tion, so that the room needed considerably less shielding than in the case of devices with no such accessory. Still, it was yet another proof of how the shortage of space impeded the development of radiotherapy. Towards the end of the 1960’s, body con­tour tracing was performed with a panto­graph (Figure 11), a mechanical device which traced the contours of the body on paper while a radiographer moved its antenna along the surface of the patient’s body. Several types of pantographs were used, from me­chanically very simple ones, to complex and precise devices. A pantograph is still part of the standard equipment of the Teleradio-therapy Department. Being a mechanical in­strument, it can hardly ever fail. It serves as a back-up device to more modern and complex appliances (such as CTs or lasers). In spite of the fact that everything pointed to the Oncological Institute moving to a new location towards the end of the 1960s, the early 1970s clearly revealed that other proj­ects had priority. The Act on the Construction of the University Clinical Centre (passed by the Assembly of the SR Slovenia in May 1981) stipulated the construction of the TRT on the left bank of the Ljubljanica river, on the premises of the old auxiliary units of the Clinical Centre. The postponement was a huge blow to the Oncological Institute, cur­tailing its spatial perspectives. To compen­sate for that, the Oncological Institute was given the old building of the Internal Clinic, which was renovated with the funds for the construction of the new Oncological Institute - the renovation cost 500 million dinars.3 The Teleradiotherapy Department moved to the renovated building of the Internal Clinic, which became the new building C of the Oncological Institute. All existing equip­ment was transported there, except the Gamatron, which went out of use. Two new roentgen devices were bought. The first one was the simulator Ximatron TEM (Figure 12), used for the preparation and planning of ra­diotherapy. The apparatus simulated the con­ditions on irradiation devices, determined the irradiation field in the patient, and tested the shielding. One of the major advantages of simulators is that the irradiation field is im­mediately visible on the screen. Since the table with the patient is movable as well, any aberration can be immediately detected and rectified, without the time-consuming radiog­raphy. This must be done only at the end, for verification, possible fabrication of shields, and records. The second new device was a Japanese Toshiba Transversal Tomograph (Figure 13). It changed completely the treatment plan­ning and the body contour tracing procedure. It made it possible to locate the precise site of the tumour in relation to all internal organs in the vicinity, which was of crucial importance for the treatment planning. The body contour tracing became contactless, the wrapping of the patient with a wire, or pressing metal needles against the patient’s body became su­perfluous. In the middle of the 1970s fixation masks re­placed cardboard profiles in securing the pa­tient’s position during the treatment (Figure 14). The procedure was long, uncomfortable and difficult for the patient. The masks were made in a special workshop, and their fabri­cation took one whole day. After the disappointment in 1971, the struggle to overcome the shortage of space continued. The staff of the Institute of Oncology became more optimistic on November 1974, when the cornerstone for the new teleradiotherapy building was laid (Figure 15). The construction took three years, and the Teleradiotherapy Department moved to new premises in 1977. All the existing equipment, except the Gamatron, were moved to the new building. Two new supervoltage radiotherapy devices were installed there as well - a new Phillips lin­ear accelerator SL75/20 (Figure 16), which was used until the year 2000, and a sealed source radiation (Co60) apparatus of the same pro­ducer, which is still being used (Figure 17),. In addition to the Ximatron simulator, a new Phillips simulator was installed (Figure 18), which went out of use in 1999. The Teleradiotherapy Department of the Institute of Oncology is still confined to the same premises, although it has outgrown them already. The number of patients has greatly increased since the 1980s, and so has the staff. The purchase of new equipment pro­motes the efficiency of the Institute, but the fi­nalization of the new Institute of Oncology will have to be next step. Although the telera­diotherapy will remain in the same building, some additional room will be provided in the new building, reducing the shortage of space, especially in view of the imminent purchase of three new linear accelerators. Acknowledgement We thank all the staff of the Institute of Oncology for the help rendered in the collec­tion of historical data. A special thank-you goes to Boris Sekereš. References 1. Stepinšek M. Pionirji radioterapije. Raopis 1996; (4): 2-7. 2. Novak F. Banovinski inštitut za raziskovanje in zdravljenje novotvorb v Ljubljani. Radiol Glas 1938; 2(3-4): 122-4. 3. Ravnihar B. Ob 35-letnici Onkološkega inštituta v Ljubljani. 35. obletnica dela Onkološkega inštituta v Ljubljani. Ljubljana: Onkološki inštitut; 1973. p. 7-13. 4. Seme M. Razvoj zavoda v prvih desetih letih ob-stoja. In: Šavnik L, editor. Zdravljenje raka. Ljubljana: Državna založba Slovenije; 1949. p. 233-44. 5. Ravnihar B. An outline of the development of ra­diotherapy in Slovenia. Radiol Oncol 1992; 26(1): 77-8. 6. Ravnihar B. Povzetek iz zgodovine Onkološkega inštituta v Ljubljani 1938-1978. Ljubljana: Onko­sloški inštitut; 1978. p.3-11. Radio/ Oncol 2003; 37(4): 213-6. Možganske metastaze pri bolnikih s pljucnim rakom. Vpliv prognosticnih dejavnikov na preživetje Smrdel U, Zwitter M, Kovac V Izhodišca. Pljucni rak pogosto zaseva v možgane. V prispevku smo ugotavljali, pri katerih bol­nikih s pljucnim rakom se pojavljajo možganske metastaze in kako razlicni napovedni dejavni­ki vplivajo na preživetje bolnikov. Bolniki in metode. V letu 1998 je bilo v Sloveniji ugotovljenih 974 novih bolnikov s pljucnim rakom, 615 med njimi je bilo obravnavanih tudi na Onkološkem inštitutu v Ljubljani. Med potekom bolezni smo pri 137 (22,3 %) od 615 bolnikov odkrili možganske metastaze. Rezultati. Srednje preživetje pri 12 bolnikih s solitarnimi možganskimi metastazami (pri vecini je bila narejena metastazektomija) je bilo 7,6 mesecev, srednje preživetje pri bolnikih z multi­plimi možganskimi metastazami pa je bilo 2,8 meseca (p = 0.0018). Od 137 bolnikov jih je 45 (32,8 %) imelo drobnocelicni pljucni rak, 43 (31,4 %) žlezni rak in 19 (13,9 %) skvamoznocelicni pljucni rak. Bolniki, ki so imeli stanje splošne zmogljivosti po WHO­ju manj kot 2, so imeli srednje preživetje 3,7 meseca, bolniki s stanjem splošne zmogljivosti 2 ali vec pa 2,7 meseca (p=0.0448). Zakljucki. Bolniki s solitarnimi možganskimi metastazami imajo statisticno znacilno boljše preživetje kot tisti z multiplimi možganskimi metastazami. Preseneca velik odstotek bolnikov z žleznim pljucnim rakom, pri katerih odkrivamo možganske metastaze skoraj v enakem odstotku kot pri bolnikih z drobnocelicnim pljucnim rakom. Tako moramo ponovno razmisliti, ali ni in­dicirano profilakticno obsevanje glave tudi pri bolnikih z žleznim pljucnim rakom? Tudi pri naših bolnikih se je pokazalo, da je stanje splošne zmogljivosti odlocilen napovedni dejavnik za preživetje. Radio/ 011co/ 2003; 37(4): 217-9. Endobronhialna metastaza kot prvi znak ledvicnega karcinoma Kaneko Y, Haraguchi N, Kodama T, Kagohashi K, Ishii Y, Satoh H, Sekizawa K Izhodišca. Najbolj pogosto najdemo endobronhialne metastaze po diagnosticiranju primarnega tumorja. Prikazujemo pa redek primer, ko je bila endobronhialna metastaza odkrita pred pri­marnim tumorjem oz. je bila prvi znak ledvicnega karcinoma. Prikaz primera. 61-letni bolnik se je ob sprejemu v našo bolnišnico pritoževal zaradi 3 mesece trajajocega kašlja. CT preiskava prsnega koša je pokazala polipoidno maso v bronhiju za desni zgornji pljucni reženj. Odvzeli so material za biobsijo in mikroskopski pregled je pokazal, da ima bolnik metastazo ledvicnega karcinoma na steni bronhija. Zakljucki. Ko odkrijemo endobronhialne tumorozne spremembe brez klinicnih znakov pri­marnega tumorja, moramo pomisliti tudi na asimptomatski primarni tumor izven prsnega koša. V takšnih primerih so potrebne vse ustrezne diagnosticne preiskave. Radio 011col 2003; 27(4): 221-4. Metastaza sramne kosti kot prvi znak pljucnega raka Kodama T, Satoh H, Ueno T, Homma S, Sekizawa K Izhodišca. Metastaze sramne kosti so kot prvi znak pljucnega raka zelo redke. Napacno jih lahko ocenimo kot parasimfizealne osteoporoticne frakture, ki so pogoste pri postmenopauzalnih ženah in pri starostnikih. Prikaz primera. 65-letna bolnica je tožila zaradi bolecin v predelu desnega boka, ki so se širile v prednji del stegna. Z roentgenskim slikanjem medenice smo odkrili osteoliticno spremembo v predelu zgornjega ramusa sramne kosti. Roentgenska slika prsnih organov pa je pokazala nodu­larni infiltrat v predelu srednjega režnja desnih pljuc. S pomocjo trans bronhialne biobsije smo ugotovili, da ima bolnica bronhialni adenokarcinom. Bolnici smo obsevali prizadeti del kosti, bolecine pa dodatno umirili z morfinskim zdravilom. Zakljucki. Ob odkritju neobicajne kostne metastaze, ko še nismo odkrili primarnega tumorja, moramo najprej pomisliti na pljucni karcinom in narediti rentgensko slikanje prsnih organov. Radio/ Oncol 2003; 37(4): 225-32. Prikaz apoptoticnih celic v tumorskih parafinskih rezinah Pižem J, Cor A Apoptoza je oblika celicne smrti z znacilnimi morfološkimi spremembami, ki jo uravnavajo zapleteni molekularni mehanizmi. Motnje v uravnavanju apoptoze so pomembne za rast tu­morjev, hkrati pa razlicne metode zdravljenja tumorjev zavirajo njihovo rast pretežno s sprožan­jem apoptoze tumorskih celic. Ugotavljanje apoptotske aktivnost v tumorju je lahko pomembno za napoved poteka bolezni in njenega zdravljenja, zato je pomemben razvoj metod za rutinski prikaz apoptoticnih celic v tumorskem tkivu, ki je bilo fiksirano v formalinu in vklopljeno v parafin. Pri uravnavanju apoptoze sodelujejo zapletene molekularne poti. Razlicni proapoptoticni signali sprožijo aktivacijo kaspaz, slednje pa cepijo tarcne beljakovine. Njihova cepitev je odgovorna za morfološke spremembe apoptoticnih celic in znacilno cepitev jedrne DNK. V zadnjem desetletju je prikaz apoptoticnih celic v tumorskem tkivu fiksiranem v formalinu temeljil pretežno na mor­foloških znacilnostih in znacilni fragmentaciji DNK. V zadnjem casu omogoca zanesljiv prikaz apoptoticnih celic imunohistokemicna reakcija na prikaz aktiviranih kaspaz in cepljenih tarcnih beljakovin (citokeratin 18, aktin, PARP). Radio/ Oncol 2003; 37(4): 233-40. Izražanje katepsina B je povezano s tumorigenostjo celicnih linij raka dojke Zajc I, Frangež L, Lah TT Izhodišce. Menimo, da lizosomski cisteinski proteazi katepsina (Cat) B in L ter njuna endogena inhibitorja stefina (St) A in B sodelujejo pri napredovanju raka na dojki pri cloveku. V prejšnji raziskavi na celicnem modelu raka dojke smo pokazali1 , da v literaturi opisana tumorigenost teh celicnih linij ne sovpada neposredno z njihovo invazivnostjo in vitro. Izražanje CatL je narašca­lo z invazivnostjo celic, in bilo v obratnem sorazmerju z izražanjem StA. V tej raziskavi prever­jamo hipotezo, da je porušeno ravnotežje med CatB in stefinoma povezano bodisi z invazivnos­tjo bodisi s tumorigenostjo izbranih celicnih linij raka dojke. Rezultati. Raziskovali smo izražanje CatB na nivoju mRNA, proteina in njegove aktivnosti v celicnih linijah humanega raka dojke, katerih in vivo tumorigenost narašca v vrstnem redu MCF­7 < MDA-MB468 < MDA-MB231 < MDA-MB435, najbolj invazivna od omenjenih pa je MDA­MB231. Izražanje CatB na vseh treh nivojih je medseboj dobro sovpadalo in narašcalo hkrati z rastoco tumorigenostjo celic. Razmerje med CatB in stefini je bilo na strani CatB v bolj tumori­genih celicnih linijah. Zakljucki. Ker smo predhodno pokazali, da je CatL povezan z invazivnostjo, v tej raziskavi pa ugotovili, da je izražanje CatB povezano s tumorigenostjo istih celicnih linij, menimo, da izražan­je obeh katepsinov v teh linijah ni regulirano na enak nacin. Izražanje CatB, kot tudi razmerja med CAtB in stefinoma, narašca s tumorigenostjo celic, kar morda zrcali podobno situacijo v cloveških tumorjih in vivo. Radio/ Oncol 2003; 37(4): 241-8. Cisteinski in aspartatni proteazi katepsina B in D dolocata invazivnost MCFlOA neoT celic Premzl A in Kos J Izhodišca. Znano je, da imata lizosomska katepsina B in D pomembno vlogo v razlicnih proce­sih, ki vodijo do napredovanja malignih bolezni. V ras-transformiranih MCFlOA neoT celicah oba encima kažeta podobno vezikularno razporeditev, tako v perinuklearnih kot v perifernih citoplazemskih regijah. Rezultati. V nekaterih veziklih je bila s pomocjo konfokalne mikroskopije dolocena njuna kolokalizacija, kar potrjuje odvisno delovanje v proteolitski kaskadi. Za stefin A, endogeni zno­trajcelicni inhibitor cisteinskih proteaz, pa smo pokazali da se ne kolokalizira s katepsinom B in predvidoma ni udeležen pri regulaciji njegove aktivnosti znotraj veziklov. Znotrajcelicna lokalizacija obeh katepsinov se ujema z lokalizacijo razgradnih produktov DQ-kolagena IV, bo­disi v posameznih celicah ali v celicnih sferoidih. Sposobnost katepsinov Bin D, da razgrajujeta kolagen in druge komponente zunaj celicnega matriksa, potrjujejo tudi rezultati testa razgradnje matrigela. Zakljucki. Pokazali smo, da specificna inhibitorja katepsina B ( znotrajcelicni CA-074 Me in zu­najcelicni CA-074) in pepstatin A, inhibitor katepsina D, znacilno zmanjšajo invazijo MCFlOA neoT celic. Naši rezultati tudi kažejo, da v nasprotju z nekaterimi prejšnjimi študijami aktivaci­jski peptid pro-katepsina Dni mitogen na MCFlOA neoT, MCF-7 in HEK-293 celice. Radio/ Oncol 2003(4): 249-55. Doživljanje preobremenjenosti medicinskih sester, radioloških inženirjev in zdravnikov na Onkološkem inštitutu v Ljubljani Škufca Smrdel AC Izhodišca. Izraz sindrom izgorevanja uporabljamo v psihologiji od leta 1974. Predstavlja možni koncni izid delovanja stresogenih dejavnikov. Maslachova ga je opredelila s pomocjo treh os­novnih dimenzij: custvena izcrpanost, depersonalizacija ter zmanjšana osebna vpletenost. V prispevku želimo opredeliti nekatere znacilnosti sindroma izgorevanja, kolikor se kažejo pri zaposlenih na Onkološkem inštitutu v Ljubljani. Subjekti in metode. Uporabili smo vprašalnik poklicnih obremenitev, avtoric Žunter Nagyeve in Kocmurjeve. Sodelovalo je 137 zdravstvenih delavcev iz 4 poklicnih skupin: zdravniki, diplomi­rane oz. višje medicinske sestre, srednje medicinske sestre ter radiološki inžinirji; kar pred­stavlja 38% vseh povabljenih k sodelovanju. Rezultati. Izsledki raziskave kažejo, da sodelujoci iz vseh štirih poklicnih skupin na zelo podoben nacin doživljajo poklicno obremenjenost. V ospredju so obcutja izcrpanosti, razdražljivosti in preoobremenjenosti z delom. Znakov razoseblenja, kot jih opisuje Maslachova, nismo zasledili. Pri srednjih medicinskih sestrah ter radioloških inženirjih je stiska statisticno pogosteje povezana z manjšim osebnim dohodkom ter slabšim materialnim stanjem. Medicinske sestre tudi statisticno pogosteje izražajo namero, da bi zamenjale delovno mesto (51%), ustanovo (57%) ali poklic (47%). Zakljucki. Obremenitve na delovnem mestu že vplivajo na doživljanje in mišljenje sodelujocih v študiji. Izražene znake lahko opišemo kot znake sindroma izgorevanja. Odpirajo pa se tudi no­va vprašanja ter potreba po longitudinalnem pristopu k raziskovanju tega vse bolj pomembne­ga podrocja. Radio/ Oncol 2003; 37(4): 257-66. Pregled razvoja radioterapije na Onkološkem inštitutu v Ljubljani od pricetkov do osemdesetih let 20. stoletja Oklješa Lukic A, Hiibscher K Izhodišca. Clanek obravnava dogajanja na radioterapevtskem oddelku Onkološkega inštituta od njegovega nastanka do osemdestih let dvajsetega stoletja. Podatki o razvoju oddelka so pri­dobljeni predvsem iz publikacij, ki jih je Onkološki inštitut izdajal ob obletnicah svojega delo­vanja, iz razlicnih porocil s strokovnih seminarjev in s pomocjo pogovorov z zaposlenimi na Onkološkem inštitutu. Cilj zgodovinske raziskave je bil kronološko predstaviti razvoj radioter­apevtskega oddelka Onkološkega inštituta, saj so dosedanja zgodovinska porocila razdrobljena in nepopolna. Zakljucki. Ugotovili sva, da si nekateri zapisi iz dosegljivih virov celo nasprotujejo, kljub temu pa je bilo moc iz zbranega gradiva izlušciti najpomembnejše prelomnice v razvoju Onkološkega inštituta in njegovega radioterapevtskega oddelka. Notices Notices submitted far publication slwuld contain a mailing address, phone and/or Jax number and/or e-mail oj a Contact person ar department. Radiation oncology March, 2004 The ISRO international teaching course on »Radiation Oncology in the 21'' Century« will take place in Cape Town, South Africa. See http://www.isro.be Tobacco counters health March 7-11, 2004 The conference »3"1 World Assembly on Tobacco Counters Health (3rd WATCH -2004)« will take place in New Delhi, India. Contact Major General Dr. Avnish K. Varma, World Assembly on Tobacco Counters Health, M 38 A, Rajouri Garden, New Delhi 110027, India; or call +91 11 2544 7395; or fax +91 11 2510 9397; or e-mail cancerak@del6.vsnlnet.in; or see http://www.watch­2000.org Radiotherapy March 7-11, 2004 The ESTRO course »Radiotherapy Treatment Planning: Principles and Practice« will take placc in Dublin, Ireland. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Brachytherapy March 21-25, 2004 The ESTRO course »Modem Brachytherapy Techniques« will take place in Bled, Slovenia. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Radiology Marc/z 26-28, 2004 The seminar »Annual Advanced Topics in CI Scanning: CT Angiography, 3D Imaging, Virtual Imaging« will take place in Los Angeles, California, USA. Contact Conference Coordinator, Office of Continuing medica! Education, John 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.hopkinsme. org/cme Surgical oncology March 31 -April 3, 2004 The 12th ESSO Congress will be held in Budapest, Hungary. See http://www.fecs.be/conferences/esso2004 Radiotherapy April 1-3, 2004 The »2nd ESTRO Meeting on Radiotherapy for Non­Malignant Diseases« will take place in Nice, France. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Oncology April 15-17, 2004 The European Oncology Nursing Society EONS Spring Convention will be held in Edinburg, UK. See http://www.fecs.be/conferences/eons4 Notices 275 Radiotherapy April 18-22, 2004 The ESTRO course »Imaging far Target Volume Determination in Radiotherapy« will take place in Mtinchen, Germany. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http://w,vw.estro.be Radiation oncology May 2-6, 2004 The ESTRO course »Radiation Oncology: a Molecular Approach« will take place in Giardini Naxos, Italy. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http://vvww.estro.be Radiotherapy May 2-6, 2004 The ESTRO course »Dose Determination in Radiotherapy: Beam Characterisation, Dose Calculation and Dose Verification« will take place in Nice, France. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http://wv,,w.estro.be Brachytherapy May 13-15, 2004 The Annual Brachytherapy Meeting GEC-ESTRO will take place in Barcelona, Spain. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http:/ ;,vww.estro. be Radiology jwze 6-8, 2004 The UK Radiological Congress will be held inManchester, U.K. Contact Ms. Rebecca Gladdish, UKRC 2003 Secretariat, PO Box 2895, London WlA SRS, U.K., or call +44(0) 20 7307 1410/20, or fax +44(0) 20 7307 1414; or e-mail conference@ukrc.org.uk/exhibition@ ukrc.org.uk; or see "\\'WW.ukrc.org.uk Radiation oncology June 13-18, 2004 The ESTRO course »Evidence-Bases Radiation Oncology: Methodological Basis and Clinical Application« will take place in Moscow, Russia. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http://,vww.estro.be Radiotherapy ]wze 20-24, 2004 The ESTRO course »IMRT and Other Confarmal Techniques in Practice« will take place in Amsterdam, The Netherlands. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http://www.estro.be Prostate cancer June 27-29, 2004 The ESTRO course »Brachytherapy far Prostate Cancer« will take place in Leeds, U.K .. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http://www.estro.be Oncology July 3-6, 2004 The 18th EACR (European Association far Cancer Research) Congress will be held in Innsbruck, Austria. See http://www.fecs.be/conferences/eacr18 Gynaecological cancer Augusl 26-28, 2004 The ESTRO advanced teaching course on »Brachyherapy far Gynaecological Cancer« will take place in Vienna, Austria. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail infa@estro.be; or see http://W,\'W.estro.be 276 Notices Medica! physics August 29 -September 2, 2004 The ESTRO course »Physics for Clinical Radiotherapy« will take place in Leuven, Belgium. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://wvvvv.estro.be Paediatric oncology September, 2004 The International Society of Paediatric Oncology ­SIOP Annual Meeting will be held in Oslo, Norway. See http://www.siop.nl Radiobiology September 19-23, 2004 The ESTRO course »Basic Clinical Radiobiology« will take place in Lausanne, Switzerland. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Lung cancer September 23-25, 2004 The »9th Central European Lung Cancer Conference« will be offered in Gdansk, Poland. »9th Contact Conference Secretariat, Central European Lung Cancer Conference«, Via Medica, ul. Swietokrzyska 73, 80 180, Gdansk, Poland; or call/fax +48 58 349 2270; or e-mail celcc@amg.gda.pl; or see www.lungcancer.pl 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://v.rww.as­tro.org Therapeutic radiology and oncology October 24-28, 2004 The 23",1 ESTRO Meeting will be held 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://vvww. estro.be Medica! oncology October 29 -November 2, 2004 The 28th ESMO Congress will be held in Vienna, Austria. See http://vv,vw.esmo.org Radiation oncology November 7-12, 2004 The ESTRO course »Evidence-Based Radiation Oncology: Methodological Basis and Clinical Application« will take place in Cyprus. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://,vww.estro.be Radiation oncology November 25-28, 2004 The ISRO international teaching course on »Practical Radiation and Molecular Biology with Mayor Emphasis on Clinical Application« will take place in Chiangmai Thailand. See http://www.isro.be Radiation oncology March, 2005 The ISRO international teaching course on »Palliative Care in Cancer Treatment« will take place in Dar es Salaam, Tanzania. See http://www.isro.be Notices 277 Lung cancer ]11/y 3-6, 2005 The » 11 th World Conference on Lung Cancer« will be offered in Barcelona, Spain. Contact Heather Drew, lmedex, !ne., 70 Technology Drive, Alpharetta, GA 30005 USA; or call +1 770 751 7332, or fax +1 770 751 7334; or e-mail h.drew@ imedex.com, or see w,vvv.imedex.com/calenders/on­cology/htm Radiation oncology September October, 2005 The ISRO international teaching course on »Rational Developments frorn developing to devel­oped Countries« will take place in Lombok, lndonesia. See http://www.isro.be Oncology October 30 -November 3, 2005 The ESTRO 24 / ECCO 13 Conference will take place in Paris, France. 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 As a service to our readers, notices of rneetings or courses will be inserted free of charge. Please send inforrnation to the Editorial office, Radiology and Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Radio/ Oncol 2003; 37(3): 274-7. Ajdukovic R: 3/175-81 Baczuk L: 1/17-22 Bagovic D: 3/175-81 Baranowska A: 3/167-74 Berkmen F: 1/23-7 Bielecki K: 1/17-22 Boric !: 3/155-9 Brnic Z: 1/5-8 Budfkova M: 2/79-88 Burger J: 2/127-31 Ciesielski A: 1/17-22 Car A: 3/195-202 ; 4/225-32 Cemažar M: 1/43-8; 2/101-7 Dimov A: 2/109-13 Dolenc VV: 2/89-99 Filippou DC: 1/1-3 Frangež L: 4/233-40 Gamulin M: 3/175-81 Georgievska-Kuzmanovska S: 1/9-12 Groše! A: 2/101-7 Grzelewska-Rzymowska !: 3/167-74 Haraguchi N: 3/183-6, 4/217-9 Hebrang A: 1/5-8 Herceg T: 3/175-81 Hodacova L: 1/37-42 Homma S: 4/221-4 Homma T: 3/183-6 Horiguchi N: 4/ Horova H: 2/79-88 Horova I: 2/79-88 Hristov D: 2/115-26 Hi.ibscher K: 4/257-66 Ishii Y: 4/217-9 Jakubowski W: 1/17-22; 3/161-5 Jamar B: 2/67-72 Janevik-Ivanovska E: 1/9-12 Juretic A: 3/175-81 Južnic P: 3/141-53 Kagohashi K: 4/217-9 Kaneko Y: 4/217-9 Kocijancic !: 1/13-6 Kodama T: 4/217-9; 4/221-4 Kolodziejczak M: 3/161-5 Kos J: 4/241-8 Kovac V: 4/213-8 Kovkarova E: 1/9-12; 2/109-13 Kranjc S: 2/101-7 Krawczyk M: 2/167-74 Kropivnik M: 2/67-72 Radio/ 011col 2003; 37(3): 278-9. Author Index 2003 Kucan D: 3/175-81 Kwiatkowska S: 3/167-74 Lah TT: 2/89-99 ; 4/233-40 Legan M: 3/187-94 Markovic J: 1/29-35 Maruši<' A: 3/155-9 Miklavcic D: 1/43-8 Musek M: 3/141-53 Naumovski J: 1/9-12; 2/109-13 Nissiotis A: 1/1-3 Noweta K: 3/167-74 Obad-Kovacevi<' D: 1/5-8 Oklješa-Lukic A: 4/257-66 Oven M: 3/141-53 Pipan Ž: 2/101-7 Pižem J: 3/195-202; 4/225-32 Posaric V: 3/155-9 Premzl A: 4/241-8 Rizos S: 1/1-3 Roic G: 3/155-9 Rudolf Z: 1/49-51 Satoh H: 3/183-6; 4/217-9; 4/221-4 Sekizawa K: 3/183-6; 4/ 217-9; 4/221-4 Serša G: 1/ 43-8 ; 2/101-7 Simova N: 1/9-12 Smrdel U: 4/213-6 Soumarova R: 2/79-88 Stavrev P: 2/115-26 Stefanovski T: 2/109-13 Stefar\ski R: 3/161-5 Sudol-Szopiriska !: 1/17-22; 3/161-5 Šeneklova Z: 2/79-88 Škufca Smrdel AC: 4/249-55 Štabuc B: 1/29-35 Tarnowski W: 1/17-22 Trinkaus M: 2/89-99 Ueno T: 4/221-4 Veingerl B: 2/73-8 Vlahovic T: 3/155-9 Vranic A: 2/89-99 Vrlicek K: 3/155-9 Zajc !: 4/233-40 Zeljko Ž: 3/175-81 ZiE;ba M: 3/167-74 Zwitter M: 4/213-6 Živkovic M: 3/175-81 Subject Index 2003 adenocarcinoma: 4/221-4 anus-ultrasonography: 1/17-22 apoptosis: 3/187-94; 4/225-32 -apoptosis-drug effects: 3/195-202 barium sulfate: 2/67-72 bibliometrics: 3/141-53 bleomycin: 1/43-8 bone cysts, aneurismal-ultrasonography: 3/155-9 bone neoplasms-secondary: 4/221-4 brachytherapy: 2/127-31 brain neoplasms secondary: 4/213-6 breast diseases: 1/1-3 breast neoplasms: 1/37-42 ; 4/233-40 -breast neoplasms-surgery: 2/79-88 bronchial neoplasms-seconda1y: 4/217-9 bronchoscopy: 2/109-13 burnout, professional: 4/249-55 carcinoembryonic antigen-blood: 2/73-8 carcinoma, non-small-cell lung: 3/183-6 carcinoma, renal cell-diagnosis: 4/217-9 caspases: 3/195-202 ; 4/225-32 cathepsin B: 4/233-40; 4/241-8 cathepsin D: 4/241-8 cathepsin L: 2/89-99 Chron disease: 2/67-72 cisplatin: 1/43-8; 2/101-7 coli tis, ulcerative: 1/J 7-22 colorectal neoplasrns: 2/73-8 cyclooxygenase inhibitors: 3/187-94 Czech Republic: 1/37-42 disease free survival: 3/183-6 DNA fragmentation: 4/225-32 dose fractionation: 2/115-26 drug delivery systems: 1/43-8; 2/101-7 electroporation: 1/43-8; 2/101-7 enerna: 2/67-72 fernale genital: 1/1-3 follow-up studies: 2/73-8 germinoma: 1/23-7 granuloma, giant cell: 3/155-9 hidradenitis suppurativa-ultrasonography: 3/161-5 intestine, small-radiology: 2/67-72 isotope labelling: 1/9-12 Ljubljana: 4/257-66 lung neoplasms: 3/167-74; 4/213-6; 4/221-4 -lung neoplasrns-diagnosis: 2/109-13 -Jung neoplasms-radiotherapy: 3/183-6 Radio! 011col 2003; 37(4): 278-9. lymphorna, non-Hodgkin: 1/23-7 ; 3/175-81 rnammography: 1/37-42 -mammography, radiation dosage: 1/5-8 manometry: 1/17-22 mastectomy: 2/79-88 medica] oncology: 3/141-53; 4/249-55; 4/257-66 meningioma-pathology: 2/89-99 neoplasms: 3/195-202; 4/225-32 -neoplasms-drug therapy-physiology: 3/187-94 -neoplasms invasiveness: 2/89-99 ; 4/233-40 ; 4/241-8 -neoplasms recurrence, local: 2/79-88 peptic ulcer-radionuclide imaging: 1/9-12 periodicals: 3/141-53 plasminogen activator inhibitor 1: 1/29-35 plasminogen activator inhibitor 2: 1/29-35 pleural effusion-ultrasonography: 1/13-6 pneumonia-rnortality: 3/167-74 polymerase chain reaction: 2/89-99 proctocolectorny, restorative: 1/17-22 prognosis: 1/29-35 ; 2/73-8 ; 2/79-88 prostatic neoplasms-radiotherapy: 2/115-26 protein p53: 3/195-202 pubic bone: 4/221-4 radiation protection: 2/127-31 radiation tolerance: 2/101-7 radioisotopes: 2/127-31 radiology: 3/141-53 radiotherapy, adjuvant: 2/79-88 radiotherapy, conformal: 2/115-26 radiotherapy dosage: 2/115-26 radiotherapy-history-trends: 4/257-66 rectal fistula: 3/161-5 reverse transcriptase, polymerase chain reaction: 2/89-99 sarcoma experimental-drug therapy-blood supply: 1/43-8 skin neoplasms-melanoma: 1/29-35 stress, psychological: 4/249-55 sucralfate: 1/9-12 survival analysis: 2/79-88 ; 4/213-6 telornerase: 2/109-13 testicular neoplasms: 3/175-81 testicular neoplasms, lymphorna: 1/23-7 therrnoluminiscent dosimetry: 1/5-8 thoracic radiography: 1/13-6 tornography, X ray computed: 3/155-9 tuberculosis: 1/1-3 turnour cells, cultured: 4/233-40; 4/241-8 tumour cells cultured-drug effects: 2/101-7 urokinase plasrninogen activator: 1/29-35 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 0 1 51 9 1 2 77 FAKS 0 1 251 81 1 3 ŽR: 50100-620-133-05-1033115-214779 Activity of »Dr. J. Cholewa« Foundation for Cancer Research and Education -A Report f or the Final Quarter of 2003 In the final report far the year 2003 it is important to emphasise some other aspects of the meeting of the Administrative and Supervising Boards of the Dr. J. Cholewa Foundation far Cancer Research and Education Foundation and the report by the Health experts Commission of the Foundation. As was stressed in our previous report, several new members joined the Foundation, research activity with the cooperation of a major pharmaceutical company was discussed, and the graceful and important do­nation to the Foundation by Dr Ana Hinterlechner Ravnik was gratefully acknowl­edged. Other topics were also discussed, especially those concerning the day-to-day ac­tivity of the Foundation and were also presented at the annual meeting of the general assembly. As reported, the reports also included the detailed overview of the Foundation's activity in supporting and sustaining research in cancer in Slovenia. With this in mind, the Foundation will continue to support the regular publication of »Radiology and Oncology« international scientific journal, which is edited, published and printed in Ljubljana, Slovenia. The Foundation will also strive to continue in its ac­tivity to promote cancer biology research, research in cancer epidemiology and clinical cancer research in their many different pathways. The Dr. J. Cholewa Foundation far Cancer Research and Education is optimistic about the prospects in the coming year 2004. Republic of Slovenia is one of the ten accessing countries that will join the European Union in 2004, and this important fact may help the Foundation to gain more infarmation, to expand its existing framework of activi­ties and to find and deal with new challenges in the new economic and political sur­roundings. Ali of these changes will probably have a serious impact on the cancer re­search and education activity in Slovenia and will present it with new challenges. Ali this may help the Foundation to find the ways to collaborate with similar institutions ali over Europe and elsewhere, and the Foundation may in this way further expand its scope and goals. Andrej Plesnicar, MD, MSc Tomaž Benulic, MD, MSc Borut Štabuc, MD, PhD kapsule raztopina za intravensko infundiranje Kontraindikacije: Preobtutljivost za flukonazol, pomožne sestavine zdravila in za druge azole. Sotasno jemanje flukonazola s terfenadinom ali cisapridom. Stranski ucinki: Lahko se pojavijo slabost, napenjanje, bruhanje, bolei'ine v trebuhu, driska. Možni so glavobol, krci in alopecija. Zelo redke so preobcutljivostne reakcije. Pri bolnikih s hudimi glivicnimi obolenji lahko pride do levkopenije, trombocitopenije, povecane aktivnosti jetrnih encimov ter hujše motnje v delovanju jeter. Oprema in nacin izdajanja: 7 kapsul po 50 mg, 28 kapsul po 100 mg, 1 kapsula po 150 mg -samo na zdravniški recept. 1 viala s 100 ml raztopine za intravensko infundiranje {200 mg[100 ml) -uporaba samo v bolnišnicah. Datum priprave besedila: marec 2003 • sistemske kandidoze • mukozne kandidoze • vaginalna kandidoza • kriptokokoze • dermatomikoze • preprecevanje kandidoze Ucinkovit antimikotik, ki ga bolniki dobro prenašajo. Sanolabor zastopa naslednja podjetja 1 v Kottermann (Nemcija): INTEGRA BIOSCIE CES (Svica): laboratorijsko pohištvo, t laboratorijska oprema za mikrobiologijo, varnostne omare za kisline, biologijo celic, molekwlarno biologijo luge, topila, pline in strupe, inl biotehnologijo ventilacijska tehnika in digestorji CORNING (ZDA): DAKO (Danska): specialna laboratbrijska plastika testi za aplikacijo v imunohistokerniji, za aplikacijo v imu.ologiji, mikro­patologiji, mikrobiologiji, virologiji, biologiji-virologiji, ipd., fehanske eno­mono-in poliklonalna protitelesa in veckanalne pipete in nastavki l SVANOVA Biotech (Švedska): EVL (t,lizozemska): Elisa testi za diagnostiko v veterini diagnosticni testi za uporabo v veteriharski medicini NOVODIRECT BIOBLOCK (Francija): .. 1 v •• kompletna oprema in pripomocki HURNER (Nemc11a): za delo v laboratoriju ventiibcijska tehnika GFL (Nemcija): CSL . Biosciences: laboratorijski aparati, omare in diagnosticni testi za uporabo skrinje za globoko zamrzovanje v veteriharski medicini ANGELANTONI SCIENTIFICA (Italija): BIOM.RICA (ZDA): hladilna tehnika in aparati za laboratorije, hitri testi h diagnostiko, transfuzijo, patologijo in sodno medicino 1 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 industriji;aparati za za laboratorijsko vzrejo živali -kletke procesno kontrolo in ko1 trolo kvalitete ROSYS -ANTHOS (Avstrija): fotometri, avtomatski pralni sistem za mikrotitrine plošce LABORMED d.o.o. LABORMED, razstavni salon Zg. Pirnice 96/c Bežigrajski dvor SI -1215 Medvode Periceva 29, Ljubljana Tel.: (0)136214 14 Tel.: (0)1 436 49 01 Fax: (0)136214 15 i n f o @ 1 o bor m e d . si Fax: (0)1 436 49 05 w w w abormed ' s AstraZeneca ·. Vaš partner pri zdravljenju raka dojke in prostate Casodex Zoladex®LA 10.8mg goserelin AstraZeneca UK Limited, Podružnica v Sloveniji, Einspielerjeva 6, Ljubljana www.astrazeneca.com PE: Stritarjeva 5, 4000 Kranj, Slovenija tel.: (0)4/ 2015 050, fax: (0)4/ 2015 055 e-mail: kemomed@siol.net, KEMOMED www.kemomed.si o ¦gmmomo Promega IZDELKI ZA MOLEKULARNO BIOLOGIJO DOKUMENTACIJA PLASTIKA ZA CELICNE KULTURE IN ANALIZA GELOV SKRINJE IN HLADILNIKI ..,...m-,u, --:-e--:-rv---:a---lt .,-,c,a BIOHfT biolob1 Biosciences ELEKTRONSKE IN MEHANSKE AVTOMATSKE PIPETE DIAGNOSTIKA SEKVENATORJI MIKOPLAZEM IN LEGIONEL 3rd Conference on Experimentaland Translational Oncology Kranjska gora, Slovenia, March, 18-21, 2004 Organised by: Tamara Lah, Gregor Serša and Janko Kos Topics: • Mechanisms of tumour progression • Tumour markers • Delivery systems in cancer therapy • New drugs and therapeutic targets Location: Hotel Lek 4280 Kranjska Gora, Sloveniahttp://www.hotel-lek.si Correspondence: Conference Secretary: Phone: +386 1 423 1867\ 1' O nf erenca O Fax: +386 1 423 50382., . T1, 0l„ Email: milena.kiso.ec@nib.si ·;._,it'iel: )' .S http://www.oplto-i.si'lradiolog/rno.html 1.r. , :X) Organised under patronage ]r1'l 1/;f. ( of Association of Radiology and Oncology .P.! ;IJ: . ct.., on ).6 i' .{)p' ?c 6-...(,(;\ - Ľ.i European Society tor Therapeutic Radiology and Oncology The GEC-ESTRO Teaching course on Modern Brachytherapy Techniques Bled, Slovenia 21-25 March, 2004 Course Director: Vice Director: Teaching Staff: Guest Lecturer: Local Organiser: Course Co-ordinator: Grand Hotel Toplice E. Van Limbergen J.J. Mazeron C. Haie-Meder, J.J. Mazeron, R. Potter, E. Van Limbergen, J. Venselaar A.Gerbaulet H. Lesnicar, Matjas Zwitter, Primos Strojan, Janez Burger. Roselinne Nisin ESTRO Office, Ave. E. Mounierlaan 83, 1200 Brussels, Belgium 'il +32.2.775.93.40 -1!1 +32.2.779.54.94 -e-mail: roselinne.nisin@estro.be