RadiologtJ and Oncology is a journal devoted to publication of original contributions in diagnostic and interventional radiologij, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. Editor-in-Chiefo Gregor Serša Ljubljana, Slovenia Executive Editoro Viljem Kovac Ljubljana, Slovenia Editor-in-Chief Emerituso Tomaž Benulic Ljubljana, Slovenia Editorial boardo Marija Auersperg Be1aFornet Maja Osmak Ljubljana, Slovenia Budapest, Hungary Zagreb, Croatia Nada Bešenski Tullio Giraldi Branko Palcic Zagreb, Croatia Trieste, Italy Vancouver, Canada Karl H. Bohuslavizki Andrija Hebrang JuricaPapa Hamburg, Germany Zagreb, Croatia Zagreb, Croatia HarisBoko Ltiszl6 Horvath Dušan Pavcnik Zagreb, Croatia Pecs, Hungary Portland, USA Nataša V. Budihna Berta Jereb Stojan Plesnicar Ljubljana, Slovenia Ljubljana, Slovenia Ljubljana, Slovenia Marjan Budihna Vladimir Jevtic Ervin B. Podgoršak Ljubljana, Slovenia Ljubljana, Slovenia Montreal, Canada Malte Clausen H. Dieter Kogelnik Jan C. Roos Hamburg, Gennany Salzburg, Austria Amsterdam, Netherlands Christoph Clemm Jurij Lindtner Slavko Šimunic Miinchen, Germany Ljubljana, Slovenia Zagreb, Croatia Mario Corsi Ivan Lovasic Lojze Šmid Udine, Italy Rijeka, Croatia Ljubljana,Slovenia Christian Dittrich Marijan Lovrencic Borut Štabuc Vienna, Austria Zagreb, Croatia Ljubljana, Slovenia Ivan Drinkovic LukaMilas Andrea Veronesi Zagreb, Croatia Houston, USA Gorizia, Italy Gillian Duchesne Metka Milcinski Živa Zupancic Melbourne, Australia Ljubljana, Slovenia Ljubljana, Slovenia Publishers Slovenian Medical Association -Slovenian Association of Radiology, Nuclear Medicine Society,Slovenian Society for Radiotherapy and Oncologtj, and Slovenian Cancer SociettJCroatian Medical Association -Croatian Society of Radiology Affiliated with Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups oj S.I.R.M.(Italian Society oj Medica/ Radiologij) Correspondence address Radiologij and OncologtJ Institute of OncologyVrazov trg4 SI-1000 LjubljanaSlovenia Tei: +386 61 132 00 68 Tel/Fax: +386 61133 74 10o Reader far English Olga Shrestha Design Monika Fink-Seršao Key words Eva Klemencico Secretaries Milica HarischBetka Savski Printed by Imprint d.o.o., Ljubljana, Slovenia Published quarterly in 1000 copies Bank account number 50101 678 48454Foreign currency account number 50100-620-133-27620-5130/ 6 NLB -Ljubljanska banka d.d. -Ljubljana Subscription fee far institutions 100 $, individuals 50 $Single issue far institutions 30 $, individuals 20 $ The publication of this journal is subsidized by the Ministry oj Science and Technologtj of the Republicof Slovenia. According to the opinion oj the Government of the Republic oj Slovenia, Public Relation and MediaOffice, the journal Radiology and Oncology is a publication of informative value, and as such subject totaxation by 5% sales tax. Indexed and abstracted by: BIOMEDICINA SLOVENICACHEMICAL ABSTRACTSEXCERPTA MEDICA This journal is printed on acid-free paper Radiology and Oncologtj is now available on the internet at: http:/www.onko-i.si/radiolog/rno.htmo FOREWORD The international conference LIFE SCIENCES '97 & 2nd SLOVENIAN-CROATIAN MEETING ON MOLECU­LAR ONCOLOGY TODAY was held at Gozd Martuljek, Slovenia, October 16-19, 1997. This confer­ence, one in a series of tradicional Life Sciences conferences was, devoted to the topic of Bio­ physics and Biology oj Tumors.o The conference gathered 180 scientists from Slovenia and abroad working on basic as well as clinical aspects of cancer. There were three key-note lectures, 55 oral presentations and 65 poster presentations. The main topics touched upon the issues of molecular oncology, electro­chemotherapy, biological diagnostic and prognostic factors, immune system and biological response modifiers, experimental oncology, membranes, MR imaging and EPR spectroscopy, and new technologies. This special issue of Radiology and Oncology is bringing selected scientific reports presented at LIPE SCIENCES '97 & 2nd SLOVENIAN-CROATIAN MEETING ON MOLECULAR ONCOLOGY TODAY to the sci­entific community as peer-reviewed papers. The issue is organized according to the topics of the submitted papers into several sections. It starts with molecular oncology, followed by the papers on experimental oncology, MR imaging and EPR spectroscopy, membranes and radiophysics. The organization of the conference and this special issue of Radiology and Oncology were made possible by financial assistance of Ministry of Science and Technology of the Republic of Slove­nia and other sponsors. We would like to thank the key-note lecturers, invited speakers, partici­pants, all chairmen and co-chairmen, and those who reviewed the manuscripts. We hope that this compilation of papers presented at the conference will contribute to continua­tion and intensification of research in preclinical oncology, as well as its application in clinical practice. Gregor SeršaoDamijan Miklavcico f. LIFE SCIENCES '97 & 2nd SL0VENIAN-CR0ATIAN MEETING ON M0LECULAR ONC0L0GY T0DAY Gozd Martuljek, Slovenia, 0ctober 16-19, 1997 Organised by: Slovenian Biophysical Society & Institute of Oncology, Ljubljana In co-operation with: Physiological Society of Slovenia, Slovenian Genetic Society, Slovenian Immunological Society, Slovenian Medica! Association, Oncology Section, Slovenian Pharmaco­logical Society, Slovenian Society for Medica! and Biological Engineering, Society of Stereology and Quantitative Image Analysis, Faculty of Electrical Engineering, University of Ljubljana, Institute Rudjer Boškovic, Zagreb Organising comrnittee: Gregor Serša, president; Damijan Miklavcic, secretary; Janez Škrk, Maja Cemažar, Tomaž Jarm, Jani Pušenjak, Srdjan Novakovic Scientific comrnittee: T. Bajd (SLO), D.J. Chaplin (UK), M. Carman-Kržan (SLO), F. Demšar (SLO), N.J.F. Dodd (UK), P. Dovc (SLO), I. Eržen (SLO), D. Gabel (D), D. Glavac (SLO), R. Golouh (SLO), R. Heller (USA), M. Kirschfink (D), R. Komel (SLO), V. Kotnik (SLO), T. Lah (SLO), J. Lindtner (SLO), L.M. Mir (F), E. Neumann (D), M. Osmak (CRO), Z. Rudolf (SLO), M. Schara (SLO), F. Sevšek (SLO), G. Stanta (I), S. Svetina (SLO), M. Šentjurc (SLO), B. Štabuc (SLO), A. Štalc (SLO), M. Štefancic (SLO), M. Us-Kraševec (SLO), T. Valentincic (SLO), L. Vodovnik (SLO), R. Zorec (SLO), N. Zovko (CRO) Sponsored by: Ministry of Science and Technology of the Republic of Slovenia, Slovenian Acacl­emy of Sciences and Arts, Institute for Rehabilitation of the Republic of Sloveni3c, Muscular Dys­trophy Association of Slovenia, The Bioelectrochemical Society, Electroinstitute Milan Vidmar, Dr. J. Cholewa Foundation, Adriamed d.o.o. -Hoffmann La Roche Diagnostics Systems, Becton Dickinson -Kemomed d.o.o., BIA d.o.o., Birografika BORI d.o.o., Bristol-Myers Squibb d.o.o., DZS d.d., Karanta d.o.o., Kemofarmacija d.d., Krka d.d., Labormed d.o.o., Lek d.d., Ljubljanske mlekarne d.d., Mikro + Polo d.o.o., PETROL d.d., SALUS d.d., STOP Slovenian Trave! Agency d.d.t CONTENTS MOLECULAR ONCOLOGYo Digression on membrane electroporation and electroporative delivery of drugsandogenes Neumann E, Kakorin So7 Molecular alterations induced in drug-resistant cells OsmakMo19 Genetic polymorphisms of xenobiotic metabolizing enzymes in humancolorectal cancer Dolžan V, Ravnik-Glavac M, Breskvar Ko35 In vitro generation of cytotoxic T lymphocytes against mutated ras peptidesJuretic A, Šamija M, Krajina Z, Eijuga D, Turic M, Heberer M, Spagnoli CCo41 Differential expression of Bcl-2 protein in non-irradiated or UVC-irradiatedmurine myleoid (ML) cells Popovic-Hadžija M, Poljak-Blaži Mo47 EXPERIMENTAL ONCOLOGYo Direct delivery of chemotherapeutic agents for the treatment of hepatomasand sarcomas in rat models Pendas S, Jaroszeski M], Gilbert R, Hyacinthe M, Dang V, Hickey ], Pottinger C,Illingworth P, Heller Ro53 Longitudinal study of malignancy associated changes in progressive cervicalodysplasia Fležar M, Lavrencak], Žganec M, Us-Krašovec Mo65 Image cytometry analysis of normal buccal mucosa smears: influence of smokingand sex related differences Lavrencak], Fležar M, Žganec M, Us-Krašovec Mo71oThe number of mitoses in simple and complex type carcinomas of themammary gland in dogs ]untes Po77 Influence of UV-B radiation on Norway spruce seedlings (Picea abies (L.) Karst.)oBavcon], Gogala N, Gaberšcik Ao83 MR IMAGING AND EPR SPECTROSCOPYo Monitoring drug release and polymer erosion from therapeutically usedbiodegradable drug carriers by EPR and MRI in vitro and in vivoMiideroKoBone marrow toxicity and antitumor action of adriamycin in relation to theantioxidant effects of melatonin Rapozzi V, Perissin L, Zorzet S, Comelli M, Mavelli I, Šentjurc M, Pregelj A,oSchara M, Giraldi ToDiffusion-weighted magnetic resonance imaging in the early detection of tumorresponse to therapy Dodd N]F, Zhao S, Moore ]Vo 89 95 103o MEMBRANESoPlasma membrane fluidity alterations in cancerous tissueŠentjurc M, Sok M, Serša GoOn mechanisms of cell plasma membrane vesiculation Kralj-Iglic V, Batista U, Hiigerstrand H, Iglic A, Majhenc ], Sok Mo 109o119 RADIOPHYSICSoTertiary collimator system for stereotactic radiosurgery with linear acceleratorCasar Bo 125 SLOVENIAN ABSTRACTSo 129 NOTICESo137 revtew Digression on membrane electroporation and electroporative delivery of drugs and genes Eberhard Neumann and Sergej Kakorin Physical and Biophysical Chemistry, Faculty oj Chemistry,University ofBielefeld,Germany Introduction A new kind of cell surgery has recently beendeveloped by a combination of drugs andgenes with electric voltage pulses. The novelsurgery operates on the level of the cell mem­brane and uses membrane electroporation asa scalpel to greatly facilitate the penetrationof drugs, especially chemotherapeuticals andgenes through electroporated membraneopatches of a cell. The phenomenon of membrane electropo­ration (ME) is methodologically an electricotechnique which renders lipid membranesporous and permeable, transiently andoreversibly, by external high voltage pulses. Itis of practical importance that the primarystructural changes induced by ME, conditionthe electroporated membrane for a variety ofsecondary processes such as, for instance, thepermeation of otherwise impermeable sub­stances. The structural concept of ME was derivedofrom ftmctional changes; explicitly from theo Key words: membrane electroporation; gene trans­fer; drug delivery; lipid vesicle Correspondence to: Prof Eberhard Neumann, Physical and Biophysical Chemistry, Faculty of Chemistry, University of Bielefeld, P. O. Box 100 131, D-33501 Bielefeld, Germany, Fax: +49 521 106 29 81; E-mail: eberhard.neuman@post.uni-bielefeld.de electrically induced permeability changes,indirectly judged from the partial release ofintracellular components1 or from the uptakeof macromolecules such as DNA.2,3 The elec­trically facilitated uptake of foreign genes iscalled the direct electroporative gene transferor electrotransformation of cells. Similarly,electrofusion of single cells to large syncytia4 and electroinsertion of foreign membraneproteins5 into electroporated membranes arebased on electrically induced structural cha­nges of ME. For the tirne being the method of ME iswidely used to manipulate all kinds of cells,organelles and even intact tissue. ME isoapplied to enhance iontophoretic drug trans­port through skin, see, e.g., Pliquett et al.6 or to introduce chemotherapeuticals into can­1 cer tissue, an approach pioneered by L. Mir.7oIt is fair to say that, despite the attractivefeatures of the various ME phenomena, thedetails of the molecular mechanism of MEoitself are not yet known. On the same line,the mechanisms of various secondary pro­cesses coupled to ME have not been clarifiedyet. Therefore, reliable directives can not begiven for specific analytical and cell-manipu­lative applications. However, model studieson cells and lipid vesicles have providedsome insight into guidelines for the planningo Neumann E and Kakorin S of trials and for the optimization of existing procedures. In this review a few fundamental aspects are selected which are important to consider when ME is to be applied to cells and tissue. We shall discuss some details of electric field induced structural changes leading to chemi­cally specific pore states in the membrane (Figure 1). The rigorous thermodynamic and kinetic analysis of electroporation > Eu the formation of aqueous pores is strongly favored in the presence of a cross-membrane potential dif­ference Llcprn induced by the interfacial Maxwell-Wagner polarization. We use here the approximation Ern -LlcpnJd for the mem­ = brane field valid for the small pores of low to obtain the mean pore radius rp from the field dependence of K or of y (the degree of poration). We recall that the actual data always reflect 8 angle averages (Figure 1).17 Since [P] defines a surface area So= N/mi of NP ef · pores with maximally SP = N P ·:rtor;, the fraction of porated area is given by 1 [P] SP l K(8) f =o--=o-=o-J---sm8d8 conductance.15 The stationary value of the induced poten­ S. 2rr ol+K(8) . (12) tial difference Llcprn in the spherical mem­brane of a vesicle of radius a is dependent on the positional angle 8 between the membrane site considered and the direction of the exter­nal field vector E (Figure 1): Llcprn = -23 a · E · f('A.111 )pos8I (10) The conductivity factor ('A.rn) can be generally expressed in terms of a and d and the con­ductivities "-rn, "-i, 'A.0 of the membrane, the cell (vesicle) interior and the external solu­tion, respectively. 2° Commonly, d << a and "-rn « 'A.0 , "-i such that f('A.rn) = [1 + "-rn (2 + Ai / 'A.0) / (2 \ d/a)J-1. For "-rn "" O, f('A.rn) = 1. It is readily seen from Eq. (10) that the field amplification is quantified as E= -Llcp/ d = (3 / 2)(a / d) · E · fU,.)1cos8I , where the ratio a/ d is the geometric amplifi­cation factor of interfacial membrane polar­ization. The final expression of the electrical ener­gy term (at 8) is obtained by sequential inser­ 111 111 111 17 tions and integration of Eq. (8). Explicitly16, , where f P is the 8-average of y = K(8) / (1 + K(8)), with [P rnaxl = [C0]. It is found that f P is usually very small21 , e.g., f:S 0.003, i.e. p 0.3%. This value certainly corresponds to a small pore density, reguired for a low value of "-rn· Curvature energy term The explicit expression for the curvature energy term of vesicles of radius a is given by:16 f 11,. dH = NAJ.r -.c}dHo"" 64-NA ·:rt2a·K·r;o-c;;o_(!._+ H.1 . ,,,,o_ d a 2:rr,. (13) Note that the aqueous pore part has no curvature, hence the curvature term is .P -.c = -.C . H is the curvature inclusively the spontaneous curvature H0. If H0 = O, then in the case of spherical vesicles we have H 1/a. H.1 is the electrical part of the spon­ = taneous curvature16, K is the elastic module, a is a material constant, 1;; is a geometric fac­ tor characterizing the pore conicity. 9 It is noted that the molar curvature term f ,1,(3 dH can be as large as 10 RT. 8 There­fore, for small vesicles or small organelles and cells, the curvature term is very impor­tant for the energetics of ME. Eq. (13) shows that the larger the curva­ture and the larger the H.1 term, the larger is the energetically favorable release of Gibbs energy during pore formation. Strongly curved membranes appear to be electroporat­ed easier than planar membrane parts. Pore edge energij and surface tension In Eq. (7), y is the line tension or pore edge energy density and L is the edge length, r is the surface energy density and S is the pore surface in the surface plane of the mem­brane. Explicitly, for cylindrical pores we obtain the pore edge energy term: L L J,1,ydLt= NAJ(yP -yc}dL= 2nNA ·y ·rP (14) o o where Yp = y since Yc = O (no edge) and L = 2nris the circumference line. p The surface pressure term f ,1,rdS = NAJ (rp -rc) dS (15) is usually negligibly small because the differ­ence in r between the states P and C is in the order of s 1 mNm-1; see, e.g., Steiner and Adam22 . We recall that the conventional chemical term covering concentration changes of lipids and water in the pore edge and pore volume is given by ,1„G0 = -RT In K0. Apply­ing this relation and Eqs. (11) -(14) to Eq. (6), we obtain the explicit expression: 1 H'' f i'.. M dE K-K11 ·exp --2m ·y-f1· ( -+-11 ) } +. ] (16) RT r a 2rra RT l N" { Experimentally, K can be determined from the fraction y of the porated surface as a function of the field strength. It remarked that K is exponentially dependent on the square of E, see Eq. (11). Therefore, the dependence of K or y on E is much stronger than linear such that the plot of y or f P versus E (see Figure 2) shows an initial part of almost no change in y. This "lag phase" is very frequently qualified to indicate a thresh­old of the field strength. The thermodynamic analysis shows that ME is highly non-linear, yet continuous in E. Thus the structural aspect inherent in our membrane electropo­ration model is not associated with a thresh­old of the field strength. However, the pore density necessary to permit a secondary phe­nomenon such as massive ion conduction, release and uptake of substances, may very well be operationally described in terms of a threshold field strength (Figure 2). The chemical thermodynamical concept has turned out to be applicable to the analy­sis of ME of vesicles, cells and organelles. For instance, it has been found that the station­ary value of the mean pore radius within the pulse duration of 10 µs is rather small: .' = 035 ± O.OS nm, just permitting free pas­sage of small ions.17 At higher field intensi­ties and longer pulse durations the pore radius may increase up to f P s 1.2 nm, lead­ing to the influx of large drug-like dye mole­cules into the cell interior, 23 see below. Electroporative cell deformations Using lipid vesicles filled with electrolyte as a model for cells and organelles, it has been shown that ME is causing appreciable increases in the rate and extent of electro­ 21 mechanical shape deformations10,. The overall shape deformation under the field­induced Maxwell stress is associated with several kinetically distinct phases. In the case of vesicles, the initial very rapid phase in the µs tirne range is the electroporative elonga­ Neumann E and Kakorin S tion from the spherical shape to an ellipsoid in the direction of the field vector E. In this phase, called phase O (Figure 3), there is no measurable release of salt ions, hence the internal volume of the vesicle remains con­stant. Elongation is therefore only possible if, in the absence of membrane undulations in Rather, the reorganization of the lipids in the pore wall leads to a local cluster structure defining an aqueous pore which has a larger electric dipole moment, and thus a higher orientational order than the equivalent space of lipids. small vesicles, the membrane surface can increase by ME. The formation of aqueous pores means entrance of water and increase in the membrane surface. In the second, slower phase (ms tirne range), called phase I (Figure 3), there is efflux of salt ions under Maxwell stress through the electropores created in phase O, leading to a decrease in the vesicle volume under practically constant membrane surface (including the surfaces of the aqueous pores). The kinetic analysis of the volume decrease yields the membrane bending rigidity K = 3.0 ± 0.3 x 10-20 J.21 At the field strength E = 1.0 MV m-1 and in the range of pulse duration 5 s tJms s; 60, the number of water-permeable electropores is found to be NP = 35 ± 5 per vesicle of radius a = 50 nm, with mean pore radius r= 0.9 ± 0.1 nm. p The kinetic analysis developed for vesicles is readily applied to cell membranes. The results aim at physical-chemical guidelines to optimize the membrane electroporation tech­niques for the direct transfer of drugs and genes into tissue cells. Interestingly, there is appreciable efflux of salt ions in the after-field period lasting sev­eral seconds. This very important observa­tion suggests that there are not only long­lived open pores but also that the structural basis of the longevity cannot be simple hydrophobic (HO) pores (Figure 1). More complicated higher order structures must have been created by ME, which face higher activation barriers for annealing in the absence of the electric field. A candidate for the higher order structure is the so called hydrophilic (HI) pore. In this sense, ME can hardly be called a breakdown phenomenon. Electroporative transport of drugs and genes Contrary to the electroporative transport of small salt ions, the transport kinetics of larg­er macromolecules such as drug-like dyes and DNA, reflects transient interactions with larger pores. The pore size seems to indicate the size of the macromolecule or parts of it which are transiently located within the membrane during the transport process. For instance, the mean pore radius fP = 1.2 ± 0.1 nm, derived from the analysis of the trans­port of the drug-like dye Serva blue G (SBG), appears to be rather large, although it is in line with previous estimates of possible pore sizes.15 An open pore of this size should lead to a significant increase in the transmem­brane conductivity, reducing locally the transmembrane voltage,16 eventually causing leakage of cell components and finally cell death. It should be noted that the detection of the dye-permeable pore state is only possi­ble when the dye molecules are (interactively) passing through the pore (Figure 4). There­ ., . o v oi.o Figure 3. Sequence of events in the electromechanical deformation of a membrane system of unilamellar lipid vesicles or biological cells. Phase O: Fast (µs) membrane electroporation rapidly coupled to electroporative defor­mation at constant volume and slight (0.01 -0.3%) increase in membrane surface area. Phase I: Slow (ms ­min) electromechanical deformation at constant mem­brane surface area and decreasing volume due to efflux of the interna! solution through the electropores. Theory and application oj membrane e/ectroporation fore, the pore is temporarily occluded by the dye molecule, reducing the conductivity for small ions compared with a dye-free pore of the same radius. Similar arguments apply for the leak pores associated with the transport of DNA. 24,25 Dye uptake by mouse B cells As an example for the transport of dye-like drugs, the color change of electroporated intact FcyR-mouse B cells (line IIAl.6) after direct electroporative transfer of the dye SBG (Mr 854) into the cell interior is shown to be prevailingly due to diffusion of the dye after the electric field pulse. Hence, the dye trans­port is described by the First Fick' s law where, as a novelty, time-integrated flow coefficients were introduced.23 The chemical­kinetic analysis suggests three different pore states (P) in the reaction cascade (c..,,p1 ..,,p2 ..,, P 3) to model the sigmoid kinetics of pore for­mation as well as the biphasic pore resealing. The rate coefficient for pore formation kp is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm-1 and tE = 200 µs, kp = 2.4 ± 0.2 x 103 s-1 at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are kf = 1.0 ± 0.1 x 10-2 s-1 and po = 2 cm s-1, respectively. (KD ) D+ ( .) . o•(.) (k pen ) The maximum value of the fractional sur­face area of the dye-conductive pores is 0.035 ± 0.003 % and the maximum pore number is NP = 1.5 ± 0.1 x 105 per average cell. The dif­fusion coefficient for SBG, D = 10-6 cm2 s-1, is slightly smaller than that of free dye diffu­sion indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are f (P) = 0.7 ± 0.1 nm, f (Po) = 1.0 ± 0.1 nm, f 12 (P 3) = 1.2 ± 0.1 nm, respectively. The resealing rate coefficients are k_2 = 4.0 ± 0.5 x 10-2 s-1 and k_3 = 4.5 ± 0.5 x 10-3 s-1 , independent of E. At zero field, the overall equilibrium con­stant of the pore states (P) relative to closed membrane states (C) is K. = [(P)] / [C] = 0.02 ± 0.002, indicating 2.0 ± 0.2 % water associated with the lipid membrane.23 Finally, the results of SBG cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs which are similar in structure to SBG, for instance, bleomycin successfully used in the new disci­pline of electrochemotherapy. 7 Kinetics oj DNA up take by yeast cells In a detailed kinetic study it was found that the direct transfer of plasmid DNA (YEv 351, 5.6 kbp, supercoiled, Mr "" 35 · 106 ) by (ko -(k ) f ) · b · DP ==>PD. Dlll . Dr Figure 4. Scheme for the cgupling of the binding of a macromolecule (D), either a dye-like drug or DNA (described by the equilibrium constant Kil of overall binding), electrodiffusive penetration (rate coefficient k) into the outer sur­ pen face of the membrane and translocation across the membrane, in terms of the Nernst-Planck transport coefficiant ( k7 ) ; and the binding of the internalized DNA or dye molecule (Din) to a celi component b to yield the interaction complex D . b as the starting point for the actual genetic celi transformation or celi coloring, respectively. Neumann E and Kakorin S membrane electroporation of yeast cells ( Sac­charomyces cerevisiae, strain AH 215 ) is basi­cally due to (electro)diffusive processes.t26 The rate-limiting step for the cell transforma­tion, however, is a bimolecular DNA binding interaction in the cell interior. Both the adsorption of DNA, directly measured with 32P-dC DNA, and the number of transfor­mants are collinearly enhanced with increas­ing total concentrations [Dtl and [Cat] of DNA and of Ca2+ , respectively. At [Cat] = 1 mM, the half-saturation or equilibrium bind­ing constant is K D = 15 ± 1 nM at 293 K (20°C). The optimal transformation frequen­cy is TF opt = 4.1 ±0.4 x 10-5 if a single expo­nential pulse of initial field strength E0 = 4 kV cm-1 and decay tirne constant ,;E = 45 ms is applied at [Dtl = 2.7 nM and 108 cells in 0.1 ml. The dependence of TF on [Cat] yields the equilibrium constants K." = 1.8 ± 0.2 mM (in the absence of DNA) and K." = 0.8 ± 0.1 mM (at 2.7 nM DNA) well comparable with K.a = 23 ± 0.2 mM and K.a = 1.0 ± 0.1 mM derived from electrophoresis data.26 In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At E0 = 4.0 kV cm-1 and T = 293 K, the flow coeffi­cient of DNA through the porous membrane patches is k. = 7.0 ± 0.1 x 103 s-1 and the elec­trodiffusion (D) of DNA is about 10 times more effective than simple diffusion (D0): the diffusion coefficient ratio is D / D0 = 10.3. The mean radius of these pores is r= 0.39 ± p 0.05 nm and the mean number of pores per cell (diameter 5.5 µm) is NP = 2.2 ± 0.2 x 104• The maximum membrane area which is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023 ± 0.002 % of the total cell surface. The surface penetration is followed either by fur­ther electrodiffusive, or by passive (after field) diffusive, translocation of the inserted DNA into the cell interior. For practical purposes of optimum trans­ formation efficiency, 1 mM Ca2+ is necessary for sufficient DNA binding and the relatively long pulse duration of 20 -40 ms is required to achieve efficient electrodiffusive transport across the cell wall and into the outer surface of electroporated cell membrane patches. Acknowledgments We thank the Deutsche Forschungsgemein­schaft for grant Ne 227 /9-2 to E. Neumann. References l. Neumann E, Rosenheck K. Permeability changes induced by electric impulses in vesicular mem­branes. J Membrane Biol 1972; 10: 279-90. 2. Wong TK, Neumann E. Electric field mediated gene transfer. Biophys Biochem Res Commun 1982; 107: 584-7. 3. Neumann E, Schaefer-Ridder M, Wang Y, Hof­schneider PH: Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1982; 1: 841-5. 4. Neumann E, Gerisch G, Opatz K. Cel! fusion induced by electric impulses applied to dic­tyostelium. Naturwissenschaften 1980; 67: 414-5. 5. Mouneimne Y, Tosi PF, Gazitt Y, Nicolau C. Elec­tro-insertion of xenoglycophorin into the red blood cel! membrane. Biochem Biophys Res Com­mun 1989; 159: 34-40. 6. Pliquett U, Zewert TE, Chen T, Langer R, Weaver JC. Imaging of fluorescent molecule and small ion­transport through human stratum-corneum dur­ing high-voltage pulsing-localized transport regions are involved. Biophys Chem 1996; 58: 185­204. 7. Mir LM, Orlowski S, Belehradek JJr, Teissie J, Rols MP, Serša G, Miklavcic D, Gilbert R, Heller R. Bio­medical applications of electric pulses with special emphasis on antitumor electrochemotherapy. Bio­electrochem Bioenerg 1995; 38: 203-7. 8. Ti:insing K, Kakorin S, Neumann E, Liemann S, Huber R. Annexin V and vesicle membrane elec­troporation. Eur Biophys J 1997; 26: 307-18. 9. Seifert U, Lipowsky R. Morphology of Vesicles. In Lipowsky R, Sackmann E, ed. Structure and Dynamics of Membranes 1A. Amsterdam: Elsevier; 1995: 403-63. Tlzeory and applicntion of membrane electroporation 10. Kakorin S, Redeker E, Neumann E. Electropora­tive deformation of salt filled lipid vesicles. Eur Biophys J 1998; 27: 43-53. 11. Winterhalter M, Klotz K-H, Benz R, Arnold WM. On the dynamics of the electric field induced breakdown in lipid membranes. IEEE Trans Ind Appl 1996; 32: 125-8. 12. Chang C. Structure and dynamics of electric field­induced membrane pores as revealed by rapid­freezing electron microscopy. in Chang C, Chassy M, Saunders J, Sowers A. ed. Guide to electropora­tion and eleclrofusion. San Diego: Academic Press, 1992: 9-28. 13. Hibino M, Itoh H, Kinosita K. Time courses of celi electroporation as revealed by submicrosecond imaging of transmembrane potential. Biophys J 1993; 64: 1789-800. 14. Weaver JC. Molecular-basis for celi-membrane electroporation. Annals of the New York Acndem11 of Sciences 1994; 720: 141-52. 15. Weaver J, Chizmadzhev Yu. Theory of electropora­tion: A review. Biolectrochem Bioenerg 1996; 41: 135-60. 16. Neumann E, Kakorin S. Electrooptics of mem­brane electroporation and vesicle shape deforma­tion. Curr Opin Colloid Inte1face Sci 1996; 1: 790-9. 17. Kakorin S, Stoylov SP, Neumann E. Electro-optics of membrane electroporation in diphenylhexa­triene-doped lipid bilayer vesicles. Biophys Chem 1996; 58: 109-16. 18. Neumann E. Chemical electric field effects in bio­logical macromolecules. Prog Bioplzys molec Biol 1986; 47: 197-231. 19. Abidor IG, Arakelyan VB, Chernomordik LV, Chizmadzhev YA, Pastuchenko VP, Tarasevich MR. Electric breakdown of bilayer lipid mem­brane. I. The main experimental facts and their theoretical discussion. Bioelectrochem Bioenerg 1979; 6: 37-52. 20. Neumann E. The Relaxation Hysteresis of Mem­brane Electroporation. In: Neumann E, Sowers AE, Jordan C, eds. Electroporation and Electrofusion in Celi Biology. New York: Plenum Press, 1989: 61­82. 21. Kakorin S, Neumann E. Kinetics of electropora­tion deformation of lipid vesicles and biological cells in an electric field. Ber Bunsenges Phys Chem 1998; 102: 1-6. 22. Steiner U, Adam G. Interfacial properties of hydrophilic surfaces of phospholipid films as determined by method of contact angles. Celi Bio­physics 1984; 6: 279-99. 23. Neumann E, Toensing K, Kakorin S, Budde P, Frey J. Mechanism of electroporative dye uptake by mouse B cells. Biophys J 1998; 74: 98-108. 24. Spassova M, Tsoneva I, Petrov AG, Petkova JI, Neumann E. Dip patch clamp currents suggest electrodifusive transport of the polyelectrolyte DNA through lipid bilayers. Biophys Clzem 1994; 52: 267-74. 25. Hristova NI, Tsoneva I, Neumann E. Sphingosine­mediated electroporative DNA transfer through lipid bilayers. FEBS Lett 1997; 415: 81-6. 26. Neumann E, Kakorin S, Tsoneva I, Nikolova B, Tomov T. Calcium-mediated DNA adsorption to yeast cells and kinetics of celi transformation. Bio­phys J 1996; 71: 868-77. Molecular alterations induced in drug-resistant cells Maja Osmak Instiut Rudjer Bošlcovic, Zagreb, Croatia The major obstacle to the ultimate success in cancer therapy is the ability of tumor cells to develop resis­tance to anticancer drugs. Severa/ molecular mechanisms have been suggested to be involved in drug­resistance: a) decrease in the intracel/ular drug accumulation (increased activity of membrane trans­porters such as P-glycoprotein ar multidrug-resistance-associated protein), b) changes in intracellular detoxification system (increased concentrations of glutathione ar metallothioneins, ar increased activity of related enzymes), c) alteration in nuclear enzymes (enhanced DNA repair and/or better tolerance of DNA damage, decreased activity of topoisomerases), d) altered expression of oncogenes (inducing increased leve/ of protective molecules in cells ar the inhibition of apoptosis). Drug resistance is a multi­factorial phenomenon. The complexity of molecular alterations in drug-resistant cells is and will stay the main problem far the successful treatment of cancer. Key words: drug resistance, tumor cells; cancer chemotherapy Introduction Chemotherapy is one of the praven strategies against malignant tumors, especially if the lesions are spread systematically. In many patients, first regimens are successful in reducing tumor size and are sometimes even able to eliminate all clinically detectable tumor masses. But most often, the successful treatments are relatively short lasting. In a vast majority, a certain number of tumor cells will survive and thus become a source of recurrent disease. Chemotherapy of cancer may fail for vari- Correspondence to: Maja Osmak Ph.D., Rudjer Boškovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia. Te!: +385 1 456 11 45; Fax: +385 1 456 11 77; E-mail: osmak@olimp.irb.hr ous reasons. Among these, drug resistance is the most important one. This phenomenon was first observed by Sidney Farber (who introduced chemotherapy into cancer treat­ment) in 1948.1 Almost fifty years later the molecular mechanisms involved in this pro­cess have been unravelled. Resistance may be primary (intrinsic): the tumor cells do not respond from the start. Drug-resistance may be secondary (acqui­red): under the selection pressure of cytotox­ic drugs tumor cells are able to develop cer­tain mechanisms which render them resis­tant to these drugs. The tumor initially responds to therapy, but tumor growth resumes and the patient relapses. Knowledge regarding the genetic nature and biochemical nature of drug resistance has been derived largely from cellular sys­tems. By step-wise increase in the drug dose, highly resistant cell lines can be obtained. The mechanisms of drug resis­tance can be defined by comparing the bio­chemical and biological characteristics of parental and resistant cells. A search for the cause or causes of drug resistance mechanisms has been occupying the attention of cancer researchers for more than four decades. Today it is known that severa! molecular mechanisms can be involved in drug-resistance. The most important one will be presented in this review. Reduced intracellular accumulation: transport proteins A broad spectrum resistance to cytotoxic drugs, termed multidrug resistance (MDR), involves simultaneous resistance to a wide array of natura!, semisynthetic and synthetic compounds. The most common of them are shown in Table l. They do not have similar structure or the same cytotoxic intracellular target, but are amphipathic and are preferen­tially soluble in lipid. Multidrug resistance is caused by overex­pression of a 170 kDa plasma membrane ­associated glycoprotein (P-glycoprotein, Pgp; Table 1). It is an energy dependent efflux pump that decreases intracellular drug accu­mulation. 2-4 Pgps are coded by MDR gene family. The number of members varies between species. Human possess two MDR genes, MDR1 and MDR2. Of these, only MDR1 can confer a drug resistance pheno­type.5 Multidrug resistant cells, particularly those which display high levels of resistance, often possess an increased copy number of the MDR1 gene.2A It was noted that Pgp bore a remarkable Table l. Characteristics of transport proteins: P-glycoprotein and MRP protein Name P-glycoprotein (Pgp) MRP (multidrug resistance -associated protein) Encoded by Mol. weight Length Number of amino acid MDRl gene 170akDa 4.5akbamRNA 1268 MRPagene 190akDa 6.5 kbamRNA 1531 Discovered in Resistance to Normal tissues distribution (high levels) Tumor tissues distribution Functions 1970 year Anthracyclines, Vinca alkaloids, Podophyllotoxins, Colchicine, paclitaxel Adrenal gland, kidney, !iver, large intestine, pancreas, bile duet, Jung, breast, prostate, gravid uterus Colonic, renal, hepatoma, adrenocortical, phaeochromocytoma Transport of xenobiotics Transport of hormones 1992 year Anthracyclines, Vinca alkaloids Arsenic and antimony-centered oxyanions Testes, skeleta! muscle, heart, kidney, Jung Leukemias ( acute myeloid, chronic lymphocytic, acute myeloid, B­chronic lymphocytic),lung (NSCLC), anaplastic thyroid, neuroblastoma Transport of leukotriene Transport of GSH-conjugates Transport of heavy metal oxyanions "MOAT" (multispecific organic anion transporter) According to references 24 and 9 structural similarity to bacterial transport proteins, particularly those transporting haemolysin. 6 Presumably, in evolutionary terms, it represents a highly conserved com­ponent of the cell. P-glycoprotein is dete­ctable at a high concentration in certain nor­mal tissues (Table 1). The disposition of the Pgp on the luminal surface of kidney brash border, on the mucosal surface of the large intestine, and on the bile canalicular surface of hepatocytes, indicates a normal role in transport and/ or protection against exoge­nous toxins. High levels of Pgp were found in different tumors as well, specially in those arising from the normal tissue with a high Pgp leve1.2-4 Pgp overexpression has been associated with multidrug resistance in many drug­selected cell lines.7 The final evidence that MDR gene is involved in multidrug resistance came from transfection studies: MDRl gene inserted into retroviral expression vector con­fers a complete multidrug resistance pheno­type.5 Recently, another member of the ATP­binding cassette transporter superfamily was isolated from non-Pgp small cell lung carci­noma cells, multidrug resistance -associated protein (MRP).8 It also lowers intracellular drug accumulation, conferring a pattern of drug resistance similar to that of the resis­tance-conferring Pgps.9 However, there may be some differences. For example, MRP con­fers only low resistance to paclitaxel and colchicine, which are reported among the best "substrates" for Pgp. Another notable difference is the ability of MRP to confer low resistance to arsenic and antimony-centered oxyanions. The characteristics of this protein are given in Table l. MRP has been identified in non-Pgp mul­tidrug resistant cell lines from a variety of tumor types.9 Transfection of an MRP expression vector into HeLa cells demon­strated conclusively that the protein con­ferred resistance to drugs.t10 Some recent observations suggest that ele­vated MRP expression may occur prior to MDR.11,12 Decreased drug uptake Decreased intracellular drug accumulation may occur due to decreased drug uptake, for drugs that enter the cells by the help of a cel­lular transport system. Loss or inactivation of this transport system may cause drug resis­tance, as it was observed for melphalan, 13 methotrexate14 or cisplatin.15 Glutathione Glutathione (GSH) is a simple tripeptide that contributes to more than 90% of intracellular non-protein sulphydryl compounds.16,17 It is present in virtually all eucariotic cells. It is also synthesized by tumors, some of which exhibit high cellular levels of glutathione and high capacity for the synthesis of glu­tathione. Glutathione plays an important role in cel­lular metabolism and in the protection of cells against free radicals induced oxidant injury (Table 2). It has been implicated in cell resistance to a number of cytotoxic drugs, particularly to alkylating agents and cis­platin.18-22 There are a number of potential mechanisms by which GSH may affect cellu­lar response to cytostatics. These include conjugation of electrophilic compounds, fre­quently catalyzed by the glutathione S­trasnferases (GST). In addition, GSH can detoxify oxygen-induced free radicals and organoperoxides using GSH-peroxidases. 23 GSH may participate in the resistant phe­notype in two ways. In cytoplasm it may bind electrophilic compounds, thus making them less dangerous. In nucleus, GSH may sup­port the repair of the damage induced in DNA: by maintaining functional repair Table 2. Functions of glutathione in metabolism and immune response Functions Antioxidant Conjugation with different compounds (exogenous, the products of metabo­lism) Amino acid transport Support of primary antibody response Regulation of T-lymphocyte prolifera­tion Co-enzyme for multiple enzymatic reactions Thiol-disulfide exchange in protein synthesis and degradation DNA precursor synthesis Enzyme activation Regulation of microtubule formation Negative control of NF-kB activation Deficiency Increased sensitivity to irradiation and different toxic compounds Oxidant stress Cataract formation Impaired function of both T and B lym­phocyte function and immune function in general According to references 16 and 17 enzymes or by maintaining deoxyribonu­cleotide triphosphate pool size.t24 An argument for the potential role of GSH in resistance is based on the observation that the toxicity of many cytotoxic agents can be increased by lowering cellular GSH (as by adding specific inhibitor of GSH synthesis­buthionine sulfoximine).25,26 Glutathione S-transferases Glutathione S-transferases (GST) are an important part of the Phase II detoxification system that metabolizes many lipophilic drugs and other foreign compounds, includ­ing anticancer drugs.27-29 The overall result of this system is the conversion of lipophilic chemicals to more polar derivates, thus facili­tating their inactivation and elimination. GST are abundant and together may constitute up to about 5 % of soluble cellular protein. Their importance supports the finding that they perform specific detoxification, structural and transport function in many phila: from bacteria to humans. GST catalyze direct coupling of GSH to electrophilic drugs, thus making a less toxic and more readily excreted metabolic com­pound. R-X + GSH -,. R-SG + H-X Figure l. Conjugation reaction catalyzed by GSTs Further, they may exhibit ligand binding function, by non-covalent binding of non­substrate hydrophobic ligands (such as heme, bilirubin, some steroids, and some lipophilic cytostatics. 27-29 The diverse biological functions of GSH/GST system are mediated by multiple GST enzymes. The genome of most species encode severa! different isoenzymes of GSTs. In eucaryotic cells, there are five classes of GST. Four are found in cytosol, while the fifth class, the microsomal GST, is found pri­marily in the hepatic endoplasmic reticulum. The microsomal GST are functional trim­mers with molecular weights of 17 kDa. Cytosol GST are both mono-and het­erodimeric complexes formed of GST sub­units that range in size from 23 to 28 kDa. The classification of the cytosolic GST into alpha, pi and mu, and recently identified theta classes was originally based on physical and catalytic properties. Among them, GST pi was found at elevated levels in many tumor tissues relative to matched normal tis­sues.30,31 Severa! anticancer drugs have been defini­tively identified as GST substrates (Table 3). Therefore, it is not surprising that elevated levels of GST were found in cells resistant to some of these drugs like cisplatin, doxoru­bicin, melphalan etc.27-29 The final confirmation of GST involve­ment in drug resistance came from the trans­fection experiments. 32 The tranfection with Table 3. Anticancer drugsa: substrates for glutathione S-transferases Direct evidence for involvement Indirect evidence for involvement in drug resistance in drug resistance Chlorambucil Melphalan Nitrogen mustard Phosphoramide mustard Acrolein BCNU Hydroxyalkenals Ethacrinic acid Steroids Bleomycin Hepsulfam Mitomycin C Adriamycin Cisplatin Carboplatin According to reference 27 GST imparts a small but significant (and clin­ically relevant) increase in resistance to cis­platin (GST mu), doxorubicin (GST pi) or chlorambucil and melphalan (GST alpha). Glutathione peroxidase Glutathione peroxidase catalyzes the reduc­tion of potentially toxic peroxides to alcohols by oxidizing GSH to its disulfide form (GSSG). GSSG is returned to GSH with the concomitant oxidation of coenzyme NADPH to NADP+. GST enzyme can catalyze a seleni­um-independent GSH peroxidase activity leading to the detoxification of lipid and nucleic acid hydroperoxides. This redox cycle may play an essential role in protecting cells from damage of lipid peroxidation generated during normal metabolism or by redox recy­cling of many drugs. Doxorubicin is one of the agents known to generate free radicals and peroxides. GST as well as the selenium -dependent enzyme GSH peroxidase can deactivate these metabolites through peroxidative mecha­nisms, resulting in decreased cytotoxicity. In tumor cells resistant to doxorubicin, increased levels of selen -dependent GSH peroxidase were found.33,34 Metallothionein Metallothioneins (MT) were first discovered as a family of inducible proteins involved in Zn2+ and Cu2+ homeostasis and in the detoxification of heavy metals.35,36 They are evolutionary conserved low molecular weight intracellular proteins with unusually high level of cystein content, that constitute the major fraction of the intracellular protein thi­ols. Today is known that metallothioneins are a part of generalized cell response to environ­mental stress: the abundant nucleophilic thiol-rich groups in MT can react with many electrophilic toxins, participate in controlling the intracellular redox potential, and act as scavengers of oxygen radicals generated dur­ing the metabolism of xenobiotics. They can be induced by environmental stimuli such as epinephrine, glucocorticoids, thermal injury, cytokines, cyclic nucleotides, phorbol esters, UV light, etc., suggesting a protective func­tion and a role in cell growth and prolifera­tion_ 35,36 Metallothioneins are attractive candidates as modulators of cellular sensitivity to anti­cancer drugs. Elevated levels of MT have been observed in some malignant cells with acquired resistance to antineoplastic drugs, such as cisplatin.3740 Increases in intracellu­lar MT by gene-transfer-produced resistance to cisplatin, melphalan and chlorambucil, and less to doxorubicin and bleomycin38 or N-methyl-N -nitro-N-nitrosoguanidine (MN­NG) and methyl nitrosourea (MNU).41 In most MT overexpressing cell lines, however, induction of MT did not cause a parallel increase in resistance. Therefore, the resis­tance associated with MT overexpression was not due to direct binding of the drug to MT. The study of Kaina and co-workers suggest that MT may participate as a cofactor or regu­latory element in the repair or tolerance of toxic alkylating drugs.41 Nevertheless, increases in MT do not always result in a phenotype that is less sen­sitive to the toxic effects of antineoplastic drugs.42 Thus, the protective role of MT has been questioned. Recently, transgenic mice lacking functional MT have been produced by homologous recombination of disrupted MTI and II genes. The embryonic cells of these mice exhibit enhanced sensitivity to cisplatin, melphalan, bleomycin, cytarabin or MNNG, confirming the protective function of metallothioneins against cytotoxic drugs.43 Gene amplification One of the important mechanisms of drug resistance is gene amplification. The first observation of this phenomenon was noted by Biedler and Spengler. They found chromo­some elongation in cultured hamster cells resistant to methotrexate (MTX) and called this extra DNA "homogeneously staining regions" (HSR).44 Schimke and co-workers then showed that the extra DNA contains extra copies of the gene for the enzyme dihy­drofolate reductase (DHFR), explaining the increased enzyme levels in the resistant cell.45A6 The amplified DNA may either be present in chromosomes as HSRs or free, as minute chromatin particles usually present in metaphase spreads as pair minutes, called double minutes (DM). After the initial demonstration that cells can overcome the MTX inhibition of DHFR by overproducing the enzyme by means of gene amplification, numerous other exam­ples of this mechanism have been reported: for MDR, Z,4 for metallothioneins 35, 36 etc. DNAtrepair The resistance to some cytotoxic drugs can be caused by enhanced ability of cells to repair the DNA induced damage or to toler­ate their presence. One of the most studied phenomena in this respect is resistance to cisplatin. It has been well established that cisplatin binds to DNA and that these adducts con­tribute to cellular toxicity. In a number of cis­platin resistant cell lines, an enhanced repair of DNA lesions has been demonstrated.21,22 Thus, Eastman and Schulte provided direct evidence for increased repair showing that the predominant lesions, cis-GG adducts, were more rapidly removed from resistant than sensitive cells.47 These resistant L1210 cells can also reactivate a cisplatin-damage plasmid more readily than sensitive parental cells.48 However, a correlation of repair activ­ity with drug-resistance has not always been demonstrated: L1210 cells with 100-fold resis­tance to cisplatin, removed cis-GG intra­strand adducts only slightly better than 20­fold resistant cells.47 Repair of platinum damage in very specif­ic regions of the genome is a possible charac­teristic of enhanced repair in resistant cells. If only active genes are more efficiently repaired in resistant cells, then it is not likely that a significant change in overall platinan­tion levels or repair rates will occur. Enhanced gene-specific repair could explain some of the controversial results found in such investigations. While preferential repair of the interstrand cross-link in active versus inactive regions was not found in Chinese hamster ovary cells,49 it was demonstrated in resistant human ovarian 2008 cells.t50 It was observed that some cisplatin-resis­tant cell may have higher DNA platination than parental cells,51 or may tolerate severa! fold more platinum on their DNA at equitox­ic concentrations as sensitive cells.52,53 Con­sidering these facts, the concept of enhanced tolerance to DNA damage was suggested as a potential mechanism of resistance. However, the basis of this phenomenon is not well understood. Severa! groups have described DNA-bind­ing proteins that retard the mobility of cis­platin-damaged DNA fragments in non-dena­turing polyacrylamide gels. It has been hypothesized that these proteins are either involved in DNA repair by shielding adducts from repair, or are involved in transcrip­tion. 54 A number of cisplatin-resistant cells have been investigated for changes in these DNA damage-recognition proteins.22 Howev­er, no obvious correlation between the expression of DNA damage-recognition pro­teins and resistance to cisplatin was found. Some of the recent papers suggest that resistance to DNA damage can be acquired via the loss of DNA mismatched repair activi­ty. The DNA mismatch repair system acts after DNA replication and corrects non-Wat­son-Crick base pair and other replication errors. Human cells lacking mismatch repair activity have high spontaneous mutation rates. Also, they may be resistant to certain cytostatics, such as etoposide,55 cisplatin56 or N-methyl-N-nitro-N-nitrosoguanidine.t57 DNA toposiomerase Besides MDR, some resistant cell lines exhib­it atypical multidrug resistance (at-MDR). At­MDR is distinguished from the MDR in the following ways: a) lack of cross-resistance to the Vinca alkaloids,58 b) absence of a drug accumulation defect,58 c) relative insensitivi­ty to modulation of resistance by verapamil or chloroquine typical inhibitors of P-glyco­protein, 59 and d) lack of overexpression of the MDRl gene or its product, Pgp.59 At-MDR involves altered activity of topoi­somerases II. Topoisomerases II are enzymes that catalyze changes in the secondary and tertiary structures of DNA. They are neces­sary for replication, recombination and tran­scription, as well as in mitotic chromosome condensation and segregation. Topoiso­merases II act via introduction of a transient double-stranded break in one segment of a DNA molecule through which a second DNA duplex is passed before religation of the break.60 The levels of these enzymes are markedly higher in exponentially growing than in qui­escent cell lines. Two distinct forms of topoi­somerase II exist in human cells, termed a (170 kDa form) and f3 (180 kDa form).61 They differ not only in molecular weight but also in their patterns of expression and their apparent sensitivity to anticancer drugs.62 In cell lines the expression of the a isoform has been shown to be strictly proliferation depen­dent, whereas the f3 isoform is presented in both dividing and non-dividing cells. There are some inhibitors of topoiso­merase II (doxorubicin, epirubicin, mitox­antrone, etoposide, teniposide) that trap the "cleavable complex" resulting in increased DNA scissions and inhibition of rejoin­ing. 60,63,64 These protein-associated DNA lesions are directly toxic to cells. The cells with a high leve! of topoisomerase II are gen­erally more sensitive to inhibitors than cells with a low leve! of these enzymes. There is a number of rodent and human tumor cells lines in which resistance to topoi­somerase II inhibitors are connected with decreased leve! of the topoisomerase II a and /or f3. The resistance mechanisms appear to be the result of a decrease in the activity of topoisomerase II.t63-68 Beside topoisomerase II, drug resistance may involve the altered activity of topoiso­merase I. Topoisomerase I is an enzyme abundant in actively transcribing gene regions. It has important role in DNA replica­tion and elongation step of transcription. Contrary to topoisomerase II, toposiomerase I introduce a transient single-stranded nick in DNA and is ATP independent. Severa! cyto­statics, such as camptothecins and actino­mycin D are the poisons of topoisomerase r_60,63,64 Drug induced accumulation of topoi­somerase I-DNA cleavable complex is direct­ly proportional to drug cytotoxicity and anti­tumor activity. Resistance to topoisomerase I inhibitors involves altered activity of this enzyme, that may be caused by mutation(s) in the gene coding for topoisomerase I. 69 Oncogenes and tumor suppresser genes: sig­nal transduction pathway and apoptosis In last few years interest has been focused on oncogenes and their role in drug-resistance. The direct evidence that oncogenes can be involved in drug-resistance came from trans­fection studies. The transfection of murine NIH3T3 cells 7o,71 or kerytocytes72 with ras oncogene resulted in resistance to cisplatin. Ras oncogene may induce resistance to dox­orubicin as well.73 Moreover it was found that the degree of cisplatin resistance corre­lated directly with the level of c-myc expres­sion,74,75 while the re-establishment of the normal level of c-myc transcription restored original sensitivity.74 C-myc oncogene was also involved in resistance to methotrexate.76 Using ribosome mediated cleavage of c-fos mRNA, the role of c-Jas oncogene in resis­tance to cisplatin was proved.t77 Resistance to cisplatin was achieved by the transfection of src oncogene as well. 78 The mechanisms by which oncogenes cause drug-resistance in not quite clear. 79 It has been suggested that c-myc oncogene binds to DNA, and thus directly or indirectly regulates a process of DNA repair.74 Ras oncogene might induce resistance by regulat­ing the expression of other genes involved in the protection of cell against cytostatics. It was shown for glutathione transferase pi,80 topopisomerase II, 81 c-jun, 82 glutathione, 83 MDR,84 or altered membrane potential.85 Scanlon hypothesized that fos expression is the trigger that causes the resistance response (primary DNA reparability, as indi­cated by DNA polymerase a, DNA poly­merase .thymidilate synthase, DHFR and ' topoiosmerase I expression). Consistent with this concept is the observation that transfec­tion of sensitive cells with c-fos generated eithold resistance to cisplatin,77 while attenu­ ation of the elevated c-fos expression returned the cisplatin toxicity to that of par­ent population. Another oncogene, mutated p53, may confer resistance to many hydrophobic drugs by stimulating specifical­ly MDRl promoter.84 It must be mentioned, however, that not always an increased expression of ras, myc or other oncogenes caused an increased resis­tance to cytostatics.75,86-88 In many cases the cellular damage caused by active doses of drug is not sufficient to explain the observed toxicity. Therefore, it is possible that some determinants of inherent drug sensitivity and resistance may be inde­pendent of those which involve the formation of the drug-target complex and its immediate biochemical sequel, such as commitment to cell death. Cell death is activated by natura! control processes whose function is to allow repair of low level damage to DNA while eliminating those cells in which repair is not possible. There are two modes of cell death: apoptosis and necrosis. They differ morpho­logically and biochemically. Necrosis is asso­ciated with cell swelling, rupture of mem­branes and dissolution of organized struc­ture. That is a consequence of the loss of osmoregulation. DNA degradation occurs at a late stage. In contrast, in apoptosis internu­cleosomal cleavage of genomic DNA and chromatin condensation precedes the loss of membrane integrity (Figure 2). Necrosis mostly results from a major cell insult such as that caused by serious mechanical, ische­mic, or toxic damage. Apoptosis generally occurs as a response to less severe injury and is also involved in the development and remodeling of normal tissue.89, 90 Apoptosis induces a wide variety of cell stresses and cytotoxic chemicals,89,90 among them anticancer drugs.91-94 Deregulation of normally integrated cell cycle progression appears a central signalling event in most forms of apoptosis.90 Apoptosis is a highly conserved active mechanism that requires the expression of several specific genes. Also their exact func­tion is not quite understood, certain genes have been proposed as positive ( p53, c-fos, c­myc, interleukin-1 converting enzyme etc.) or negative regulatory elements (bcl-2 or glu­tathione redox cycle). They induce or prevent the onset of apoptosis.95-103 Among them, p53 and bcl-2 are the most important and most studied. p53 protein can function as a genetic switch capable of activating G1 arrest, result­ing in the repair of DNA damage.94 Also, it is Figure 2. HeLa cells obtained 48 hours after a 1-hour treatment with 150 r1M cisplatin. Apoptotic cells showed typical chromatin condensation, fragmented nuclei and cellular shrinkage (arrowhead), while intact nuclei exhibit "mottled" fluorescence. Fram reference 91. required for efficient activation of apoptosis following irradiation or treatment with chem­icals. Loss of p53 function has been reported to increase resistance of tumor cells to a vari­ety of cytotoxic drugs.95,96 Recently it was shown that cells with mutated p53 gene dis­play perturbed Garrest or apoptosis. This 1 defect appears to reduce the sensitivity to DNA-damaging agents, suggesting that inhi­bition of apoptosis may represents a mecha­nism by which tumor cells may acquire drug­resistance. 95,96 By transfer of normal p53 into p53-defective non-small cell cancer line, an important increase in sensitivity to cisplatin was determined, which was related to the promotion of apoptosis.97 However, the paper recently published by Wosikowski et al. suggests that alterations in p53 gene sta­tus or protein functions are not critical for the development of multidrug resistance.98 On the other hand, Bcl-2 protein inhibits apoptosis 99,ioo and increases cell resistance to drugs.101 Recently, bcl-2 related gene prod­ucts have been reported. One of them, Bax, homodimerizes as well as heterodimerizes with Bcl-2 protein. The Bcl-2:Bax ratio may determine survival or death after an apoptot­ic stimulus.102 Therefore, oncogenes and tumor supres­sor genes may be involved in drug resistance in two ways: by increasing the level of protec­tive molecules in cells or by inhibiting apop­tosis. Other factors In doxorubicin treated cells an altered pat­tern of intracellular drug distribution was observed. The initial accumulation of drug in perinuclear location was followed by the development of a punctate pattern of the drug scattered throughout the cytoplasm. This pattern was suggestive of a process of drug sequestration, possibly followed by vesi­de transport. In resistant cells, alteration in the intracellular drug distribution was acco-­mpanied by a decrease in nuclear versus cyto-­ 105 plasm drug ratio.104 , There is more and more evidence, that drug resistance is a multifactorial phenome­non (only for very low doses of the drug a single mechanism can be involved in drug­resistance. Thus, for instance, in cells resis­tant to cisplatin altered drug accumulation, increased levels of glutathione and related enzymes, metallothloneins, increased repair and altered expression of oncogenes could be 2239 40 observed.21 ,,, In methotrexate resistant cells, decreased uptake of this drug, decre­ased polyglutamation, decreased affinity to DHFR and increased levels of target enzyme are the most common cause of drug resis­tance to MTX.106 It should be mentioned, however, that all of these mechanisms need not be induced in drug resistant cells. So, in cisplatin resistant human laryngeal carcino­ma cells only decreased platinum accumula­tion was connected with resistance to cis­platin, while no alteration in oncogene expression, no involvement of glutathione, glutathione transferase or metallothioneins was determined.40,87 Table 4. Drug sensitivity pattern of resistant cell lines Due to induction of several protective mol­ecular mechanisms, resistant cells obtained after treatment with a single drug, can become resistant to various unrelated drugs (Table 4). The schedule of drug-resistance development can also influence the resis­tance pattern. Even with the same treatment schedule, clones with different cross-resis­tance patterns occur.109 It is generally accepted that the resistance to drugs can be induced by treatment with chemicals. However, in last severa! years it became obvious that also ionizing irradiation can induce drug resistance in irradiated cells no-114 by the same mechanisms that are involved in resistance development induced by cytostatics.112,114-120 Thls fact, if supported in vivo, and specially in clinic, is of the out­most importance for the patients. Namely, if irradiation precedes chemotherapy, it can reduce the success of combined therapy. In conclusion, drug resistance is a com­plex, multifactorial phenomenon, which may involve decreased intracellular drug accumu­lation, increased detoxification, increased DNA decreased activity of topo­siomerases, gene amplification, altered onco- Celi line Drug used far Treatment Resistant Sensitive resistance development schedule to to Laryngeal carcinoma 1 vincristine acute DOX, MTX CDDP continuous DOX, MTX, 5-FU CDDP Cervical carcinoma2 cisplatin acute VCR, DOX, ETO, MTX, 5-FU continuous VCR, MTX Laryngeal cardnoma3 cisplatin acute VCR, I\IIMC ETO, DOX continuous VCR, MMC, 5-FU Breast adeno-carcinoma4 doxorubicin continuous VCR, VBL, CDDP, CBDCA, (MMC, 5-FU)* 1 reference 107, 2 reference 39, 3 reference 108, 4 reference 88. * Significant resistance only at higher doses. Acute 1 hour treatment; continuous 24 hours treatment VCR= vincristine, VBL = vinblastine, DOX doxorubicin, ETO = etoposide, MTX methotrexate, 5-FU = 5-fluorouracil, CDDP= cisplatin, CBDCA= carboplatin, MMC= mitomycin C. gene and tumor supressor gene expression, as well as inhibition of apoptosis. Resistance pattern of anticancer drugs is determined by the a) genotype of the cells, b) genotoxic agent involved in resistance development, and c) treatment schedule. The complexity of drug-resistance mechanisms, as well as sometimes conflicting experimental 80% have been obtained in both animal and human tria/s far severa/ types oj skin malignancies using ECT with bleomycin. This study was initiated to determine if ECT could be used to effectively treat interna/ tumors such as hepatomas in an animal model and human rhabdomyosarcomas in athymic rats. Bleomycin, cisplatin, doxorubicin, 5-fluorouracil, and taxol were used in conjunction with electric pulses. Following an intra tumor injection oj a single drug, electric puls­es were administered directly to the tumor. Far the hepatoma model, ECT worked the best with cisplatin and bleomycin, yielding complete response rates oj about 70%. The other drugs used to treat hepatomas were ineffective. Bleomycin combined with electric pulses resulted in a 100% response rate far sarcoma; response rates with cisplatin and doxorubicin were low. These studies indicate that ECT is a technically feasible procedure for visceral tumors and soft tissue sarcomas. Key words: /iver neoplasms, experimental; rhabdomyosarcoma; electroporation; bleomycin; cisplatin; doxorubicin; fluorouracil lntroduction The delivery of drugs to cancerous tissue is an important modality in the potential treat­ment of various tumors. Most anti-tumor drugs have an intracellular mode of action. Correspondence to: Richard Heller, Ph.D., University of South Florida, College of Medicine Department of Surgery, MDC Box 16, 12901 Bruce B. Downs Blvd., Tampa, FL 33612-4799, Tei: 813 974-3065, Fax: 813 974-2669, E-mail: rheller@coml.med.usf.edu However, for many of these drugs, the cell membrane is often times a significant barrier which reduces the effectiveness by restricting intracellular access. As a result, it is essential to find a mechanism to deliver the drugs through the cell membrane more efficiently. Therefore, it is possible to increase the thera­peutic potential of these drugs by increasing the permeability of the tumor cell mem­branes. Pendas S et al. Electric pulses can be used to temporarily and reversibly permeabilize cell mem­branes.1-4 The transient alteration of the cell membranes permeability using electric puls­es is known as electroporation. Over the past twenty years electric fields have been used successfully as a method of targeting mole­cules to tissues, 5,6 electrofusing cells to tis­sues7-9 and increasing the uptake of certain drugs by cells.10,n Recently, work has been performed demonstrating that electroporation could be used to enhance the effectiveness of chemo­therapeutic agents. This combination of elec­tric pulses and anti-tumor agents is known as electrochemotherapy (ECT).t12, 13 The incre­ased effectiveness of these anti-tumor agents is a direct result of electroporation facilitating the uptake of drugs through the cell mem­brane which has been made transiently more permeable. Electric pulses delivered to the tumor are non-cytotoxic, and cell membrane permeability returns to baseline levels sever­a! minutes after the treatment with electric pulses. Bleomycin has been the drug most often used for ECT for several reasons. Bleomycin is a very potent cytotoxic molecule when introduced inside the cell. The drug works by causing single stranded and double stranded breaks in DNA.14-17 In addition, only a few hundred molecules are sufficient to be cytotoxic.10,18 Since bleomycin has an intra­cellular mechanism of action, the drug must be able to enter the cell to be effective. How­ever, bleomycin is a relatively nonpermeant drug10 showing minimal intracellular concen­tration with a systemic dose. Thus, bleomycin cytotoxicity is dependent upon membrane permeability. Several studies have been performed in both mice and rats and have shown that when bleomycin is administered in combina­tion with electroporation its effectiveness as an anti-tumor agent is greatly enhanced. These studies were done with a variety of tumor types including, melanoma, hepatocel­lular carcinoma, lung carcinoma, breast carci­noma, fibrosarcoma, glioma and cervical car­cinoma.12,13,19-32 In addition, the combination of electroporation with other chemotherapeu­ 33 tic agents has also been tested.23,One agent that has shown promise is cisplatin. Although cisplatin is a more permeant drug than bleomycin its effectiveness was aug­mented by electroporation of cells in vitro as well as tumors in vivo. 33 Severa! clinical studies have shown the potential of electrochemotherapy as an anti­tumor treatment for a variety of cutaneous malignancies.t34-40 Initial trials utilized bleomycin administered intravenously fol­lowed by local administration of electric puls­es directly to the tumor. Response rates for the treatment of squamous cell carcinoma of the head and neck were 70% with complete responses of 50-60%. 34,35,37 The treatment of melanoma and basal cell carcinoma yielded response rate of 70% with a complete response rate of 33%.36,39 Subsequent trials for the treatment of melanoma and basal cell carcinoma utilized intratumor administration of bleomycin in conjunction with electric pulses. Response rates in this trial were as high as 99% with a complete response rate of 40 90%.39, The results of the animal and human stud­ies have been extremely encouraging. Since electroporation is based on general physical principles and has been shown to work on most mammalian cells, studies have been ini­tiated to examine if ECT could be used to treat other tumor types. The study reported here, examines the use of this antitumor ther­apy for the treatment of hepatoma and soft tissue sarcoma in rat models. The effects of ECT with bleomycin, cisplatin, taxol, 5-FU and doxorubicin on established hepatomas was investigated first. Sarcomas were then treated with ECT using bleomycin, cisplatin and doxorubicin. Taxol and 5-FU were not used to treat sarcomas because they were Direct delivery oj chemotherapeutic agents jor the treatment oj hepatomas and sarcomas in ral mode/s found to be ineffective in the hepatoma model. Materials and methods Cell lines and culture methods Visceral tumor study: NlSl rat hepatoma cells (ATCC CRL-1604; American Type Culture Collection, Rockville, MD, USA) were grown in Swimms S-77 medium modified to con­tain, 4mM L-glutamine, 0.01 % Pluronic F68, 9% fetal calf serum, and 90.tg/ml gentamycin sulfate. Cells were maintained in humidified air that contained 5% CO2. In addition, the cells used for this study were greater than 95% viable. Soft tissue sarcoma tumor study: Human A204 rhabdomyosarcoma cells (HTB 82; American Type Culture Collection, Rockville, MD, USA) were used to induce tumors in nude rats. The cell line was grown in McCoy's 5A medium (Mediatech, Washing­ton, DC, USA) supplemented with 10% (v/v) fetal bovine serum (PAA Laboratories, New­port Beach, CA, USA) and 90µg/ml gen­tamycin sulphate (Gibco, Grand Island, NY, USA). Cells were grown in a humidified atmosphere that contained 5% CO2. Conflu­ent cultures were prepared for use by detach­ing with a nonenzymatic cell dissociation solution (Sigma, St. Louis, MO, USA). The trypan blue exclusion dye method was used to determine the viability of all harvested cell batches. Celi viability was greater than 95% for all batches used in this study. Animals and tumor induction Tumors were induced in both male Sprague­Dawley rats using NlSl rat hepatoma cells and nude rats using human A204 sarcoma cells. General anesthesia was administered using isoflurane. Rats were first placed in an induction chamber that was charged with a mixture of 5% isoflurane in oxygen for sever­a! minutes. These rats were subsequently fit­ted with a standard rodent mask and kept under general anesthesia using 3% isoflu­rane. Hepatoma study: the right median lobe of the rat was surgically exposed and injected with 1X106 viable NlSl cells, suspended in 0.5 ml of saline. The animals were closed with surgical staples immediately after injec­tion with tumor cells. The tumors were allowed to grow for 7-10 days. This procedure yielded hepatomas that were approximately 0.75cm in diameter. Sarcoma study: male athymic rats (Harlan Sprague Dawley, Inc., Indianapolis, IN, USA) that were 3-4 weeks old at the tirne of tumor induction were used for the sarcoma tumor study. Tumors were induced by injecting 8X106 cells, contained in 70µ1 of saline, into the biceps femoris muscle of each rear limb of the athymic rats. Tumors were allowed to grow for 7 to 10 days resulting in sarcomas that were 6 to 8mm in diameter for the case of small sarcomas. Large sarcomas were allowed to grow for greater than 35 days which produced tumors that were 18 to 20 mm in diameter. Tumor treatment Treatment of hepatoma: After the establishing tumors in the right median lobes, ECT was performed. Bleomycin, cisplatin, taxol, 5-FU, or doxorubicin were injected directly into tumors using different doses in order to determine the effect of each drug separately after the delivery of electric pulses. All doses were administered in a volume of 100 µl. Control animals that did not receive drug therapy received a 100 µl saline injection. Electric pulses were administered ninety sec­onds after the intratumor injection by insert­ing a circular array of 6 needle electrodes 41,42 (BTX 878-2a; Genetronics, Inc., San Diego, CA, USA) to a depth of 5 mm around the Pendas S et al. peripheral tissue of all tumors so that the entire tumor was contained within the array of needles. The tirne between drug injection and electric pulse administration was reduced to 90 seconds from the standard of ten minutes, which was used in previous studie s, 25 due to the highly vascular nature of hepatoma tumors. Six electric pulses with a field strength of 1000 V/ cm were delivered via the inserted needle electrodes in a man­ner that rotated the applied field around the treatment site.t29A1 A lower field strength was used to treat the hepatoma tumors vs the sar­coma tumors because of the lower impedence of !iver tissue compaired to skin tissue. These pulses were administered using a DC genera­tor (BTX T820 generator; Genetronics, Inc., San Diego CA, USA), and the pulses were 99µs in duration with a one second interval between the initiation of each pulse. Treatment oj sarcoma: After injection of human sarcoma cells in the rear limbs of athymic rats, all animals developed firm pal­pable tumors. All drugs were administered by intratumor injection in a volume that was equal to 25% of the tumor volume. The chemotherapeutic agents were administered at the following concentrations: bleomycin 5 units/ml, cisplatin 1 mg/ml and doxorubicin 20 mg/ml. Control animals that did not receive a chemotherapeutic agent were given an intratumor injection of saline that was equal to 25% of the tumor volume. The delivery of electric fields to sarcoma tumors was similar to hepatoma electrical treatment except that the electric pulses were administered ten minutes after injection with the chemotherapeutic agent or saline. In addition, the electrode was placed around the perimeter of each tumor to a maximum depth of 1 cm so that the entire tumor was encom­passed within the needle array. The ratio of the applied voltage for each pulse to the elec­trode spacing was 1300 V/cm.29A1 A larger field strength was used in the treatment sar­coma tumors due to higher tissue impedence. Large sarcomas were too big to fit within the volume delineated by the needle array elec­trode. These tumors were electrically treated by multiple insertions of the electrode until the entire tumor volume received pulses. Protocols for the treatment of small and large sarcomas were designed for a single ECT treatment and multiple ECT treatments. Electrochemotherapy was administered once for single treatment experiments and a maxi­mum of three times for multiple treatment scenarios. All single treatment animals received ECT on the same day. Similarly, all multiple treatment animals received their first ECT treatment on the same day. For ani­mals that received multiple treatment, ECT was administered again when palpable tumor within the original treatment site was first detected. Tumor measurements Hepatoma study: At days 7 and 14 post ECT treatment, all the animals induced with hepatoma tumors were surgically explored, and the tumors were examined for evidence of response to treatment. Tumor volumes were measured prior to and after treatment using the formula V=abc n/6. Measurements were made using a digital Vernier caliper. Objective responses to ECT treatment was determined based on reduction in tumor vol­ume. A complete response was when no visi­ble tumor was evident. Greater than 50% reduction in tumor volume was considered a partial response, and stable disease was less than 50% reduction in tumor volume. Pro­gressive disease was when the tumor volume continued to increase in size. An objective response was defined as the sum of complete and partial responses. For long term studies, the animals were checked every 14-21 days after day 14. Sarcoma study: Another study was con­ducted to confirm the efficacy of ECT in the treatment of highly aggressive human sarco­ Direct delivery of clze111otherapeutic age11ts for tl1e treat111ent of lzepatomas and sarcomas in ral 111odels ma tumors induced in the rear limbs of male athymic rats. Response to electrochemothera­py treatment was also based on tumor vol­ume. Tumor volume was determined on 3 mutually orthogonal measurements (a,b,c) of the nodule, and the tumor volume was based on the formula V= abc rr/6. Tumors were mea­sured prior to treatment and then at 7 day intervals after treatment. Each tumor was categorized as a complete response, partial response, stable disease, or progressive dis­ease at 28 days post treatment. Animals were considered cured if complete responses were maintained for 100 days. Histologic analysis Tissue specimens were fixed overnight in 10% formalin and then processed for routine histopathological examination. Briefly, speci­mens were dehydrated through a sequence of 50, 70, 95 and 100% ethanol, cleared in xylene and then embedded in paraffin wax. Sections were cut with a microtome (three sections per specimen) and stained with hematoxylin-eosin. The overall condition of the tissue was examined with respect to cel­lular integrity. Statistical analysis The Fisher's test for 2 X 2 contigency tables was used to determine the statistical signifi­cance of the complete response rates between the treatment and the control groups. For this test, partial response, stable disease and pro­gressive disease were considered incomplete responses. Results A total of 223 established hepatoma tumors were treated in the visceral tumor study and 89 tumors were treated in the sarcoma study. Four different treatment groups were exam­ined. These groups included those with no treatment (D-E-), electrical treatment (D-E+), drug treatment (D+E-), and combined drug and electric pulses (D+E+). ECT far hepatomas Treatment with bleomycin: Objective responses were obtained in 84.5% of the tumors treated with both bleomycin (O.S unitjtumor) and electric pulses (D+ E+ group). This group also had a 69% complete response rate (Table 1). Tumors that received drug only (D+E-) or only electric pulses (D-E+) or no treatment (D-E-), were found to have 100% progressive disease (Table 1). The response was based on tumor measurements taken 14 days after treatment. The complete response rate for the D+E+ group differed significantly (p< 0.01) from the other groups. Incomplete responses were considered to be those animals which had progressive disease, stable disease, and partial responses. The number of complete responses for the D+E+ treatment group was significantly greater ( P< 0.01) than the num­ber of complete responses in each of the con­trol (D-E-, D-E+, and D+E-) groups. In addi­tion, no adverse effects from the treatment were observed in any of the animals. Treatment with other chemotherapeutic agents: The ability to augment the effective­ness of other chemotherapeutic agents when Table 1. Treatment of rat hepatomas with bleomycin Treatment n %PD" %SDb %PW %CRd D-E-9 100 o o o D-E+ 9 100 o o o D+E-10 90 o o 10 D+E+ 13 15.5 o 15.5 69 a: PD = Progressive disease = tumor increasing in size Day 14 compared to Day O b: SD = Stable disease = tumor decreasing less than 50% in size Day 14 compared to Day O c: PR = Partial response = tumor decreasing in size more than 50% Day 14 compared to Day O d: CR = Complete Response = no tumor present on Day 14 Pendas S et al. combined with electric fields was tested in the NlSl rat hepatoma model. The treatment was performed as described above using vari­ous concentrations of cisplatin, doxorubicin, taxol and 5-FU. Examination of responses at day 14 showed that hepatomas treated with 0.0357 mg of cisplatin and electric fields resulted in a complete response rate of 67% (Table 2) which is similar to the result obtained with bleomycin. The higher tested cisplatin 35 days (treatment of large tumors) Pendas S et al. were taken at day 28. All tumors in the con­trol group showed evidence of malignant sar­coma cells with a high mitotic rate and mini­mal necrosis of the tumor. However, tumors in the D + E + group showed a few anucleated tumor cell associated with abundant necrotic tissue suggestive of no residual viable tumor. To examine if the therapy would work on large tumors, tumors of the D+E-group, showing no response 35 days after treatment were treated with ECT. The treatment of these large tumors resulted in a 50% com­plete response rate and a 37.5% partial response rate (fable 4). In addition, treat­ment of these large tumors only had a cure rate of 12.5%. The low cure rate obtained with both small and large tumors was surprising due to the high complete response rate. It is possible that the single treatment was not sufficient to eliminate all tumor cells. Previous work in mouse models with subcutaneous tumors had demonstrated an increased cure rate when multiple treatments were performed.31 Therefore, an additional experiment was per­formed to determine if multiple ECT treat­ments with bleomycin would be beneficial. Small tumors that received multiple ECT treatments showed a cure rate of 83.3% (Table 5) compared to the 42% cure rate obtained with single treatment (Table 4). Treatment of large tumors with multiple treatments resulted in a cure rate of 100% (Table 5). Treatment with other chemotherapeutic agents:The effectiveness of treating sarcoma with cisplatin or doxorubicin in combination with electric pulses was studied. Cisplatin was administered at a dose of 1 mg/ml via intratumor injection. The injection volume was equivalent to 50% of the tumor volume. Two treatment groups were used, drug alone (D+E-) or drug with electric pulses (D+E+). The D+E+ group had a 33% complete response at 28 days and a 33% cure rate (Table 6). The D+E-group had a 17% com­plete response rate and 17% cure rate (Table 6). Doxorubicin was administered at a dose of 20 mg/ml via intratumor injection. The injection volume was equivalent to 100% of the tumor volume. The D+E+ group had a 17% complete response at 28 days and a 0% cure rate (Table 6). The D+E-group had a 0% complete response rate and 0% cure rate (Table 6). Doxorubicin treatment had a high toxicity as 5 of 6 animals died in each group. All deaths occurred prior to day 14. However, tumor volumes were obtained on day 7 and at tirne of death. Discussion Primary hepatocellular carcinoma is associat­ed with chronic hepatitis B infection and liver cirrhosis. Approximately 80% of patients who develop hepatomas are positive for hepatitis B surface antigen. 43 These patients have Table 5. Multiple treatment of sarcomas with bleomycin (5 U/ml); injection volume is 25% tumor volume Group n Volume Initial Fina! %PDa %SDb %PRC %CR .t) Po.mer erosion mass loss) simultaneous with drug release after drug release after drug release Size of the polymer matrix decreases simultaneously with drug release remains constant during tirne of drug release increases during tirne of drug release Time of drug solubilization inside the matrix zero of very short long long prior to release Danger of drug decomposition prior to release low high high Biodegi-adable drug carries noninvasiveness. The drawbacks of the ana­lytical methods currently employed (size exclusion chromatography, differential scan­ning calorimetry, electron microscopy) neces­sitate sample separation from the biological surrounding which is difficult for micro-and nanoparticulate systems and may lead to arti­facts. Isotopic labelling does not permit the characterization of key processes of drug delivery, such as water penetration and poly­mer degradation. Magnetic resonance based techniques are promising candidates to fol­low BDDS in vivo due to their noninvasive­ness and their sensitivity to water concentra­tion and water mobility. The development of low frequency EPR spectrometers makes it now feasible to conduct noninvasive mea­surements on living mammals.t2 The sensitiv­ity is high enough to detect free radicals derived from xenobiotics3 or drugs4 and reac­ 6 tive intermediates of metal ions.5, Spin trap­ping techniques can be used to detect and 89 image radicals with short half lives.7, , The following examples illustrate how electron paramagnetic resonance spectroscopy (EPR) and nuclear magnetic resonance imaging (MRI) give unique information about the processes of drug delivery and polymer degradation in vitro and in vivo. Results and discussion Gamma irradiation is widely used to sterilize BDDS. Some drug or polymer derived radi­cals which are formed during irradiation are very stable at room temperature under dry conditions. However, they will decay immedi­ately after water induced solubility of the sur­rounding matrix. Therefore, these endoge­nous signals may be used to compare the velocity of water penetration between in vitro and in vivo. The realization of this concept has been demonstrated on gentamicin loaded polyanhydrides, which were subcutaneously implanted in mice.t10 The application of EPR can be expanded to diamagnetic BDDS by the introduction of nitroxyl radicals. Low molecular weight nitroxides may serve as model drugs. Anoth­er possibility is to use spin labelled drugs (for example spin labeled peptides) or spin labelled polymers. A large variety of nitrox­ides permits the choice of the compound with the most appropriate characteristics (mol. weight, hydrophilicity, acidity etc.). The EPR spectra give information about: l. nitroxide concentration (by double integra­tion of the EPR spectra) 2. micropolarity (by the hyperfine coupling constants) 3. microviscosity (by the shape of the EPR spectra) which can be used to elucidate the release mechanism. Figure 2 demonstrates how the percentage of water solubilized and nonsolu­bilized nitroxides can be estimated by spec­tral simulation. The percentage of undis­solved nitroxides is easily underestimated in the EPR spectrum due to the large line width which leads to small signal amplitudes. Therefore, the integration of spectra is desir­able. It is possible to indicate on the mecha­nism of drug release directly from the infor­mation of the EPR spectra. Results of previ­ous studies demonstrate that diffusion con­trolled processes contribute to the release 12 13 mechanism of clinically used polymers.t11,, Erosion controlled release was observed only in the case of the polyanhydride bis­carboxyphenoxypropane, a polymer which is used clinically to deliver BCNU against glioma.13 The EPR method is also able to fol­low complexe release mechanisms: Drug release from poly(fatty acid dimer-sebacic acid) polymers involves water penetration, polymer degradation with precipitation of the monomers and incorporated drug mole­cules, resolubility and diffusion.12 Further information on the release processes can be 100 % mobile 50 % mobile 33.3 % mobile Figure 2. Spectral simulation of the EPR spectra (l. derivative of microwave absorption, left) and their inte­grated form (right) of the superposition of water solubi­lized, mobile nitroxides and non solubilized nitroxide molecules. Note that the signal amplitude of the water solubilized nitroxides is much higher than the signal amplitudes of the nonsolubilized form due to the narrow lineawidth. achieved by simultaneous monitoring of dis­tinct polymer layers which can be realized in vitro by spectral-spatial EPR-imaging.14 The spatial resolution of the current in vivo EPR imaging machines is in the range of few mil­limeters 15 and not sufficient to resolve het­erogeneity within a millimeter sized implant. However, a distinct regions of the implant can be separated by means of different nitroxide isotopes.13 An important parameter is the pH inside the degrading polymer matrix. The acidity influences the polymer degradation rate, sol­ubility of the incorporated drug and drug sta­bility. An acidic pH may result from the poly­mer degradation, because the polymers which are clinically used degrade into a­hydroxyacids (polyesters) or dicarboxylic -x _NH . \_+/ x.:x -H+ :o -H+ N)(_ :Q: g = 2.0054 g =2.0057 aN = 1.555 mT aN 1.43 mT 2aN = 3.ll mT 2aN = 2.86 mT pH 2.5 4.7 8.0 Figure 3. Top: Basic principle of the pH-measurement by imidazolidine nitroxides. Protonation of the nitrogen in position 3 decreases the spin density of the nitrogen atom of the radical moiety, which results in a decreased hyperfine splitting constant and an increased g-value. Bottom: Influence on the pH on the experimental 1.1 GHz EPR-spectra (left) and their integrated form of the pH-sensitive nitroxide 2,2,3,4,5,5-Hexamthyl-imidazoli­dine-1-oxid. The EPR spectrum at pH = pKa = 4.7 results from a superposition of the protonated and the nonpro­tonated form of the radical. Biodegradable drug earries acids (polyanhydrides). However, the acidity inside the matrix is difficult to predict due to an uncertain monomer concentration and possible influences of incorporated drugs or penetrating ions. There was no information available about the acidity of the microenvi­ronment inside BDDS in vivo due to the lack of suitable techniques. The development of pH-sensitive nitroxides 16 made it possible to study the pH inside degrading polymers non­invasively and continuously in vivo (Figure 3) A pH drop from 5 to 2 was observed in biodegradable polyester implants in mice.t11 Clearly, such an acidic microenvironment may lead to drug decomposition prior to drug release. Complementary information can be obtained by the combination of the EPR stud­ies with nuclear magnetic resonance imag­ing.12,13 MRI provides information concern­ing the implant shape and size, edema and encapsulation. The cause of incomplete nitroxide release was found by the MRI detection of the encapsulation of the implant.t12 Care must be taken to conclude from low MRI contrast to the absence of water inside the implant, because small pore sizes may lead to very short relaxation times. Therefore, drug release may be completed, although no increase in MRI signal intensity was observed.12,13 This water can be detected indirectly by EPR using spin probes. In summary, drug release from BDDS is a promising approach in the field of cancer treatment. Magnetic resonance techniques EPR and MRI can provide unique and addi­tional information needed to understand the mechanisms of drug release and polymer degradation. They permit also the in vivo characterization of submicron sized delivery systems,17 which are otherwise only detected by radiolabeling. Ongoing developments in the field of EPR-imaging will result in new opportunities to monitor the localization and physical state of the delivery system (hydra­tion, microviscosity, micro-pH). Acknowledgment The author acknowledges the cooperation with the laboratory of Prof. HM Swartz (Dartmouth Medica! School, NH, USA) and Prof. A. Domb (Hebrew University Jeru­salem, Israel). This work was supported by grants from the German Academic Exchange Service (DAAD) and the German Research Foundation (DFG MA 1648/1-1). References l. Wu MP, Tamada JA, Brem H, Langer R. In vivo versus in vitro degradation of eontrolled release polymers for intracranial surgery. J Biomed Mat Res 1994; 28: 387-95. 2. Eaton GR, Eaton SS, Ohno K. EPR-Imaging and in vivo EPR. Boca Raton: CRC press, 1991. 3. Fuji H, Zhao B, Koscielniak J, Berliner J. In vivo EPR studies of the metabolic fate of nitrosoben­zene. Magn Res Med 1994; 31: 77-80. 4. Miider K, Bacic G, Swartz HM. In vivo detection of anthralin derived free radicals in the skin of hair­!ess mice by low frequency electron paramagnetic resonance spectroscopy. J Invest Dermatol 1995; 104: 514-7. 5. Liu K, Jiang J, Swartz HM, Shi X. Low frequency detection of chromium-(V) formation by chromi­um-(VI) reduction in whole mice. Are/z Bioehem Biophys 1994; 313: 248-52. 6. Liu KJ, Miider K, Shi X, Swartz HM. Reduction of carcinogenic Cr(VI) on the skin of !iving rat. Magn Res Med 1997; 38: 524-6. 7. Halpern HJ, Yu C, Barth E, Peric M, Rosen GM. In situ detection, by spin trapping, of hydroxyl radi­cal markers produced from ionizing radiation in tumor of a living mice. Proc Nat Aead Sci 1995; 92: 796-800. 8. Jiang JJ, Liu KJ, Jordan SJ, Swartz HM, Mason RP. Detection of free radical metabo!ite formation using in vivo EPR spectroscopy -evidence for rat hemoglobin thiyl radical formation following administration of phenylhydrazine. Are/z Bioehem Biophys 1996; 330: 266-70. 9. Yoshimura T, Yokoyama H, Fujii S, Takayama F, Oikawa K, Kamada H: In-vivo EPR detection and imaging of endogenous nitric-oxide in lipopolysac­charide-treated mice. Nature Biotechnol 1996; 14: 992-4. 10. Mader K, Domb A, Swartz HM. Gamma steriliza­tion induced radicals in biodegradable drug deliv­ery systems. Appl Rad Isot 1996; 47: 1669-74. 11. Mader K, Gallez B, Liu KJ, SwartzHM. Noninva­sive in vivo characterization of release processes in biodegradable polymers by low frequency Elec­tron Paramagnetic Resonance Spectroscopy. Bio­materials 1996; 17: 459-63. 12. Mader K, Cremmilleux Y, Domb A, Dunn JF, Swartz HM: In vitro / in vivo comparison of drug release and polymer erosion from biodegradable P(FAD-SA) polyanhydrides -a noninvasive approach by the combined use of Electron Para­magnetic Resonance Spectroscopy and Nuclear Magnetic Resonance Imaging. Phannaceut Res 1997; 14: 820-6. 13. Mader K, Bacic G, Domb A, Elmalak O, Langer R, Swartz HM. Noninvasive in vivo monitoring of drug release and polymer erosion from biodegrad­able polymers by EPR spectroscopy and NMR imaging. J Phann Sci 1997; 86: 126-34. 14. Mader K, Nitschke S, Stosser R, Borchert HH, Domb A. Nondestructive and localised assesment of acidic microenvironments inside biodegradable polyanhydrides by spectral spatial Electron Para­magnetic Resonance Imaging (EPRI). Polymer 1997; 38: 4785-94. 15. Alecci M, Ferrari M, Quaresima V, Sotgiu A, Ursi­ni CL. Simultaneous 280 MHz EPR imaging of rat organs during nitroxide radical clearance.Bioplzys J 1994; 67: 1274-9. 16. Khramtsov VV, Weiner LM: Proton exchange in stable nitroxyl radicals: pH sensitive spin probes, In: Volodarsky LB (ed.) Imidazoline nitroxides. Boca Raton: CRC press 1988: Vol. 2, 37-80. 17. Yamagnchi T, Itai S, Hayashi H, Soda S, Hamada A, Utsumi H. In vivo ESR studies on pharmacoki­netics and metabolism of parenteral lipid emul­sion in living mice. Plzannaceut Res 1996; 13: 729­33. Bone marrow toxicity and antitumor action of adriamycin in relation to the antioxidant effects of melatonin Valentina Rapozzi1, Laura Perissin1 , Sonia Zorzet2 , Marina Comelli1, Irene Mavelli1 , Marjeta Sentjurc3 , Alja Pregelj3 , Milan Schara3 and Tullio Giraldi1 1 Department of Biomedical Sciences and Teclmologies, University of Udine,2 Department of Biomedical Sciences, University of Trie ste, Italy, 3 ]ozef Stefan Institute, Ljubljana, Slovenia Melatonin has been reported to possess numerous properties, including antioxidant ejjects. Some antitu­mor drugs, such as anthracyclines, display a pro-oxidant activity which is held responsible jor their toxi­city to normal tissues oj the host. The aim oj this work was, therejore, to preliminarily examine the ejjects oj melatonin on the bone marrow toxicity caused by the treatment with adriamycin in CBA mice bearing TLX5 lymphoma. Ajter a single treatment with adriamycin (28-40 mg/kg i. v.), the administration oj a single pharmacological dose oj melatonin (10 mg/kg s.c.) reduced the acute mortality oj the hosts Jrom 9/16 to 2/16. The antitumor action oj adriamycin, consisting in the increase in survival time oj ani­mals which were not ajjected by the acute toxicity oj the drug, was not reduced by melatonin. Melatonin also attenuated the reduction in the number oj bone marrow GM-CFU caused by adriamycin, and signifi­cantly restored the reduced and total glutathione leve/s. Moreover, the use oj Fenton reaction and jree radical determination via spin trapping, show that melatonin acts as a direct jree radical scavenger. The data reported indicate that melatonin attenuates the bone marrow toxicity oj adriamycin with a mecha­nism consistent with its antioxidant properties. Key words: lymphoma; doxorubicin-adverse ejjects; bone marrow; melatonin; mice Introduction The pineal gland and its indole hormone, melatonin, have been shown in numerous experimental studies to be involved in cancer progression. In the 30's, Engel suggested a link between the pineal gland and cancer.1,2 Cancer treatment with pineal extracts has Correspondence to: Prof. Tullio Giraldi, Department of Biomedical Sciences, University of Trieste, Via L. Giorgieri 7, 34100 Trieste, Italy. Te!: +39-40-6763539; Fax: +39-40-577435; E-mail: giraldi@univ.etrieste.it been performed later in the clinic, resulting in a reported retardation in the progression of the disease and in an improvement of the quality of life of the patients.3 The role of pineal gland and of melatonin for cancer growth has been investigated rather extensively in laboratory animals. Sur­gical pinealectomy resulted in the increased growth in vivo of different types of experi­mental tumors.4-8 Tumor growth was corre­spondingly attenuated in pinealectomized animals by the administration of exogenous Rapozzi Vet a/. melatonin.t9-11 Tumor growth inhibition in vivo and in vitro following treatment with melatonin in non pinealectomized animals has been de seri bed in some instance s, 1 2-16 although contrasting reports showing a stim­ulation of tumor growth are also available in 18 the literature.17, In Lewis lung carcinoma bearing mice melatonin has been shown to increase the therapeutic index of the antitumor drugs cyclophosphamide and etoposide, since it protects the bone marrow stem cells from the apoptosis induced by these drugs while it does not reduce their antitumor action. This effect of melatonin was suggested to occur via interaction with its receptors on T-helper lymphocytes in the bone marrow, 19 leading to the stimulation of the production of a Th cell factor constituted of two cytokines named MIO (melatonin induced opioids). In turn, this factor would act on bone marrow stromal cells inducing the release of hematopoietic growth factors.20 On the other hand, melatonin has been shown to cause potent direct antioxidant 22 effects, rapidly scavenging hydroxyl21,and peroxyl radicals.23 Additionally, melatonin can also upregulate endogenous antioxidant defenses, as shown for glutathione peroxi­ 25 dase activity.24, The antioxidant action of melatonin is also supported by experiments indicating that it decreases the DNA damage caused by ionizing radiation in cultured cells,26 the in vivo cataract formation induced 28 by BSO in rats,27,the DNA damage caused by chemical carcinogen safrol, 29 as well as the kainate excytotoxicity in cerebellar gran­ular neurons. 30 The anthracycline antitumor drug, adri­amycin, is being widely used in the clinic. The most serious adverse effects limiting the applicable 600 mm3 . PDT involved i.p. injection of 40 mg kg-1 of haematopor­phyrin esters in saline, followed 24 h later by illumination with 630 nm light at 100 m W cm-2 from a laser, to a dose of 80-150 J cm-2. Electrotherapy involved insertion of a gold needle electrode into the tumour while the animal lay on a copper plate counter-elec­trode, covered with a conducting gel (Drac­ard, Maidstone, UK) and a current of 5 mA was passed for 15-30 min. For radiotherapy, tumours were exposed to 300 kV X-rays at a