Characterization of cell death induced by vinflunine, the most recent Vinca alkaloid in clinical development.

Vinflunine, the most recent Vinca alkaloid in clinical development, demonstrated superior antitumour activity to other Vincas in preclinical tumour models. This study aimed to define its molecular mechanisms of cell killing in both parental sensitive and vinflunine-resistant P388 leukaemia cells. Vinflunine treatment of these cells resulted in apoptosis characterized by DNA fragmentation and proteolytic cleavage of poly-(ADP-ribose) polymerase. Apoptosis-inducing concentrations of vinflunine caused c-Jun N-terminal kinase 1 stimulation, as well as caspases-3/7 activation. This activation of caspases and the induction of apoptosis could be inhibited by the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde. Interestingly, the apoptosis signal triggered by vinflunine in these P388 cells was not mediated through Bcl-2 phosphorylation. In addition, when vinflunine resistance was developed in P388 cells, it was associated with resistance to vinflunine-induced apoptosis, as reflected by a loss of capacity to induce DNA fragmentation and PARP degradation, and characterized by increased levels of Bcl-2 and Bfl-1/A1. Therefore, these data indirectly implicate Bcl-2 and Bfl-1/A1 in vinflunine-induced cell death mechanisms.

Markedly diminished drug resistance-inducing properties of vinflunine (20',20'-difluoro-3',4'-dihydrovinorelbine) relative to vinorelbine, identified in murine and human tumour cells in vivo and in vitro.

PURPOSE: Vinflunine (VFL) is a novel Vinca alkaloid with markedly superior experimental in vivo antitumour activity to its parent molecule, vinorelbine (Navelbine, NVB), against a panel of murine and human tumours. The aim of this study was to establish whether there are differences in the rate and extent of development of resistance, both in vivo and in vitro, to these two newer Vinca alkaloids under identical selection conditions. METHODS: Using P388 leukaemia cells in vivo, it was evident that VFL induced drug resistance far less readily than NVB, as shown by the number of passages required to select for total resistance. Under in vitro conditions, using A549 human lung carcinoma cells, it was also clearly shown by drug sensitivity determinations that VFL was a less-potent inducer of drug resistance than NVB. Resistance resulting from either in vivo or in vitro selection was associated with a classic multidrug resistance profile. Further characterization of the drug-resistance phenotype of the most highly resistant A549 sublines showed that the level of total beta-tubulin expression appeared to be modified exclusively in the NVB-resistant cells. CONCLUSION: The clear demonstration that resistance to VFL developed far less readily than resistance to NVB both in vivo and in vitro may have potential clinical implications.

Preclinical antitumour activity of F 11782, a novel dual catalytic inhibitor of topoisomerases.

F 11782 is a novel inhibitor of topoisomerases I and II, with an original mechanism of action (Perrin et al, 2000). This study, aimed to define its anticancer efficacy against a series of murine and human tumour models, has provided evidence of major antitumour activity for F 11782. This was demonstrated as a high level of activity against the P388 leukaemia, as reflected by increased survival of 143-457%, when administered i.p., p.o. or i.v. as single or multiple doses, and proved consistently superior to etoposide or camptothecin tested concurrently. Single or multiple i.p. doses of F 11782 also proved highly active against the s.c. grafted B16 melanoma, significantly increasing survival (P < 0.001) and inhibiting tumour growth (T/C of 0.3%), again superior to etoposide tested concurrently. Furthermore, F 11782 inhibited the number of pulmonary metastatic foci of the B16F10 melanoma by 99%. In human tumour xenograft studies, multiple i.p. doses of F 11782 resulted in major inhibitory activity against MX-1 (breast) tumours (T/C of 0.1%), as well as causing definite tumour regressions, whereas none resulted from similar experimental treatments with etoposide. Significant activity was also recorded with F 11782 against the relatively refractory LX-1 (lung) xenografts, with an optimal T/C value of 19%. It was notable that the antitumour activity of F 11782 was consistently demonstrated over a wide range of 2-6 dose levels, providing evidence of its good overall tolerance. In conclusion, these results emphasize the preclinical interest of this novel molecule and support its further preclinical development.

Vinca alkaloid-induced tubulin spiral formation correlates with cytotoxicity in the leukemic L1210 cell line.

The ability of a class of C-20' modified vinca alkaloid congeners to induce tubulin spiral formation was investigated relative to their ability to inhibit microtubule assembly, their cytotoxicity against a leukemic cell line, L1210, and their measured and calculated partition coefficients. These studies were prompted by the observation that the energetics of vinca alkaloid-induced tubulin spiral polymers, or spiraling potential, is inversely related to their clinical dosage and are aimed at the long-term goal of developing the ability to predict the cytotoxic and antineoplastic properties of antimitotic drugs. We demonstrate here that vinca-induced tubulin-spiraling potential is significantly correlated with cytotoxicity against L1210 cells. This is consistent with the size of spirals formed being proportional to the relaxation time for polymer redistribution, the lifetime of cell retention, and effects on microtubule ends and dynamics. Spiraling potential also correlates with calculated but not measured partition coefficients. Surprisingly, spiraling potential does not correlate with the ability to inhibit microtubule formation with purified tubulin or microtubule protein. For the set of C-20' modified compounds studied, the largest inhibitory effects on spiraling potential and cytotoxicity are caused by multiple sites of halogen (-F, -Cl) substitution with the introduction of increased rigidity in the ring. This suggests the C-20' position interacts with a hydrogen bond acceptor or an electrophilic region on the protein that electrostatically disfavors halogen substitutions. These studies are discussed in terms of the cellular mode of action of antimitotic drugs, particularly the importance of microtubule dynamics during mitosis and the factors that regulate those dynamics.

F 11782, a dual inhibitor of topoisomerases I and II with an original mechanism of action in vitro, and markedly superior in vivo antitumour activity, relative to three other dual topoisomerase inhibitors, intoplicin, aclarubicin and TAS-103.

PURPOSE: F 11782 (2",3"-bis pentafluorophenoxyacetyl-4",6"-ethylidene-beta-D-glucoside of 4'-phosphate-4'-dimethylepipodophyllotoxin, di-N-methyl glucamine salt) is a newly synthesized dual catalytic inhibitor of topoisomerases I and II with major in vivo antitumour activity. In this study, we compared and contrasted F 11782 with three other known inhibitors of both these nuclear enzymes, namely aclarubicin. intoplicin and TAS-103, and established its novel mechanism of action. METHODS: In vitro growth-inhibitory effects against a panel of murine and tumour cell lines were measured by cell counting, clonogenicity or tetrazolium metabolic dye (MTT) assays. In vivo antitumour activities were evaluated against two murine tumour models (i.v. P388 leukaemia and s.c. B16 melanoma). Finally, interactions with either DNA or DNA-topoisomerases were determined using various methodologies: DNA-intercalator displacement, pBR322 DNA relaxation, kDNA decatenation, topoisomerase II extractability measurements, stabilization of topoisomerase-induced cleavable complexes (CC) in vitro and in cells, and gel retardation assays. RESULTS: F 11782 had a different profile of sensitivities and proved generally less cytotoxic than the other dual inhibitors tested in vitro, while showing significantly superior antitumour activity in vivo. F 11782, which did not stabilize CC either in vitro or in cells, was the only compound of this series capable of inhibiting the catalytic activity of both DNA-topoisomerases without interacting with DNA, and of completely impairing the binding of these nuclear proteins to DNA. Moreover, only cotreatment of cells in vitro with F 11782 enhanced the cytotoxic activity of etoposide. CONCLUSION: These results emphasize the novel mechanism of action of F 11782 vis-a-vis the other dual inhibitors of topoisomerases I and II and so augur well for its future clinical development.

Low-dose twice-daily fractionated X-irradiation of ovarian tumor cells in vitro generates drug-resistant cells overexpressing two multidrug resistance-associated proteins, P-glycoprotein and MRP1.

Failure of chemotherapy is frequently observed in patients previously treated with radiotherapy. To establish a cellular model for examining this resistance phenotype a series of mammalian tumor cell lines were exposed in vitro to fractionated X-irradiation and were then shown to express resistance to multiple antitumor drugs, including vincristine, etoposide and cisplatin. In these experiments the radiation was delivered as 10 fractions of 5 Gy (dose resulting in 1 log cell kill) given intermittently over several months. We now report that a comparable multidrug-resistance profile is expressed by human SK-OV-3 human ovarian tumor cells exposed in vitro to low dose (2 Gy) twice-daily fractions of X-rays given for 5 days on two consecutive weeks, essentially mimicking clinical practice, involving an overexpression of two MDR-associated proteins, P-glycoprotein and the multidrug resistance protein 1 (MRP1), with the latter being readily detectable by immunocytochemistry.

In vitro synergistic effects of vinflunine, a novel fluorinated vinca alkaloid, in combination with other anticancer drugs.

PURPOSE: Vinflunine (20'-20'-difluoro-3',4'-dihydrovinorelbine), a novel derivative of vinorelbine characterized by marked antitumour activity in vivo in a series of experimental murine and human tumours is currently undergoing phase I evaluation. To investigate its potential for inclusion in combination chemotherapy regimens, this preclinical study was undertaken. The in vitro cytotoxicity of vinflunine incubated simultaneously with one of the following drugs was investigated: camptothecin, cisplatin, doxorubicin, etoposide, 5-fluorouracil, gemcitabine, mitomycin C, paclitaxel or vinorelbine. METHODS: The combinations were first evaluated in vitro against the A549 human non-small-cell lung cancer cell line using median-effect analyses. RESULTS: The results revealed synergistic cytotoxicity when vinflunine was combined with cisplatin, mitomycin C, doxorubicin or 5-fluorouracil. Synergy was also observed when testing similar combinations against CCRF-CEM human leukaemia cells. Finally, these findings were comparable with those resulting from such combinations involving vinorelbine instead of vinflunine. CONCLUSION: Vinflunine appears a promising candidate for combining with other anticancer drugs.

F 11782, a novel epipodophylloid non-intercalating dual catalytic inhibitor of topoisomerases I and II with an original mechanism of action.

F 11782, a novel epipodophylloid, proved a potent inhibitor of the catalytic activities of both topoisomerases I and II. Unlike classical inhibitors such as camptothecin or etoposide, F 11782 did not stabilise cleavable complexes induced by either topoisomerases I or II nor did it preferentially inhibit the religation step of the catalytic cycle of either enzyme. F 11782 neither intercalated DNA nor bound in its minor groove, and showed only weak inhibition of the ATPase activity associated with topoisomerase II. F 11782 appeared to act by inhibiting the binding of topoisomerases I and II to DNA in a manner dependent both on drug and enzyme concentrations, via a mechanism not previously described or shared by other known topoisomerase 'poisons' or inhibitors. In contrast, F 11782 had only a weak effect or none at all on various other DNA-interacting enzymes. In conclusion, F 11782, as a non-intercalating, specific catalytic inhibitor of both topoisomerases I and II with an original mechanism of action, may be considered to represent the first of a new class of topoisomerase-interacting agents.

Characterization of the biological and biochemical activities of F 11782 and the bisdioxopiperazines, ICRF-187 and ICRF-193, two types of topoisomerase II catalytic inhibitors with distinctive mechanisms of action.

F 11782 is a newly identified catalytic inhibitor of topoisomerases I and II, without any detectable interaction with DNA. This study aimed to establish whether its catalytic inhibition of topoisomerase II was mediated by mechanisms similar to those identified for the bisdioxopiperazines. In vitro combinations of F 11782 with etoposide resulted in greater than additive cytotoxicity in L1210 cells, contrasting with marked antagonism for combinations of etoposide with either ICRF-187 or ICRF-193. All three compounds caused a G2/M blockade of P388 cells after an 18-h incubation, but by 40 h polyploidization was evident only with the bisdioxopiperazines. Gel retardation data revealed that only F 11782, and not the bisdioxopiperazines, was capable of completely inhibiting the DNA-binding activity of topoisomerase II, confirming its novel mechanism of action. Furthermore, unlike ICRF-187 and ICRF-193, the cytotoxicity of F 11782 appeared mediated, at least partially, by DNA damage induction in cultured GCT27 human teratoma cells, as judged by a fluorescence-enhancement assay and monitoring p53 activation. Finally, the major in vivo antitumor activity of F 11782 against the murine P388 leukemia (i.v. implanted) and the B16 melanoma (s.c. grafted) contrasted with the bisdioxopiperazines' general lack of activity. Overall, F 11782 and the bisdioxopiperazines appear to function as quite distinctive catalytic topoisomerase II inhibitors.

Detection of DNA-strand breaks in cells treated with F 11782, a catalytic inhibitor of topoisomerases I and II.

F 11782, or 2", 3"-bis pentafluorophenoxyacetyl-4',6'-ethylidene-beta-D glucoside of 4'-phosphate-4'-dimethylepipodophyllotoxin 2N-methyl glucamine salt, is a novel fluorinated lipophylic epipodophylloid which has proven cytotoxic activity in vitro and has shown markedly superior antitumour activity in vivo compared to etoposide in various experimental tumour models. However, the precise mechanism(s) of cytotoxicity of F 11782 remains to be defined. In this study, the DNA damaging activity of F 11782 was investigated in GCT27 and C6S cells using, respectively the fluorescence enhancement assay and the technique of DNA alkaline elution. All the results obtained were consistent with induction of DNA damage by F 11782. No evidence of any stabilisation of DNA-topoisomerase cleavable complexes though was obtained with this catalytic inhibitor. Furthermore, such induction of DNA damage has not been reported with other known catalytic topoisomerase inhibitors and so it appears to be unique to F 11782.

Novel artificial endonucleases inhibit base excision repair and potentiate the cytotoxicity of DNA-damaging agents on L1210 cells.

A series of molecules with apurinic/apyrimidic (AP) endonuclease activity targeted to abasic sites in DNA, which incorporate an intercalating moiety linked to a purine base by a polyamino chain and recognize and cleave abasic sites in DNA with high efficiency, has been studied. The aim was first to establish whether these compounds were inhibitors of base excision DNA repair, since abasic sites are generated during this process. Using an extension of a recently established methodology, two members of this series have been identified as definite repair inhibitors. Secondly, the potential of using such compounds as sensitizers of alkylating agents has been investigated by determining the cytotoxic activity of these compounds on L1210 cells in culture. A concentration-dependent potentiation of nitrosoureas has been demonstrated, but interpretation is complicated by the inherent cytotoxic properties of the test compounds themselves. Such molecules, however, provide interesting lead compounds for new strategies aimed at enhancing the cytotoxic potential of clinically useful DNA-damaging agents.

Synthesis and antitumor activity of new glycosides of epipodophyllotoxin, analogues of etoposide, and NK 611.

A series of 3-amino- and 3-alkylamino-2-deoxy-beta-D-ribo- and beta-D-arabino-glycosides of 4'-demethylepipodophyllotoxin have been synthesized by means of an improved trimethylsilyliodide procedure for the podophyllotoxin-4'-demethylepipodophyllotoxin conversion, an efficient and high yielding synthesis of silyl glycoside donors of 3-azido-2,3-dideoxy-beta-D-ribo- and beta-D-arabino-hexopyranosides and stereoselective glycosylations. In vitro evaluation of cytotoxic effects against murine L1210 leukemia critically demonstrates the essential role played by a 4,6-acetal for biological activity. Among the most cytotoxic compounds, 3-amino-2,3-dideoxy- and 3-N, N-(dimethylamino)-2,3-dideoxy etoposide analogues, 17 and 27-29 are at least as potent as etoposide on the in vivo P388 (iv/ip) murine leukemia models. However, surprisingly enough, none of these compounds inhibits the human DNA topoisomerases I or II or binds to tubulin to prevent its polymerization and microtubule assembly. Therefore, their mechanism of action remains to be cleared up.

Vinflunine (20',20'-difluoro-3',4'-dihydrovinorelbine), a novel Vinca alkaloid, which participates in P-glycoprotein (Pgp)-mediated multidrug resistance in vivo and in vitro.

Vinflunine (VFL) is a novel derivative of vinorelbine (NVB, Navelbine), which has shown markedly superior antitumor activity to NVB, in various experimental animal models. To establish whether this new Vinca alkaloid participates in P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), VFL-resistant murine P388 cells (P388/VFL) were established in vivo and used in conjunction with the well established MDR P388/ADR subline, to define the in vivo resistance profile for VFL. P388/VFL cells proved cross-resistant to drugs implicated in MDR (other Vinca alkaloids, doxorubicin, etoposide), but not to campothecin or cisplatin and showed an increased expression of Pgp, without any detectable alterations in topoisomerase II or in glutathione metabolism. The P388/ADR cells proved cross-resistant to VFL both in vivo and in vitro, and this VFL resistance was efficiently modulated by verapamil in vitro. Cellular transport experiments with tritiated-VFL revealed differential uptake by P388 sensitive and P388/ADR resistant cells, comparable with data obtained using tritiated-NVB. In various in vitro models of human MDR tumor cells, whilst full sensitivity was retained in cells expressing alternative non-Pgp-mediated MDR mechanisms, cross resistance was identified in Pgp-overexpressing cells. Differences were, however, noted in terms of the drug resistance profiles relative to the other Vinca, with tumor cell lines proving generally least cross-resistant to VFL. Overall, these results suggest that VFL, like other Vinca alkaloids, participates in Pgp-mediated MDR, with tumor cells selected for resistance to VFL overexpressing Pgp, yet MDR tumor cell lines proved generally less cross resistant to VFL relative to the other Vinca alkaloids.

Requirements for P-glycoprotein recognition based on structure-activity relationships in the podophyllotoxin series.

Podophyllotoxin and epipodophyllotoxin react with tubulin at the same binding site as colchicine, but in contrast to colchicine, do not appear to exert their cytotoxicities by mechanisms dependent on P-glycoprotein (Pgp) expression. To investigate structural requirements for Pgp recognition a series of podophyllotoxin and epipodophyllotoxin derivatives have been synthesized. Their interactions with the multidrug resistance-related protein Pgp have been studied by evaluating their relative cytotoxicities versus P388-sensitive murine leukemic cells and a classic multidrug-resistant (MDR) Pgp-overexpressing subline (P388/ADR), and their relative tubulin polymerization inhibitory activities against microtubular proteins have been determined. Based on tridimensional structure-activity relationships within this series of compounds, structural requirements for Pgp recognition have been identified. Moreover, proposals are made for extending these criteria to other chemical classes of anticancer drugs.

Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid.

This study aimed to define the mechanism of action of vinflunine, a novel Vinca alkaloid synthesised from vinorelbine using superacidic chemistry and characterised by superior in vivo activity to vinorelbine in preclinical tumour models. In vitro vinflunine cytotoxicity proved dependent on concentration and exposure duration, with IC50 values (72-hr exposures) generally ranging from 60-300 nM. Vinflunine induced G2 + M arrest, associated with mitotic accumulation and a concentration-dependent reduction of the microtubular network of interphase cells, accompanied by paracrystal formation. These effects, while comparable to those of vincristine, vinblastine or vinorelbine, were achieved with 3- to 17-fold higher vinflunine concentrations. However, vinflunine and the other Vincas all inhibited microtubule assembly at micromolar concentrations. Vinflunine, like vinblastine, vincristine and vinorelbine, appeared to interact at the Vinca binding domain, as judged by proteolytic cleavage patterns, and induced tubulin structural changes favouring an inhibition of GTP hydrolysis. However, vinflunine did not prevent [3H]vincristine binding to unassembled tubulin at concentrations < or = 100 microM, and only weakly inhibited binding of [3H]vinblastine or [3H]vinorelbine. Indeed, specific binding of [3H]vinflunine to tubulin was undetectable by centrifugal gel filtration. Thus, the comparative capacities of these Vincas to bind to or to interfere with their binding to tubulin could be classified as: vincristine > vinblastine > vinorelbine > vinflunine. By monitoring alkylation of sulfhydryl groups, differential effects on tubulin conformation were identified with vinflunine and vinorelbine acting similarly, yet distinctively from vinblastine and vincristine. Overall, vinflunine appears to function as a definite inhibitor of tubulin assembly, while exhibiting quantitatively different tubulin binding properties to the classic Vinca alkaloids.

Influence of culture media on the morphological differentiation of the PC-3 and DU145 prostatic neoplastic cell lines.

The concept of differentiation of prostate cancer in terms of morphonuclear characteristics and population dynamics was investigated on the PC-3 and DU145 cell lines. A software based on the concept of Voronoi paving was set up in order to characterize the structure of these cell lines growing in vitro on histological slides. The morphonuclear characteristics were assessed by means of the digital cell image analyses of Feulgen-stained nuclei. The in vitro "morphonuclear" and "pseudo-tissular" differentiations of the PC-3 and DU145 cells were described in terms of the use of various culture media, i.e., media supplemented with either 10% (F10 medium) or 1% (F1 medium) fetal calf serum and with (or without) platelet-derived growth factor and dihydrotestosterone (PA10 and PA1 media). The present data reveal that the PC-3 cell line would be more hormone-sensitive than the DU145 one. Indeed, decreasing the FCS concentration in the culture medium while adding DHT and PDGF led to marked modifications to the morphonuclear characteristics of the PC-3 cells, but not to the DU145 cells. These modifications corresponded to an increase in nuclear size occurring concomitantly with chromatin decondensation. In the same way, spectacular modifications in terms of medium-induced pseudo-tissular differentiation were observed in the PC-3 cell line, but not in the DU145 one. Such modifications corresponded to an increase in clone size related to an increase in the mean distances between neighboring cell nuclei in a given clone. Thus, according to the criteria defined in this study, the PC-3 cell line would seem to maintain a higher degree of differentiation than the DU145 cell line.

Characterization of the differentiation of human colorectal cancer cell lines by means of Voronoi diagrams.

This paper describes differentiation in terms of population dynamics through the medium of Voronoi paving which enables (via digital cell image analysis) the structure of human LOVO and HCT-15 colorectal neoplastic cell colonies growing on histological slides to be characterized. Two other tests were also used, i.e., the colorimetric MTT assay that enables the cell growth level to be determined, and a test allowing the assessment of the proliferation index, i.e., the percentage of cells in the S phase of the cell cycle. The results show that these colorectal neoplastic cells exhibited a comparatively high level of organisation in terms of the topographical distribution of nuclei within the clones when the cells were cultivated in media containing even small amounts of fetal calf serum. On the other hand, certain chemically defined media completely overturned this "pseudo-tissular" architecture. Furthermore, the colorectal cells growing in media including fetal calf serum exhibited relatively large and dense clones, undergoing an increase in the density of these clones when hormones were added to the culture medium and, concomitantly, a decrease in their proliferation. In contrast, the cells growing in chemically defined media generally exhibited smaller clones whose cell proliferation was paradoxically greater than that of the cells referred to above. This seems to bring out the importance of the part played by the cell loss factor in this cell population dynamic.

Monitoring of morphonuclear characteristics of hormone-sensitive and insensitive HCT-15 and LOVO human colorectal cells by means of digital cell image analysis.

We recently set up in vitro several human colorectal neoplastic cell lines that we labelled hormone-sensitive (HS) in comparison with the original cell lines which appeared to be rather "hormone-insensitive" (HI). We describe here the cell proliferation rate and the morphonuclear characteristics of the HS and HI variants of the HCT-15 and LOVO human neoplastic colorectal cell lines which were cultured either in serum-supplemented or chemically-defined media. Morphonuclear characteristics were monitored by means of the digital cell image analyses of Feulgen-stained nuclei, while the proliferation activity of the various cell types was assessed by means of the tetrazolium-based compound (MTT) assay. The results show that it is possible to culture human LOVO and HCT-15 colorectal cells in chemically defined media. This said, growth in a chemically defined medium is difficult for these cells and is markedly less sustained than when they are cultured in serum-supplemented media. The transition of the culture from a serum-supplemented medium to a chemically defined one is accompanied by a very marked drop in cell proliferation and a number of profound changes in terms of morphonuclear characteristics. These changes basically involve chromatin decondensation, which occurs as the result of the drop in the number of large chromatin clumps in the nucleus.

Digital cell image analysis of verapamil-induced effects in chemosensitive and chemoresistant neoplastic cell lines.

We used chemosensitive and chemoresistant variants of the neoplastic mouse MXT mammary and human J82 and T24 bladder cell lines to characterize verapamil-induced cell proliferation and morphonuclear modifications in drug-treated and untreated cells. Chemoresistance to vinorelbine (Navelbine, a Vinca alkaloid derivative), to DIAM3 (an investigational alkylating compound) and to Adriamycin (an intercalating agent) in the presence or absence of verapamil was monitored by means of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that verapamil restored a significant level of chemosensitivity in doses such as 1 microM or 10 microM in the three chemoresistant variants. The digital cell image analysis of Feulgen-stained T24-resistant cell nuclei revealed that verapamil restored the drug-treated cell kinetics and morphonuclear features observed in the sensitive counterpart especially with respect to the effects of Adriamycin. Interestingly, verapamil induced a highly significant chromatin decondensation in resistant but not in sensitive variants. Such verapamil-induced decondensation may favour the accessibility of drugs to their DNA targets. Therefore, in addition to the well-known action of the drug on the influx of a cytotoxic compound from the cellular to the intracellular compartment, verapamil might also favour the accessibility of the nucleus, to the drug.

Influence of suramin alone or in combination with DHT and PDGF on the cell proliferation of benign and malignant human prostatic tissues in organ cultures.

We studied the suramin-induced influence on the cell proliferation of 16 benign and 6 malignant lesions of the human prostate maintained in vitro as organ cultures. The cell proliferation was assessed by nuclear labeling with tritiated thymidine autoradiography. We also studied the dihydrotestosterone (DHT)-and platelet-derived growth factor (PDGF)-induced modulation of suramin influence on such prostate organ culture cell proliferation. Our results indicate that more than half of the benign prostatic tissues showed cell proliferation which was modulated by DHT and/or PDGF, while none of the six carcinomas responded to such hormonal stimulation. Suramin alone inhibited the cell proliferation of only 19% of the prostate organ culture under study, while in combination with DHT and/or PDGF this inhibition level reached 48%. However, we occasionally observed that S alone or in combination with DHT and/or PDGF was also able to stimulate prostate cell proliferation. We think that organ cultures of human prostatic tissues might represent a helpful pre-clinical tool to study the anti-tumoral influence of suramin, which is a new antineoplastic generative compound.