Total tissue lactate dehydrogenase activity in endometrial carcinoma.

Lactate dehydrogenase (LDH) is essential for continuous glycolysis necessary for accelerated tumor growth. The aim of this study was to reconsider if assay of total tissue activity of this enzyme could be useful as marker for endometrial carcinoma (EC). Activity of LDH was measured spectrophotometrically in homogenate supernatants of uterine tissue samples of 40 patients (10 normal endometria, 27 normal myometria, and 33 EC), including 30 matched pairs. Data obtained were analyzed in relation to clinical and histopathologic findings and compared with our previously published results on the tissue levels of the same enzyme in ovarian cancer and on the proteolytic activity of dipeptidyl peptidase III (DPP III) in EC (suggested biochemical indicator of this malignancy). Significantly increased (1.8-3.0 times; P < 1 x 10(-4)) LDH activity was observed in EC samples if compared with normal uterine tissues. This rise was not related to the clinicopathologic findings, however. In contrast to previous results on LDH in ovarian carcinomas, a significant rise in LDH activity was found already in grade 1 EC. Using the cutoff value of 1.06 U/mg, diagnostic sensitivity of 82%, specificity of 100%, and accuracy of 91% for total tissue LDH assay have been calculated. A correlation of tissue's LDH and DPP III activities was found, and their combined assay for EC showed increased diagnostic sensitivity (94%) and accuracy (96%).

Selective cytotoxicity of diazenecarboxamides towards human leukemic cell lines.

In this study, the cytotoxicity of 16 diazenes towards four human leukemic cell lines was tested. Regarding their structure these 16 diazenes belong to three subclasses: diazenecarboxamides (11 compounds), diazenedicarboxamides (4 compounds) and alkyl aminocarbonyldiazenecarboxylate (1 compound). The leukemic cell lines used in this study were NALM-1, JURKAT, HL-60 and K-562. Fifteen out of 16 tested diazenes were cytotoxic towards the leukemic cell lines: 11 with high efficacy (IC(50)<50 microM) at least towards two to three leukemic cell lines, and 4 with medium efficacy (IC(50)>50 microM). Ten out of these 11 diazenes have a common structure and belong to the subclass of diazenecarboxamides. Five diazenes (SB-681, LK-34, UP-39, JK-1197, UP-11) were highly cytotoxic (IC(50) values 3.3-38.9 microM) towards all four leukemic cell lines. The selectivity of the cytotoxicity towards leukemic cells was tested by using resting and Con-A-stimulated peripheral blood mononuclear cells (PBMC) isolated from healthy donors and towards normal mouse fibroblast cell line, 3T3. The diazenes cytotoxic towards leukemic cells, did not affect the viability of the resting PBMC suggesting selectivity of their action. Moreover, eight diazenes did not affect the normal dividing cells (Con-A-stimulated PBMC and fibroblasts). Thus, we present eight diazenes which are selectively cytotoxic towards leukemic cells, not affecting normal cells even when activated to proliferation. These compounds may represent new potential agents for the treatment of leukemia patients.

Diazene JK-279 induces apoptosis-like cell death in human cervical carcinoma cells.

Diazene N-phenyl-2-(2-pyridinyl)diazenecarboxamide (JK-279) is a newly synthesized compound, cytotoxic for several tumor cell lines and their drug-resistant sublines. In human cervical carcinoma cells (HeLa), this compound reduced intracellular glutathione content and increased sensitivity to cisplatin. The aim of the present study was to elucidate the molecular mechanisms involved in the cytotoxic effect of diazene JK-279 on HeLa cells. Cytotoxicity was determined by the MTT method. Flow cytometry analysis showed that diazene JK-279 induces G(2)/M phase arrest, mediated by the increase in p21 expression, and accompanied by an alteration in the expression of survivin. The highest concentration of JK-279 altered nuclear morphology in intact cells, showing "apoptosis-like" features. No cleavage of procaspase-3, procaspase-9 and PARP, or altered expression of apoptotic proteins Bcl-2 and Bax were detected. At the same time, PS externalization and internucleosomal DNA cleavage were observed. Partial necrosis was detected as well. Our results demonstrate that cytotoxicity of diazene JK-279 is mostly the consequence of caspase-independent cell death, which is in some aspects "apoptosis-like". Taking into account the multiplicity of mechanisms used by cancer cells to prevent apoptosis, the drugs (like diazene JK-279) that would activate alternative cell death pathways could provide a useful tool for new types of cancer therapy.

The influence of mouse sera, regenerating liver extracts and bacterial products on the abilities of different cells in vitro.

In the complexity of host tumor relations, the regeneration of the tissue in which the tumor is growing, or in some other tissue in the organism, could influence the maturation of tumor cells, i.e. tumor reversion. Clinical observations and experiments on plants, lower animals, or animal embryos, performed by several authors, and our results on the influence of regenerating mouse liver on the abilities of tumor transplanted there or elsewhere in the organism led us to study the in vitro growth of different cells or bacteria exposed to the extracts of normal or regenerating liver and/or sera from these animals. Further, sterile used bacterial media were added to bacterial or cell cultures, respectively. Depending on the model, liver extracts-particularly extracts and sera from mice with regenerating liver-were shown to inhibit radioactive thymidine incorporation in the cells. In these experiments, the number of bacteria or cells per culture was lower than in otherwise treated corresponding cultures. Further, used sterile media of bacterial cultures stimulated the growth of bacteria but inhibited thymidine incorporation into fibrosarcoma cells in vitro. Whether this means that one or several common regulators exist in nature appears as an intriguing, but still completely open question. The idea of controlling tumor growth by using such regulatory growth factors seems very provocative.

Dipeptidyl peptidase III in malignant and non-malignant gynaecological tissue.

Exopeptidases, in contrast to endopeptidases (proteinases) have been much less studied in relation to cancer. The aim of this study was to investigate one such enzyme, dipeptidyl peptidase III (DPP III), in gynaecological tissues, by measuring both the enzyme activity and enzyme content. DPP III activity was assessed in normal (n = 65), benign (n = 9) and malignant (n = 51) gynaecological tissues. A statistically significant higher DPP III activity was observed in endometrial (n = 40, P = 4.6 x 10(-7)) and ovarian (n = 11, P = 8.1 x 10(-4)) malignant tumours, whereas no significant difference was detected for leiomyomas (n = 8), if compared to the activity in normal tissue. A matched pair analysis of normal and cancerous endometrial tissue confirmed the significance of the DPP III activity increase in the transformed tissue (n = 7, P = 0.022). Western blot analysis revealed a significantly (P = 0.014) increased level of DPP III in endometrial cancer. Further, regression analysis showed a positive correlation between the activity and the content of DPP III in normal tissue (r = 0.637, P = 0.047) and in endometrial cancer (r = 0.574, P < 0.007). The increase of the DPP III activity was observed in the endometrial carcinomas of various histological types, grade or the depth of myometrial invasion. The easy-to-perform determination of this exopeptidase activity may serve as a potential indicator of endometrial and ovarian malignancies.

Multiple fractions of gamma rays induced resistance to cis-dichloro-diammineplatinum (II) and methotrexate in human HeLa cells.

Previous irradiation could induce changes in the cell-sensitivity to additional cytotoxic agents. In this study we examined whether the sensitivity to additional cytotoxic agents was affected in cells irradiated with multiple fractions of gamma rays if these agents were given at the time when the lesions induced in DNA by radiation have already been repaired. Human cervix carcinoma HeLa cells were irradiated daily with 0.5 Gy of gamma rays five times a week for 6 weeks. When the fractionation regimen was completed, that is when the cells had accumulated the total dose of 15 Gy of gamma rays, the sensitivity of these cells to gamma rays, UV light, cis-dichlorodiammineplatinum (II) (cis-DDP), methotrexate (MTX), and hydroxyurea (HU) was examined and compared to control cells. Results revealed that preirradiated cells did not change sensitivity to gamma rays and UV light, but that they increased the resistance to cis-DDP, and MTX (especially for higher concentrations of MTX), and increased sensitivity to HU (for lower concentrations of HU). The increased resistance to cis-DDP was also measurable up to 30 days after the last dose of gamma rays. The results indicate that preirradiation of HeLa cells with multiple fractions of gamma rays could change their sensitivity to additional cytotoxic agents, and that this is a relatively long-lasting effect. Our results suggest that caution is needed in medical application of radiation combined with chemical treatment.

The influence of cathepsin B and leupeptin on potentially lethal damage repair in mammalian cells.

Cell response to irradiation depends on many micro-environmental and intracellular factors. It is known that proteinases control many physiological functions and are also involved in progression of the cell cycle. They also could be involved in cell response to irradiation. In this work the influence of cathepsin B, which is one of the important lysosomal proteinases, and one of its inhibitors, leupeptin, on the potentially lethal damage repair (PLDR) was studied. Chinese hamster V79 cells were irradiated with gamma rays in the plateau-phase of growth. Immediately after irradiation cathepsin B or leupeptin were added to the growth medium. Four hours later, a determined sufficient period of time for maximal PLDR, the cells were replated to assess survival and mutation induction. Mutation frequency was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus using resistance to 6-thioguanine (6-TG). Simultaneously, the activity of cysteine, aspartic and serine proteinases were determined at different postirradiation intervals. The results show that when plateau-phase cells were incubated with cathepsin B during the postirradiation interval strong inhibition of PLDR was observed, accompanied with a reduced number of 6-TG resistant mutants. If leupeptin was added, more modest inhibition of PLDR was observed, accompanied with only slight reduction in the mutation frequency. The addition of cathepsin B or leupeptin to irradiated cells modified the activities of intracellular proteinases. As the highest alterations in proteinase activities were observed at the time when maximum repair of DNA lesions occurred, the biological consequences could involve a series of sequential steps in intracellular proteinase activities.

Alteration of NK- and ADCC-activities in rats genetically selected for low or high platelet serotonin level.

By selective breeding we have recently obtained two discrete sublines of rats that differ in serotonin content in their platelets. As both serotonin and platelets may influence, or even take part, in immune reactions, we tested in this work the natural cytotoxicity in rats with constitutionally different platelet serotonin levels (PSL). Rats with low platelet serotonin level (mean +/- SD, 1.26 +/- 0.14 micrograms 5HT/mg protein; 81% vs. controls) had significantly higher (P less than 0.001) natural killer (NK) activity (mean +/- SD, 9.1 +/- 3.9%) than control rats with average PSL (1.57 +/- 0.18 micrograms 5HT/mg protein). On the contrary, rats with constitutionally high PSL (2.42 +/- 0.21 micrograms 5HT/mg protein, 154% vs. controls) had somewhat lower (P less than 0.02) NK activity (4.1 +/- 1.7%) than control animals (5.7 +/- 1.9%). Antibody-dependent cellular cytotoxicity (ADCC) against nucleated targets of the RCH line, detecting lymphoid effectors, as well as ADCC against chicken red blood cells (CRBC), detecting predominantly non-lymphoid effectors, were also significantly higher (P less than 0.001) in rats with low PSL (19.6 +/- 6.8% vs. 6.6 +/- 3.1% in controls for lymphoid effectors, and 71.8 +/- 6.1% vs. 48.7 +/- 8.8% in control rats for non-lymphoid effectors). However, no significant alteration of either ADCC was determined in rats with high PSL. The results suggest in vivo regulation of natural cytotoxicity by serotonin.

Repeated doses of gamma rays induce resistance to N-methyl-N'-nitro-N-nitrosoguanidine in Chinese hamster cells.

Chinese hamster V79 cells were preirradiated repeatedly with gamma rays and then exposed to ultraviolet (uv) light or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The cell killing and induction of mutation at the hypoxanthine-guanine phosphoribosyltransferase locus were examined following these treatments. Cells preirradiated with multiple fractions of gamma rays exhibit the same sensitivity to uv light as the control cells with respect to cell survival and mutation induction. Following treatment with MNNG, resistance to cell killing was observed along with a decreased frequency of mutations induced. These results indicate that the progeny of cells irradiated with multiple fractions of gamma rays could display subsequent changes in sensitivity to lethal and mutagenic effects of additional treatment with DNA-damaging agents.

Multiple, small exposures of far-ultraviolet or mid-ultraviolet light change the sensitivity to acute ultraviolet exposures measured by cell lethality and mutagenesis in V79 Chinese hamster cells.

Chinese hamster V79 cells (subline MI2G) were exposed repeatedly to fractionated doses of germicidal 254 nm light (far-uv) at 6 J.m-2/fraction/day or sunlight-simulating 290-330 nm (mid-uv) at 150 J.m-2/fraction/day and sensitivities to cell killing action and mutation of far-uv and mid-uv were examined. As the number of exposure fractions increased, the cell cultures became resistant to cell killing induced by both far-uv and mid-uv. Increases in both Do and Dq were observed. Treatment with exposures of 6 J.m-2 far-uv is more efficient in yielding cell cultures that are resistant than exposures of 150 J.m-2 mid-uv. In contrast to the cells exposed to repeated far-uv, the cells exposed to repeated mid-uv were relatively more resistant to cell killing effects of mid-uv than far-uv, suggesting a possible role of photolesions other than pyrimidine dimers. When mutants resistant to 6-thioguanine were assayed during repeated exposure to far- or mid-uv light, the yield was initially linear with accumulating dose. At high total accumulated doses, the frequency decreased gradually (6 J.m-2 mid-uv) or reached a plateau (150 J.m-2 mid-uv). The sensitivity of N80 cells (exposed to 80 fractions of mid-uv) to mutation induction by uv light is higher than that of the original MI2G cells, whereas U81 cells (exposed to 81 fractions of far-uv) have a sensitivity similar to that of the original cells. Although an initial decrease in resistance to cell killing was observed, resistant cells retained their characteristics after 100 days in culture without further exposure. Cross-resistance to X rays was not shown. The data in this paper suggest that the capacity for repair of photolesions in DNA by repair processes was enhanced in cell cultures by repeated exposure to far-uv or mid-uv and that this altered the cells' ability to cope with lethal and mutagenic lesions. It remains to be seen if these changes in cell sensitivity were brought about by selective or inductive processes or a combination of both.

Modulation of resistance to cisplatin by amphotericin B and aphidicolin in human larynx carcinoma cells.

The aim of this study was to examine whether resistance to cisplatin [cis-diamminedichloroplatinum (II)] (CDDP) could be overcome by amphotericin B, cyclosporin A and aphidicolin in two sublines of human larynx carcinoma HEp2 cells. The sensitivity of parental and cisplatin-resistant CA3 and CK2 cells to amphotericin B, cyclosporin A and aphidicolin, and also the effects of these drugs (given in maximal nontoxic concentrations) on cisplatin sensitivity were determined by clonogenic survival assay. CA3 and CK2 cells were sensitive to amphotericin B, and resistant to cyclosporin A and aphidicolin, compared with their parental cells. Amphotericin B increased cisplatin toxicity 2-fold in CA3 cells and 2.7-fold in CK2 cells, while it had no effect in parental HEp2 cells. Cyclosporin A did not influence the sensitivity of examined cells to cisplatin. The sensitizing effect of aphidicolin was more obvious in cisplatin-resistant cells. Cisplatin toxicity was increased by aphidicolin: 1.5-fold in HEp2 cells, 2-fold in CA3 cells, and 1.9-fold in CK2 cells. Therefore, the resistance to cisplatin in human larynx carcinoma CA3 and CK2 cells can be partially reversed by amphotericin B and aphidicolin.

Resistance of human and mouse myeloid leukemia cells to UV radiation.

Sensitivity of mouse bone marrow and myeloid leukemia cells as well as the sensitivity of human myeloid leukemia cells to UV light was tested. Criteria were the in vivo colony-forming ability of UV exposed cells and the inhibition of DNA synthesis during post-irradiation incubation for 24 h in vitro. Mouse bone marrow cells irradiated with a small dose of UV light (5 J/m2) and injected into x-irradiated animals did not form hemopoietic colonies on the recipients' spleens, and the recipients died. However, mouse leukemia cells, after irradiation with higher doses of UV light, retained the ability to form colonies on the spleens, and all recipient mice died with typical symptoms of leukemia. In vitro, mouse bone marrow cells exhibited high sensitivity to UV light as compared to mouse myeloid leukemia cells. Human leukemia cells were also resistant to UV light, but more sensitive than mouse leukemia cells. These results indicate that myeloid leukemia cells are resistant to UV light as compared with normal bone marrow cells.

In vivo bidirectional regulation of mouse natural killer (NK) cell cytotoxic activities by Leu-enkephalin: reversibility by naloxone.

Intraperitoneal injection of Leu-enkephalin (LENK, 10 or 7.5 mg/kg) induced bidirectional modulation of natural cytotoxic activities in spleens of CBA mice (suppression followed by enhancement). NK-cytotoxic activity was more affected than the ADCC. Early suppression of NK activity could be reversed by 4 x M excess of naloxone injected 20 min before LENK, suggesting that the suppression was mediated by opioid receptors. Subsequent increase of NK activity could not be abrogated by naloxone, at least not completely. Naloxone itself decreased NK activity 12 hours after treatment, but enhanced ADCC at 24 and 48 hours. This increase was abrogated by LENK. In addition to functional alterations, LENK also induced phenotypic changes of spleen cells, i.e. a decrease in the percentage of asialo-GM-1+ cells 24 hours posttreatment. There was no correlation between LENK-induced alterations of cytotoxic function and the percentage of cells with NK phenotype (GM-1+). Thus, LENK modulates cytolytic functions and the phenotype of NK cells in vivo in a complex way, which besides opioid mechanisms may also include non-opioid ones.

Drug-resistant human laryngeal carcinoma cells have increased levels of cathepsin B.

In our previous work we showed that the drug-resistance of cervical carcinoma, laryngeal carcinoma and glioblastoma cells may be accompanied by increased levels of tumor markers for invasion and metastasis (i.e. urokinase-type plasminogen activator, plasminogen activator inhibitor type 1, and/or cathepsin D). In the present study we examined the concentration of cathepsins B, L and H in three drug-resistant clones isolated from human laryngeal carcinoma (HEp2). The basal levels of cathepsins B, L and H were determined by enzyme linked immunoabsorbent assay (ELISA). Our results showed that all three clones had an increased level of cathepsin B (in two clones an almost 4-fold increase was determined). The level of cathepsin L was altered (increased) only in VK2 clone, while the levels of cathepsin H were similar in parental cells and drug-resistant clones. Thus, our results suggest that drug-resistance may be accompanied by an increased level of cathepsin B, i.e. tumor associated protease, involved in invasion and metastasis.

Drug resistant tumor cells have increased levels of tumor markers for invasion and metastasis.

The association between drug-resistance and three markers for invasive capacity: cathepsin D (Cath D), urokinase type plasminogen activator (uPA) and inhibitor of plasminogen activator type 1 (PAI-1) was examined in nine cervical and laryngeal carcinoma cell lines resistant to different cytostatics. The level of Cath D was measured by solid phase two-site immunoradiometric assay, while uPA and PAI-1 concentrations were determined by use of ELISA. All drug resistant cell lines had increased concentration of cathepsin D. uPA levels were similar in parental and drug resistant cervical carcinoma cells, but significantly higher in all examined drug resistant laryngeal carcinoma cells. In cervical carcinoma cells, PAI-1 concentrations were similar in parental and cisplatin resistant, but significantly higher in doxorubicin resistant cells. In laryngeal carcinoma cells, no increase in concentrations of PAI-1 was determined in the three from five resistant cell lines. There was no uPA in conditioned medium of parental or drug resistant cells. PAI-1 was detected in conditioned medium. Its levels were significantly increased in the medium of two cervical and three laryngeal drug resistant carcinoma cells. Thus, our results suggest that drug-resistance may be accompanied by increased levels of tumor associated proteases and/or its inhibitor.

Multiple small exposures of filtered mid-UV radiation increase the resistance of Chinese hamster cells to far-UV, mid-UV and filtered mid-UV radiation.

The response of Chinese hamster cells (V79-MI2G) to multiple, low doses of filtered mid-UV radiation (wavelengths longer than 300 nm) were examined over an exposure period of 30 days. Cell survival and the induction of mutation at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus using resistance to 6-thioguanine (TG) were the endpoints in this study. With increasing total accumulated dose given at 500 J/m2/day as a single short exposure, an increased resistance to cell killing was observed. This increase in resistance to cell killing was accompanied by a gradual decrease in sensitivity to the induction of mutants resistant to 6-TG. Above total accumulated doses of 5000 J/m2 the frequency of 6-TG resistance did not increase. After multiple doses of filtered mid-UV radiation the cells became more resistant to subsequent challenges with acute doses of far-UV, mid-UV or filtered mid-UV. The increased resistance to the cell killing action and to the mutation induction by UV suggests that during exposure to low, multiple doses of filtered mid-UV radiation the cells become adapted to the damaging effects of filtered mid-UV radiation.

Modification of potentially lethal damage repair by some intrinsic intra- and extracellular agents: I. Proteinases and proteinase inhibitors.

The effects of nine intra- and extracellular proteinases and six proteinase inhibitors on the repair of potentially lethal damage (PLDR) induced by gamma-rays in plateau-phase V79 cells were examined. It was demonstrated that these agents, which are intrinsic factors produced within mammalian cells, can modify PLDR activity. A stimulatory effect on PLDR was seen with calf liver neutral proteinase, and to a lesser extent, with inhibitor pepstatin A. Other proteinases which belong to serine, cysteine and aspartic superfamilies, as well as proteinase inhibitors, inhibited PLDR to different degrees. The effects of some of these agents, present during the PLDR period, on the rate of tritiated thymidine incorporation into the acid-insoluble cell fraction was also examined. They can modify the DNA synthesis of cells when subcultured from plateau phase for the assessment of colony-forming ability. There is no clear evidence that the effects observed are entirely attributed to the alteration of cellular proliferative processes. It seems more likely that many serine and cysteine proteinases and their inhibitors can adversely affect the PLDR process by modulating the activity of proteinase(s) and other enzymes involved more directly in PLDR because of interrelationships of the entire intracellular proteinase system.

Chromosomal analysis of Chinese hamster V79 cells exposed to multiple gamma-ray fractions: induction of adaptive response to mitomycin C.

Chinese hamster V79 cells were irradiated daily with 0.3 Gy of gamma-rays 5 times per week for 12 weeks (total 18 Gy). These cells were challenged with an additional dose of 15. Gy gamma-rays or treated with 5 micrograms/ml of mitomycin C (MMC) for 2 h. In spite of the high total accumulated dose of gamma-rays, the number of chromosomal aberrations and sister-chromatid exchanges (SCEs) did not significantly increase in the preirradiated cells, as compared to control cells. If preirradiated cells were challenged with an additional 1.5 Gy of gamma-rays, an insignificant decrease in the yield of chromatid aberrations was observed. In contrast, preirradiated cells became significantly more resistant to the induction of chromosomal damage when challenged with mitomycin C. Our results suggest that multiple fractions of gamma-rays can induce the adaptive response to mitomycin C in preirradiated cells.

Low doses of gamma-rays can induce the expression of mdr gene.

We reported earlier that human cervical carcinoma HeLa cells exposed to 30 fractions of 0.5 Gy gamma-rays became resistant to cisplatin, methotrexate and vincristine, but retained the same sensitivity to gamma-rays and ultraviolet light. The aim of this study was to examine whether a small number of gamma-ray fractions, with a lower daily dose, may also change the sensitivity of preirradiated cells to different cytotoxic drugs. Using the modified MTT staining procedure, we found that cells preirradiated with 5 or 10 daily fractions of only 0.17 Gy gamma-rays did not alter their sensitivity to mitomycin, cisplatin, methotrexate, 5-fluorouracil, etoposide and doxorubicin. However, 10 fractions of gamma-rays induced resistance to vincristine and vinblastine. Our immunocytochemical experiments using monoclonal antibody JSB-1 show that the plasma membrane P-glycoprotein is involved in the induced resistance to Vinca alkaloids.

Characterization of carboplatin-resistant sublines derived from human larynx carcinoma cells.

In human larynx carcinoma cells, resistance to carboplatin (CBDCA) was induced by continuous five-day exposure of parental lines to the increasing CBDCA concentrations in culture medium, reaching the clinical level of 9.23 micrograms/ml. Three clones were selected and characterized: CBP-3, CBP-6 and CBP-7, CBP-3 clone was 2.0-fold, CBP-6 2.1-fold, and CBP-7 2.9-fold more resistant to carboplatin. The response of these sublines to different cytostatics was compared to the response of the parental cell lines to the same drug. CBP-7 and CBP-6 clones exhibited cross-resistance to cisplatin (cis-DDP), CBP-7 clone became markedly more sensitive and CBP-3 slightly more sensitive to 5-fluorouracil (5-FU), CBP-6 became sensitive to etoposide (Et), CBP-6 became sensitive and CBP-7 resistant to vinblastine (VBL). Other clones did not change change their sensitivity to cis-DDP, 5-FU, Et or VBL. None of the three clones did alter the sensitivity to mitomycin C, doxorubicin (Dox) or vincristine (VCR). There was no change in the growth rate. Glutathione (GHS) levels were elevated in all three clones, but the increase was significant only for CBP-7 clone. Similarly, the activity of glutathione transferase (GST) was elevated in all clones, but this increase was not significant for CBP-7 clone. The analysis of the of c-myc, c-Ha-ras and c-fos genes reveal no change in the c-myc expression, induction of the c-Ha-ras oncogene in CBP-6 and CBP-7 cells, and biochemistry and oncogene expression indicate that the acquired resistance to carboplatin is a complex, multifactorial process in these cells.