Summary A collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in 2 ovarian cancer cell lines with experimentally induced resistance to cisplatin. Loss of mismatch repair proteins (hMLH1, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to BBR 3464. Moreover, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N-ras) level were observed between BBR 3464 and CDDP.
Summary The effect of taxol (TX) and cisplatin (CDDP), singly or in association, was assessed on two human ovarian cancer cell lines, one sensitive (A2780) and one resistant (A2780 cp8) to CDDP. Cell lines showed a similar sensitivity to TX, whereas different cytotoxicity results were obtained in the two cell lines as a function of TX and CDDP sequence. Specifically, TX followed by CDDP induced simply additive effects in both cell lines, whereas the opposite sequence produced antagonistic effects in A2780 cells and synergistic effects in A2780 cp8 cells. TX, with or without CDDP, induced oligonucleosomal DNA fragmentation typical of the apoptotic process, but the biochemical mechanisms undergoing apoptosis were different in the two cell lines. In fact, in A2780 cells, TX (with or without CDDP) treatment markedly increased p53 as well as p2lwafl protein expression. In A2780 cp8 cells, drug treatment enhanced p53 levels, whereas the expression of p21waf1 was always undetectable at mRNA and protein levels. In the latter cell line, a premature activation of p34cdc2 kinase was observed in correspondence with the drug-induced increase in the S-phase cell fraction. Such an activation was not ascribable to an increase in the overall expression of p34cdc2 or cyclin B1 proteins, but to a dephosphorylation of p34cdc2 kinase. Overall, our results indicate that TX-induced apoptosis in human ovarian cancer cells may be sustained by different events at the cell cycle-control level.Keywords: taxol; cisplatin; apoptosis; cell cycle-related proteins; ovarian cancer Taxol (TX), an antimicrotubule agent that stabilizes mitotic spindles, has a well-documented clinical efficacy in a variety of human neoplasms, including ovarian (Einzig et al, 1992), breast cancer (Nabholts et al, 1993) and cutaneous melanoma (Legha et al, 1990). The encouraging responses as a single agent have prompted the activation of trials of TX in association with cisplatin (CDDP) or doxorubicin. The greatest effect of the CDDP-TX combination in experimental systems was observed when TX preceded the alkylator (Jekunen et al, 1994; Leibmann et al, 1994). However, the type of interaction between the two drugs in the different treatment schedules is controversial (Milross et al, 1995;Mohith et al, 1996), and the biochemical mechanisms responsible for such effects have not been clearly identified.Preclinical studies on the mechanisms of CDDP-TX activity have been carried out only on CDDP-sensitive cells. In the present study, we analysed the effect of the two-drug combination in a CDDP-resistant ovarian cancer cell line (A2780 cp8) in comparison with that observed in the parental CDDP-sensitive cell line (A2780) to ascertain whether CDDP resistance interferes with the pattern of the CDDP-TX interaction. Moreover, as previous reports have indicated that cell death induced by TX is sustained by an apoptotic process (Donaldson et al, 1994;Liu et al, 1994;Danesi et al, 1995;Haldar et al, 1996;Wahl et al, 1996), we analysed the occurrence of apoptosis after CDDP-TX expo...
The accumulation and repair of radiation-induced DNA double-strand breaks (dsbs) were determined by neutral filter elution on 20 primary cultures obtained from ovarian cancer and malignant melanoma clinical specimens. The initial frequency of DNA dsbs after exposure to 50 Gy gamma-irradiation varied greatly for the individual cultures. However, melanomas were generally more efficient than ovarian cancers in repairing these DNA lesions (mean percentage of DNA dsb rejoined after 2 h: 83 versus 62%). In 13 of 20 cultures radiosensitivity was also assessed by the Courtenay clonogenic assay. The mean +/- SD of the surviving fraction at 2 Gy (SF2) was slightly higher for melanomas (0.56 +/- 0.25) than for ovarian carcinomas (0.43 +/- 0.23). No correlation was observed between SF2 and in vitro plating efficiencies or any biological characteristics of the tumour cell population, such as proliferative activity and DNA ploidy. Similarly, we failed to find any relation between the initial frequencies of DNA dsbs and SF2 in individual tumours. In contrast, a significant and direct relationship (r = 0.86, p < 0.01) was observed between SF2 and the percentages of DNA dsbs rejoined 2 h after irradiation. In agreement with reported data on human tumour established cell lines, our results indicate that the ability to repair DNA dsbs is an important determinant for radiation response even in primary cultures of clinical tumours.
Mechanisms of resistance to Tomudex include increased thymidylate synthase activity, as well as reduced intracellular drug uptake and polyglutamation. However, little is known about other mechanisms of resistance, such as a possible protection against Tomudex-induced apoptosis mediated by bcl-2 . We transfected the MDA-MB-435 human breast cancer cell line, which is characterized by a mutated p53 gene, with cDNA of the bcl-2 gene and generated two clones (MDA-bcl4 and MDA-bcl7) characterized by bcl-2 expression twofold and fourfold that observed in the control cell clone (MDA neo ). A concomitant overexpression of p21 wafl was also detected in the MDA-bcl7 clone. The MDA-bcl4 clone was three times more resistant to a 24-h Tomudex exposure than the MDA neo clone, whereas the MDA-bcl7 clone was as sensitive to Tomudex as the control cell clone. A lower sensitivity of the MDA-bcl4 clone than MDA neo and MDA-bcl7 clones to 5-fluorouracil and gemcitabine was also observed. No significant difference was noted in the susceptibility of clones to fludarabine and methothrexate. Basal levels of thymidylate synthase activity were superimposable in the three clones. Tomudex induced a marked accumulation of cells in the S phase in all the clones. However, an apoptotic hypodiploid DNA peak and the characteristic nuclear morphology of apoptosis were observed only in the MDA-bcl7 clone after exposure to Tomudex. No difference in the treatment-induced modulation of proteins involved in cell cycle progression (cyclin A, cdk2, pRB, E2F-1) and apoptosis ( bcl-2 , bax ) was observed in the three clones. The only exception was that the expression of p21 wafl in the MDA-bcl4 clone was inducible at a Tomudex concentration much higher than that required to induce the protein in the other clones. Overall, the results indicate that bcl-2 and p21 wafl proteins concur in determining the cellular profile of sensitivity/resistance to Tomudex. © 1999 Cancer Research Campaign
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