We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G 2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G 2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the nonpermissive temperature for Cdc2 function. Failure to abrogate G 2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G 2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G 2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.

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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.

The ability of lonidamine, an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxicity of cisplatin was investigated in human ovarian-cancer cell lines sensitive (A2780) or with experimentally induced resistance (A2780/cp8) to the alkylating agent. A 24-hr post-incubation with 300 microM lonidamine significantly potentiated the activity of a 1-hr cisplatin treatment in both cell lines. In particular, the cisplatin IC50 value was reduced 4-fold in the sensitive line and 5-fold in the resistant line. Flow cytometric analysis showed that, in the resistant cell line, lonidamine alone did not affect cell kinetics, but when given after cisplatin it was able to transform the temporary G2 + M cell accumulation induced by the alkylating agent to a persistent block in S/G2 + M. In the A2780/cp8 cell line, lonidamine was also able to significantly enhance the accumulation of cisplatin-induced DNA interstrand cross-links. Our results suggest that lonidamine can positively modulate the anti-tumor activity of cisplatin in ovarian cancer cells and also indicate that the drug is potentially useful in combination therapy including the alkylating agent for ovarian cancer patients.