Lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in testicular germ cell tumour cell lines

We investigated the role of p53 and of the Bcl-2 family proteins in the apoptotic response of a panel of testicular tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53 gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility to cisplatin-induced apoptosis. In contrast to wild-type p53-containing NT2 and 2102 EP cells, NCCIT (mutant p53) and S2 (no p53 protein) cells were shown to be p53-transactivation defective. However, NCCIT and S2 cells with non-functional p53 were readily triggered into apoptosis by cisplatin, whereas p53-transactivation competent 2102 EP cells failed to undergo cisplatin-induced apoptosis. The defective apoptotic pathway in 2102 EP cells was reflected by a 4-fold decreased sensitivity to cisplatin in the MTT assay. We further demonstrated that the p53-independent differential cisplatin sensitivity among the testicular germ cell tumour (TGCT) cell lines was not due to differences in cellular cisplatin accumulation or DNA platination. The pattern of endogenous expression levels of Bax, Bcl-2, Bcl-x and Bak, which was not modulated by cisplatin treatment, demonstrated that these Bcl-2 family proteins are not involved in drug-induced apoptosis in the TGCT cell lines. Our results suggest a lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in our panel of TGCT cell lines. We conclude that the cisplatin-induced apoptotic pathway in TGCT cell lines might be p53-independent and is probably not associated with differences in the Bcl-2/Bax rheostat.     

p53, mutation frequency and apoptosis in the murine small intestine

Normal function of the p53 gene is integral to the cellular response to genotoxic stress. One prediction arising from this is that p53 deficiency results in an increased mutation frequency. However, limited evidence has been produced in support of this idea. In order to further investigate the in vivo role of p53 in surveillance against mutation, and particularly to address the significance of p53-dependent apoptosis, we scored mutation frequency at the Dlb-1 locus within cells of the intestinal epithelium of animals which were wild type, heterozygous or null for p53 and heterozygous (a/b) at the Dlb-1 locus. Using this assay we have shown that loss of a p53-dependent apoptotic pathway is associated with the detectable acquisition of mutations, but only at high levels of DNA damage. These results question the significance of the immediate 'wave' of p53-dependent apoptosis seen in this tissue, particularly as there was a delayed p53-independent apoptotic pathway. We conclude that loss of p53 function only becomes relevant to the in vivo acquisition of mutations and thus tumorigenesis in certain circumstances.     

Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.     

Insulin and insulin-like growth factor-I signal transduction requires p21ras

We have investigated the role of cellular p21ras protein in insulin and insulin-like growth factor-I (IGF-I) signaling pathways. Insulin stimulation increased Ras-GTP formation in Rat-1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), far greater than in parental Rat-1 fibroblasts, indicating that competent insulin receptors mediate this response. Cellular microinjection of a dominant-negative mutant p21ras protein (N17 ras) or anti-p21ras monoclonal antibody (Y13-259) into HIRc-B cells reduced insulin- and IGF-I-stimulated DNA synthesis by 75-90%. Insulin-induced c-fos protein expression was also inhibited by 74%. Microinjection of oncogenic p21ras (T-24 ras) into HIRc-B cells activated the mitogenic pathway, and coinjection of N17 ras and T-24 ras showed that oncogenic p21ras rescued the cells from the N17 ras blockade. This later finding indicates that T-24 ras acts downstream of N17 ras. In conclusion, 1) microinjection of a dominant interferring ras mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) oncogenic ras protein rescued cells from the N17 ras blockade, indicating that T24 ras action is downstream of the site of N17 inhibition; and 3) p21ras is an intermediate signaling molecule in the insulin/IGF-I signal transduction pathway and is required for gene expression and DNA synthesis.     

UCN-01 in ovary cancer cells: effective as a single agent and in combination with cis-diamminedichloroplatinum(II)independent of p53 status

Our goal was to determine the cytotoxicity of 7-OH-hydroxystaurosporine (UCN-01) as a single agent and in combination with cis-diamminedichloroplatinum(II) (CDDP) in a panel of ovarian carcinoma cells. We sought to examine the role of p53 gene function and alterations in cell cycle progression or other mechanisms of action of UCN-01 including perturbation of the apoptosis pathway mediated by NF-kappaB and Bcl-2/Bax. Cytotoxicity was determined from dose-response curves established by the Alamar blue vital dye indicator assay. Restoration of wild-type p53 in a p53 null cell line, SKOV 3, was achieved by transfection of a p53 expression vector. Cell cycle distribution was measured by fluorescence-activated cell sorting analysis of ethidium bromide-stained nuclei. Apoptosis was measured by quantitative fluorescence microscopy. NF-kappaB DNA binding activity was measured by electrophoretic mobility shift assay. Bcl-2 and Bax levels were determined by Western immunoblotting. UCN-01 was effective as a cytotoxic agent alone and in combination with CDDP in all cell lines studied, regardless of p53 status. The degree of sensitization to CDDP conferred by UCN-01, however, was found to correlate with p53 gene status. p53 wild-type cells seem to be more sensitive to the cytotoxic effects of the combination of UCN-01 + CDDP than the p53 mutant cells. This was confirmed in cells in which p53 wild-type function was restored by transfection of p53 cDNA, but these cells are also significantly more sensitive to CDDP alone. The effects of UCN-01 on cell cycle progression also appear to be p53 dependent but may not be the primary mechanism of action. The rate of apoptosis is increased 4-fold in UCN-01 + CDDP-treated cells compared to either agent alone. UCN-01 does not effect NF-kappaB DNA binding activity or Bcl-2 and Bax levels. UCN-01 enhances CDDP cytotoxicity and apoptosis in ovary cancer cells. This occurs regardless of p53 status, but wild-type p53 seems to increase the degree of sensitization.     

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Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.     

A phase I study of adenovirus-mediated wild-type p53 gene transfer in patients with advanced non-small cell lung cancer

Mutations of the tumor suppressor gene p53 are the most common genetic alterations observed in human cancer. Loss of wild-type p53 function impairs cell cycle arrest as well as repair mechanisms involved in response to DNA damage. Further, apoptotic pathways as induced by radio- or chemotherapy are also abrogated. Gene transfer of wild-type p53 was shown to reverse these deficiencies and to induce apoptosis in vitro and in preclinical in vivo tumor models. A phase I dose escalation study of a single intratumoral injection of a replication-defective adenoviral expression vector encoding wild-type p53 was carried out in patients with incurable non-small cell lung cancer. All patients enrolled had p53 protein overexpression as a marker of mutant p53 status in pretreatment tumor biopsies. Treatment was performed either by bronchoscopic intratumoral injection or by CT-guided percutaneous intratumoral injection of the vector solution. Fifteen patients were enrolled in two centers, and were treated at four different dose levels ranging from 10(7) to 10(10) PFU (7.5 x 10(9) to 7.5 x 10(12) particles). No clinically significant toxicity was observed. Successful transfer of wild-type p53 was achieved only with higher vector doses. Vector-specific wild-type p53 RNA sequences could be demonstrated in posttreatment biopsies of six patients. Transient local disease control by a single intratumoral injection of the vector solution was observed in four of those six successfully transduced patients. There was no evidence of clinical responses at untreated tumor sites. Wild-type p53 gene therapy by intratumoral injection of a replication-defective adenoviral expression vector is safe, feasible, and biologically effective in patients with advanced non-small cell lung cancer.     

Dexamethasone-mediated protection from drug cytotoxicity: association with p21WAF1/CIP1 protein accumulation?

Dexamethasone (DEX)-mediated inhibition of drug-induced, but not CD95 ligand-induced, apoptosis in malignant glioma cells correlates with wild-type p53 status. Here, we examined mechanisms underlying DEX-mediated protection from apoptosis. DEX did not induce p53 expression in two p53 wild-type cell lines (U87MG, LN-229) and did not alter drug-induced p53 accumulation. Forced expression of temperature-sensitive p53val135 in mutant conformation failed to prevent accumulation of endogenous wild-type p53 but acted in a transdominant negative manner to inhibit p53-mediated, camptothecin-induced p21WAF1/CIP1 expression. p53val135-transfected cells retained responsiveness to DEX at restrictive temperature, suggesting that p53 activity is not required for cytoprotection. Forced expression of wild-type p53val135 abrogated the protective effect of DEX, suggesting redundant cytoprotective effects of DEX and p53. Indeed, DEX induced moderate accumulation of p21WAF1/CIP1 in U87MG, LN-229 and p53 mutant LN-18 cells, but not in p53 mutant LN-308 or T98G cells. LN-18 is also the p53 mutant cell line with the best cytoprotective response to DEX. p21WAF1/CIP1 accumulation occurred in the absence of changes in p21WAF1/CIP1 mRNA expression. Wild-type p53 was not required for this DEX effect since DEX induced p21WAF1/CIP1 accumulation in p53val135-transfected LN-229 cells, too. DEX failed to induce p21WAF1/CIP1 expression or cytoprotection in untransformed rat astrocytes. The same lack of modulation of p21WAF1/CIP1 expression and drug toxicity was observed in p21(+/+), p21(+/-) and p21(-/-) human colon carcinoma cells. Paradoxically, while only p21(+/+) and p21(+/-) mouse embryonic fibroblasts showed enhance p21WAF1/CIP1 levels after exposure to DEX, only p21(-/-) fibroblasts were protected from drug toxicity by DEX. The present study links DEX-mediated protection from cancer chemotherapy to a p53-independent pathway of regulating p21WAF1/CIP1 expression in glioma cells but this effect appears to cell type-specific.     

Low dose desensitisation does not reduce the toxicity of sulphasalazine in rheumatoid arthritis

To investigate the apoptosis-inducing effect of glucocorticoid on gastric epithelial cell expressing different p53 genes, a human gastric epithelial cell line-GES-1 was transfected with either wild type or mutant p53 cDNA in vitro. The cells were treated with hydrocortisone in a concentration range of 0.2-0.8 g/L. Apoptotic cells were found in those transfectants. The apoptotic response of the wild-type p53-transfected GES-1 cells was much more marked than that of the mutant p53 transfected ones. It can be inferred that the glucocorticoid secreted not only suppress the immunological response during inflammation, but induce gastric epithelial cell apoptosis as well. If the gastric epithelial cell contains mutant p53 genes, the glucocorticoid confer a selective growth advantage which advance the malignant process.     

Reversal left diastolic ventriculo-atrial flow induced by asynchronism in a patient with dilated cardiopathy and VVI pacemaker]

Current therapy for glioma is suboptimal. The transfer of apoptosis genes to tumors constitutes one of the most promising strategies for cancer gene therapy. We have previously shown that massive apoptosis occurs when wild-type p53 or E2F-1 expression is induced in glioma. However, the mechanism of action and the efficiency in inducing apoptosis of these two proteins are not similar. Adenovirus-mediated p53 gene transfer is ineffective in causing apoptosis in glioma cells that retain wild-type p53 genotype or overexpress the p21 protein. The p16/Rb/E2F pathway is the most frequent target of genetic alterations in gliomas, and therefore constitutes a suitable target for gene therapy strategies. However, the transfer of either the p16 or Rb gene to glioma cells results in cytostatic effect. The E2F-1 protein is able to induce generalized apoptosis in gliomas independently of the p53, p16 or Rb status. In addition, p21- or p16-mediated growth arrest did not protect glioma cells from E2F-1-mediated apoptosis. The apoptotic molecule bax is induced in p53-mediated apoptosis, but bax is not induced in E2F-1-mediated apoptosis in glioma cells. Careful selection of patients may be necessary before designing therapeutic strategies using either p53 or E2F-1 as a therapeutic tools for glioma patients.