p53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells.

Induction of CD95 (Fas/APO-1) and CD95 ligand during chemotherapeutic treatment may contribute to the death by apoptosis of some tumor cells. In this study, we have analyzed the role of the CD95 system in genotoxic drug-induced death of human breast tumor cells. Incubation of the breast tumor cell lines MCF-7 and EVSA-T with doxorubicin or methotrexate caused apoptosis after 48 h of treatment. These drugs induced a marked increase in the level of CD95 mRNA and protein in wild-type p53-expressing MCF-7 cells. On the contrary, the breast cancer cell line EVSA-T that expresses high levels of an inactive form of p53, did not up-regulate CD95 upon drug treatment. Elevation of CD95 expression by DNA-damaging drugs was notably blocked in MCF-7 cells expressing the human papillomavirus type 16 E6 protein (E6 cells) which prevented p53 accumulation upon DNA damage. However, E6 cells were still killed by the drugs. Furthermore, the genotoxic drugs did not induce the expression of CD95 ligand in MCF-7 cells at doses that caused apoptosis in these breast tumor cells. Moreover, drug-induced apoptosis of breast tumor cells was not prevented in the presence of either a CD95 antagonistic antibody or a CD95 ligand blocking antibody. We also observed a strong synergism between lower doses of DNA-damaging drugs and CD95 agonistic antibody in the induction of apoptosis in MCF-7 cells. In summary, our data indicate that drug-induced apoptosis of breast tumor cells occurs by a CD95/CD95L-independent mechanism although by elevating the tumor suppressor proteins p53 and CD95, genotoxic drugs may sensitize breast tumor cells to CD95-mediated apoptosis.

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.

Activation of protein kinase C attenuates early signals in Fas-mediated apoptosis.

Activation of protein kinase C (PKC) has been reported to inhibit Fas (APO-1, CD95)-mediated apoptosis in different cellular systems. Human Jurkat leukemic T cells express the Fas antigen in the cell membrane and undergo apoptosis upon cross-linking by anti-Fas monoclonal antibodies (mAb). Cleavage of the apoptosis-associated protease CPP32 and its substrate poly(ADP-ribose)polymerase are observed after the engagement of Fas antigen with mAb. In this report, we show that all these effects are substantially inhibited by the activation of PKC with a phorbol ester. Bisindolylmaleimide, an inhibitor of PKC, prevents phorbol ester-induced down-regulation of Fas signaling. Inhibition of Fas-mediated cell death by phorbol ester is also observed in other human leukemic T cell lines. Cross-linking of Fas antigen by mAb results in the rapid increase in tyrosine phosphorylation of several protein substrates which is further elevated in the presence of the protein tyrosine phosphatase inhibitor, orthovanadate. Furthermore, orthovanadate markedly enhances the cell death response to Fas mAb in different human leukemic T cell lines and human T cell blasts. These effects of orthovanadate on early tyrosine phosphorylation and cell death are clearly diminished by PKC activation. These results strongly suggest that tyrosine phosphorylation is involved in Fas signaling in apoptosis and that PKC plays a negative role in Fas-mediated apoptosis by counteracting at a very early stage the signals generated following cross-linking of this receptor.

Reactive oxygen intermediate(s) (ROI): common mediator(s) of poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis.

HL-60 acute myeloblastic and U937 monoblastoid leukaemic cell lines both cleave poly(ADP-ribose)polymerase (PARP), at the onset of apoptosis, in response to a wide range of cytotoxic agents. This appears to be a common feature of leukaemic cell apoptosis. However, in the chronic myelogenous leukaemic (CML) derived cell line, K562, no such cleavage was detectable. This correlated with previous findings that this cell line is particularly resistant to apoptosis induced by cytotoxic agents. Proteolytic cleavage of PARP and the subsequent progression to apoptosis was inhibited by two protease inhibitors NEM and IOD. As both PARP cleavage and DNA fragmentation appeared closely linked in these cell lines, anti-oxidants (previously shown to be effective inhibitors of DNA fragmentation and apoptosis) were also demonstrated to prevent PARP cleavage. These results combine to suggest that ROI may mediate PARP cleavage, DNA fragmentation and the eventual apoptosis of these cells following cytotoxic insult.

Stimulation of phosphatidylinositol turnover is a key event for Fas-dependent, activation-induced apoptosis in human T lymphocytes.

We have analyzed the requirements for activation-induced apoptosis in Jurkat T cells expressing the heterologous human muscarinic type 1 receptor (HM1R; J-HM1-2.2 cells) that is coupled to phosphatidylinositol turnover through a protein tyrosine kinase-independent, G protein-regulated mechanism. Triggering of HM1R with the agonist carbachol is sufficient to induce apoptosis in J-HM1-2.2 cells. Apoptosis is also induced in J-HM1-2.2 cells by triggering of the TCR. Calcium influx, intracellular Ca2+ increase, calcineurin function, and de novo protein synthesis are necessary for receptor-controlled apoptosis. However, blocking protein kinase C with a specific inhibitor does not abrogate receptor-induced apoptosis. Furthermore, HM1R-induced apoptosis is inhibited by blocking Fas ligand/Fas interaction with an antagonist anti-Fas Ab, and Fas ligand mRNA and protein are expressed in J-HM1-2.2 cells stimulated through the HM1R. Therefore, protein tyrosine kinase activation is not an absolute requirement for receptor-controlled Fas ligand expression. Taken together, the results demonstrate that stimulation of phosphatidylinositol turnover can induce apoptosis through a Fas-dependent mechanism that requires calcineurin stimulation, but not protein kinase C activation.

Activation-induced apoptosis in Jurkat cells through a myc-independent mechanism.

Apoptosis can be induced in the human leukemic T-cell line Jurkat when these cells are stimulated by several known T-cell activators. Among the stimuli that activate Jurkat cells, calcium ionophore A23187, phytohemagglutinin and cross-linked monoclonal antibody anti-CD3 are the best inducers of apoptosis. The activator of protein kinase C phorbol-12,13-dibutyrate causes a rapid but transient stimulation of DNA fragmentation and cell death, and soluble monoclonal antibody anti-CD3 has no effect. Furthermore, apoptosis is delayed when Jurkat cells are stimulated simultaneously with calcium ionophore and phorbol ester. We have analysed the mechanism underlying the induction of apoptosis by these agents and found that down-regulation of c-myc expression by pretreatment with the DNA binding antibiotic mithramycin, an inhibitor of c-myc transcription, does not prevent the activation of the apoptotic process. In the light of these results we can conclude that in these leukemic T-cells, activation of apoptosis may occur by a mechanism independent of c-myc expression.