Mutant p53-carrying tumors are often more resistant to chemotherapeutical drugs. We demonstrate here that the mutant p53-reactivating compound PRIMA-1 MET acts synergistically with several chemotherapeutic drugs to inhibit tumor cell growth. Combined treatment with cisplatin and PRIMA-1 MET resulted in a synergistic induction of tumor cell apoptosis and inhibition of human tumor xenograft growth in vivo in SCID mice. The induction of mutant p53 levels by chemotherapeutic drugs is likely to increase the sensitivity of tumor cells to PRIMA-1 MET . Thus, the combination of PRIMA-1 MET with currently used chemotherapeutic drugs may represent a novel and more efficient therapeutic strategy for treatment of mutant p53-carrying tumors.

The ability of H P O P and tributyltin (TBT) to trigger pro-caspase activation via export of cytochrome c from mitochondria to the cytoplasm was investigated. Treatment of Jurkat T lymphocytes with H P O P resulted in the appearance of cytochrome c in the cytosol within 2 h. This was at least 1 h before caspase activation was observed. TBT caused cytochrome c release already after 5 min, followed by caspase activation within 1 h. Measurement of mitochondrial membrane potential (v v8 8 m ) showed that both H P O P and TBT dissipated v v8 8 m , but with different time courses. TBT caused a concomitant loss of v v8 8 m and release of cytochrome c, whereas cytochrome c release and caspase activation preceded any apparent v v8 8 m loss in H P O P -treated cells. Thus, our results suggest that different mechanisms are involved in triggering cytochrome c release with these apoptosis-inducing agents.z 1998 Federation of European Biochemical Societies.

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The toxicity of tributyltin chloride (TBT) involves Ca(2+) overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 microM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATP-dependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATP-containing cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBT-treated Jurkat cells at both the mitochondrial and caspase activation levels.

p53 mutations occur frequently in human tumors. The low-molecular-weight compound PRIMA-1 MET reactivates mutant p53, induces apoptosis in human tumor cells and inhibits tumor xenograft growth in vivo. Here, we show that PRIMA-1 MET induces mutant p53-dependent mitochondria-mediated apoptosis through activation of caspase-2 with subsequent cytochrome c release and further activation of downstream caspase-9 and caspase-3. Inhibition of caspase-2 by a selective inhibitor and/or siRNA prevents cytochrome c release on PRIMA-1 MET treatment and causes a significant reduction in PRIMA-1 MET -induced cell death. Our findings highlight a chain of cellular events triggered by MET that lead to apoptotic cell death. This should facilitate further development and optimization of efficient PRIMA-1 MET -based anticancer drugs.

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In the present study, we show that the immunotoxicant, tributyltin (TBT), induces a dose-dependent activation of caspases followed by typical apoptotic morphology in resting human peripheral blood lymphocytes. TBT also caused an early loss of mitochondrial membrane potential (Delta(Psi)(m)) and release of cytochrome c, suggesting that apoptosis was triggered by the mitochondrial pathway. When CD4+ T-cells were sorted from peripheral blood and exposed to TBT for 30 min, caspase activation and apoptosis were induced. Interestingly, in the sorted CD8+ T-cell population, caspase activation was not observed until 2 h of TBT exposure, suggesting that these cells were more resistant toward TBT. Moreover, a time-dependent induction of caspase activity was also detected in CD3-stimulated peripheral blood lymphocytes. This caspase activation was not associated with cytochrome c release or loss of mitochondrial Delta(Psi) and did not lead to apoptotic morphology, although it did lead to both PARP and DFF cleavage. We also noticed a concomitant induction of Hsp27, and it awaits to be seen if this chaperone may interfere with the processing of nuclear protein substrates downstream from these primary caspase-3 substrates. Moreover, no increase in caspase activation or induction of apoptosis was observed after TBT treatment in these cells. Instead, the cells were directed toward necrotic deletion. Taken together, these data suggest that TBT-induced deletion of peripheral lymphocytes is likely to be a component in the overall risk for immunotoxic responses in exposed humans.

Background: Sarcoidosis is a chronic granulomatous lung disease of unknown origin. The accumulation of activated T cells at sites of inflammation represents an early stage in granuloma formation. Since mechanisms governing the normal resolution of inflammatory processes are poorly understood, this study aimed to investigate the apoptotic phenotype of peripheral blood and lung T lymphocytes from patients with sarcoidosis. Methods: Bronchoalveolar lavage (BAL) was performed in 10 patients with active sarcoidosis and five healthy controls. Results: Virtually no lymphocyte apoptosis, as determined by annexin V or Hoechst staining, was seen in either patients or controls. Sustained caspase-3 activity in non-apoptotic BAL fluid lymphocytes of the patients was detected, however, in agreement with in vitro studies demonstrating caspase activation after T cell receptor (TCR) triggering as a physiological response required for efficient T cell activation. Only 11.0% (range 7.7-17.6) of the BAL lymphocytes from sarcoidosis patients were annexin V positive after exposure to the apoptotic stimulus tributyltin compared with 55.0% (range 42.0-62.0) of BAL lymphocytes from healthy controls (p<0.001). After anti-Fas treatment only 8.5% (range 6-10) of BAL fluid lymphocytes from patients but 45.5% (range 38-62) from healthy controls were apoptotic. Conclusion: BAL fluid lymphocytes from patients with sarcoidosis display a non-apoptotic morphology associated with endogenous caspase-3 activity. They seem to be resistant to apoptosis, which might contribute to the accumulation of inflammatory cells in the lungs, persistence of inflammation, and the development and maintenance of granuloma.