Treatment with the DNA topoisomerase inhibitors etoposide, doxorubicin, and camptothecin, and with the alkylating agents cisplatin and melphalan, caused peroxide accumulation and apoptosis in U-937 human promonocytic cells. Preincubation with the reduced glutathione (GSH) synthesis inhibitor L-buthionine-(S,R)-sulfoximine (BSO) always potentiated peroxide accumulation. However, although GSH depletion potentiated the toxicity of cisplatin and melphalan, occasionally switching the mode of death from apoptosis to necrosis, it did not affect the toxicity of the other antitumor drugs. Hypoxia or preincubation with antioxidant agents attenuated death induction, apoptotic and necrotic, by alkylating drugs. The generation of necrosis by cisplatin could not be mimicked by addition of exogenous H 2 O 2 instead of BSO and was not adequately explained by caspase inactivation nor by a selective fall in ATP content. Treatment with cisplatin and melphalan caused a late decrease in mitochondrial transmembrane potential (⌬⌿m), which was much greater during necrosis than during apoptosis. The administration of the antioxidant agents N-acetyl-L-cysteine and butylated hydroxyanisole after pulse treatment with cisplatin or melphalan did not affect apoptosis but attenuated necrosis. Under these conditions, both antioxidants attenuated the necrosis-associated ⌬⌿m decrease. These results indicate that oxidation-mediated alterations in mitochondrial function regulate the selection between apoptosis and necrosis in alkylating drug-treated human promonocytic cells.

Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 M) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK ) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38 MAPK -specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium-provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione-depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H 2 O 2 accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38 MAPK phosphorylation. In a similar manner, p38 MAPK phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38 MAPK activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells.Cadmium and other heavy metals are frequent environmental contaminants with well known mutagenic, carcinogenic, and teratogenic effects (1). Another property of heavy metals (and of other physical and chemical agents, such as heat and inhibitors of energy metabolism) is the capacity to induce the stress response, characterized by the synthesis and accumulation of heat-shock proteins (HSPs)

Infection of HeLa cells with adenovirus-carrying HSF1؉ cDNA, which encodes a mutated form of HSF1 with constitutive transactivation capacity, increased multidrug resistance 1 (MDR1) mRNA level and P-glycoprotein (P-gp) cell surface content and stimulated rhodamine 123 accumulation and vinblastine efflux activity. On the other hand, infection with adenovirus-carrying HSP70 and HSP27 cDNAs did not increase MDR1/P-gp expression. HSF1 regulates MDR1/P-gp expression at the transcriptional level, since HSF1؉ bound the heat-shock consensus elements (HSEs) in the MDR1 gene promoter and also activated the expression of an MDR1 promoterdriven reporter plasmid (pMDR1(؊1202)). In addition, heat-shock increased pMDR1(؊1202) promoter activity but not the activity of a similar reporter plasmid with point mutations at specific HSEs, and the heat-induced increase was totally inhibited by co-transfection with an expression plasmid carrying HSF1 ؊ , a dominant negative mutant of HSF1. The stress inducers arsenite, butyrate, and etoposide also increased pMDR1(؊1202) promoter activity, but the increase was not inhibited (in the case of butyrate) or was only partially inhibited (in the case of arsenite and etoposide) by HSF1 ؊ . These results demonstrate that HSF1 regulates MDR1 expression, and that the HSEs present in the ؊315 to ؊285 region mediate the heat-induced activation of the MDR1 promoter. However, other factors may also participate in MDR1 induction by stressing agents.

Treatment with 0.2 mM hydrogen peroxide (H 2 O 2 ) or with 0.5 mM cisplatin caused caspase-9 and caspase-3 activation and death by apoptosis in U-937 human promonocytic cells. However, treatment with 2 mM H 2 O 2 , or incubation with the glutathione suppressor DL-buthionine-(S,R)-sulfoximine (BSO) prior to treatment with cisplatin, suppressed caspase activation and changed the mode of death to necrosis. Treatment with 2 mM H 2 O 2 caused a great decrease in the intracellular ATP level, which was partially prevented by 3-aminobenzamide (3-ABA). Correspondingly, 3-ABA restored the activation of caspases and the execution of apoptosis. By contrast, BSO plus cisplatin did not decrease the ATP levels, and the generation of necrosis by this treatment was not affected by 3-ABA. On the other hand, while all apoptosisinducing treatments and treatment with 2 mM H 2 O 2 caused Bax translocation from the cytosol to mitochondria as well as cytochrome c release from mitochondria to the cytosol, treatment with BSO plus cisplatin did not. Treatment with cisplatin alone caused Bid cleavage, while BSO plus cisplatin as well as 0.2 and 2 mM H 2 O 2 did not. Bcl-2 overexpression reduced the generation of necrosis by H 2 O 2 , but not by BSO plus cisplatin. These results indicate the existence of different apoptosis/necrosis regulatory mechanisms in promonocytic cells subjected to different forms of oxidative stress.