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We investigated whether the dissipation of mitochondrial transmembrane potential (Delta(Psi)(m)) was involved in apoptosis of cultured human aortic endothelial cells (HAECs) exposed to hyperglycemic conditions (30 mmol/L glucose). In parallel experiments, N-acetyl-L-cysteine (NAC) was added to the culture medium to verify whether this antioxidant may prevent apoptosis in these cells. The binding of annexin V and DNA fragmentation were measured, in addition to the production of reactive oxygen species (ROS), the number of cells with depolarized mitochondria, and the intracellular glutathione (GSH) content. As compared to the control (5 mmol/L glucose), high-glucose treatment increases both ROS generation and the number of cells binding annexin V. Moreover, a simultaneous decrease of intracellular GSH content was observed, which was accompanied by an increased number of cells showing both depolarized mitochondria and fragmented DNA. Incubation of HAECs with high glucose in the presence of 10 mmol/L NAC prevented the drop of intracellular GSH content, and decreased both ROS generation and the number of cells committed to apoptosis. These results suggest that high glucose triggers the same cascade of molecular events as do other apoptosis inducers in other cells. Among these events, the disruption of mitochondrial membrane barrier function might be decisive because it causes the release of soluble proteins from intermembrane space, which then induce nuclear apoptotic changes., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

It is accepted that apoptosis is a gene-controlled process of cellular self-destruction. It occurs during physiological regulation and in pathological situations in the life of a cell. In the immune system, several different intracellular and extracellular factors have been associated with the induction of apoptosis, and the final responses depend on the cell system and the acquired signals. In lymphoid cells, dexamethasone-induced apoptosis is associated with c-myc downregulation in cells that remain in G0-G1 until the point of death. Ornithine decarboxylase (ODC), a key enzyme involved in polyamine biosynthesis, is regulated by c-myc, which is a transcriptional activator implicated not only in the control of cell proliferation and differentiation but also in programmed cell death. As dimethylsulphoxide (DMSO) induces apoptosis in the RPMI-8402 human pre-T cell line, the present study analysed the involvement of the c-myc proto-oncogene and polyamine pathway as mediators of apoptosis. Cell growth, programmed cell death, c-myc expression, ODC activity and intracellular polyamine content were detected after DMSO and difluoromethylornithine (DFMO) treatment. DMSO-treated cells exhibit a decrease in ODC activity and polyamine levels associated with cell growth arrest and programmed cell death induction. The expression of c-myc proto-oncogene, as its mRNA or protein, is specifically down-regulated. DFMO, a well defined polyamine biosynthesis inhibitor, completely blocks ODC activity, resulting in growth inhibition but not apoptosis. Moreover, in these samples no evidence of changes of c-myc expression were found. The results obtained suggest that, in RPMI-8402 cells, DMSO provokes a c-myc-dependent decrease of ODC activity followed by a depletion of intracellular polyamine levels, associated with programmed cell death and cell growth arrest.

The regulation of cell proliferation or cell death by extracellular factors are the most intensely studied subjects in cell biology. Many conceptual problems remain to be clarified concerning the mechanisms that regulate the programmed cell death. In this work, we focus our attention on the possible role of protein kinase C activation during dimethyl sulfoxide (DMSO)-induced cell death. The present results suggest that the frequency of DMSO-dependent apoptosis of RPMI 8402 thymic lymphoma cells is increased by phorbol ester acetate supplementation. Enhancement of apoptosis can be abolished by cotreatment with the bisindolylmaleimide, a specific PKC inhibitor. The association between PMA and DMSO treatment provokes an early activation of an intracellular signaling mechanism that results, via sustained diacylglycerol elevation, in a possible long-term PKC activation.