David W. Voehringer

Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols

The mechanism by which Bcl-2 oncogene expression inhibits radiation-induced apoptosis has been investigated in two mouse lymphoma cell lines: line LY-as is radiation sensitive, displays substantial radiaton-induced apoptosis, and expresses low levels of Bcl-2; line LY-ar is radiation-resistant, displays a low apoptosis propensity, and expresses 30-fold higher amount of Bcl-2 protein than does the sensitive line. We observed that upon incubation in cystine/methionine-free (C/M−) medium, radiation-induced apoptosis in the LY-ar cells was restored to levels comparable to that seen in the LY-as cells. Intracellular glutathione (GSH) concentrations in LY-ar cells incubated in C/M− medium plummeted to 50% of control values within 2 h. LY-ar cells treated with diethyl maleate (DEM) or diamide, agents that deplete cellular thiols, had increased susceptibility to radiation-induced apoptosis in a manner similar to C/M− medium. These results are consistent with the general idea that Bcl-2 expression blocks apoptosis through an antioxidant pathway that involves cellular thiols. That Bcl-2-expressing tumor cells can be sensitized by exogeneous agents that modify cellular thiols offers strategies for overcoming such resistance.

Redox aspects of Bcl-2 function.

The oncogene Bcl-2 has attracted recent research attention as recognition of the importance of Bcl-2 control over apoptosis commitment in disease development and clinical response to therapy has been targeted for pharmacological intervention. Much of the basic science research regarding Bcl-2 has focused on the role that Bcl-2 plays in directly regulating mitochondrial function. This has come about because of Bcl-2s localization to mitochondrial membranes and its reported interaction with the mitochondrial megachannel. During the time that the mitochondrial function of Bcl-2 was being investigated, a smaller, yet potentially as important, role for Bcl-2 was being pursued by investigators who were following up the initial study of Bcl-2 knockout mice. These mice expressed a phenotype consistent with that of mice exposed to chronic oxidative stress. This research into the redox aspects of Bcl-2 function has led to a hypothesis that Bcl-2-expressing cells have enhanced antioxidant capacities that suppress oxidative stress signals generated during the initiation phase of many apoptotic pathways. This review will further develop the idea of Bcl-2s role in regulating cellular redox pathways associated with apoptosis, as well as integrate recently reported evidence that ties the antioxidant effects of Bcl-2 to mitochondrial function, thereby unifying both mitochondrial and redox aspects of Bcl-2 function.

The Serine/Threonine Kinase Nek6 Is Required for Cell Cycle Progression through Mitosis

The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.

L-asparaginase kills lymphoma cells by apoptosis

Microscopic examination of histological sections of lymph nodes from a canine case of malignant lymphoma at 4 h after treatment with L-asparaginase revealed massive destruction of neoplastic cells by what was consistent with apoptosis morphologically. Apoptosis as the mode of cell death after asparaginase treatment was confirmed in a mouse lymphoma cell line (LY-TH) by the characteristic fragmentation of DNA into oligonucleosome-sized pieces and by the morphological changes consistent with apoptosis following treatment in vitro. Applied to these cells, asparaginase was found to be most cytotoxic over the range of 1–10 IU/ml. Even after 4 h of asparaginase treatment at 100 IU/ml, protein synthesis was reduced by only one-half, yet DNA fragmentation reached 40%. Other agents that affect protein synthesis (cycloheximide and actinomycin D) caused apoptosis as well; however, agents (radiation, prednisolone, and VP-16) whose mechanisms are different from inhibition of protein synthesis also caused apoptosis. As such, it seems unlikely that protein depletion per se and/or the elimination of specific shortlived proteins is the triggering event that leads to cell death. It is more likely that the suspension of cellular proliferation commits cells to apoptosis after asparaginase treatment.

Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system

In this study, we investigated the importance of redox and Bcl-2 status on cytochrome c-mediated apoptosis. Two mouse lymphoma cell lines, LYas and LYar that express Bcl-2 protein at different levels, were used to reconstitute a cell-free system. Cytoplasmic extracts made from apoptosis-sensitive LYas cells 2.5 h after exposure to 5 Gy γ-radiation were able to induce apoptosis in isolated nuclei, whereas extracts made from LYas cells at time points earlier than 2.5 h, or from Bcl-2-overexpressing, apoptosis-resistant LYar cells at all time points after irradiation were inactive. Apoptotic activity was restored to inactive extracts by the addition of oxidized but not reduced cytochrome c. Cytochrome c reductase was able to inhibit apoptosis in extracts made from LYas cells 2.5 h after irradiation and LYar extracts activated by addition of oxidized cytochrome c. Antioxidants, but not oxidant defensive enzymes, blocked apoptosis implying that antioxidants might alter the redox state of factors important in mediating apoptosis. These findings confirm the importance of cellular redox state during apoptosis and are consistent with a role for Bcl-2 in regulating this redox state.

Biochemical Modulation of Radiation-Induced Apoptosis in Murine Lymphoma Cells

Considerable effort in our laboratory has been directed toward characterizing the role of apoptosis as a mode of cell death in model tumors irradiated in vivo. These studies have shown that apoptosis is an important response in some tumors, correlating with tumor growth delay and tumor cure. However, the response is heterogeneous among both the various tumors examined and the cells in a given tumor, suggesting that the propensity for cells to undergo apoptosis upon irradiation is regulated by unknown factors in tumors. To develop a model system for investigating these regulatory pathways in vitro at the molecular and biochemical levels, we have established cells from a tumor that displays a dramatic apoptotic response in vivo, the TH lymphoma, in cell culture. In this article, we review some of the results of our studies using this model system. To date, we have shown that the dose-response relationship and kinetics of the development of apoptosis for these cells in culture are similar to what we observed for the tumor response in vivo. Moreover, the roles of calcium and signal transduction pathways as important regulatory factors in radiation-induced apoptosis have been defined in this system. Ultimately such investigations may yield the insight necessary for designing protocols to modulate apoptosis biochemically in irradiated normal and tumor tissues to therapeutic advantage.

Modulation of radiation-induced apoptosis by lazaroids

Lazaroids (21-aminosteroids) are a novel class of compounds specifically designed to inhibit the iron-dependent lipid peroxidation associated with reperfusion injury. In the present study, we compared the effect of several lazaroids on radiation-induced apoptosis in explanted rat thymocytes and cultured murine lymphoma cells irradiated in vitro. An assay for the DNA fragmentation specific for apoptosis indicated that the lazaroids U-74500A, U-75412E, and U-74389F when dissolved in ethanol protected rat thymocytes and that U-75412E when dissolved in 10% cyclodextrin protected lymphoma cells. Additional experiments were conducted to ascertain which of the mechanisms associated with lazaroids were responsible for the inhibition of apoptosis. Other, nonsteroid inhibitors of lipid peroxidation—cysteamine, Desferal, and α-tocopherol—displayed no ability to inhibit apoptosis, suggesting that the steroid moiety of the lazaroid molecule was the active component. This was confirmed by showing that the nonglucocorticoid steroid testosterone inhibited radiation-induced apoptosis in these cell systems. Thus, lazaroids may inhibit apoptosis by interfering with specific functions of the cell membrane that are required for the manifestation of apoptosis and not by the inhibition of lipid peroxidation. We conclude that lazaroids may be useful agents for blocking radiation injury in normal tissues, but this will require confirmation in animal systems. Radiat Oncol Invest 1996;4:66–73.