BCL-2 is involved in preventing oxidant-induced cell death and in decreasing oxygen radical production.

It has been hypothesized that programmed cell death is mediated, in part, through the formation of free radicals via oxidative pathways. Furthermore, it has been proposed that BCL-2 acts to inhibit cell death by interfering with the production of oxygen-derived free radicals induced by a wide variety of stimuli. In order to examine the antioxidant function of BCL-2, we transfected mouse epidermal cells JB6 clone 41 with the expression vector pD5-Neo-BCL-2 and studied the effect of BCL-2 overexpression on oxidant-induced cell death and on the production of reactive oxygen species. Compared to Neo control cells, BCL-2-expressing cells are more resistant to the killing and growth retardation induced by hydrogen peroxide, superoxide, or by the oxygen radical-generating quinone-containing compounds menadione, diaziquone and adriamycin. The latter compounds generate reactive oxygen species during bioreductive metabolism. In addition, the exposed cells die by necrosis rather than apoptosis. Hydroxyl radical levels generated by the quinone-containing agents were low in BCL-2-expressing JB6 cells compared to control Neo cells. BCL-2, however, does not change the activities of the major cellular antioxidant enzymes superoxide dismutase, catalase or glutathione peroxidase. On the other hand, the glutathione concentrations increased in BCL-2 overexpressing cells after oxidative challenge, while the opposite was true for control cells. Thus, our results suggest that BCL-2 inhibition of oxidant-induced cell death is mediated, at least in part, through an antioxidant pathway, and that this pathway involves glutathione.

Studies on the mechanisms and kinetics of apoptosis induced by microinjection of cytochrome c in rat kidney tubule epithelial cells (NRK-52E).

Recent reports substantiating the role of cytochrome c in the induction of apoptosis led us to examine the kinetics and mechanisms involved in this process as an extension of our ongoing studies of cell injury and cell death. Microinjection of cytochrome c into NRK-52E kidney cells produced rapid apoptosis, which usually began within 30 minutes and reached a maximum of 60-70% by 3 hours. The changes that occurred included four phases: an initial shrinkage phase, an active phase, a spherical phase, and a necrotic phase. For morphological purposes, the progressive changes were followed by phase-contrast and fluorescence microscopy, transmission and scanning electron microscopy, and time-lapse video microscopy. Cells first showed shrinkage, then displayed multiple pseudopods, which rapidly extended and retracted, giving the cells a bosselated appearance. During this active phase there was chromatin condensation, mitochondria were swollen but retained membrane potential, and the endoplasmic reticulum was dilated. Within 2-4 hours, active-phase cells became spherical and smooth-surfaced but were still alive, the nuclei showed chromatin clumping, the mitochondria underwent high-amplitude swelling but retained membrane potential, the endoplasmic reticulum was highly dilated, and many large apical vacuoles were present. Elevation of [Ca(2+)](i) was seen at the late spherical phase, shortly before cell death. Pretreatment with the caspase 3 inhibitor (Ac-DEVD-CHO) prevented apoptosis, whereas overexpression of Bcl-2 did not. Depletion of cellular ATP by cyanide inhibition of energy metabolism prevented cytochrome c from inducing the active and later phases of apoptosis. The results clearly indicate that cytochrome c-induced apoptosis is a dynamic and energy-requiring process that has a distinct active and spherical phase before cell death.

Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca2+ mobilization.

The mechanism by which Bcl-2 inhibits cell death is unknown. It has been suggested that Bcl-2 functions as an antioxidant. Because Bcl-2 is localized mainly to the membranes of the endoplasmic reticulum (ER) and the mitochondria, which represent the main intracellular storage sites for Ca2+, we hypothesized that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca2+ homeostasis. To test this hypothesis, we examined the effect of oxidant treatment on viability in normal rat kidney (NRK) cells and in NRK cells stably transfected with Bcl-2 in the presence or absence of intracellular Ca2+, and we compared the effect of Bcl-2 expression on oxidant-induced intracellular Ca2+ mobilization and on ER and mitochondrial Ca2+ pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant to H2O2-induced cytotoxicity than control cells. EGTA-AM, an intracellular Ca2+ chelator, as well as the absence of Ca2+ in the medium, reduced H2O2-induced cytotoxicity in both cell lines. Compared with controls, cells overexpressing Bcl-2 showed a delayed rise in intracellular Ca2+ concentration ([Ca2+]i) after H2O2 treatment. After treatment with the Ca2+ ionophore ionomycin, Bcl-2-transfected cells showed a much quicker decrease after the maximal rise than control cells, suggesting stronger intracellular Ca2+ buffering, whereas treatment with thapsigargin, an inhibitor of the ER Ca2+-ATPases, transiently increased [Ca2+]i in control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation show that NRK-Bcl-2 cells have a higher capacity for mitochondrial Ca2+ storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity of mitochondria to store Ca2+.

bcl-2 enhancement of malignant transformation in mouse epidermal JB6 cells.

Increased bcl-2 expression is a common feature of many types of human malignancies, which implies that bcl-2 plays an important role in tumorigenesis. To better understand the molecular mechanisms of bcl-2-induced oncogenesis, we examined the effects of bcl-2 expression on transformation of mouse epidermal JB6 cells induced by the tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA). Promotion-sensitive JB6 clone41 cells were transfected with the bcl-2-containing expression vector pD5-neo/bcl-2, and the soft agar growth of bcl-2-transfected cells and control cells were compared. bcl-2 overexpression in JB6 clone41 cells caused a TPA-induced soft-agar growth fivefold greater than the growth of nontransfected or vector-transfected (neo control) cells. bcl-2 expression in the absence of TPA did not lead to colony formation in soft agar. Because the level of the transcription factor activator protein 1 (AP-1) has been shown to be critical for the responsiveness of JB6 cells to TPA-induced transformation, we compared c-jun and c-fos expression as well as the AP-1-binding activity and the AP-1-mediated transactivation of the reporter construct TRE-CAT between bcl-2-expressing cells and control cells. When compared with control cells, bcl-2-transfected cells expressed significantly more c-fos but not c-jun after TPA treatment. Furthermore, the levels of AP-1 and AP-1-induced transactivation of TRE-CAT were greater in bcl-2-transfected cells than in control cells after TPA treatment. These results showed that bcl-2 cooperates with a tumor promoter such as TPA in the induction of malignant transformation in mouse epidermal cells and that bcl-2 enhances soft-agar growth by stimulating signaling pathways that led to increased AP-1 expression.

Manganese superoxide dismutase expression inhibits soft agar growth in JB6 clone41 mouse epidermal cells.

Manganese superoxide dismutase (MnSOD) has been found to be depleted in a variety of tumor cells as well as in in vitro transformed cell lines, suggesting that MnSOD may function as an anticarcinogen by protecting the cell from oxidant-induced carcinogenesis. The relationship between MnSOD expression and tumor promotion was studied by transfection of a human MnSOD cDNA into the promotable mouse epidermal cell line JB6 clone41. The effect of MnSOD overexpression on the promotion-sensitive phenotype of JB6 cells was assessed by measuring growth characteristics such as growth rate and the ability to form colonies in soft agar. Compared with the parental and vector-transfected (gpt) control cells, MnSOD-overexpressing cells had a slower growth rate and their ability to form colonies in soft agar was significantly decreased in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Since the transformation-sensitive phenotype of JB6 clone41 cells is associated with increased expression of the transcription factor AP-1, we compared c-jun and c-fos mRNA expression in MnSOD-transfected and control JB6 cells. Overexpression of MnSOD led to a significant decrease in c-jun and c-fos expression in response to treatment with TPA or the oxidant promoter superoxide. These findings indicate that the promotion-sensitive phenotype of JB6 clone41 cells can be reverted by increasing MnSOD intracellularly. A possible mechanism is that elevated MnSOD expression might change the intracellular redox state by altering the balance of reactive oxygen species. This could lead to a modulation of TPA and oxidant-induced signal transduction pathways controlling cell growth and differentiation.

The pathways of cell death: oncosis, apoptosis, and necrosis.

The pathways and identification of cell injury and cell death are of key importance to the practice of diagnostic and research toxicologic pathology. Following a lethal injury, cellular reactions are initially reversible. Currently, we recognize two patterns, oncosis and apoptosis. Oncosis, derived from the Greek word "swelling," is the common pattern of change in infarcts and in zonal killing following chemical toxicity, e.g., centrilobular hepatic necrosis after CC14 toxicity. In this common reaction, the earliest changes involve cytoplasmic blebbing, dilatation of the endoplasmic reticulum (ER), swelling of the cytosol, normal or condensed mitochondria, and chromatin clumping in the nucleus. In apoptosis, the early changes involve cell shrinkage, cytosolic shrinkage, more marked chromatin clumping, cytoplasmic blebbing, swollen ER on occasion, and mitochondria that are normal or condensed. Following cell death, both types undergo postmortem changes collectively termed "necrosis." In the case of oncosis, this typically involves broad zones of cells while, in the case of apoptosis, the cells and/or the fragments are often phagocytized prior to their death by adjacent macrophages or parenchymal cells. In either case, the changes converge to a pattern that involves mitochondrial swelling, mitochondrial flocculent densities and/or calcification, karyolysis, and disruption of plasmalemmal continuity. The biochemical mechanisms of cell death are currently under intense study, particularly concerning the genes involved in the process. Pro-death genes include p53, the ced-3/ICE proteases, and the Bax family. Anti-death genes include ced-9/Bcl-2 and the adenovirus protein EIB. It is clear that ion deregulation, particularly that of [Ca2+]i plays an important role in cell death following either apoptosis or oncosis. Genetic evidence strongly indicates that activation of proteases is an important step, possibly very near to the point where cell death occurs.

The role of altered [Ca2+]i regulation in apoptosis, oncosis, and necrosis.

Understanding the processes and events that occur when a cell undergoes a prelethal injury or that lead the cell to death following a lethal injury has been the aim of our research for a number of years. Throughout this period much has been learned, recently at rapid rates, not only by us but by many other investigators as well. Based on the data gathered, we proposed a working hypothesis over a decade ago and have since continually updated it as new experimentation is performed. Our laboratory has focused particularly on the role of cytoplasmic ionized calcium ([Ca2+]i) and the effects of its deregulation on prelethal events, including oncosis and apoptosis, and lethal events (necrosis) following cell death. [Ca2+]i appears to be a major link and signalling event. Understanding the mechanisms involved by using a variety of in vivo and in vitro models, coupled with state-of-the-art methodologies, should now allow us to prevent cell death by killing cells when necessary through gene therapy and cancer chemotherapy.

H-ras transfection of the rat kidney cell line NRK-52E results in increased induction of c-fos, c-jun and hsp70 following sulofenur treatment.

The effect of the antineoplastic drug sulofenur on the induction of the immediate-early genes (IEG) c-fos and c-jun and the stress gene hsp70 was compared in the rat kidney epithelial-like cell line NRK-52E and a derivative H-ras-transfected (H/1.2NRK-52E) cell line. Fold induction for each gene after sulofenur (500 microM) treatment was greater in H/1.2NRK-52E. The maximum increases for NRK-2E and H/1.2NRK-52E were as follows: c-fos, approximately 10-fold and approximately 18-fold; c-jun, approximately 2.5-fold and approximately 3.6-fold; hsp70, approximately 13-fold and approximately 30-fold. In cells loaded with EGTA/AM or treated in low or no Ca2+ HBSS, c-fos induction was reduced similarly in both cell types. However, inhibition of protein kinases with staurosporin and calphostin C reduced c-fos by 80% in NRK-52E but by only 10-20% in H/1.2NRK.52E. These results indicate that sulofenur-induced IEG elevation is Ca(2+)-dependent and that the requirement for protein kinase C activation is bypassed in H-ras-transfected cells.

The mechanisms of calcium-mediated cell injury and cell death [correcgted].

It has been known for some time that all pathophysiologic changes in shock and trauma have their basis at the cellular and molecular levels. These changes have been and continue to be studied in vivo in human patients and animal models and in vitro using a number of human and animal cell cultures. Cell injury and cell death have been studied in our laboratories for many years with reversible and irreversible cell responses having been characterized by functional, morphological, and biochemical methodologies. Recently, new technologies such as molecular biological experimentation for gene expression studies and digital imaging fluorescence microscopy to measure and localize ions, specifically the concentration of intracellular ionized calcium ([Ca2+i]), have been undertaken. Data from these studies have led us to propose a working hypothesis for a number of principle subcellular events that occur following a lethal injury. Dysregulation of [Ca2+i] is central to this hypothesis, since the effects of such dysregulation appear to affect many phenomena either positively or negatively. These phenomena are briefly discussed here. Much additional experimentation needs to be performed which should lead to further understanding of these events and to improved therapy, including modifiers of Ca(2+)-mediated events, new regulators of gene expression, and even gene therapy itself.

Studies on the mechanism of sulofenur and LY295501 toxicity: effect on the regulation of cytosolic calcium in relation to cytotoxicity in normal and tumorigenic rat kidney cell lines.

Treatment of NRK-52E (normal) and H/1.2-NRK-52E (Harvey-ras transfected NRK-52E) rat kidney epithelial-like cells with two Eli Lilly antitumor compounds, sulofenur and LY295501 (15.6 microM-1000 microM) resulted in concentration- and time-dependent cell killing. Cytosolic Ca2+ became elevated in both cell lines in the presence of extracellular Ca2+ but only minimally in its absence. Both drugs were more toxic to the tumorigenic cells than to the normal cells, but LY295501 was significantly more toxic to both cells. The similarity in toxic response by both cell lines suggests a similar mechanism of toxic action for both drugs. Since LY295501 is highly toxic to tumorigenic cells but has a manageable dose-limiting toxicity it shows excellent potential for use in chemotherapy.

Changes in [Ca2+]i in cultured rat proximal tubular epithelium: an in vitro model for renal ischemia.

Two components of ischemia, oxygen deprivation and glycolytic inhibition, were studied in primary cultures of rat proximal tubular epithelial cells (PTE). Changes in cytosolic Ca2+ ([Ca2+]i) and its relationship to loss of mitochondrial membrane potential (delta psi m) and cell killing were characterized in single cells whereas ATP and LDH release were determined in populations of monolayer PTE. (1) Inhibition of mitochondrial respiration with KCN or anoxia resulted in little decrease in ATP or cell killing and slight change in [Ca2+]i over many hours. (2) Inhibition of respiration and glycolysis with anoxic HBSS minus glucose resulted in decreased ATP (54.4%) and cell killing (20%) during 5 h anoxic exposure. In all cases, but at highly variable times (113 +/- 62 min), [Ca2+]i initially rose to > 1 microM. In some cases it immediately dropped, stabilizing at about 500 nM for up to 1 h and rising again just prior to cell death. (3) Inhibition with anoxia + 1 mM IAA resulted in rapid depletion of ATP and cell killing, with increases in [Ca2+]i to > 1 microM by 20 +/- 2 min. (4) Depletion of glycolytic metabolites by depriving cells of substrate for 12 h (in HBSS minus glucose) before subjecting to anoxia minus glucose resulted in increases in [Ca2+]i at 40 +/- 17 min followed by cell killing. (5) Injury with anoxic HBSS minus glucose was reversed by reaeration before or during the initial rise in [Ca2+]i. Later reaeration resulted in rapid cell killing. In all cases, delta psi m was dissipated only after [Ca2+]i was significantly elevated.

Calcium-mediated cell injury and cell death.

The effect of intracellular ion deregulation, particularly of [Ca2+], on the events following acute cell injury and the progression of change from initiation (reversible) to maintenance (reversible-irreversible) phases and finally to cell death has been the major thrust of experimentation in our laboratory for over 20 years. Cell death, which plays an important role in both normal and pathological phenomena, has been classified into two principal types, accidental and programmed. Recent exploration of programmed cell death (or apoptosis) has revealed extensive data showing it is an important mechanism for the normal maintenance and also differentiation of a variety of cell types and organs. From the results from our laboratory and those of others, we continue to expand and refine our working hypothesis: deregulation of [Ca2+] results in a number of phenomena from activation of signaling mechanisms and alterations in cellular structure to alterations in gene expression, all of which contribute to or play a critical role in cellular toxicity, including carcinogenesis and cell death. Therefore, although much more experimentation is needed to clarify some of these phenomena, the implications of such data for understanding the mechanisms and processes involved in carcinogenesis and the chemotherapeutic killing of cancer cells are extremely exciting. These relationships between [Ca2+], cell injury, and cell death are briefly reviewed here within the framework of our hypothesis.

Sulofenur cytotoxicity and changes in cytosolic calcium and mitochondrial membrane potential in human colon adenocarcinoma cell lines.

Sulofenur treatment (12.5 microM-1 mM) of colon adenocarcinoma cell lines resulted in dose- and time-dependent cell killing. LYc5 cells were viable longer than GC3/c1 cells. Each concentration resulted in elevation of cytosolic calcium [Ca2+]i) for both cell lines. At lower doses, elevation was delayed for LYc5 cells. GC3/c1 cells after 1 mM treatment in Ca(2+)-free HBSS showed no rise of [Ca2+]i. GC3/c1 cells after carbonyl cyanide-m-chlorophenylhydrazone rapidly lost rhodamine 123 fluorescence from mitochondria; after 1 mM sulofenur, fluorescence faded slowly. Following treatment, cells became rounded, blebs formed and the cells died. Results suggest that elevated [Ca2+]i plays an important role in sulofenur cytotoxicity.

Differential cytotoxicity in mouse epidermal JB6 cells: a potential mechanism for oxidant tumor promotion.

It has been suggested that superior antioxidant defense systems protect promotion-sensitive (p+t) mouse epidermal JB6 clone 41 cells from excessive deleterious effects of oxidants, allowing their clonal expansion in contrast to that of promotion-resistant (p-) clone 30 cells. In support of this concept, we report that oxidants produced by xanthine/xanthine oxidase cause more cytotoxicity, cellular damage, and cell death in p-cells. Cell surface blebbing, an early morphological consequence of oxidative injury, was detected in cultures grown on glass coverslips. While a rise in cytosolic ionized calcium ([Ca2+]i) preceding bleb formation was observed in both p+ and p- cells by digital imaging fluorescence microscopy, elevated levels of [Ca2+]i were sustained longer in p- cells. This increase was dependent on the levels of extracellular ionized calcium ([Ca2+]e) in p+ but not p- cells. We conclude that the superior antioxidant defense or improved Ca2+ buffering of promotable clone 41 cells protects them from more severe deregulation of [Ca2+]i and, as a consequence, from excessive cytotoxicity after exposure to oxidant promoters.

Role of cytosolic Ca2+ and protein kinases in the induction of the hsp70 gene.

The role of cytosolic Ca2+ ([Ca2+]i) and protein kinases in the hsp70 induction following heat shock was investigated in cultured rat proximal tubular epithelial (PTE) cells. Changes in [Ca2+]i were measured by digital imaging fluorescence microscopy using fura 2. Steady state levels of hsp70 mRNA were examined by either Northern or dot blot analyses. [Ca2+]i increased within 10 minutes and continued to increase following heat shock. The increases in [Ca2+]i were reduced in nominally Ca(2+)-free media with or without EGTA. [Ca2+]i also increased within 0.5 minutes following ionomycin, but then declined to normal levels by 1.0 to 1.5 minutes. Heat shock induced hsp70 mRNA within 15 minutes, which continued to increase up to three hours. Ionomycin also induced hsp70 mRNA, which peaked at 30 minutes, and gradually decreased thereafter. The hsp70 induction following heat shock was attenuated when extracellular Ca2+ was reduced. Chelation of [Ca2+]i by quin-2 also reduced the hsp70 induction. Inhibitors of protein kinases, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), calphostin C, genistein, and 2-aminopurine, also had inhibitory effects on the hsp70 induction. In contrast, a calmodulin inhibitor, chlorpromazine, had little effect. These results suggest that heat shock increases [Ca2+]i in rat PTE cells and that [Ca2+]i and protein kinases are involved in the hsp70 induction following heat shock.

The relationship between [Ca2+]i and cell death using an in vivo model: a study using the ced-1 mutant strain of C. elegans.

The ced-1 mutant of the free-living nematode, Caenorhabitis elegans, was used to study cell injury and cell death in relation to changes in intracellular ionized calcium ([Ca2+]i). This animal, which is being genetically characterized, may prove to be extremely useful for certain toxicologic studies because of its small size, optical transparency, rapid generation time, and the morphologic and genetic data currently available. During the development of this animal, 131 of 1,090 ultimate somatic cells undergo programmed cell death. Using mutagenesis techniques, several genes responsible for this death have been identified. In this study, we have taken advantage of the ced-1 mutant in which dead cells accumulate, as they cannot be phagocytized and removed. Although changes in [Ca2+]i have been studied in relation to cell injury and cell death, observations have been essentially restricted to in vitro monolayer cultures because of the methodology involved. To study the relationship between changes in [Ca2+]i and injury in vivo, we selected this animal model for further study and report here the morphological changes following the effects of ionomycin treatment in relation to increases of [Ca2+]i and cell death as measured using the fluorescent probes Fluo-3/AM and propidium iodide, respectively. The technique of confocal laser scanning microscopy is ideally adapted to such measurements in these living animals, and the results can be readily correlated with those made with Nomarski differential interference contrast microscopy as well as with transmission electron microscopy. The results support previous in vitro observations and show that early increases of [Ca2+]i accompany early reactions to injury. Furthermore, the results also show that changes in this small invertebrate metazoan parallel those seen in mammalian systems, including human. Thus, the current study indicates that ced-1 C. elegans can potentially serve as an in vivo model not only for evaluating the possible temporal relationship of [Ca2+]i elevation with cell death but also for evaluating the [Ca2+]i elevation observed in relation to other phenomena and in evaluating toxic agents.

Induction of immediate early and stress genes in rat proximal tubule epithelium following injury: the significance of cytosolic ionized calcium.

This study was designed to investigate the influence of intracellular ionized calcium ([Ca2+]i) on the induction of c-fos, c-jun, c-myc, and hsp70 genes after oxidant stress induced by xanthine/xanthine oxidase (X/XOD) treatment or after heat shock using primary cultures of rat proximal tubule epithelium (PTE). X/XOD (500 microM/25 mU/mL) induced all of these genes; ionomycin also resulted in similar kinetics of induction of all genes. The expression of both c-fos following X/XOD treatment and hsp70 following heat shock was markedly decreased through chelation of [Ca2+]i by Quin 2/AM. The c-fos expression following X/XOD treatment was partly reduced by a protein kinase C inhibitor, staurosporine (ST), and markedly inhibited by another protein kinase inhibitor, 2-aminopurine (2AP), while both ST and 2AP markedly reduced hsp70 expression. The ADP-ribosylation transferase inhibitor 3-aminobenzamide had no effect on either c-fos or hsp70 expression. These results suggest that cell injuries leading to increased [Ca2+]i in PTE result in induction of c-fos, c-jun, c-myc, and hsp70; and that the activation of c-fos and hsp70 genes may be regulated by [Ca2+]i and [Ca2+]i-dependent protein kinases.

The role of cytosolic Ca2+ in cell injury, necrosis and apoptosis.

Increases in cytosolic Ca2+ are believed to be a pivotal signal in the regulation of cell injury, cell death, cell proliferation, cellular differentiation and cellular aging. Changes in the concentration of cytosolic Ca2+ are involved in both acute and chronic cell injury, as well as in accidental or programmed cell death. Signalling in all of these phenomena is dependent on mediated activities of a number of intracellular factors, including phospholipases, proteases and endonucleases. The coordinate regulation of these factors, as well as of oncogene activation, seems to play a role both in the processes of cell injury and cell death, and in the recovery from injury in sublethally injured cells.