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.

"Thanatosomes": a unifying morphogenetic concept for tumor hyaline globules related to apoptosis.

Hyaline globules (HG) have been observed in a large variety of unrelated categories of tumors and benign tissues. Different explanations for their formation have been proposed, varying according to tumor type and anatomic location. We have studied 80 tumor cases containing HG, by light microscopy, electron microscopy, immunohistochemical stains for various plasma proteins, and in situ DNA-end labeling for apoptosis. On light microscopy, HG were invariably related to areas of increased apoptosis and often contained apoptotic nuclear fragments. The HG stained as expected, with variable intensity with acidic stains. Most cells containing HG stained with immunohistochemical stains for all plasma proteins examined (alpha-1-antitrypsin, ferritin, C3, kappa and lambda light chains, and IgG), indicating an increased plasma membrane permeability. A morphologic change associated with the increased permeability was cytoplasmic blebbing. In the HG themselves, immunohistochemical stains for the plasma proteins were either diffusely positive or stained only the periphery of the larger HG. Double stains for apoptosis and plasma proteins confirmed the increased plasma membrane permeability to proteins of apoptotic cells in general and cells containing HG in particular. Free hyaline globules were often linked to the extracellular matrix by fibrin fibrils. Ultrastructurally, the HG appeared as phagosomes/secondary lysosomes or areas of cytoplasmic condensation surrounded by rough endoplasmic reticulum whorls. These were always associated with intense cytoplasmic blebbing. We conclude that HG reflect stages of cell injury, which in most instances relate to apoptotic cell death. They are specifically associated with the cytoplasmic blebbing and condensation typically seen in this form of cell death. These phenomena are accompanied by plasma protein influx (insudated plasma) and formation of distinct intracellular "hyalinized" cellular fragments. With cell fragmentation, the HG become extracellular and are likely to be ultimately disposed of by a process of "remodeling" and incorporation into the extracellular matrix, followed by collagenization. The latter process partly occurs by the initial linking of all components (HG, collagen fibers, cellular fragments, etc.) by a network of fibrin. The process of formation of HG follows a stereotypical pathway independent of cell type. Because it results mostly from apoptotic cell death, it is more pronounced in rapidly growing tumors or posttreatment. We propose the term thanatosomes for the entire spectrum of HG to emphasize their relationship to cell death. HUM PATHOL 31:1455-1465.

H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo.

We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells. H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.

The nomenclature of cell death: recommendations of an ad hoc Committee of the Society of Toxicologic Pathologists.

The last several years have seen considerable confusion regarding the terms "apoptosis" and "necrosis" in pathology. This situation prompted the Society of Toxicologic Pathologists to charter the Committee on the Nomenclature of Cell Death, which was charged with making recommendations about the use of the terms "apoptosis" and "necrosis" in toxicity studies. The Committee recommends use of the term "necrosis" to describe findings comprising dead cells in histological sections, regardless of the pathway by which the cells died. The modifiers "apoptotic" and "oncotic" or "mixed apoptotic and oncotic" are recommended to specify the predominant morphological cell death pathway or pathways, when appropriate. Other standard modifiers, indicating the lesion distribution and severity, may also be used in conjunction with these. "Individual cell necrosis" (also known as "single cell necrosis") may be either of the apoptotic, oncotic, or mixed types. In many cases, more traditional terms such as "coagulation necrosis" may be used to convey a meaning similar to oncotic necrosis. It is important that pathologists use terms that accurately and concisely convey the level of information appropriate to the study's needs. Furthermore, toxicologic pathologists should actively help to disseminate these recommendations to other biologists and to regulatory authorities.

Cytotoxic effects of eosinophils on renal proximal tubular epithelial cells: implications for renal allograft rejection.

Eosinophils are inflammatory cells that actively participate in the defense against large, multicellular parasites. Eosinophils are also a prominent component in the inflammatory infiltrates seen in acute allograft rejection. Their role in that process, however, remains obscure. In this study we examined in vitro the direct cytotoxicity of eosinophils against renal proximal tubular epithelial cells (PTEC) in the presence or absence of antibody and complement. Eosinophils were able to attach to the PTEC surface in both circumstances, i.e., irrespective of opsonization, or other mediation by humoral factors. The release of eosinophil granule contents (including their characteristic crystalloids) on the target cell surface and resulting PTEC injury occurred also irrespective of the presence or absence of opsonization. Thus, based on these results, the potential of direct cytotoxicity of eosinophils for PTEC exists, particularly in situations where both of these cell types are 'activated'.

Interspecies differences in renal localization of cyclooxygenase isoforms: implications in nonsteroidal antiinflammatory drug-related nephrotoxicity.

Cyclooxygenase (COX) exists in 2 related but unique isoforms: one is constitutive (COX-1) and functions in normal cell physiology, and the other is inducible (COX-2) and is expressed in response to inflammatory stimuli. Nonsteroidal antiinflammatory drugs (NSAIDs) cause renal toxicity following inhibition of renal cyclooxygenases. Humans and animals exhibit differences in susceptibility to NSAID-related renal toxicity, which may be associated with differences in expression of 1 or both isoforms of COX in the kidney. In this study, we evaluated COX-1 and COX-2 expression in the kidneys of mixed-breed dogs, Sprague-Dawley rats, cynomolgus monkeys, and humans. In addition, the effect of volume depletion on renal COX expression was investigated in rats, dogs, and monkeys. COX expression was evaluated using 1 or more of the following procedures: reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. We demonstrated that both COX isoforms are expressed in the kidneys of all species examined, with differences in the localization and level of basal expression. COX-1 is expressed at high levels in the collecting ducts and renal vasculature of all species and in a small number of papillary interstitial cells in rats, monkeys, and humans. Basal levels of COX-2 are present in the maculae densa, thick ascending limbs, and papillary interstitial cells in rats and dogs and in glomerular podocytes and small blood vessels in monkeys and humans. COX-2 expression is markedly increased in volume-depleted rats and dogs but not monkeys. These results indicate that significant interspecies differences exist in the presence and distribution of COX isoforms, which may help explain the difference in species susceptibility to NSAID-related renal toxicity.

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.

Interaction between cytolytic T-cells and immobilized renal tubular epithelial cells: evidence of polarity on both effector and target cells.

The mode by which cytolytic T-lymphocytes (CTL) deliver the lethal hit to their targets has been studied extensively. The CTL exhibits a characteristic polarization, with the attacking end formed by the uropod (containing the Golgi complex and associated granules). The effect on the target cell (TC) has been considered as a form of apoptosis or of both necrosis. In this communication we describe the particular morphological steps that relate to the participation of the target renal epithelial cell to its own destruction. Thus, the TC forms tight membrane contacts with the CTL that are invested by a dense network of stress fibers and microtubules within the TC cytoplasm in a manner similar to the areas of attachment between the epithelial cells or of the latter to the underlying surface. The TC mitochondria at the site of the CTL attachment are markedly swollen, in contrast to the more distally located ones that appear initially normal. The TC plasma membrane surrounding the CTL attachment site shows focally intense blebbing. In later stages, associated with overt apoptosis, global TC blebbing is present. In summary, the TC shows a polarization pattern, that corresponds roughly to the polarity exhibited by the attacking cell (uropod-protopod), and to some degree treats the latter cell as a junctional partner.

Human breast cancer cell growth inhibition and deregulation of [Ca2+]i by estradiol.

Estradiol inhibits the growth of human breast cancer MCF-7 cells at supramicromolar concentrations. The mechanism of such phenomenon remains to be unravelled. Confocal laser scanning microscopic studies suggest elevation of [Ca2+]i preceding bleb formation and cellular injury following acute and chronic treatment of ionomycin and estradiol at supramicromolar concentration. Phase contrast morphological study demonstrates metaphase-arrested cells and giant multinuclear cells possibly due to lack of cytokinesis caused by estradiol. There is a striking similarity between the morphological changes caused by estradiol and enforced overexpression of cyclin-dependent kinase inhibitor p21waft/cip1. Such similarity together with the reported key role of intracellular ionized calcium [ca2+]i in regulating cyclin and deregulation of [Ca2+]i by estradiol raises the possibility of deregulation of gene expression leading to inhibition of cyclin-dependent proliferative signals participating in inhibition of growth in MCF-7 cells.

Tamoxifen induces deregulation of [Ca2+] in human breast cancer cells.

Tamoxifen is a clinically useful estrogen antagonist at or below 10(-6) M concentration. However, above this concentration tamoxifen exerts non-ER mediated cytotoxicity. Such cytotoxic effects are lethal or sublethal. The lethal effects lead to cell death while the sublethal effects may lead to cellular transformation and response modification participating in the process of tumor resistance or even tumor stimulation. Deregulation of intracellular ionized calcium ([Ca2+]i) could lead to genomic instability and deregulation of oncogene expression which might participate in the process of carcinogenesis and/or tumor promotion. Tamoxifen may cause tumor stimulation due to deregulation of [Ca2+]i or its consequences such as activity of protein kinase C, calmodulin and related protein kinases. Precise understanding of such mechanism is important for avoiding tamoxifen induced tumor resistance or tumor stimulation. The deregulation of [Ca2+]i was studied on fluo-3/AM loaded MCF-7 human breast cancer cells following acute and chronic treatment of tamoxifen and calcium ionophore ionomycin. The elevation of [Ca2+]i preceded the death of MCF-7 cells following treatment with ionomycin as previously reported on other cells. Tamoxifen above 10(-6) M also caused an increase in [Ca2+]i preceding the death of MCF-7 cells. However, below this concentration, tamoxifen caused a decrease in [Ca2+]i without any signs of cytotoxicity. The present data clearly demonstrate a tamoxifen-induced increase in [Ca2+]i and cell death only at the concentration-range in which non-E R mediated cytotoxicity is reported.

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.

Differential sensitivity of normal and H-ras oncogene-transformed rat kidney epithelial cells to okadaic acid-induced apoptosis.

H-ras oncogenes have been identified in greater than 50% of the most common forms of human neoplasia. Ras-related proteins have been postulated to mediated signal transduction pathways involving mitogen-activated protein (MAP) kinases and nuclear responses that may be involved in the induction of apoptosis. We examined whether expression of H-ras oncogene conferred resistance or susceptibility to the morphologic effects of the protein phosphatase inhibitor, okadaic acid, using a tumorigenic H-ras-transformed normal rat kidney epithelial cell line, NRK-H/6.1. We also examined whether okadaic acid induced apoptosis correlated with a differential effect on kinase activity in H-Ras-transformed cells as compared to the nontransformed NRK-52E cells. Treatment with various concentrations of okadaic acid produced rapid and extensive morphologic changes characteristic of apoptosis in both cell types. Equimolar okadaic acid concentrations for 2 or 4 hr resulted in cell detachment and loss of membrane integrity (as measured by propidium iodide uptake) in 74% (0.5 microM) and 78% (1.0 microM) of the H-Ras-transformed cells as compared to 8 and 25%, respectively, in the non-transformed cells. Furthermore, a higher basal level of kinase activity was observed in the H-Ras-transformed cells as compared to the nontransformed cells. Okadaic acid-induced apoptosis correlated with activation of members of the MAP kinase family, raf-1 and protein kinase C (PKC). These studies show that H-ras oncogene expression imparts selective susceptibility to cell death induced by phosphatase inhibition. The observed increase in susceptibility to okadaic acid-induced apoptosis appears to involve the modulation of raf-1, PKC, and MAP kinase activities. These findings may be significant in the elucidation of mechanisms for selective induction of cell death in tumor cells expressing H-ras oncogene.

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.