Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin-1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin-1 but contain normal amounts of caveolin-2. NIH 3T3 cells harboring antisense caveolin-1 exhibit anchorage-independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin-1 downregulation is reversed when caveolin-1 protein levels are restored to normal by loss of the caveolin-1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin-1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin-1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade.     

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.     

Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.     

Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response

Apoptosis serves as a protective mechanism by eliminating damaged cells through programmed cell death. After apoptotic cells pass critical checkpoints, including mitochondrial fragmentation, executioner caspase activation, and DNA damage, it is assumed that cell death inevitably follows. However, this assumption has not been tested directly. Here we report an unexpected reversal of late-stage apoptosis in primary liver and heart cells, macrophages, NIH 3T3 fibroblasts, cervical cancer HeLa cells, and brain cells. After exposure to an inducer of apoptosis, cells exhibited multiple morphological and biochemical hallmarks of late-stage apoptosis, including mitochondrial fragmentation, caspase-3 activation, and DNA damage. Surprisingly, the vast majority of dying cells arrested the apoptotic process and recovered when the inducer was washed away. Of importance, some cells acquired permanent genetic changes and underwent oncogenic transformation at a higher frequency than controls. Global gene expression analysis identified a molecular signature of the reversal process. We propose that reversal of apoptosis is an unanticipated mechanism to rescue cells from crisis and propose to name this mechanism "anastasis" (Greek for "rising to life"). Whereas carcinogenesis represents a harmful side effect, potential benefits of anastasis could include preservation of cells that are difficult to replace and stress-induced genetic diversity.     

Hypoxia‐induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase‐1

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia‐induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl₂). Both the hypoxic gas mixture (1% O₂) and chemical hypoxia‐induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia‐mediated activation of caspase‐1. Active caspase‐1 induced the classical caspase‐dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase‐3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase‐1/classical caspase‐dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase‐3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.     

c-Myc induces cellular susceptibility to the cytotoxic action of TNF-alpha.

Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine which is cytotoxic for some tumor cells and transformed cells. The molecular mechanisms which render transformed and tumor cells sensitive to the cytotoxic action of TNF are unclear. We show here that an increased expression of the c-Myc oncoprotein strongly increases cellular sensitivity to TNF cytotoxicity. In Rat1A fibroblasts, which are resistant to TNF, the addition of TNF with a concomitant activation of a hormone-inducible c-Myc-estrogen receptor chimera (MycER) resulted in apoptotic cell death. Similarly, c-Myc overexpression enhanced the sensitivity of NIH3T3 fibroblasts to TNF-induced death. The c-Myc and TNF-induced apoptosis was inhibited by ectopic expression of the Bcl2 oncoprotein and by the free oxygen radical scavenging enzyme Mn superoxide dismutase. Furthermore, in highly TNF-sensitive fibrosarcoma cells, antisense c-myc oligodeoxynucleotides caused a specific inhibition of TNF cytotoxicity. Our results suggest that the deregulation of c-Myc, which is common in human tumors and tumor cell lines is one reason why these cells are TNF sensitive.     

Caveolin-1 expression enhances endothelial capillary tubule formation.

The level of caveolin-1 expression closely correlates with the oncogenic transformation of NIH 3T3 cells, the proliferation of human cancer cells, and the differentiation of adipocytes and muscle cells. However, the role of caveolin-1 in endothelial cell proliferation and differentiation remains unknown. Here, we have shown that angiogenic growth factors that stimulate endothelial cell proliferation lead to dramatic reductions in caveolin-1 expression. In addition, using an in vitro Matrigel assay system, we studied the potential role of caveolin-1 in capillary-like tubule formation (i.e. endothelial cell differentiation) using human microvascular endothelial cells (HMEC-1). We showed that the level of endogenous caveolin-1 expression increased in a time-dependent manner when endothelial cells underwent differentiation and that the maximum level of caveolin-1 expression occurred just prior to the formation of capillary-like tubules. Interestingly, overexpression of caveolin-1, via an adenoviral gene delivery system, clearly accelerated endothelial cell differentiation/tubule formation and led to a dramatic approximately 3-fold increase in the number of capillary-like tubular structures. Conversely, down-regulation of caveolin-1 expression, via an antisense adenoviral approach, reduced the number of capillary-like tubules formed by >10-fold. Consistent with the unique function of caveolin-1 in interacting with key signaling molecules, delivery of the caveolin-1 scaffolding domain into the cytoplasm of living endothelial cells was also sufficient to enhance capillary-like tubule formation. Taken together, these results clearly demonstrate that caveolin-1 and the caveolin-1 scaffolding domain play an important positive role in the regulation of endothelial cell differentiation, a prerequisite step in the process of angiogenesis.     

Identification of a novel 300-kDa factor termed IkappaB alphaE3-F1 that is required for ubiquitinylation of IkappaB alpha

Intracellular superoxide (O(2)*- was manipulated in M14 melanoma cells by overexpression or repression of Cu/Zn SOD using a tetracycline-inducible expression system. Scavenging intracellular O(2)*- increased tumor cell sensitivity to daunorubicin, etoposide, and pMC540, whereas expression of the antisense SOD mRNA significantly decreased cell sensitivity to drug treatment. Whereas Cu/Zn SOD overexpressing cells exhibited higher activation of the executioner caspase 3 upon drug exposure, caspase 3 activation was significantly lower when Cu/Zn SOD was repressed by antisense expression. These data show that intracellular O(2)*- regulates tumor cell response to drug-induced cell death via a direct or indirect effect on the caspase activation pathway.     

Cellular apoptosis is associated with increased caveolin-1 expression in macrophages

Macrophage apoptosis is an important factor in determining the efficiency of the immune response, atherosclerotic lesion stability, and clearance of aged cells by phagocytosis. The involvement of caveolin-1 in the regulation of apoptosis has been previously suggested in fibroblasts and epithelial cells. Here we show that treatment of thioglycollate-elicited mouse peritoneal macrophages with various unrelated apoptotic agents, including simvastatin, camptothecin, or glucose deprivation, is associated with a specific and large increase in caveolin-1 expression. In contrast, caveolin-2 levels remain unaffected. Induction of apoptosis was measured by changes in cell morphology, annexin V-labeling, and DNA fragmentation. We demonstrate that caveolin-1 in macrophages is present in lipid rafts and colocalizes with phosphatidylserine (PS) at the cell surface of apoptotic macrophages. Our data suggest that caveolin-1 increase is an early event, closely accompanied by PS externalization and independent of caspase activation and nuclear DNA fragmentation. The increase in caveolin-1 levels does not require new protein synthesis, as cycloheximide does not prevent the apoptosis-mediated increase in caveolin-1 levels. We propose that increased levels of caveolin-1 characterize the apoptotic phenotype of macrophages. Caveolin-1 may be involved in the efficient externalization of PS at the surface of the apoptotic cells.     

TGF-beta1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production

Serum deprivation induces apoptosis in NIH3T3 cells, which is associated with increased intracellular ceramide generation and with the activation of p38 mitogen-activated protein (MAP) kinase. Treatment of cells with transforming growth factor-beta1 (TGF-beta1) activated the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2), inhibited the serum deprivation-induced p38 activation and the increase in intracellular ceramide formation, leading to the stimulation of cell proliferation and the suppression of apoptosis. Inhibition of p38 MAP kinase by SB203580 significantly reduced the serum-deprivation-induced apoptosis. Overexpression of p38 increased the cell apoptosis and reduced the antiapoptotic effect of TGF-beta1. Inhibition of ERK1/ERK2 by PD98059 completely inhibited the TGF-beta1-stimulated proliferation and partially inhibited the antiapoptotic effects of TGF-beta1. Neither SB203580 nor PD98059 has obvious effect on TGF-beta1-mediated inhibition of the increased ceramide generation. Serum-deprivation-induced apoptosis in NIH3T3 cells can also be blocked by broad-spectrum caspase inhibitor. TGF-beta1 treatment has an inhibitory effect on caspase activities. Our results indicate that ceramide, p38, and ERK1/ERK2 play critical but differential roles in cell proliferation and stress-induced apoptosis. TGF-beta1 suppresses the serum-deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 activation and increased ceramide generation.     

Functional discontinuities in prothymosin alpha caused by caspase cleavage in apoptotic cells

Our study examines the effect of apoptosis on prothymosin alpha, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin alpha levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of prothymosin alpha. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact prothymosin alpha. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified prothymosin alpha. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated prothymosin alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of prothymosin alpha is a significant event in apoptosis. J. Cell. Physiol. 182:256-268, 2000. Published 2000 Wiley-Liss, Inc.     

Suppression of apoptosis by UVB irradiation: survival signaling via PI3-kinase/Akt pathway

UVB irradiation induces apoptosis in several cell types. However, we report here that UVB irradiation prevents induction of apoptosis in cells detached from the extracellular matrix under serum-free conditions. NIH3T3 cells cultured in bovine serum albumin-coated dishes (detached from the extracellular matrix) underwent apoptosis under serum-free conditions, which was inhibited by UVB (<0.1 J/cm(2)) irradiation, keeping suspension conditions, as determined by chromatin condensation and the appearance of a subG1 DNA fraction. Furthermore, UVB irradiation decreased caspase-3/7, -8/6, and -9 activation and eliminated loss of mitochondrial inner transmembrane potential, suggesting suppression upstream of the caspase cascade. Treatment with PI3-kinase inhibitors, wortmannin, and LY294002 partly eliminated the UV-mediated inhibition of cell death and recovered the inhibited caspase-3/7 activity. Phosphorylation of Akt was observed from 15 min after UVB irradiation. These results suggested that UVB irradiation transduced a survival signal via PI3 kinase activation and phosphorylation of Akt, and induced some apoptosis inhibition factors upstream of the caspase cascade.     

Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: Relation to drug cytotoxicity and caspase-3 activation

We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase-3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.     

Downregulation of bcl-xL is relevant to UV-induced apoptosis in fibroblasts

Exposure to ultraviolet light (UV) induces apoptosis in mammalian cells. The caspase group of proteases is required for the apoptosis. This pathway is initiated by a release of cytochrome c from the mitochondria into the cytosol. Several Bcl-2 family proteins can regulate the release of cytochrome c by stabilizing the mitochondrial membrane. Here we show that expression of the endogenous bcl-xL was strongly downregulated in NIH3T3 cells within 2 h after UV-C irradiation, and that of bax was upregulated from 8 h after irradiation. Apoptosis was induced in more than 50% of the NIH3T3 cells 48 h after irradiation. Constitutive overexpression of bcl-xL in NIH3T3 cells protected the UV-induced apoptosis by preventing the loss of mitochondrial membrane potential and the activation of caspase 9. These results suggest that downregulation of Bcl-xL is relevant to UV-induced apoptosis of fibroblasts.     

E1A oncogene enhancement of caspase-2-mediated mitochondrial injury sensitizes cells to macrophage nitric oxide-induced apoptosis

The adenovirus E1A oncogene induces innate immune rejection of tumors by sensitizing tumor cells to apoptosis in response to injuries, such as those inflicted by macrophage-produced TNF alpha and NO. E1A sensitizes cells to TNF by repressing its activation of NF-kappaB-dependent, antiapoptotic defenses. This suggested the hypothesis that E1A blockade of the NF-kappaB activation response might be the central mechanism of E1A induced cellular sensitivity to other proapoptotic injuries, such as macrophage-produced NO. However, creation of E1A-positive NIH-3T3 mouse cell variants with high-level, NF-kappaB-dependent resistance to TNF did not coselect for resistance to apoptosis induced by either macrophage-NO or chemical-NO, as the hypothesis would predict. E1A expression did block cellular recovery from NO-induced mitochondrial injury and converted the reversible, NO-induced cytostasis response of cells to an apoptotic response. This viral oncogene-induced phenotypic conversion of the cellular injury response of mouse and human cells was mediated by an E1A-related increase in NO-induced activation of caspase-2, an apical initiator of intrinsic apoptosis. Blocking caspase-2 activation or expression eliminated the NO-induced apoptotic response of E1A-positive cells. These results define an NF-kappaB-independent pathway through which the E1A gene of human adenovirus sensitizes mouse and human cells to apoptosis by enhancement of caspase-2-mediated mitochondrial injury.