Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.     

Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: roles of p53 and caspase activation

We and others have previously reported that human papillomavirus (HPV)-16 E6 protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explore the mechanism(s) of sensitization, we infected human keratinocytes (HKCs) with retroviruses containing HPV-6 E6, HPV-16 E6, HPV-16 E7, or HPV-16 E6/E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct observation of nuclei in cells stained with DAPI. Exposure of HKCs to etoposide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resulted in a time and dose-dependent induction of apoptosis. Expression of HPV-16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensitivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to normal HKCs, and resulted in cytoplasmic localization of wt p53. P53 induction occurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to cisplatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs, and no induction of p21 was seen in these cells following exposure to cisplatin or MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression of wt CrmA or treatment of HPV-16 E6 expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 E6-induced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV-16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etoposide and MMC, but not atractyloside or sodium butyrate. The data also suggest that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents.     

Ras signaling in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H-ras oncogene converted non-tumorigenic, TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF-induced apoptosis. This finding suggested that Ras activation may play a role in TNF-induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF-induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras-transformed 10TEJ cells inhibited TNF-induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF-induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF-sensitive 10TEJ cells, TNF treatment increased Ras-bound GTP in TNF-sensitive L929 cells but not in TNF-resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF-induced Ras-GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF-induced apoptosis in mouse fibroblasts.     

Multiple Functions of Human Papillomavirus Type 16 E6 Contribute to the Immortalization of Mammary Epithelial Cells

The E6 proteins from cervical cancer-associated human papillomavirus (HPV) types such as HPV type 16 (HPV-16) induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. We have previously shown that human mammary epithelial cells (MECs) immortalized by HPV-16 E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). To explore the potential functional significance of these interactions, we created and analyzed a series of E6 mutants for their ability to interact with E6-AP, p53, and E6BP in vitro. While there was a similar pattern of binding among these E6 targets, a subset of mutants differentiated E6-AP binding, p53 binding, and p53 degradation activities. These results demonstrated that E6 binding to E6-AP is not sufficient for binding to p53 and that E6 binding to p53 is not sufficient for inducing p53 degradation. The in vivo activity of these HPV-16 E6 mutants was tested in MECs. In agreement with the in vitro results, most of these p53 degradation-defective E6 mutants were unable to reduce the p53 level in early-passage MECs. Interestingly, several mutants that showed severely reduced ability for interacting with E6-AP, p53, and E6BP in vitro efficiently immortalized MECs. These immortalized cells exhibited low p53 levels at late passage. Furthermore, mutants defective for p53 degradation but able to immortalize MECs were also identified, and the immortal cells retained normal levels of p53 protein. These results imply that multiple functions of HPV-16 E6 contribute to MEC immortalization.     

The human papillomavirus 16 E6 protein binds to tumor necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that transfection of HPV 16 E6 into the tumor necrosis factor (TNF)-sensitive LM cell line protects expressing cells from TNF-induced apoptosis in a p53-independent manner, and the purpose of this study was to determine the molecular mechanism underlying this protection. Caspase 3 and caspase 8 activation were significantly reduced in E6-expressing cells, indicating that E6 acts early in the TNF apoptotic pathway. In fact, E6 binds directly to TNF R1, as shown both by co-immunoprecipitation and mammalian two-hybrid approaches. E6 requires the same C-terminal portion of TNF R1 for binding as does TNF R1-associated death domain, and TNF R1/TNF R1-associated death domain interactions are decreased in the presence of E6. HA-E6 also blocked cell death triggered by transfection of the death domain of TNF R1. Together, these results provide strong support for a model in which HPV E6 binding to TNF R1 interferes with formation of the death-inducing signaling complex and thus with transduction of proapoptotic signals. They also demonstrate that HPV, like several other viruses, has developed a method for evading the TNF-mediated host immune response.     

E1A sensitizes cells to tumor necrosis factor-induced apoptosis through inhibition of IkappaB kinases and nuclear factor kappaB activities.

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.     

E1A oncogene-induced cellular sensitization to immune-mediated apoptosis is independent of p53 and resistant to blockade by E1B 19 kDa protein.

E1A oncogene expression sensitizes mammalian cells to apoptosis triggered by cytolytic lymphocytes (CL) [16]. Most studies suggest that E1A-induced apoptosis involves a p53-dependent cellular pathway that is blocked by the E1B 19 kDa gene product. In this study, the roles of p53 and E1B 19 kDa were tested for E1A sensitization to CL-induced apoptosis in contrast with apoptosis triggered by TNF alpha or chemical injuries. E1A sensitization to immune-mediated (CL- or TNF-induced) apoptosis was independent of p53 expression and was resistant to blockade by E1B 19 kDa protein in mouse and hamster cells. In contrast, the p53 requirement for chemically induced apoptosis of E1A-sensitized cells varied with the agent used to treat cells. Apoptosis induced by diverse chemical agents (hygromycin, beauvericin, etoposide, H(2)O(2)) was blocked by E1B 19 kDa expression. Therefore, both the p53-dependence and the E1B 19 kDa blockade of E1A-induced cellular sensitization to apoptotic injury depend on the type of proapoptotic injury tested. These data suggest that the mechanisms by which E1A sensitizes tumor cells to immune-mediated apoptosis and to rejection by immunocompetent animals do not require cellular expression of wild-type p53 and can function independently of the Bcl-2-like, antiapoptotic mechanisms of E1B 19 kDa.     

Distinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells

Tumor necrosis factor (TNF) alpha is a cytokine capable of inducing caspase-dependent (apoptotic) cell death in some cells and caspase-independent (necrosis-like) cell death in others. Here, using a mutagenesis screen for genes critical in TNF-induced death in L929 cells, we have found that H-ferritin deficiency is responsible for TNF resistance in a mutant line and that, upon treatment with TNF, this line fails to elevate levels of labile iron pool (LIP), critical for TNF-induced reactive oxygen species (ROS) production and ROS-dependent cell death. Since we found that TNF-induced LIP in L929 cells is primarily furnished by intracellular storage iron, the lesser induction of LIP in H-ferritin-deficient cells results from a reduction of intracellular iron storage caused by less H-ferritin. Different from some other cell lines, the H-ferritin gene in L929 cells is not TNF inducible; however, when H-ferritin is expressed in L929 cells under a TNF-inducible system, the TNF-induced LIP and subsequent ROS production and cell death were all prevented. Thus, LIP is a common denominator of ferritin both in the enhancement of cell death by basal steady-state H-ferritin and in protection against cell death by induced H-ferritin, thereby acting as a key determinant of TNF-induced cell death.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Rac1 inhibits TNF-alpha-induced endothelial cell apoptosis: dual regulation by reactive oxygen species.

Reactive oxygen species (ROS) have been implicated as mediators of tumor necrosis factor-alpha (TNF) -induced apoptosis. In addition to leading to cell death, ROS can also promote cell growth and/or survival. We investigated these two roles of ROS in TNF-induced endothelial cell apoptosis. Human umbilical vein endothelial cells (HUVECs) stimulated with TNF produced an intracellular burst of ROS. Adenoviral-mediated gene transfer of a dominant negative form of the small GTPase Rac1 (Rac1N17) partially suppressed the TNF-induced oxidative burst without affecting TNF-induced mitochondrial ROS production. HUVECs were protected from TNF-induced apoptosis. Expression of Rac1N17 blocked TNF-induced activation of nuclear factor-kappa B (NF-kappaB), increased activity of caspase-3, and markedly augmented endothelial cell susceptibility to TNF-induced apoptosis. Direct inhibition of NF-kappaB through adenoviral expression of the super repressor form of inhibitor of kappaBalpha (I-kappaB S32/36A) also increased susceptibility of HUVECs to TNF-induced apoptosis. Rotenone, a mitochondrial electron transport chain inhibitor, suppressed TNF-induced mitochondrial ROS production, proteolytic cleavage of procaspase-3, and apoptosis. These findings show that Rac1 is an important regulator of TNF-induced ROS production in endothelial cells. Moreover, they suggest that Rac1-dependent ROS, directly or indirectly, lead to protection against TNF-induced death, whereas mitochondrial-derived ROS promote TNF-induced apoptosis.     

Adenovirus genes that modulate the sensitivity of virus-infected cells to lysis by TNF

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G₀ cells into S-phase, and induces apoptosis and susceptibility to TNF-induced apoptosis. The adenovirus E1B-19K protein inhibits both E1A- and TNF-induced apoptosis. The E3-14.7K protein and the E3-10.4K/14.5K complex of proteins inhibit TNF- but not E1A-induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A₂ (cPLA₂), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA₂) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.     

Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins.

The E6 and the E7 proteins of the oncogenic human papillomavirus types 16 and 18 can stably associate with p53 and the retinoblastoma protein, respectively. The E6-p53 interaction results in the accelerated degradation of p53 in vitro via the ubiquitin-dependent proteolysis system. In this study we demonstrate that a fusion protein consisting of the N-terminal half of the HPV-16 E7 protein and the full length HPV-16 E6 protein promotes the in vitro degradation of the retinoblastoma protein. This indicates that the property of the HPV-16 E6 protein to stimulate the degradation of p53 can be targeted to other proteins. Unlike the HPV-16 or HPV-18 E6 protein, the E6 proteins of HPV-6 and 11 do not bind to p53 and consequently do not target p53 for degradation. Analogous E7-E6 fusion proteins using the E6 proteins of HPV-6 and HPV-11, however, also have the ability to promote the degradation of the retinoblastoma protein, indicating that the property to target associated proteins for degradation is shared by the anogenital specific HPV E6 proteins.     

cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.     

Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling

Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-1 mutant were more sensitive to TNF than wild-type cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death.     

Interaction of the human papillomavirus type 16 E6 oncoprotein with wild-type and mutant human p53 proteins.

The E6 oncoproteins encoded by the cancer-associated human papillomaviruses (HPVs) can associate with and promote the degradation of wild-type p53 in vitro. To gain further insight into this process, the ability of HPV-16 E6 to complex with and promote the degradation of mutant forms of p53 was studied. A correlation between binding and the targeted degradation of p53 was established. Mutant p53 proteins that bound HPV-16 E6 were targeted for degradation, whereas those that did not complex HPV-16 E6 were not degraded. Since the HPV-16 E6-promoted degradation involves the ubiquitin-dependent proteolysis pathway, specific mutations were made in the amino terminus of p53 to examine whether the E6 targeted degradation involved the N-end rule pathway. No requirement for destabilizing amino acids at the N terminus of p53 was found, nor was evidence found that HPV-16 E6 could provide this determinant in trans, indicating that the N-terminal rule pathway is not involved in the E6-promoted degradation of p53.     

Redox regulation of TNF signaling

TNF is produced during inflammation and induces, among other activities, cell death in sensitive tumour cells. We previously reported an increased generation of ROS in TNF-treated L929 fibrosarcoma cells prior to cell death. These ROS are of mitochondrial origin and participate in the cell death process. Presently, we focus on the identification of parameters that control ROS production and subsequent cytotoxicity. From the cytotoxic properties and susceptibility to scavenging of TNF-induced ROS as compared to pro-oxidant-induced ROS we conclude that TNF-mediated ROS generation and their lethal action are confined to the inner mitochondrial membrane. Oxidative substrates, electron-transport inhibitors, glutathione and thiol-reactive agents but also caspase inhibitors modulate TNF-induced ROS production and imply the existence of a negative regulator of ROS production. Inactivation of this regulator by a TNF-induced reduction of NAD(P)H levels and/or formation of intraprotein disulfides would be responsible for ROS generation.     

A protein serologically and functionally related to the group C E3 14,700-kilodalton protein is found in multiple adenovirus serotypes.

A 14.7-kilodalton protein (14.7K protein) encoded by the E3 region of group C adenoviruses has been shown to protect virus-infected fibroblasts from lysis by tumor necrosis factor (TNF) (L.R. Gooding, L.W. Elmore, A.E. Tollefson, H.A. Brady, and W.S.M. Wold, Cell 53:341-346, 1988). In this study we show that adenoviruses of other groups are also protected from TNF-induced cytolysis. Representative serotypes of groups A, B, D, and E produce a protein analogous to the 14.7K protein found in human group C adenoviruses. Deletion of this protein in group C viruses permits virus infection to induce cellular susceptibility to TNF killing. As with group C adenoviruses, cells infected with wild-type adenoviruses of other serotypes are not killed by TNF and are protected from lysis induced by TNF plus cycloheximide. However, cells are susceptible to TNF-induced lysis when infected with adenovirus type 4 mutants from which the 14.7K gene has been deleted. Although all known adenovirus serotypes infect epithelial cells, adenoviruses cause several diseases with various degrees of pathogenesis. Our findings suggest that the 14.7K protein provides a function required for the in vivo cytotoxicity of many adenoviruses independent of the site of infection or degree of pathogenesis.     

Human papillomavirus (HPV) 16 E6 sensitizes cells to atractyloside-induced apoptosis: Role of p53, ICE-like proteases and the mitochondrial permeability transition

Infection of cervical epithelial cells with certain high risk HPV genotypes is thought to play an etiologic role in the development of cervical cancer. In particular, HPV type 16 and 18 early protein 6 (E6) is thought to contribute to epithelial transformation by binding to the tumor suppressor protein p53, targeting it for rapid proteolysis, resulting in loss of its cell cycle arrest and apoptosis-inducing activities. Recent data indicate that factors responsible for triggering apoptosis reside in the cytoplasm of cells, and not in the nucleus. In particular, the findings that mitochondria are required in certain cell-free models for induction of apoptosis and that bcl-2 is localized to mitochondria have focused attention on the role of the mitochondrial membrane permeability transition (MPT) in apoptosis. Here we present data to indicate that HPV 16 E6 expression sensitizes cells to MPT-induced apoptosis. We also report that HPV 16 E6 sensitization of cells to MPT-induced apoptosis occurs only in the presence of wildtype (wt) p53 expression. The extent of apoptosis induced by atractyloside (an inducer of the MPT) in normal, temperature-sensitive (ts) p53, and HPV-16 E6 transfected J2-3T3 cells, and the HPV expressing cervical carcinoma cell lines SiHa, Hela and CaSki was determined. C33A cells, which express mutant p53 but not HPV, were also exposed to atractyloside in the presence or absence of HPV 16 E6 expression. Dose-dependent apoptosis induced by atractyloside in normal J2-3T3 cells and cervical carcinoma cells was measured by loss of cell viability, nuclear fragmentation and DNA laddering. The sensitivity of cells to atractyloside-induced apoptosis was found to be: HPV 16 E6-J2-3T3 > CaSki > normal-J2-3T3 cells ≈ ts p53-J2-3T3 ≈ vector-J2-3T3 cells > Hela > SiHa > C33A ≈ C33A 16 E6. Cyclosporin A (CsA), an inhibitor of the MPT, and ICE-I, a protease inhibitor, provided protection against atractyloside-induced apoptosis. These findings indicate that: 1) high risk HPV 16 E6 protein is capable of sensitizing cells to apoptosis; 2) HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis occurs in a p53-dependent fashion; 3) the target of HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis is the mitochondria; and 4) HPV 16 E6 sensitization of cells to atroctycoside-induced apoptosis involves an ICE-like protease-sensitive mechanism, regulating the onset of the MPT. These findings constitute the first evidence that mitochondria play a role in HPV 16 E6 modulation of apoptosis. J. Cell. Biochem. 66:245-255. © 1997 Wiley-Liss, Inc.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.