Inhibition of Bim Enhances Replication of Varicella-Zoster Virus and Delays Plaque Formation in Virus-Infected Cells

Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.     

ERBB2 is a target for USP8-mediated deubiquitination

Overexpression and poor downregulation of ErbB receptor tyrosine kinases are associated with enhanced signaling and tumorigenesis. Attenuation of EGF-receptor (EGFR) signaling is mediated by endocytosis and ubiquitination by the E3-ligase Cbl. En route to lysosomes, but before incorporation of the EGFR into internal vesicles of MVBs, the EGFR undergoes Usp8-mediated deubiquitination. ErbB2 displays enhanced recycling back to the cell surface, and therefore we hypothesized that Usp8 is not part of the ErbB2 trafficking pathway. Here, we demonstrate, in the context of a chimeric EGFR-ErbB2 receptor, that (i) EGF induces pY1091 Cbl binding site-dependent K63-polyubiquitination of EGFR-ErbB2, (ii) Cbl is tyrosine phosphorylated upon stimulation of EGFR-ErbB2 wt and Y1091F mutant receptor, (iii) EGF-induced activation of EGFR-ErbB2 induces Usp8 tyrosine phosphorylation, and (iv) ubiquitination of the EGFR-ErbB2 wt and Y1091F mutant is enhanced upon coexpression of catalytically inactive Usp8-C748A in the presence and absence of EGF. We further show that Usp8 tyrosine phosphorylation upon stimulation of EGFR-ErbB2 is (a) independent of Y1091, (b) dependent on Src- and EGFR-ErbB2-kinase activity, (c) enhanced upon coexpression of Usp8-C748A, and (d) partly dependent on the Microtubule Interacting and Transport (MIT) domain of Usp8. Our findings demonstrate that Usp8 is part of the ErbB2 endosomal trafficking pathway.     

Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.     

Bim is reversibly phosphorylated but plays a limited role in paclitaxel cytotoxicity of breast cancer cell lines

The chemotherapeutic drug, paclitaxel, induces mitotic arrest and then activates the cellular apoptotic program. Although paclitaxel has been in clinical use for over 10 years for the treatment of breast, ovarian, and lung cancer, the molecular mechanisms of paclitaxel-induced cytotoxicity are ill defined. We decided to investigate the regulatory mechanism of the pro-apoptotic BH3-only protein Bim, which is known to play a role in paclitaxel cytotoxicity. We discovered that paclitaxel induces reversible phosphorylation of Bim. Bim initially displays enhanced phosphorylation during paclitaxel-induced mitotic arrest, and then undergoes de-phosphorylation as cells become apoptotic. This dynamic phosphorylation is dependent on mitotic checkpoint signaling. However, while these results suggest that reversible phosphorylation of Bim may contribute to the transmission of a mitotic checkpoint-to-apoptosis signal, we did not observe a strong correlation between Bim protein levels and cellular sensitivity to paclitaxel. Indeed, in contrast to the well-defined role of Bim in paclitaxel-induced cell death in mouse model cells, our depletion studies demonstrate that Bim is not absolutely required for paclitaxel cytotoxicity in breast cancer cell lines. Clearly it is imperative to define the contribution of Bim in paclitaxel-induced apoptosis of clinically relevant targets in order to rationally develop enhanced treatment strategies.     

Activation of Raf1 and the ERK pathway in response to l-ascorbic acid in acute myeloid leukemia cells

L-ascorbic acid (LAA) shows cytotoxicity and induces apoptosis of malignant cells in vitro, but the mechanisms by which such effects occur have not been elucidated. In the present study, we provide evidence that the ERK MAP kinase pathway is activated in response to LAA (< 1 mM) in acute myeloid leukemia cell lines. LAA treatment of cells induces a dose-dependent phosphorylation of extracellular signal-regulated kinases (ERK) and results in activation of its catalytic domain. Our data also demonstrate that the small G protein Raf1 and MAPK-activated protein kinase 2 are activated by LAA as an upstream and a downstream regulator of ERK, respectively. Although the ERK pathway has been known to activate cell proliferation, pharmacologic inhibition of ERK reduces LAA-dependent apoptosis and growth inhibitory response of acute myeloid leukemia cell lines, suggesting that this signaling cascade positively regulates induction of apoptotic response by LAA.     

Non‐canonical Raf‐1/p70S6K signalling in non–small‐cell lung cancer

Lung cancer is the leading cause of cancer‐related death globally, with non–small‐cell lung cancer (NSCLC) being the predominant subtype. Overall survival remains low for NSCLC patients, and novel targets are needed to improve outcome. Raf‐1 is a key component of the Ras/Raf/MEK signalling pathway, but its role and downstream targets in NSCLC are not completely understood. Our previous study indicated a possible correlation between Raf‐1 levels and ribosomal protein S6 kinase (p70S6K) function. In this study, we aimed to investigate whether p70S6K is a downstream target of Raf‐1 in NSCLC. Raf‐1 was silenced in NSCLC cell lines by using small hairpin RNA, and Raf‐1 and p70S6K protein levels were measured via Western blot. p70S6K was then overexpressed following Raf‐1 knock‐down; then, cell proliferation, apoptosis and the cell cycle in NSCLC cell lines were examined. Tumour xenografts with NSCLC cells were then transplanted for in vivo study. Tumours were measured and weighed, and Raf‐1 and p70S6K expression, cell proliferation and apoptosis were examined in tumour tissues by Western blot, Ki‐67 staining and TUNEL staining, respectively. When Raf‐1 was silenced, p70S6K protein levels were markedly decreased in the A549 and H1299 NSCLC cell lines. A significant decrease in NSCLC cell proliferation, a profound increase in apoptosis and cell cycle arrest were observed in vitro following Raf‐1 knock‐down. Overexpression of p70S6K after Raf‐1 depletion effectively reversed these effects. Xenograft studies confirmed these results in vivo. In conclusion, Raf‐1 targets p70S6K as its downstream effector to regulate NSCLC tumorigenicity, making Raf‐1/p70S6K signalling a promising target for NSCLC treatment.     

Regulation of Bim by TCR signals in CD4/CD8 double-positive thymocytes

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3epsilon injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that Bim(EL) is rapidly phosphorylated in thymocytes in response to CD3epsilon cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-x(L) bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-x(L) in immature thymocytes.     

Phosphorylation of heat shock protein 27 antagonizes TNF-α induced HeLa cell apoptosis via regulating TAK1 ubiquitination and activation of p38 and ERK signaling

Tumor necrosis factor (TNF)-α is a potent cytokine that regulates critical cellular processes including apoptosis. TNF-α usually triggers both survival and apoptotic signals in various cell types. Heat shock protein 27 (HSP27), an important cellular chaperone, is believed to protect cells from apoptosis. HSP27 can be phosphorylated and changed its cellular function according to different stimuli. However, available reports on the role of HSP27 phosphorylation in apoptosis remain elusive. In this study, we investigated the role of HSP27 phosphorylation in TNF-α induced apoptosis in human cervical carcinoma (HeLa) cells. We found that TNF-α induced apoptosis was enhanced if we suppressed the TNF-α induced HSP27 phosphorylation by specific inhibitor CMPD1 or MAPKAPK2 (MK2) knockdown or by overexpression of non-phosphorylatable mutant HSP27-3A. Through co-immunoprecipitation and confocal microscopy, we observed that HSP27 associated with transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) in response to TNF-α stimulation. By blocking MK2 activity or overexpressing phospho-mimetic mutant Hsp27-3D, we further showed that HSP27 phosphorylation facilitated the TNF-α induced ubiquitination and phosphorylation of TAK1 and the activations of p38 MAPK and ERK, the TAK1 downstream pro-survival signaling. In addition, we also found that increased HSP27 phosphorylation inhibited TRADD ubiquitination but did not influence the binding between TRADD and FADD in a pro-apoptotic complex. Taken together, our data indicated that HSP27 phosphorylation was involved in modulating the TNF-α induced apoptosis via interacting with TAK1 and regulating TAK1 post-translational modifications in HeLa cells. This study demonstrates that HSP27 phosphorylation serves as a novel regulator in TNF-α-induced apoptosis, and provides a new insight into the cytoprotective role of HSP27 phosphorylation.     

H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib

Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.     

CDK4/6 inhibition suppresses tumour growth and enhances the effect of temozolomide in glioma cells

In adults, glioma is the most commonly occurring and invasive brain tumour. For malignant gliomas, the current advanced chemotherapy includes TMZ (temozolomide). However, a sizeable number of gliomas are unyielding to TMZ, hence, giving rise to an urgent need for more efficient treatment choices. Here, we report that cyclin‐dependent kinases 4 (CDK4) is expressed at significantly high levels in glioma cell lines and tissues. CDK4 overexpression enhances colony formation and proliferation of glioma cells and extends resistance to inhibition of TMZ‐mediated cell proliferation and induction of apoptosis. However, CDK4 knockdown impedes colony formation and cell proliferation, and enhances sensitivity of glioma cells to TMZ. The selective inhibition of CDK4/6 impedes glioma cell proliferation and induces apoptotic induction. The selective inhibitors of CDK4/6 may enhance glioma cell sensitivity to TMZ. We further showed the possible role of RB phosphorylation mediated by CDK4 for its oncogenic function in glioma. The growth of glioma xenografts was inhibited in vivo, through combination treatment, and corresponded to enhanced p‐RB levels, reduced staining of Ki‐67 and enhanced activation of caspase 3. Therefore, CDK4 inhibition may be a favourable strategy for glioma treatment and overcomes TMZ resistance.     

c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression

Overexpression of the oncogene c-Myc sensitizes many apoptotic signals through the activation of mitochondrial apoptosis pathway. However, the underling mechanism has not been clearly defined. Here, we investigated the effect of c-Myc expression on histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)-induced apoptosis in rat fibroblast cells possessing various c-Myc levels. In Rat 1a cells overexpressing c-Myc, SAHA-induced enhanced the cell death response relative to the parental cells; whereas Rat 1a cells lacking c-Myc were refractory to SAHA treatment. We demonstrated that SAHA selectively induced the expression of pro-apoptotic BH3-only protein Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA, but is required for subsequent Bax activation. We further show that the expression levels of anti-apoptotic Bcl-2/Bcl2-xL were much elevated in Myc-null cells compared with the c-Myc-expressing cells; furthermore, depletion of Bcl-2/Bcl-xL in these cells restored the ability of SAHA to induce apoptosis by enhancing Bax activation. These data indicate that SAHA induces apoptosis through Bim-triggered Bax activation and that c-Myc regulates this process by modulating Bcl-2/Bcl-xL. Our results provide novel insight into the mechanism whereby Myc sensitizes the apoptotic signals; furthermore, our data suggest that cancer cells with deregulated Myc might be more sensitive to SAHA treatment.     

Parathyroid hormone inhibits phosphorylation of mitogen‐activated protein kinase (MAPK) ERK1/2 through inhibition of c‐Raf and activation of MKP‐1 in osteoblastic cells

Parathyroid hormone (PTH) regulation of mitogen‐activated protein kinases (MAPK) ERK1/2 contributes to PTH regulation of osteoblast growth and apoptosis. We investigated the mechanisms by which PTH inhibits ERK1/2 activity in osteoblastic UMR 106‐01 cells. Treatment with PTH significantly inhibited phosphorylated ERK1/2 between 5 and 60 min. Transient transfection of cells with a cDNA encoding MAPK phosphatase‐1 (MKP‐1) resulted in 30–40% inhibition of pERK1/2; however MKP‐1 protein levels were only significantly stimulated by PTH after 30 mins, suggesting another mechanism for the early phase of pERK1/2 inhibition. The active upstream kinase c‐Raf phosphorylation at serine 338 (ser³³⁸) was significantly inhibited by PTH treatment within 5 min and transfection of the cells with constitutively‐active c‐Raf blocked PTH inhibition of pERK1/2. Inhibition of pERK1/2 and phosphor‐c‐Raf were seen when cells were treated with PTH(1‐34) or PTH(1‐31) analogues that stimulate cAMP, but not with PTH(3‐34), PTH(7‐34) or PTH(18‐48) that do not stimulate cAMP. Stimulation of the cells with forskolin or 8BrcAMP also inhibited pERK1/2 and c‐Raf.p338. Our results suggest that rapid PTH inhibition of ERK1/2 activity is mediated by PKA dependent inhibition of c‐Raf activity and that stimulation of MKP‐1 may contribute to maintaining pERK1/2 inhibition over prolonged time. Copyright © 2009 John Wiley & Sons, Ltd.     

TIEG1 induces apoptosis through mitochondrial apoptotic pathway and promotes apoptosis induced by homoharringtonine and velcade

Overexpression of TGFbeta inducible early gene (TIEG1) mimics TGFbeta action and induces apoptosis. In this study, we found that TIEG1 was significantly up-regulated during apoptosis induced by homoharringtonine or velcade. Overexpression of TIEG1 could induce apoptosis in K562 cells and promote apoptosis induced by HHT or velcade. TIEG1-induced apoptosis was shown to involve Bax and Bim up-regulation, Bcl-2 and Bcl-XL down-regulation, release of cytochrome c from mitochondria into the cytosol, activation of caspase 3 and disruption of the mitochondrial membrane potential (DeltaPsim). We concluded that TIEG1 is a key regulator which induces and promotes apoptosis through the mitochondrial apoptotic pathway.