Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.     

X-linked inhibitor of apoptosis protein expression and the regulation of apoptosis during human placental development

In this study, we have examined the expression and potential role of X-linked inhibitor of apoptosis protein (XIAP), Fas, and Fas ligand (FasL) in the regulation of apoptosis throughout placental development. Protein expression was determined by Western blot analysis and immunohistochemistry, whereas apoptotic cell death was assessed by DNA fragmentation analysis and TUNEL. The XIAP was present in trophoblast throughout placental development, but its content significantly decreased during late pregnancy, when apoptosis was maximal. The FasL content was low during early placental development but increased coincidentally to the decrease in XIAP during the third trimester. Our data also suggest that placental apoptosis is the culmination of the relative expression of these cell-death and -survival proteins, a phenomenon that is cell type-specific and dependent on cytodifferentiation and the stage of placental development. Moreover, the induction of syncytiotrophoblast apoptosis may involve the concomitant up-regulation of FasL for Fas activation and the removal of downstream inhibition of the apoptotic cascade by XIAP.     

Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-kappaB-dependent XIAP up-regulation

The aggressiveness of a tumor is partly attributed to its resistance to chemotherapeutic agent-induced apoptosis. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. Here we established a cell model system to examine whether stable expression of Cyr61 in MCF-7 cells can confer resistance to apoptosis and identify possible participating mechanisms. We showed that stable cell lines overexpressing Cyr61 had acquired a remarkable resistance to apoptosis induced by paclitaxel, adriamycin, and beta-lapachone. Most interesting, gel shift and reporter assays showed that the Cyr61-overexpressing cells had significantly increased NF-kappaB activity compared with neo control cells. Blockage of NF-kappaB activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-IkappaB or with an NF-kappaB decoy rendered them more susceptible to anti-cancer drugs-induced apoptosis. In addition, several NF-kappaB-regulated anti-apoptotic genes were examined, and we found that only XIAP showed a significant 3-4-fold increase in mRNA and protein in Cyr61-overexpressing cells but not in neo control cells. Treatment with inhibitor of apoptosis protein (XIAP)-specific antisense, but not sense, oligonucleotides abolished the apoptosis resistance of the Cyr61-overexpressing cells. At the same time, transfection of these stable cells with DN-IkappaB to block NF-kappaB activity also effectively reduced the elevated XIAP level. Function-neutralizing antibodies to alpha(v)beta(3) and alpha(v)beta(5) could inhibit Cyr61-mediated NF-kappaB activation as well as XIAP expression. Taken together, our data suggested that Cyr61 plays an important role in resistance to chemotherapeutic agent-induced apoptosis in human breast cancer MCF-7 cells by a mechanism involving the activation of the integrins/NF-kappaB/XIAP signaling pathway.     

Sensitization of human bladder tumor cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis with a small molecule IAP antagonist

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of bladder tumors are non-muscle invasive at diagnosis, but even after local surgical therapy there is a high rate of local tumor recurrence and progression. Current treatments extend time to recurrence but do not significantly alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein TNF-related apoptosis-inducing ligand (TRAIL) with a small molecule inhibitor of apoptosis proteins (IAP) antagonist to interfere with intracellular regulators of apoptosis in human bladder tumor cells. Our results demonstrate that the IAP antagonist Compound A exhibits high binding affinity to the XIAP BIR3 domain. When Compound A was used at nontoxic concentrations in combination with TRAIL, there was a significant increase in the sensitivity of TRAIL-sensitive and TRAIL-resistant bladder tumor lines to TRAIL-mediated apoptosis. In addition, modulation of TRAIL sensitivity in the TRAIL-resistant bladder tumor cell line T24 with Compound A was reciprocated by XIAP small interfering RNA-mediated suppression of XIAP expression, suggesting the importance of XIAP-mediated resistance to TRAIL in these cells. These results suggest the potential of combining Compound A with TRAIL as an alternative therapy for bladder cancer.     

Role and gonadotrophic regulation of X-linked inhibitor of apoptosis protein expression during rat ovarian follicular development in vitro

Although FSH up-regulates follicular cell X-linked inhibitor of apoptosis protein (XIAP) expression and suppresses apoptosis in vivo, if these events are coincidental or causally related remains to be investigated. The present study examined the role and gonadotrophic regulation of XIAP expression during follicular development in vitro. Follicles (160-210 microm) cultured for 0-6 days with FSH (100 ng/ml) showed significant growth, as evidenced by increases in follicular size, cell number, and DNA contents. Follicular XIAP content was low in the absence of FSH but was increased by the addition of gonadotropin. Apoptosis was evident in follicles cultured without FSH but was suppressed in the presence of gonadotropin. At low FSH concentration (5 ng/ml), adenoviral XIAP sense cDNA expression increased XIAP and DNA contents, reduced apoptosis, and enhanced follicular growth. Infection of the FSH-stimulated follicles with XIAP antisense elicited opposite responses. In primary granulosa cell cultures, FSH significantly increased XIAP content, inhibited apoptosis, and decreased cell number, a response potentiated by XIAP sense expression. In conclusion, the present studies demonstrated, to our knowledge for the first time, that XIAP plays an important role in the regulation of ovarian follicular development. In addition, a follicle culture system coupled to an adenoviral gene-manipulation procedure has been established and may prove to be a useful approach in assessing the role of specific genes in follicular development and atresia.     

Unique expression of a small IL-32 protein in the Jurkat leukemic T cell line

Interleukin (IL)-32 was recently identified as a new cytokine which induces various proinflammatory cytokines in human monocytes and macrophages. Therefore, IL-32 has been primarily studied in inflammatory models such as rheumatoid arthritis and inflammatory bowel diseases. The regulation of endogenous IL-32 in other immune cells remains unknown. In the present study, we stimulated Jurkat T cells with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and examined IL-32 expression at both the mRNA and protein levels. All mRNAs of the four IL-32 isoforms and the 12-15 kDa IL-32 protein were independent of PHA and PMA stimulation, however a 9 kDa molecular weight IL-32 protein in the cell culture supernatant was induced by PHA and PMA after 16 h of stimulation. Compared to other human cell lines, the Jurkat cell line constitutively expressed a 12-15 kDa molecule of IL-32, which is smaller than the known IL-32 isoforms. We used IL-32 shRNA to examine the specificity of the 12-15 kDa molecule. Upon IL-32 shRNA transfection, the 12-15 kDa band was decreased specifically as compared to the control scrambled clone. Thus, the constitutive expression of IL-32 mRNA as well as the predominant production of a smaller sized IL-32 isoform in Jurkat cells may implicate a role for IL-32 in human T cell leukemia.     

Backbone and side-chain 1H, 13C and 15N assignments of the ubiquitin-associated domain of human X-linked inhibitor of apoptosis protein

X-linked inhibitor of apoptosis protein (XIAP), a leading member of the family of inhibitor of apoptosis (IAP) proteins, is considered as the most potent and versatile inhibitor of caspases and apoptosis. It has been reported that XIAP is frequently overexpressed in cancer and its expression level is implicated in contributing to tumorigenesis, disease progression, chemoresistance and poor patient-survival. Therefore, XIAP is one of the leading targets in drug development for cancer therapy. Recently, based on bioinformatics study, a previously unrecognized but evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs was identified. The UBA domain is found to be essential for the oncogenic potential of IAP, to maintain endothelial cell survival and to protect cells from TNF-α-induced apoptosis. Moreover, the UBA domain is required for XIAP to activate NF-κB. In the present study, we report the near complete resonance assignments of the UBA domain-containing region of human XIAP protein. Secondary structure prediction based on chemical shift index (CSI) analysis reveals that the protein is predominately α-helical, which is consistent with the structures of known UBA proteins.     

IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells

Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.     

Antisense depletion of death-associated protein kinase promotes apoptosis

Death-associated protein kinases (DAPK) are serine/threonine protein kinases that have an important role in regulating cell death. In this study two antisense approaches were employed to down-regulate expression of the endogenous DAPK-alpha and DAPK-beta proteins. Transient expression of an antisense DAPK cDNA or antisense morpholino oligonucleotides in HeLa, 3T3, or primary human vascular smooth muscle cells demonstrate that decreased DAPK expression promotes a spontaneous, caspase-mediated apoptosis as evidenced by increased activities of caspases-3 and -9. Clonal HeLa cell lines with attenuated levels of DAPK expression, obtained following selection in the presence of antisense DAPK cDNA, are more sensitive to tumor necrosis factor-induced caspase-mediated apoptosis, and their sensitivity is inversely related to DAPK expression. In contrast, HeLa cells with reduced DAPK expression are moderately resistant to cell death induced by interferon-gamma. This finding is consistent with previous studies showing that DAPK has a role in promoting caspase-independent cell death. Together, these studies demonstrate that the cellular activities of DAPK are critical for antagonizing caspase-dependent apoptosis to promote cell survival under normal cell growth conditions.     

Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells both initiate apoptotic signaling, resulting in caspase activation, after treatment with anti-cancer agents. However, in contrast to SCLC cells, NSCLC cells do not fully execute apoptosis. The apoptotic process in NSCLC cells seems to be blocked downstream of caspase activation, thus the failure of NSCLC cells to execute apoptosis could result from inhibition of active caspases by inhibitor of apoptosis proteins (IAPs). Here we investigate the mRNA and protein expression of IAPs in a panel of SCLC and NSCLC cell lines. The NSCLC cell lines had a stronger cIAP-2 expression at both mRNA and protein levels, while the SCLC cell lines had a higher level of XIAP protein. Expression of cIAP-1, cIAP-2, and XIAP, the most potent caspase inhibitors, was further investigated in three lung carcinoma cell lines after treatment with 8 Gy of ionizing radiation or etoposide (VP16). In response to treatment, the level of IAPs was not altered in a way that explained the differences in cellular chemo- and radiosensitivity. The intracellular localization of IAPs was analyzed in untreated and treated lung cancer cells. Surprisingly, we found that cIAP-2 was mainly detected in the mitochondrial fraction, although the function of this protein in mitochondria is unknown. No major relocalization of IAPs was observed after treatment. Taken together, these results indicate that IAPs alone are not the main factor responsible for the resistance of NSCLC cells to treatment.     

Effect of hepatocyte growth factor (HGF) on the level of Survivin & XIAP expression in several human cancer cell lines, after treating with DNA damaging agent

Hepatocyte growth factor (HGF) has opposite biological activities in regulating apoptosis, also underlying molecular mechanisms are not clearly defined. We investigated HGF ability to inhibit cell death, which was induced by Doxorubicin, a DNA damaging agent. Also Survivin and XIAP mRNA levels were compared in HGF treated and non-treated cells. Cell proliferation and death were assessed using MTT assay and dye exclusion tests. Quantitative real-time PCR was used to evaluate Survivin and XIAP expression levels after treatment with HGF. ELISA was performed to quantify HGF secretion in the selected cancer cell lines media. HGF appeared to have inhibitory effect on Doxorubicin induced cell death in all of the studied cell lines. It had minimal effect on XAIP and Survivin expression levels in MRC-5, MOLT-4 and AGS cell lines; except for XIAP expression level in AGS cell line, which was increased substantially after treatment. Surprisingly, in KG-1 cell line, XIAP and Survivin expression levels were significantly reduced after HGF treatment. Although several members of IAP gene family are reported to play role in HGF mediated cytoprotective pathway, we showed that XIAP and Survivin do not seem to be involved.     

Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants.

Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines.     

X-linked inhibitor of apoptosis regulates T cell effector function

To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice with experimental autoimmune encephalomyelitis, correlating with disease severity. Daily administration (10 mg/kg/day i.p.) of a 19-mer antisense oligonucleotide specific for XIAP (ASO-XIAP) abolished disease-associated XIAP mRNA and protein expression, and given from day of onset, alleviated experimental autoimmune encephalomyelitis and prevented relapses. Prophylactic treatment also reduced XIAP expression and prevented disease. Random or 5-base mismatched ASO was not inhibitory, and ASO-XIAP did not affect T cell priming. In ASO-XIAP-treated animals, infiltrating cells and inflammatory foci were dramatically reduced within the CNS. Flow cytometry showed an 88-93% reduction in T cells. The proportion of TUNEL(+) apoptotic CD4(+) T cells in the CNS was increased from <1.6 to 26% in ASO-XIAP-treated mice, and the proportion of Annexin V-positive CD4(+) T cells in the CNS increased. Neurons and oligodendrocytes were not affected; neither did apoptosis increase in liver, where XIAP knockdown also occurred. ASO-XIAP increased susceptibility of T cells to activation-induced apoptosis in vitro. Our results identify XIAP as a critical controller of apoptotic susceptibility of effector T cell function.     

c-FLIPR, a new regulator of death receptor-induced apoptosis

c-FLIPs (c-FLICE inhibitory proteins) play an essential role in regulation of death receptor-induced apoptosis. Multiple splice variants of c-FLIP have been described on the mRNA level; so far only two of them, c-FLIP(L) and c-FLIP(S,) had been found to be expressed at the protein level. In this report, we reveal the endogenous expression of a third isoform of c-FLIP. We demonstrate its presence in a number of T and B cell lines as well as in primary human T cells. We identified this isoform as c-FLIP(R), a death effector domain-only splice variant previously identified on the mRNA level. Impor-/tantly, c-FLIP(R) is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex upon CD95 stimulation. Several properties of c-FLIP(R) are similar to c-FLIP(S): both isoforms have a short half-life, a similar pattern of expression during activation of primary human T cells, and are strongly induced in T cells upon CD3/CD28 costimulation. Taken together, our data demonstrate endogenous expression of c-FLIP(R) and similar roles of c-FLIP(R) and c-FLIP(S) isoforms in death receptor-mediated apoptosis.     

Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out. The results indicate that the expression of a limited, distinctive set of genes was altered in the two apoptosis-prone clones, not in the resistant clone. That clone showed altered expression of different sets of genes, suggesting that a molecular switch converted patterns of gene expression between the two phenotypes: apoptosis-prone and apoptosis-resistant. The results are consistent with the hypothesis that altered expression of a distinctive network of genes after glucocorticoid administration ultimately triggers apoptosis of leukemic lymphoid cells. The altered genes identified provide new foci for study of their role in cell death.     

Zinc chelation induces rapid depletion of the X-linked inhibitor of apoptosis and sensitizes prostate cancer cells to TRAIL-mediated apoptosis

The X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane-permeable zinc chelator, N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the post-translational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family, including cIAP1, cIAP2 and survivin. The downregulation of XIAP in TPEN-treated cells occurs via proteasome- and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via downregulation of XIAP.