Upregulation Of X-linked Inhibitor Of Apoptosis Protein Research Articles

Down-regulated HSA_circ_0003528 inhibits hepatocellular carcinoma aggressiveness via the miR-212-3p/XIAP axis

ABSTRACT Hepatocellular carcinoma (HCC) is characterized by a high mortality rate. Dysregulated circular RNAs (circRNAs) play a vital role in HCC. We aimed to study the role of circ_0003528 in HCC and its fundamental molecular mechanisms. HSA_circ_0003528 was identified through bioinformatics dataset analysis. The binding sites between mRNA and miRNA were predicted using online bioinformatics tools. The interaction between miR-212-3p and X-linked inhibitor of apoptosis protein (XIAP) or circ_0003528 was confirmed through the luciferase reporter assay. RT-qPCR and western blot assays were used to analyze the expression of all miRNAs/mRNAs and proteins. Cellular functions were evaluated using the MTT and TUNEL assays. A xenograft model was established to evaluate the function of circ_0003528 in vivo. Circ_0003528 was dramatically overexpressed in HepG2 and HUH7 cells. However, knockdown of circ_0003528 suppressed the aggressiveness of HCC cells and tumor growth both in vitro and in vivo. Furthermore, binding of miR-212-3p to circ_0003528 and XIAP was verified. Downregulation of miR-212-3p abrogated the effects of si-circ_0003528 on cell viability and apoptosis, and upregulation of XIAP antagonized the functions of the miR-212-3p mimic in HCC cells. circ_0003528 contributes to the development of HCC in vitro and in vivo via the miR-212-3p/XIAP axis. Hence, circ_0003528 knockdown may be a potential therapeutic strategy for HCC treatment.

Circular RNA circ0005276 promotes the proliferation and migration of prostate cancer cells by interacting with FUS to transcriptionally activate XIAP

Prostate cancer (PCa) is one of the major men’s malignancies with high mortality worldwide. Circular RNAs (circRNAs) have been shown to serve as essential regulators in human cancers. CircRNA can exert their functions by cooperating with their host genes. In the present study, microarray analysis revealed an upregulated mRNA in PCa samples. X-linked inhibitor of apoptosis protein (XIAP), a key regulator in the progression of human cancers. Through bioinformatics analysis, we determined that XIAP is a host gene for circRNA0005276. Therefore, this study focused on the interaction between circ0005276 and XIAP as well as their functions in PCa progression. The upregulation of XIAP and circ0005276 was determined in PCa tissues and cell lines. Moreover, we confirmed the positive regulation of circ0005276 on XIAP expression. Functionally, we validated that circ0005276 and XIAP promoted cell proliferation, migration and epithelial–mesenchymal transition. Mechanistically, we verified that circ0005276 interacted with FUS binding protein (FUS) so as to activate the transcription of XIAP. Rescue assays were conducted to determine the crucial role of XIAP in circ0005276 and FUS-mediated PCa cellular processes. Collectively, our study revealed the mechanism and function of circ0005276 and its host gene XIAP in PCa progression.

MiR-122 Targets X-Linked Inhibitor of Apoptosis Protein to Sensitize Oxaliplatin-Resistant Colorectal Cancer Cells to Oxaliplatin-Mediated Cytotoxicity

Background/Aims: Although oxaliplatin is one of the most effective chemotherapeutic drugs used to treat colorectal cancer (CRC), long-term administration usually induces acquired drug resistance during the course of treatment. Thus, there is an urgent need to explore novel strategies to improve the efficiency of cancer therapy. The aim of this study was to explore the effect of microRNA-122 (miR-122) on reversing oxaliplatin resistance in CRC. Methods: The expression of miR-122 in CRC cells was examined by quantitative reverse transcriptase real-time PCR. The cytotoxicity of oxaliplatin against CRC cells was evaluated by Cell Counting Kit-8 assays. Mitochondrial membrane potentials and cell apoptotic rates were measured by flow cytometry. Cellular protein expression and interactions were detected by western blot and co-immunoprecipitation. Results: Established oxaliplatin-resistant SW480 and HT29 cells (SW480/OR and HT29/OR) expressed significantly higher levels of X-linked inhibitor of apoptosis protein (XIAP) and lower levels of miR-122 compared with normal SW480 and HT29 cells, respectively. Our results showed that the downregulation of miR-122 was responsible for the overexpression of XIAP in these oxaliplatin-resistant CRC cells. We then found that the recovery of miR-122 expression can sensitize SW480/OR and HT29/OR cells to oxaliplatin-mediated apoptosis through the inhibition of XIAP expression. Conclusion: Upregulation of XIAP in CRC cells is responsible for the acquired resistance to oxaliplatin. Furthermore, miR-122 reversed oxaliplatin resistance in CRC by targeting XIAP.

Immunohistochemical expression of X-linked inhibitor of apoptosis in eyelid sebaceous gland carcinoma predicts a worse prognosis.

Purpose:Overexpression of the inhibitors of apoptosis proteins have been demonstrated in a variety and of solid tumors including melanomas and nonmelanomas skin cancers. X-linked inhibitor of apoptosis protein (XIAP) is an inhibitor of apoptosis which prevents apoptosis by inhibiting caspases 9, 7, and 3. The prognostic value of XIAP in sebaceous gland carcinoma (SGC) remains unexplored.Methods:The immunohistochemical expression of XIAP was evaluated in 29 SGC cases.Results:The cytoplasmic overexpression of XIAP was detected in 62% SGC cases. XIAP expression was found to be significantly associated with advanced age, large tumor size, and with reduced disease-free survival (P = 0.0174). XIAP expression and advance tumor Grade III emerged as significant risk factors on univariate analysis. On stepwise multivariate analysis, both increased cytoplasmic XIAP expression and high tumor grade were found to be significantly associated with recurrence. Patients with low XIAP immunoexpression had a longer disease-specific survival than those with high expression in the 5-year follow-up.Conclusion:The present study demonstrates at the immunohistochemical level that XIAP is overexpressed in SGC and that high expression could be of biological significance in the development of eyelid SGC. Our finding suggests that up-regulation of XIAP may aggravate tumor metastasis in SGC.

Embelin prevents LMP1-induced TRAIL resistance via inhibition of XIAP in nasopharyngeal carcinoma cells.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in the majority of tumor cells, whilst sparing normal cells. However, the potential use of TRAIL in the treatment of cancer is limited by the inevitable emergence of drug resistance. The present study reports the upregulation of latent membrane protein 1 (LMP1)-induced TRAIL resistance via the enhanced expression of X-linked inhibitor of apoptosis protein (XIAP) in nasopharyngeal carcinoma (NPC) cells. LMP1-positive NPC cells were indicated to be more sensitive to TRAIL compared with LMP1-negative NPC cells in three NPC cell lines. CNE-1 is a LMP1-negative NPC cell line that was transfected with pGL6-LMP1; following which, sensitivity to TRAIL decreased. LMP1-induced TRAIL resistance was associated with the decreased cleavage of caspase-8,-3 and -9, BH3 interacting domain death agonist (Bid) and mitochondrial depolarization, without any effects on the expression of the death receptors, B-cell lymphoma (Bcl)-2 and Bcl-extra long. Knockdown of XIAP with small interfering RNA increased caspase-3 and -9 and Bid cleavage, and prevented LMP1-induced TRAIL resistance. Furthermore, embelin, the inhibitor of XIAP, prevented LMP1-induced TRAIL resistance in the Epstein-Barr virus (EBV)-positive CNE-1-LMP1 and C666-1 NPC cell lines. However, embelin did not enhance TRAIL-induced apoptosis in NP-69, which was used as a benign nasopharyngeal epithelial cell line. These data show that LMP1 inhibits TRAIL-mediated apoptosis by upregulation of XIAP. Embelin may be used in an efficacious and safe manner to prevent LMP1-induced TRAIL resistance. The present study may have implications for the development and validation of novel strategies to prevent TRAIL resistance in EBV-positive NPC.

Reciprocal Complementation of the Tumoricidal Effects of Radiation and Natural Killer Cells

The tumor microenvironment is a key determinant for radio-responsiveness. Immune cells play an important role in shaping tumor microenvironments; however, there is limited understanding of how natural killer (NK) cells can enhance radiation effects. This study aimed to assess the mechanism of reciprocal complementation of radiation and NK cells on tumor killing. Various tumor cell lines were co-cultured with human primary NK cells or NK cell line (NK-92) for short periods and then exposed to irradiation. Cell proliferation, apoptosis and transwell assays were performed to assess apoptotic efficacy and cell viability. Western blot analysis and immunoprecipitation methods were used to determine XIAP (X-linked inhibitor of apoptosis protein) and Smac (second mitochondria-derived activator of caspase) expression and interaction in tumor cells. Co-culture did not induce apoptosis in tumor cells, but a time- and dose-dependent enhancing effect was found when co-cultured cells were irradiated. A key role for caspase activation via perforin/granzyme B (Grz B) after cell-cell contact was determined, as the primary radiation enhancing effect. The efficacy of NK cell killing was attenuated by upregulation of XIAP to bind caspase-3 in tumor cells to escape apoptosis. Knockdown of XIAP effectively potentiated NK cell-mediated apoptosis. Radiation induced Smac released from mitochondria and neutralized XIAP and therefore increased the NK killing. Our findings suggest NK cells in tumor microenvironment have direct radiosensitization effect through Grz B injection while radiation enhances NK cytotoxicity through triggering Smac release.

XIAP: a potential determinant of ovarian follicular fate

X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis protein family, is involved in regulating a number of functions including receptor-mediated intracellular signalling and early development. Its role as an endogenous caspase inhibitor, however, is the most highly characterised. Consequently, this protein has been implicated as an anti-apoptotic factor in the ovary. In vitro and in vivo studies have begun dissecting the stimuli and signalling networks that lead to XIAP upregulation in granulosa cells. The objective of this review is to briefly summarise the current knowledge concerning XIAP and its interactions with different caspases. Furthermore, XIAP's emerging role in the mammalian ovary will be explored and comparison is made with its functions in the mammary gland. Finally, the idea that XIAP may act as a molecular signalling switch in granulosa cells following detachment from underlying layers to promote follicular atresia will be introduced.

Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein

AbstractUrokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up-regulation and partially by mRNA stabilization of inhibitor of apoptosis proteins, most prominently the X-linked inhibitor of apoptosis protein (XIAP). The antiapoptotic activity of uPA was dependent on its protease activity, the presence of uPA receptor (uPAR) and low-density lipoprotein receptor-related protein (LRP), but independent of the phosphatidylinositol 3 (PI3) kinase pathway, whereas vascular endothelial growth factor (VEGF)–induced antiapoptosis was PI3 kinase dependent. uPA-induced cell survival involved phosphorylation of p21-activated kinase 1 (Pak1) and the IκB kinase α that leads to nuclear factor κB (NF-κB) p52 activation. Indeed, blocking NF-κB activation by using specific NF-κB inhibitors abolished uPA-induced cell survival as it blocked uPA-induced XIAP up-regulation. Furthermore, down-regulating XIAP expression by small interfering RNA (siRNA) significantly reduced uPA-dependent endothelial cell survival. This mechanism is also important for VEGF-induced antiapoptosis because VEGF-dependent up-regulation of XIAP was found defective in uPA−/− endothelial cells. This led us to conclude that uPA is part of a novel NF-κB–dependent cell survival pathway.

Concomitant Transitory Up-Regulation of X-Linked Inhibitor of Apoptosis Protein (XIAP) and the Heterogeneous Nuclear Ribonucleoprotein C1–C2 in Surviving Cells During Neuronal Apoptosis

Although cap-dependent translation initiation is the prevalent mode of ribosome binding to mRNAs in eukaryotes, some mRNAs exhibit the ability to bypass the requirement for the cap structure. The translation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA is controlled by an internal ribosome entry site (IRES) element, which requires the interaction of the heterogeneous nuclear ribonucleoprotein C1-C2 (hnRNP-C1/C2). We analyze, at the protein level, the time course and distribution of XIAP and hnRNP-C1/C2 upon ischemia in mice or staurosporine (STP)-induced apoptosis in HT22 cells. Both ischemia and STP induced a parallel upregulation of XIAP and hnRNP-C1/C2 protein levels in the penumbra and in HT22 cells. These results suggest that the increased levels of hnRNP C1/C2 may modulate XIAP translation, probably by interacting with the XIAP-IRES. The up-regulation of hnRNP-C1/C2 may foster the synthesis of XIAP as a protective pathway by which neurons try to counteract the initial deleterious effects of apoptosis.

The association of up‐regulation of X‐linked inhibitor of apoptosis protein with cell adhesion‐mediated drug resistance in U937 cells

An increasing body of evidence indicates that environmental factors may contribute to the drug resistance of acute myeloid leukaemia (AML). CAM-DR (cell adhesion-mediated drug resistance) is a reversible, de novo drug resistance induced by adhesion of tumour cell lines to fibronectin (FN). Adhesion was demonstrated to directly regulate the apoptotic machinery. And it was observed in previous studies that high levels of X-linked inhibitor of apoptosis protein (XIAP) were related to resistance to chemotherapeutics in many cancer cell lines. However, whether XIAP is relevant to CAM-DR of AML cells is unknown. In this report, we demonstrated that the mRNA and protein levels of XIAP were increased by 96.15% and 120.92%, respectively in U937 cells cocultured with FN as compared with controls. Antisense oligonucleotides targeting XIAP down-regulated the expression of XIAP and sensitized U937 cells to daunorubicin. In addition, we investigated the signalling pathway involved in the upregulation of XIAP. The levels of phosphorylated Akt (Ser473) were elevated in U937/FN cells and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed XIAP expression and restored the chemosensitivity to daunorubicin. Our findings suggested that adhesion-dependent activation of the PI3K/Akt/XIAP pathway may be one of the factors involved in the CAM-DR of U937 cells. Targeting this pathway may be a useful approach to improve the therapeutic responsiveness of leukaemia cells.

Transforming Growth Factor-β3 Increases the Invasiveness of Endometrial Carcinoma Cells through Phosphatidylinositol 3-Kinase-dependent Up-regulation of X-linked Inhibitor of Apoptosis and Protein Kinase C-dependent Induction of Matrix Metalloproteinase-9

Tumor cells often acquire intrinsic resistance to the growth inhibitory and pro-apoptotic effects of transforming growth factor-beta (TGF-beta); moreover, TGF-beta can confer invasive properties to established tumor cells. In the present study, we show that TGF-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) trigger proper Smad signaling in human endometrial carcinoma cell lines and efficiently inhibit cellular proliferation. These cells, however, exhibit a high degree of resistance to TGF-beta pro-apoptotic effects; we found that this resistant phenotype would be acquired through up-regulation of X-linked inhibitor of apoptosis protein (XIAP) levels. In addition, using RNA interference and pharmacological inhibitors, we show that TGF-beta increases cellular invasiveness via two distinct signaling pathways in endometrial carcinoma cells: phosphatidylinositol 3-kinase/AKT-dependent up-regulation of XIAP and protein kinase C-dependent induction of matrix-metalloproteinase-9 (MMP-9) expression. Additionally, these findings were correlated with clinical observations showing abundant TGF-beta immunoreactivity in human endometrial carcinoma tumors in vivo, extending from the epithelial compartment to the stroma upon acquisition of an invasive phenotype (gradually from grades I to III). Collectively our results describe for the first time a role for TGF-beta3 in tumor invasiveness.

The X-linked inhibitor of apoptosis protein (XIAP) is up-regulated in metastatic melanoma, and XIAP cleavage by Phenoxodiol is associated with Carboplatin sensitization

XIAP up-regulation is associated with chemotherapy resistance. Phenoxodiol causes XIAP degradation and chemotherapy sensitization in ovarian cancer. Here we assessed XIAP expression in melanomas, using tissue microarrays containing 436 melanomas and 336 nevi by a novel method of automated, quantitative analysis (AQUA). We used S100 to define pixels as melanoma (tumor mask) within the array spot, and measured XIAP expression using Cy5-conjugated antibodies within the mask. XIAP expression was significantly higher in melanomas than nevi (P < 0.0001), and higher in metastatic than primary lesions (P < 0.0001). We then assessed a panel of melanoma cell lines for XIAP expression, and found high expression in all cell lines. Three of the cell lines were assessed for Phenoxodiol and Carboplatin sensitivity; all were resistant to Carboplatin and showed variable sensitivity to Phenoxodiol. Pre-treating Phenoxodiol sensitive cells with Phenoxodiol prior to Carboplatin resulted in XIAP degradation, associated with Carboplatin sensitization and apoptosis, whereas exposing Phenoxodiol resistant cells to Phenoxodiol resulted in less XIAP degradation and minimal Carboplatin sensitization. We conclude that XIAP levels in clinical specimens are significantly higher in melanomas than their benign counterparts, and higher in metastatic than in primary specimens, suggesting an association with malignant progression and disease aggression. Melanoma resistance to Carboplatin is possibly due to XIAP over-expression. Phenoxodiol can sensitize melanoma cells to Carboplatin in vitro with corresponding XIAP degradation, although the precise target and mechanism of action of Phenoxodiol are subject to further assessment. Targeting XIAP warrants additional investigation as a therapeutic approach for metastatic melanoma.

IFN-γ sensitizes MIN6N8 insulinoma cells to TNF-α-induced apoptosis by inhibiting NF-κB-mediated XIAP upregulation

Although X-linked inhibitor of apoptosis protein (XIAP) is an important intracellular suppressor of apoptosis in a variety of cell types, its role in cytokine-induced pancreatic β-cell apoptosis remains unclear. Here, we found that: (i) XIAP level was inversely correlated with tumor necrosis factor (TNF)-α-induced apoptosis in MIN6N8 insulinoma cells; (ii) adenoviral XIAP overexpression abrogated the TNF-α-induced apoptosis through inhibition of caspase activity; (iii) downregulation of XIAP by antisense oligonucleotide or Smac peptide sensitized MIN6N8 cells to TNF-α-induced apoptosis; (iv) XIAP expression was induced by TNF-α through a nuclear factor-κB (NF-κB)-dependent pathway, and interferon (IFN)-γ prevented such an induction in a manner independent of NF-κB, which presents a potential mechanism underlying cytotoxic IFN-γ/TNF-α synergism. Taken together, our results suggest that XIAP is an important modulator of TNF-α-induced apoptosis of MIN6N8 cells, and XIAP regulation in pancreatic β-cells might play an important role in pancreatic β-cell apoptosis and in the pathogenesis of type 1 diabetes.

Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone.

The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.

Activation of the MEK/MAPK Pathway Is Involved in Bryostatin1-Induced Monocytic Differenciation and Up-regulation of X-Linked Inhibitor of Apoptosis Protein

Induction of monocytic differentiation by bryostatin1 (bryo1) conferred on THP-1 leukemia cells the ability to resist Z-LLL-CHO-induced apoptosis. The mechanism of resistance developed during this process was investigated. Apoptosis resistance was associated with an enhanced expression of X-linked inhibitor of apoptosis protein (XIAP), an endogenous caspase inhibitor, in differentiated THP-1 cells. Bryo1 also increased the level of c-IAP-1, yet decreased the level of c-IAP-2 in THP-1 cells, indicating that distinct regulatory mechanisms are operative. In addition, treatment of THP-1 cells with bryo1 induced a rapid and sustained activation of MEK, prior to the upregulation of XIAP and monocytic differentiation. Pretreatment of THP-1 cells with MEK inhibitors (U0126 and PD98059) prior to bryo1 induction blocked the expression of both XIAP and the c-fms product (M-CSF receptor), a hallmark of monocytic differentiation, but not Bcl-2. In addition, the expression of XIAP in bryo1-treated cells was inhibited by CAPE, a NF-κB-specific inhibitor, indicating that its expression is under the transcriptional regulation of NF-κB downstream of the MEK/MAPK pathway. The importance of XIAP in mediating apoptosis resistance was illustrated in cells transiently transfected with XIAP, which conferred on THP-1 cells the ability to resist Z-LLL-CHO-induced apoptosis. These findings suggest that the expression of XIAP is linked to monocytic differentiation in bryo1-treated THP-1 cells and represents one of the potential antiapoptotic mechanisms acquired during this process.

Resistance of bone marrow-derived macrophages to apoptosis is associated with the expression of X-linked inhibitor of apoptosis protein in primary cultures of bone marrow cells

In this study we investigated the underlying mechanisms that confer resistance on mature macrophages with the use of macrophage colony-stimulating factor (M-CSF)-induced bone marrow-derived macrophages (BMDM). In the presence of M-CSF, immature precursor cells were induced to undergo proliferation and differentiation into mature macrophages in vitro with cell morphology similar to that of tissue macrophages by day 7Ő10. Immunoblot analyses showed that bone marrow precursors express appreciable levels of caspase-3 and caspase-9 but no or very low levels of c-fms (M-CSF receptor) and the apoptosis regulators X-linked inhibitor of apoptosis protein (XIAP), c-IAP-1, Bcl-2 and Bax. The differentiation of BMDM is associated with a steady and gradual increase in the levels of c-fms, XIAP, c-IAP-1, Bcl-2 and Bax, reaching maximal levels by day 7. However, the levels of caspase-3 and caspase-9 stayed essentially unchanged even after prolonged incubation (more than 10 days) with M-CSF. Unlike bone marrow precursor cells, mature BMDM (day 7Ő10) were resistant to apoptosis induced by M-CSF depletion, which includes the activation of caspase-3 and caspase-9 and the degradation of XIAP, Bcl-2 and Bax proteins in the process. Treatment of day 7 BMDM with XIAP anti-sense oligonucleotides (oligos), but not sense oligos, partly abolished their resistance to apoptosis. By using a gel-shift assay and a specific nuclear factor κB (NF-κB) inhibitor, we demonstrated that NF-κB activity is responsible for the up-regulation of XIAP in M-CSF-treated macrophages. In addition, treatment of starved macrophages with M-CSF induced a rapid phosphorylation of Akt kinase before the activation of NF-κB. Our results showed that XIAP is one of the anti-apoptotic regulators that confer resistance on mature macrophages by M-CSF.

Resistance of bone marrow-derived macrophages to apoptosis is associated with the expression of X-linked inhibitor of apoptosis protein in primary cultures of bone marrow cells.

In this study we investigated the underlying mechanisms that confer resistance on mature macrophages with the use of macrophage colony-stimulating factor (M-CSF)-induced bone marrow-derived macrophages (BMDM). In the presence of M-CSF, immature precursor cells were induced to undergo proliferation and differentiation into mature macrophages in vitro with cell morphology similar to that of tissue macrophages by day 7-10. Immunoblot analyses showed that bone marrow precursors express appreciable levels of caspase-3 and caspase-9 but no or very low levels of c-fms (M-CSF receptor) and the apoptosis regulators X-linked inhibitor of apoptosis protein (XIAP), c-IAP-1, Bcl-2 and Bax. The differentiation of BMDM is associated with a steady and gradual increase in the levels of c-fms, XIAP, c-IAP-1, Bcl-2 and Bax, reaching maximal levels by day 7. However, the levels of caspase-3 and caspase-9 stayed essentially unchanged even after prolonged incubation (more than 10 days) with M-CSF. Unlike bone marrow precursor cells, mature BMDM (day 7-10) were resistant to apoptosis induced by M-CSF depletion, which includes the activation of caspase-3 and caspase-9 and the degradation of XIAP, Bcl-2 and Bax proteins in the process. Treatment of day 7 BMDM with XIAP anti-sense oligonucleotides (oligos), but not sense oligos, partly abolished their resistance to apoptosis. By using a gel-shift assay and a specific nuclear factor kappaB (NF-kappaB) inhibitor, we demonstrated that NF-kappaB activity is responsible for the up-regulation of XIAP in M-CSF-treated macrophages. In addition, treatment of starved macrophages with M-CSF induced a rapid phosphorylation of Akt kinase before the activation of NF-kappaB. Our results showed that XIAP is one of the anti-apoptotic regulators that confer resistance on mature macrophages by M-CSF.