Interleukin-2 (IL-2)-dependent T cell clone CTLL-2 underwent apoptosis by deprivation of IL-2 from culture medium. The decrease in the anti-apoptotic Bcl-X L protein level was observed during apoptosis after IL-2 withdrawal. We found that Bcl-X L protein was cleaved to produce two 18 kDa fragments during CTLL-2 cell apoptosis. When the activation of caspases was suppressed by overexpressing human Bcl-2 protein or by the addition of caspase inhibitors, cleavage of Bcl-X L protein was suppressed in vivo. Bcl-X L protein cleavage by incubation with apoptosed CTLL-2 cell lysate was suppressed by the caspase-3/CPP32-speci®c tetrapeptide inhibitor in vitro. Therefore, caspase-3/CPP32-like proteases were activated and involved in the cleavage of Bcl-X L protein during CTLL-2 cell apoptosis. We found that Bcl-X L protein was cleaved by caspase-3/ CPP32 at two sites in the loop domain (i.e., HLAD A). The transfection of the carboxy-terminal 18 kDa Bcl-X L fragment increased the sensitivity to apoptosis. These results indicate that caspase-3/CPP32-like proteases cleaved anti-apoptotic Bcl-X L protein and resulted in accelerated apoptotic cell death.

Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)-and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/ protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML.protein modification | DNA damage T he p53 protein functions as a key regulator of pathways mediating cellular responses by inducing myriad target genes that regulate diverse cellular processes including cell-cycle arrest, apoptosis, and genomic stability (1-4). Regulation of p53 transcriptional activities is crucial for genotoxic stress because of the varieties of cellular responses that are mediated by p53, which, in some cases, can be mutually exclusive (e.g., arrest and apoptosis) (5). p53 has been detected in discrete nuclear speckles, known as promyelocytic leukemia nuclear bodies (PML-NBs), in which CREB binding protein (CBP)/p300, Tip60, and pRB are also found (6-8).PML was originally identified as a t(15, 17) chromosomal translocation partner with the retinoic acid receptor-α (RARα) in acute promyelocytic leukemia, in which fusion genes encoding the PML-RARα fusion protein are generated (9-11). The ability of PML to interact with activators such as CBP/p300 in the nuclear body suggests that PML could modulate transcription through its ability to stabilize complexes of cofactors (12, 13). Overexpression of PML, γ-irradiation of cells, or oncogenic signals such as Ras overexpression can recruit p53 into PML-NBs (14, 15). The resulting ternary p53-PML-CBP complex then promotes the acetylation of p53 and harmonically coordinates critical tumorsuppressive functions such as apoptosis, senescence, and growth arrest (16,17). Unlike bona fide coactivators such as CBP and p300, PML does not possess intrinsic histone acetylase activity. PML-mediated recruitment of both coactivators promotes p53 modification, such as acetylation and phosphorylation (16,17). Acetylation levels of p53 are known to be significantly enhanced in response to stress and are involved in p53 activation and stabilization (18,19). Given that p300 −/− mouse ...

Cancer-associated fibroblasts (CAFs) have been shown to play an important role in angiogenesis, invasion, and metastasis. In the present study, we determined whether CAFs within the tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) contributed to promoting immunosuppression and evasion from immune surveillance. Six pairs of CAFs and normal fibroblasts (NFs) were established from the resected tumor tissues of patients with HNSCC. The effects of CAFs and NFs on the functions of T cells were comparatively analyzed. CAFs expressed the co-regulatory molecules, B7H1 and B7DC, whereas NFs did not. The expression levels of cytokine genes, including those for IL6, CXCL8, TNF, TGFB1, and VEGFA, were higher in CAFs. T cell proliferation was suppressed more by CAFs or their supernatants than by NFs. Moreover, PBMCs co-cultured with the supernatants of CAFs preferentially induced T cell apoptosis and regulatory T cells over those co-cultured with the supernatants of NFs. A microarray analysis revealed that the level of genes related to the leukocyte extravasation and paxillin signaling pathways was higher in CAFs than in NFs. These results demonstrated that CAFs collaborated with tumor cells in the TME to establish an immunosuppressive network that facilitated tumor evasion from immunological destruction.

Upon DNA damage, p53 can induce either cell-cycle arrest or apoptosis. Here we show that monocytic leukemia zinc finger (MOZ) forms a complex with p53 to induce p21 expression and cell-cycle arrest. The levels of the p53-MOZ complex increased in response to DNA damage to levels that induce cell-cycle arrest. MOZ(-/-) mouse embryonic fibroblasts failed to arrest in G1 in response to DNA damage, and DNA damage-induced expression of p21 was impaired in MOZ(-/-) cells. These results suggest that MOZ is involved in regulating cell-cycle arrest in the G1 phase. Screening of tumor-associated p53 mutants demonstrated that the G279E mutation in p53 disrupts interactions between p53 and MOZ, but does not affect the DNA binding activity of p53. The leukemia-associated MOZ-CBP fusion protein inhibits p53-mediated transcription. These results suggest that inhibition of p53/MOZ-mediated transcription is involved in tumor pathogenesis and leukemogenesis.

The AML1 transcription factor complex is the most frequent target of leukemia-associated chromosomal translocations. Homeodomain-interacting protein kinase 2 (HIPK2) is a part of the AML1 complex and activates AML1-mediated transcription. However, chromosomal translocations and mutations of HIPK2 have not been reported. In the current study, we screened mutations of the HIPK2 gene in 50 cases of acute myeloid leukemia (AML) and in 80 cases of myelodysplastic syndrome (MDS). Results indicated there were two missense mutations (R868W and N958I) in the speckle-retention signal (SRS) domain of HIPK2. Subcellular localization analyses indicated that the two mutants were largely localized to nuclear regions with conical or ring shapes, and were somewhat diffused in the nucleus, in contrast to the wild type, which were mainly localized in nuclear speckles. The mutations impaired the overlapping localization of AML1 and HIPK2. The mutants showed decreased activities and a dominant-negative function over wild-type protein in AML1-and p53-dependent transcription. These findings suggest that dysfunction of HIPK2 may play a role in the pathogenesis of leukemia.

Chemotherapeutic drugs exhibit their cytotoxic effect by inducing apoptosis in tumor cells. Because the serine/threonine kinase Akt is involved in apoptosis suppression, we investigated the relationship between Akt activity and drug sensitivity. We discovered that certain chemotherapeutic drugs induced apoptosis with caspase activation only when Akt was inactivated after drug treatment, while inactivation of Akt was not observed when tumor cells showed resistance to the drug-induced caspase activation. So, turn-off of the Akt-mediated survival signal is correlated with the sensitivity of the cells to chemotherapy. With a cDNA microarray, we revealed that tumor necrosis factor receptor-associated death domain (tradd) gene expression was elevated in response to Akt inactivation. Reportedly, Forkhead family transcription factors are phosphorylated by Akt, which results in their nuclear exit and inactivation. Analysis of the tradd promoter revealed that it contains at least one potential Forkhead family transcription factor-responsive element, and we confirmed that this element was involved in chemotherapeutic drug-induced TRADD expression. Overexpression of mutant TRADD proteins to block its apoptosis-inducing capability attenuated chemotherapeutic drug-induced apoptosis. Thus, chemotherapeutic drugs exhibited their cytotoxic effects in part by down-regulating Akt signaling following TRADD expression. These results indicate that Akt kinase activity after drug treatment is a hallmark of sensitivity of the cells to chemotherapeutic drugs.Many growth factors and cytokines have been reported to promote cell survival. The characterization of survival signal transduction pathways stimulated by these factors has revealed that phosphatidylinositide-3-OH kinase (PI3K) is involved in apoptosis suppression (16). PI3K is a heterodimeric lipid kinase consisting of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. After growth factor stimulation, PI3K is activated and generates phospholipid second-messenger molecules, phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P 2 ], that raise a diverse set of cellular responses. The major targets of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 are pleckstrin homology domain-containing proteins.One target of PI3K is the pleckstrin homology domaincontaining serine/threonine kinase Akt (also known as protein kinase B) (16), and some pharmacological PI3K inhibitors, such as wortmannin and LY294002, suppress Akt kinase activity. Akt was originally identified as the cellular homolog of the viral oncogene v-akt (45). Akt mediates many PI3K-regulated biological responses, including glucose uptake, protein synthesis, cell cycle progression, and inhibition of apoptosis. Catalytically inactive Akt resides within the cytosol. By stimulation with growth factors and cytokines, the PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 proteins generated lead to the recruitment of Akt to the plasma membrane. Akt is then phosphorylated at two specific ph...

Levels of the N-terminally truncated isoform of p63 (ΔN p63), well documented to play a pivotal role in basal epidermal gene expression and epithelial maintenance, need to be strictly regulated. We demonstrate here that the anaphase-promoting complex/cyclosome (APC/C) complex plays an essential role in the ubiquitin-mediated turnover of ΔNp63α through the M-G1 phase. In addition, syntaxin-binding protein 4 (Stxbp4), which we previously discovered to bind to ΔNp63, can suppress the APC/C-mediated proteolysis of ΔNp63. Supporting the physiological relevance, of these interactions, both Stxbp4 and an APC/C-resistant version of ΔNp63α (RL7-ΔNp63α) inhibit the terminal differentiation process in 3D organotypic cultures. In line with this, both the stable RL7-ΔNp63α variant and Stxbp4 have oncogenic activity in soft agar and xenograft tumor assays. Notably as well, higher levels of Stxbp4 expression are correlated with the accumulation of ΔNp63 in human squamous cell carcinoma (SCC). Our study reveals that Stxbp4 drives the oncogenic potential of ΔNp63α and may provide a relevant therapeutic target for SCC.

Purpose Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be a SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis. Experimental design We carried out a clinicopathological analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells. Results Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman’s rank correlation ρ=0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P<0.001), disease stage (P=0.030) and pleural involvement (P=0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death and survival in cancer. Platelet-Derived Growth Factor Receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft agar and xenograft assays. Conclusions STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC.