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The endoplasmic reticulum (ER), a complex membrane structure, has important roles in all eukaryotic cells. Catastrophe of its functions would lead to ER stress that causes various diseases such as cancer, neurodegenerative diseases, diabetes and so on. Prolonged ER stress could trigger apoptosis via activation of various signal transduction pathways. To investigate physiological roles of histone acetyltransferase GCN5 in regulation of ER stress, we analyzed responses of homozygous GCN5-deficient DT40 mutants, ΔGCN5, against ER stress. GCN5-deficiency in DT40 caused drastic resistance against apoptosis induced by pharmacological ER stress agents (thapsigargin and tunicamycin). Pharmaceutical analysis using specific Bcl-2 inhibitors showed that the drastic resistance against prolonged ER stress-induced apoptosis is, in part, due to up-regulation of Bcl-2 gene expression in ΔGCN5. These data revealed that GCN5 is involved in regulation of prolonged ER stress-induced apoptosis through controlling Bcl-2 gene expression. [ABSTRACT FROM AUTHOR]

Highlights: [•] Treatment with a cytotoxic anti-Fas antibody caused cross-link of Fas antigen. [•] Inhibition of factor XIII caused disappearance of the cross-link of Fas. [•] Apoptotic cell death was concurrently hastened by inhibition of factor XIII. [•] The cross-link of Fas was seen only in fetal serum but not in other sera tested. [•] Factor XIII may play a key role in fetal development via cross-link of Fas. [ABSTRACT FROM AUTHOR]

Abstract: Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton’s tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H2O2) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H2O2, were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H2O2-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5′-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H2O2. [Copyright &y& Elsevier]

Abstract: The membrane bound cytochrome b558 composed of large gp91-phox and small p22-phox subunits, and cytosolic proteins p40-, p47- and p67-phox are important components of superoxide ()-generating system in phagocytes and B lymphocytes. A lack of this system in phagocytes is known to cause serious life-threatening infections. Here, we describe that curcumin, a polyphenol responsible for the yellow color of curry spice turmeric, dramatically activates the -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and curcumin, the -generating activity increased more than 4-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and curcumin slightly enhanced gene expressions of the five components compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and curcumin caused remarkable accumulation of protein levels of p47-phox (to 7-fold) and p67-phox (to 4-fold) compared with those of the RA-treatment alone. These results suggested that curcumin dramatically enhances RA-induced -generating activity via accumulation of cytosolic p47-phox and p67-phox proteins in U937 cells. Therefore, it should have the potential as an effective modifier in therapy of leukemia and/or as an immunopotentiator. [Copyright &y& Elsevier]

The membrane bound cytochrome b 558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O 2 − )-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, dramatically activates the O 2 − -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and resveratrol, the O 2 − -generating activity increased more than 5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and resveratrol strongly enhanced transcription of the gp91-phox compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and resveratrol caused remarkable accumulation of protein levels of gp91-phox (to 4-fold), p22-phox (to 5-fold) and p47-phox (to 4-fold) compared with those of the RA-treatment alone. In addition, ChIP assay suggested that resveratrol participates in enhancing the gene expression of gp91-phox via promoting acetylation of Lys-9 residues and Lys-14 residues of histone H3 within chromatin around the promoter regions of the gene. These results suggested that resveratrol strongly enhances the RA-induced O 2 − -generating activity via up-regulation of gp91-phox gene expression in U937 cells. [ABSTRACT FROM AUTHOR]

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells. • TLR4 is upregulated in bladder cancer cells. • S100A8/A9-TLR4 axis plays a crucial role in bladder cancer progression. • TPL2 is required for bladder cancer progression under TLR4 upon S100A8/A9 binding. • TPL2 requires TIRAP adaptor protein for its activation. [ABSTRACT FROM AUTHOR]

Histone acetyltransferase p300/CBP-associated factor (PCAF) belonging to GCN5 family regulates various epigenetic events for transcriptional regulation through alterations in the chromatin structure. During normal development of B cells, gene expressions of numerous transcription factors are strictly regulated by epigenetic mechanisms including histone acetylation and deacetylation to complete their development pathways. Here, by analyzing PCAF-deficient DT40 mutants, ΔPCAF, we report that PCAF takes part in transcriptional activation of B cell lymphoma-6 (Bcl-6) and Paired box gene 5 (Pax5), which are essential transcription factors for normal development of B cells. PCAF-deficiency caused drastic decrease in mRNA levels of Bcl-6 and Pax5, and remarkable increase in that of B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, chromatin immunoprecipitation assay showed that PCAF-deficiency caused remarkable decrease in acetylation levels of both H3K9 and H3K14 residues within chromatin surrounding the 5’-flanking regions of Bcl-6 and Pax5 genes in vivo , suggesting that their gene expressions may be regulated by PCAF. These results revealed that PCAF is involved in transactivation of Bcl-6 and Pax5 genes, resulting in down-regulation of Blimp-1 gene expression, and plays a key role in epigenetic regulation of B cell development. [ABSTRACT FROM AUTHOR]

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway–dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling–dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR–Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling–dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche. • Coculture with endothelial cells inhibits the drug-induced apoptosis of AML cells. • The chemoresistance is not reversed by the inhibition or knockout of VEGFR-2. • The VEGFR-2 pathway is dispensable for endothelial cells-induced AML chemoresistance. [ABSTRACT FROM AUTHOR]

Abstract: The transcription factor, early B cell factor 1 (EBF1) with an atypical zinc-finger and helix-loop-helix motif, is essential for development and differentiation of lymphocytes. In mice, EBF1 is involved in the generation of pre-pro B cells (the first specified progenitors of B cells) from common lymphoid progenitors (CLPs) and transcription regulations of various genes involved in B cell-development, for instance, mb-1 and Pax5. During B lymphopoiesis, interestingly, EBF1 is detected throughout from CLPs to mature B cells. However, in immature B cells, the physiological role of EBF1 remains to be elucidated. Here, by analyzing EBF1-deficient DT40 cells, EBF1 −/−, generated by us, we show that EBF1-deficiency caused significant increases (to ∼800%) in both mRNA and protein levels of B lymphocyte-induced maturation protein-1 (Blimp-1), the master gene for plasma cell differentiation. In addition, both transcription and protein synthesis of Blimp-1 were remarkably down-regulated (to ∼20%) by re-expression (over-expression) of EBF1. Chromatin immunoprecipitation assay revealed that EBF1 binds to proximal 5′-upstream regions around two putative EBF1 binding motifs of the gene in vivo. These results suggest that EBF1 takes part in transcriptional regulations of the Blimp-1 gene in immature B cells, and may play a key role in B cell differentiation. This is the first report on a novel EBF1 function in immature B cells as a powerful repressor of Blimp-1 gene expression. [Copyright &y& Elsevier]