Reprogramming of arachidonate metabolism confers temozolomide resistance to glioblastoma through enhancing mitochondrial activity in fatty acid oxidation.

BACKGROUND: Sp1 is involved in the recurrence of glioblastoma (GBM) due to the acquirement of resistance to temozolomide (TMZ). Particularly, the role of Sp1 in metabolic reprogramming for drug resistance remains unknown. METHODS: RNA-Seq and mass spectrometry were used to analyze gene expression and metabolites amounts in paired GBM specimens (primary vs. recurrent) and in paired GBM cells (sensitive vs. resistant). ω-3/6 fatty acid and arachidonic acid (AA) metabolism in GBM patients were analyzed by targeted metabolome. Mitochondrial functions were determined by Seahorse XF Mito Stress Test, RNA-Seq, metabolome and substrate utilization for producing ATP. Therapeutic options targeting prostaglandin (PG) E2 in TMZ-resistant GBM were validated in vitro and in vivo. RESULTS: Among the metabolic pathways, Sp1 increased the prostaglandin-endoperoxide synthase 2 expression and PGE2 production in TMZ-resistant GBM. Mitochondrial genes and metabolites were obviously increased by PGE2, and these characteristics were required for developing resistance in GBM cells. For inducing TMZ resistance, PGE2 activated mitochondrial functions, including fatty acid ß-oxidation (FAO) and tricarboxylic acid (TCA) cycle progression, through PGE2 receptors, E-type prostanoid (EP)1 and EP3. Additionally, EP1 antagonist ONO-8713 inhibited the survival of TMZ-resistant GBM synergistically with TMZ. CONCLUSION: Sp1-regulated PGE2 production activates FAO and TCA cycle in mitochondria, through EP1 and EP3 receptors, resulting in TMZ resistance in GBM. These results will provide us a new strategy to attenuate drug resistance or to re-sensitize recurred GBM.

HDAC6 involves in regulating the lncRNA-microRNA-mRNA network to promote the proliferation of glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is the most aggressive and lethal brain tumor. Although the histone deacetylase (HDAC)/transcription factor axis promotes growth in GBM, whether HDACs including HDAC6 are involved in modulating long non-coding RNAs (lncRNAs) to affect GBM malignancy remains obscure. METHODS: Integrative analysis of microarray and RNA-seq was performed to identify lncRNAs governed by HDAC6. Half-life measurement and RNA-protein pull-down assay combined with isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis were conducted to identify RNA modulators. The effect of LINC00461 on GBM malignancy was evaluated using animal models and cell proliferation-related assays. Functional analysis of the LINC00461 downstream networks was performed comprehensively using ingenuity pathway analysis and public databases. RESULTS: We identified a lncRNA, LINC00461, which was substantially increased in stem-like/treatment-resistant GBM cells. LINC00461 was inversely correlated with the survival of mice-bearing GBM and it was stabilized by the interaction between HDAC6 and RNA-binding proteins (RBPs) such as carbon catabolite repression-negative on TATA-less (CCR4-NOT) core exoribonuclease subunit 6 and fused in sarcoma. Targeting LINC00461 using azaindolylsulfonamide, an HDAC6 inhibitor, decreased cell-division-related proteins via the lncRNA-microRNA (miRNA)-mRNA networks and caused cell-cycle arrest, thereby suppressing proliferation in parental and drug-resistant GBM cells and prolonging the survival of mice-bearing GBM. CONCLUSIONS: This study sheds light on the role of LINC00461 in GBM malignancy and provides a novel therapeutic strategy for targeting the HDAC6/RBP/LINC00461 axis and its downstream effectors in patients with GBM.

RalA, PLD and mTORC1 Are Required for Kinase-Independent Pathways in DGKß-Induced Neurite Outgrowth.

Diacylglycerol kinase ß (DGKß) is an enzyme that converts diacylglycerol to phosphatidic acid and is mainly expressed in the cerebral cortex, hippocampus and striatum. We previously reported that DGKß induces neurite outgrowth and spinogenesis, contributing to higher brain functions, including emotion and memory. To elucidate the mechanisms involved in neuronal development by DGKß, we investigated the importance of DGKß activity in the induction of neurite outgrowth using human neuroblastoma SH-SY5Y cells. Interestingly, both wild-type DGKß and the kinase-negative (KN) mutant partially induced neurite outgrowth, and these functions shared a common pathway via the activation of mammalian target of rapamycin complex 1 (mTORC1). In addition, we found that DGKß interacted with the small GTPase RalA and that siRNA against RalA and phospholipase D (PLD) inhibitor treatments abolished DGKßKN-induced neurite outgrowth. These results indicate that binding of RalA and activation of PLD and mTORC1 are involved in DGKßKN-induced neurite outgrowth. Taken together with our previous reports, mTORC1 is a key molecule in both kinase-dependent and kinase-independent pathways of DGKß-mediated neurite outgrowth, which is important for higher brain functions.

Histone deacetylase 6 acts upstream of DNA damage response activation to support the survival of glioblastoma cells.

DNA repair promotes the progression and recurrence of glioblastoma (GBM). However, there remain no effective therapies for targeting the DNA damage response and repair (DDR) pathway in the clinical setting. Thus, we aimed to conduct a comprehensive analysis of DDR genes in GBM specimens to understand the molecular mechanisms underlying treatment resistance. Herein, transcriptomic analysis of 177 well-defined DDR genes was performed with normal and GBM specimens (n = 137) from The Cancer Genome Atlas and further integrated with the expression profiling of histone deacetylase 6 (HDAC6) inhibition in temozolomide (TMZ)-resistant GBM cells and patient-derived tumor cells. The effects of HDAC6 inhibition on DDR signaling were examined both in vitro and intracranial mouse models. We found that the expression of DDR genes, involved in repair pathways for DNA double-strand breaks, was upregulated in highly malignant primary and recurrent brain tumors, and their expression was related to abnormal clinical features. However, a potent HDAC6 inhibitor, MPT0B291, attenuated the expression of these genes, including RAD51 and CHEK1, and was more effective in blocking homologous recombination repair in GBM cells. Interestingly, it resulted in lower cytotoxicity in primary glial cells than other HDAC6 inhibitors. MPT0B291 reduced the growth of both TMZ-sensitive and TMZ-resistant tumor cells and prolonged survival in mouse models of GBM. We verified that HDAC6 regulated DDR genes by affecting Sp1 expression, which abolished MPT0B291-induced DNA damage. Our findings uncover a regulatory network among HDAC6, Sp1, and DDR genes for drug resistance and survival of GBM cells. Furthermore, MPT0B291 may serve as a potential lead compound for GBM therapy.

LY6D-induced macropinocytosis as a survival mechanism of senescent cells.

Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients.

mTORC1 is involved in DGKß-induced neurite outgrowth and spinogenesis.

Diacylglycerol kinase ß (DGKß) is an enzyme converting DG to phosphatidic acid (PA) and is specifically expressed in neurons, especially those in the cerebral cortex, hippocampus and striatum. We previously reported that DGKß induces neurite outgrowth and spinogenesis, contributing to higher brain function including emotion and memory, and plasma membrane localization of DGKß via the C1 domain and a cluster of basic amino acids at the C-terminus is necessary for its function. To clarify the mechanisms involved in neuronal development by DGKß, we investigated whether DGKß activity induces neurite outgrowth using human neuroblastoma SH-SY5Y cells. DGKß induced neurite outgrowth by activation of mammalian target of rapamycin complex 1 (mTORC1) through a kinase-dependent pathway. In addition, in primary cultured cortical and hippocampal neurons, inhibition of mTORC1 abolished DGKß induced-neurite outgrowth, branching and spinogenesis. These results indicated that DGKß induces neurite outgrowth and spinogenesis by activating mTORC1 in a kinase-dependent pathway.

d-amino acid oxidase promotes cellular senescence via the production of reactive oxygen species.

d-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase metabolizing neutral and polar d-amino acids. Unlike l-amino acids, the amounts of d-amino acids in mammalian tissues are extremely low, and therefore, little has been investigated regarding the physiological role of DAO. We have recently identified DAO to be up-regulated in cellular senescence, a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Because DAO produces reactive oxygen species (ROS) as byproducts of substrate oxidation and the accumulation of ROS mediates the senescence induction, we explored the relationship between DAO and senescence. We found that inhibition of DAO impaired senescence induced by DNA damage, and ectopic expression of wild-type DAO, but not enzymatically inactive mutant, enhanced it in an ROS-dependent manner. Furthermore, addition of d-amino acids and riboflavin, a metabolic precursor of FAD, to the medium potentiated the senescence-promoting effect of DAO. These results indicate that DAO promotes senescence through the enzymatic ROS generation, and its activity is regulated by the availability of its substrate and coenzyme.

The story of PKC: A discovery marked by unexpected twists and turns.

Protein kinase C (PKC) is activated by 1,2-diacylglycerol as a second messenger in the signaling mechanism coupled with the hydrolysis of membrane inositol phospholipids, although it was not found by screening for a 1,2-diacylglycerol-dependent enzyme. PKC is also a receptor for the tumor-promoting phorbol esters, but it was not identified by its property of binding phorbol esters, either. Instead, the discovery and characterization of PKC, now known to comprise a family with multiple isoforms, was through a circuitous voyage filled with unexpected twists and turns. This review summarizes the discovery and the initial experiments of PKC as a historical perspective of the enzyme family in the context of the progress in the studies on protein phosphorylation. © 2018 IUBMB Life, 71(6):697-705, 2019.

Proline dehydrogenase promotes senescence through the generation of reactive oxygen species.

Cellular senescence is a complex stress response characterized by permanent loss of proliferative capacity and is implicated in age-related disorders. Although the transcriptional activity of p53 (encoded by TP53) is known to be vital for senescence induction, the downstream effector genes critical for senescence remain unsolved. Recently, we have identified the proline dehydrogenase gene (PRODH) to be upregulated specifically in senescent cells in a p53-dependent manner, and the functional relevance of this to senescence is yet to be defined. Here, we conducted functional analyses to explore the relationship between PRODH and the senescence program. We found that genetic and pharmacological inhibition of PRODH suppressed senescent phenotypes induced by DNA damage. Furthermore, ectopic expression of wild-type PRODH, but not enzymatically inactive forms, induced senescence associated with the increase in reactive oxygen species (ROS) and the accumulation of DNA damage. Treatment with N-acetyl-L-cysteine, a ROS scavenger, prevented senescence induced by PRODH overexpression. These results indicate that PRODH plays a causative role in DNA damage-induced senescence through the enzymatic generation of ROS.

Identification of cellular senescence-specific genes by comparative transcriptomics.

Cellular senescence is defined as permanent cell cycle arrest induced by various stresses. Although the p53 transcriptional activity is essential for senescence induction, the downstream genes that are crucial for senescence remain unsolved. Here, by using a developed experimental system in which cellular senescence or apoptosis is induced preferentially by altering concentration of etoposide, a DNA-damaging drug, we compared gene expression profiles of senescent and apoptotic cells by microarray analysis. Subtraction of the expression profile of apoptotic cells identified 20 genes upregulated specifically in senescent cells. Furthermore, 6 out of 20 genes showed p53-dependent upregulation by comparing gene expression between p53-proficient and -deficient cells. These 6 genes were also upregulated during replicative senescence of normal human diploid fibroblasts, suggesting that upregulation of these genes is a general phenomenon in senescence. Among these genes, 2 genes (PRODH and DAO) were found to be directly regulated by p53, and ectopic expression of 4 genes (PRODH, DAO, EPN3, and GPR172B) affected senescence phenotypes induced by etoposide treatment. Collectively, our results identified several proteins as novel downstream effectors of p53-mediated senescence and provided new clues for further research on the complex signalling networks underlying the induction and maintenance of senescence.

Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation.

ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.

Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family.

BACKGROUND: Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC. RESULTS: The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells. CONCLUSION: Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex.

The TSC1-TSC2-TBC1D7 complex is an important negative regulator of the mechanistic target of rapamycin complex 1 that controls cell growth in response to environmental cues. Inactivating TSC1 and TSC2 mutations cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterised by the occurrence of benign tumours in various organs and tissues, notably the brain, skin and kidneys. TBC1D7 mutations have not been reported in TSC patients but homozygous inactivation of TBC1D7 causes megaencephaly and intellectual disability. Here, using an exon-specific deletion strategy, we demonstrate that some regions of TSC1 are not necessary for the core function of the TSC1-TSC2 complex. Furthermore, we show that the TBC1D7 binding site is encoded by TSC1 exon 22 and identify amino acid residues involved in the TSC1-TBC1D7 interaction.

Novel antiviral host factor, TNK1, regulates IFN signaling through serine phosphorylation of STAT1.

In response to viral infection, the host induces over 300 IFN-stimulated genes (ISGs), which are the central component of intracellular antiviral innate immunity. Inefficient induction of ISGs contributes to poor control and persistence of hepatitis C virus infection. Therefore, further understanding of the hepatocytic ISG regulation machinery will guide us to an improved management strategy against hepatitis C virus infection. In this study, comprehensive genome-wide, high-throughput cDNA screening for genes regulating ISG expression identified a tyrosine kinase nonreceptor 1 (TNK1) as a unique player in the ISG induction pathway. The immune-modulatory function of TNK1 has never been studied, and this study characterizes its significance in antiviral innate immunity. TNK1 is abundantly expressed in hepatocytes and maintains basal ISG expression. More importantly, TNK1 plays a critical role in type I IFN-mediated ISG induction. We discovered that the activated IFN receptor complex recruits TNK1 from the cytoplasm. TNK1 is then phosphorylated to enhance its kinase activity. The activated TNK1 potentiates JAK-STAT signaling through dual phosphorylation of STAT1 at tyrosine 701 and serine 727 amino acid positions. Our loss-of-function approach demonstrated that TNK1 governs a cluster of ISG expression that defines the TNK1 pathway effector genes. More importantly, TNK1 abundance is inversely correlated to viral replication efficiency and is also a determinant factor for the hepatocytic response to antiviral treatment. Taken together, our studies found a critical but unidentified integrated component of the IFN-JAK-STAT signaling cascade.

Branched-chain amino acids enhance premature senescence through mammalian target of rapamycin complex I-mediated upregulation of p21 protein.

Branched-chain amino acids (BCAAs) have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR) links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer's ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.

A positive role of mammalian Tip41-like protein, TIPRL, in the amino-acid dependent mTORC1-signaling pathway through interaction with PP2A.

Target of rapamycin complex 1 (TORC1) has a key role in cellular regulations in response to environmental conditions. In yeast, Tip41 downregulates TORC1 signaling via activation of PP2A phosphatase. We show here that overexpression of TIPRL, a mammalian Tip41, suppressed dephosphorylation of mechanistic TORC1 (mTORC1) substrates under amino acid withdrawal, and knockdown of TIPRL conversely attenuated phosphorylation of those substrates after amino acid refeeding. TIPRL associated with the catalytic subunit of PP2A (PP2Ac), which was required for the TIPRL action on mTORC1 signaling. Collectively, unlike yeast TIP41, TIPRL has a positive effect on mTORC1 signaling through the association with PP2Ac.

Association of CAD, a multifunctional protein involved in pyrimidine synthesis, with mLST8, a component of the mTOR complexes.

BACKGROUND: mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. RESULTS: CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. CONCLUSION: The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8.

Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells.

BACKGROUND: Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. METHODS AND RESULTS: In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death. CONCLUSION: Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.

Functional and direct interaction between the RNA binding protein HuD and active Akt1.

The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

Contribution of caspase(s) to the cell cycle regulation at mitotic phase.

Caspases have been suggested to contribute to not only apoptosis regulation but also non-apoptotic cellular phenomena. Recently, we have reported the involvement of caspase-7 to the cell cycle progression at mitotic phase by knockdown of caspase-7 using small interfering RNAs and short hairpin RNA. Here we showed that chemically synthesized broad-spectrum caspase inhibitors, which have been used to suppress apoptosis, prevented the cell proliferation in a dose-dependent manner, and that the subtype-specific peptide-based caspase inhibitor for caspase-3 and -7, but not for caspase-9, inhibited cell proliferation. It was also indicated that the BIR2 domain of X-linked inhibitor of apoptosis protein, functioning as an inhibitor for caspase-3 and -7, but not the BIR3 domain which plays as a caspase-9 inhibitor, induced cell cycle arrest. Furthermore, flow cytometry revealed that the cells treated with caspase inhibitors arrested at G(2)/M phase. By using HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, the prevention of the cell proliferation by caspase inhibitors induced cell cycle arrest at mitotic phase accompanying the accumulation of the substrates for APC/C, suggesting the impairment of the APC/C activity at the transition from M to G(1) phases. These results indicate that caspase(s) contribute to the cell cycle regulation at mitotic phase.