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DNA methylation is a common method of gene expression regulation, and this form of regulation occurs in the neurodevelopmental disorder Prader-Willi syndrome (PWS). Gene expression regulation via methylation is important for humans, although there is little understanding of the role of methylation in neuronal differentiation. We characterized the cellular differentiation potential of iPS cells derived from a patient with PWS with abnormal methylation (M-iPWS cells). A comparative genomic hybridization (CGH) array revealed that, unlike iPWS cells (deletion genes type), the abnormally methylated M-iPWS cells had no deletion in the15q11.2-q13 chromosome region. In addition, methylation-specific PCR showed that M-iPWS cells had strong methylation in CpG island of the small nuclear ribonucleoprotein polypeptide N (SNRPN) on both alleles. To assess the effect of abnormal methylation on cell differentiation, the M-iPWS and iPWS cells were induced to differentiate into embryoid bodies (EBs). The results suggest that iPWS and M-iPWS cells are defective at differentiation into ectoderm. Neural stem cells (NSCs) and neurons derived from M-iPWS cells had fewer NSCs and mature neurons with low expression of NSCs and neuronal markers. We conclude that expression of the downstream of genes in the PWS region regulated by methylation is involved in neuronal differentiation.

DNA methylation is a common method of gene expression regulation, and this form of regulation occurs in the neurodevelopmental disorder Prader Willi syndrome (PWS). Gene expression regulation via methylation is important for humans, although the role of methylation in neuronal differentiation is unknown. We characterized the cellular differentiation potential of iPS cells derived from a patient with PWS with abnormal methylation (M-iPWS cells). A CGH array revealed that, unlike iPWS cells, the abnormally methylated M-iPWS cells had no deletion in the15q11.2-q13 chromosome region. In addition, methylation-specific PCR showed that M-iPWS cells had strong methylation of the SNRPN CpG island. To assess the effect of abnormal methylation on cell differentiation, the M-iPWS cells and iPWS cells were induced to differentiate into embryoid bodies, which contain endoderm, mesoderm, and ectoderm. The results suggest that iPS cells from patients with PWS are defective at differentiation into ectoderm. When directionally differentiated into neuronal cells, the expression of neural stem cells (NSCs) markers in M-iPWS cells was relatively low compared to those from control cells. Nestin and Musashi-1 immunoreactivity was also low in NSCs from M-iPWS cells. MAP2 and βIII-tubulin immunoreactivity was also sparse, and marker expression was low in neuronal cells differentiated from M-iPWS cells. We conclude that abnormal methylation in the PWS region is involved in neuronal differentiation.

Adult T-cell leukemia/lymphoma (ATL), an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1), requires new treatments. Drug repositioning, reuse of a drug previously approved for the treatment of another condition to treat ATL, offers the possibility of reduced time and risk. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate peroxisome proliferator-activated receptor-γ, which plays various roles in lipid metabolism, cellular differentiation, and apoptosis. Here, telmisartan reduced cell viability and enhanced apoptotic cells via caspase activation in ex vivo peripheral blood monocytes from asymptomatic HTLV-1 carriers (ACs) or via caspase-independent cell death in acute-type ATL, which has a poor prognosis. Telmisartan also induced significant growth inhibition and apoptosis in leukemia cell lines via caspase activation, whereas other angiotensin II receptor blockers did not induce cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation and autophagy. Thus, telmisartan simultaneously caused caspase activation and autophagy. A hypertension medication with antiproliferation effects on primary and leukemia cells is intriguing. Patients with an early diagnosis of ATL are generally monitored until the disease progresses; thus, suppression of progression from AC and indolent ATL to acute ATL is important. Our results suggest that telmisartan is highly effective against primary cells and leukemia cell lines in caspase-dependent and -independent manners, and its clinical use may suppress acute transformation and improve prognosis of patients with this mortal disease. This is the first report demonstrating a cell growth-inhibitory effect of telmisartan in fresh peripheral blood mononuclear cells from leukemia patients.

Sirtuin involves in cellular processes to adapt to starvation in the endocrine and metabolic systems. We examined Sir2B function during early Dictyostelium development upon starvation. GFP-tagged Sir2B was expressed and the immunoreactivity was detected a punctate pattern near cell surface. Sir2B mRNA was expressed in vegetative cells and peaked at 2 h after starvation. Ectopic expression of Sir2B enhanced aggregate formation with increased expressions of cell adhesion molecules such as cadA, csaA, TgrB1, and TgrC1 at 8h compared to those in control cells. Sir2B null-mutant cells (Sir2B KO cells) were generated and they formed aggregates smaller than those of wild-type cells after 24 h. Because cell-cell adhesion affects aggregation size, cell cohesion assay of Sir2B KO cells showed cell-cell adhesion impairment at 8 and 10 h during development, and the expressions of four cell adhesion molecules were reduced in Sir2B KO cells compared to wild-type cells until 8 h after starvation. When RNAi-mediated cadA or csaA knockdown cells (cadA KD or csaA KD cells) were generated, csaA KD cells formed smaller aggregates and affected the expressions of other cell adhesion molecules to reduce, but cadA KD cells did not show to affect aggregation size and cell adhesion molecules expressions apparently. These results suggest that Sir2B is involved in csaA expression and affects other cell adhesion molecules during early development upon starvation.

Sirtuin interacts with many regulatory proteins involved in energy homeostasis, DNA repair, cell survival, and lifespan extension. We investigated the functional roles of Sir2D during early Dictyostelium development upon starvation. We found that ectopic expression of Sir2D accelerated development among three Sirtuins containing highly homologous catalytic domain sequences to mouse Sirt1. Sir2D expression upregulated adenylate cyclase A (aca) mRNA expression 2, 4 and 6 h after starvation. We have previously reported that nicotinamide, a Sirt1 inhibitor, treatment delayed the development and decreased the expression of aca at 4 h after starvation. Sir2D expressing cells showed resistance against the nicotinamide effect. RNAi-mediated Sir2D knockdown cells were generated, and their development was also delayed. Aca expression was decreased 4 h after starvation. Sir2D expression restored the developmental impairment of Sir2D knockdown cells. The induction of aca upon starvation starts with transcriptional activation of MybB. The ectopic expression of MybB accelerated the development and increased the expression of aca 2 and 4 h after starvation but did not restore the phenotype of Sir2D knockdown cells. Sir2D expression had no effects on MybB-null mutant cells during early development. Thus, MybB is necessary for the upregulation of aca by Sir2D, and Sir2D is necessary for the full induction of aca after 4 h by MybB. MybB was coimmunoprecipitated with Sir2D, suggesting an interaction between MybB and Sir2D. These results suggest that Sir2D regulates aca expression through interaction with the MybB transcription factor early in Dictyostelium development upon starvation.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by a lack of expression of paternally inherited genes located in the15q11.2-q13 chromosome region. An obstacle in the study of human neurological diseases is the inaccessibility of brain material. Generation of induced pluripotent stem cells (iPSC cells) from patients can partially overcome this problem. We characterized the cellular differentiation potential of iPS cells derived from a PWS patient with a paternal 15q11-q13 deletion. A gene tip transcriptome array revealed very low expression of genes in the 15q11.2-q13 chromosome region, including SNRPN, SNORD64, SNORD108, SNORD109, and SNORD116, in iPS cells of this patient compared to that in control iPS cells. Methylation-specific PCR analysis of the SNRPN gene locus indicated that the PWS region of the paternal chromosome was deleted or methylated in iPS cells from the patient. Both the control and patient-derived iPS cells were positive for Oct3/4, a key marker of pluripotent cells. After 11 days of differentiation into neural stem cells (NSCs), Oct3/4 expression in both types of iPS cells was decreased. The NSC markers Pax6, Sox1, and Nestin were induced in NSCs derived from control iPS cells, whereas induction of these NSC markers was not apparent in NSCs derived from iPS cells from the patient. After 7 days of differentiation into neurons, neuronal cells derived from control iPS cells were positive for βIII-tubulin and MAP2. However, neuronal cells derived from patient iPS cells only included a few immunopositive neurons. The mRNA expression levels of the neuronal marker βIII-tubulin were increased in neuronal cells derived from control iPS cells, while the expression levels of βIII-tubulin in neuronal cells derived from patient iPS cells were similar to those of NSCs. These results indicate that iPS cells derived from a PWS patient exhibited neuronal differentiation defects.

Sirtuin interacts with many regulatory proteins involved in energy homeostasis, DNA repair, cell survival, and lifespan extension. We investigated the functional roles of Sir2D during early Dictyostelium development upon starvation. We found that ectopic expression of Sir2D accelerated development among three Sirtuins containing highly homologous catalytic domain sequences to mouse Sirt1. Sir2D expression upregulated adenylate cyclase A (aca) mRNA expression 2, 4 and 6 h after starvation. We have previously reported that nicotinamide, a Sirt1 inhibitor, treatment delayed the development and decreased the expression of aca at 4 h after starvation. Sir2D expressing cells showed resistance against the nicotinamide effect. RNAi-mediated Sir2D knockdown cells were generated, and their development was also delayed. Aca expression was decreased 4 h after starvation. Sir2D expression restored the developmental impairment of Sir2D knockdown cells. The induction of aca upon starvation starts with transcriptional activation of MybB. The ectopic expression of MybB accelerated the development and increased the expression of aca 2 and 4 h after starvation but did not restore the phenotype of Sir2D knockdown cells. Sir2D expression had no effects on MybB-null mutant cells during early development. Thus, MybB is necessary for the upregulation of aca by Sir2D, and Sir2D is necessary for the full induction of aca after 4 h by MybB. MybB was coimmunoprecipitated with Sir2D, suggesting an interaction between MybB and Sir2D. These results suggest that Sir2D regulates aca expression through interaction with the MybB transcription factor early in Dictyostelium development upon starvation.

v-Src, an oncogene found in Rous sarcoma virus, is a constitutively active variant of c-Src. Activation of Src is observed frequently in colorectal and breast cancers, and is critical in tumor progression through multiple processes. However, in some experimental conditions, v-Src causes growth suppression and apoptosis. In this review, we highlight recent progress in our understanding of cytokinesis failure and the attenuation of the tetraploidy checkpoint in v-Src-expressing cells. v-Src induces cell cycle changes-such as the accumulation of the 4N cell population-and increases the number of binucleated cells, which is accompanied by an excess number of centrosomes. Time-lapse analysis of v-Src-expressing cells showed that cytokinesis failure is caused by cleavage furrow regression. Microscopic analysis revealed that v-Src induces delocalization of cytokinesis regulators including Aurora B and Mklp1. Tetraploid cell formation is one of the causes of chromosome instability; however, tetraploid cells can be eliminated at the tetraploidy checkpoint. Interestingly, v-Src weakens the tetraploidy checkpoint by inhibiting the nuclear exclusion of the transcription coactivator YAP, which is downstream of the Hippo pathway and its nuclear exclusion is critical in the tetraploidy checkpoint. We also discuss the relationship between v-Src-induced chromosome instability and growth suppression in v-Src-induced oncogenesis.

Genistein, an isoflavone abundantly present in soybeans, possesses anticancer properties and induces growth inhibition including cell cycle arrest and apoptosis. Although abnormal cell division, such as defects in chromosome segregation and spindle formation, and polyploidization have been described, the mechanisms underlying the induction of abnormal cell division are unknown. In this study, we examined the effect of genistein on cell division in cells that are synchronized in M phase, since genistein treatment delays mitotic entry in asynchronous cells. HeLa S3 cells were arrested at the G2 phase and subsequently released into the M phase in presence of genistein. Immunofluorescence staining showed that genistein treatment delays M phase progression. Time-lapse analysis revealed that the delay occurs until anaphase onset. In addition, genistein treatment induces cleavage furrow regression, resulting in the generation of binucleated cells. Central spindle formation, which is essential for cytokinesis, is partially disrupted in genistein-treated cells. Moreover, aberrant chromosome segregation, such as a chromosome bridge and lagging chromosome, occurs through progression of cytokinesis. RhoA, which plays a role in the assembly and constriction of an actomyosin contractile ring, is delocalized from the cortex of the ingressing cleavage furrow. These results suggest that genistein treatment induces binucleated cell formation through cleavage furrow regression, which is accompanied by chromosome bridge formation and RhoA delocalization. Our results provide the mechanism that underlies genistein-induced polyploidization, which may be involved in genistein-induced growth inhibition.

Src-family tyrosine kinases, which are expressed in various cell types, play critical roles in cell signalling at the cytoplasmic side of the plasma membrane through their lipid modifications. Src-family kinases are cotranslationally myristoylated and posttranslationally palmitoylated in the amino-terminal region. The Src-family member Lyn contains a myristoylation site at glycine-2 and a palmitoylation site at cysteine-3, whereas c-Src has a myristoylation site at glycine-2 but not any palmitoylation sites. However, little is known about the role for lipid modifications of Src-family kinases in cell division. Here, we show that non-lipid-modified Lyn and c-Src, Lyn(G2A/C3A) and c-Src(G2A), are delocalized from membranes to the cytoplasm and the nucleus, which gives rise to a significant increase in the rate of chromosome missegregation, such as chromosome lagging and anaphase chromosome bridging, in a tyrosine kinase activity-dependent manner. Treatment with the Src inhibitor PP2 shows that the kinase activity of non-lipid-modified, non-membrane-bound Src during M phase is critical for giving rise to chromosome missegregation. Given that only a fraction of Src-family kinases fails in lipid modifications during biosynthesis, these results suggest that Src’s membrane anchorage through their lipid modifications from prophase to anaphase plays a protective role against induction of chromosome missegregation.

Neurosteroidal oestrogen has been proposed to play important roles in a variety of reproductive behaviours. Aromatase, a key enzyme in oestrogen synthesis, is localised in neural nuclei of specific brain regions and is developmentally regulated, with a transient expression peak at the perinatal period. The brain-specific promoter of the aromatase gene was analysed aiming to determine the transcriptional control mechanisms that could help explain the spatiotemporal expression. We previously reported that a 202-bp sequence, which is upstream from the transcriptional initiation site, is essential for the basal transcriptional activity. The 202-bp upstream region of brain-specific exon 1 comprises at least three types of cis-acting elements: aro-AI (Arom-Aα), aro-AII (Arom-Aβ) and aro-B (Arom-B). To identify the binding proteins for the cis-acting elements, a yeast one-hybrid screen was performed with these cis-element sequences using a mouse foetal cDNA library. Lhx2, a LIM-homeodomain protein, was identified as one of the aro-B binding proteins. The identification was further confirmed using the gel shift assay, which demonstrated binding competition of nuclear proteins to the aro-B element with a typical Lhx2-binding element. In addition, a chromatin immunoprecipitation assay with an anti-Lhx2 antibody demonstrated that Lhx2 bound to the aro-B site in vivo. A reporter assay of the brain-specific promoter demonstrated increased Lhx2-dependent promoter activity. Furthermore, the time-dependent increase in aromatase mRNA in primary cultured foetal neurones was suppressed by an small-interfering RNA-mediated knockdown of Lhx2 expression. These results show that Lhx2 is involved in the transcriptional regulation of aromatase in the rodent brain.

The recent discovery of significant hydropersulfide (RSSH) levels in mammalian tissues, fluids and cells has led to numerous questions regarding their possible physiological function. Cysteine hydropersulfides have been found in free cysteine, small molecule peptides as well as in proteins. Based on their chemical properties and likely cellular conditions associated with their biosynthesis, it has been proposed that they can serve a protective function. That is, hydropersulfide formation on critical thiols may protect them from irreversible oxidative or electrophilic inactivation. As a prelude to understanding the possible roles and functions of hydropersulfides in biological systems, this study utilizes primarily chemical experiments to delineate the possible mechanistic chemistry associated with cellular protection. Thus, the ability of hydropersulfides to protect against irreversible electrophilic and oxidative modification was examined. The results herein indicate that hydropersulfides are very reactive towards oxidants and electrophiles and are modified readily. However, reduction of these oxidized/modified species is facile generating the corresponding thiol, consistent with the idea that hydropersulfides can serve a protective function for thiol proteins.

Recent reports indicate the ubiquitous prevalence of hydropersulfides (RSSH) in mammalian systems. The biological utility of these and related species is currently a matter of significant speculation. The function, lifetime and fate of hydropersulfides will be assuredly based on their chemical properties and reactivity. Thus, to serve as the basis for further mechanistic studies regarding hydropersulfide biology, some of the basic chemical properties/reactivity of hydropersulfides were studied. The nucleophilicity, electrophilicity and redox properties of hydropersulfides were examined under biological conditions. These studies indicate that hydropersulfides can be nucleophilic or electrophilic, depending on the pH (i.e. the protonation state) and can act as good one- and two-electron reductants. These diverse chemical properties in a single species make hydropersulfides chemically distinct from other, well-known sulfur containing biological species, giving them unique and potentially important biological function.

Sirtuin is an evolutionally conserved histone deacetylase. The effects of sirtuin activator, resveratrol and inhibitor, nicotinamide on early Dictyostelium development upon starvation were examined. The cell streaming was appeared at 7 h and tight aggregates was formed at 24 h in control cells under submerged culture. Resveratrol treatment accelerated the development. Cell streaming at 6 h and weak aggregates at 9 h were already observed. While, nicotinamide treatment delayed the development. The initiation of streaming was delayed at 9 h, and the development was still under a cell streaming even after 24 h. Dictyostelium development starts inducing the essential molecules adenylyl cyclase, aca and cAMP pulsing induces cell-surface cAMP receptor (carA). Resveratrol treatment significantly increased the expression level of aca at 4 and 6 h after starvation. Nicotinamide treatment decreased the aca expression level at 4 h and carA expression level at 6 h. The induction of aca upon starvation starts with the transcriptional activation by MybB, a. putative transcription factor. MybB expression recovered the initiation timing of the cell streaming against the effect of nicotinamide, but the aggregates formation was still impaired at 24 h. Resveratrol had no effects on MybB-null mutant cells. Neither streaming nor aggregates formation was observed even after 24 h with low levels of aca expression after starvation by resveratrol treatment. These findings suggest that sirtuins may participate in more than one point of the early developmental processes of Dictyostelium upon starvation.

Genistein, an isoflavone abundantly present in soybeans, possesses anticancer properties and induces growth inhibition including cell cycle arrest and apoptosis. Although abnormal cell division, such as defects in chromosome segregation and spindle formation, and polyploidization have been described, the mechanisms underlying the induction of abnormal cell division are unknown. In this study, we examined the effect of genistein on cell division in cells that are synchronized in M phase, since genistein treatment delays mitotic entry in asynchronous cells. HeLa S3 cells were arrested at the G2 phase and subsequently released into the M phase in presence of genistein. Immunofluorescence staining showed that genistein treatment delays M phase progression. Time-lapse analysis revealed that the delay occurs until anaphase onset. In addition, genistein treatment induces cleavage furrow regression, resulting in the generation of binucleated cells. Central spindle formation, which is essential for cytokinesis, is partially disrupted in genistein-treated cells. Moreover, aberrant chromosome segregation, such as a chromosome bridge and lagging chromosome, occurs through progression of cytokinesis. RhoA, which plays a role in the assembly and constriction of an actomyosin contractile ring, is delocalized from the cortex of the ingressing cleavage furrow. These results suggest that genistein treatment induces binucleated cell formation through cleavage furrow regression, which is accompanied by chromosome bridge formation and RhoA delocalization. Our results provide the mechanism that underlies genistein-induced polyploidization, which may be involved in genistein-induced growth inhibition.

Src-family tyrosine kinases are aberrantly activated in cancers, and this activation is associated with malignant tumor progression. v-Src, encoded by the v-src transforming gene of the Rous sarcoma virus, is a mutant variant of the cellular proto-oncogene c-Src. Although investigations with temperature sensitive mutants of v-Src have shown that v-Src induces many oncogenic processes, the effects on cell division are unknown. Here, we show that v-Src inhibits cellular proliferation of HCT116, HeLa S3 and NIH3T3 cells. Flow cytometry analysis indicated that inducible expression of v-Src results in an accumulation of 4N cells. Time-lapse analysis revealed that binucleation is induced through the inhibition of cytokinesis, a final step of cell division. The localization of Mklp1, which is essential for cytokinesis, to the spindle midzone is inhibited in v-Src-expressing cells. Intriguingly, Aurora B, which regulates Mklp1 localization at the midzone, is delocalized from the spindle midzone and the midbody but not from the metaphase chromosomes upon v-Src expression. Mklp2, which is responsible for the relocation of Aurora B from the metaphase chromosomes to the spindle midzone, is also lost from the spindle midzone. These results suggest that v-Src inhibits cytokinesis through the delocalization of Mklp1 and Aurora B from the spindle midzone, resulting in binucleation.

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1). Despite the recent advances in chemotherapy, allogeneic hematopoietic stem cell transplantation, and supportive care, the prognosis for patients with acute, lymphoma, or unfavorable chronic subtypes (aggressive ATL) is one of the poorest among hematological malignancies; overall survival at 3 years is only 24 % in the more aggressive subtypes of ATL. In case of favourable chronic or smoldering ATL (indolent ATL), watchful waiting until disease progression has been recommended. Some patients with indolent ATL develop infections during this period. Therefore, urgent need for therapy and prophylaxis of ATL are still required. Recently, drug repositioning offers the possibility of reduced time and risk as several stages common to novel drug discovery and development can be bypassed because repositioning candidates have frequently been through several phases of development for their original indication. With the successful clinical introduction of a number of non-cancer drugs for cancer treatment, drug repositioning now became a potent alternative strategy to discover and develop novel anticancer drug candidates from the existing drug space. On the other hand, the transcription factor PPARγ plays various roles in lipid metabolism, immune response, cellular differentiation and apoptosis. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate PPARγ. Here, we assessed how telmisartan affects ATL cells from patients and leukemia cell lines. Methods and Results: Methods used in this study were carried out in accordance with the approved guidelines by the Committees for Ethical Review of Research involving Human Subjects at Kagoshima University. The subjects were examined by standard serological testing for the presence of HTLV-1 and by haematological/Southern blotting analysis for diagnosis of ATL. The classification of ATL was performed according to the criteria of Shimoyama. Telmisartan reduced cell viability and enhanced apoptotic cells in ex vivo peripheral blood monocytes of acute-type ATL, which has a poor prognosis. Telmisartan-induced apoptosis in cells from ATL patients (acute and chronic-type) and asymptomatic HTLV-1 carriers was significantly increased compared with those from healthy donors. Telmisartan also induced significant growth inhibition and apoptosis (Annexin V+ cells and TUNEL) in leukemia cell lines (HTLV-1-related cell lines: S1T, MT-2; Jurkat and HL60), while other angiotensin II receptor blockers, irbesartan and valsartan, did not induce cell death. In apoptosis, several key events occur in mitochondria, including the release of caspase activators, such as apoptosis-induced factor, and loss of mitochondrial transmembrane potential. Telmisartan induced loss of mitochondrial transmembrane potential and generation of reactive oxygen species, although apoptosis-inducing factor level was stable. Telmisartan also inhibited cell growth via caspase activation (caspase-3, 8 and 9) in the leukemia cells. However, treatment with a caspase inhibitor did not inhibit telmisartan-induced cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Thus, Telmisartan simultaneously caused caspase activation and autophagy. Conclusion: A hypertension medication that has an anti-proliferation effect on leukemia cells is intriguing. These results suggest that telmisartan is highly effective against HTLV-1-infected cells and ATL cells in a caspase-dependent and -independent manner, and its clinical use may suppress the progression from indolent ATL or carrier status to aggressive ATL, and thus improve the prognosis of patients with this fatal disease. Disclosures No relevant conflicts of interest to declare.