Role of plant homeodomain finger protein 8 in P19 embryonic carcinoma cells revealed by genome editing and specific inhibitor.

Plant homeodomain finger protein 8 (PHF8) is a histone demethylase that regulates the expression of various genes. PHF8 targets repressor histone markers and activates gene expression. Although PHF8 has been involved in X-linked mental retardation and certain types of cancers, the role of PHF8 remains largely unknown, and its relevance to the pathogenesis of these diseases is also uncertain. In the present study, we aimed to clarify the cellular function of PHF8 in P19 cells using Phf8 knockout (KO) cells generated via the CRISPR-Cas9 system and by performing PHF8 specific inhibitor experiments, instead of using PHF8 small interfering RNA transfection. After establishing Phf8 KO cells, we analyzed the effects of PHF8 on neuronal differentiation and cell proliferation. Both PHF8 deficiency and inhibition of its activity did not considerably affect neuronal differentiation, however, they showed an increased trend of promoted neurite outgrowth. Moreover, we found that PHF8 regulated cell proliferation via the MEK/ERK pathway. PHF8 deficiency and activity inhibition reduced the phosphorylation of ERK and MEK. The MEK expression level was associated with PHF8 expression, as revealed by chromatin immunoprecipitation analysis. These results suggested that PHF8 regulates cell proliferation via the MEK/ERK pathway in P19 embryonic carcinoma cells.

v-Src delocalizes Aurora B by suppressing Aurora B kinase activity during monopolar cytokinesis.

c-Src tyrosine kinase plays roles in a wide range of signaling events and its increased activity is frequently observed in a variety of epithelial and non-epithelial cancers. v-Src, an oncogene first identified in the Rous sarcoma virus, is an oncogenic version of c-Src and has constitutively active tyrosine kinase activity. We previously showed that v-Src induces Aurora B delocalization, resulting in cytokinesis failure and binucleated cell formation. In the present study, we explored the mechanism underlying v-Src-induced Aurora B delocalization. Treatment with the Eg5 inhibitor (+)-S-trityl-L-cysteine (STLC) arrested cells in a prometaphase-like state with a monopolar spindle; upon further inhibition of cyclin-dependent kinase (CDK1) by RO-3306, cells underwent monopolar cytokinesis with bleb-like protrusions. Aurora B was localized to the protruding furrow region or the polarized plasma membrane 30 min after RO-3306 addition, whereas inducible v-Src expression caused Aurora B delocalization in cells undergoing monopolar cytokinesis. Delocalization was similarly observed in monopolar cytokinesis induced by inhibiting Mps1, instead of CDK1, in the STLC-arrested mitotic cells. Importantly, western blotting analysis and in vitro kinase assay revealed that v-Src decreased the levels of Aurora B autophosphorylation and its kinase activity. Furthermore, like v-Src, treatment with the Aurora B inhibitor ZM447439 also caused Aurora B delocalization at concentrations that partially inhibited Aurora B autophosphorylation. Given that phosphorylation of Aurora B by v-Src was not observed, these results suggest that v-Src causes Aurora B delocalization by indirectly suppressing Aurora B kinase activity.

Abnormal DNA methylation in pluripotent stem cells from a patient with Prader-Willi syndrome results in neuronal differentiation defects.

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.

Cytokinesis Failure Leading to Chromosome Instability in v-Src-Induced Oncogenesis.

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.

Protective role for lipid modifications of Src-family kinases against chromosome missegregation.

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.

Angiotensin II type 1 receptor blocker telmisartan induces apoptosis and autophagy in adult T-cell leukemia cells.

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.

The chemical biology of protein hydropersulfides: Studies of a possible protective function of biological hydropersulfide generation.

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.

The chemical biology of hydropersulfides (RSSH): Chemical stability, reactivity and redox roles.

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 was 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.

Genistein induces cytokinesis failure through RhoA delocalization and anaphase chromosome bridging.

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.

v-Src causes delocalization of Mklp1, Aurora B, and INCENP from the spindle midzone during cytokinesis failure.

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.