Phosphorylation Of Histone H3 Research Articles

THSC/TREX-2 deficiency causes replication stress and genome instability in Caenorhabditis elegans.

Transcription is an essential process of DNA metabolism, yet it makes DNA more susceptible to DNA damage. THSC/TREX-2 is a conserved eukaryotic protein complex with a key role in mRNP biogenesis and maturation that prevents genome instability. One source of such instability is linked to transcription, as shown in yeast and human cells, but the underlying mechanism and whether this link is universal is still unclear. To obtain further insight into the putative role of the THSC/TREX-2 complex in genome integrity, we have used Caenorhabditis elegans mutants of the thp-1 and dss-1 components of THSC/TREX-2. These mutants show similar defective meiosis, DNA damage accumulation and activation of the DNA damage checkpoint. However, they differ from each other regarding replication defects, as determined by measuring dUTP incorporation in the germline. Interestingly, this specific thp-1 mutant phenotype can be partially rescued by overexpression of RNase H. Furthermore, both mutants show a mild increase in phosphorylation of histone H3 at Ser10 (H3S10P), a mark previously shown to be linked to DNA-RNA hybrid-mediated genome instability. These data support the view that both THSC/TREX-2 factors prevent transcription-associated DNA damage derived from DNA-RNA hybrid accumulation by separate means.

Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs.

ABSTRACTBranaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects, we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMNΔ7), and in juvenile Wistar Hannover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image-analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

Spatiotemporal coordination of the RSF1-PLK1-Aurora B cascade establishes mitotic signaling platforms

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.

Distinct roles of haspin in stem cell division and male gametogenesis

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

Mitogen-induced transcriptional programming in human fibroblasts.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA±H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

Improving precise counting of mitotic cells in mantle cell lymphoma using phosphohistone H3 (PHH3) antibody

AimsMantle cell lymphoma (MCL) has a highly heterogeneous clinical course ranging from indolent, to aggressive and rapidly progressive disease. Proliferation is a strong predictor for disease outcome. In routine clinical...

Association of Caspase 3 Activation and H2AX γ Phosphorylation in the Aging Brain: Studies on Untreated and Irradiated Mice.

Phosphorylation of H2AX is a response to DNA damage, but γH2AX also associates with mitosis and/or apoptosis. We examined the effects of X-rays on DNA integrity to shed more light on the significance of H2AX phosphorylation and its relationship with activation of caspase 3 (CASP3), the main apoptotic effector. After administration of the S phase marker BrdU, brains were collected from untreated and irradiated (10 Gray) 24-month-old mice surviving 15 or 30 min after irradiation. After paraffin embedding, brain sections were single- or double-stained with antibodies against γH2AX, p53-binding protein 1 (53BP1) (which is recruited during the DNA damage response (DDR)), active CASP3 (cCASP3), 5-Bromo-2-deoxyuridine (BrdU), and phosphorylated histone H3 (pHH3) (which labels proliferating cells). After statistical analysis, we demonstrated that irradiation not only induced a robust DDR with the appearance of γH2AX and upregulation of 53BP1 but also that cells with damaged DNA attempted to synthesize new genetic material from the rise in BrdU immunostaining, with increased expression of cCASP3. Association of γH2AX, 53BP1, and cCASP3 was also evident in normal nonirradiated mice, where DNA synthesis appeared to be linked to disturbances in DNA repair mechanisms rather than true mitotic activity.

Oxcarbazepine induces mitotic catastrophe and apoptosis in NRK-52E proximal tubular cells

Certain medicines including anticancer drugs, NSAIDs and antiepileptic drugs are known to cause drug-induced nephropathy. For example, antiepileptic drugs such as carbamazepine (CBZ) and valproic acid have been reported to cause damage to the proximal tubular cells. Although there has been a great deal of research concerning the nephrotoxicity of CBZ, little is known about that of oxcarbazepine (OXC), a derivative of CBZ. To investigate the molecular mechanism underlying renal proximal tubular cell death caused by OXC, we examined alterations in the gene expression profile of NRK-52E proximal tubular cells during OXC exposure. DNA microarray analysis revealed that the levels of genes related to mitotic processes including chromosomal and cytoplasmic segregation, progression to G2/M phase, and formation of the mitotic spindle are increased after exposure to 50 μM OXC for 6 h. Cell cycle analysis by flow cytometry showed that OXC at concentrations between 25 and 100 μM induces G2/M arrest. We also found that OXC significantly increases histone H3 phosphorylation, indicative of mitotic cells. These results imply that OXC induces cell cycle arrest at the mitotic phase. Immunofluorescence analysis showed monopolar spindles, which are formed in response to centrosome separation defects, in OXC-treated cells. We also show that OXC suppresses the phosphorylation of PLK1, which is involved not only in the activation of the kinesin family of motor proteins for centrosome separation and bipolar spindle assembly, but also in the cleavage of centrosomal proteins. Thus, our results indicate that OXC inhibits centrosome separation by reducing the activation of PLK1, which leads to the formation of an abnormal spindle and induces mitotic catastrophe and apoptosis in NRK-52E cells.

Microtubule disruption upon CNS damage triggers mitotic entry via TNF signaling activation.

Repair after traumatic injury often starts with mitotic activation around the lesion edges. Early midline cells in the Drosophila embryonic CNS can enter into division following the traumatic disruption of microtubules. We demonstrate that microtubule disruption activates non-canonical TNF signaling by phosphorylation of TGF-β activated kinase 1 (Tak1) and its target IkappaB kinase (Ik2), culminating in Dorsal/NfkappaB nuclear translocation and Jra/Jun expression. Tak1 and Ik2 are necessary for the damaged-induced divisions. Microtubule disruption caused by Tau accumulation is also reported in Alzheimer's disease (AD). Human Tau expression in Drosophila midline cells is sufficient to induce Tak1 phosphorylation, Dorsal and Jra/Jun expression, and entry into mitosis. Interestingly, activation of Tak1 and Tank binding kinase 1 (Tbk1), the human Ik2 ortholog, and NfkappaB upregulation are observed in AD brains.

Mechanistic Targets of Diallyl Trisulfide in Human Breast Cancer Cells Identified by RNA-seq Analysis.

Diallyl trisulfide (DATS), a metabolic by-product of processed garlic, is highly effective in inhibiting growth of human breast cancer cells in vitro and in vivo, but the underlying mechanisms are still not fully understood. In this study, we performed RNA-seq analyses using luminal-type (MCF-7) and basal-like (MDA-MB-231) human breast cancer cells to identify mechanistic targets of DATS. The Reactome Pathway Analysis revealed upregulation of genes associated with SLIT/ROBO tumor suppressor signaling following DATS treatment in both MCF-7 and MDA-MB-231 cells. However, the expression of SLIT2 and ROBO1 proteins or their downstream target C-X-C motif chemokine receptor 4 was not affected by DATS treatment in both cell lines. The Reactome as well as the Gene Ontology Pathways Analyses of the RNA-seq data from DATS-treated cells indicated downregulation of genes associated with G2/M phase cell cycle arrest in comparison with vehicle-treated control cells. Consistent with the RNA-seq data, DATS treatment caused a significant increase in the fraction of the G2/M population in both cell lines when compared to corresponding control cells. In addition, Ser10 phosphorylation of histone H3, a mitotic marker, was also increased significantly following DATS treatment in MCF-7 and MDA-MB-231 cells. These results indicate that while SLIT/ROBO signaling is not affected by DATS treatment, cell cycle arrest likely contributes to the antitumor effect of this phytochemical.

Gene Regulation and Suppression of Type I Interferon Signaling By STAT3 in Diffuse Large B-Cell Lymphoma

The Janus kinas (JAK)/signal transducer and activator of transcription (STAT) pathway is central to signaling by receptors of diverse cytokines, growth factors and related molecules, which is critical for normal hematopoiesis and immune response. However, this signaling pathway is deregulated in several B cell lymphomas. In activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL), autocrine production of IL-6 and IL-10 can result from high NF-κB activity caused by a variety of genetic alterations, such as mutations in MYD88 and CARD11. Our recent work has demonstrated that JAK1 is an active kinase downstream of IL-6 and IL-10 signaling that activates STAT3. Interestingly, JAK1 can function in the nucleus to regulate gene expression through phosphorylating histone H3 on tyrosine 41. There are nearly 3,000 JAK1 epigenetic target genes including MYD88, IRF4 and MYC, of which expression is essential for cancer cell survival and proliferation.Here we have investigated the canonical gene regulatory mechanism by STAT3 in ABC DLBCL cells. We performed genome-wide analysis and identified 2,251 STAT3 direct target genes, all of which involve B cell activation, survival, proliferation, differentiation and migration. Whole transcriptome profiling revealed that STAT3 acts as both a transcriptional activator and suppressor, with a comparable number of up- and down-regulated genes. STAT3 regulates multiple oncogenic signaling pathways, including NF-κB, cell cycle checkpoint, PI3K/AKT/mTORC1 and STAT3 itself. These results suggest that STAT3 is involved in the positive feedback regulation of IL-6/IL-10 signaling and shows crosstalk with NF-κB signaling pathways in ABC DLBCL cells. In addition, STAT3 negatively regulates the lethal type I interferon signaling pathway by inhibiting expression of IRF7, IRF9, STAT1 and STAT2, all of which are critical transcription factors in the type I interferon pathway. Inhibition of STAT3 activity by ruxolitinib synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro . In an ABC DLBCL xenograft mouse model, combination treatment using ruxolitinib and lenalidomide completely inhibits the tumor growth during the period of treatment.In summary, we demonstrate that STAT3 is a critical transcriptional regulator of ABC DLBCL. Gene regulation by STAT3 in ABC DLBCL accentuates survival signaling pathways while dampening the lethal type I interferon pathway. Knowledge of these STAT3 regulated genes has led to our demonstration that a small molecule inhibitor in the JAK1-STAT3 signaling pathway synergizes with lenalidomide, suggesting a new therapeutic strategy for ABC DLBCL, a subtype that is particularly difficult to treat and has poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of DNA Methyltransferases By Volasertib in HMA Resistant Cell Lines

Introduction: Resistance to hypomethylating agents (HMA) in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is a concerning problem. We established HMA resistant cell lines with overexpression of DNA methyltransferases (DNMTs), which were not suppressed by azacitidine or decitabine. Polo-like kinase 1 (PLK1) is a key cell cycle modulator and is associated with regulation of the PIK3/Akt pathway, which was reported to stabilize DNMTs. We hypothesized that volasertib, a PLK inhibitor, might overcome HMA resistance through regulation of PIK3/Akt pathway and DNMTs.Methods: Cell viability was determined in HMA resistant cell lines (MOLM/AZA-1 and MOLM/DEC-5) and MOLM-13. Specific antibodies including phosphorylated-histone H3 (PHH3), caspase 3, PARP, and XIAP were used to evaluate apoptosis or DNA damage. We also analyzed phosphorylated-Akt (p-Akt), Akt, and DNMTs (DNMT1, 3A, and 3B) by immunoblot assay.Results: Volasertib effectively inhibit proliferation of MOLM/AZA-1 and MOLM/DEC-5 cells as well as MOLM-13 cells in a dose dependent manner. It also showed superior anti-leukemic effects compared to cytarabine, azicitidine or decitiabine. Volasertib showed a dose dependent increased expression of PHH3, caspase 3, and PARP, and decrease of XIAP. Treatment of volasertib caused decreased expression of p-AKT and all DNMTs (DNMT1, 3A, and 3B). GDC-0941, a PI3K inhibitor, suppressed DNMT1 and DNMT3A as well as p-Akt, but it could not decrease DNMT3B. A combination of volasertib and decitabine showed synergistic effects in MOLM/DEC-5. Anti-proliferative effects of volasertib were sustained in ex vivo AML or MDS samples.Conclusion: In our HMA resistant models, volasertib effectively inhibited all DNMTs. DNMT1 and 3A seemed to be suppressed via regulation of PI3K/Akt, but suppression of DNMT3B by volasertib might have separate mechanisms. Our data suggest that volasertib with or without decitabine has potential role in overcoming the HMA resistance in patients with AML and MDS. DisclosuresKo:Boehringer Ingelheim: Research Funding. Hur:Boehringer Ingelheim: Research Funding. Goo:Boehringer Ingelheim: Research Funding. Moon:Boehringer Ingelheim: Research Funding. Choi:Boehringer Ingelheim: Research Funding. Park:Boehringer Ingelheim: Research Funding. Lee:Boehringer Ingelheim: Research Funding. Lee:Boehringer Ingelheim: Research Funding.

TRPM7 kinase-mediated immunomodulation in macrophage plays a central role in magnesium ion-induced bone regeneration

Despite the widespread observations on the osteogenic effects of magnesium ion (Mg2+), the diverse roles of Mg2+ during bone healing have not been systematically dissected. Here, we reveal a previously unknown, biphasic mode of action of Mg2+ in bone repair. During the early inflammation phase, Mg2+ contributes to an upregulated expression of transient receptor potential cation channel member 7 (TRPM7), and a TRPM7-dependent influx of Mg2+ in the monocyte-macrophage lineage, resulting in the cleavage and nuclear accumulation of TRPM7-cleaved kinase fragments (M7CKs). This then triggers the phosphorylation of Histone H3 at serine 10, in a TRPM7-dependent manner at the promoters of inflammatory cytokines, leading to the formation of a pro-osteogenic immune microenvironment. In the later remodeling phase, however, the continued exposure of Mg2+ not only lead to the over-activation of NF-κB signaling in macrophages and increased number of osteoclastic-like cells but also decelerates bone maturation through the suppression of hydroxyapatite precipitation. Thus, the negative effects of Mg2+ on osteogenesis can override the initial pro-osteogenic benefits of Mg2+. Taken together, this study establishes a paradigm shift in the understanding of the diverse and multifaceted roles of Mg2+ in bone healing.

Effects of Prolonged Emotional-Pain Stress on Histone H3 Phosphorylation in the Medial Prefrontal Cortex and Basolateral Area of the Amygdala in Rats with Genetic Differences in Nervous System Arousability

Effects of Prolonged Emotional-Pain Stress on Histone H3 Phosphorylation in the Medial Prefrontal Cortex and Basolateral Area of the Amygdala in Rats with Genetic Differences in Nervous System Arousability

Ontogenetic Changes in the Reptilian Heart: An Investigation of Cardiomyocyte Proliferation and Hyperplasia in the Leopard Gecko ( Eublepharis macularius )

Relative rates of cardiomyocyte proliferation vary among and within species. Whereas embryonic and fetal mammals demonstrate robust rates of cardiomyocyte proliferation, this capacity sharply declines within days following birth. Continued growth of the heart in mammals primarily involves cardiomyocyte hypertrophy. In contrast, cardiomyocytes in some teleost fish and salamanders retain a relative high proliferative capacity, even as adults. In these species, continued growth involves increasing the relative population of cardiomyocytes (cardiomyocyte hyperplasia). Previously, we have demonstrated that subadult leopard geckos (Eublepharis macularius, hereafter ‘geckos’) demonstrate relatively high rates of cardiomyocyte proliferation. As subadult geckos are actively undergoing somatic growth, we sought to determine if the rates of cardiomyocyte proliferation changed in adult geckos. We hypothesized that the elevated rate of cardiomyocyte proliferation observed in subadults is a function of ontogeny, and contributes to overall heart growth through cardiomyocyte hyperplasia. To document cardiomyocyte proliferation, we performed double immunofluorescence on heart ventricles from subadult and adult geckos. To identify proliferating cardiomyocytes we immunostained for the motor protein marker myosin heavy chain with either the DNA synthesis (S) phase marker proliferating cell nuclear antigen or the mitotic (M) phase marker phosphorylated histone H3. We found that there were significantly fewer cardiomyocytes in S phase (~0.1%) and M phase (~0%) in adult geckos as compared to subadult geckos (~11% and ~0.5%, respectively). This decline in proliferation likely reflects a shift from tissue growth to maintenance and remodeling. We also determined that the ventricle of adult geckos had more than twice as many cardiac cells when compared to subadult geckos. These data indicate that, similar to zebrafish (and unlike mammals), ontogenetic growth of the gecko heart involves continued cardiomyocyte proliferation and hyperplasia.

Invitro induction of mitotic catastrophe as a therapeutic approach for oral cancer using the ethanolic extract of Juniperus squamata.

Mitotic catastrophe, a cell death mechanism characterized by abnormal mitosis, has been regarded as a therapeutic approach for the development of anti‑cancer drug candidates. The aim of the present study was to investigate the potential effect of the ethanolic extract of Juniperus squamata(EEJS) on the occurrence of mitotic catastrophe in human oral cancer cell lines. The effect of EEJS on the occurrence of mitotic catastrophe was evaluated by measuring cytotoxicity, observing phase‑contrast or transmission electron microscope findings, evaluating the appearance of microtubule or chromosome abnormalities, and detecting the phosphorylation of histoneH3(Ser10). The apoptotic effect of EEJS was assessed by detecting cleaved PARP, analyzing the sub‑G1 population, AnnexinV‑FITC/PI double staining, western blot analysis, and the transient transfection of myeloid cell leukemia‑1(Mcl‑1) overexpression vectors. EEJS treatment was effective in inhibiting cell proliferation in human oral cancer cell lines. EEJS resulted in the enrichment of enlarged multinucleated cells, the disturbance of microtubule formation, and increased phosphorylation of histoneH3 (Ser10), which demonstrates the occurrence of mitotic catastrophe. Additionally, the multinucleated cells underwent apoptotic cell death in a cell context‑dependent manner, which was associated with the reduction of Mcl‑1 protein levels. Findings of the present study indicate that EEJS could be effective for treating human oral cancer by promoting mitotic catastrophe linked to apoptotic cell death.

Evaluation of the antiproliferative effects of the HASPIN inhibitor CHR-6494 in breast cancer cell lines.

HASPIN is a serine/threonine kinase that regulates mitosis by phosphorylating histone H3 at threonine 3. The expression levels of HASPIN in various cancers are associated with tumor malignancy and poor survival, suggesting that HASPIN inhibition may suppress cancer growth. As HASPIN mRNA levels are elevated in human breast cancer tissues compared with adjacent normal tissues, we examined the growth-suppressive effects of CHR-6494, a potent HASPIN inhibitor, in breast cancer cell lines in vitro and in vivo. We found that HASPIN was expressed in breast cancer cells of all molecular subtypes, as well as in immortalized mammary epithelial cells. HASPIN expression levels appeared to be correlated with the cell growth rate but not the molecular subtype of breast cancer. CHR-6494 exhibited potent antiproliferative effects against breast cancer cell lines and immortalized mammary epithelial cells in vitro, but failed to inhibit the growth of MDA-MB-231 xenografted tumors under conditions that have significant effects in a colorectal cancer model. These results imply that CHR-6494 does have antiproliferative effects in some situations, and further drug screening efforts are anticipated to identify more potent and selective HASPIN inhibition for use as an anticancer agent in breast cancer patients.

\u201cMetalloestrogenic\u201d effects of cadmium downstream of G protein-coupled estrogen receptor and mitogen-activated protein kinase pathways in human uterine fibroid cells

Cadmium (Cd) is a toxic metal reported to act as an estrogen “mimic” in the rat uterus and in vitro. We have reported that Cd stimulates proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM; fibroid) cells through nongenomic signaling involving the G protein-coupled estrogen receptor (GPER), with activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (pMAPK44/42). In this study, we explored Cd-induced mechanisms downstream of MAPK and whether Cd could stimulate phosphorylation of Histone H3 at serine 10 (H3Ser10ph) through activated Aurora B kinase (pAurora B), a kinase important in activation of histone H3 at serine 10 during mitosis, and if this occurs via Fork head box M1 (FOXM1) and cyclin D1 immediately downstream of MAPK. We found that Cd increased proliferating cell nuclear antigen (PCNA) and H3Ser10ph expression by immunofluorescence, and that H3ser10ph and pAurora B were coexpressed along the metaphase plate in ht-UtLM cells. In addition, Cd-exposed cells showed higher expression of pMAPK44/42, FOXM1, pAurora B, H3ser10ph, and Cyclin D1 by western blotting. Immunoprecipitation and proximity ligation assays further indicated an association between FOXM1 and Cyclin D1 in Cd-exposed cells. These effects were attenuated by MAPK kinase (MEK1/2) inhibitor. In summary, Cd-induced proliferation of ht-UtLM cells occurred through activation of Histone H3 and Aurora B via FOXM1/Cyclin D1 interactions downstream of MAPK. This provides a molecular mechanism of how Cd acts as an “estrogen mimic” resulting in mitosis in hormonally responsive cells.

Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

A novel histone deacetylase inhibitor LT-548-133-1 induces apoptosis by inhibiting HDAC and interfering with microtubule assembly in MCF-7 cells.

Many studies have indicated that histone deacetylase inhibitors (HDACis) have a significant antitumor effect in cancer. Here we report a compound named LT-548-133-1 that not only acts as an HDAC inhibitor but also interferes with microtubule assembly to inhibit MCF-7 cell proliferation and induce apoptosis. Consistent with Chidamide, LT-548-133-1 inhibited HDAC activity and increased histone H3 acetylation. But the difference is that it significantly induced cell cycle G2/M arrest while Chidamide caused G0/G1 arrest in MCF-7 cells. By Western blotting, we found the accumulation of CyclinB1 and phosphorylated histone H3 in LT-548-133-1 treated cells. Immunofluorescence based microtubule-repolymerization experiments and immunofluorescence staining of cell microtubules and nuclei showed that LT-548-133-1inhibited microtubule-repolymerization and induced mitotic abnormalities. The decreased expression of Bcl-2 and the increased expression of Bax, p53, p21, and cleaved-Caspase3 indicated the occurrence of apoptosis. Flow cytometry results also showed an increase in the proportion of apoptotic cells after administration of LT-548-133-1 or Chidamide. Therefore, we demonstrated that LT-548-133-1 could act as an HDAC inhibitor while inhibiting microtubule-repolymerization, causing mitosis to be arrested in G2/M. These two effects ultimately lead to proliferation inhibition and apoptosis of MCF-7 cells.