Given that ovarian stimulation is vital for assisted reproductive technology (ART) and results in elevated serum estrogen levels, exploring the impact of elevated estrogen exposure on oocytes and embryos is necessary. We investigated the effects of various ovarian stimulation treatments on oocyte and embryo morphology and gene expression using a mouse model and estrogen-treated mouse embryonic stem cells (mESCs). Female C57BL/6J mice were subjected to two types of conventional ovarian stimulation and ovarian hyperstimulation; mice treated with only normal saline served as controls. Hyperstimulation resulted in high serum estrogen levels, enlarged ovaries, an increased number of aberrant oocytes, and decreased embryo formation. The messenger RNA (mRNA)‍-sequencing of oocytes revealed the dysregulated expression of lysine-specific demethylase 6b (Kdm6b), which may be a key factor indicating hyperstimulation-induced aberrant oocytes and embryos. In vitro, Kdm6b expression was downregulated in mESCs treated with high-dose estrogen; treatment with an estrogen receptor antagonist could reverse this downregulated expression level. Furthermore, treatment with high-dose estrogen resulted in the upregulated expression of histone H3 lysine 27 trimethylation (H3K27me3) and phosphorylated H2A histone family member X (γ-H2AX). Notably, knockdown of Kdm6b and high estrogen levels hindered the formation of embryoid bodies, with a concomitant increase in the expression of H3K27me3 and γ-H2AX. Collectively, our findings revealed that hyperstimulation-induced high-dose estrogen could impair the demethylation of H3K27me3 by reducing Kdm6b expression. Accordingly, Kdm6b could be a promising marker for clinically predicting ART outcomes in patients with ovarian hyperstimulation syndrome.
Female ; Mice ; Demethylation/drug effects* ; Embryonic Stem Cells ; Estrogens/administration & dosage* ; Gene Expression/drug effects* ; Histones/metabolism* ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Mice, Inbred C57BL ; Oocytes ; Ovary/drug effects* ; Reproductive Techniques, Assisted ; Animals

Diffuse intrinsic pontine glioma (DIPG) is the main cause of brain tumor-related death among children. Until now, there is still a lack of effective therapy with prolonged overall survival for this disease. A typical strategy for preclinical cancer research is to find out the molecular differences between tumor tissue and para-tumor normal tissue, in order to identify potential therapeutic targets. Unfortunately, it is impossible to obtain normal tissue for DIPG because of the vital functions of the pons. Here we report the human fetal hindbrain-derived neural progenitor cells (pontine progenitor cells, PPCs) as normal control cells for DIPG. The PPCs not only harbored similar cell biological and molecular signatures as DIPG glioma stem cells, but also had the potential to be immortalized by the DIPG-specific mutation H3K27M in vitro. These findings provide researchers with a candidate normal control and a potential medicine carrier for preclinical research on DIPG.
Animals ; Brain Stem Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cellular Senescence ; Female ; Glioma ; genetics ; metabolism ; pathology ; Histones ; genetics ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Transplantation ; Neoplastic Stem Cells ; drug effects ; metabolism ; pathology ; Neural Stem Cells ; drug effects ; metabolism ; pathology ; Pons ; embryology ; metabolism ; pathology ; Primary Cell Culture

There are large knowledge gaps regarding how to control stem cells growth and differentiation. The limitations of currently available technologies, such as growth factors and/or gene therapies has led to the search of alternatives. We explore here how a cell's epigenome influences determination of cell type, and potential applications in tissue engineering. A prevalent epigenetic modification is the acetylation of DNA core histone proteins. Acetylation levels heavily influence gene transcription. Histone deacetylase (HDAC) enzymes can remove these acetyl groups, leading to the formation of a condensed and more transcriptionally silenced chromatin. Histone deacetylase inhibitors (HDACis) can inhibit these enzymes, resulting in the increased acetylation of histones, thereby affecting gene expression. There is strong evidence to suggest that HDACis can be utilised in stem cell therapies and tissue engineering, potentially providing novel tools to control stem cell fate. This review introduces the structure/function of HDAC enzymes and their links to different tissue types (specifically bone, cardiac, neural tissues), including the history, current status and future perspectives of using HDACis for stem cell research and tissue engineering, with particular attention paid to how different HDAC isoforms may be integral to this field.
Acetylation ; drug effects ; Histone Deacetylase Inhibitors ; pharmacology ; Histone Deacetylases ; metabolism ; Histones ; isolation & purification ; metabolism ; Humans ; Tissue Engineering

OBJECTIVETo examine the effect of icariin (ICA) on the cognitive impairment induced by traumatic brain injury (TBI) in mice and the underlying mechanisms related to changes in hippocampal acetylation level.

METHODSThe modifified free-fall method was used to establish the TBI mouse model. Mice with post-TBI cognitive impairment were randomly divided into 3 groups using the randomised block method (n=7): TBI (vehicle-treated), low-dose (75 mg/kg) and high-dose (150 mg/kg) of ICA groups. An additional sham-operated group (vehicle-treated) was employed. The vehicle or ICA was administrated by gavage for 28 consecutive days. The Morris water maze (MWM) test was conducted. Acetylcholine (ACh) content, mRNA and protein levels of choline acetyltransferase (ChAT), and protein levels of acetylated H3 (Ac-H3) and Ac-H4 were detected in the hippocampus.

RESULTSCompared with the sham-operated group, the MWM performance, hippocampal ACh content, mRNA and protein levels of ChAT, and protein levels of Ac-H3 and Ac-H4 were signifificantly decreased in the TBI group (P<0.05). High-dose of ICA signifificantly ameliorated the TBI-induced weak MWM performance, increased hippocampal ACh content, and mRNA and protein levels of ChAT, as well as Ac-H3 protein level compared with the TBI group (P<0.05).

CONCLUSIONICA improved post-TBI cognitive impairment in mice by enhancing hippocampal acetylation, which improved hippocampal cholinergic function and ultimately improved cognition.

Acetylation ; Acetylcholine ; metabolism ; Animals ; Brain Injuries, Traumatic ; complications ; Choline O-Acetyltransferase ; genetics ; metabolism ; Cognitive Dysfunction ; drug therapy ; etiology ; Flavonoids ; chemistry ; pharmacology ; therapeutic use ; Hippocampus ; pathology ; Histones ; metabolism ; Homeostasis ; drug effects ; Male ; Maze Learning ; drug effects ; Mice ; RNA, Messenger ; genetics ; metabolism

PURPOSE: When integrating molecularly targeted compounds in radiotherapy, synergistic effects of the systemic agent and radiation may extend the limits of patient tolerance, increasing the demand for understanding the pathophysiological mechanisms of treatment toxicity. In this Pelvic Radiation and Vorinostat (PRAVO) study, we investigated mechanisms of adverse effects in response to the histone deacetylase (HDAC) inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) when administered as a potential radiosensitiser. MATERIALS AND METHODS: This phase I study for advanced gastrointestinal carcinoma was conducted in sequential patient cohorts exposed to escalating doses of vorinostat combined with standard-fractionated palliative radiotherapy to pelvic target volumes. Gene expression microarray analysis of the study patient peripheral blood mononuclear cells (PBMC) was followed by functional validation in cultured cell lines and mice treated with SAHA. RESULTS: PBMC transcriptional responses to vorinostat, including induction of apoptosis, were confined to the patient cohort reporting dose-limiting intestinal toxicities. At relevant SAHA concentrations, apoptotic features (annexin V staining and caspase 3/7 activation, but not poly-(ADP-ribose)-polymerase cleavage) were observed in cultured intestinal epithelial cells. Moreover, SAHA-treated mice displayed significant weight loss. CONCLUSION: The PRAVO study design implemented a strategy to explore treatment toxicity caused by an HDAC inhibitor when combined with radiotherapy and enabled the identification of apoptosis as a potential mechanism responsible for the dose-limiting effects of vorinostat. To the best of our knowledge, this is the first report deciphering mechanisms of normal tissue adverse effects in response to an HDAC inhibitor within a combined-modality treatment regimen.
Animals ; Apoptosis* ; Cells, Cultured ; Clinical Trials, Phase I as Topic ; Cohort Studies ; Drug-Related Side Effects and Adverse Reactions ; Epithelial Cells ; Gene Expression ; Histone Deacetylase Inhibitors* ; Histone Deacetylases* ; Histones* ; Humans ; Hydroxamic Acids ; Mice ; Microarray Analysis ; Radiotherapy* ; Weight Loss

OBJECTIVETo investigate the effect of histone acetylation/deacetylation imbalances on embryonic hearts of mice and its effect on key genes of planar cell polarity (PCP) pathway-Vangl2, Scrib and Rac1 in H9C2 cells.

METHODSForty pregnant C57/B6 mice were randomly assigned into three groups: blank group (n=10), vehicle group (n=10), and valproic acid (VPA)-treated group (n=20). In the VPA-treated group, VPA, a histone deacetylase (HDAC) inhibitor, was administered to each individual dam intraperitoneally at a single dose of 700 mg/kg on embryonic day 10.5 (E10.5). The vehicle and blank groups received equivalent saline or no interventions, respectively. Dams were sacrificed on E15.5, and death rates of embryos were evaluated. Subsequently, embryonic hearts of survival fetus were removed to observe cardiac abnormalities by hematoxylin-eosin (HE) staining. H9C2 cells were cultured and allotted to the blank, vehicle, and VPA-treated groups: the VPA treated group received VPA exposure at concentrations of 2.0, 4.0 and 8.0 mmol/L; the vehicle and blank groups received equivalent saline or no interventions, respectively. HDAC1-3 as well as Vangl2, Scrib and Rac1 mRNA and protein expression levels were determined by quantitative real-time PCR and Western blot, respectively. The total HDAC activity was analyzed by colorimetric assay.

RESULTSThe fetus mortality rate after VPA treatment was 31.7%, with a significantly higher rate of cardiac abnormalities in comparison with the controls (P<0.05). In comparison with the blank and vehicle groups, HDAC1 mRNA was significantly increased at various concentrations of VPA treatment at all time points of exposure (P<0.05), together with a reduction of protein level after 48 and 72 hours of exposure (P<0.05). The inhibition of HDAC2 mRNA after various concentrations of VPA incubation was pronounced at 24 hours of exposure (P<0.05), while the protein levels were reduced at all time points (P<0.05). HDAC3 mRNA was prominently induced by VPA (4.0 and 8.0 mmol/L) at all time points of treatment (P<0.05). In contrast, the protein level was inhibited after VPA treatment (P<0.05). In comparison with the blank and vehicle groups, Vangl2 mRNA as well as Scrib mRNA/protein expression levels were markedly reduced after 48 and 72 hours of VPA treatment (P<0.05), together with a reduction of protein level in Vangl2 at 72 hours (P<0.05). Compared with the blank and vehicle groups, a significant repression in the total HDAC activity was observed in the VPA-treated group at concentrations of 4.0 and 8.0 mmol/L after 24 hours of treatment (P<0.05), and the effect persisted up to 48 and 72 hours, exhibiting pronounced inhibition at all concentrations (P<0.05).

CONCLUSIONSVPA might result in acetylation/deacetylation imbalances by inhibiting HDAC1-3 protein expression and total HDAC activity, leading to the down-regulation of mRNA and protein expression of Vangl2 and Scrib. This could be one of the mechanisms contributing to congenital heart disease.

Acetylation ; Animals ; Cell Polarity ; Cells, Cultured ; Fetal Heart ; drug effects ; metabolism ; Heart Defects, Congenital ; etiology ; Histone Deacetylase 1 ; genetics ; Histone Deacetylase 2 ; genetics ; Histones ; metabolism ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; genetics ; RNA, Messenger ; analysis ; Valproic Acid ; pharmacology

OBJECTIVETo investigate the protective effect of histone acetylation against hypoxic-ischemic cortical injury in neonatal rats.

METHODSA total of 90 neonatal rats aged 3 days were divided into three groups: sham-operation, cortical injury model, and sodium butyrate (a histone deacetylase inhibitor) treatment. The rats in the model and the sodium butyrate treatment groups were intraperitoneally injected with lipopolysaccharide (0.05 mg/kg), and then right common carotid artery ligation was performed 2 hours later and the rats were put in a hypoxic chamber (oxygen concentration 6.5%) for 90 minutes. The rats in the sham-operation group were intraperitoneally injected with normal saline and the right common carotid artery was only separated and exposed without ligation or hypoxic treatment. The rats in the sodium butyrate treatment group were intraperitoneally injected with sodium butyrate (300 mg/kg) immediately after establishment of the cortical injury model once a day for 7 days. Those in the sham-operation and the model groups were injected with the same volume of normal saline. At 7 days after establishment of the model, Western blot was used to measure the protein expression of histone H3 (HH3), acetylated histone H3 (AH3), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (BAX), cleaved caspase-3 (CC3), and brain-derived neurotrophic factor (BDNF). Immunofluorescence assay was used to measure the expression of 5-bromo-2'-deoxyuridine (BrdU) as the cortex cell proliferation index.

RESULTSThe sodium butyrate treatment group had a significantly lower HH3/AH3 ratio than the model group (P<0.05), which suggested that the sodium butyrate treatment group had increased acetylation of HH3. Compared with the model group, the sodium butyrate treatment group had a significant increase in Bcl-2/Bax ratio, a significant reduction in CC3 expression, and a significant increase in BDNF expression (P<0.05). The sodium butyrate treatment group had a significant increase in the number of BrdU-positive cells in the cortex compared with the model group (P<0.05), and BrdU was mainly expressed in the neurons.

CONCLUSIONSIncreased histone acetylation may protect neonatal rats against cortical injury by reducing apoptosis and promoting regeneration of neurons. The mechanism may be associated with increased expression of BDNF.

Acetylation ; Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Brain-Derived Neurotrophic Factor ; analysis ; Butyric Acid ; therapeutic use ; Cerebral Cortex ; pathology ; Female ; Histones ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of monoamine oxidase inhibitor phenelzine on proliferation, apoptosis and histone modulation in acute lymphoblastic leukemia cell line Molt-4 cells.

METHODSThe effect of Phenelzine on cell proliferation was detected by MTT. Apoptotic rate was measured by flow cytometry. The variation of apoptosis associated proteins Caspase-3, Bcl-2 and Bax, cyclin-dependent kinase inhibitor p21, tumor suppressor protein p15, and the expression level of histone methylation of H3K4, H3K9 and histone acetylation of H3, DNMT1 were detected by Western Blot.

RESULTS① Molt-4 cell proliferation rates were (87.68±3.54)%, (67.84±3.24)%, (51.48±3.37)%, (28.72±2.56)% respectively after exposured to phenelzine at 5, 10, 15, 20 μmol/L for 24 h, P<0.05. ② After 10 μmol/L of phenelzine exposure for 24, 48, 72 h, cell proliferation rates were (67.84±3.24)%, (50.24±2.01)%, (40.31±2.25)%, P<0.05. ③ The apoptotic rates were (13.64±2.58)%, (31.24±3.42)%, (56.37±4.26)% after phenelzine treatment at 5, 10, 20 μmol/L for 24 h, which was concentration dependent. ④ Phenelzine could upregulate the expression of Bax, caspase-3, p21, and downregulate Bcl-2 expression. Phenelzine upregulated the methylation level of histone H3K4me1, H3K4me2 and histone acetylated H3, while it didn't change the level of histone H3K4me3, H3K9me1, H3K9me2. ⑤ Phenelzine inhibited DNMT1 expression and promoted p15 expression.

CONCLUSIONSPhenelzine increased the methylation of histone H3K4me1, H3K4me2, acetylation of histone H3 and p21, and decreased the expression of DNMT1 and p15, and ultimately inhibited the proliferation and apoptosis of Molt-4 cells.

Acetylation ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; Histones ; metabolism ; Humans ; Methylation ; Phenelzine ; pharmacology

The molecular mechanism of DNA damage induced by hydroquinone (HQ) remains unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor (AATF), PARP-1, and phosphorylated H2AX (γ-H2AX) were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-1-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, γ-H2AX expression increased to 265%. Co-immunoprecipitation (co-IP) assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly(ADP-ribosyl)ation (PARylation) regulated AATF expression. In conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation.
Antioxidants ; toxicity ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Line ; DNA Damage ; drug effects ; Gene Expression Regulation ; drug effects ; Gene Silencing ; Histones ; genetics ; metabolism ; Humans ; Hydroquinones ; toxicity ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism ; Protein Transport ; Repressor Proteins ; genetics ; metabolism

BACKGROUNDClaudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported.

METHODSIn this study, HCMECs were stimulated with CoCl2to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs.

RESULTSWe found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription.

CONCLUSIONSThe results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.

Cell Hypoxia ; Cells, Cultured ; Claudins ; analysis ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Histones ; metabolism ; Humans ; Myocardium ; cytology ; metabolism ; Promoter Regions, Genetic