63 Bovine embryo-secreted microRNA-30c negatively regulates cell cycle progression through downregulation of CDK12

2019 ◽  
Vol 31 (1) ◽  
pp. 157
Author(s):  
X. Lin ◽  
E. Beckers ◽  
S. Mc Cafferty ◽  
J. P. Catani ◽  
K. J. Szymanska ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Differentially Expressed ◽  
Bovine Embryo ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Cycle Progression ◽  
Novel Mirna ◽  
Differentially Expressed Mirna

Identifying microRNA (miRNA) differentially secreted by intermediate (cleaved between 26.6-31.4 hpi) and slow cleaving bovine embryos (cleaved after 31.4 hpi), investigating how they influence embryo development and consequently if they can be used as biomarkers for bovine embryo development. MicroRNA collected from conditioned media (CM) of 167 individually cultured embryos were sequenced on an Illumina Miseq (Illumina, San Diego, CA, USA) after small RNA library construction with the Tailormix v2 kit (SeqMatic, Fremont, CA, USA) and compared with miRBase, using cow as primary organism, to identify known miRNA in all samples. To predict potential novel miRNA, the sequences were aligned against the annotated cow genome (GCA_000003055.3), structurally analysed, and compared with all other mammalian data in miRBase. Quantitative RT-PCR was used to validate miRNA sequencing results. Apoptosis staining was performed for functional analysis of the differentially expressed miRNA-30c (miR-30c). Luciferase reporter assay, Western blotting, and quantitative RT-PCR were used to validate one of the predicted target genes of miR-30c, CDK12. The DNA content analysis using propidium iodide staining followed by flow cytometry and a water-soluble tetrazolium-1 proliferation assay were performed together with ELISA to assess the effect of miR-30c on cell progression. Quantitative RT-PCR was used to assess mRNA levels of DNA damage response (DDR) genes. In total, 114 known and 180 potentially novel miRNA were found in bovine embryo CM. Seven differentially expressed miRNA were identified in the CM from embryos with different cleavage patterns and different quality: miR-30c, miR-10b, and miR-novel-66 were differentially expressed between slow and intermediate developing embryos; miR-10b, miR-novel-113, miR-novel-44, miR-novel-45, and miR-novel-139 were differentially expressed between blastocysts and degenerate embryos. We further focused on functional analysis of miR-30c. Exogeneous delivery of miR-30c mimics to cultured embryos resulted in an increased cell apoptosis. Additionally, transfer of miR-30c mimics to Madin-Darby bovine kidney cells (MDBK) reduced cell cycle progression/cell proliferation, while inhibition of miR-30c resulted in the opposite. In addition, knockdown of CDK12 caused significant decreases in mRNA levels of DDR genes BRCA1, FANCD2, FANCI, and ATR. These data were derived from 3 independent experiments and were considered significant with a P-value<0.05. In vitro-cultured bovine embryos secrete miRNA in the culture medium depending on their developmental capacity. One of these miRNA, miR-30c, increases bovine embryo apoptosis when exogeneously delivered. In addition, miR-30c directly targets and down-regulates CDK12, indicating the possibility that delivery of miR-30c mimics influences cell cycle progression by inhibiting DDR pathways through regulating CDK12. These findings contribute to a better understanding of the mechanisms of secreted miRNA participation in intercellular communication.

Effects of methylglyoxal on human cardiac fibroblast: roles of transient receptor potential ankyrin 1 (TRPA1) channels

2014 ◽  
Vol 307 (9) ◽  
pp. H1339-H1352 ◽  
Author(s):  
Gaku Oguri ◽  
Toshiaki Nakajima ◽  
Yumiko Yamamoto ◽  
Nami Takano ◽  
Tomofumi Tanaka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Diabetic Cardiomyopathy ◽  
Cell Cycle Progression ◽  
Transient Receptor Potential ◽  
Cardiac Fibroblasts ◽  
Receptor Potential ◽  
Rt Pcr ◽  
Cycle Progression ◽  
Transient Receptor ◽  
Human Cardiac Fibroblasts

Cardiac fibroblasts contribute to the pathogenesis of cardiac remodeling. Methylglyoxal (MG) is an endogenous carbonyl compound produced under hyperglycemic conditions, which may play a role in the development of pathophysiological conditions including diabetic cardiomyopathy. However, the mechanism by which this occurs and the molecular targets of MG are unclear. We investigated the effects of MG on Ca2+ signals, its underlying mechanism, and cell cycle progression/cell differentiation in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, Western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell cycle progression was assessed using the fluorescence activated cell sorting. MG induced Ca2+ entry concentration dependently. Ruthenium red (RR), a general cation channel blocker, and HC030031 , a selective transient receptor potential ankyrin 1 (TRPA1) antagonist, inhibited MG-induced Ca2+ entry. Treatment with aminoguanidine, a MG scavenger, also inhibited it. Allyl isothiocyanate, a selective TRPA1 agonist, increased Ca2+ entry. The use of small interfering RNA to knock down TRPA1 reduced the MG-induced Ca2+ entry as well as TRPA1 mRNA expression. The quantitative real-time RT-PCR analysis showed the prominent existence of TRPA1 mRNA. Expression of TRPA1 protein was confirmed by Western blotting and immunocytochemical analyses. MG promoted cell cycle progression from G0/G1 to S/G2/M, which was suppressed by HC030031 or RR. MG also enhanced α-smooth muscle actin expression. The present results suggest that methylglyoxal activates TRPA1 and promotes cell cycle progression and differentiation in human cardiac fibroblasts. MG might participate the development of pathophysiological conditions including diabetic cardiomyopathy via activation of TRPA1.

scholarly journals Anti-Tumor Effect of Rutin on Human Neuroblastoma Cell Lines through Inducing G2/M Cell Cycle Arrest and Promoting Apoptosis

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Hongyan Chen ◽  
Qing Miao ◽  
Miao Geng ◽  
Jing Liu ◽  
Yazhuo Hu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Apoptosis ◽  
Cell Cycle Progression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Human Neuroblastoma ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Flow Cytometric

Aims. To further investigate the antineuroblastoma effect of rutin which is a type of flavonoid.Methods. The antiproliferation of rutin in human neuroblastoma cells LAN-5 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chemotaxis of LAN-5 cells was assessed using transwell migration chambers and scratch wound migration assay. The cell cycle arrest and apoptosis in a dose-dependent manner was measured by flow cytometric and fluorescent microscopy analyses. The apoptosis-related proteins BAX and BCL2 as well as MYCN mRNA express were determined by RT-PCR analysis. Secreted TNF-αlevel were determined using specific enzyme-linked immunosorbent assay kits.Results. Rutin significantly inhibited the growth of LAN-5 cells and chemotactic ability. Flow cytometric analysis revealed that rutin induced G2/M arrest in the cell cycle progression and induced cell apoptosis. The RT-PCR showed that rutin could decrease BCL2 expression and BCL2/BAX ratio. In the meantime, the MYCN mRNA level and the secretion of TNF-αwere inhibited.Conclusion. These results suggest that rutin produces obvious antineuroblastoma effects via induced G2/M arrest in the cell cycle progression and induced cell apoptosis as well as regulating the expression of gene related to apoptosis and so on. It supports the viability of developing rutin as a novel therapeutic prodrug for neuroblastoma treatment, as well as providing a new path on anticancer effect of Chinese traditional drug.

Pro-Apoptotic and Proliferative Effects of Bone Marrow Adipocytes on Myeloid Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4572-4572
Author(s):  
Linhua Jin ◽  
Marina Konopleva ◽  
Yixin Zhou ◽  
Akimichi Osaka ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Lipid Vesicles ◽  
Mrna Levels ◽  
U937 Cells ◽  
Mature Adipocyte ◽  
Cycle Progression ◽  
Induced Apoptosis ◽  
Pai 1

Abstract Abstract 4572 Bone marrow stromal cells (MSCs) from elderly subjects have a reduced capacity to differentiate into osteoblasts and an increased capacity to differentiate into adipocytes, which leads to progressive accumulation of fat in the bone marrow space with increasing age. Adipocytes are the prevalent stromal cell type in adult BM that play an important role in the leukemic bone marrow microenvironment (Tabe et al., Blood 2004 103:1815-22). In this study, we examined the role of BM-derived adipocytes at different stages of differentiation on proliferation and apoptosis of AML cells. U937 cells were co-cultured with BM-derived MSC, MSC-differentiated pre-adipocytes (containing few small lipid vesicles), and mature adipocytes (with multiple hypertrophic lipid vesicules). Under serum-starved conditions, MSC and premature/mature adipocytes induced cell cycle progression of U937 cells with increase in the proportion of cells in S- and G2/M-phase fractions, and inhibited spontaneous cell death with decrease in subG1 fractions. However, only pre-adipocytes inhibited Ara-C-induced cell killing (Table 1). We next focused on lepin and plasminogen activator inhibitor 1(PAI-1) as potential mediators of these effects by adipocytes. Leptin mRNA and protein levels were upregulated during adipocytic differentiation (mRNA relative expression to GAPDH (PCR): MSC 0, premature adipocyte 2.0±0.5, mature adipocyte 123.3± 35.0; leptin secretion: MSC 23.1±2.9, premature adipocyte 49.3±11.3, mature adipocyte 110.0±4.6 pg/mL (ELISA). PAI-1 mRNA levels were increased in mature adipocyte (relative expression to GAPDH: MSC 314.9±46.5, premature adipocyte 215.1, mature adipocyte 3766.1±656.2). Since PPARÿ activation is known to promote maturation and re-generation of fat-derived adipocytes, we next examined the potential of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) on the BM adipocytes, leptin and PAI-1 production and the survival of the leukemic cells. CDDO induced adipocyte re-generation with significant increase of the number of Oil-Red(+) small sized lipid vesicles without apoptosis induction. This resulted in a markedly enhanced leptin release from adipocytes (10-fold and 23-fold increase at 0.5 μM and 1.0 μM CDDO, respectively, at 72 hrs) without change in leptin mRNA transcription. On the contrary, PAI-1 mRNA levels were significantly decreased by CDDO (6 fold decrease in MSC, 4 fold decrease in premature adipocyte, 6 fold decrease in mature adipocyte). Co-culture of U937 cells with CDDO-primed premature adipocytes and mature adipocytes resulted in increased spontaneous apoptosis of U937 cells compared to adipocytes not exposed to CDDO (% specific apoptosis, U937 co-cultured with CDDO-primed premature adipocytes 26.9 %, mature adipocytes 20.9 %). At the same time, CDDO-primed premature adipocytes induced significant cell cycle progression with decreased proportion of G0/G1-phase and increase in S-phase fractions in U937 cells (Table 2). Co-culture with CDDO-primed premature adipocytes or with premature adipocytes co-treated with recombinant leptin increased subG1- and S-phase fractions in Ara-C-treated U937 cells compared to U937 cells co-cultured with premature adipocytes (Table 3). In mature adipocytes, which already produce high levels of leptin, CDDO or leptin treatment failed to modulate anti-apoptotic or proliferative effects of AraC on U937 cells. In contrast, human recombinant PAI-1 effectively inhibited spontaneous and Ara-C induced apoptosis of U937 cells (decreased % of Annexin V: spontaneous apoptosis 11.2±1.1%, Ara-C induced apoptosis 15.1± 1.7%). In summary, these results suggest that BM pre-adipocytes support proliferation and survival of myeloid leukemia cells in part through complementary effects of leptin and PAI-1. Our findings indicate that secretion of leptin during MSC differentiation or through PPARg ligation promotes cell cycle progression, while PAI-1 primarily inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of AML cells to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

HMGA1 Drives Inflammatory Pathways, Cell Cycle Progression, and Embryonic Stem Cell Genes During Leukemic Transformation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1371-1371
Author(s):  
Andrew Schuldenfrei ◽  
Amy Belton ◽  
Jeanne Kowalski ◽  
C. Conover Talbot ◽  
Francescopaolo Di Cello ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Differentially Expressed Genes ◽  
Cell Cycle Progression ◽  
Embryonic Stem ◽  
Differentially Expressed ◽  
Lymphoid Leukemia ◽  
Cycle Progression ◽  
Hmga1 Expression ◽  
Cellular Pathways ◽  
Late Stages

Abstract Abstract 1371 Although the high mobility group AT-hook 1 (HMGA1) gene functions as a potent oncogene in experimental models and high expression of HMGA1 portends a poor prognosis in diverse tumors, its role in leukemogenesis has remained elusive. We showed previously that HMGA1 induces leukemic transformation in cultured cells and causes aggressive lymphoid leukemia in transgenic mice. Inhibiting HMGA1 expression blocks colony formation in human lymphoid leukemia cells in vitro. Moreover, high levels correlate with relapse in childhood acute lymphoblastic leukemia (ALL), suggesting that it plays an important role in ALL. Because HMGA1 functions as a chromatin remodeling protein that modulates gene expression, we hypothesized that it drives leukemogenesis by dysregulating specific genes and pathways. To identify genes and cellular pathways induced by HMGA1 that could be targeted in therapy, we performed global gene expression profile analysis from lymphoid cells from the HMGA1 transgenic mice at different stages in tumorigenesis. All HMGA1 transgenics succumb to lymphoid malignancy with complete penetrance by 8–12 months. Pooled RNA samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) were analyzed for differential expression of >20,000 unique genes by microarray analysis (Affymetrix) using both a parametric and nonparametric approach. A subset of differentially expressed genes was confirmed using quantitative, RT-PCR. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis (IPA; www.ingenuity.com) and Gene Set Enrichment Analysis. To determine if these genes and pathways were relevant in human ALL, we knocked down HMGA1 expression in human ALL cells and assessed expression of a subset of the differentially expressed genes. Early in leukemogenesis (at 2 months), 113 genes were differentially expressed in the HMGA1 transgenics compared to controls. In established leukemia (12 months), 715 genes were differentially expressed. In established tumors, the dysregulated genes are involved in cancer, cell cycle regulation, and cell-mediated immune response by Ingenuity Pathway Analysis. Geneset enrichment showed that embryonic stem cell genes are enriched in the established leukemic cells. At both early and late stages in leukemogenesis, differentially regulated genes are involved in cellular development, hematopoiesis, and hematologic development. Early in leukemogenesis, most of the significantly dysregulated genes are involved in the inflammatory response and included NF-kappaB as a major node. In human ALL cells, knock-down of HMGA1 also resulted in knock-down of genes identified in our transgenic model, suggesting that these HMGA1 regulated genes are also relevant to human ALL. In summary, we found that HMGA1 induces inflammatory pathways early in leukemogenesis and pathways involved in embryonic stem cells, cell cycle progression, and cancer in established tumors. HMGA1 also dysregulates genes involved in cellular development and hematopoiesis at both early and late stages of tumorigenesis. Some of these HMGA1 pathways were also present in human ALL cells. Moreover, these results provide mechanistic insight into HMGA1 function at different stages in leukemogenesis and point to cellular pathways that could serve as therapeutic targets in ALL. Disclosures: No relevant conflicts of interest to declare.

IGFBP7 Gene Promoting Cell Proliferation in Acute Myeloid Leukemia Through Activation of AKT3 and CCND1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1447-1447
Author(s):  
Shaoyan Hu ◽  
Shui-yan Wu ◽  
Jian-nong Cen ◽  
Jian Pan ◽  
Xiaofei Qi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
K562 Cells ◽  
S Phase ◽  
G1 Phase ◽  
Rt Pcr ◽  
Cycle Progression ◽  
Childhood Aml

Abstract Abstract 1447 Insulin-like growth factor binding protein 7 (IGFBP7) has been ascribed properties of both tumor suppressor and enhancer of cell proliferation. In solid tumors the important role of IGFBP7 as a tumor suppressor was revealed in several studies. In acute T-lymphoblastic leukemia (T-ALL), high IGFBP7 expression is associated with a more immature phenotype of early T-ALL, inferior survival, and predicts primary chemotherapy resistance. In a separate study, IGFBP7 acts as a positive regulator of ALL and bone marrow stromal cells growth, and significantly enhances in-vitro resistance to asparaginase. Higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (P=0.003) in precursor B-cell Ph negative ALL patients (n=147) treated with a contemporary polychemotherapy protocol. In acute myeloid leukemia, the role of IGFBP7 is largely unknown. In our previous published study [Hu et al, 2011], we demonstrated that IGFBP7 overexpressed in majority of childhood AML (n=66) at diagnosis and upon relapsed, but not at remission stage. We now further explore its mechanism in promoting AML cells proliferation. Compared with control, transfection of full length IGFBP7 in K562 cells [V-BP7] resulted in 23% increased of proliferation in 48 hours. Cell cycle analysis by flow cytometry showed decreased G0/G1 phase and increased S phase in V-BP7 comparing with control, suggesting enhanced cell cycle progression. While transfection of IGFPB7 siRNA produced an opposite effect of reducing the cell growth in K562 cells. In consistent with the nature of a secretory protein, the extracellular IGFBP7 level in the condition media from v-BP7 was significantly higher than that from vector control or parental K562 cells measured by ELISA. Incubation parental K562 cells in V-BP7 derived conditioned medium resulted in significant growth enhancement. Gene expression profiling (GEP) was performed on V-BP7 in contrast to parental K562 cells. Genes which were up-regulated or down-regulated more than 2 folds were regarded as significant difference. Among 10 verified genes, AKT3 showed the highest extent of up-regulation and IGFBP7 siRNA transfection reduced its expression. Cyclin D1 (CCND1) expression was also significantly up-regulated and validated by RT-PCR and Western blot. V-BP7 treated with an AKT inhibitor (Triciribine) at 2.5μM for 72 hours showed 50% reduction of cell viability. The cell cycle analysis indicated that triciribine reversed cell cycle progression in V-BP7, by increasing cells in G0/G1 phase and reducing cells in S phase. Western blot demonstrated that both phospho-AKT3 and CCND1 were down regulated after treatment with triciribine. Using real time RT-PCR, we further identified that IGFBP7 and AKT3 expression were significantly correlated (p=0.001; r=0.255) in 39 newly diagnosed childhood AML. Conclusions IGFBP7 aberrantly overexpressed in majority of childhood AML. IGFBP7 promotes proliferation of K562 cells and AML via overexpression/activation of AKT3 and CCND1. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation.

1996 ◽  
Vol 16 (4) ◽  
pp. 1500-1508 ◽  
Author(s):  
P C Goswami ◽  
J L Roti Roti ◽  
C R Hunt
Keyword(s):  
Cell Cycle ◽  
Heat Shock ◽  
Ionizing Radiation ◽  
Mrna Stability ◽  
Cell Cycle Progression ◽  
Dna Supercoiling ◽  
S Phase ◽  
Mrna Levels ◽  
Cycle Progression ◽  
Topoisomerase Iialpha

Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase IIalpha and IIbeta genes. In HeLa cells synchronized by mitotic shake-off, topoisomeraseII alpha mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G1 phase. Also detected was a corresponding increase in topoisomerase IIalpha protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G2 phases. Topoisomerase IIalpha expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase IIalpha expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, the half-life of topoisomerase IIalpha mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase IIalpha mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45 degrees C for 15 min) caused the accumulated topoisomerase IIalpha mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase IIalpha expression and cell cycle progression delays in late S/G2.

scholarly journals The Saccharomyces cerevisiae RNase Mitochondrial RNA Processing Is Critical for Cell Cycle Progression at the End of Mitosis

Genetics ◽  
2002 ◽  
Vol 161 (3) ◽  
pp. 1029-1042 ◽  
Author(s):  
Ti Cai ◽  
Jason Aulds ◽  
Tina Gill ◽  
Michael Cerio ◽  
Mark E Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mitochondrial Rna ◽  
Mrna Levels ◽  
Cycle Progression ◽  
Rnase Mrp ◽  
Processing Step ◽  
A Cell

Abstract We have identified a cell cycle delay in Saccharomyces cerevisiae RNase MRP mutants. Mutants delay with large budded cells, dumbbell-shaped nuclei, and extended spindles characteristic of “exit from mitosis” mutants. In accord with this, a RNase MRP mutation can be suppressed by overexpressing the polo-like kinase CDC5 or by deleting the B-type cyclin CLB1, without restoring the MRP-dependent rRNA-processing step. In addition, we identified a series of genetic interactions between RNase MRP mutations and mutations in CDC5, CDC14, CDC15, CLB2, and CLB5. As in most “exit from mitosis” mutants, levels of the Clb2 cyclin were increased. The buildup of Clb2 protein is not the result of a defect in the release of the Cdc14 phosphatase from the nucleolus, but rather the result of an increase in CLB2 mRNA levels. These results indicate a clear role of RNase MRP in cell cycle progression at the end of mitosis. Conservation of this function in humans may explain many of the pleiotropic phenotypes of cartilage hair hypoplasia.

scholarly journals Melatonin improves the first cleavage of parthenogenetic embryos from vitrified–warmed mouse oocytes potentially by promoting cell cycle progression

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bo Pan ◽  
Izhar Hyder Qazi ◽  
Shichao Guo ◽  
Jingyu Yang ◽  
Jianpeng Qin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Mrna Levels ◽  
Blastocyst Formation ◽  
Cycle Progression ◽  
Maternal Mrna ◽  
Early Apoptosis ◽  
G2 Phase

Abstract Background This study investigated the effect of melatonin (MT) on cell cycle (G1/S/G2/M) of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II (MII) oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos. Results After vitrification and warming, oocytes were parthenogenetically activated (PA) and in vitro cultured (IVC). Then the spindle morphology and chromosome segregation in oocytes, the maternal mRNA levels of genes including Miss, Doc1r, Setd2 and Ythdf2 in activated oocytes, pronuclear formation, the S phase duration in zygotes, mitochondrial function at G1 phase, reactive oxygen species (ROS) level at S phase, DNA damage at G2 phase, early apoptosis in 2-cell embryos, cleavage and blastocyst formation rates were evaluated. The results indicated that the vitrification/warming procedures led to following perturbations 1) spindle abnormalities and chromosome misalignment, alteration of maternal mRNAs and delay in pronucleus formation, 2) decreased mitochondrial membrane potential (MMP) and lower adenosine triphosphate (ATP) levels, increased ROS production and DNA damage, G1/S and S/G2 phase transition delay, and delayed first cleavage, and 3) increased early apoptosis and lower levels of cleavage and blastocyst formation. Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming, recovery, PA and IVC media with 10− 9 mol/L MT before the embryos moved into the 2-cell stage of development. Conclusions MT might promote cell cycle progression via regulation of MMP, ATP, ROS and maternal mRNA levels, potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified–warmed mouse oocytes and their subsequent development.

Phosphorylation of Runx1 by Cyclin-Dependent Kinases Regulates Its Interaction with HDAC1 and HDAC3.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1381-1381 ◽  
Author(s):  
Hong Guo ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cyclin D3 ◽  
Mrna Levels ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Cdk Phosphorylation ◽  
Regulation Of Cell Cycle

Abstract Runx1/AML1 is a key regulator of hematopoiesis and leukemic transformation, as RUNX1(−/−) mice do not develop definitive hematopoietic stem cells, and sever alleukemic oncogenes, e.g. AML1-ETO, CBFβ-SMMHC, or TEL-AML1, inhibit Runx1activities. We have investigated regulation of cell cycle progression by Runx1. Runx1stimulates G1 to S cell cycle progression in hematopoietic cell lines and in transduced myeloid progenitors, and inhibition of Runx1 by CBFβ-SMMHC or AML1-ETO slows G1 progression. Runx1 induces cdk4 and cyclin D3 transcription, and exogenous cdk4, cyclin D2, or c-Myc overcomes inhibition of G1 progression by CBF oncoproteins. In addition to regulating cell cycle progression, Runx1 protein levels are themselves increased as hematopoietic cells progress from G1 to S to G2/M, though mRNA levels remain constant. Runx1 contains three consensus cdk sites, (S/T)PX(R/K), S48, S303, and S424, and using phospho-specific antisera we find that each of these is modified in hematopoietic cells. Mutation of these serines to aspartic acid, mimicking phosphorylation, increases trans-activation of a reporter containing four CBF sites or the TCRβ promoter, whereas mutation to alanine reduces trans-activation. p300 interacts similarly with Runx1(tripleA) and Runx1(tripleD). We have now evaluated interaction of HDACs1–8 with these variants and Runx1 and find that both HDAC1 and HDAC3 have reduced affinity for RUNX1(tripleD), as assessed by co-immunoprecipitation from transiently transfected 293T cells. Evaluation of single serine residue mutants (S48D, S303D, and S424D) demonstrates reduced affinity of HDAC1 or HDAC3 specifically for the Runx1(S424D) mutant, consistent with previous mapping of the Runx1:HDAC1 and Runx1:HDAC3 interactions to this region of Runx1. Thus, cdk phosphorylation of Runx1 S424 reduces affinity for HDAC1 and HDAC3, increasing Runx1 trans-activation potency. Regulation of Runx1 activity by cdks may control key developmental processes, including expansion of definitive HSC during development and regulation of the balance between adult HSC quiescence and proliferation.

scholarly journals Transcription feedback dynamics in the wake of cytoplasmic degradation shutdown

2021 ◽  
Author(s):  
Alon Chappleboim ◽  
Daphna Joseph-Strauss ◽  
Omer Gershon ◽  
Nir Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Transcriptional Response ◽  
Degradation Pathway ◽  
Feedback Mechanism ◽  
Mrna Levels ◽  
Cycle Progression ◽  
A Cell ◽  
Nascent Mrna ◽  
Global Reduction

AbstractIn the last decade, multiple studies have shown that cells maintain a balance of mRNA production and degradation in different settings, but the mechanisms by which cells implement this balance remain poorly understood. Here, we monitored cells’ mRNA and nascent mRNA profiles immediately following an acute depletion of Xrn1 - the main 5’-3’ mRNA exonuclease - that was previously implicated in balancing mRNA levels. We captured the detailed dynamics of the cells’ adaptation to rapid degradation of Xrn1 and observed a significant accumulation of mRNA, followed by global reduction in nascent transcription and a return to baseline mRNA levels. We present evidence that this transcriptional response is linked to cell cycle progression, and that it is not unique to Xrn1 depletion; rather, it is induced earlier when upstream factors in the 5’-3’ degradation pathway are perturbed. Using the detailed dynamic measurements we hypothesize a cell-cycle-linked feedback mechanism that monitors the accumulation of inputs to the 5’-3’ exonucleolytic pathway rather than its outputs.

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