scholarly journals c-Myb Contributes to G2/M Cell Cycle Transition in Human Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression

2007 ◽  
Vol 27 (6) ◽  
pp. 2048-2058 ◽  
Author(s):  
Yuji Nakata ◽  
Susan Shetzline ◽  
Chizuko Sakashita ◽  
Anna Kalota ◽  
Ravikumar Rallapalli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Biological Significance ◽  
Cyclin B1 ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
M Cell ◽  
Cell Cycle Transition

ABSTRACTMyb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G1/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G2/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-mybexpression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G2/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-mybfunctions in regulating human hematopoiesis.

Myb Family Transcription Factors Contribute to G2/M Cell Cycle Transition in Normal and Malignant Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1115-1115
Author(s):  
Yuji Nakata ◽  
Susan Shetzline ◽  
Chizuko Sakashita ◽  
Anna Kalota ◽  
Stephen I. Rudnick ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cyclin B1 ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
M Cell ◽  
Human Leukemia Cells ◽  
M Phase ◽  
Cell Cycle Transition

Abstract Myb family transcription factors are ubiquitously expressed, and are known to play a critical role in regulating G1/S cell cycle transition. Recently, Myb-like proteins have been found to regulate G2/M transit in plants, yeast, and Drosophila. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, but not c-Myb, regulated cyclin B1 expression directly. However, in hematopoietic cells Myb’s role in regulating cell cycle check points other than G1/S is less well defined. Herein we report that c-Myb, as well as B-Myb, up-regulates cyclin B1 expression in normal and malignant human hematopoietic cells, thereby contributing to G2/M cell cycle progression. Our initial experiments revealed a direct relationship between Myb and cyclin B1 expression. We then attempted to show causality using a variety of experimental approaches. First, ChIP assays demonstrated that c-Myb protein directly bound the cyclin B1 promoter in K562 and Mo7e cells. Second, a cycle 3 GFP reporter construct, driven by the cyclin B1 promoter, was upregulated in cells co-tranfected with a c-myb expression vector. Third, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide in 6 hours. All these assays strongly suggest that c-myb directly regulates cyclin B1. Finally, cyclin B1 expression decreased by 85–90 % in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A. The biologic significance of this relationship was revealed by two independent lines of experimentation. First, silencing B-myb resulted in a delay of cell cycle progression from S to G2/M, and an accumulation of cells in M phase, in HCT116 cells and K562 cells respectively. These abnormalities could be rescued, at least partially, by expression of exogenous c-myb. This observation conflicts with the report that c-Myb does not regulate cyclin B1 or G2/M progression in T98G cells suggesting that Myb functions could well be cell type specific. Additional analysis using PCR array showed that the absence of B-myb decreased the expression of 19 of 84 cell cycle related genes. Exogenous c-myb expression partially rescued 11 genes including cyclin B1, cyclin B2, cdc2, cdc20, CKS1B, p15INK4b and Ki-67, but not cyclin D1. In another experiment an inducible dominant negative c-Myb protein decreased cyclin B1 expression in K562 human leukemia cells, and the expected consequence of this, accelerated exit from the M phase, was observed. In activated primary human T-lymphocytes with IL-2 and CD34+ bone marrow cells, expression of c-Myb and cyclin B1 increased concordantly and silencing c-myb expression resulted in decreased cyclin B1 expression. We conclude from these studies that c-Myb in addition to B-myb plays a heretofore unappreciated role in G2/M cell cycle transition in normal and malignant human hematopoietic cells by directly regulating cyclin B1 expression.

Abundance of cyclin B1 regulates γ-radiation–induced apoptosis

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2645-2650 ◽  
Author(s):  
Lisa A. Porter ◽  
Gurmit Singh ◽  
Jonathan M. Lee
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Hematopoietic Cells ◽  
Cyclin B1 ◽  
Regulatory Subunit ◽  
Potent Inducer ◽  
Γ Radiation ◽  
Radiation Induced ◽  
Induced Apoptosis

Abstract γ-Radiation is a potent inducer of apoptosis. There are multiple pathways regulating DNA damage-induced apoptosis, and we set out to identify novel mechanisms regulating γ-radiation–induced apoptosis in hematopoietic cells. In this report, we present data implicating the cyclin B1 protein as a regulator of apoptotic fate following DNA damage. Cyclin B1 is the regulatory subunit of the cdc2 serine/threonine kinase, and accumulation of cyclin B1 in late G2 phase of the cell cycle is a prerequisite for mitotic initiation in mammalian cells. We find that abundance of the cyclin B1 protein rapidly increases in several mouse and human hematopoietic cells (Ramos, DP16, HL60, thymocytes) undergoing γ-radiation–induced apoptosis. Cyclin B1 accumulation occurs in all phases of the cell cycle. Antisense inhibition of cyclin B1 accumulation decreases apoptosis, and ectopic cyclin B1 expression is sufficient to induce apoptosis. These observations are consistent with the idea that cyclin B1 is both necessary and sufficient for γ-radiation-induced apoptosis.

c-Myb Plays a Role InG2/M Cell Cycle Transition by Direct Regulation of Cyclin B1 Expression in Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1355-1355 ◽  
Author(s):  
Yuji Nakata ◽  
Susan Shetzline ◽  
Chizuko Sakashita ◽  
Anna Kalota ◽  
Andrzej Ptasznik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Hematopoietic Cells ◽  
Cyclin B1 ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Human Leukemia Cells ◽  
M Phase ◽  
Cell Cycle Transition ◽  
Myb Protein

Abstract Myb family transcription factors are found throughout the phyla, and recent studies have demonstrated that Drosophila myb, as well as plant and yeast c-myb-like transcription factors, play an important role in regulating transition though the G1/S and G2/M phases of the cell cycle. Myb’s ability to regulate passage through G2/M is due at least in part to its ability to induce Cyclin B1 expression. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, regulated cyclin B1 expression. Though c-myb was expressed in these cells, it was not found in immunoprecipitated E2F-B-Myb protein complexes and for this reason was felt not to participate in cyclin B1 expression in these cells. Since c-myb plays such a critical role in regulating hematopoietic cell proliferation, and its role in regulating G2/M in blood cells has not previously been explored, we investigated whether c-myb was important is regulating this phase of the cell cycle using K562 and Mo7e cells, as well as PHA stimulated human T lymphocytes. In distinct contrast to findings reported for T98G cells, we now report that in normal and malignant human hematopoietic cells, c-Myb directly upregulates cyclin B1 expression. Several lines of evidence support this claim. First, cyclin B1 expression decreased in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A in these cells. As expected, siRNA targeted against c-myb or B-myb impaired Mo7e cell proliferation. Simultaneous exposure to both siRNA blocked proliferation completely. Second, using an alternative strategy, an inducible dominant negative c-Myb protein also decreased cyclin B1 expression in K562 human leukemia cells. The expected consequence of this, accelerated exit from the M phase, was also observed. Third, we examined c-Myb expression in human T cells by western and Real Time PCR, pre and post PHA stimulation. c-Myb expression began to gradually increase in the G1 phase of cell cycle, continued to increase after S phase, with the maximal protein level being found in G2/M phase, and concordant with cyclin B1 expression. These results indicated a correlation between c-Myb and cyclin B1 expression but did not indicate if c-Myb regulated cyclin B1 expression directly. To address this question, several additional experiments were carried out. A CAT assay showed that overexpressing c-Myb protein could increase activity when driven by a cyclin B1 promoter construct ~5X compared to K562 control cells. Next, examination of the cyclin B1 promoter showed eight potential c-Myb binding sites. Two were canonical [5′-pyrimidine AACG/TG-3′] and located upstream of 6 others which were [5′-AACNG-3′] in type. An in vitro c-Myb binding assay revealed that c-Myb bound the canonical sites. We then performed a Chromatin Immunoprecipitation (ChIP) Assay with anti-c-Myb antibody and specifically enriched cyclin B1 promoter DNA sequences which strongly suggested that c-Myb bound the cyclin B1 promoter in vivo. A control antibody was inactive. Finally, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide within 6 hours. Therefore, in addition to its role in regulating G1/S cell cycle transition, c-Myb also regulates cyclin B1 expression and therefore transition through the G2/M phase in human hematopoietic cells.

Abundance of cyclin B1 regulates γ-radiation–induced apoptosis

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2645-2650 ◽  
Author(s):  
Lisa A. Porter ◽  
Gurmit Singh ◽  
Jonathan M. Lee
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Hematopoietic Cells ◽  
Cyclin B1 ◽  
Regulatory Subunit ◽  
Potent Inducer ◽  
Γ Radiation ◽  
Radiation Induced ◽  
Induced Apoptosis

γ-Radiation is a potent inducer of apoptosis. There are multiple pathways regulating DNA damage-induced apoptosis, and we set out to identify novel mechanisms regulating γ-radiation–induced apoptosis in hematopoietic cells. In this report, we present data implicating the cyclin B1 protein as a regulator of apoptotic fate following DNA damage. Cyclin B1 is the regulatory subunit of the cdc2 serine/threonine kinase, and accumulation of cyclin B1 in late G2 phase of the cell cycle is a prerequisite for mitotic initiation in mammalian cells. We find that abundance of the cyclin B1 protein rapidly increases in several mouse and human hematopoietic cells (Ramos, DP16, HL60, thymocytes) undergoing γ-radiation–induced apoptosis. Cyclin B1 accumulation occurs in all phases of the cell cycle. Antisense inhibition of cyclin B1 accumulation decreases apoptosis, and ectopic cyclin B1 expression is sufficient to induce apoptosis. These observations are consistent with the idea that cyclin B1 is both necessary and sufficient for γ-radiation-induced apoptosis.

scholarly journals The positive circadian regulators CLOCK and BMAL1 control G2/M cell cycle transition through Cyclin B1

Cell Cycle ◽  
2018 ◽  
Vol 18 (1) ◽  
pp. 16-33 ◽  
Author(s):  
Elham Farshadi ◽  
Jie Yan ◽  
Pierre Leclere ◽  
Albert Goldbeter ◽  
Inês Chaves ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin B1 ◽  
M Cell ◽  
Cell Cycle Transition

scholarly journals E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

1997 ◽  
Vol 17 (12) ◽  
pp. 7268-7282 ◽  
Author(s):  
R Verona ◽  
K Moberg ◽  
S Estes ◽  
M Starz ◽  
J P Vernon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Critical Role ◽  
Biological Properties ◽  
Dependent Manner ◽  
Restriction Point ◽  
Cycle Dependent ◽  
Almost All

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

Signal transducer and activator of transcription 5a (STAT5a) is required for eosinophil differentiation of human cord blood–derived CD34+ cells

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 134-142 ◽  
Author(s):  
Miranda Buitenhuis ◽  
Belinda Baltus ◽  
Jan-Willem J. Lammers ◽  
Paul J. Coffer ◽  
Leo Koenderman
Keyword(s):  
Cord Blood ◽  
Target Genes ◽  
Ex Vivo ◽  
Critical Role ◽  
Ectopic Expression ◽  
Hematopoietic Cells ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Human Cord Blood ◽  
Cd34 Cells

Abstract Signal transducers and activators of transcription (STATs) have been reported to play a critical role in the differentiation of several myeloid cell lines, although the importance of STATs in the differentiation of primary human hematopoietic cells remains to be established. Terminal eosinophil differentiation is induced by interleukin-5 (IL-5), which has also been demonstrated to activate STAT5. We have investigated whether STAT5 plays a critical role during eosinophil differentiation using umbilical cord blood–derived CD34+ cells. In this ex vivo system, STAT5 expression and activation are high early during differentiation, and STAT5 protein expression is down-regulated during the final stages of eosinophil differentiation. Retroviral transductions were performed to ectopically express wild-type and dominant-negative STAT5a (STAT5aΔ750) in CD34+ cells. Transduction of cells with STAT5a resulted in enhanced proliferation compared with cells transduced with empty vector alone. Interestingly, ectopic expression of STAT5a also resulted in accelerated differentiation. In contrast, ectopic expression of STAT5aΔ750 resulted in a block in differentiation, whereas proliferation was also severely inhibited. Similar results were obtained with dominant-negative STAT5b. Forced expression of STAT5a enhanced expression of the STAT5 target genes Bcl-2 andp21WAF/Cip1, suggesting they may be important in STAT5a-mediated eosinophil differentiation. These results demonstrate that STAT5 plays a critical role in eosinophil differentiation of primary human hematopoietic cells.

scholarly journals miR-548b-3p Regulates Proliferation, Apoptosis, and Mitochondrial Function by Targeting CIP2A in Hepatocellular Carcinoma

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Lin Lin ◽  
Yong Wang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cell Growth ◽  
Mitochondrial Function ◽  
Luciferase Reporter ◽  
Target List ◽  
Downstream Target ◽  
Normal Tissues ◽  
Cisplatin Sensitivity ◽  
Cell Cycle Transition

The roles of miR-548b-3p in the progression of hepatocellular carcinoma (HCC) remain undiscovered. This study aims to explore the roles and mechanisms of miR-548b-3p in HCC. Using TCGA database, we found that miR-548b-3p expression was lower in HCC compared to the normal tissues, which was further confirmed by RT-qPCR of 20 cases of surgically resected HCC and corresponding normal tissues. miR-548b-3p mimic and inhibitor were transfected into Huh7 and SK-Hep-1 cells, respectively. MTT, colony formation, and cell cycle assays showed that miR-548b-3p mimic suppressed cell growth and G1/S cell cycle transition. In contrast, miR-548b-3p inhibitor facilitated cell growth and cell cycle transition. miR-548b-3p mimic also increased cisplatin sensitivity by upregulating apoptosis rate. JC-1 staining showed that miR-548b-3p mimic downregulated mitochondrial membrane potential, while miR-548b-3p inhibitor showed the opposite effects in SK-Hep-1 cells. Using prediction software, we found that CIP2A was on the target list of miR-548b-3p. miR-548b-3p mimic downregulated CIP2A and its downstream target protein c-Myc. Luciferase reporter assay demonstrated that CIP2A was as a direct target of miR-548b-3p. CIP2A depletion partly reduced the effect of miR-548b-3p mimic/inhibitor on c-Myc. CIP2A depletion also reduced the effect of miR-548b-3p mimic/inhibitor on proliferation. In conclusion, our data demonstrated that miR-548b-3p was downregulated in HCC. miR-548b-3p regulates proliferation, apoptosis and mitochondrial function by targeting CIP2A in HCC.

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