Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.

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Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.6725-6738.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 6725-6738 ◽

Cited By ~ 48

Author(s):

Yi Cao ◽

Zhefeng Zhao ◽

Joanna Gruszczynska-Biegala ◽

Anna Zolkiewska

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Transmembrane Domain ◽

Myogenic Differentiation ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Differentiation Markers ◽

Cycle Arrest

ABSTRACT Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated “reserve cells” that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin α7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation.

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Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

Molecular Biology of the Cell ◽

10.1091/mbc.01-03-0104 ◽

2002 ◽

Vol 13 (2) ◽

pp. 480-492 ◽

Cited By ~ 24

Author(s):

Tom D. Wolkow ◽

Tamar Enoch

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Fission Yeast ◽

Complex Analysis ◽

Kinase Activity ◽

Regulatory Subunit ◽

Kinase Activation ◽

Checkpoint Proteins ◽

Cycle Arrest ◽

Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

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Novel benzylidene-thiazolidine-2,4-diones inhibit Pim protein kinase activity and induce cell cycle arrest in leukemia and prostate cancer cells

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-08-1037 ◽

2009 ◽

Vol 8 (6) ◽

pp. 1473-1483 ◽

Cited By ~ 60

Author(s):

Zanna Beharry ◽

Marina Zemskova ◽

Sandeep Mahajan ◽

Fengxue Zhang ◽

Jian Ma ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Cycle ◽

Protein Kinase ◽

Cell Cycle Arrest ◽

Cancer Cells ◽

Kinase Activity ◽

Protein Kinase Activity ◽

Prostate Cancer Cells ◽

Induce Cell Cycle Arrest ◽

Cycle Arrest

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Characterization of the Akt2 Domain Essential for Binding Nuclear p21cip1 to Promote Cell Cycle Arrest during Myogenic Differentiation

PLoS ONE ◽

10.1371/journal.pone.0076987 ◽

2013 ◽

Vol 8 (10) ◽

pp. e76987 ◽

Cited By ~ 4

Author(s):

Lisa Heron-Milhavet ◽

Celine Franckhauser ◽

Anne Fernandez ◽

Ned J. Lamb

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Myogenic Differentiation ◽

Cycle Arrest

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Critical role of PP2A-B56 family protein degradation in HIV-1 Vif mediated G2 cell cycle arrest

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2020.04.123 ◽

2020 ◽

Vol 527 (1) ◽

pp. 257-263

Author(s):

Kayoko Nagata ◽

Keisuke Shindo ◽

Yusuke Matsui ◽

Kotaro Shirakawa ◽

Akifumi Takaori-Kondo

Keyword(s):

Cell Cycle ◽

Protein Degradation ◽

Cell Cycle Arrest ◽

Critical Role ◽

Cycle Arrest ◽

Family Protein ◽

G2 Cell Cycle Arrest ◽

Hiv 1

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Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c

Oncogene ◽

10.1038/sj.onc.1206498 ◽

2003 ◽

Vol 22 (26) ◽

pp. 4074-4082 ◽

Cited By ~ 26

Author(s):

Noriko Nishimura ◽

Yusuke Furukawa ◽

Krittaya Sutheesophon ◽

Mitsuru Nakamura ◽

Kenji Kishi ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Kinase Activity ◽

Cdk Inhibitor ◽

Cycle Arrest

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Cdc7 inhibition as a novel approach for pancreas cancer therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e15059 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e15059-e15059

Author(s):

Mark G. Frattini ◽

Lucia Regales ◽

Ruth Santos ◽

Diana Carrillo

Keyword(s):

Cell Cycle ◽

Pancreatic Cancer ◽

Dna Damage ◽

Cell Viability ◽

Cell Cycle Arrest ◽

Western Blotting ◽

Kinase Activity ◽

S Phase ◽

Cycle Arrest ◽

Cdc7 Kinase

e15059 Background: Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, 43,920 people will be diagnosed and 37,390 people will die of this disease. 95% of tumors reveal loss of the p16 protein, a regulator of the G1 to S phase transition. Cdc7 is a conserved kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Downregulation of Cdc7 kinase activity resulted in slowing of S-phase and cell cycle arrest followed by accumulation of DNA damage. Cdc7 has been shown to be over-expressed in many different tumors including the majority of solid and liquid tumors. In our laboratory a novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be efficacious in cell based cytotoxicity assays and multiple animal models of cancer. Methods: We have examined the efficacy of Cdc7 kinase inhibition as a therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in Capan-1, BxPC3, and PANC-1 cell lines. These cells were treated with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS), and western blotting. Results: Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. Conclusions: We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease.

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