scholarly journals Genetic Evidence for the Interactions of Cyclin D1 and p27Kip1 in Mice

2001 ◽  
Vol 21 (4) ◽  
pp. 1319-1328 ◽  
Author(s):  
Wei Tong ◽  
Jeffrey W. Pollard
Keyword(s):  
Cell Cycle ◽  
Body Weight ◽  
Cyclin D1 ◽  
Cultured Cells ◽  
Cyclin E ◽  
Negative Regulator ◽  
Luteal Cell ◽  
Null Mouse ◽  
Cdk2 Activity

ABSTRACT The cell cycle of cultured cells appears to be regulated by opposing actions of the cyclins together with their partners, the cyclin-dependent kinases (Cdk), and their inhibitors (Cki). Consistent with this situation null mutations in the genes for cyclin D1 and Cki p27Kip1 in mice give opposite phenotypes of dwarfism and gigantism. To test their genetic interactions, we generated mice nullizygous for both genes. Correction of cyclin D1 or p27 null to wild-type phenotypes was observed for many but not all traits. These included, for cyclin D1−/− mice, body weight, early lethality, retinal hypoplasia, and male aggressiveness and, for p27−/− mice, body weight, retinal hyperplasia, and embryo implantation. p27−/− traits that were not corrected were the aberrant estrus cycles, luteal cell proliferation, and susceptibility to pituitary tumors. This mutual correction of these phenotypes is the first genetic demonstration of the interaction of these inhibitory and stimulatory cell cycle-regulatory molecules in vivo. The molecular basis for the correction was analyzed in the neonatal retina. Retinal cellularity was rescued in the cyclin D1 null mouse by loss of p27 with only a partial restoration of phosphorylation of retinoblastoma protein (Rb) and Cdk4 activity but with a dramatic elevation of Cdk2 activity. Our data provide in vivo genetic validation of cell culture experiments that indicated that p27 acts as a negative regulator of cyclin E-Cdk2 activity and that it can be titrated away by cyclin D-Cdk4 complexes. It also supports the suggestion that the cyclin E/Cdk2 pathway can largely bypass Rb in regulating the cell cycle in vivo.

scholarly journals Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function

2001 ◽  
Vol 21 (3) ◽  
pp. 794-810 ◽  
Author(s):  
James S. Foster ◽  
Donald C. Henley ◽  
Antonin Bukovsky ◽  
Prem Seth ◽  
Jay Wimalasena
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Inhibitory Activity ◽  
Cell Cycle Progression ◽  
Estrogen Action ◽  
Cycle Progression ◽  
Cdk2 Activity ◽  
Mcf 7

ABSTRACT Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G1/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16INK4a to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16INK4a inhibited G1/S transition induced in MCF-7 cells by 17-β-estradiol (E2) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G1 and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21Cip1 and p27Kip1 was decreased, however, in both control and p16INK4a-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E2 in control and p16INK4a-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16INK4a. Inhibition of Cdc25A activity in p16INK4a-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E2-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisenseCDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16INK4a-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21Cip1 and p27Kip1.

scholarly journals Novel localization and possible functions of cyclin E in early sea urchin development

2002 ◽  
Vol 115 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Bradley J. Schnackenberg ◽  
William F. Marzluff
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Sea Urchin ◽  
Kinase Activity ◽  
Cyclin E ◽  
Sperm Head ◽  
Mitotic Spindles ◽  
Paternal Genome ◽  
Cell Cycles ◽  
Cdk2 Activity

In somatic cells, cyclin E-cdk2 activity oscillates during the cell cycle and is required for the regulation of the G1/S transition. Cyclin E and its associated kinase activity remain constant throughout early sea urchin embryogenesis, consistent with reports from studies using several other embryonic systems. Here we have expanded these studies and show that cyclin E rapidly and selectively enters the sperm head after fertilization and remains concentrated in the male pronucleus until pronuclear fusion, at which time it disperses throughout the zygotic nucleus. We also show that cyclin E is not concentrated at the centrosomes but is associated with condensed chromosomes throughout mitosis for at least the first four cell cycles. Isolated mitotic spindles are enriched for cyclin E and cdk2, which are localized to the chromosomes. The chromosomal cyclin E is associated with active kinase during mitosis. We propose that cyclin E may play a role in the remodeling of the sperm head and re-licensing of the paternal genome after fertilization. Furthermore, cyclin E does not need to be degraded or dissociated from the chromosomes during mitosis; instead, it may be required on chromosomes during mitosis to immediately initiate the next round of DNA replication.

Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis

1997 ◽  
Vol 110 (12) ◽  
pp. 1373-1386 ◽  
Author(s):  
G.R. Walker ◽  
C.B. Shuster ◽  
D.R. Burgess
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Immunoblot Analysis ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Apparatus ◽  
Cortical Cytoskeleton

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

Expression of Cell Cycle Proteins P21 waf1/Cip1 , P27 kip1 , Cyclin D1 and CDK4 in Blood Vessels of Angiotensin II-Infused Rats. Role of AT 1 Receptors.

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 707-707
Author(s):  
Quy N Diep ◽  
Mohammed El Mabrouk ◽  
Rhian M Touyz ◽  
Ernesto L Schiffrin
Keyword(s):  
Cell Cycle ◽  
Angiotensin Ii ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Blood Vessels ◽  
Thymidine Incorporation ◽  
Mesenteric Arteries ◽  
Ang Ii

P79 Angiotensin II (Ang II) is an important modulator of cell growth via AT 1 receptors, as demonstrated both in vivo and in vitro . Here, we investigated the role of different proteins involved in the cell cycle, including cyclin D1, cyclin-dependent kinase 4 (cdk4) and cdk inhibitors p21 and p27 in blood vessels of Ang II-infused rats and the effect therein of the AT 1 receptor antagonist losartan. Male Sprague Dawley rats were infused for 7 days with Ang II (120 ng/kg/min s.c.) and/or treated with losartan (10 mg/kg/day orally). DNA synthesis in mesenteric arteries was evaluated by radiolabeled 3 H-thymidine incorporation. The expression of p21, p27, cyclin D1, cdk4 and E2F, which play critical roles during G1-phase of the cell cycle process, was examined by Western blot analysis. Tail cuff systolic blood pressure (mmHg) was elevated (p<0.05, n=9) in Ang II-infused rats (161.3±8.2) vs. controls (110.1±5.3) and normalized by losartan (104.4±3.2). Radiolabeled 3 H-thymidine incorporation (cpm/100 μg DNA) showed that Ang II-infusion significantly increased DNA synthesis (152±5 vs. 102±6, p<0.05). Expression of p21 and p27 was significantly decreased in the Ang II group to 23.2±10.4% and 10.3±5.3% of controls, respectively, whereas expression of cyclin D1 and cdk4 was significantly increased in the Ang II group to 213.7±8% and 263.6±37% of controls, respectively. These effects induced by Ang II infusion was normalized in the presence of losartan. Ang II had no effect on the expression of E2F. Thus, when AT 1 receptors are stimulated in vivo , DNA synthesis is enhanced in blood vessels by activation of cyclin D1 and cdk4. Reduction in cell cycle kinase inhibitors p21 and p27 may contribute to activation of growth induced by in vivo AT 1 receptor stimulation.

scholarly journals A Novel Long Non-Coding RNA-01488 Suppressed Metastasis and Tumorigenesis by Inducing miRNAs That Reduce Vimentin Expression and Ubiquitination of Cyclin E

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1504
Author(s):  
Syuan-Ling Lin ◽  
Yang-Hsiang Lin ◽  
Hsiang-Cheng Chi ◽  
Tzu-Kang Lin ◽  
Wei-Jan Chen ◽  
...  
Keyword(s):  
Human Cancer ◽  
Cyclin E ◽  
Survival Rates ◽  
Negative Regulator ◽  
Vimentin Expression ◽  
Protein Coding ◽  
Tumorigenic Activity ◽  
Underlying Mechanisms

Long intergenic non-coding RNAs (lincRNAs) play important roles in human cancer development, including cell differentiation, apoptosis, and tumor progression. However, their underlying mechanisms of action are largely unknown at present. In this study, we focused on a novel suppressor lincRNA that has the potential to inhibit progression of human hepatocellular carcinoma (HCC). Our experiments disclosed long intergenic non-protein coding RNA 1488 (LINC01488) as a key negative regulator of HCC. Clinically, patients with high LINC01488 expression displayed greater survival rates and better prognosis. In vitro and in vivo functional assays showed that LINC01488 overexpression leads to significant suppression of cell proliferation and metastasis in HCC. Furthermore, LINC01488 bound to cyclin E to induce its ubiquitination and reduced expression of vimentin mediated by both miR-124-3p/miR-138-5p. Our results collectively indicate that LINC01488 acts as a tumor suppressor that inhibits metastasis and tumorigenesis in HCC via the miR-124-3p/miR-138-5p/vimentin axis. Furthermore, LINC01488 interacts with and degrades cyclin E, which contributes to its anti-tumorigenic activity. In view of these findings, we propose that enhancement of LINC01488 expression could be effective as a potential therapeutic strategy for HCC.

scholarly journals Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 596-604 ◽  
Author(s):  
Ian S. Hitchcock ◽  
Norma E. Fox ◽  
Nicolas Prévost ◽  
Katherine Sear ◽  
Sanford J. Shattil ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Platelet Function ◽  
Therapeutic Benefit ◽  
Negative Regulator ◽  
Null Mouse ◽  
Wild Type ◽  
Platelet Recovery ◽  
Lyn Kinase

Focal adhesion kinase (FAK) plays a key role in mediating signaling downstream of integrins and growth factor receptors. In this study, we determined the roles of FAK in vivo by generating a megakaryocyte lineage–specific FAK-null mouse (Pf4-Cre/FAK-floxed). Megakaryocyte and platelet FAK expression was ablated in Pf4-Cre/FAK-floxed mice without affecting expression of the FAK homologue PYK2, although PYK2 phosphorylation was increased in FAK−/− megakaryocytes in response to fibrinogen. Megakaryopoiesis is greatly enhanced in Pf4-Cre/FAK-floxed mice, with significant increases in megakaryocytic progenitors (CFU-MK), mature megakaryocytes, megakaryocyte ploidy, and moderate increases in resting platelet number and platelet recovery following a thrombocytopenic stress. Thrombopoietin (Tpo)–mediated activation of Lyn kinase, a negative regulator of megakaryopoiesis, is severely attenuated in FAK-null megakaryocytes compared with wild-type controls. In contrast, Tpo-mediated activation of positive megakaryopoiesis regulators such as ERK1/2 and AKT is increased in FAK-null megakaryocytes, providing a plausible explanation for the observed increases in megakaryopoiesis in these mice. In Pf4-Cre/FAK-floxed mice, rebleeding times are significantly increased, and FAK-null platelets exhibit diminished spreading on immobilized fibrinogen. These studies establish clear roles for FAK in megakaryocyte growth and platelet function, setting the stage for manipulation of this component of the Tpo signaling apparatus for therapeutic benefit.

Depsipeptide in the Treatment of Relapsed and Refractory Multiple Myeloma (MM): A Prospective Evaluation of the Cell Cycle.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1497-1497
Author(s):  
Zoe Goldberg* ◽  
Scott Ely ◽  
Selina Chen-Kiang ◽  
Martha Chesi ◽  
Peter L. Bergsagel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Phase Ii ◽  
Caspase 3 ◽  
Gene Array ◽  
Cyclin D3 ◽  
Proliferation Index ◽  
G1 Arrest

Abstract Background: Dysregulation of the cell cycle and apoptosis are two critical events in the pathophysiology of MM. This notion is supported by: 1)A high tumor burden is often present despite a low rate of tumor cell proliferation. 2)G1 arrest is common in MM cells while normal plasma cells are permanently withdrawn from cell cycle. 3) Cyclin D1 is often overexpressed without a defined genetic substrate. Herein, we show that cell cycle evaluation in vivo is feasible and that the histone-deacetylase inhibitor depsipeptide might be effective in selected patients with MM. Patients and Methods: In vitro studies were performed in 12 human MM cell lines with defined cytogenetic abnormalities. The IC50 for depsipeptide was determined by evaluation of apoptosis by standard methods. In vivo studies where done as correlates in a phase II protocol. These include: Immunohistochemistry (IHC) for co-expression of CD138/Ki-67 as a proliferation index (PCPI), cyclin D1, D3, caspase 3 cleavege, CD31 and bcl-2 before treatment and at 24 hrs and 30 days after treatment. Gene array studies are being performed on selected patients at those timepoints. To date, four stage III patients (PTS) with relapsed MM with four or fewer prior lines of therapy have been treated with one to three cycles of depsipeptide at a dose of 13mg/m2,as a 4-hour infusion on days 1, 8, and 15, repeated every 28 days. Mean age was 63 years (range, 56 to 72). KPS of >80%. Mean albumin was 3.5, (range, 3.2 to 4), mean LDH was 243 (range, 179 to 315). Results: 1)Depsipeptide induces apoptosis in several MM cell lines. All lines were susceptible to depsipeptide, however, differential sensitivities were noted. Three cell lines (ie U266) that contained 11q13 translocation (cyclin D1 overexpression) were the most sensitive with IC50s at least 2 fold lower than other lines. 2) Cell cycle changes are induced by depsipeptide: In 2/4 PTS, a significant increase of the PCPI was seen, whereas a marked reduction in the PCPI in a patient with cyclin D3 overexpression (27% to 16%) was also noted. One patient had an increase of cyclin D1 post treatment. No changes where seen in bcl-2, CD-31, or cleaved caspase-3 expression. 3) Depsipeptide is safe in a limited cohort of MM PTS: Grade 2 fatigue and anorexia were the most common toxicities. Mild thrombocytopenia (mean of 67) did not require transfusions. One patient had stable disease after 3 cycles of treatment, one patient had progression of disease after 3 cycles, one patient progressed after the 1st cycle, and one patient is too early for evaluation. Conclusions: 1)Patients with 11q13 translocation should be a target for treatment with depsipeptide. 2)Depsipeptide given on this schedule is safe and can stabilize tumor-mass in PTS with otherwise progressive relapsed and refractory disease.3) Evidence of cell cycle modulation can be seen during treatment with depsipeptide. No profound changes in apoptosis is evident.4)Further studies may help to understand the mechanism of transcriptional regulation by depsipeptide and will help design rational therapy and combinations. This study continues to accrue patients as part of New York Phase II Consortium. Supported by NCI grant (SAIC1N01-CO-12400-02) and a SCOR for Myeloma grant from the Leukemia and Lymphoma Society of America.

Interaction and Regulation of HSC with Osteopontin Is Mediated through α4β1 and α9β1Integrins, Which Are Differentially Expressed in the HSC Pool.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1330-1330
Author(s):  
David N. Haylock ◽  
Genevieve A. Whitty ◽  
Brenda Williams ◽  
Melonie J. Storan ◽  
Susie K. Nilsson
Keyword(s):  
Cord Blood ◽  
Critical Role ◽  
Negative Regulator ◽  
Hemopoietic Stem Cell ◽  
Null Mouse ◽  
Dependent Manner ◽  
Independent Manner ◽  
Hsc Niche

Abstract Osteoblasts are a key cellular component of the hemopoietic stem cell (HSC) niche and directly regulate the HSC pool. Molecules synthesised by osteoblasts both promote or inhibit HSC proliferation. Osteopontin (Opn) is an osteoblast produced, RGD containing protein with roles in cell adhesion and migration. Until recently, the role of Opn in hemopoiesis was seen as restricted to the regulation of bone turnover. However, from analysis of hemopoiesis in the Opn null mouse, we have demonstrated that Opn plays a critical role in regulating the HSC pool. Furthermore Opn is critical in trans-marrow migration and lodgement of HSC within the BM after transplantation. When added to in vitro HSC cultures, exogenous thrombin-cleaved Opn also inhibits cell proliferation and potently suppresses HSC differentiation. We have now demonstrated that this interaction occurs in an RGD-independent manner via the cryptic SVVYGLR epitope revealed on the N-terminal fragment of Opn following thrombin cleavage. This epitope has previously been shown to bind to α4β1 and α9β1. HSC are known to express α4β1, but we have now shown that within the HSC pool this occurs in a differential manner, mimicking that of CD38, with more committed CD34+CD38+ cord blood progenitors having the highest levels of expression. In addition, we have shown the previously unrecognised characteristic of human marrow and cord blood HSC, the expression of α9β1, which also occurs in a differential manner, but mimicking CD34. Expression of α9β1 is highest on cord blood CD34+CD38− cells, a population highly enriched for HSC. Using the synthetic SVVYGLR peptide in culture, we re-capitulated the thrombin-cleaved Opn induced suppression of HSC differentiation in a dose dependent manner. Antibody blocking experiments demonstrated that binding to this peptide was occurring through both α4β1 and α9β1. In contrast, suppression of HSC proliferation and differentiation did not occur through the upstream alternate α4β1 binding site. Furthermore, we have now demonstrated endogenous binding of Opn to α4β1 and α9β1 to cord blood HSC in vivo. Together, these data provide strong evidence that Opn is an important component of the HSC niche which acts as a physiological negative regulator. Furthermore, our studies identify the previously unrecognised characteristic of HSC, the expression of α9β1, which together with α4β1 provides two receptors on HSC with differing expression signatures and potentially a mechanism for fine tunning the physiological effects of Opn.

Cyclin D: CDK4/6 Kinase Activity, Regulated by GATA-1, Is Required for Megakaryocyte Polyploidization.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 780-780
Author(s):  
Andrew G. Muntean ◽  
Liyan Pang ◽  
Mortimer Poncz ◽  
Steve Dowdy ◽  
Gerd Blobel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Cyclin D1 ◽  
Kinase Activity ◽  
Fetal Liver ◽  
Cyclin D3 ◽  
Luciferase Reporter ◽  
Cell Cycle Regulators ◽  
Wild Type

Abstract Megakaryocytes, which fragment to give rise to platelets, undergo a unique form of cell cycle, termed endomitosis, to become polyploid and terminally differentiate. During this process, cells transverse the cell cycle but the late stages of mitosis are bypassed to lead to accumulation of DNA up to 128N. While the mechanisms of polyploidization in megakaryocytes are poorly understood, a few cell cycle regulators, such as cyclin D3, have been implicated in this process. Hematopoietic transcription factors, including GATA-1 and RUNX1 are also essential for polyploidization, as both GATA1-deficient and RUNX1-null megakaryocytes undergo fewer rounds of endomitosis. Interestingly, GATA-1 deficient megakaryocytes are also smaller than their wild-type counterparts. However, the link between transcription factors and the growth and polyploidization of megakaryocytes has not been established. In our studies to identify key downstream targets of GATA-1 in the megakaryocyte lineage, we discovered that the cell cycle regulators cyclin D1 and p16 were aberrantly expressed in the absence of GATA-1: cyclin D1 expression was reduced nearly 10-fold, while that of p16ink4a was increased 10-fold. Luciferase reporter assays revealed that GATA-1, but not the leukemic isoform GATA-1s, promotes cyclinD1 expression. Consistent with these observations, megakaryocytes that express GATA-1s in place of full-length GATA-1 are smaller than their wild-type counterparts. Chromatin immunoprecipitation studies revealed that GATA-1 is bound to the cyclin D1 promoter in vivo, in primary fetal liver derived megakaryocytes. In contrast, GATA-1 is not associated with the cyclin D1 promoter in erythroid cells, which do not become polyploid. Thus, cyclin D1 is a bona fide GATA-1 target gene in megakaryocytes. To investigate whether restoration of cyclin D1 expression could rescue the polyploidization defect in GATA-1 deficient cells, we infected fetal liver progenitors isolated from GATA-1 knock-down mice with retroviruses harboring the cyclin D1 cDNA (and GFP via an IRES element) or GFP alone. Surprisingly, expression of cyclin D1 did not increase the extent of polyploidization of the GATA-1 deficient megakaryocytes. However, co-overexpression of cyclin D1 and Cdk4 resulted in a dramatic increase in polyploidization. Consistent with the model that cyclinD:Cdk4/6 also regulates cellular metabolism, we observed that the size of the doubly infected cells was also significantly increased. Finally, in support of our model that cyclin D:Cdk4/6 kinase activity is essential for endomitosis, we discovered that introduction of wild-type p16 TAT fusion protein, but not a mutant that fails to interact with Cdk4/6, significantly blocked polyploidization of primary fetal liver derived megakaryocytes. Taken together, our data reveal that the process of endomitosis and cell growth relies heavily on cyclinD:Cdk4/6 kinase activity and that the maturation defects in GATA-1 deficient megakaryocytes are due, in part, to reduced Cyclin D1 and increase p16 expression.

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