Table of Contents

Volume 5, Issue 19

  October 1, 2006

  Pages 2179 - 2289

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EXTRA VIEWS

Open Access Article

Caveolae Internalization Regulates Integrin-Dependent Signaling Pathways

Asier Echarri and Miguel A. Del Pozo
Pages 2179 - 2182
http://dx.doi.org/10.4161/cc.5.19.3264
Abstract | PDF

Integrin-mediated adhesion regulates trafficking of cholesterol-enriched membrane microdomains (CEMM). Upon cell detachment from the extracellular matrix (ECM), CEMMs undergo rapid internalization and are cleared from the plasma membrane. This pathway regulates integrin-mediated Rac membrane targeting, allowing coupling of Rac to downstream effectors. Internalization of CEMMs is mediated by Dynamin-2, a regulator of caveolae dynamics, and caveolin-1, an essential caveolae coat protein. Translocation of tyrosine phosphorylated caveolin-1 from focal adhesions to caveolae upon cell detachment induces CEMM internalization. Notably, integrin-mediated regulation of Erk, phosphatidylinositol-3-OH kinase (PI3K) and Rac pathways is dependent on caveolin-1. These results describe a novel pathway in which integrins prevent downregulation of Erk, PI3K and Rac-dependent pathways by inhibiting caveolin-1-dependent endocytosis. This pathway define a novel molecular mechanism for regulated cell growth and tumor suppression by caveolin-1.

Open Access Article

Calmodulin-Mediated Cell Cycle Regulation: New Mechanisms for Old Observations

Jaehyun Choi and Mansoor Husain
Pages 2183 - 2186
http://dx.doi.org/10.4161/cc.5.19.3265
Abstract | PDF

The significance of divalent calcium ions (Ca2+) to cell cycle progression has been a subject of study for several decades, with a regulatory role for Ca2+ suggested in distinct cell types and multiple organisms. Our interest in proliferative vascular diseases led us to focus on mammalian vascular smooth muscle cells (VSMC) in particular, in which we and others had shown that a coordinate elevation in the intracellular free Ca2+ concentration is required for G1 to S phase cell cycle progression. However, the molecular basis for this Ca2+-sensitive cell cycle transition was not known. Our recent discovery of a functional protein-protein interaction between the late G1-active cyclin E1 and the major calcium signal-transducing factor Calmodulin (CaM) sheds new light on the mechanism(s) through which Ca2+ concentrations regulate cell cycle. Having identified a CaM-binding site on cyclin E1, our studies support a direct role for CaM in mediating Ca2+-sensitive cyclin E/cdk2 activity and G1 to S phase transitions in VSMC. The CaM binding site identified on cyclin E1 has a Kd for CaM consistent with that of known CaM-binding proteins, and is composed of a 22 amino acids N-terminal sequence that is highly conserved across several mammalian species. Deletion of this binding site abolished CaM binding and Ca2+-sensitive cyclin E/cdk2 activity. Here we provide our perspectives on the literature supporting a role for Ca2+ in cell cycle regulation, focusing on the evidence implicating CaM in this functionality, and discuss the potential for therapeutic modulation of CaM-dependent cell cycle machinery.

Open Access Article

ASPP2: A Gene that Controls Life and Death in Vivo

Virginie Vives, Elizabeth A. Slee and Xin Lu
Pages 2187 - 2190
http://dx.doi.org/10.4161/cc.5.19.3266
Abstract | PDF

The fundamental role of apoptosis in tumor prevention and the important role of p53 in this process are now universally recognized. Recently, several families of p53-binding proteins have been shown to influence p53’s decision to direct the cells either in the apoptotic pathway or in cell cycle arrest. Among them, the ASPP family specifically regulate p53-dependent apoptosis. Its member ASPP2 was discovered more than 10 years ago as a binding partner of p53 and its role as a positive regulator of p53 mediated apoptosis has been clearly established in vitro. However, its physiological importance in vivo has just emerged through the generation and characterisation of the ASPP2-deficient mice. We now know that ASPP2 is a haploinsufficient tumor suppressor and an important activator of p53 during mouse development and tumor suppression in vivo. ASPP2 might be a novel target for future cancer therapy.

Open Access Article

Myc Overexpression Enhances Apoptosis Induced by Small Molecules

Hao Mo, Marina Vita, Marianne Crespin and Marie Henriksson
Pages 2191 - 2194
http://dx.doi.org/10.4161/cc.5.19.3320
Abstract | PDF

The Myc oncogene is deregulated in a wide variety of human tumors hence the Myc pathway is regarded as an attractive target for tailored cancer treatment. We have recently identified two small molecules, MYRAs (Myc-pathway response agents), that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation. Here, we show that these compounds in addition have prominent effects in MYCN overexpressing neuroblastoma cells. A third compound, NSC308848, also induced apoptosis in Myc-overexpressing cells and inhibited Myc-induced cellular transformation. However, in contrast to the MYRAs, NSC308848 treatment resulted in decreased Myc protein levels and gave rise to inhibitory effects also on other transcription factors than Myc. Taken together, our findings suggest that these three small molecules can elicit a similar biological response by interfering with the Myc pathway at different levels.

Open Access Article

Cells That Trigger Fever

Andrej A. Romanovsky, Alexandre Steiner and Kiyoshi Matsumura
Pages 2195 - 2197
http://dx.doi.org/10.4161/cc.5.19.3321
Abstract | PDF

Bacterial lipopolysaccharide (LPS) is recognized by several receptors, including the toll-like receptor (TLR) 4, on various cells. Among many biological responses to LPS is fever, an often polyphasic rise in body temperature that is thought to be mediated by prostaglandin (PG) E2. Which receptors on which cells are linked to fever production is unknown. It is also unknown which cells produce PGE2 that triggers the earliest (first) phase of fever. Two recent studies from our group answer these questions. In the first one, we studied LPS-induced fever in mouse chimeras selectively lacking the TLR4 in hematopoietic or nonhematopoietic cells. We found that the first phase of fever is triggered via the TLR4 on hematopoietic cells. In the second study, we investigated LPS fever in rats. We found that the number of cells expressing cyclooxygenase (COX)-2, a PGE2-synthesizing enzyme, surged at the onset of fever in the lung and liver (but not in the brain), and that most of these cells were macrophages. Because LPS-induced PGE2 production in macrophages is TLR4-dependent, it is tempting to speculate that the TLR4-bearing, bone marrow-derived cells implicated in fever pathogenesis by the first study are the same as the COX-2-positive macrophages identified in the second study. Hence, pulmonary and hepatic macrophages that recognize LPS via the TLR4 and rapidly produce PGE2 are likely triggers of the fever response.




PERSPECTIVES

Open Access Article

Proliferation: the Most Prominent Predictor of Clinical Outcome in Breast Cancer

Christine Desmedt and Christos Sotiriou
Pages 2198 - 2202
http://dx.doi.org/10.4161/cc.5.19.3254
Abstract | PDF

We recently identified a gene expression cassette of 97 unique genes that were consistently differentially expressed between low and high grade breast carcinomas. The majority of these genes were overexpressed in high grade tumors and, as expected, they were associated with cell cycle progression and proliferation. Interestingly, by applying this gene expression cassette to several datasets, we demonstrated that intermediate grade tumors were composed of a mixture of well- and poorly- differentiated tumors with statistically distinct clinical outcome similar to those of low and high grade carcinomas. Furthermore, these proliferation-related genes appear to be a common denominator of several existing prognostic gene expression signatures. This recapitulates their prognostic power far beyond the estrogen receptor (ER) status and highlights the importance of proliferation genes in breast cancer biology. Importantly, their weight seems to be far more important in ER-positive than in ER-negative disease.

Open Access Article

Evidence That the ATR/Chk1 Pathway Maintains Normal Replication Fork Progression during Unperturbed S Phase

Eva Petermann and Keith W. Caldecott
Pages 2203 - 2209
http://dx.doi.org/10.4161/cc.5.19.3256
Abstract | PDF

If cells are treated with DNA damaging agents or inhibitors that interfere with ongoing DNA replication, the intra-S and S/M checkpoints delay progression through S phase and mitotic entry, respectively, to allow time for DNA repair and replication restart. In vertebrates, these checkpoint responses to replication blocks are largely mediated by the sensor kinase ATR and its major downstream effector kinase Chk1. Increasing evidence suggests that the ATR pathway is also vital in the absence of exogenous stresses, i.e. during “unperturbed” replication. Both ATR and Chk1 are essential proteins in vertebrates, and lack of components of the ATR/Chk1 pathway can result in impaired replication and spontaneous DNA damage. Here we give an overview of how the ATR/Chk1 pathway responds to exogenously blocked replication and then describe evidence for roles of this pathway during replication in an unperturbed S phase.

Open Access Article

Regulation of MAP Kinases by the VHR Dual-Specific Phosphatase – Implications for Cell Growth and Differentiation

Fabio Cerignoli, Souad Rahmouni, Ze’ev Ronai and Tomas Mustelin
Pages 2210 - 2215
http://dx.doi.org/10.4161/cc.5.19.3267
Abstract | PDF

Although it is well established that a transient activation of the mitogenactivated protein kinases Erk and Jnk is a crucial step in most growth promoting signaling pathways, it has also been demonstrated that a prolonged activation of these kinases can induce differentiation, cell cycle arrest, and cell senescence. We recently found that the expression of the 21-kDa human Vaccinia H1-related (VHR) dual-specific phosphatase fluctuates during cell cycle progression and affects Erk and Jnk activity in a cell cycle-dependent manner. Cells lacking VHR arrested at the G1/S and G2/M transitions of the cell cycle and exhibited senescence phenotypes. Cells lacking VHR upregulated p21Cip/Waf1 and downregulated many genes for cell cycle regulators, DNA replication, transcription, and mRNA processing. In the absence of VHR, the serum-induced activation of Jnk and Erk was further elevated and was required for the G1/S and G2/M blocks, which were attenuated upon Jnk and Erk inhibition. Collectively, VHR provides a long sought layer in the regulation of Jnk and Erk during cell cycle progression thereby contributing to cell cycle arrest, differentiation or senescence.

Open Access Article

MicroRNAs: A New Insight into Cancer Genome

Lin Zhang and George Coukos
Pages 2216 - 2219
http://dx.doi.org/10.4161/cc.5.19.3319
Abstract | PDF

Cancer is a disease involving multi-step dynamic changes in the genome. However, studies on cancer genome so far have focused most heavily on protein-coding genes, and our knowledge on alterations of the functional non-coding sequences in cancer is largely absent. MicroRNAs (miRNA) are ~22 nt non-coding RNAs, which regulate gene expression in a sequence-specific manner via translational inhibition or mRNA degradation. Mounting evidence is showing that miRNAs may play important roles in tumor development, and a better understanding of their alteration in cancer genome and oncogenic property should contribute to the diagnosis and treatment of cancer.

Open Access Article

Epigenetic Activation of Tumor Suppressor MicroRNAs in Human Cancer Cells

Yoshimasa Saito and Peter M. Jones
Pages 2220 - 2222
http://dx.doi.org/10.4161/cc.5.19.3340
Abstract | PDF

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous post-transcriptional silencers of target genes. miRNAs are expressed in a tissue specific manner and play important roles in cell proliferation, apoptosis, and differentiation during animal development. Recent studies have shown a distinct connection between aberrant expression of miRNAs and the development of cancer, suggesting that miRNAs can be potential therapeutic targets. Our recent study has shown that some miRNAs are controlled by epigenetic alterations such as DNA methylation and histone modification in human cancer cells. Activation of tumor suppressor miRNAs by chromatin modifying drugs may cause downregulation of target oncogenes and could be a novel strategy for the prevention and treatment of human cancer.




REPORTS

Open Access Article

Regulation and Targeting of Eg5, a Mitotic Motor Protein in Blast Crisis CML: Overcoming Imatinib Resistance

Bing Z. Carter, Duncan H. Mak, Yuexi Shi, Wendy D Schober, Rui-Yu Wang, Marina Konopleva, Erich Koller, Nicholas M. Dean and Michael Andreeff
Pages 2223 - 2229
http://dx.doi.org/10.4161/cc.5.19.3255
Abstract | PDF

Patients with blast crisis (BC) CML frequently become resistant to Imatinib, a Bcr-Abl tyrosine kinase-targeting agent. Eg5, a microtubule-associated motor protein has been described to be highly expressed in BC CML by microarray analysis (Nowicki et al, Oncogene 22:3952-3963, 2003). We investigated the regulation of Eg5 by Bcr-Abl tyrosine kinase and its potential as a therapeutic target in BC CML. Eg5 was highly expressed in all Philadelphia chromosome positive (Ph+) cell lines and BC CML patient samples. Inhibition of Bcr-Abl by Imatinib downregulated Eg5 expression in Imatinibsensitive KBM5 and HL-60p185 cells, but not in Imatinib-resistant KBM5-STI571, harboring a T315I mutation, and Bcr-Abl-negative HL-60 cells. Blocking Eg5 expression with antisense oligonucleotide (Eg5-ASO) or inhibiting its activity with the smallmolecule Eg5 inhibitor, S-trityl-L-cysteine induced G2/M cell cycle block and subsequent cell death in both Imatinib-sensitive and -resistant cells. Further, Eg5-ASO treatment of SCID mice harboring KBM5 cell xenografts significantly prolonged the median survival of the animals (p=0.03). Our findings suggest that Eg5 is downstream of and regulated by Bcr-Abl tyrosine kinase in Philadelphia chromosome positive cells. Inhibition of Eg5 expression or its activity blocks cell cycle progression and induces cell death independent of the cellular response to Imatinib. Therefore, Eg5 could be a potential therapeutic target for the treatment of BC CML, in particular Imatinib-resistant BC CML.

Open Access Article

Cyclin A1/Cdk2 is Sufficient but not Required for the Induction of Apoptosis in Early Xenopus laevis Embryos

Ayesha D. Carter, Brian N. Wroble and Jill C. Sible
Pages 2230 - 2236
http://dx.doi.org/10.4161/cc.5.19.3262
Abstract | PDF

The role of cyclin-dependent kinases in cell proliferation is well characterized, whereas their somewhat paradoxical role in catalyzing apoptosis is less understood. One Cdk complex implicated in both cell proliferation and cell death is cyclin A/Cdk2. During early embryonic development of Xenopus laevis, distinct isoforms of cyclin A are expressed at different times. From fertilization through gastrulation, cyclin A1 is the predominant isoform. Cyclin A1 dimerizes with Cdk2 but not Cdk1. In contrast, cyclin A2 is expressed at a low level until gastrulation, when it becomes the major A-type cyclin and associates with both Cdk1 and Cdk2. When Xenopus embryos are treated with ionizing radiation (IR) prior to the midblastula transition (MBT), cyclin A1 protein persists beyond the MBT and forms an active complex with Cdk2. During this window of cyclin A1/Cdk2 activity, the embryo undergoes apoptosis. To test the hypothesis that cyclin A1-associated activity is a mediator of apoptosis, cyclin A1 protein level and associated kinase activity were measured in embryos treated with aphidicolin to induce apoptosis. Both cyclin A1 content and associated kinase activity were sustained after the MBT as embryos underwent apoptosis. To determine whether cyclin A1/Cdk2 was sufficient to induce apoptosis, recombinant cyclin A1/Cdk2 complex was injected into single-celled embryos, which induced apoptosis after the MBT. However, morpholinos targeting translation of cyclins A1 and A2 did not block apoptosis in embryos treated with X-rays or aphidicolin. These data indicate that cyclin A1/Cdk2 is sufficient, but not required for apoptosis during early development.

Open Access Article

Dual PPARα/γ Ligand TZD18 Either Alone or in Combination with Imatinib Inhibits Proliferation and Induces Apoptosis of Human CML Cell Lines


Pages 2237 - 2243
http://dx.doi.org/10.4161/cc.5.19.3259
Abstract | PDF

Despite progress in the treatment of early-stage chronic myeloid leukemia (CML), the accelerated and blastic phases of CML still remain a therapeutic challenge. Persistence of BCR-ABL-positive (bcr-abl+) cells or secondary resistance during imatinib therapy frequently occurs. In this study, we investigated the activity of a novel dual ligand specific for peroxisome proliferator-activated receptor α and γ (PPARα/γ) against CML blast crisis cell lines. Exposure of these cell lines (K562, KU812 and KCL22) to TZD18 resulted in a growth inhibition in a dose- and time-dependent manner. This effect may not be mediated through PPARγ and/or PPARα activation, since antagonists of PPARg and/or PPARa could not reverse this inhibition. Western blotting analysis showed that expression of the cyclin dependent kinase inhibitor (CDKI) p27kip1 was enhanced, whereas levels of cyclin E, cyclin D2 and cyclin dependent kinase 2 (CDK-2) were decreased when these cells were treated with TZD18. Most interestingly, TZD18 synergistically enhanced the antiproliferative and pro-apoptotic effects of imatinib. Overall, our findings strongly suggest that TZD18, either alone or in combination with imatinib may be beneficial for the treatment of CML in myeloid blast crisis.

Open Access Article

E1B55K-Deleted Adenovirus (ONYX-015) Overrides G₁/S and G₂/M Checkpoints and Causes Mitotic Catastrophe and Endoreduplication in p53-Proficient Normal Cells

Gioia Cherubini, Tatiana Petouchoff, Milena Grossi, Stefania Piersanti, Enrico Cundari and Isabella Saggio
Pages 2244 - 2252
http://dx.doi.org/10.4161/cc.5.19.3263
Abstract | PDF

In order to take advantage of cell replication machinery, viruses have evolved complex strategies to override cell cycle checkpoints and force host cells into S phase. To do so, virus products must interfere not only with the basal cell cycle regulators, such as pRb or Mad2, but also with the main surveillance pathways such as those controlled by p53 and ATM. Recently, a number of defective viruses has been produced which, lacking the latter ability, are incapable of replicating in normal cells but should be able to grow and finally lyse those cells that, such as the tumor cells, have lost their surveillance mechanisms. A prototype of these oncolytic viruses is the E1B55K-defective Adenovirus ONYX-015, which was predicted to selectively replicate and kill p53-deficient cancer cells. We found that, despite wt p53 and notwithstanding the activation of the checkpoint regulators p53, ATM, and Mad2, ONYX-015 actively replicated in HUVEC cells. Furthermore, ONYX-015 replication induced a specific phenotype, which is distinct from that of the E4-deleted adenovirus dlE4 Ad5, although both viruses express the main regulatory region E1A. This phenotype includes overriding of the G1/S and G2/M checkpoints, over-expression of MAD2 and retardation of mitosis and accumulation of polyploid cells, suggesting the occurrence of alterations at the mitotic-spindle checkpoint and impairment of the post-mitotic checkpoint. Our data suggest that viral E1A and E4 region products can override all host cell-checkpoint response even at the presence of a full activation of the ATM/p53 pathway. Furthermore, the E4 region alone seems to act independently of the E1B55K virus product in impairing the ATM-dependent, p53-independent G2/M checkpoint since dlE4 Ad5-infected cells arrested in G2 while ONYX-015-infected cells did enter mitosis.

Open Access Article

Oligomerization of p53 Precedes its Association with Dynein and Nuclear Accumulation

Shana Trostel, Dan L. Sackett and Tito Fojo
Pages 2253 - 2259
http://dx.doi.org/10.4161/cc.5.19.3291
Abstract | PDF

Previous studies have identified several proteins that associate with microtubules and the dynein motor complex including p53, the glucocorticoid and the vitamin D receptors, and the APC (adenomatous polyposis coli) protein; but neither the residues important for this interaction nor the physical state of the proteins involved have been clarified. We observed in SN12C cells harboring a mutant p53 truncated at amino acid 336, impaired nuclear localization and impaired association with dynein. This finding was confirmed and extended by examining a series of truncated p53 proteins that identified residues 336 to 348 as crucial for association with dynein and nuclear transport. Point mutations identified the importance of residues involved in p53 oligomerization in this process, establishing a p53 oligomer as the cargo for dynein transport. The association of cytosolic p53 oligomers with dynein occurs independent of microtubules indicating that following this association, the p53/dynein complex then associates with microtubules and is transported to the peri-nuclear region. These studies suggest that mutations or modifications that affect p53 oligomerization not only interfere with DNA binding but also with its intracellular distribution. They also highlight the importance of an intact microtubule network in the trafficking of crucial cellular proteins.

Open Access Article

Condensin Function in Mitotic Nucleolar Segregation is Regulated by rDNA Transcription

Bi-Dar Wang, Pavel Butylin and Alexander Strunnikov
Pages 2260 - 2267
http://dx.doi.org/10.4161/cc.5.19.3292
Abstract | PDF

Chromosome condensation is established and maintained by the condensin complex. The mechanisms governing loading of condensin onto specific chromosomal sites remain unknown. To elucidate the molecular pathways that determine condensin binding to the nucleolar organizer, a key condensin binding site, we analyzed the properties of condensin-bound sites within the rDNA repeat in budding yeast and demonstrated that the bulk of mitotic condensin binding to rDNA is reduced or eliminated when Pol I transcription is elevated. Conversely, when Pol I transcription is repressed either by rapamycin treatment or by promoter shut-off, condensin binding to rDNA is increased. This novel potential role for constitutive and/or periodic repression of Pol I transcription in rDNA condensin loading is an important factor in determining the segregation proficiency of NOR-containing chromosomes.

Open Access Article

Aurora-A Kinase and Inhibitor-2 Regulate the Cyclin Threshold for Mitotic Entry in Xenopus Early Embryonic Cell Cycles

David L. Satinover, David L. Brautigan and P. Todd Stukenberg
Pages 2268 - 2274
http://dx.doi.org/10.4161/cc.5.19.3316
Abstract | PDF

The abrupt activation of CDK1 during mitotic entry requires suppression of CDK activity until a threshold concentration of cyclin B is synthesized, triggering the activation of a large pool of CDK. The cellular mechanisms that define the concentration of cyclin B at which the threshold occurs are unknown. Here we demonstrate that this threshold is regulated by Aurora-A kinase and phosphatase Inhibitor-2. In Xenopus CSF extracts that actively translate cyclin B1, immunodepletion of either endogenous xInhibitor-2 or endogenous xAurora-A caused delayed mitotic entry and normal timing was restored by addition of the respective recombinant proteins. Aurora-A depleted extracts also could be rescued by the addition of full-length xInhibitor-2, but not an xInhibitor-2 truncated of its PP1 binding motif. This demonstrates that inhibition of PP1 was required to compensate for the absence of Aurora-A. To test the hypothesis that the delays in mitotic entry in CSF extracts were due to increases in cyclin B thresholds, we employed interphase extracts, which are driven into mitosis by the addition of recombinant cyclin B in a non-linear (threshold) dose-response. Neutralization of endogenous xInhibitor-2 or xAurora with antibodies increased the cyclin B threshold concentration. Alternatively, the addition of exogenous Aurora-A or Inhibitor-2 lowered the concentration of cyclin B that triggered CDK activation. Because the cyclin B threshold could be raised or lowered by changing the amount of either Aurora-A or Inhibitor-2, the results demonstrate these regulatory proteins are involved in a signaling loop required to create the switching behavior characteristic of mitotic entry.

Open Access Article

The Role of Vpr in HIV-1 Disease Progression is Independent of Its G2 Arrest Induction Function

Maoyi Lai and Junjie Chen
Pages 2275 - 2280
http://dx.doi.org/10.4161/cc.5.19.3317
Abstract | PDF

Vpr (viral protein R) is a vital HIV-1 accessory protein with multiple functions in the viral life cycle, including nuclear import of preintegration complex, induction of apoptosis and G2 cell cycle arrest. The cell cycle perturbation activity of Vpr requires activation of the ATR (Ataxia-Telangiectasia and Rad3-related) pathway and the integrity of Vpr C-terminal motif that is crucial for chromatin binding. Recent studies also demonstrated Vpr as one of the viral factors that influence HIV disease progression, as mutations in Vpr were overrepresented in some cohorts of long-term nonprogressors (LTNP). The LTNP-associated mutations of Vpr are frequently observed in the C-terminal domain. This raises the question whether the LTNP phenotype of Vpr is the result of the loss its ability to induce G2 arrest. Here we report that the LTNP-associated mutants of Vpr function normally in the induction of G2 arrest. No defects in ATR activation and direct binding to chromatin are observed. These mutants also show similar levels of apoptosis induction as wild-type Vpr. These data differentiate the LTNP-associated mutations of Vpr with those defective in inducing G2 arrest. We propose that the G2 arrest function of Vpr is separated from the LTNP phenotype, and the role of Vpr in HIV disease progression may involve other functions of Vpr.

Open Access Article

p27Kip1 and Cyclin Dependent Kinase 2 Regulate Passage Through the Restriction Point

Masahiro Hitomi, Ke Yang, Yang Guo, Jonathan Fretthold, Jyoti Harwalkar and Dennis Stacey
Pages 2281 - 2289
http://dx.doi.org/10.4161/cc.5.19.3318
Abstract | PDF

When quiescent cells are stimulated to re-enter the cell cycle, growth factors are required only until the restriction point in G1 phase. After this point the cell no longer requires growth factors, proliferative signaling molecules, or even protein synthesis in order to initiate DNA synthesis, which starts several hours later. Consequently, understanding the molecular nature of the restriction point constitutes one of the major goals in studies of growth regulation. We recently demonstrated that p27Kip1 (p27) regulates passage through G1 phase in actively proliferating cultures, and initiated these studies to determine if it is also involved in passage through the restriction point following stimulation of quiescent cells. In support of this suggestion, we found that passage through the restriction point requires mitogen-dependent suppression of the high p27 levels normally present in quiescent cells. Moreover, as the culture progresses to mid-G1 phase, the proportion of cells that pass the restriction point is increased by artificial suppression of p27 levels, while this proportion is reduced by elevation of p27 levels. p27 performs this critical function by regulating the subsequent activating phosphorylation of cyclin dependent kinase (CDK)2, which we also show is necessary for and closely associated with the initiation of DNA synthesis. We conclude that the p27 expression level at mid-G1 phase determines when a cell passes through the restriction point, and does so by regulating subsequent CDK2 activation.