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1.  Vitamin E δ-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism 
PLoS ONE  2013;8(2):e52526.
Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G1 cell-cycle arrest and increased p27Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27Kip1 expression. To determine whether p27Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27Kip1 mRNA expression but not its protein degradation. p27Kip1 gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.
PMCID: PMC3564846  PMID: 23393547
2.  Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1 
Respiratory Research  2007;8(1):77.
Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.
We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.
Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.
Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.
PMCID: PMC2164950  PMID: 17974037
3.  p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes. 
Molecular Biology of the Cell  1997;8(9):1815-1827.
p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.
PMCID: PMC305739  PMID: 9307976
4.  Hes1 Directly Controls Cell Proliferation through the Transcriptional Repression of p27Kip1 
Molecular and Cellular Biology  2005;25(10):4262-4271.
A transcriptional regulator, Hes1, plays crucial roles in the control of differentiation and proliferation of neuronal, endocrine, and T-lymphocyte progenitors during development. Mechanisms for the regulation of cell proliferation by Hes1, however, remain to be verified. In embryonic carcinoma cells, endogenous Hes1 expression was repressed by retinoic acid in concord with enhanced p27Kip1 expression and cell cycle arrest. Conversely, conditional expression of a moderate but not maximal level of Hes1 in HeLa cells by a tetracycline-inducible system resulted in reduced p27Kip1 expression, which was attributed to decreased basal transcript rather than enhanced proteasomal degradation, with concomitant increases in the growth rate and saturation density. Hes1 induction repressed the promoter activity of a 5′ flanking basal enhancer region of p27Kip1 gene in a manner dependent on Hes1 expression levels, and this was mediated by its binding to class C sites in the promoter region. Finally, hypoplastic fetal thymi, as well as livers and brains of Hes1-deficient mice, showed significantly increased p27Kip1 transcripts compared with those of control littermates. These results have suggested that Hes1 directly contributes to the promotion of progenitor cell proliferation through transcriptional repression of a cyclin-dependent kinase inhibitor, p27Kip1.
PMCID: PMC1087711  PMID: 15870295
5.  A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition 
Biochemical Journal  2014;463(Pt 3):383-392.
p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.
An inverse relationship between p27Kip1 and EGFR expression in parental T24 human bladder cancer cells and various human cancer tissues was found. Depletion of p27Kip1 in cells markedly elevated EGFR expression through transcriptional repression of Egfr by p27Kip1 via the JNK/c-Jun cascade.
PMCID: PMC4209780  PMID: 25121353
bladder cancer; c-Jun N-terminal kinase (JNK)/c-Jun pathway; epidermal growth factor receptor (EGFR); p27Kip1; signal transduction pathway; AP-1, activator protein 1; BME, basal medium Eagle; CDK, cyclin-dependent kinase; DMEM, Dulbecco’s modified Eagle’s medium; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSF-1, heat-shock factor 1; Hsp, heat-shock protein; IHC, immunohistochemistry; JNK, c-Jun N-terminal kinase; MEF, mouse embryonic fibroblast; RT, reverse transcription; SP1, specificity protein 1
6.  A novel role for the cyclin-dependent kinase inhibitor p27Kip1 in angiotensin II–stimulated vascular smooth muscle cell hypertrophy 
Journal of Clinical Investigation  1999;104(6):815-823.
Angiotensin II (Ang II) has been shown to stimulate either hypertrophy or hyperplasia. We postulated that the differential response of vascular smooth muscle cells (VSMCs) to Ang II is mediated by the cyclin-dependent kinase (Cdk) inhibitor p27Kip1, which is abundant in quiescent cells and drops after serum stimulation. Ang II treatment (100 nM) of quiescent VSMCs led to upregulation of the cell-cycle regulatory proteins cyclin D1, Cdk2, proliferating cell nuclear antigen, and Cdk1. p27Kip1 levels, however, remained high, and the activation of the G1-phase Cdk2 was inhibited as the cells underwent hypertrophy. Overexpression of p27Kip1 cDNA inhibited serum-stimulated [3H]thymidine incorporation compared with control-transfected cells. This cell-cycle inhibition was associated with cellular hypertrophy, as reflected by an increase in the [3H]leucine/[3H]thymidine incorporation ratio and by an increase in forward-angle light scatter during flow cytometry at 48 hours after transfection. The role of p27Kip1 in modulating the hypertrophic response of VSMCs to Ang II was further tested by antisense oligodeoxynucleotide (ODN) inhibition of p27Kip1 expression. Ang II stimulated an increase in [3H]thymidine incorporation and the percentage of S-phase cells in antisense ODN–transfected cells but not in control ODN–transfected cells. We conclude that p27Kip1 plays a role in mediating VSMC hypertrophy. Ang II stimulation of quiescent cells in which p27Kip1 levels are high results in hypertrophy but promotes hyperplasia when levels of p27Kip1 are low, as in the presence of other growth factors.
PMCID: PMC408428  PMID: 10491417
7.  ErbB2 Potentiates Breast Tumor Proliferation through Modulation of p27Kip1-Cdk2 Complex Formation: Receptor Overexpression Does Not Determine Growth Dependency 
Molecular and Cellular Biology  2000;20(9):3210-3223.
Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G1 arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27Kip1, and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27Kip1 onto Cdk2 complexes, an event preceding increased p27Kip1 expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27Kip1 sequestration) and the loss of p27Kip1 from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27Kip1 redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27Kip1 protein levels remained constant. Antisense-mediated inhibition of p27Kip1 expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27Kip1 accumulation, p27Kip1 redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G1 block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27Kip1 sequestration proteins, thus deregulating the G1/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.
PMCID: PMC85615  PMID: 10757805
8.  A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype 
PLoS Genetics  2013;9(3):e1003350.
The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.
Author Summary
Gene expression can be modulated at different steps on the way from DNA to protein including control of transcription, translation, and post-translational modifications. An abnormality in the regulation of mRNA and protein expression is a hallmark of many human diseases, including cancer. In some eukaryotic genes translation can be influenced by small DNA sequences termed upstream open reading frames (uORFs). These elements located upstream to the gene start codon may either negatively influence the ability of the translational machinery to reinitiate translation of the main protein or, much less frequently, stimulate protein translation by enabling the ribosomes to bypass cis-acting inhibitory elements. CDKN1B, which encodes the cell cycle inhibitor p27KIP1, includes an uORF in its 5′UTR sequence. p27KIP1 expression is often reduced in cancer, and germline mutations have been identified in CDKN1B in patients affected with a syndrome (MEN4) characterized by varying combinations of tumors in endocrine glands. Here we show that a small deletion in the uORF upstream to CDKN1B reduces translation reinitiation efficiency, leading to underexpression of p27KIP1 and coinciding with tumorigenesis. This study describes a novel mechanism by which p27KIP1 could be underexpressed in human tumors. In addition, our data provide a new insight to the unique pathogenic potential of uORFs in human diseases.
PMCID: PMC3605397  PMID: 23555276
9.  MyoD regulates p57kip2 expression by interacting with a distant cis-element and modifying a higher order chromatin structure 
Nucleic Acids Research  2012;40(17):8266-8275.
The bHLH transcription factor MyoD, the prototypical master regulator of differentiation, directs a complex program of gene expression during skeletal myogenesis. The up-regulation of the cdk inhibitor p57kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57kip2 displays a highly specific expression pattern and is subject to a complex epigenetic control driving the imprinting of the paternal allele. However, the regulatory mechanisms governing its expression during development are still poorly understood. We have identified an unexpected mechanism by which MyoD regulates p57kip2 transcription in differentiating muscle cells. We show that the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We also show that differentiation-dependent regulation of p57kip2, while involving a region implicated in the imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings highlight a novel mechanism, involving the modification of higher order chromatin structures, by which MyoD regulates gene expression. Our results also suggest that chromatin folding mediated by KvDMR1 could account for the highly restricted expression of p57kip2 during development and, possibly, for its aberrant silencing in some pathologies.
PMCID: PMC3458561  PMID: 22740650
10.  CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6 
Cell Cycle  2012;11(24):4633-4641.
Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57Kip2. CSN6 associates with p57Kip2, and its overexpression can decrease the steady-state expression of p57Kip2; accordingly, CSN6 deficiency leads to p57Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57Kip2. Loss of Skp2 compromised CSN6-mediated p57Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57Kip2. CSN6’s negative impact on p57Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57Kip2 and result in promoted cancer cell growth.
PMCID: PMC3562308  PMID: 23187808
COP9; CSN6; Skp2; cell cycle; p57
11.  Differential effects of hepatocyte growth factor and keratinocyte growth factor on corneal epithelial cell cycle protein expression, cell survival, and growth 
Molecular Vision  2014;20:24-37.
Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are secreted in the cornea in response to injury. In this study, we investigated the HGF- and KGF-mediated effect on the expression of cell cycle and apoptosis controlling proteins, cell survival, and growth in the corneal epithelium to better understand the possible role of their signaling mechanisms in repairing epithelial injuries.
The cell survival capability of HGF and KGF in epithelial primary cultures was evaluated by using a staurosporine-induced apoptosis model. Apoptosis was quantified with image analysis following nuclear staining with Hoechst fluorescent dye and DNA laddering. Western immunoblotting was used to study the effect of growth factors on the expression of cell cycle- and apoptosis-regulating proteins.
HGF and KGF protected cells from apoptosis for a short duration (10 h), but only KGF exhibited cell survival capability and maintained cell growth for a longer period (24 h). The onset of apoptosis was accompanied by a significant increase in cell cycle inhibitor p27kip. HGF and KGF suppressed p27kip levels in the apoptosis environment; however, KGF- but not HGF-dependent downregulation in p27kip expression was sustained for a longer duration. Inhibition of phosphatidylinositol 3-kinase/Akt activation blocked HGF- and KGF-mediated control of p27kip expression. Further, when compared to HGF, the presence of KGF produced significant downregulation of p53 and poly(adenosine diphosphate-ribose) polymerase, the key proteins involved in apoptosis and blocked the degradation of G1/S cell cycle progression checkpoint protein retinoblastoma. HGF and KGF upregulated the levels of p21cip, cyclins A, D, and E and cyclin-dependent kinases (CDK2 and CDK4) as well, but the KGF-mediated effect on the expression of these molecules lasted longer.
Sustained effect of KGF on cell survival and proliferation could be attributed to its ability to inhibit p53, retinoblastoma, caspases, and p27kip functions in apoptosis and cell cycle arrest and promote the expression of cell cycle progressing molecules for longer duration. Designing therapeutic strategies targeting cell cycle control through KGF may be beneficial for repairing difficult-to-heal corneal epithelial injuries that require sustained growth and cell survival promoting signals.
PMCID: PMC3888494  PMID: 24426773
12.  DFMO/eflornithine inhibits migration and invasion downstream of MYCN and involves p27Kip1 activity in neuroblastoma 
International Journal of Oncology  2013;42(4):1219-1228.
Neuroblastoma (NB) is the most common extracranial pediatric tumor. NB patients over 18 months of age at the time of diagnosis are often in the later stages of the disease, present with widespread dissemination, and often possess MYCN tumor gene amplification. MYCN is a transcription factor that regulates the expression of a number of genes including ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Inhibiting ODC in NB cells produces many deleterious effects including G1 cell cycle arrest, inhibition of cell proliferation, and decreased tumor growth, making ODC a promising target for drug interference. DFMO treatment leads to the accumulation of the cyclin-dependent kinase inhibitor p27Kip1 protein and causes p27Kip1/Rb-coupled G1 cell cycle arrest in MYCN-amplified NB tumor cells through a process that involves p27Kip1 phosphorylation at residues Ser10 and Thr198. While p27Kip1 is well known for its role as a cyclin-dependent kinase inhibitor, recent studies have revealed a novel function of p27Kip1 as a regulator of cell migration and invasion. In the present study we found that p27Kip1 regulates the migration and invasion in NB and that these events are dependent on the state of phosphorylation of p27Kip1. DFMO treatments induced MYCN protein downregulation and phosphorylation of Akt/PKB (Ser473) and GSK3-β (Ser9), and polyamine supplementation alleviated the DFMO-induced effects. Importantly, we provide strong evidence that p27Kip1 mRNA correlates with clinical features and the survival probability of NB patients.
PMCID: PMC3622674  PMID: 23440295
DFMO; Kaplan-Meier survival plot; metastasis; MYCN; neuroblastoma; ornithine decarboxylase; polyamines; p27Kip
13.  Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells ▿ †  
Molecular and Cellular Biology  2010;30(21):5057-5070.
The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
PMCID: PMC2953065  PMID: 20805359
14.  A Receptor-Specific Function for Notch2 in Mediating Vascular Smooth Muscle Cell Growth Arrest Through p27kip1 
Circulation research  2013;113(8):10.1161/CIRCRESAHA.113.301272.
Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype.
Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared to uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMC.
Methods and Results
In human primary VSMC, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27kip1 were observed downstream of Jag-1/Notch2 signaling, and required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination and prolonged half-life of p27kip1. Jag-1/Notch2 signaling robustly decreased S-phase kinase associated protein (Skp2), an F-box protein that degrades p27kip1 during G1. Over expression of Skp2 prior to Notch activation by Jag-1 suppressed the induction of p27kip1. Additionally, increased Notch2 and p27kip1 expression was co-localized to the non-proliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen (PCNA), whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation.
We propose a receptor specific function for Notch2 in regulating Jag-1-induced p27kip1 expression and growth arrest in VSMC. During vascular remodeling, co-localization of Notch2 and p27kip1 to the non-proliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus Notch2 is a potential target for control of VSMC hyperplasia.
PMCID: PMC3882755  PMID: 23965337
Smooth muscle cell; Notch receptor; proliferation; neointima; neointimal hyerplasia
15.  Differential Regulation of P27Kip1 Expression by Mitogenic and Hypertrophic Factors 
The Journal of Cell Biology  2000;148(3):543-556.
Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G1 cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G1 phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27Kip1 expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27Kip1 protein. The time course of p27Kip1 decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27Kip1 synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-β1 by aortic SMC. These results identify p27Kip1 as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.
PMCID: PMC2174813  PMID: 10662779
growth factors; cell cycle; CDK inhibitors; gene expression; smooth muscle cells
16.  Patterns of p57Kip2 Expression in Embryonic Rat Brain Suggest Roles in Progenitor Cell Cycle Exit and Neuronal Differentiation 
Developmental neurobiology  2009;69(1):1-21.
In developing central nervous system, a variety of mechanisms couple cell cycle exit to differentiation during neurogenesis. The cyclin-dependent kinase (CDK) inhibitor p57Kip2 controls the transition from proliferation to differentiation in many tissues, but roles in developing brain remain uncertain. To characterize possible functions, we defined p57Kip2 protein expression in embryonic day (E) 12.5 to 20.5 rat brains using immunohistochemistry combined with markers of proliferation and differentiation. p57Kip2 was localized primarily in cell nuclei and positive cells formed two distinct patterns including wide dispersion and laminar aggregation that were brain region-specific. From E12.5 to E16.5, p57Kip2 expression was detected mainly in ventricular (VZ) and/or mantle zones of hippocampus, septum, basal ganglia, thalamus, hypothalamus, midbrain and spinal cord. After E18.5, p57Kip2 was detected in select regions undergoing differentiation. p57Kip2 expression was also compared to regional transcription factors, including Ngn2, Nkx2.1 and Pax6. Time course studies performed in diencephalon showed that p57Kip2 immunoreactivity co-localized with BrdU at 8 hr in nuclei exhibiting the wide dispersion pattern, whereas co-localization in the laminar pattern occurred only later. Moreover, p57Kip2 frequently co-localized with neuronal marker, β-III tubulin. Finally, we characterized relationships of p57Kip2 to CDK inhibitor p27Kip1: In proliferative regions, p57Kip2 expression preceded p27Kip1 as cells underwent differentiation, though the proteins co-localized in substantial numbers of cells, suggesting potentially related yet distinct functions of Cip/Kip family members during neurogenesis. Our observations that p57Kip2 exhibits nuclear expression as precursors exit the cell cycle and begin expressing neuronal characteristics suggests that the CDK inhibitor contributes to regulating the transition from proliferation to differentiation during brain development.
PMCID: PMC2967216  PMID: 18814313
Cyclin-Dependent Kinase Inhibitor p57Kip2; Embryonic Development/physiology; Nervous System/cytology/*embryology; Brain/embryology; Neuronal Differentiation
17.  The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist 
Cell Cycle  2011;10(9):1435-1447.
The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27Kip1 in hESC lead to a G1 phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27Kip1 protein occupies the Twist1 gene promoter and manipulation of p27Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.
PMCID: PMC3685623  PMID: 21478681
cell cycle; embryonic stem cell; induced pluripotent stem cell; self renewal; pluripotency; differentiation
18.  Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells 
PLoS ONE  2012;7(9):e44502.
The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.
PMCID: PMC3435274  PMID: 22970236
19.  A single-nucleotide polymorphism in the human p27kip1 gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction 
BMC Biology  2004;2:5.
Excessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27kip1 in the control of neointimal hyperplasia. For instance, p27kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke).
In this study we found three common single-nucleotide polymorphisms in the human p27kip1 gene (+326T>G [V109G], -79C>T, and -838C>A). The frequency of -838A carriers was significantly increased in myocardial infarction patients compared to healthy controls (odds ratio [OR] = 1.73, 95% confidence interval [95%CI] = 1.12–2.70). In addition, luciferase reporter constructs driven by the human p27kip1 gene promoter containing A at position -838 had decreased basal transcriptional activity when transiently transfected in Jurkat cells, compared with constructs bearing C in -838 (P = 0.04).
These data suggest that -838A is associated with reduced p27kip1 promoter activity and increased risk of myocardial infarction.
PMCID: PMC400507  PMID: 15061869
myocardial infarction; p27kip1; single-nucleotide polymorphisms
20.  Contact inhibition modulates intracellular levels of miR-223 in a p27kip1-dependent manner 
Oncotarget  2014;5(5):1185-1197.
MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression.
Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers.
Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression.
PMCID: PMC4012735  PMID: 24727437
p27kip1; miR-223; RNA binding; contact inhibition
21.  p27Kip1, a double-edged sword in Shh-mediated medulloblastoma 
Cell Cycle  2010;9(21):4307-4314.
Medulloblastoma, a brain tumor arising in the cerebellum, is the most common solid childhood malignancy. The current standard of care for medulloblastoma leaves survivors with life-long side effects. Gaining insight into mechanisms regulating transformation of medulloblastoma cells-of-origin may lead to development of better treatments for these tumors. Cerebellar granule neuron precursors (CGNPs) are proposed cells of origin for certain classes of medulloblastoma, specifically those marked by aberrant Sonic hedgehog (Shh) signaling pathway activation. CGNPs require signaling by Shh for proliferation during brain development. In mitogen-stimulated cells, nuclear localized cyclin-dependent kinase (Cdk) inhibitor p27Kip1 functions as a checkpoint control at the G1- to S-phase transition by inhibiting Cdk2. Recent studies have suggested that cytoplasmically localized p27Kip1 acquires oncogenic functions. Here, we show that p27Kip1 is cytoplasmically localized in CGNPs and mouse Shh-mediated medulloblastomas. Transgenic mice bearing an activating mutation in the Shh pathway and lacking one or both p27Kip1 alleles have accelerated tumor incidence compared to mice bearing both p27Kip1 alleles. Interestingly, mice heterozygous for p27Kip1 have decreased survival latency compared to p27Kip1-null animals. Our data indicate that this may reflect the requiremen of at least one copy of p27Kip1 for recruiting cyclin D/Cdk4/6 to promote cell cycle progression, yet insufficient expression in the heterozygous or null state to inhibit cyclin E/Cdk2. Finally, we find that mislocalized p27Kip1 may play a positive role in motility in medulloblastoma cells. Together, our data indicate that the dosage of p27Kip1 plays a role in cell cycle progression and tumor suppression in Shh-mediated medulloblastoma expansion.
PMCID: PMC3055184  PMID: 21051932
p27; Kip1; medulloblastoma; cerebellum; cell cycle; Sonic hedgehog; tumor; motility; RhoA
22.  Regulation of p27Kip1 by Sox2 maintains quiescence of inner pillar cells in the murine auditory sensory epithelium 
Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27Kip1, a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27Kip1, p27Kip1-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27Kip1 gradually declined with age. In addition, deletion of Sox2 or p27Kip1 did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27Kip1 promoter support that Sox2 directly activates p27Kip1 transcription in postmitotic IPCs. Hence, in contrast to the well-known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27Kip1 to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27Kip1 expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.
PMCID: PMC3427024  PMID: 22855803
23.  Regulation of p27Kip1 by Sox2 maintains quiescence of inner pillar cells in the murine auditory sensory epithelium 
Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27Kip1, a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27Kip1, p27Kip1-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27Kip1 gradually declined with age. In addition, deletion of Sox2 or p27Kip1 did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27Kip1 promoter support that Sox2 directly activates p27Kip1 transcription in postmitotic IPCs. Hence, in contrast to the well-known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27Kip1 to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27Kip1 expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.
PMCID: PMC3427024  PMID: 22855803
24.  Skp2 Regulates G2/M Progression in a p53-dependent Manner 
Molecular Biology of the Cell  2008;19(11):4602-4610.
Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27Kip1 degradation.
PMCID: PMC2575176  PMID: 18716061
25.  E Proteins and Id2 Converge on p57Kip2 To Regulate Cell Cycle in Neural Cells 
Molecular and Cellular Biology  2006;26(11):4351-4361.
A precise balance between proliferation and differentiation must be maintained during neural development to obtain the correct proportion of differentiated cell types in the adult nervous system. The basic helix-loop-helix (bHLH) transcription factors known as E proteins and their natural inhibitors, the Id proteins, control the timing of differentiation and terminal exit from the cell cycle. Here we show that progression into S phase of human neuroblastoma cells is prevented by E proteins and promoted by Id2. Cyclin-dependent kinase inhibitors (CKI) have been identified as key effectors of cell cycle arrest in differentiating cells. However, p57Kip2 is the only CKI that is absolutely required for normal development. Through the use of global gene expression analysis in neuroblastoma cells engineered to acutely express the E protein E47 and Id2, we find that p57Kip2 is a target of E47. Consistent with the role of Id proteins, Id2 prevents activation of p57Kip2 expression, and the retinoblastoma tumor suppressor protein, a known Id2 inhibitor, counters this activity. The strong E47-mediated inhibition of entry into S phase is entirely reversed in cells in which expression of p57Kip2 is silenced by RNA interference. During brain development, expression of p57Kip2 is opposite that of Id2. Our findings identify p57Kip2 as a functionally relevant target recruited by bHLH transcription factors to induce cell cycle arrest in developing neuroblasts and suggest that deregulated expression of Id proteins may be an epigenetic mechanism to silence expression of this CKI in neural tumors.
PMCID: PMC1489106  PMID: 16705184

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