P27 Kip1 Protein Expression Research Articles

Increased expression of Cks1 protein is associated with lymph node metastasis and poor prognosis in nasopharyngeal carcinoma

BackgroundThe Cks1 protein is an essential factor in regulating cell cycle by mediating the ubiquitination of CDK inhibitor p27kip1. It has been reported that aberrant expression of Cks1 and p27kip1 proteins was found in various tumors and related to initiation and progression of carcinomas. However, the potential roles which Cks1 and p27KIP1 proteins play in NPC remain unclear. This study aims to examine the expression status of Cks1 and p27kip1 and their possible prognostic significance in NPC.MethodsParaffin-embedded specimens with NPC (n = 168) and non-tumor nasopharyngeal tissues (n = 49) were analyzed by IHC.ResultsExpression of Cks1 increased in NPC tissues compared with non-tumor nasopharyngeal tissues (P < 0.05), whereas p27kip1 protein frequently expressed in non-tumor nasopharyngeal tissues compared with NPC tissues (P < 0.05). There was a significant reverse correlation between Cks1 and p27kip1 protein expression in NPC (r = −0.189, P < 0.05).In addition, Kaplan-Meier survival curve showed that there was a significant tendency of shorter overall survival (OS) in NPC patients with Cks1 positive expression compared to negative ones, especially in patients with lymph node metastasis (P < 0.001, respectively). But there was no significance between p27kip1 expression and survival viability of NPC patients. Multivariate Cox regression analysis further identified increased expression of Cks1 was the independent poor prognostic factor for NPC (p = 0.13).ConclusionOur research found expression of Cks1 increased and was inverse to the expression of p27KIP1. High expression of Cks1 was significantly associated with lymph node metastasis and survival status in NPC. In addition, the abnormally high level of Cks1 protein was proved to be an independent poor prognostic factor in NPC. These results may provide novel clue for NPC therapy method.

MiR-155 effectively induces apoptosis in K562 Philadelphia positive cell line through upregulation of p27kip1.

Introduction: Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia chromosome translocation, at (9; 22), which results in BCR-ABL fusion tyrosine kinase oncoprotein. This fusion induces down-regulation of miR-155. Upregulation of miR-155 can influence cell fate via the effect on p27kip1 and apoptosis. The aim of this study was to induce apoptosis in K562 CML cell line by overexpression of miR-155. Methods: The K562 cell line was transfected with pLenti-III-pre mir155-GFP constructs through electroporation. Then, overexpression of miR-155 as well as the expression level of p27kip1 and c-Myc was analyzed by quantitative PCR (qPCR). The level of p27 (Kip1) protein expression was measured by Western blot and the Annexin V method was carried out to investigate apoptosis. Results: Flow cytometric analysis results of K562 cells transfected with pLenti-III-pre mir155-GFP construct showed a significant increase in cell apoptosis. Gene expression and protein level of p27kip1 were upregulated. However, there was no change in c-Myc expression profile. Conclusion: miR-155 could be a promising approach to aid in the treatment of CML. However, further studies are required in this respect.

Correlation between cyclin-dependent kinase inhibitor p27kip1 and trastuzumab-resistance in gastric cancer

To investigate the correlation between cyclin-dependent kinase inhibitor p27kip1 and trastuzumab-resistance in gastric cancer. We selected HER2-overexpressed human gastric cancer cell line NCI-N87 to establish trastuzumab-resistant NCI-N87/TR cell line by stepwise exposure to different doses of trastuzumab. The 50% inhibitory concentration (IC(50)) of trastuzumab and resistance index (RI) were calculated or analyzed by MTT assay. The expression levels of cdk2 and p27kip1 were detected by Western blot. After the treatment with cdk2 inhibitor (Purvalanol A), the expression levels of relevant proteins in NCI-N87/TR cells were detected by Western blot, and the sensitivity to trastuzumab was analyzed by MTT assay. Compared with NCI-N87 cells, the expression of cdk2 was significantly increased in NCI-N87/TR cells (P<0.001), while the expression of p27kip1 showed a significant decrease (P<0.001). Restoration of the p27kip1 protein expression by cdk2 inhibitor (Purvalanol A) increased the sensitivity of NCI-N87/TR to trastuzumab. Down-regulation of p27kip1 might be a mechanism for triggering trastuzumab resistance to gastric cancer cell line NCI-N87.

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.

Effects of Rapamycin and Rapamycin-loaded Poly(lactic-co-glycolic)Acid Nanoparticles on Apoptosis and Expression of bcl-2 and p27(kip1) Proteins of Human Umbilical Arterial Vascular Smooth Muscle Cell

To investgate the effects of rapamycin(RPM)and RPM-loaded poly(lactic-co-glycolic)acid(PLGA)nanoparticles(NPs)on the apoptosis of human umbilical arterial vascular smooth muscle cells(HUASMCs)in vitro and expression of bcl-2 and p27(kip1) protein. HUASMCs were cultured in vitro and divided to RPM and RPM-PLGA-NPs groups treated at 3 different concentration by 12 and 24 hours,with M231-smooth muscle growth supplements medium and null-PLGA-NPs treated groups as controlled. The apoptosis of HUASMCs was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining and flow cytometry. The expressions of bcl-2 and p27(kip1) were detected by streptacidin/peroxidase immunohistochemical method. The effect on cellular proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidecolorimetry. The proliferation of HUASMCs was inhibited by RPM and RPM-PLGA-NPs in a dose-dependent manner. DNA electrophoresis showed DNA ladder in RPM and RPM-PLGA-NPs groups and classical scalar strips in control groups. The apoptotic indexes of RPM 100 ng/ml group and RPM-PLGA-NPs 500 ng/ml group detected by flow cytometry were(45.45<2.36)% and(35.04<5.64)%,respectively,which were significantly higher than that of M231-smooth muscle growth supplements control group [(2.60<0.95)%,all P<0.01]. The apoptotic indexes of groups incubated with RPM and RPM-PLGA-NPs for 24 hours were significantly higher than those of groups which incubated for 12 hours(P<0.05,P<0.01). The positive expression indexes(PEI)of p27(kip1) and bcl-2 protein were higher in RPM and RPM-PLGA-NPs groups than that of control groups. The Spearman's rank correlation coefficient test showed that there was no significant correlation between the PEI of p27(kip1) and the apoptotic indexes in the RPM group and RPM-PLGA-NPs group(P>0.05). Rapamycin-loaded PLGA nanoparticles and rapamycin have similar effects in inhibiting proliferation and inducing apoptosis;meanwhile,they upregulate the expression of p27(kip1) protein without downregulating the expression of bcl-2 protein in HUASMCs in vitro. RPM-PLGA-NPs has more potent pro-apoptotic effect than equivalent dose of RPM but is not linearly correlated with the p27(kip1) expression level.

Forkhead Box Transcription Factor (FOXO3a) mediates the cytotoxic effect of vernodalin in vitro and inhibits the breast tumor growth in vivo.

BackgroundNatural compounds have been demonstrated to lower breast cancer risk and sensitize tumor cells to anticancer therapies. Recently, we demonstrated that vernodalin (the active constituent of the medicinal herb Centratherum anthelminticum seeds) induces apoptosis in breast cancer cell-lines. The aim of this work was to gain an insight into the underlying anticancer mechanism of vernodalin using in vitro and in vivo model.MethodsVernodalin was isolated through the bioassay guided fractionation from the seeds. The protein expression of p-Akt, PI3K, FOXO3a, Bim, p27kip1, cyclinD1, and cyclinE was examined by the Western blot analysis. Immunoprecipitation assays were performed to analyse Akt kinase activity. Small interfering RNA (siRNA) was used to study the role of FOXO3a upregulation and their targets during vernodalin treatment. Immunofluorescence, subcellular localisation of FOXO3a by Western blot was performed to analyse FOXO3a localisation in nucleus of breast cancer cells. Immunohistochemical analysis of PCNA, Ki67, p27kip1, FOXO3a and p-FOXO3a in the LA7-induced mammary gland tumor model was performed.ResultsOur results showed that vernodalin regulates cancer cell apoptosis through activation of FOXO transcription factors and its downstream targets (Bim, p27Kip1, p21Waf1/cip1, cyclin D1, cyclin E) as examined by Western blots. Furthermore, we showed that FOXO3a/PI3K-Akt played a significant role in vernodalin induced apoptosis in breast cancer cells. Immunoprecipitation assays showed Akt kinase activity was downregulated. Immunofluorescence, subcellular fractionation and Western blot showed FOXO3a accumulation in the nucleus of breast cancer cells after vernodalin treatment. Silencing of FOXO3a protected breast cancer cells against vernodalin induced apoptosis. The anti-tumor action of vernodalin was further confirmed by examining cell proliferative markers, PCNA and Ki67 in the LA7-induced mammary gland tumor model. We also corroborated our findings in vivo by showing upregulation of p27Kip1, FOXO3a and decrease in the p-FOXO3a level in vernodalin-treated breast tumor tissue.ConclusionsOur results suggest that PI3K-Akt/FOXOa pathway is a critical mediator of vernodalin-induced cytotoxicity and this compound could be further developed as a potential chemopreventive or chemotherapeutic agent for breast cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0266-y) contains supplementary material, which is available to authorized users.

MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.

BackgroundOverexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.Methods and ResultssiRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.ConclusionsMARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

Antimicrobial Peptide, Lumbricusin, Ameliorates Motor Dysfunction and Dopaminergic Neurodegeneration in a Mouse Model of Parkinson's Disease.

We recently reported that the antimicrobial peptide Lumbricusin (NH2-RNRRWCIDQQA), isolated from the earthworm, increases cell proliferation in neuroblastoma SH-SY5Y cells. Here, we investigated whether Lumbricusin has neurotropic activity in mouse neural stem cells (MNSCs) and a protective effect in a mouse model of Parkinson's disease (PD). In MNSCs isolated from mouse brains, Lumbricusin treatment significantly increased cell proliferation (up to 12%) and reduced the protein expression of p27(Kip1) through proteasomal protein degradation but not transcriptional regulation. Lumbricusin inhibited the 6-OHDA-induced apoptosis of MNSCs, and also showed neuroprotective effects in a mouse PD model, ameliorating the motor impairments seen in the pole, elevated body swing, and rotation tests. These results suggest that the Lumbricusin-induced promotion of neural cell proliferation via p27(Kip1) degradation has a protective effect in an experimental PD model. Thus, the antimicrobial peptide Lumbricusin could possibly be developed as a potential therapeutic agent for the treatment of PD.

Expression of Skp2 and p27kip1 proteins in hypopharyngeal squamous cell carcinoma and its clinical significance.

The aim of the present study was to determine the effect of S-phase kinase-associated protein 2 (Skp2) and cyclin-dependent kinase inhibitor p27kinase-interacting protein 1 (p27kip1) protein expression on the occurrence and development of hypopharyngeal squamous cell carcinoma. The protein expression levels of Skp2 and p27kip1 were detected in 42 hypopharyngeal squamous cell carcinoma and 15 normal hypopharyngeal mucous membrane specimens using the an immunohistochemical PV-9000 two-step method. The expression levels of Skp2 protein were significantly different in hypopharyngeal squamous cell carcinomas and normal hypopharyngeal mucous membranes (61.90 vs. 26.67%; P<0.05). By contrast, the protein expression levels of Skp2 were significantly positively correlated with tumor T stage (rs=0.329, P<0.05) and cervical lymph node metastasis (rs=0.402, P<0.05). Furthermore, the expression levels of p27kip1 protein were significantly different in hypopharyngeal squamous cell carcinomas and normal hypopharyngeal mucous membranes (11.9 vs. 53.33%; P<0.05), while p27kip1 protein expression was significantly negatively correlated with tumor T-stage (rs=-0.351, P<0.05) and cervical lymph node metastasis (rs=-0.371, P<0.05). Notably, a significant negative correlation was observed between the expression levels of Skp2 and p27kip1 proteins in hypopharyngeal squamous cell carcinoma (P<0.05). In addition, abnormal expression levels of Skp2 and p27kip1 proteins were observed in hypopharyngeal squamous cell carcinoma tissues. Thus, Skp2 and p27kip1 proteins may be involved in the development of hypopharyngeal squamous cell carcinoma. The current study proposed that combined detection of Skp2 and p27kip1 may be useful for assessing the characteristics and prognosis of hypopharyngeal squamous cell carcinoma.

P27Kip1 signaling: Transcriptional and post-translational regulation

p27Kip1 is an inhibitor of a broad spectrum of cyclin-dependent kinases (CDKs), and the loss of a single p27Kip1 allele is thereby sufficient to increase tumor incidence via CDK-mediated cell cycle entry. As such, down-regulation of p27Kip1 protein levels, in particular nuclear expressed p27Kip1, is implicated in both disease progression and poor prognosis in a variety of cancers. p27Kip1 expression is positively regulated by the transcription factor MENIN, and inhibited by oncogenic transcription factors MYC and PIM. However, regulation of p27Kip1 protein expression and function is predominantly through post-translational modifications that alter both the cellular localization and the extent of E3 ubiquitin ligase-mediated degradation. Phosphorylation of p27Kip1 at Thr187 and Ser10 is a prerequisite for its degradation via the E3 ubiquitin ligases SKP2 (nuclear) and KPC (cytoplasmic), respectively. Additionally, Ser10 phosphorylated p27Kip1 is predominantly localized in the cytoplasm due to the nuclear export protein CRM1. Another E3 ubiquitin ligase, PIRH2, degrades p27Kip1 in both the cytoplasm and nucleus independent of phosphorylation state. As such, inhibition of cell cycle entry and progression in a variety of cancers may be achieved with therapies designed to correct p27Kip1 localization and/or block its degradation.

Abstract LB-251: p27Kip1 suppression of colitis-associated colon cancer mediated through cell intrinsic function in both mucosal epithelia and mucosal T cells

Abstract The cyclin-dependent kinase inhibitor p27Kip1 is a negative regulator of the cell cycle. Reduced expression of p27Kip1 has prognostic significance in several human epithelial cancers. Loss of p27Kip1 expression in cancer cells correlates with tumor aggressiveness, depth of tumor cell invasion and with lack of tumor cell differentiation. However, p27Kip1 also promotes apoptosis and contraction of effector CD4+ T cells, suggesting the potential that suppression of epithelial tumorigenesis may be linked to an intrinsic function of p27Kip1 in T cells. Here we show both mRNA and protein expression of p27Kip1 are significantly decreased in a murine model wherein a T cell-restricted deletion of the gene encoding the TGF-β intermediate, Smad4 (Smad4TKO), results in spontaneous colitis that invariably progresses to colon cancer by the age of 8 months. This phenotype is associated with a consistent decline in the mucosal expression of p27Kip1. To further investigate the role of p27Kip1 in the maintenance of intestinal mucosal homeostasis, we generated a ‘double knockout’ (Smad4TKO / p27Kip1-/- or ‘DKO’) mouse model in which a germ line p27Kip1 gene deletion (p27Kip1-/-) is combined with the T cell-restricted deletion of the Smad4 gene (Smad4TKO). All DKO mice develop spontaneous gastrointestinal inflammation and colon carcinoma by 1-2 months and 3 months of age, respectively. The p27Kip1 gene deletion, when combined with the T cell-restricted deletion of the Smad4 gene, leads to progressive inflammatory cytokine production (TNF-α, IFN-γ, iNOS, IL-6, IL-1β), activation of Stat1 and Stat3 and suppression of the expression of the tumor suppressor, 15-PGDH. Collectively our data suggest that p27Kip1 deficiency promotes gastrointestinal epithelial malignancy through both enhancement of proliferation of mucosal epithelial cells and through expansion of mucosal effector memory T cells. Citation Format: Sung Hee Choi, Byung-Gyu Kim, Letterio J. John. p27Kip1 suppression of colitis-associated colon cancer mediated through cell intrinsic function in both mucosal epithelia and mucosal T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-251. doi:10.1158/1538-7445.AM2014-LB-251

Activation of the receptor for advanced glycation end products induces nuclear inhibitor of protein phosphatase-1 suppression

The activation of the receptor for advanced glycation end products (RAGE) is involved in the development of diabetic nephropathy. Analysis of protein phosphatase-1 indicated that advanced glycation end products did not affect its expression, but increased its phosphatase activity. Using differential display analysis we previously demonstrated that stimulation of RAGE in podocytes modulates the expression of numerous genes, among others nuclear inhibitor of protein phosphatase-1 (NIPP1). Here we found that silencing of NIPP1 induced podocyte hypertrophy, cell cycle arrest, and significantly increased protein phosphatase-1 activity. NIPP1 downregulation was associated with increased p27(Kip1) protein expression. Reporter assays revealed a transcriptional activation of nuclear factor-κB in podocytes after suppression of NIPP1. The protein level of NIPP1 was also significantly reduced in podocytes of diabetic mice. Blocking the RAGE in vivo by a soluble analog elevated the NIPP1 protein in podocytes of diabetic mice. Thus, activation of the RAGE by advanced glycation end products or other ligands suppresses NIPP1 expression in diabetic nephropathy, contributes to podocyte hypertrophy, and glomerular inflammation.

Effects of melittin on the cell cycle of bladder cancer cell line T24 and its mechanism

Objective To investigate the effects of melittin on the cell cycle of bladder cancer cell line T24 and the related regulatory mechanisms.Methods In vitro cultured bladder cancer T24 cells were treated with melittin (0,7.5,10.0,and 12.5 mg/L).Flow cytometry was applied to examine the cell cycle of T24 cells.Western blotting method was applied to detect the protein expression of Cyclin A,cyclindependent kinase 2 (CDK2),CDK inhibitor p21 cip1 and cyclin dependent kinase inhibitor protein 27 (p27kip1).Results Corresponding to different concentration groups of melittin,the proportion of T24 cells in S phase was (25.73 ±2.11)％,(31.57 ±1.91)％,(45.37±0.74)％,and (53.67±1.12)％,showing significant difference between melittin-treated groups and control group (P ＜ 0.01).The protein expression of p21cip1 and p27kip1 in melittin-treated T24 cells was increased (p21cip1:0.156 1 ±0.011 6,0.209 2 ±0.010 8,0.287 9 ±0.012 9,0.351 8 ±0.013 0;p27kip1:0.250 3 ±0.012 3,0.342 1 ±0.012 3,0.400 3 ±0.009 9,0.469 2 ±0.013 0),while that of Cyclin A and CDK2 significantly decreased as compared with control group (Cyclin A:0.950 7 ± 0.017 8,0.870 6 ± 0.011 9,0.806 5 ± 0.012 0,0.726 7 ± 0.018 4;CDK2:0.965 4 ±0.014 5,0.868 3 ±0.015 3,0.755 4 ±0.008 5,0.652 6 ±0.018 3,P ＜0.01).Conclusion Melittin can cause S phase arrest of T24 cells,which may be associated with the down-regulation of Cyclin A and CDK2 expression while up-regulation of p21cip1 and p27kip1. Key words: Bladder neoplasm; Melittin; Cell cycle

Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221

MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a oligoarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR-221. In addition, the effects of R8-PNA-a221 on p27(Kip1) (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27(Kip1) mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27(Kip1) mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27(Kip1) protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR-221 might be proposed for experimental treatment of human gliomas.

C-myc Regulates Cell Proliferation during Lens Development

Myc protooncogenes play important roles in the regulation of cell proliferation, growth, differentiation and survival during development. In various developing organs, c-myc has been shown to control the expression of cell cycle regulators and its misregulated expression is detected in many human tumors. Here, we show that c-myc gene (Myc) is highly expressed in developing mouse lens. Targeted deletion of c-myc gene from head surface ectoderm dramatically impaired ocular organogenesis, resulting in severe microphtalmia, defective anterior segment development, formation of a lens stalk and/or aphakia. In particular, lenses lacking c-myc presented thinner epithelial cell layer and growth impairment that was detectable soon after its inactivation. Defective development of c-myc-null lens was not caused by increased cell death of lens progenitor cells. Instead, c-myc loss reduced cell proliferation, what was associated with an ectopic expression of Prox1 and p27Kip1 proteins within epithelial cells. Interestingly, a sharp decrease in the expression of the forkhead box transcription factor Foxe3 was also observed following c-myc inactivation. These data represent the first description of the physiological roles played by a Myc family member in mouse lens development. Our findings support the conclusion that c-myc regulates the proliferation of lens epithelial cells in vivo and may, directly or indirectly, modulate the expression of classical cell cycle regulators in developing mouse lens.

Mechanisms in cardiac fibroblast growth: an obligate role for Skp2 and FOXO3a in ERK1/2 MAPK-dependent regulation of p27kip1

Cardiac fibroblast hyperplasia associated with enhanced matrix deposition is a major determinant of tissue remodeling in several disease states of the heart. However, mechanisms controlling cell cycle progression in cardiac fibroblasts remain unexplored. Identification of cell cycle regulatory elements in these cells is important to develop strategies to check adverse cardiac remodeling under pathological conditions. This study sought to probe the mechanisms underlying ERK1/2-mediated p27(Kip1) regulation in mitogenically stimulated cardiac fibroblasts. Addition of 10% fetal calf serum to quiescent cultures of adult rat cardiac fibroblasts promoted ERK1/2 activation, as evidenced by its phosphorylation status. Reduction in [(3)H]thymidine incorporation into DNA increased population doubling time, flow cytometry, and Western blot analysis showing reduced levels of cyclins D and A, p27(Kip1) induction, and retinoblastoma protein (Rb) hypophosphorylation in ERK1/2-inhibited cells indicated ERK1/2 dependence of G1-S transition in cardiac fibroblasts. Lack of p27(Kip1) protein in serum-stimulated, ERK1/2-active cells was associated with increased levels of Skp2, an E3 ubiquitin ligase for p27(Kip1), whose knockdown by RNA interference induced p27(Kip1) expression. Further, forced expression of Skp2 in ERK1/2-inhibited cells downregulated p27(Kip1). Transcriptional upregulation of p27(Kip1) mRNA in ERK1/2-inhibited cells, demonstrated by real-time PCR, correlated with forkhead box O 3a (FOXO3a) transcription factor activation, shown by gel shift assay. FOXO3a knockdown attenuated p27(Kip1) mRNA and protein expression in ERK1/2-inhibited cells. We provide evidence for the first time that, in cardiac fibroblasts, activated ERK1/2 regulates p27(Kip1) expression transcriptionally and posttranslationally via FOXO3a- and Skp2-dependent mechanisms. Additionally, this study uncovers interesting interactions between critical cell cycle regulatory elements that are only beginning to be understood.

In Vitro and In Vivo Effects of Xanthorrhizol on Human Breast Cancer MCF-7 Cells Treated With Tamoxifen

This study investigated the herb-drug interaction of xanthorrhizol and tamoxifen in human breast cancer cells. Using MCF-7 cell line as an in vitro model, the herb-drug interaction between xanthorrhizol and tamoxifen was measured by MTT assay, luciferase reporter assay, and cell cycle analysis. The effects of xanthorrhizol on growth/autophagy related signaling were determined by immunostaining, western blotting, and real time RT-PCR. Additionally, the in vivo effect of xanthorrhizol and tamoxifen on athymic nude mice implanted with MCF-7 cells was evaluated. When MCF-7 cells were co-treated with tamoxifen and xanthorrhizol, there were no significant changes in terms of cell number, luciferase activity, percentage S-phase cells and LC3-II expression. However, using the MCF-7 implanted nude mice model, it was possible to detect significantly increased tumor volumes, a larger tumor size, and increased protein expression of P38 and P27(Kip1) in the xanthorrhizol + tamoxifen group compared to the tamoxifen-alone group. It can be concluded that while there is no significant herb-drug interaction between xanthorrhizol and tamoxifen in vitro, there is such an interaction in tumor-bearing mice, which provides important information that affects breast cancer treatment translational research.

Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway.

The far-upstream element-binding protein-interacting repressor (FIR) is a c-myc transcriptional suppressor. FIR is alternatively spliced to lack the transcriptional repression domain within exon 2 (FIRΔexon2) in colorectal cancers. FIR and FIRΔexon2 form homo- or heterodimers that complex with SAP155. SAP155, a subunit of the essential splicing factor 3b subcomplex in the spliceosome, is required for proper P27Kip1 pre-mRNA splicing, and P27Kip1 arrests cells at G1. In contrast, FIR was co-immunoprecipitated with Ku86 and DNA-PKcs. siRNA against Ku86/Ku70 decreased FIR and P27Kip1 expression, whereas siRNA against FIR decreased Ku86/XRCC5 and P27Kip1 expression. Thus the mechanical interaction of FIR/FIRΔexon2/SAP155 bridges c-myc and P27Kip1 expression, potentially integrates cell-cycle progression and c-myc transcription in cell. Bleomycin(BLM) is an anticancer agent that introduces DNA breaks. Because DNA breaks generate the recruitment of Ku86/Ku70 to bind to the broken DNA ends, the possible involvement of FIR and Ku86/Ku70 interaction in the BLM-induced DNA damage repair response was investigated in this study. First, BLM treatment reduced SAP155 expression and increased FIR and FIRΔexon2 mRNA expression as well as the ratio of FIRΔexon2:FIR in hepatoblastoma cells (HLE and HLF). Second, FIR or FIRΔexon2 adenovirus vectors (Ad-FIR or Ad-FIRΔexon2) increased Ku86/Ku70 and P27Kip1 expression in vitro. Third, BLM decreased P27Kip1 protein expression, whereas increased P27Kip1 and γH2AX expression with Ad-FIRΔexon2. Together, the interaction of FIR/SAP155 modulates FIR splicing and involves in cell-cycle control or cell fate via P27Kip1 and c-myc in BLM-induced DNA damage pathway. This novel function of FIR splicing will contribute to clinical studies of cancer management through elucidating the mechanical interaction of FIR/FIRΔexon2/SAP155 as a potential target for cancer treatment.

Effects of platelet rich plasma and platelet lysate on proliferation of rat bone marrow mesenchymal stem cells: a comparative study

Objective To compare the effects of platelet rich plasma(PRP)and platelet lysate(PL)on proliferation and cell cycle of cultured rat bone marrow stem cells(BMSCs).Methods BMSCs were obtained from twenty 4-week-old SD rats using the whole bone marrow isolation and cultivation method and were identified with flow cytometry.Blood samples were taken from the hearts of thirty 12-week-old SD rats and gradient centrifugation was used to prepare PRP,and PL was obtained after three times of centrifugation and repeated freezing and thawing.The third generation of BMSCs with good growth state were divided into seven groups according to different culture media:ordinary complete medium(A group),1% PRPconditioned medium(B group),1% PL-conditioned medium(C group),5% PRP-conditioned medium(D group),5% PLconditioned medium(E group),10% PRP-conditioned medium(F group),and 10% PL-conditioned medium(G group). The proliferation of BMSCs was assessed by MTT assay.The PCNA protein expression was assessed by immunofluorescence method.Cell cycle of BMSCs was tested by flow cytometry.Western blotting analysis was used to analyze CyclinD1and p27 Kip1protein expression of BMSCs.Results The proliferation of BMSCs was significantly promoted by 1%,5%,10% PRPand 1%,5%,10% PL-conditioned media after cultured for 24h,48h,and 72hin a time-and dose-dependent manner(P 0.05),with the effect of PRP and PL being similar at the same concentration(P0.05).Immunofluorescence assay showed that PRP and PL both significantly promoted PCNA protein expression in a dose-dependent manner(P0.01).Flow cytometry showed that different concentrations of PRP- and PL-conditioned media,compared with the ordinary complete medium,resulted in gradually reduced cells in G0/G1 phase,gradually increased cells in S,G2 /M phase,and significantly increased PI(P0.05),with the changes in a dose-dependent manner.However,the effects of the same concentration of PRP and PL on cell cycle were not significantly different(P0.05).Western blotting analysis showed that 5% PRP and 5% PL significantly up-regulated CyclinD1and down-regulated the expression of p27Kip1.Conclusion Different concentrations of PL and PRP can accelerate cell cycle progression of cultured rat BMSCs by up-regulation CyclinD1and inhibiting p27Kip1, promoting the proliferation of BMSCs in a time-and dose-dependent manner.PL and PRP at the same concentration have the same proliferation-promoting effect,indicating that PL may be used as an alternative of PRP to promote the repair of bone defects.

Abstract 5353: EZH2-mediated, but not BMI-1, epigenetic alteration may be associated with the accelerated cell proliferation and malignant step in IPMN.

Abstract Backgrounds. Histologic examination of IPMNs shows the co-existence of benign and malignant lesions within a single specimen of the pancreas represented by multifocality. Thus, IPMNs of pancreas are characterized by multifocal and malignant potential represented by an adenomacarcinoma sequence, whereas its molecular mechanism of oncogenesis remains largely unknown. Recently, epigenetic gene silencing in cancer is now increasingly recognized as a novel therapeutic target. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is associated with transcriptional repression. B-lymphoma moloney murine leukemia virus insertion-1 (BMI-1). is a proto-oncogene that belongs to polycom group gene family. The role of epigenetic alteration mediated by EZH2 and BMI-1 in IPMNs progression is unclear. Method. From 2002 to 2011, 54 patients with IPMN underwent curative pancreatic resection. The total 133 IPMN lesions and 48 normal duct epithelial lesions within IPMN lesion were analyzed in this study. In the present study, a total of 133 IPMN lesions were classified as either benign (adenoma and borderline tumors)or malignant (carcinoma in situ or invasive cancer). Result. Fifty-four cases of IPMNs consists of 23 IPMC (91 lesions) and 31 IPMA (90 lesions). We assessed to a proliferative activity in IPMNs by Ki-67 immunostainig. The number of Ki-67 positive cell was significantly increased during the progression of IPMNs. In malignant IPMN, Ki-67 positive cell was significantly increased compared with that in benign IPMN. Thus, the increased proliferative activity plays an important role in an adenoma-carcinoma sequence of IPMNs. The expression of cell-cycle related suppressor protein, p27Kip1 expression was inversely down-regulated during the progression of IPMNs. To further assess the molecular mechanism of the downregulated p27KIP1 expression during the progression of IPMNs, we focused the epigenetic epigenetic gene silencing during the tumor progression. The EZH2 expression was increased step by step during the progression of the IPMNs. In benign IPMN (IPMA), EZH2-negative cells expressed p27KIP1 protein. In contrast, in malignant IPMN (CIS lesion), EZH2-positive cells unexpressed p27KIP1 protein, suggesting that the increased EZH2 expression during the progression of IPMNs suppress the p27KIP1 protein expression. Furthermore, EZH2 expression level in IPMNs showed the significantly positive correlation with the Ki-67 positive nuclear ratio (p&amp;lt;0.0001). However, there was no significant association between BMI-1 expression and Ki-67 positive nuclear ratio. Conclusion. Here, we show EZH2-mediated, but not BMI-1, epigenetic alteration as a novel oncogenetic mechanism in IPMNs. EZH2-mediated epigenetic alteration may be associated with the accelerated cell proliferation and malignant step in IPMN via a downregulation of p27KIP1. Citation Format: Hideyuki Kuroki. EZH2-mediated, but not BMI-1, epigenetic alteration may be associated with the accelerated cell proliferation and malignant step in IPMN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5353. doi:10.1158/1538-7445.AM2013-5353