P27 Kip1 Protein Research Articles

Urushiol Induces Apoptosis via a p53-dependent Pathway in Human Gastric Cancer Cells.

Background:Urushiols are mixtures of olefinic catechols which is isolated from the sap of Korean lacquer tree (Rhus vernicifera Stokes). The aim of this study was to determine the anticancer effects of urushiol in human gastric adenocarcinoma cell lines.Methods:The cytotoxicity of urushiols was assessed by MTT assays on the two gastric adenocarcinoma cell lines, MKN-45 (wild type of p53) and MKN-28 (mutant type of p53). We also examined the action mechanisms of urushiol by analyzing its effects on cell cycle progression and apoptosis induction.Results:The cytotoxic results from MTT assays indicated that urushiol inhibited human gastric cancer cell growth in a dose-dependent manner, with IC50 values of approximately 15 and 20 μg/ml on MKN-45 and MKN-28 cells, respectively. Urushiol mediated cell death on these two cancer cell lines through different pathways. Urushiol induced apoptosis on MKN-45 cells, concomitant with apoptotic nuclear change, DNA fragmentation, poly (ADP-ribose) polymerase cleavage and apoptotic body formation via extrinsic pathway of apoptosis. However, no apoptotic features were induced by urushiol treatment on MKN-28 cells. Urushiol induced cytostatic cell growth inhibition via upregulation of the cyclin-dependent kinase inhibitors, p21WAF1/CIP1 and p27KIP1 proteins and down-regulation of cyclin-dependent kinase 2 and 4 proteins in a p53-independent manner.Conclusions:These data provide evidence that urushiol has the potential to be used as a chemotherapeutic agent in human gastric cancer.

Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31±2% in human neuroblastoma SH-SY5Y cells, and up to 29±2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

Extracellular ATP regulates FoxO family of transcription factors and cell cycle progression through PI3K/Akt in MCF-7 cells

BackgroundForkhead Box-O (FoxO) transcription factors regulate the expression of many genes involved in suppression. Released nucleotides can regulate intracellular signaling pathways through membrane-bound purinergic receptors, to promote or prevent malignant cell transformation. We studied the role of extracellular ATP in the modulation of Forkhead Box O (FoxO) transcription factors and of cell cycle progression in MCF-7 breast cancer cells. MethodsWestern blot analysis, cell transfections with siRNA against Akt, immunocytochemistry, subcellular fractionation studies and flow cytometry analysis were performed. ResultsATP induced the phosphorylation of FoxO1/3a at threonine 24/32, whereas reduced the expression of FoxO1. In addition, ATP increased the expression of the cyclins D1 and D3 and down-regulated the cell cycle inhibitory proteins p21Cip1 and p27Kip1. The use of the phosphatidylinositol 3 kinase (PI3K) inhibitor, Ly294002, and/or of siRNA to reduce the expression of the serine/threonine kinase Akt showed that these effects are mediated by the PI3K/Akt signaling pathway. ATP induced the translocation of FoxO3a from the nucleus to the cytoplasm. Also, ATP increased the number of cells in the S phase of cell cycle; this effect was reverted by the use of Ly294002 and the proteasome inhibitor bortezomib. ConclusionExtracellular ATP induces the inactivation of FoxO transcription factors and cell cycle progression through the PI3K/Akt pathway in MCF-7 cells. General significanceThese findings provide new molecular basis for further understanding the mechanisms involved in ATP signal transduction in breast cancer cells, and should be considered for the development of effective breast cancer therapeutic strategies.

In vivo and in vitro demonstration of herb-drug interference in human breast cancer cells treated with tamoxifen and trastuzumab

In recent trends, patients with breast cancer seek integrative medical treatment when receiving either hormonal (tamoxifen [Tam]) or target (trastuzumab) therapy. Our previous in vitro studies demonstrated that the Chinese medicine Si-Wu-Tang (SWT) stimulates MCF-7 cell growth via activation of estrogen receptor α and human epidermal growth factor receptor 2 (HER2) signaling. The present study demonstrates herb-drug interference with cell proliferation in tumor-bearing mice treated with SWT and Tam in vivo and with proliferation capacity in breast cancer cells treated with SWT and trastuzumab in vitro. To assess in vivo SWT + Tam interference, we randomly separated female MCF-7-implanted athymic nude mice into five groups, namely, vehicle (n = 11), estradiol (n = 8), SWT (n = 8), Tam (n = 11), and SWT + Tam (n = 8). All mice were killed after 21 days of treatment. Body weight, uterine weight, tumor volume, and tumor weight were measured. To assess in vitro SWT-trastuzumab interference, we cotreated BT-474 and SK-BR-3 breast cancer cells with SWT and trastuzumab. This was followed by (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays and cell cycle analysis to measure cell proliferation and by Western blot analysis to analyze protein expression in growth-related signal pathways. SWT reversed Tam-induced antiproliferative effects on tumor weight and tumor volume and increased estrogen receptor α and N-cadherin expression in the SWT + Tam-treated group compared with the Tam-treated group. Furthermore, SWT reversed trastuzumab-induced antiproliferative activity in HER2 cell lines (SK-BR-3 and BT-474) through increased phosphorylation of the cell cycle regulatory protein p27(Kip1) and possibly of the antiapoptosis protein P38. Based on the in vivo and in vitro demonstration of herb-drug interference in breast cancer cells, we conclude that physicians should pay more attention to such interference when treating patients with receptor-positive (estrogen receptor-positive, progesterone receptor-positive, or HER2) breast cancers.

Dysregulation of the Mammalian Target of Rapamycin and p27Kip1 Promotes Intimal Hyperplasia in Diabetes Mellitus

The proliferation and migration of vascular smooth muscle cells (VSMCs) in the intima of an artery, known as intimal hyperplasia, is an important component of cardiovascular diseases. This is seen most clearly in the case of in-stent restenosis, where drug eluting stents are used to deliver agents that prevent VSMC proliferation and migration. One class of agents that are highly effective in the prevention of in-stent restenosis is the mammalian Target of Rapamycin (mTOR) inhibitors. Inhibition of mTOR blocks protein synthesis, cell cycle progression, and cell migration. Key to the effects on cell cycle progression and cell migration is the inhibition of mTOR-mediated degradation of p27Kip1 protein. p27Kip1 is a cyclin dependent kinase inhibitor that is elevated in quiescent VSMCs and inhibits the G1 to S phase transition and cell migration. Under normal conditions, vascular injury promotes degradation of p27Kip1 protein in an mTOR dependent manner. Recent reports from our lab suggest that in the presence of diabetes mellitus, elevation of extracellular signal response kinase activity may promote decreased p27Kip1 mRNA and produce a relative resistance to mTOR inhibition. Here we review these findings and their relevance to designing treatments for cardiovascular disease in the presence of diabetes mellitus.

Nuclear Expression of p27Kip1 Is Associated with In Vivo Differentiation of Adult Human Odontoblasts

IntroductionOdontoblasts are terminally differentiated cells of ectomesenchymal origin that produce the dentin. Differentiated odontoblasts cannot be identified yet by a single phenotypic marker protein; therefore, a combination of markers is currently used. Up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 has been associated with exit from the cell cycle and terminal differentiation of mammalian cells. Immunoreactivity for p27Kip1 protein was shown in many adult mouse tissues, but no information is available on the expression of p27Kip1 in mammalian dental pulp. MethodsHealthy and carious adult human molars with reparative dentin formation were decalcified, cryoprotected, frozen embedded, and frozen sectioned. The expression of p27Kip1 and nestin in cells of adult human dental pulp was analyzed by immunohistochemistry using free floating sections. Resultsp27Kip1 showed strong nuclear expression in many differentiated human molar odontoblasts at the odontoblastic layer. Most cells of the cell-rich zone displayed low levels of p27Kip1 despite the fact that preodontoblasts localized in the cell-rich zone of the subodontoblastic layer have been identified as quiescent cells. The nuclear expression of p27Kip1 in stromal cells of the dental pulp was variable, indicating that subpopulations of these cells were in distinct states of differentiation. Odontoblasts generating reparative dentin showed comparable nuclear expression of p27Kip1 in comparison with odontoblasts synthesizing primary/secondary dentin. This result indicates that odontoblasts synthesizing primary/secondary or reparative dentin exhibit a similar differentiation status. ConclusionsOur findings show that increased expression of nuclear p27Kip1 occurred during differentiation from preodontoblasts to odontoblasts in adult healthy and carious molars. p27Kip1 can be used as a novel nuclear marker protein for differentiated human odontoblasts in vivo.

C-Jun N-Terminal Kinase Phosphorylation Is a Biomarker of Plitidepsin Activity

Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin.

The expression of PML in chronic myeloid leukemia and effect on cell proliferation

Objective To investigate whether PML is expressed differently in chronic myeloid leukemia (CML) patients and healthy controls,then explore the effect of PML on proliferation in leukemia cell lines K562. Methods Real-time PCR was used to detect the PML expression in peripheral blood mononuclear cells from 30 chronic myeloid leukemia patients and 24 healthy controls.Plasmids containing full length PML were constructed and transfected into K562 cell line to screen clones stably expressing PML,the clones stably expressing empty vector as a control.MTT assay and flow cytometry were used to detect the effect of PML on K562 cell proliferation and cell cycle.Furthermore,RT-PCR and western blot were used to detect c-myc and p27kip1 (p27) mRNA and protein expression.Lastly,the mRNA levels of c-myc and p27 were detected in CML patients and healthy controls. Results The mRNA level of PML in CML patients (ΔCt=9.02±0.74) was significantly lower than the healthy controls (ΔCt=7.91±0.34) and the difference was statistically significant (t=5.07,P<0.001).PML over-expression could inhibit the proliferation of K562 cells obviously and cause G0/G1 cell cycle arrest.In addition,c-myc mRNA and protein expression decreased while p27 mRNA and protein expression increased in K562 cells with stably expressed PML compared with vector control.The mRNA level of c-myc in CML patients (ΔCt=7.13±0.43) was significantly higher than the healthy controls (ΔCt=9.35±0.82) and the difference was statistically significant (t=6.78,P<0.001),while the m RNA level of p27 in CML patients (ΔCt=4.56±0.58) was significantly lower than the healthy controls (ΔCt=3.29±0.92) and the difference was statistically significant (t=2.93,P<0.01). Conclusions PML protein is expressed highly in healthy controls,whereas its expression is declined significantly in chronic myeloid leukemia patients.PML may inhibit leukemia cell proliferation through regulating c-myc and p27 expression. (Chin J Lab Med,2013,36:395-399) Key words: Nuclear proteins; Transcription factors; Tumor suppressor proteins; Leukemia; myelogenous; chronic; BCR-ABL prositive; Cell proliferation

The influence of high glucose on the Cip/Kip family expression profiles in HRECs

Neovascularization is the main characteristic of the proliferative stage of diabetic retinopathy. It has been proven that cell cycle regulation is involved in angiogenesis. The cell cycle regulators, Cip/Kip protein family, belong to the cyclin-dependent kinase inhibitors, are versatile proteins, and except for their function in cell cycle regulation, they also participate in transcription, apoptosis and migration. The expression profiles of the Cip/Kip family in human retina microvascular endothelial cells (HRECs) under normal or high glucose conditions has not been described before. This study was undertaken to determine the expression profiles of the Cip/Kip family proteins, e.g., proteins which are influenced by high glucose and in what manner. Western blot and immunofluorescence analyses were used to investigate the protein expression profiles. Only p21(cip1) and p27(kip1) were detected in HRECs, and they were located in the nucleus. P21(cip1) protein abundance was higher than p27(kip1) in HRECs. Incubation of HRECs in medium containing 30 mM D-glucose for 48 h resulted in downregulation of p21(cip1) protein expression, but had no influence on p27(kip1) protein levels or p21(cip1) mRNA abundance. These results were accompanied by cell cycle G1 phase exit and a lower cell survival rate. Our data show for the first time that high glucose changes the Cip/Kip family expression profiles in HRECs, which may be the foundation for the investigation of the role of the Cip/Kip family in the pathogenesis of diabetic retinopathy.

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

STAT1 requirement for PKR-induced cell cycle arrest in vascular smooth muscle cells in response to heparin

Interferons (IFNs) are a family of cytokines that exhibit antiviral, antiproliferative, and immunomodulatory properties. PKR (protein kinase, RNA activated) is of central importance in mediating the antiproliferative actions of IFNs. Our research has established that PKR inhibits vascular smooth muscle cell (VSMC) proliferation by regulating G1 to S transition. Many cardiovascular diseases result from complications of atherosclerosis, a chronic and progressive inflammatory condition often characterized by excessive proliferation of VSMC. Thus, an effective method for inhibiting VSMC proliferation is likely to arrest atherosclerosis and restenosis at early stages. Our research establishes that PKR activation in VSMC leads to a G1 arrest brought about by an inhibition of cyclin-dependent kinase 2 (Cdk2) activity by p27kip1. In quiescent VSMC, p27kip1 levels are high and when stimulated by serum/growth factors, p27kip1 levels drop by destabilization of the protein. Under conditions that lead to activation of PKR, there is a marked inhibition of p27kip1 down-regulation due to increased stability of p27kip1 protein. In order to understand the mechanism of heparin-induced stabilization of p27kip1 in VSMC, we examined the involvement of the Signal Transducer and Activator of Transcription-1 (STAT1), which is an important player in mediating antiproliferative effects of IFNs. Our results demonstrate that PKR overexpression in VSMC leads to an increase in p27kip1 protein levels and this increase requires the catalytic activity of PKR. PKR activation induced by antiproliferative agent heparin leads to phosphorylation of STAT1 on serine 727, which is essential for the cell cycle block. STAT1 null VSMCs are largely defective in heparin-induced cell cycle arrest and in PKR null cells the STAT1 phosphorylation in response to heparin was absent. These results establish that heparin causes STAT1 phosphorylation on serine 727 via activation of PKR and that this event is required for the G1 arrest in VSMC.

Abstract 5433: Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells.

Abstract Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensitivity to paclitaxel. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) is the cardiac isoform of the most important glycolytic regulator, phosphofructokinase-2, From the TCGA database of &amp;gt;300 ovarian cancers, the PFKFB2 gene ranked in the top 33% of overexpressed genes and in the top 19% of genes with copy number gain. We found that PFKFB2 mRNA was upregulated in 10 of 12 ovarian cancer cell lines when expression of the gene was compared to that in normal ovarian epithelial cells in culture. Knockdown of PFKFB2 with siRNA markedly inhibited cell proliferation and increased paclitaxel sensitivity in p53 wild type ovarian cancer cell lines as demonstrated in short-term cytotoxicity and long-term clonogenic assays. Liposome encapsulated PFKFB2 siRNA significantly inhibited growth (P&amp;lt;0.05) of human ovarian cancer xenografts in two ovarian cancer models (Hey A8 and A2780); even greater growth inhibition was observed when PFKFB2 siRNA treatment was combined with paclitaxel. Cell growth inhibition in cell culture was linked to G1 arrest and to a modest increase in apoptosis. Induction of p21Cip1 and p27Kip1 protein by PFKFB2 knockdown contributed to the G1 arrest, since silencing p21Cip1 and p27Kip1 dramatically reduced PFKFB2 knockdown-induced G1 arrest. PFKFB2 knockdown also modulated the expression of CDK2 and the phosphorylation of p27Kip1. Induction of p53 protein contributed to both PFKFB2 knockdown-induced G1 arrest and to apoptosis, since silencing p53 markedly alleviated PFKFB2 knockdown-induced G1 arrest and apoptosis. PFKFB2 knockdown inhibited expression of the ABC transporter family members ABCC4 and ABCG2 at the level of transcription, possibly contributing to enhanced paclitaxel retention and increased cytotoxicity after PFKFB2 knockdown. Taken together, these results suggest that PFKFB2 protein may regulate cell growth and sensitivity to paclitaxel by multiple mechanisms, particularly in p53 wild type ovarian cancers. As methods to deliver siRNA evolve in the clinic, PFKFB2 may provide a novel target for enhancing sensitivity to paclitaxel-based chemotherapy in patients with low grade serous ovarian cancer where p53 function remains intact. Citation Format: Shu Zhang, Weiqun Mao, Anil K. Sood, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Zong-Fang Li, Robert C. Bast, Xiao-Feng Le. Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells. [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 5433. doi:10.1158/1538-7445.AM2013-5433

CDK1 interacts with RARγ and plays an important role in treatment response of acute myeloid leukemia

Alterations in cell cycle pathways and retinoic acid signaling are implicated in leukemogenesis. However, little is known about the roles of cyclin-dependent kinases (CDKs) in treatment response of leukemia. In this study, we observed that CDK1 expression was significantly higher in bone marrow from 42 patients with acute myeloid leukemia (AML) at recurrence than that at first diagnosis (p = 0.04). AML patients had higher level of nuclear CDK1 in their leukemic blasts tended to have poorer clinical outcome compared with those with lower levels. We showed that CDK1 function is required for all-trans retinoic acid (ATRA) to achieve the optimal effect in U-937 human leukemic cells. CDK1 modulates the levels of P27kip and AKT phosphorylation in response to ATRA treatment. Further, we show, for the first time, that RARγ in concert with ATRA regulates protein levels of CDK1 and its subcellular localization. The regulation of the subcellular content of CDK1 and RARγ by ATRA is an important process for achieving an effective response in treatment of leukemia. RARγ and CDK1 form a reciprocal regulatory circuit in the nucleus and influence the function and protein stability of each other and the level of P27kip protein. In addition, expression of wee1 kinase and Cdc25A/C phosphatases also coincide with CDK1 expression and its subcellular localization in response to ATRA treatment. Our study reveals a novel mechanism by which CDK1 and RARγ coordinate with ATRA to influence cell cycle progression and cellular differentiation.

The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression

Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F‐box protein) complex and ubiquitylates many proteins (p27kip1, p21Cip1, cyclin E) that regulate the G1/S transition. Previously we demonstrated that UbcH3 interferes with UbcH10 functions, an E2 involved in the cell cycle regulation, at the G2/M transition, by competing for Anaphase‐promoting complex (APC). In this work, we aim to determine the effects of UbcH10 on the function of UbcH3. We showed by in vitro degradation assays that addition of UbcH10 inhibited UbcH3‐catalyzed degradation of a typical SCF substrate, p27kip1, while it had no effect on the degradation of a non SCF substrate, such as á‐lactalbumin. The competition for the binding to the SCF was confirmed by GST pull‐down assays using GST‐Roc1, the RING subunit of SCF that normally interacts with UbcH3. The results showed that UbcH10 was able to interact with Roc1 and decreased UbcH3/Roc1 interaction. These data suggest that a balance between UbcH3 and UbcH10 levels are critical for controlling the cell cycle progression and thus highlight a novel mechanism for regulating the progress through the cell cycle.

Placental mesenchymal dysplasia: A rare clinicopathologic entity confused with molar pregnancy

Placental mesenchymal dysplasia (PMD) is a rare placental abnormality characterised by placentomegaly and grape-like vesicles resembling partial mole by ultrasonography, but in contrast to partial mole can co-exist with a viable fetus. Although the karyotype is normal, the fetus is at increased risk for intrauterine growth restriction, intrauterine fetal demise or perinatal death and Beckwith–Wiedemann syndrome. Prenatal diagnosis is difficult and the final diagnosis is usually achieved by postpartum histological examination of the placenta. We present two recent cases of placental mesenchymal dysplasia with poor obstetric outcome. One fetus presented with reduced growth parameters, while the other fetus showed hepatosplenomegaly and early hydropic changes that appear to be associated with Beckwith–Wiedemann syndrome. In this report, the clinico-pathological features of two cases of PMD are discussed and the differentiation from a partial mole is highlighted. This study also supports the utility of cytogenetic ploidy analysis and p57KIP2 protein staining in the evaluation of pregnancies with PMD.

Ubiquitin C-terminal hydrolase-L5 is required for high glucose-induced transforming growth factor-β receptor I expression and hypertrophy in mesangial cells

Transforming growth factor-β (TGF-β) is pivotal in the pathogenesis of diabetic nephropathy. Type 1 TGF-β receptor (TGF-βR1) is degraded by Smad7-dependent ubiquitination–proteasomal pathway, which is deubiquitinated by ubiquitin C-terminal hydrolase-L5 (UCHL5). Therefore, we studied the role of UCHL5 in high glucose (27.8mM)-induced TGF-βR1 protein expression in mouse mesangial (MES13) cells. UCHL5 short hairpin RNA (shRNA) was used to knock down UCHL5 while LY294002 and the dominant-negative p85 were used to inhibit phosphatidylinositol-3-kinase (PI3K). We found that high glucose increased phospho-Akt, TGF-βR1 mRNA and protein expression. High glucose also increased UCHL5 protein expression, which was attenuated by LY294002, the dominant-negative p85 and the dominant-negative CREB. High glucose-induced TGF-βR1 protein expression and TGF-βR1 protein deubiquitination were attenuated by UCHL5 shRNA. Additionally, high glucose-induced p21WAF1, fibronectin protein expression and cell hypertrophy were attenuated by UCHL5 shRNA. However, high glucose-induced TGF-βR1 mRNA, p27kip1 protein expression and growth inhibition were not affected by UCHL5 shRNA. Finally, glomerular UCHL5 and TGF-βR1 protein expression were increased in streptozotocin-diabetic rats at 8weeks. We conclude that PI3K-dependent UCHL5 is required for high glucose-induced TGF-βR1 protein expression in mesangial cells. UCHL5 is also required for high glucose-induced TGF-βR1 protein deubiquitination, p21WAF1 and fibronectin protein expression and cell hypertrophy.

Expression of SKP2 and Thr187 phosphorylated p27(kip1) proteins in human breast carcinoma and their prognostic significance

p27kip1(简称p27)是由Polyak等1发现的一种相对分子质量为27000的热稳定蛋白,是重要的细胞周期负性调控因子,其蛋白水平的表达及功能异常与肿瘤的发生、发展及预后密切相关.肿瘤细胞中存在多种转录后调控机制调节p27蛋白的表达,磷酸化调节是最重要的途径之一[2-3].S期激酶相关蛋白2 (S-phase kinase-associated protein 2,SKI2)能特异性识别p27的第187位苏氨酸(Thr187)磷酸化位点(P-p27Thr187),使其通过泛素-蛋白酶体途径降解.目前关于P-p27Thr187在乳腺癌中表达的研究甚少.我们检测了乳腺癌组织中SKP2及P-p27Thr187的表达,探讨两者的表达与乳腺癌临床病理特征的关系,及其对预后判断的意义。

The Diagnostic Value of p27 in Comparison to p57 in Differentiation Between Different Gestational Trophoblastic Diseases

The histologic features that permit the identification of complete mole (CM) and partial mole (PM) as well as hydropic abortion (HA) may be overlapping. The cyclin-dependent kinase inhibitor p57Kip2 protein (p57) has been included among the paternally imprinted genes in humans that commonly used for diagnosis of CM. P27 is one of the cell-cycle controlling molecules that may be involved in the proliferation, differentiation and oncogenesis of trophoblastic cells. The current study tried to test the diagnostic validity of several histopathological parameters together with p57 and p27 immunostaining in differentiation between different gestational trophoblastic diseases. The current study was carried out on 13 cases product of conception, 13 cases PM, 25 cases CM and 8 cases choriocarcinoma. Maximal villous diameter at 1.5 mm cut-off point was found to be the most reliable factor in discrimination between PM and product of conception followed by presence of villous cistern, p57 expression by extravillous cytotrophoblasts at 1.5% cut-off point and p27 expression by villous cytotrophoblasts at 25% cut-off point. P27 expression by stromal cells and total trophoblastic population at 7.5% cut-off point could discriminate between PM and CM. P57 and p27 are co-parallely expressed in non-molar as well as partial molar gestations, but they did not show this coordination in choriocarcinoma. This study demonstrated diagnostic values for several cut-off points for p57 and p27 in discrimination between different categories of gestational trophoblastic diseases. The co-expression of p27 and p57 by extravillous cytotrophoblasts and their positive correlation in non-molar gestations may indicate its suppressive role on the proliferation of these cells to provide them the capacity for differentiation and invasion.

In Vitro and in Vivo Anti-tumor Activity of miR-221/222 Inhibitors in Multiple Myeloma

A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/ NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.

DFMO/eflornithine inhibits migration and invasion downstream of MYCN and involves p27Kip1 activity in neuroblastoma

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.