The forkhead box proteins (FOXO proteins) comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation, differentiation and longevity. Recently, ubiquitination and proteasome degradation of FOXO3a have been reported. In this study, we investigated the role of FOXO3a and Skp2 in human ovarian cancer. We detected the expression of FOXO3a and Skp2 in ovarian cancer by immunohistochemistry (IHC) and analyzed the relationship of FOXO3a and Skp2 with clinicopathological parameters, including prognosis. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 46 specimens in vivo. We found that the expression of FOXO3a was negatively related to Skp2 expression (r = -0.743; p < 0.05) and FOXO3a expression correlates significantly with disease stage (p = 0.007) and lymph node (p = 0.009) while Skp2 expression correlates significantly with age (p = 0.040), disease stage (p = 0.003) and lymph node (p = 0.019). Kaplan-Meier analysis revealed that survival curves of low versus high expressers of FOXO3a and Skp2 showed a highly significant separation in ovarian cancer (p < 0.01). FOXO3a and Skp2 may be considered to be important prognoses in human ovarian cancer.

Forkhead box class O 3a (FOXO3a) is an important direct target of the phosphatidylinositol 3-kinase (PI3K)/protein B(Akt) pathway, mediating signal transduction in regulating cell survival and cell-cycle progression. Recent reports have shown that FOXO3a inhibits cell-cycle progression at the G1/S transition by controlling transcription of the cyclin-dependent kinase inhibitor p27 kip1 , which is frequently down-regulated in human cancers, including human glioma. In this study we investigated the status of FOXO3a expression and related signaling in human glioma in order to test its potential value as a therapeutic target for this disease. Immunohistochemistry, western blot, RT-PCR, and immunofluorescence staining analysis were performed on specimens from 70 cases of human glioma and on U87MG and T98G glioma cells. Our data showed FOXO3a expression is directly correlated with the malignant grade of glioma. More importantly, low expression of FOXO3a was associated with poor patient outcome. In vitro, FOXO3a modulated the cell cycle by transcriptional regulation of p27 kip1 . Administration of the PI3K pharmacological inhibitor LY294002 abrogated this effect by regulating FOXO3a expression and subcellular localization. Our results suggested that FOXO3a may be a favorable independent prognostic indicator of glioma. Gene therapeutic approaches aimed at PI3K or at pharmacological inhibitors of PI3K to down-regulate P-FOXO3a expression could be developed for management of glioma.

The p27(Kip1) cyclin-dependent kinase inhibitor is a negative regulator of cell cycle progression in G(1) phase; recent studies suggested that oncogenically activated kinase Akt/PKB can also phosphorylate p27(kip1) at T157 inducing its relocalization to the cytoplasm. To evaluate the significance of p-p27 Thr157 and PI3K pathway in hepatocellular carcinoma (HCC), we studied 51 hepatocellular carcinomas along with corresponding nontumoral tissue and the HCC cell lines. Immunohistochemistry and western blot analysis suggested that p-p27 Thr157 was overexpressed in HCC, which was positively correlated with proliferation marker Ki-67. Correlation analysis was performed among immunohistochemistry-assessed level of p-p27 Thr157, survival, and major clinical and pathological variables. Overexpressed p-p27 Thr157 was correlated with histological differentiation (P < 0.05). Univariate analysis showed that p-p27 Thr157 and Ki-67 expression were correlated with tumor-specific survival. In a multivariate analysis, p-p27 Thr157 and Ki-67 protein expression were proved to be an independent prognostic for HCC. While in vitro, treatment of LY294002 and transduction of mutant p27 (T157A) could diminish the expression of p-p27 Thr157 protein and arrest cells growth. Our results suggested that p-p27 Thr157 protein expression may be a favorable independent poor prognostic parameter for HCC. Gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K and transduction of mutant p27 (T157A) to down-regulate p-p27 Thr157 expression could be developed for the management of HCC.

OBJECTIVE:Gliomas are the most common type of primary intracranial tumor. Although tumor grade predicts the clinical course of most patients, molecular characteristics of individual tumors have emerged as important prognostic factors for patients with gliomas. Reduced expression of p27 protein is known as an independent prognostic marker in a large variety of cancers and is associated with an unfavorable prognosis. It is believed that phosphorylation of p27 on Ser10 has been shown to be required for the binding of CRM1, a carrier protein for nuclear export. This study assessed whether CRM1, Ser10-phosphorylated p27, and p27 correlated with each other, with glioma pathological stage, and with patient outcome.METHODS:Immunohistochemical and Western blot analysis were performed in 70 cases of human gliomas and normal brain tissues. Survival analyses were performed using the Kaplan-Meier method.RESULTS:High CRM1 expression (80% of cancer cell nuclei stained) was observed in 70 specimens and was related to the grade of malignancy. A strong inverse correlation was evident between p27 levels and both Ser10-phosphorylated p27 (P < 0.001) and CRM1 level (P < 0.001). We also reviewed each grade of tumors separately and investigated whether CRM1 expression predicted patient survival within each subgroup. In brief, CRM1 overexpression was significantly associated with overall survival (P < 0.001).CONCLUSION:The current results showed that CRM1 and p27 expression were associated with glioma grade and that high CRM1 protein expression might be related to poor outcome.

To investigate whether Foxo3a expression is correlated with p27kip1 protein levels as well as how it might be clinically relevant, we evaluated the expression of Foxo3a in several ovarian tumors. Immunohistochemical analysis was performed in 63 cases of ovarian tumors. Ten cases were evaluated by Western blot analysis. There was a correlation observed between Foxo3a over-expression and clinic pathological parameters (p = 0.032). Kaplan-Meier survival analysis showed that Foxo3a low expression was significantly associated with poor prognosis of patients. It may be a useful prognostic marker and target in ovarian cancer.

The forkhead box proteins (FOXO proteins) comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation, differentiation and longevity. Recently, ubiquitination and proteasome degradation of FOXO3a have been reported. In this study, we investigated the role of FOXO3a and Skp2 in human hepatocellular carcinoma progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 91 specimens. Furthermore in vitro, western-blot analysis and protein stabilization studies were used to study the relationship between FOXO3a and Skp2. We found that the expression of FOXO3a was negatively related with Skp2 expression (r = -0.583; p < 0.05) and FOXO3a expression correlated significantly with histological grade (p = 0.000), cirrhosis (p = 0.015), and tumor size (p = 0.043) while Skp2 expression correlated significantly with histological grade (p = 0.000) and tumor size (p = 0.005). Kaplan-Meier analysis revealed that survival curves of low versus high expressers of FOXO3a and Skp2 showed a highly significant separation in HCC (p < 0.01). Our results suggested that FOXO3a and Skp2 may be considered to be important prognosis in human hepatocellular carcinoma. In vitro studies suggested that the degradation of FOXO3a may dependent on the expression of Skp2 in the proliferated Huh7 cells.

Purpose The aim of the present study was to examine whether Jab1 expression is correlated with p27 protein and its phosphorylation status as well as how it might be clinically relevant in epithelial ovarian carcinoma. Using ovarian carcinoma cell line HO-8910 to confirm and extend the findings. Methods Immunohistochemical and Western blot analysis were done in 70 cases of epithelial ovarian cacinoma and HO-8910 cells. Results Jab1 overexpression was detected in 84.3% (59 of 70) of malignant tumors and 31.6% (6 of 19) of benign tumors. A positive correlation between Jab1 and cytoplasmic p27 as well as Ser10 phosphorylated p27 was found in malignant ovarian tumors. In addition, patients displaying overexpression of Jab1, cytoplasmic p27 and Ser10 phosphorylated p27 were significantly associated with unfavorable clinicopathologic variables. Transfection with Jab1 in HO-8910 cells resulted in decreased p27 expression and this reduction was sensitive to 26S proteasome inhibitors. Overexpression of Jab1 caused p27 nuclear export and dissociate from Cdk2/Cyclin complex. What is more, increased expression of a phosphorylated histone H1 in the immune-complex obtained from Jab1 transfected HO-8910 cells was also observed. Conclusions Jab1, as a negative regulator of p27, may be associated with the progression and prognosis of epithelial ovarian tumors.

Objective: Recent evidences suggest that Jun activation domain-binding protein 1(Jab1) specifically interacts with the Cdk inhibitor p27kip1 and induces nuclear export and subsequent degradation of p27kip1. The purpose of this study is to investigate whether Jab1 expression is correlated with p27kip1 level in Non-Hodgkin's Lymphomas(NHLs) and how it influenced the stability of p27kip1, as well as whether Jab1 expression is associated with clinicopathologic variables and prognosis of NHLs.Methods: Immunohistochemical and/or Western blot analysis was done in 116 cases of NHLs and Jurkat cells.Results: Immunohistochemical analysis showed that Jab1 expression was negatively associated with p27kip1 level and significantly associated with unfavorable clinicopathologic variables. Overexpression Jab1 in lymphoma cell lines Jurkat resulted in decreased p27kip1 level and advanced progression from G1 to S phase of the cell cycle. Subcellular fractionation confirmed Jab1 could lead to nuclear export of p27kip1. Phosphorylation of p27kip1 at Ser10 and Thr157 was significantly increased after Jab1 transient transfected, while Thr187 phosphorylation was decreased. Immunoprecipitation demonstrated that Jab1 overexpression facilitated p27kip1 to dissociate from Cdk2 and associated with Cdk4. Finally, Survival analysis revealed that Jab1 overexpression is significantly associated with overall survival (P=0.000). When Jab1 and p27kip1 are combined, patients with Jab1(+)/p27kip1(-) revealed poorer overall survival (P=0.001). Multivariate analysis revealed that Jab1 protein is an independent prognostic indicator for overall survival.Conclusions: These findings suggest that Jab1 protein may contribute to the tumor progression through Jab1-mediated p27kip1 degradation and that control of Jab1 expression is a novel therapeutic target in patients with NHLs.

Objective To investigate the expression and relationship of P27kip1 with nuclear export factor CRM1 in lymphoma cells. Methods Cells were synchronized by serum starvation, and 10%serum stimulating proliferation. The expression of P27kip1 and CRM1 was detected by cell fractionation and Western blot. Results The albumen amount of P27kip1 increased while albumen amount of CRM1 was decreased. The reverse changes happened after serum release. The location of P27kip1 was accorded with the expression of CRM1. P27kip1 and CRM1 could form compound in Jurkat and Raji cells as detected by immunoprecipitation. Conclusion CRM1 may influence the location and expression of P27kip1 through integrating with P27kip1, and then may participate in regulating the growth of lymphoma cells.

Human hepatocellular carcinoma (HCC) remains incurable with current therapies, and novel biologically based therapies are urgently needed. Arsenic agents have long been used as anticancer agents in traditional Chinese medicine. In this study, to evaluate the effect of As(2)O(3) on HCC cells, we investigate cell growth inhibition, cell cycle arrest, and the molecular mechanism after As(2)O(3) treatment in human HCC cells in vitro. We detected the proliferation of HCC cells by the Cell Counting Kit and FACS/Calibur Flow Cytometer and analyzed the expression and localization of FOXO3a by Western blotting Analysis and Cell Fractionation. Furthermore, we study the Akt activation after As(2)O(3) treatment and the HCC cells proliferation after combination of As(2)O(3) with PI3K inhibitor Wortmannin. As(2)O(3) significantly inhibited the proliferation of all the three HCC cell lines (SMMC7721, HepG2, Hep3B) tested in this study in a dose-dependent manner. Western blotting revealed that treatment HCC cells HepG2 with As(2)O(3) resulted in the increasing of FOXO3a expression and triggered phosphorylation of FOXO3a at the Thr(32) residue decrease. This FOXO3a accumulation correlated well with the As(2)O(3)-induced reduction of active Akt. Nuclear and cytoplasmic protein extracts isolated from the HCC cell line HepG2 revealed that the amount of nuclear FOXO3a was increased by treatment with As(2)O(3), whereas the amount of cytoplasmic FOXO3a was decreased. Both As(2)O(3) and PI3K/Akt inhibitor Wortmannin induced cell cycle arrest. However, compared with As(2)O(3) alone, PI3K inhibitor Wortmannin combined with As(2)O(3) enhanced the antitumor effect of As(2)O(3) through induction of apoptosis. These findings suggest that As(2)O(3) at a clinically safe concentration may be an effective chemotherapeutic agent, and that As(2)O(3) and PI3K/Akt inhibitor Wortmannin may synergize for HCC cells. Taken together, the present study may suggest a specific molecular mechanism by which HCC cell lines are susceptible to the As(2)O(3) therapy through FOXO3a expression and localization.

Objective To investigate the expression of the phosphorylated P27kip1 and its correlation with Non-Hodgkin's lymphomas.Methods The expressions of the phosphorylated P27kip1 was detected in lymph nodes by immunohistochemical technique.Results The average labeling indexs(LIs) of phosphorylated P27kip1 in NHLs were significantly higher than those in benign lymph nodes(outside of the germinal center).The expression of phosphorylated P27kip1 in aggressive lymphoma was higher than that in indolent group;and the expression of phosphorylated P27kip1 was significantly correlated with expression of Ki-67.The expression of Thr187 phosphorylated P27kip1 was significantly correlated with expression of P27kip1 while the Ser10 phosphorylated P27kip1 negtively correlated with the expression of P27kip1.Conclusion The high expression of phosphorylated P27kip1 plays key roles in the occurrence and development of NHL;phosphorylated P27kip1 LI and P27kip1 LI are potential parameters to determine the malignance of NHL.

OBJECTIVE:Cell cycle arrest by FOXO transcription factors involves in transcriptional activation of p27(kip1), although the exact mechanism remains unclear. And it has been evidenced that reduced level of p27(kip1) which is frequently occurred in human cancers has been associated with poor prognosis. In this study, our purpose is to investigate the clinical relevance of altered patterns of Foxo3a and p27(kip1) expression in Chinese patients with localized non-Hodgkin's lymphomas (NHL).METHODS:We analyzed the Foxo3a and p27(kip1) expression of Chinese NHL patients by immunohistochemistry and protein levels using Western Blot.RESULTS:There was a direct relationship between the low level of Foxo3a and the rapid proliferation in immunohistochemical analyses. We also found a positive correlation between Foxo3a and p27(kip1) in immunohistochemical analyses and cell culture. Additionally we revealed that activation of Foxo3a could induce the accumulation of p27(kip1) at protein levels when cell cycle was arrested.CONCLUSIONS:The expression of Foxo3a may be correlated with patients' survival.

研究发现,p27kip1为细胞周期重要调控蛋白,其水平及功能异常参与了非霍奇金淋巴瘤(NHL)的发生发展.S期激酶相关蛋白2(S-phase kinase-associated protein 2,Skp2)能够特异性识别第187位苏氨酸(Thr187)磷酸化的p27kip1,并介导其多聚泛素化及降解,这一过程的异常直接影响细胞内p27kip1蛋白的绝对含量,并可能与NHL的发病机制有关[1-2].我们检测了与p27kip1降解相关的Skp2及Thr187磷酸化p27kip1蛋白在NHL中的表达,以探讨二者的表达异常与NHL发生发展的关系.

OBJECTIVE:To study the effect of all-trans retinoic acid (ATRA) on U937 cell growth and its mechanism.METHODS:Cell cycle was detected by flow cytometry (FCM), expressions of cell cycle associated protein and the p27 related protein were detected by Western blot. The binding of P27 and Skp2 was detected by immunoprecipitation.RESULTS:FCM displayed that ATRA could inhibit the proliferation of U937 cells. At 72 h on 1 micromol/L ATRA treatment, 72% of the cells were arrested at G0/G1 phase. Western blot displayed that ATRA could decrease the expression of cyclin A, up-regulate the expression of p21 and p27, and down-regulate the expression of p27 related proteins Skp2. p27 could bind with Skp2 in U937 cells as detected by immunoprecipitation.CONCLUSION:ATRA may arrest the proliferation of U937 cells through the reduction of Skp2 expression, and finally the induction of the accumulation of p27.

Background. The cyclin-dependent kinase (cdk) inhibitor p27(Kip1) is an important regulator of cell cycle progression as it negatively regulates G(0/1) progression and plays a major role in controlling the cell cycle. The screening of the p27(Kip1) sequence identified many potential phosphorylation sites. To investigate the effects of the overexpression of exogenous p27(Kip1) protein lacking the Thr157 sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27(Kip1) at Thr157 T157A p27), and transfected into the SMMC7721 cell line with Lipofectamine. Wildtype and mutant p27 plasmids T157A were transfected separately as control groups.Methods. We detected the proliferation of SMMC7721 cells by the Cell Counting Kit and FACS/Calibur Flow Cytometer and analyzed the expression and localization of p27(Kip1) by Western blotting analysis and cell fractionation. The cdk2 dependent kinase activity was determined by in vitro kinase assay.Results. Proliferation of SMMC7721 cells was greatly inhibited and cell cycle was arrested in G(0/1) phase after exogenous p27(Kip1) mutant expression much more than wild-type p27(Kip1). The expressed T157A p27(Kip1) proteins were translocated from the cytoplasm into nucleus much more compare with wild-type. Compared with pcDNA3.1-Myc control, transient transfection of T157A p27(Kip1) decreased expression of cyclin D1 and the phosphorylated form of retinoblastoma protein.Conclusions. These findings support the potential effectiveness of a PI3 K/Akt-resistant phosphorylated form of p27 in hepatocellular carcinoma gene therapy. (c) 2008 IMSS. Published by Elsevier Inc.

Objective To investigate the expression variation and significance of Skp2 and p27kip1 during the proliferation of lymphoma cell line Jurkat cells. Methods The binding of p27kip1 and Skp2 in Jurkat cells were detected by immunoprecipitation. Jurkat cells were treated with serum starvation and release synchronization. The expression variation and subcellular localization of p27kip1 and Skp2 were detected by subcellular fractionation, Western blot and double immunofluorescence labelling. Results The results of immunoprecipitation suggested that p27kip1 and Skp2 could bind each other in Jurkat cells. During the proliferation of Jurkat cells, the protein expression of p27kip1 decreased and intranuclear p27kip1 decreased significantly, while the Skp2 protein increased and cytoplasmic Skp2 increased significantly. Conclusion During the proliferation of Jurkat cells, the increased cytoplasmic synthesis of Skp2 may speed up p27kip1 degradation via the ubiquitin-proteasome pathway, then intranuclear p27kip1 decreases significantly, leading to an increased cell cycling activity.

Objective To investigate the expression and relationship of P27(superscript kip1) with nuclear export factor CRM1 in lymphoma cells. Methods Cells were synchronized by serum starvation, and 10% serum stimulating proliferation. The expression of P27(superscript kip1) and CRM1 was detected by cell fractionation and Western blot. Results The albumen amount of P27(superscript kip1) increased while albumen amount of CRM1 was decreased. The reverse changes happened after serum release. The location of P27(superscript kip1) was accorded with the expression of CRM1. P27(superscript kip1) and CRM1 could form compound in Jurkat and Raji cells as detected by immunoprecipitation. Conclusion CRM1 may influence the location and expression of P27(superscript kip1) through integrating with P27(superscript kip1), and then may participate in regulating the growth of lymphoma cells.

OBJECTIVE:To investigate the expression and relationship of p27(kip1) and its nuclear export factor Jab1 during proliferation process of lymphoma cell.METHODS:Jurkat and Raji cells were treated with serum starvation and then serum release. The protein and mRNA expression of p27(kip1), Jab1 in the cells were detected by Western blot and RT-PCR respectively. LMB were used for stimulating Jurkat cells during their proliferation process, and then the expression changes of p27(kip1) and Jab1 were detected. An eukaryotic expression plasmid(pcDNA3. 1-myc) containing Jab1 was constructed. Jurkat cell were transfected in vitro with or without pcDNA3. 1-myc-Jab1. Double immunolabelling was used to identify the localization of p27(kip1). Immunoprecipitation was used to detect the combination of p27(kip1) and Jab1.RESULTS:The growth of Jurkat and Raji cells were blocked by serum starvation. The total protein amount of p27(kip1) increased while that of Jab1 decreased. The reverse changes were happened after serum release, but the mRNA expression of p27(kip1) has no significant change. LMB could inhibit the cell proliferation caused by serum release. The expression of p27(kip1) was up-regulated and Jab1 down-regulated when Jurkat cells were treated with LMB. After pcDNA3. 1-myc-Jab1 infected Jurkat cells for 48 h, the distribution of p27(kip1) was translocated from nucleus into cytoplasma. p27(kip1) and Jab1 could form compound in Jurkat and Raji cells detected by Immunoprecipitation.CONCLUSION:Jab1 may influence the location and expression of p27(kip1) through integrating with p27(kip1), and then participates in regulating the growth of NHL cell through interfering with the function of p27(kip1).

Objective: To investigate the expression variation and significance of Skp2 and p27kip1 during serum starvation and release process of Lymphoma cell line Raji cells.Methods: The combination of p27kip1 and Skp2 were detected by Immunoprecipitation in Raji cells.Raji cells were treated by serum starvation and release assay for synchronization purpose.The expression variation of p27kip1,Skp2 were detected by Western blot.Results: The results of immunoprecipitation suggest p27kip1 and Skp2 could form compound in Raji cell.During serum starvation and release process of Raji cells,the albumen amount of Skp2 increased,while the albumen amount of p27kip1 decreased.Conclusion: During serum starvation and release process of Raji cells,the increased synthesis of Skp2 may speed up p27kip1 degradation via the ubiquitin-proteasome pathway,then p27kip1 decreased significantly,and then the cell cycle was put forward.