Studies On The Correlation Between Abnormal Phosphorylation Of P27^kip1 And Malignancy Of Non-Hodgkin's Lymphomas

Objectives1. To investigate the expressions of 2 major phosphorylation formats of p27^kip1 respectively and its relative protein S-phase kinase-associated protein 2(Skp2) and Jun activation domain binding protein 1 (Jab1) in human non-Hodgkin's Lymphomas and their clinicopathologic significance.2. To evaluate the specific effects of pThr187p27^kip1 and pSer10p27^kip1 during lymphoma cell growth.Methods1. Immunohistochemical technique was used to detect the expression levels of pThr187p27^kip1, pSer10p27^kip1p27^kip1, Skp2, Jab1 and Ki-67 in 116 cases of NHLs and 15 cases of reactive lymphoid tissue. The NHL cases characterized in this study were universal in oriental included: 11 follicular lymphomas (FL), 7 mucosa-associated lymphoid tissue B cell lymphomas (MALT), 37 diffuse large B-cell lymphomas (DLBCL), 49 Nasal natural killer (NK)/T-cell lymphomas and 12 unspecified peripheral T-cell lymphomas (PT-un).2. Human Jab1 complete cDNA was amplified by RT-PCR method, and was reconstructed into the eukaryotic expressive vector pcDNA3.1-Myc to form the Jab1 sense expressive plasmid. The reconstructed DNA was identified by enzyme digestion and DNA sequencing.3. Serum starvation and release used to synchronize lymphoma cells. Flow cytometer was used to analyze cell-cycles. The expression and subcellular localization of pThr187p27^kip1, pSer10p27^kip1, Skp2 and Jab1 were detected by western blot, immunoflurescence and subcellular fractionation. Transfection was performed using lipofectamine 2000 transfection reagent according to the manufacture's protocol. The cells were harvested 48 h after transfection and cell-cycles were detected by Flow cytometry. The expression and subcellular localization of Jab1, p27^kip1 and p-Ser10p27^kip1 were investigated by western blot, immunoflurescence, immunoprecipitation and subcellular fractionation.Results1. Immunohistochemistry analysis showed: 1) The expression level of pThr187p27^kip1 in NHLs was higher than that in reactive lymphoid tissues (except germinal center). Using a 10% cutoff for pThr187p27^kip1 expression, pThr187p27^kip1 positive expression was significantly associated with patients'age and clinical stage, but it was not associated with patients'sex and lymph node metastasis. In all cases of NHLs analyzed, pThr187p27^kip1 expression did correlate with expression of Skp2 (rs =0.594, P<0.001) and p27^kip1 (rs =0.175, P=0.062) as well as Ki-67 (rs =0.639, P<0.001). By using the Kaplan-Meier analysis, patients with pThr187p27^kip1-positive tumor were significantly associated with short overall survival (P < 0.001). 2) The expression levels of pSer10p27^kip1 and Jab1 in NHLs were higher than that in reactive lymphoid tissues (except germinal center). Using a 10% for cutoff, pSer10p27^kip1 and Jab1 overexpression were both significantly associated with advanced stage, but they were not associated with patients'age, sex and lymph node metastasis. As continuous variables, pSer10p27^kip1 expression did positively correlated with expression of Jab1(rs= 0.696, P<0.001) and negatively correlated with expression of p27^kip1 (rs = -0.457,P<0.001); Jab1 expression was negatively associated with p27^kip1 expression (rs = -0.380, P <0.001) and was positively associated with Ki-67 expression (rs = 0.870, P < 0.001) in all cases of NHLs analyzed. Survival analysis indicated that patients with pSer10p27^kip1 or Jab1-positive tumor were significantly associated with short overall survival (P < 0.001).2. The cloned DNA was confirmed to be human Jab1 cDNA.3. Advances in the cell cycle study have revealed: 1) pThr187p27^kip1 increased as the cells were treated with serum addition as well as the same kinetics were observed at cyclin E and CDK2. Subcellular fractionation showed that pThr187p27^kip1 was mainly present in the nucleus of cells released from G1, leading to the dramatically reduced p27^kip1 accumulation. Immunoprecipitation studies suggested that the enhancement of phosphorylation at Thr187 of p27^kip1 mainly occurred in the CDK2/CyclinE complex. 2) pSer10p27^kip1 and Jab1 were also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. We've found that overexpression of Jab1 could increasing the Ser10 and Thr157 phosphorylation of p27^kip1 while the Thr187 phosphorylation was significantly decreased. Our preliminary data demonstrated a significant decrease of p27^kip1 expression level and a significant increase of S phase of the cell cycle in NHL cell lines that overexpressed Jab1. Immunoprecipitation studies suggested that Jab1 could change in p27^kip1 binding preferences to CDKs and relatively major p27^kip1preferentially bound to CDK4.Conclusions1. Increased expression levels of pThr187p27^kip1 and pSer10-p27^kip1 were closely correlated with the aggressiveness of NHLs. Both 2 major phosphrylation formats may influence the expression and function of p27^kip1 through different pathways, thus may play key roles in the occurrence and development of NHLs.2. We successfully cloned human Jab1 gene and constructed its eukaryotic expressive plasmid, which was an ideal model for further study.3. Since Thr187 phosphorylation is required for p27^kip1 ubiquitination and Ser10 phosphorylation is necessary for the nuclear export of the protein mediated by the exportin Jab1, both phosphorylation formats may be involved in the control of the G1-S transition and have oncogenic potential.