Co-localization Of NS And PPP2R5A In HL-60 Leukemia Cells And The Construction Of PCMV Myc-PPP2R5A Expression Vector

Objective:Nucleostemin (NS), which is involved in indefinite proliferation of stem cells and cancer cells, is an essential protein that discovered in 2002, and NS can also keeps cells in non-differentiated state. NS is a p53-binding protein that mainly existing in the karyon or nucleoli of stem cells and cancer cells, we can change the pathological characters of cancer by controlling or inhibiting the expression of NS anthropogenicly. According to our prior research, NS is highly expressed in leukemia cell lines such as HL-60 and K562, and different kinds of acute leukemia cells. The signal transduction pathway of NS is generally regarded relating to p53, but some biological phenomenas in p53 absent leukemia and solid tumor cells couldn't be explained accordingly. Proteinphosphatase 2 A (PP2A) is a Ser/Thr phosphatases, and it is implicated in the negativecontrol of cell apoptosis, hyperplasia, differentiation and metabolism. PPP2R5A is the alpha isoform of protein phosphatase 2 regulatory subunit B, and determines the specificity between PP2A and the substrate, it also mediates the signal transduction of PP2A. Some documents shows the interaction between NS and PPP2R5A, but whether the phenomenon also exists in acute leukemia cells or not is not reported internal or abroad at present. The total objective of the research is to explore the existence of the p53-independent NS signal transduction pathway, and whether this signal transduction pathway is related with PPP2R5A protein and PP2A mediated signaling pathway. The issue is the sub-project of the total research, which is aimed to examine whether the p53 deficient cells have NS and PPP2R5A expression and co-localization, then construct the pCMV Myc-PPP2R5A expression vector and transfected into HL-60 cells to expand PPP2R5A protein expression, which lays the foundation of the co-Immunoprecipitation test for NS and PPP2R5A protein.Methods:(1) RT-PCR, immunohistochemistry and Western blot were used to detect the expression of PPP2R5A in HL-60 cells and in the peripheral blood mononuclear cell of acute leukemia cells.(2) PT-PCR method was used for detection of NS gene and p53 gene in HL-60 leukemia cells.(3) Double immune fluorescence staining method and laser scanning confocal microscope technology was used to detect the co-localization of NS and PPP2R5A in HL-60 cells.(4) pCMV Myc-PPP2R5A restructuring expression vector was constructed and transformed into DH5a, the plasmid was extracted and detected by RT-PCR technology.(5) The recombinant clones were transfected into HL-60 cells, so to strengthen the expression of PPP2R5A protein in HL-60 cells. (6)SPSS 17.0 statistical software was applied to analyse the data statistics. And (x±SD) and one-way ANOVA were used to compare the quntitative data, Chi-Square Test was used for qualitative data.α=0.05 was considered as the significance level.Results:(1) The expression of PPP2R5A in leukemia cells:RT-PCR, and immunohistochemistry results showed the expression of PPP2R5A in the cell line HL-60 and acute leukemia (M5, M2, M3, ALL, mixed leukemia) peripheral blood mononuclear cells, and in various types of acute leukemia there was no significant difference (P> 0.05).(2) The expression of p53 gene in HL-60 cells:PT-PCR results showed that p53 gene was not expressed in HL-60 cells.(3) The co- localization of NS and PPP2R5A in HL-60 cells:confocal results showed that both NS and PPP2R5A were expressed in the cytoplasm and nucleus, NS protein was mainly expressed in nucleolus, and PPP2R5A protein mainly in existed in the cytoplasm, the co-localized phenomenon of the two proteins was displayed.(4) The result of restructuring and transfection:the pCMV Myc-PPP2R5A restructuring expression vector was constructed and cloned and transformed into HL-60 cells successfully, and the positive transfection rate was (18.5±2.4)%.Conclusions:(1) PPP2R5A gene and protein were expressed in HL-60 cells and acute leukemia cells, and the two proteins NS and PPP2R5A were confirmed co-localization in HL-60 leukemia cells.(2) pCMV Myc-PPP2R5A vector was successfully constructed, which lays the foundation of the co-Immunoprecipitation test of NS and PPP2R5A protein.