| Objective:Nucleostemin (NS) is a p53-binding protein related with proliferation of stem and cancer cells,which has been found in the karyon or nucleoli of stem cells and various cancer cells,but is few in the cytoplasm and dose not express in committed and terminally differentiated cells.NS protein plays an important role in self-replication and unlimited proliferation of tumor and stem cells,and also possibly as a kind of specific regulatory factor that may be decided to across G2/M check point and may inhibit their differentiation and maturation. Our previous studies have found that NS was highly expressed in K-562 and HL-60 cell lines and varieties of leukemia,and also the expression of NS decreased gradually with the differentiation and maturation. Different concentration of all-trans retinoic acid (ATRA)were used on HL-60 cells in vitro,the NS gene and protein were found reduced. The result suggested that NS has involved in the differentiation mechanism. In this study,we based on the results of previous studies to detect the changes of NS,NF-κB,Cyclin D1 and Cyclin A gene and protein in the differentiation process by ATRA induced. The aim is to explore the relevance of NS and NF-κB,Cyclin D1,Cyclin A after induced on HL-60 leukemia cells and the mechanisms that how NS gene regulate the cell cycle.Methods:(1)HL-60 cells were cultured in vitro,and was induced with a final concentration of 0.1μmol/L,0.5μmol/L and 1μmol/L ATRA respectively in the logarithmic phase of cells. RT-PCR was used to detect the effect of ATRA on NS gene expression at 24h,48h and 72h respectively,and to find the optimal time and concentration. Then,under such a condition,to detect the expression of the NF-κB,Cyclin D1 and Cyclin A gene respectively by RT-PCR.(2) The culture state of HL-60 cells were observed in living cells under inverted microscope,the morphology changes were observed by Wright-Giemsa staining.(3)Flow cytometry was used to detected the granularity,size and the differentiation antigen on cell surface.(4) NS,NF-κB,Cyclin A and Cyclin D1 protein were detected by immunocytochemistry technology before and after induced with ATRA,and from the protein level to reflect the relevance of NS,NF-κB,Cyclin A,Cyclin D1 protein and the differentiation of leukemia cells.(5)SPSS 17.0 statistical software was applied for the data statistics.(x±s)and one-way ANOVA were used to compare the quntitative data,Chi-Square Test was used for qualitative data. a=0.05 was considered significant.Results:(1)The expression of NS gene was depressed induced by ATRA,and there was time and dose-dependent. The optimal concentration and time were 1μmol/L and 48h. In which condition,HL-60 cells appeared the characteristics of differentiation as following:①Under inverted microscope,the cell density and the level of aggregation decreased,cell size of the disparity,the grainy of cytoplasm enhanced, the cell debris increased,and some of HL-60 cells into spindle or a tail,and with obvious pseudopodia. ②Wright-Giemsa staining showed that HL-60 cells aniso-magnitude,in addition to circular and oval-shaped,there were also some irregular-shaped cells, some cells were flat or depressed at the side of nucleus,the nucleus were striction and enrich of the chromatin,the nucleolus was unclear or disappeared.③Flow cytometry results showed that the rate of CD64 differentiation antigen reduced from 31.7% to 12.5% after inducing.(2)In the condition of 1μmol/L ATRA induced for 48h,the inhibition rates of NS,NF-κB,Cyclin D1 gene were respectively 64.62%,77.99%,58.95%,the inhibition rates of NS,NF-κB,Cyclin D1 protein were respectively 62.42%,59.24%,65.49%.(3)The level of Cyclin A gene and protein had no significant changes.Conclusions:(1)The expression of NS,NF-κB,Cyclin D1 gene and protein of HL-60 cells is decreased in the process of inducing differentiation by ATRA.(2) There is may be a synergy of NS,NF-κB,Cyclin D1 in the process of re-differentiation of HL-60 leukemia cells by ATRA.
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