| JWA,a proposed cytoskeleton associate gene,was primarily found to be regulated by ATRA,13 cis-retinoic acid (13cis-RA) and TPA in human bronchial epithelial (HBE) cells. Our preliminary data showed that JWA might involve in both cellular differentiation and apoptosis induced by several chemicals,and might play an important role in development of acute myelocytic leukemia.In this study,we provided further evidences to understand how JWA involves in the regulation of cell differentiation and apoptosis in NB4 cell,a human acute promyelocytic leukemia cell line. The expression of cell surface antigens and the effects on cell cycle were analyzed for detecting of drug-induced responses in both cell differentiation and apoptosis. Both RT-PCR and Western blot assays were used for understanding of the expressions of JWA. The results showed that in the present experimental models,ATRA (1uM) and As2O3 (luM) induced cell differentiation and apoptosis separately;however,another two cell differentiation inducers,4HPR (1 uM) and TPA (0.1 uM),showed a dual-directional effect on NB4 cells. They not only triggered cellsdifferentiation but induced apoptosis at the same time. All these chemicals up-regulated JWA expression whenever they triggered cells undergo differentiation or apoptosis. Anti-sense JWA oligonucleotide (10 jiiM) could partially block the ability of TPA induced cell differentiation and apoptosis via an unknown signal pathway.The similar evidences were also found in primary APL cells. In de novo primary APL cells,the expression of JWA protein could be regulated only by TPA. However,after ATRA treatment in vivo,not only TPA,but both ATRA and As2O3 could also regulate JWA protein expression in consequent in vitro cell culture models. We assumed that ATRA pretreatment might change the cell differentiation associate signal pathway in primary APL cell. Interestingly,a higher molecular weight JWA protein,a proposed JWA fusion protein-JWA-F,was identified in de novo primary APL cells and it was also responsible to ATRA treatment. It raises questions whether the JWA-F is a novel APL-specific marker and,how does it involve in the known molecular mechanism in APL development.We also found a JWA protein similar as JWA-F in HL-60 cells. We further investigated if JWA-F is associated with cell differentiation and apoptosis in vitro. The result showed that 2 uM of As2O3 could trigger HL-60 cells undergo apoptosis and the expression of JWA protein was up-regulated in a dose dependent manner (immunohistochemical staining technique). In another hand,in these cell culture models,the normal sized JWA protein (JWA-L) showed a similar expression pattern as the caspase-3 (Western blot assay). They both were up-regulated at earlier phase and then down-regulated after treatment. JWA-F was showed a time-dependent increase pattern after As2O3 treatment,and especially,only JWA-F can be detected when cells treated by higher concentration (5uM)ofAs2O3.To understand the potential mechanism of the JWA-F formation,thepolymerase chain reaction (PCR) technique was used to investigate and compare if any differences are present among NB4 cells,HL-60 cells and controlled human lymphocytes. The genomic DNA was extracted and used as the templates of the PCR. A series of primers were designed to amplify JWA fragments between proposed promoter and cDNA region. The comparable results showed that all PCR fragment showed no differences in their sizes except one fragment (+27-+7S6 bp of cDNA) which was failed to amplify in both cell lines. It is still obscured that if the JWA-F is formed by the 5'-end of JWA fused to a fragment from other gene.In summary,JWA might involve in a potential common signal pathway which involves in APL cell differentiation and apoptosis induced by ATRA,4HPR,TPA and As2O3. TPA triggered cell differentiation and apoptosis via both direct and indirect signal pathways,JWA may as a novel molecule that regulates the direct pathway. In both primary APL cells and HL-60 cells,JWA forms a special fusion protein that ma...
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