| BackgroundAdult T-cell leukemia (adult T-cell leukemia, ATL)， directly related to ahuman T cell leukemia virus I (HTLV-I) infection，is the special type oflymphatic system in adult malignant clonal proliferation disease. the acutephase of ATL invasionhighly. Poor prognostic factors in the ATL and HTLV-1infected cells with unusually strong unlimited proliferation and inhibition ofapoptosis,also related to the ability of its innate resistance of the traditionalchemotherapy drugs, so there is an urgent need for new treatments to improvethe poor prognosis of ATL.JAK-STAT signaling pathway is a cytokine-stimulated signal transductionpathways involved in cell proliferation, differentiation, apoptosis and immuneregulation of many important biological processes found in recent years. It ismainly composed of three components, the tyrosine kinase receptor, tyrosinekinase JAK and transcription factor STAT. Many research results have shownthat abnormal activation of the JAK-STAT pathway is a key role in thepathological mechanism of ATL,and there is Widespread sustained activationof the JAK-STAT signaling pathway in ATL cells.The molecular chaperone protein Hsp90is involved in the folding,activation and assembly of a variety of proteins。Abnormal high level ofexpression of heat shock protein90(of HSP90) in ATL cells is related to thepathogenesis of the ATL. In recent years, studies have shown that many signaltransduction pathway proteins are HSP90client protein and abnormallyactivated by HSP90. In normal cells, HSP90is expressed at relatively lowlevels and does not form complexes with other chaperone proteins。Bycontrast, in neoplastic cells HSP90is expressed at much higher levels。HSP90 also forms large complexes with other chaperone proteins in neoplastic cells toform the so-called multichaper one or superchaperone complexes。stablemutations or aberrant activation of cancer gene-related protein fromdegradation of the ubiquitin-proteasome pathway to promote the abnormalproliferation of tumor cells.In this study, we select HTLV (+) the ATL cell line HUT-102。WESTERN-BLOT, immunoprecipitation, flow cytometry and other detectionmethods of modern biology to explore the ATL cell within HSP90and JAK/STAT pathway abnormalities continued to whether there is a certain linkbetween the activation of what kind of contact, in order to further deepen theunderstanding of the pathogenesis of ATL。Methods:Cell cultureusing the RP-1640+10%fetal bovine serum dubbed entirely to HUT-102cells were cultured and passaged on a regular basis, change the fluid.WESTERN-BLOT:2.1detection to HUT-102cells、Jurkat cells and peripheral blood mononuclearcells (PBMC) within the JAK3protein、STAT5protein、P-STAT5protein、HSP90expression. Observe the different amount of protein in the cells in each group.2.2dilution of different concentrations of specific HSP90inhibitor17-AAG(0-2000NM) role to HUT-102cells24and48hours were extracted from eachgroup of total cellular protein, WESTERN-BLOT detect JAK3protein, STAT5protein and P-STAT5protein expression trends.2.3use the the1000NM concentration of17AAG role to HUT-102cells0,3,6,9,12,24hours after the total protein was extracted, WESTERN-BLOTdetect JAK3protein, STAT5protein and P-STAT5protein expression changesin trends.2.4located in the no stimulation group17AAG+CP690550group,17-of AAGgroup, the CP690550group were stimulated to HUT-102cells for24hours, extraction of total cellular protein WESTERN-BLOT detect JAK3proteinexpression.3RT-PCRDifferent concentration (0,125,250,500,1000NM). Inhibitors stimulate toHUT-102cells24h after the extraction of total cellular RNA, using RT-PCRmethod to detect the expression of the cells in each group JAK3mRNA,GAPDH, to set the internal reference.4CCK8Diluted with different concentrations of17AAG(0,500,1000,2000,4000,5000,8000NM). Role to HUT-102cells, respectively12,24,48and72hours after the the CCK8method to detect cell absorbancevalues based on cell survival.5Annexin V-PIDiluted17AAG concentration to0,250,500,1000,2000the NM were toHUT-102cells after48hours of Annexin V-PI binding by flow cytometry toanalyze cell apoptosis.6, co-immunoprecipitation (CO-IP)JAK3antibody, the beads and to HUT-102cells total protein JAK3protein combination of beads-protein-antibody complex precipitated removethe beads with the antibody, WESTERN-BLOT detect whether it contains theHSP90protein in the protein precipitation.Result1HSP90protein expression in normal PBMC and two cell lines, normal humanHSP90protein expression was significantly lower than the two T-lymphoidtumor cell lines, JAK3/STAT5protein is hardly expressed. Conpered to Jurkatcells, the protein expression of HSP90、JAK3、STAT5and P-STAT5ofHUT-102cells was significantly increased.2the CCK8results show that when17AAG concentration increased,cell viability decreased gradually.17AAG concentration decreased to the lowest inthe2000NM. Then the cell survival rate reaches a minimum value andstabilized. Analysis showed that from the time, with the extension of theincubation time,17AAG inhibition of cell gradually increased to48h inhibitoryrate reached the maximum, and then to72h, and48h showed no significantdifference (P>0.05)3To examine whether induction of apoptosis accounts for the cell survivalinhibition observed in HTLV-I-infected T-cell lines, cells treated with17-AAG for48hr were examined by the Annexin V method. The results showed that17-AAG increased the proportion of cells positive for Annexin V in HUT-102.and such effect was observed in a dose-dependent fashion.4compared with the unstimulated group, treatment of stimulation group with17-AAG dose-dependently decreased the levels of the JAK3and P-STAT5protein，and P-STAT5protein is almost not be detected. Under the sameconditions, the detection of total STAT5protein found no significant differencesbetween each stimulation group and the non-stimulation group STAT5proteinexpression (P>0.05).5With the same concentration levels,17-AAG time-dependently decreasedthe levels of the JAK3and P-STAT5protein。 P-STAT5protein levels can notbe detected after4h. Total STAT5protein expression levels did not changewith the time of inhibitor increase or decrease (P>0.05).6using RT-PCR method to determined the effects of17-AAG on the mRNAlevels of JAK3in HUT-102cells. Treatment of HUT-102cells with17-AAGdid not alter the mRNA levels of JAK3.7according to the CO-IP results show that use of the JAK3antibodyprecipitate the natural conformation of the protein, then usingWESTERN-BLOT in addition to the detection of JAK3protein precipitation, butalso detected a small part of the HSP90protein precipitation.8combine treatment with CP690550and17-AAG stimulated cells, the resultsshow that a separate group of17-AAG inhibition of JAK3protein is stronger than the CP690550. Separately with17-AAG/CP690550stimulation groupcompared with the two inhibitors to stimulate a group of cells in JAK3proteinoverexpression inhibition more.Conclusion1JURKAT, to HUT-102cells compared with the expression of HSP90proteinin normal peripheral blood mononuclear cells was significantly increased. ToHUT-102cells continued abnormal activation of JAK3/STAT5pathway.2JAK3is HSP90-dependent protein.317-of AAG inhibit JAK3/STAT5signaling pathway sustained activation in adose and time dependence.417-of AAG inhibit to HUT-102cell proliferation and induce apoptosis.5CP690550and17AAG may have a synergistic effect in HUT-102cells... |
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