The Study Of Survivin In The Mechanisms Of The Apoptosis Induced By Daunorubicin In Leukemic Cells

ObjectiveTo investigate the role of survivin in the apoptosis induced by DNR in the leukemic cells by detecting the expression of survivin and caspase-3, 8, 9.MethodsDNR or VCR in different concentrations (0.1 mg/L, 1 mg/L, 10 mg/L) were added into cultured Molt-4 cells respectively. The cell culture was stopped in different time.(1) MTT was used to analyze Molt-4 cells'proliferation. (2) Flow cytometry (FCM) was performed to confirm cell apoptosis. (3) The expression of survivin mRNA was determined by semi-quantitative reverse transcription PCR (RT-PCR). (4) Survivin protein expression was detected by immunohistochemistry. (5) Western blot was done to detect the expression of caspase-3, 8, 9. The expression of survivin on protein level and its location were detected by immunohistochemical assay in 62 children of acute leukemia. The relationship between the expression of survivin and P53 was analyzed.ResultsPart One:(1) MTT assay indicated that DNR and VCR were able to inhibit the proliferation of Molt-4 cells in a time and dose dependent manner (P<0.05).(2) Flow cytometry showed that a higher apoptosis rate could be induced in Molt-4 cells by DNR as compared with that induced by VCR in the same time and same concentrations (P<0.01). (3) RT-PCR revealed that the expression of survivin mRNA decreased in DNR group in time- and dose- dependent manners (P<0.05). It was no obvious change in VCR group (P>0.05).(4) Immunohistochemistry showed that the expression of survivin protein decreased (P<0.01) and survivin shifted from cytoplasm to nucleus in DNR group in a time and dose dependent manner, but not in VCR group (P>0.05).(5) Western bolt showed that the expression of procaspase-3 and its activated fragment were found in DNA group. Procaspase-9 and its activated fragment but few activated caspase-8 were detected in DNR group in a time and dose dependent manner (P<0.05).Part Two:(1) The expression of survivin in leukemic cells was stronger than that in the normal cells (P<0.05).(2) The expression of survivin was related to the expression of P53 significantly in pediatric acute leukemia (P<0.05).(3) The complete remission rate of leukemia was higher in the patients expressed survivin in nucleus than that in cytoplasm (P<0.05).Conclusion(1) DNR induce Molt-4 cell apoptosis by mitochondrion pathway.(2) Survivin/P53 may play an important role in apoptosis induced by DNR, but not by VCR.(3) The strong expression of survivin in the children of AL showed that it would play an important role in the development and prognosis of pediatric acute leukemia. The different expression of survivin in cytoplasm and nucleus would be a marker of prognosis. Expression of survivin and de-activation of P53 may play synergetic role in the genesis of AL.