| Objesctives:To investigate the expression of mTOR in leukemia and leukemic cell lines. To analyze the effect of rapamycin on cell growth, cell cycle and expression of mTOR in leukemic cell lines.Methods:Real-time quantitative PCR was used to investigate the expression of mTOR in leukemia and leukemic cell lines. Different concertrations of rapamycin were administrated to leukemic cell lines KG1, K562 and U937.MTT was used to analyze the effect of rapamycin on cell growth inhibition and cell cycle blockage was observed by using flow cytometry.Results:1 Expression of mTOR mRNA1.1 Expression of Livin mRNA in patients with acute leukemia The mRNA expression of mTOR in acute leukemia was higher than that in normal control, with statistical significance(P<0.05). mRNA expression of mTOR in patients who recurrent was higher than that in acute leukemia without statistical significance(P>0.05) and normal control with statistical significance(P<0.05). mRNA level of mTOR in patient who had complete response was lower than that in acute leukemia and recurrent patients, with statistical significance respectively(P<0.05)and higher than that in normal control without statistical significanc(eP>0.05). Furthermore, there was difference of mRNA level of mTOR in different subtypes of lecukemia(M2-M6), but without statistical significance(P>0.05).1.2 Expression of mTOR mRNA in patients with chronic myeloid leukemiaThe mRNA expression of mTOR in chronic myelocytic leukemia was higher than that in normal control with statistical significance(P<0.05)and lower than that in acute leukemia and patients with recurrence without statistical significance(P>0.05).2 The relationship between the expression of mTOR and clinical effect (CR rate) in newly diagnosed acute leukemia patients The therapeutic efficacy in 40 of 43 newly diagnosed acute leukemia patients was evaluated (1 patient died early, 2patients gave up treatment).33 of 40 AL patients achieved complete hematological response (CR) after induction therapy, with a CR rate of 83.3%. mRNA level of mTOR in patient who have complete response was lower than that in acute leukemia and recurrent patients, with statistical significance respectively(P<0.05)and higher than that in normal control and CCR patients without statistical significance(P>0.05). 7/40 AL patients have not achieved complete hematological response (CR) after induction therapy, mRNA level of mTOR in patient who have not complete response was higher than that in acute leukemia and recurrent patients, without statistical significance respectively(P>0.05)and lower than that in normal control and CCR patients with statistical significance(P<0.05).3 The concentrations of rapamycin were inhibited proliferation of leukemic cell lines.Rapamycin inhibited proliferation of leukemic cell lines.The concentrations of rapamycin were 1nmol/l, 10nmol/l, 20nmol/l, 50nmol/l and 100nmol/l.3.1The proliferative inhibition ratio of KG1 cell line 24 hours after different concentrations of rapamycin .The proliferative inhibition ratio was 31%,42%,70%,73%,78%,81%, with dose and time dependent.There were statistical significance of ratio in 20nmol/l and 1nmol/l,10nmol/l(P<0.05),but without statistical difference in 20nmol/l and 50nmol/l, 100nmol/l, 500nmol/l(P>0.05).3.2 The proliferative inhibition ratio of K562 cell line 24 hours after different concentrations of rapamycin.The proliferative inhibition ratio was9%,13%,18%,20%,21%and 27%. There were statistical difference in 1nmol/l and 50nmol/l, 100nmol/l and 50nmol/l, respectively(P<0.05). Significant difference was observed between 10nmol/l,20nmol/l and 500nmol/l(P<0.05).No difference in the rest concentrations(P>0.05).3.3 The proliferative inhibition ratio of U937 cell line 24 hours after different concentrations of rapamycin .The proliferative inhibition ratio was4%,6%,10%,12%,21%and 26%.There were statistical difference in 1nmol/l and 100nmol/l and 50nmol/l, respectively(P<0.05).Significant difference was observed between 10nmol/l,20nmol/l and 500nmol/(lP<0.05). No difference in the rest concentrations(P>0.05).4 rapamycin induced apoptosis of KG1,K562 and U937 cell linesBefore the administration of rapamycin,leukemic cell lines had intact cell membrane with clear cytoplasm. 24 hours after the treatment of rapamycin,classical apoptotic morphology was observed,such as reduced celluar refraction, shrinked and deformed cell body. The quantity of apoptotic cells increased in a time-dependent manner 48 hours after the apply of rapamycin, celluar refraction reduced significantly and cell disruption was observed. The most significantlywas KG1 cell lines.5 Cell cycle changes of KG1, K562 and U937 cell lines after the administration of rapamycin.24 hours after the different concentrations of rapamycin induction (0nmol/l, 1nmol/l, 20nmol/l and 100nmol/l ).the G0/G1 arrest in KG1 cell line was 50.88%,68.32%,83.54%,85.31% respectively.G0/G1 arrest was 41.1%, 42.96%, 54.05%, 56.63% in K562 cell line, G0/G1 arrest was 38.17%, 48.37%, 50.54%, 54.63% in U937 cell line. All of the G0/G1 arrest were higher comparing with cell lines without rapamycin differention. 6 The mRNA levels of mTOR after the treatment of rapamycin 0nmol/l, 1nmol/l, 20nmol/l and 100nmol/l rapamycin were administrated,24hours later, the mRNA expression of mTOR in KG1 cell line decreased in dose-dependent manner with significant differdeces in different concentrations(P<0.05).Reducetion of mRNA levels of mTOR were observed in K562 and U937 cell lines, but without statistical differences between 1nmol/l rapamycin induction and control and 20nmol/l and 100nmol/l rapamycin application. The rest concentrations showed statistical differences comparing with normal control(P<0.05).Conclusions:1 The mRNA expression of mTOR in acute leukemia was significantly higher than that in normal control. mRNA levels of mTOR in recurrent patients were higher than normal control mTOR mRNA epression in patients who had complete response was lower than that in acute leukemia and recurrent patients, but without difference comparing with normal control.2 mRNA level of mTOR in patient who have complete response was lower than that in acute leukemia and recurrent patients, with statistical significance respectively and higher than that in normal control and CCR patients without statistical significance. patients have not achieved complete hematological response (CR) after induction therapy, mRNA level of mTOR in patient who have not complete response was higher than that in acute leukemia and recurrent patients,without statistical significance respectively,and lower than that in normal control and CCR patients with statistical significance.3 mTOR mRNA expression in chronic leukemia was higher than normal controls, but without significant differences comparing with acute leukemia and recurrent patients.4 Cell proliferation of KG1 cell line could be inhibited by different concentrations of rapamycin induction. 20nmol/l was optimal.Rapamycin could cause significantly G0/G1 arrest,and its effect on celluar apoptosis induction was significant. mRNA levels of KG1 mTOR mRNA decreased after rapamycin induction in dose-dependent manner, comparing with normal control.5 Cell proliferation of K562 and U937 cell line could be inhibited by different concentrations of rapamycin induction. Rapamycin could cause G0/G1 arrest, and its effect on celluar apoptosis induction was increase. mRNA levels of K562 and U937 mTOR mRNA decreased after rapamycin induction in dose-dependent manner, comparing with normal control.But it was not as significant as in KG1 cell line.
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