Effect Of Amifostine On Chemotherapeutic Drug-Amsacrine Sensitivity Of Acute Myeloid Leukemic Cells

Object:To investigate the effect of cytoprotector——Amifostine on human telomerase reverse transcriptase(hTERT)and apoptosis induced by chemotherapeutic drugs——Amsacrine in acute leukemic cells——U-937 cell lines.Methods:The object was divided into nine groups:control,Amifostine (10³Î¼mol/L,10⁴μmol/L),Amsacrine(3μmol/L,10μmol/L),and Amsacrine +Amifostine 3μmol/L+10³Î¼mol/L,3μmol/L+10⁴μmol/L,10μmol/L+10³Î¼mol/L,10μmol/L+10⁴μmol/L.The time was divided into three levels:24o'clock,48 o'clock,72o'clock.To investigate the effect of low dose Amsacrine combined with high dose Amifostine on human acute leukemia cells——U-937 cell lines.To detect the change of the correlated index.(1)The cell proliferating activity was assessed with MTT assay.(2)The cell appearance were determined by Giemsa staining techniquedis.(3)hTERTmRNA expression were measured by semi-quantitative RT-PCR.(4)The DNA content of U-937 cell lines was analysed by flow cytometry.Results:(1)MTT assay disclosed that followed by the extension of effect time Amsacrine/Amifostine group,the cell depressive effect was advanced(P(0.05).on the other hand,followed by the extension of effect time of the combination of Amsacrine and Amifostine,the U-937 cell proliferating activity was obviously inhibited(P(0.05).In 72 hours,the cell depressive effect of the combination group(Amsacrine 3μmol/L+Amifostine 10⁴μmol/L)was obviously stronger than high Amsacrine 10μmol/L group(P(0.05)(2)Apoptosis was assayed by morphological added Amsacrine/Amifostine.In morphological observation of apoptotic cells using Giemsa staining technique,cells displayed classic apoptotic changes treated with Amsacrine3μmol/L alone and Amsacrine3μmol/L plus Amifostine 10⁴μmol/L at 72h.In this part,the number of apoptotic cells treated with Amsacrine3μmol/L and Amifostine10⁴μmol/L together is obviously more than that treated with Amsacrine 10μmol/L alone.(3)Semi-quantitative RT-PCR result show the expression of hTERTmRNA has significant difference in the different density of Amsacrine or Amifostine groups in the U-937 cell(P(0.05).There is the obvious expression of hTERTmRNA in the control group.There are interaction between Amsacrine and Amifostine.The lowest expression of hTERTmRNA was the combination group(Amsacrine 3μmol/L+ Amifostine 10⁴μmol/L)in 72 hours(P(0.05).(4)DNA flow cytometry analysis show that Amsacrine and/or Amifostine induced most of the U-937 cell arrest at G₁ phase and decrease significantly in S phase.In 72 hours,the cell number of the G₁ phase of the combination group (Amsacrine 3μmol/L+Amifostine 10⁴μmol/L)was obviously larger than high dose Amsacrine 10μmol/L group(P(0.05).Conclusion1.Amifostine could inhibit hTERTmRNA of the U-937 cells and increased the Amsacrine——induced apoptosis and enhanced Amsacrine-sensitivity.2.Amsacrine and/or Amifostine induced most of the U-937 cell arrest at G1 phase and decrease significantly in S phase.3.After the combination of Amsacrine 3μmol/L°and Amifostine 10⁴μmol/L group,the cell depressive effect and the cell phase arrest effect were obviously elevated,the extension of hTERTmRNA were cut down. Compared with the high dose Amsacrine 10μmol/L group,the effect were obviously elevated.