The Role Of Kinesin EG5 In Human Chronic Myeloid Leukemia And Antileukemia Mechanism Of Target Therapy Drug S-Trityl-L-Cysteine

Objective:1. To observe expression of kinesin EG5 mRNA in normal peripheral blood and bone marrow with chronic myeloid leukemia clinical specimens. Assess the correlation of EG5 mRNA and BCR/ABL mRNA with potential diagnosis and prognosis value of CML.2. To investigate the effect of S-Trityl-L-Cysteine on the mitosis arrest and apoptosis for acute leukemia HL-60 cell,and rudimentary study the causality of mitosis arrest and apoptosis in HL-60 cell under the drug treatment.3.To investigate the effect of S-Trityl-L-Cysteine on the mitosis arrest and apoptosis in CML K562 cells, study the mechanisms of mitosis arrest and apoptosis under the drug treatment, as well as to clarity the relationship between Eg5 and bcr/abl singnal pathway .Methods:1. The EG5 mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and BCR/ABL fusion gene were detected by nested reverse transcription-polymerase chain reaction (Nested -RT-PCR).To examinated of 8 normal peripheral blood and bone marrow specimens with 62 peripheral blood samples of CML.2. HL-60 cell was exposed to various concentrations of STLC (1,2.5,5,10,50,100μmol/L) and compared with the control group.The inhibitory effects were determined by MTT assay and Trypan blue staining assay was used to examine the number of viable cells. Cell cycle arrest and sub-G1 cells was detected by flow cytometry assay and character of cell and its nuclear were observed by immunofluorescence staining, Annexin-v/PI assay was detected the apoptosis rates of normal bone marrow cells by STLC treatment.3.After K562 cell exposed to various concentrations of STLC, the proliferation of cells were determined by MTT assay, the cell cycle and Annexin-v/PI was detected by flow cytometry assay .The space character of between cell spindle and nuclear and the migration of AIF were observed by confocal microscope.The connection of Eg5 and bcr/abl were examined by micromolecule inhibitor and RT-PCR .Results:1.The EG5 mRNA were not detected in the normal peripheral blood specimens ,but low abundance of EG5 expression were detected in the normal bone marrow specimens ; The sensitivity and specificity of EG5 auxiliary diagnosis CML was 89.6% (26/29) and 84.8%(28/33); the BCR/ABL fusion gene diagnosis CML sensitivity and specificity was 96.5%(28/29) and 90.9%(30/33);The EG5 and BCR/ABL expression in CML was no significant difference and they are assured highly positively correlation. 2. MTT and Trypan blue staining assay revealed that STLC inhibited HL-60 cell growth and induced its death .The typical phenomenon of mitotic catastrophe was observed by immunofluorescence staining.STLC induced apoptosis on HL-60 positively correlated with drug concentration and exposed times,and the rate of G2/M arrest increased with drug concentration at 24h and 48h ,but no significant change at 72h.Furthermore revealed G2/M arrest and apoptosis simultaneous in early stage of drug treament The mitotic arrest was Reversibly after the drug removed,The apoptosis rates of normal bone marrow cells was slightly increase under the drug treatment.3. Results showed that STLC inhibited k562 cell growth by MTT .STLC induced G2/M arrest at 24h and the rates of apoptosis increased at 48h under the drugs treatment, 88.5% monaster spindle cells was observed at 24h , which demonstrated that the mechanism of apoptosis was not dependent on AIF. Eg5 locate at downstream of bcr/abl signal pathway and was regulated by bcr/abl in K562 cells.Conclusion:1.The expression of EG5 and BCR/ABL in chronic myelogenous leukemia patients basically consistent with disease progression, the dynamic examination of EG5 and BCR/ABL may be used as clinical auxiliary diagnosis and prognosis of CML.2.S-Trityl-L-Cysteine induce G2/M arrest and apoptosis in HL-60 cell and manifest more potent effects of antimitotic and antitumour. simultaneous G2/M arrest and apoptosis suggest that possiblely have an new mechanism in the early stage of drug treatment 3.STLC induce G2/M arrest and apoptosis in K562 cell,but wan not dependent on AIF, and manifest more potent antimitotic and antitumour effects .