Study On Killing Effect Of DAAO Gene Transfer On Highly Tumorigenic Leukemic Cell Line K562e

Modern cancer therapies have greatly improved the prognosis of cancers. Even so, treatments remain toxic, ineffective for many patients. Gene therapy provides a new way for therapy of cancer. In this study, we constructed retroviral vectors containing R. gracilis DAAO cDNA and tranfected human leukemia cell line K562e which had high tumorigenicity in nude mice,, in order..to investigate the killing efficiency and mechanism, of DAAO/D-Ala system on leukemic cellsConstruction of recombinant retroviral vector encoding DAAO geneLinearized retroviral vector pLSN was isolated from pLDCSN by digestion with restriction enzyme BamH I. DAAO cDNA with Bgl II sticky end obtained by PCR was cloned into retroviral vector pLSN and generated pLDAAOSN. The pLDAAOSN and the other vector pLDfG containing DAAO cDNA and GFP gene were transferred into retrovirus packaging cells 3>XNA respectively. liter of virus was 5. 2X 106CFU/ml, DAAO gene containing retrovirus was used to transfect human leukemia cell line K562e, which had high tumorigenicity. PCR, in situ hybridization analysis and cytotoxicity tests showed stable integration of DAAO gene in K562e genome (KDAAO^ Korco KD玀) and expression of DAAO gene at mRNA level.The killing effects and the mechanisms of D-Ala on DAAO carrying K562e cell in vitro The killing effects of D-Ala alone on DAAO*cells or on a mixture of DAAO*and DAAO'cells in different ratio were observed. H202 produced by KDrcc and KDrcd cells were measured by the phenol red oxidation assay. There was no significant difference in cell proliferation betweenK562e and DAAO'cells. When DAAO'cells were treated with D-Ala , the change of morphology of the cells could be observed. The optical density was assayed with MTT method. KDMo, l^and Korcd cells were more sensitive to D-Ala than the parental K562e cells and there was a threshold in the killing effects of D-Ala concentration on K562e Cells. The highest killing effect was observed within 24 to 48 hours. The H20j levels in the medium were consistent with the killing effects of D-Ala. This demonstrates that DAAO/D-Ala system could kill human leukemic cells and the killing effect was mediated by H202. When KDrec was mixed with K562e at different ratio, no significant bystanter effect could be found in vitro after treating with 15mmol/L D-Ala for 24 hours.The killing effect of D-Ala on DAAO transfected K562e tumors in nude mice KDfcc and K562e cells were subcutanously injected into one side of forelimb and hindlimb of nude mice respectively, thus established human leukemic tumors in situs. The tumor bearing mices were then treated with D-Ala. The results were as following: In therapeutics group, the weight of K562e tumor was 2. 44 times of that of KDrcc tumor. In control group, the weight of K562e tumor was only 1. 2 times of that of KDrcc tumor. These results indicate that D-Ala could inhibit implanting DAAO gene carrying tumor from growing. There were necrosis surrounding vessles in the KDfcc tumor which had been treated with D~Ala, suggesting the therapeutical effect of D-Ala. On the contrary , this characterization did not exist in K562e tumor. There was no damage on heart, liver and kidney in both groups.Conclusion1. The recombinant retroviral vector encoding DAAO was constructed and transferred into human leukemia cell line K562e which had high tumorigenicity in nude mice.2. DAAO gene carrying human leukemic cell K562e could be killed byD-Ala.3. There was a threshold in the killing effects of DAAO/D-Ala System on K562e Cells. The highest killing effect could be observed within 24hours to 48 hours.4. The H202 levels produced by D-Ala treated DAAO carrying cells were consistent with the killing effects.5. The growth of tumors formed by K562e transfected with DAAO gene can be inhibited by administration of D-Ala. The dosage of D-Ala used to treat implanted tumor in vivo is not harmful to major organs such as liver, heart and kidney.