Experimental Study Of Combination Gene Transfer FasL And P21～(WAF1/CIP1)Therapy For Human Gliomas

OBJECTiVE Fas ligand (FasL) belongs to the tumor necrosis factor family and has ability to induce apoptosis in susceptive target cells by binding to its receptor, Fas. P21WAF1/CIP1 is a downstream mediator of P53 and mediates growth arrest by inhibiting the action of G1 cyclin-dependent kinases. It has been demonstrated recently that the FasL/Fas system plays a pivotal role in the cytocidal activity of T lymphocytes in the immune system. Overexpression of P21 WAF1/CIP1 induces growth suppression and differentiation in p53-defective human tumor cells.To evaluate effects of gene transfer FasL and br P21 WAFI/CIP1 in vitro and in vivo for treatment of human malignant gliomas. METHODS We constructed two recombinant advenoviral vectors ----one containing human FasL cDNA under the control of a cytomegalovirus(CMV) promoter (Ad-FL), which encodes human Fas ligand, the other containing human P21 cDNA under the control a CMV promotor (Ad-P2 1), which encodes human P21 cyclin dependent kinase inhibitor(Ad-P2 1). In vitro Ad-FL or Ad-p21 was transfected into six glioma cells, respectively. MTT, TUNEL assay were used to analysis glioma cells apoptosis and RT-PCR.. flow cytometric analysis identified the expression of Fas/FasL and p21WAF1/CIP1. We also constructed the gliorna tumor model of BALB/c nude mice and SD rats in order to use Ad-FL and/or Ad-p2 1 to identify its effect through examination of pathology, assessment of MRI ,observation of tumor size and evaluation of the mean survival time of mice bearing tumors.We used one-way ANOVA (analysis of variance) of SPSS 8.0 software to analyze the results. LII Uk RESULTS (1)Six glioma cells surface except SKN-SH expressed Fas, which level were not correlated with the sensitivity of apoptosis induced by anti-Fas antibody, whereas none express FasL detected by RT-PCR and flow cytometric analysis , while tumor cells transducted by Ad-FL can express high level of FasL; (2)Both of six glioma cells surface did not express P21 protein detected by FACScan, but P21 mRNA expressed detected by RT-PCR, meanwhile tumor cells transducted by Ad-p21 can overexpress P21 detected by FACScan; (3)Both Ad-FL and Ad-p21 alone could induce six glioma cells apoptosis or suppress its growth in vitro, which was significant difference comparing with Ad-LacZ, respectively(P<0.01); (4)In vitro combining Ad-FL and Ad-P21 introduce glioma cells apoptosis ,which was very difference comparing with Ad-FL or Ad-P21 alone (P