Antisense-mediated inhibition of the bcl-2 gene induces apoptosis in humanmalignant glioma

BACKGROUND The bcl-2 protooncogene represses a number of cellular apoptotic pathways a nd is known to be expressed in increasing amounts in glial tumors of higher malignancy. We tested whether antisense oligonucleotides to the bcl-2 gene would affect glioma cell viability. METHODS Antisense oligonucleotides directed to the first six codons of the human bc l-2 gene, and nonsense oligonucleotides as a control, were transfected into malignant glioma cells. Two human Bcl-2 positive glioblastoma cell lines f rom our tumor bank (Jon52 and Roc) were both transfected in vitro with bcl- 2 antisense (AS) and nonsense (NS) oligonucleotides at 1 mu m and 5 mu m co ncentrations for 5 and 24 hr. Cell viability was assessed at 2, 4, 5, and 7 days by using an MTT mitogenic assay and by cell counting via direct visua lization using a hemocytometer. RESULTS There was up to a log-fold decrease in cell growth of the bcl-2 AS treated cells compared to the NS transfected cells for both Roc (p = 0.007 and p = 0.004) and Jon52 (p = 0.02 and p = 0.004) at 5 and 24 hr of transfection. T here was as much as 50% cytotoxicity in both glioblastoma cell lines at 1 m u m and 5 mu m concentrations after 24 hr transfection with AS bcl-2 oligon ucleotides (all p < 0.01). Western blot analysis demonstrated a decrease in the expression of the Bcl-2 protein in one cell line, whereas there was a statistically significant increase in the apoptotic index of both cell line s (p < 0.05 by chi square analysis). CONCLUSIONS Our results suggest that transfection of human glioma cells with antisense bcl-2 results in an increase in apoptotic death. This provides evidence tha t Bcl-2 plays a role in tumor progression of glioma by acting as an oncogen e, and suggests that inhibition of the bcl-2 gene could have a therapeutic effect. (C) 2000 by Elsevier Science Inc.