Antisense-mediated inhibition of the bcl-2 gene induces apoptosis in human malignant glioma.
BACKGROUND
The bcl-2 protooncogene represses a number of cellular apoptotic pathways and 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 bcl-2 gene, and nonsense oligonucleotides as a control, were transfected into malignant glioma cells. Two human Bcl-2 positive glioblastoma cell lines from our tumor bank (Jon52 and Roc) were both transfected in vitro with bcl-2 antisense (AS) and nonsense (NS) oligonucleotides at 1 microm and 5 microm concentrations 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 visualization 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. There was as much as 50% cytotoxicity in both glioblastoma cell lines at 1 microm and 5 microm concentrations after 24 hr transfection with AS bcl-2 oligonucleotides (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 lines (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 that Bcl-2 plays a role in tumor progression of glioma by acting as an oncogene, and suggests that inhibition of the bcl-2 gene could have a therapeutic effect.
Julien
T
T
Molecular Neurosurgery Laboratory, Department of Neurosurgery, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
Frankel
B
B
Longo
S
S
Kyle
M
M
Gibson
S
S
Shillitoe
E
E
Ryken
T
T
D000368:Aged; D017209:Apoptosis / Q000235:genetics*; D001932:Brain Neoplasms / Q000235:genetics* / Q000473:pathology; D002470:Cell Survival / Q000235:genetics; D018389:Codon, Nonsense / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000235:genetics; D005910:Glioma / Q000235:genetics* / Q000473:pathology; D006801:Humans; D008297:Male; D008875:Middle Aged; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D018389:Codon, Nonsense; D004273:DNA, Neoplasm; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2