Adenovirus - Mediated gene Transfer of Wild - Type p53 Results in Restoration of Tumor - Suppressor Function in Glioma Cell Lines

Kim, Mi Sook; Kwon, Hee Chung; Hong, Seok Il; Lee, Choon Taek; Lee, Seung Hoon

Abstract

PURPOSE
The replacement of functional genes into cells that lack genes or mutant genes is the basis of gene therapy. In cancer, where cells often have multiple genetic defects, the replacement of critical genes may suffice to suppress cell growth or induce cell death. In malignant brain tumors, p53 mutation are among the most frequently observed genetic findings and inactivation p53 suggests that p53 plays a critical role in carcinogenesis and tumor progression. Therefore, we study the successful transfer of the wild-type p53 gene using a replicative deficient adenovirus vector into human glioma and medulloblastoma c~ell lines. Meterials and Methods: The human glioma cell line T-98G, U-87MG, U-373MG were used. To determine the efficiency of the adenovirus vector, cell lines were transfected with the Ad-p gal and analysed with X-Gal staining. Cell viability was determined by trypan blue exclusion every day after infection and Westem blot analysis was used to conform the expression of the exogenous p53 protein.
RESULTS
Cell growth of the Ad-CMV-p53 infected U-373MG, and U-87MG was significantly suppressed. It appeared that exogenous p53 protein expression had an earlier ad more profound suppressive effect on U-373MG having a mutated p53 gene than on U-87MG having a wild-type p53. The expression of the exogenous p53 was more than 10 times higher than the expression of the endogenous p53. To examine the decreased viability, U-373MG was stained with Hochest 33258 and detected nuclear condensation and apoptic body. Staining results suggest that cells undergo apoptosis.
CONCLUSION
The replicative deficient adenoviral vector can transfer and express p53 in human glioma cell lines in vitro, restoring wild-type p53 tumor suppressor functions. The restoration of normal p53-encoded protein in the mutant ceil lines induced cell death. The high expression of the newly transduced protein had different effects on the growth rate of the infected cell lines depending on the p53 status of the cells.