Generalized Death of Human Glioma Cells via Apoptosis Gene Produces Rapid p 53 Adenovirus-mediated Transfer of the Updated Version

In this study, an adenovirus (Ad5CMV-p533; Ref. 5) was used to transfer wild-type p53 cDNA into human malignant glioma cell lines. The exogenous p53 gene was transferred efficiently, and the exoge nous p53-encoded protein was expressedhighly in six human glioma cell lines. Apoptosis was induced in cell lines possessing mutant p53 genes, and glioma cell lines possessing wild-type p53 genes under went growth inhibition but no apoptosis. This report presents a strat egy for the delivery of the p53 gene into human glioma cells using a recombinant adenovirus, and our in vitro data suggest that a new approach to brain tumor treatment based on tumor suppressor gene transfer strategies is possible and effective. described previously (6). The sequencing reactions were carried out using the terminationmixtureof a Sequenase kit (UnitedStatesBiochemical, Cleveland, OH) andanalyzedon a 6% polyacrylamidegel containing7 M ureafor 2 h at 40 W, and the dried gel was exposed to Kodak XAR5 film (Eastman Kodak Co., Rochester, NY) overnight.The procedurefor automatedsequencing has been reported already (6). The experiments were repeated three times using three different PCR reactions to confirm every mutation. Restriction Enzyme Analysis. A portion of the PCR-amplified product (1.5 pJ)fromtheU-138, LG, andA-172 cell lineswassubjected to A1wNI (GIBCO-BRL, GrandIsland, NY) restriction enzymedigestionat 37°C for 4 h. All samples were analyzed by 3% NuSieve agarose (FMC BioProducts, Rockland, ME) gel electrophoresis. The enzyme did not cut the PCR product if the transversionfrom G:C to T:A at codon 242 was present, renderingtwo different possiblepatterns:(a) homozygousfor the mutation, only one fragmentof 563 nucleotides;and (b) heterozygousfor the muta tion, two fragments of 346 and 217 nucleotides each.