Combined radiation and p53 gene therapy of malignant glioma cells Behnam Badie, 1 Chern Sing Goh, 1 Jessica Klaver, 1 Hans Herweijer, 2 and David A. Boothman 3 1 Neuro-oncology Laboratory, Department of Neurological Surgery, 2 The Waisman Center, and 3 Department of Human Oncology, University of Wisconsin School of Medicine, Madison, Wisconsin 53792. More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene. Because p53 plays a key role in the cellular response to DNA-damaging agents, we investigated the role of p53 gene therapy before ionizing radiation in cultured human glioma cells containing normal or mutated p53. Three established human glioma cell lines expressing the wild-type (U87 MG, p53 wt ) or mutant (A172 and U373 MG, p53 mut ) p53 gene were transduced by recombinant adenoviral vectors bearing human p53 (Adp53) and Escherichia coli -galactosidase genes (AdLacZ, control virus) before radiation (0 –20 Gy). Changes in p53, p21, and Bax expression were studied by Western immunoblotting, whereas cell cycle alterations and apoptosis were investigated by flow cytometry and nuclear staining. Survival was assessed by clonogenic assays. Within 48 hours of Adp53 exposure, all three cell lines demonstrated p53 expression at a viral multiplicity of infection of 100. p21, which is a p53-inducible downstream effector gene, was overexpressed, and cells were arrested in the G 1 phase. Bax expression, which is thought to play a role in p53-induced apoptosis, did not change with either radiation or Adp53. Apoptosis and survival after p53 gene therapy varied. U87 MG (p53 wt ) cells showed minimal apoptosis after Adp53, irradiation, or combined treatments. U373 MG (p53 mut ) cells underwent massive apoptosis and died within 48 hours of Adp53 treatment, independent of irradiation. Surprisingly, A172 (p53 mut ) cells demonstrated minimal apoptosis after Adp53 exposure; however, unlike U373 MG cells, apoptosis increased with radiation dose. Survival of all three cell lines was reduced dramatically after 10 Gy. Although Adp53 transduction significantly reduced the survival of U373 MG cells and inhibited A172 growth, it had no effect on the U87 MG cell line. Transduction with AdLacZ did not affect apoptosis or cell cycle progression and only minimally affected survival in all cell lines. We conclude that responses to p53 gene therapy are variable among gliomas and most likely depend upon both cellular p53 status and as yet ill-defined downstream pathways involving activation of cell cycle regulatory and apoptotic genes. Key words: Adenoviral vectors; brain neoplasm; gene therapy; glioma; p53; radiation. M utations of the p53 tumor suppressor gene have been reported in more than half of all human cancers, including brain, lung, breast, prostate, thyroid, bladder, colon, liver, leukemia, head and neck, and ovarian cancers. 1,2 p53 may play a central role in cell cycle regulation, DNA repair, and programmed cell death after DNA damage. Alteration of its function can also lead to genomic instability, which is thought to be an early event in glioma oncogenesis. 3 Normal p53 function is important in the cellular response to genotoxic agents causing DNA damage. The p53 protein acts as a tran- scription factor, inducing growth arrest by modulating specific downstream target genes, including p21, which arrests cells in late G 1 by inhibiting cyclin-dependent kinase activity, 4,5 or by up-regulating GADD45, which inhibits DNA synthesis and may activate nucleotide excision repair. 6 Alternatively, p53-mediated regulation of apoptosis is not well understood, and some evidence implies that transactivation of the bax gene (a member of the Bcl-2 family that, in contrast with Bcl-2, has the ability to induce apoptosis) plays a role. 7 Loss of these p53-dependent regulatory functions may, therefore, lead to tumor radioresistance and account for the association between p53 mutations and the poorer prognosis ob- served in some tumors. Despite recent advances in p53 biology, the relation- ship between p53 gene expression and cellular radiosen- sitivity remains unclear. Reports of increased, de- creased, and no apparent differences in radiosensitivities have all been associated with p53 function. 8 –10 In a normal cell, irradiation or other DNA-damaging agents can induce a p53-dependent cell cycle arrest, which presumably allows enough time to repair the damaged cellular genome. Alternatively, if DNA damage is be- yond repair, p53-dependent apoptosis may serve a pro- tective role and prevent the transfer of new mutations to daughter cells. In a neoplastic cell bearing the wild-type Received November 5, 1997; accepted May 4, 1998. Address correspondence and reprint requests to Dr. Behnam Badie, H4/330 Clinical Science Center, Department of Neurological Surgery, University of Wisconsin, 600 Highland Avenue, Madison, WI 53792-3232. E-mail address: badie@neuro-novell.neurosurg.wisc.edu © 1999 Stockton Press 0929-1903/99/$12.00/+0 Cancer Gene Therapy, Vol 6, No 2, 1999: pp 155–162 155