We used isogenic human tumor cell lines to investigate the specific and direct effects of wild-type (wt) p53 on the expression of
O(6)-methylguanine-DNA methyltransferase (MGMT),
a DNA repair
protein that confers
tumor resistance to many anticancer
alkylating agents. A p53-null, MGMT-proficient lung tumor cell line (H1299) was engineered to express wt p53 in a
tetracycline-regulated system. High levels of p53 induction achieved by
tetracycline withdrawal were accompanied by G(1) cell cycle arrest without significant apoptosis in this cell line. p53 accumulation resulted in a gradual and dramatic loss of MGMT
mRNA,
protein, and
enzyme activity, whose levels were undetectable by day 3 of induction. The loss of MGMT
protein was, however, not due to its degradation because the
ubiquitin-promoted in vitro degradation of MGMT, which mediates the cellular disposal of the repair
protein, was not altered by p53. Run-on transcription assays revealed a significant reduction in the rate of MGMT gene transcription. The negative regulation of MGMT expression by wt p53 was confirmed in two other human isogenic cell lines, namely, the GM47.23
glioblastoma, which contains a
dexamethasone-inducible wt p53, and the H460
lung cancer cell line, in which wt p53 had been inactivated by the human papillomavirus E6
protein. Furthermore, a panel of four human tumor cell lines, including
gliomas with wt p53 status, displayed markedly lower levels of MGMT gene transcripts than those having p53 mutations. Induction of wt p53 in these models led to a 3- and 2-fold increase in sensitivity to
1,3-bis(2-chloroethyl)-1-nitrosourea and
temozolomide, respectively, which generate the MGMT-repairable O(6)-alkyl adducts in
DNA. These results demonstrate that p53 is a negative regulator of MGMT gene expression and can create a MGMT-depleted state in human
tumors similar to that achieved by
O(6)-benzylguanine, a potent inhibitor of MGMT currently undergoing clinical trials. Thus, our study exposes an additional benefit associated with p53 gene
therapy and provides a strong biochemical rationale for combining the MGMT-directed
alkylators with p53 gene transfer to achieve improved antitumor efficacy.