Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents.
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.
Srivenugopal
K S
KS
Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. ksrivenu@mdanderson.org
Shou
J
J
Mullapudi
S R
SR
Lang
F F
FF
Rao
J S
JS
Ali-Osman
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004354:Drug Screening Assays, Antitumor; D005455:Fluorescent Antibody Technique / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014452:Ubiquitins / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010447:Peptide Hydrolases