Resistance of glioblastoma cells to alkylating agents: Role of tumor suppressor p53 and DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT)

Glioblastoma multiforme is among the most malignant class of CNS tumors, and resistance of a fraction of tumor cells surviving chemo and radiotherapy is the primary reason for failure of therapy. Chemotherapeutic agents cause death of tumor cells through DNA-damage induced activation of apoptosis, and wild type p53 (WT-p53) plays an important role in this response. Here, we demonstrate that p53-status is a key factor that modulates resistance to monofunctinal alkylating agent Temozolomide (TMZ), but does not affect resistance to bifunctional alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We have used a collection of glioblastoma (GBM) lines with WT- and mutant-p53 (mut-p53), and have characterized their O6-methyguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) activities, and their resistance to alkylating agents. Based on these preliminary results we hypothesized that resistance of these cell lines involves a deficiency of p53-dependent apoptosis, and to investigate this effects we used WT- and mut-p53 transfected glioblastoma cells, derived from the cell lines described above; these nearly isogenic pairs of cell lines have allowed us to determine differences in the mechanisms of response to DNA damage. Expression of MGMT, a ubiquitous DNA repair protein that reverses mutagenic, and cytotoxic O 6-alkylguanine adducts induced by chemotherapeutic N-alkyl N-nitrosourea and procarbazine type drugs, is downregulated by wild type p53 (WT-p53) in human tumor cells. In this study, we report that the downregulation is caused by interference of p53 binding of the three Sp1 consensus sequences in the MGMT promoter closest to the transcription initiation site. Transient co-transfection of a p53-null colon carcinoma cell line (HCT116 −/−) with a MGMT promoter-luciferase reporter (containing 1 Kb of the region 5 ′ to the promoter sequence) and WT-p53 induced a 10-fold decrease in the activity of the MGMT promoter, while two mutant forms of p53 were unable to downregulate MGMT promoter activity in a similar manner. Co-transfection of WT-p53 with Sp1 transcription factor overcame the inhibition of MGMT promoter activity by p53 in a dose dependent manner. This effect was also observed with a minimal MGMT promoter (containing only the three Sp1 binding sequences 5' to the initiation site). P53 was able to co-immunoprecipitate with Sp1 from lysate of HCT116 −/− cells transiently transfected with p53 and Sp1, suggesting a direct interaction between the two. Moreover, overexpression of p53 affected binding of nuclear extract from transiently transfected HCT116 −/− cells to a labeled Sp1 consensus binding sequence, even though recombinant p53 did not bind to the same sequence in vitro. These results indicate that the interaction between Sp1 and p53 may cause the downregulation of MGMT promoter activity, and suggest a mechanism for this effect.; A better understanding of the interlocking roles of p53, and repair pathways, and their effects in tumor resistance is essential to develop improved treatments for this highly malignant class of tumors.