O⁶‐methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Abstract

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O 6 -methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O 6 -alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X L . We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r 2 ¼ 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O 6 -benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X L protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ. Keywords: BCL-X L , glioblastoma, O 6 -methylguanine DNA methyltransferase, p53, temozolomide. Temozolomide (TMZ) is a cytotoxic alkylating agent which has shown activity in recurrent anaplastic glioma and glioblastoma (Yung et al. 1999(Yung et al. , 2000. Moreover, the recent European Organisation for Research and Treatment of Cancer/National Cancer Institute of Canada trial on concomitant and adjuvant TMZ in addition to radiotherapy as the first-line treatment of glioblastoma produced an increase in median survival from 12.1 to 14.6 months and an increase in the 2-year survival rate from 10 to 26% compared with radiotherapy alone (Stupp et al. 2005).Temozolomide is rapidly and completely absorbed after oral administration and undergoes spontaneous hydrolysis at physiological pH to its active metabolite 5-(3-methyltriazeno)-imidazole-4-carboxamide. 5-(3-methyltriazeno)-imidazole-4-carboxamide causes DNA damage by methylation of the O 6 position of guanine. However, DNA adducts different from O 6 -methylguanine might also be involved in the cytotoxicity induced by methylating agents and poly (ADP-ribose) polymerase (PARP) inhibitors may also sensitize tumour cells to TMZ-induced cell death (Tentori e...

p53 promotes cellular survival in a context-dependent manner by directly inducing the expression of haeme-oxygenase-1

“…Similarly, p53 was shown to function as a survival factor in the gastrointestinal tract under conditions of severe irradiation, which was attributed to increased sensitivity of vascular endothelial cells (Komarova et al, 2004;Burdelya et al, 2006). Furthermore, other reports have identified conditions in which absence of p53 led to elevated apoptosis, but a direct causal role for p53 in providing survival signals has not been described (Lassus et al, 1996;Lackinger and Kaina, 2000;Hermisson et al, 2006;Batista et al, 2007;Roepke et al, 2007;Roos et al, 2007). Notwithstanding these occasional reports, the major observations that p53 activation often leads to apoptosis or DNA repair have been overwhelming, and hence, have overshadowed the exploration of the survival-promoting properties of p53.…”

“…For example, absence of p53 has been shown to promote taxol-mediated death owing to failure of cell arrest in the G 1 phase of the cell cycle, and hence, allowing cells to accumulate in the G 2 /M phase where they undergo mitotic cell death (Vikhanskaya et al, 1998). In addition, cells with wild-type p53 have been shown to be more resistant to cytotoxic drug treatment in some cases (Hermisson et al, 2006;Batista et al, 2007;Roos et al, 2007). Other reports have also shown that absence of p53 leads to sensitization to UV irradiation and alkalyting agents in mouse embryonic fibroblasts (MEFs), and to thymoquinone-induced apoptosis in human cells (Lackinger and Kaina, 2000;Roepke et al, 2007).…”

MGMT promoter methylation in malignant gliomas: ready for personalized medicine?

“…Neither dexamethsone nor irradiation induced MGMT gene transcription in glioma cells in vitro. 68 In tissue culture and animal studies, however, temozolomide strongly induced MGMT protein expression in temozolomide-resistant glioma cells lacking MGMT promoter methylation. 69 Loss of MGMT promoter methylation might represent a key mechanism by which patients with initially methylated tumors eventually acquire resistance to temozolomide, leading to progression or relapse.…”

Downregulation of PRDX1 by promoter hypermethylation is frequent in 1p/19q-deleted oligodendroglial tumours and increases radio- and chemosensitivity of Hs683 glioma cells in vitro

“…For assessment of TMZ concentrations and doses of radiation the protocol of Hermisson et al (2006) was adapted. Cytotoxicity assays were done afterwards to optimize the doses for Hs683 cells (data not shown).…”