Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients
Sameer Agnihotri, … , Monika Hegi, Abhijit Guha
Sameer Agnihotri, … , Monika Hegi, Abhijit Guha
Published December 12, 2011
Citation Information: J Clin Invest. 2012;122(1):253-266. https://doi.org/10.1172/JCI59334.
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Research Article Oncology

Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients

Abstract

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O6-methylguanine–DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O6-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine–DNA–N-glycosylase (APNG), which repairs the cytotoxic lesions N3-methyladenine and N7-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

Authors

Sameer Agnihotri, Aaron S. Gajadhar, Christian Ternamian, Thierry Gorlia, Kristin L. Diefes, Paul S. Mischel, Joanna Kelly, Gail McGown, Mary Thorncroft, Brett L. Carlson, Jann N. Sarkaria, Geoffrey P. Margison, Kenneth Aldape, Cynthia Hawkins, Monika Hegi, Abhijit Guha

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Figure 4

APNG affects in vitro transformation.

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APNG affects in vitro transformation. (A) Anchorage-independent growth a...
(A) Anchorage-independent growth assay of A172 cells grown in soft agar with 100 μM TMZ. Scale bar: 100 μm. (B) Summary of anchorage-independent growth assay. Black bars, colony number; white bars, colony size. (C and D) Direct measurement of alkylated (methylated) N7-guanine DNA adducts in A172 and T98G cells. (E and F) Cell viability assay of A172 cells expression and T98G cells with knockdown of APNG, MGMT, or both, treated with varying concentrations of MMS. MMS generated N7-guanine and N3-adenine, but not O6-guanine, alkylated bases. *P < 0.05, **P < 0.01, ***P < 0.001.