Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients
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
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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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 1

APNG expression in GBM.

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APNG expression in GBM. (A) Western blot of APNG and MGMT in several GBM...
(A) Western blot of APNG and MGMT in several GBM cell lines demonstrating variable expression levels. NHA, normal human astrocytes. (B) IF of A172 and T98G GBM cells (APNG-negative and -positive, respectively) stained with DAPI (blue) for nuclei. APNG expression (green), which was seen only in T98G cells, colocalized to the nucleus (yellow in merged image). (C) Cell viability of GBM cell lines in response to TMZ. (D) DNA damage, measured as the number of AP sites in response to TMZ. (E) Real-time qRT-PCR of APNG and MGMT. ΔCt values are reported by subtracting the Ct value for β-ACTIN housekeeping gene from that for APNG or MGMT. Pearson r2 values and P values are shown. (F) Chemiluminescent densitometric analysis of APNG and MGMT protein levels, normalized to housekeeping gene β-ACTIN. Pearson r2 values and P values are shown. *P < 0.05, **P < 0.01.