Human glioma cell sensitivity to the sequence-specific alkylating agent methyl-lexitropsin
Michael S Bobola1, Sridhar Varadarajan, Nolan W Smith
1Department of Neurological Surgery, University of Washington, Seattle, WA 98105, USA.
Purpose:
Defining the cytotoxicity of individual adducts in DNA is necessary for mechanistic understanding of human brain tumor resistance to therapeutic alkylating agents and for design of DNA repair-related antiresistance strategies. Our purpose is to characterize the sensitivity of human glioma cells to methyl-lexitropsin (Me-lex), a sequence-specific alkylator that produces 3-methyladenine (3-meA) as the predominant (>90%) DNA lesion.
Experimental Design:
We quantitated the Me-lex cytotoxicity of 10 human glioma cell lines that differ in O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT) and mismatch repair activity. We used antisense suppression of alkyladenine DNA glycosylase (AAG) and Ape1 to assess the contribution of 3-meA and abasic sites to lethality and measured abasic sites.
Results:
(a) The LD(10) for Me-lex varied widely among the cell lines. (b) MGMT-proficient lines were more resistant than MGMT-deficient lines, an unexpected finding because Me-lex produces very little O(6)-meG. (c) Suppression of AAG increased Me-lex killing and reduced abasic site content. (d) Suppression of Ape1 increased Me-lex killing and increased abasic site content. (e) Ablation of MGMT had no effect on Me-lex cytotoxicity.
Conclusions:
(a) Me-lex is cytotoxic in human glioma cells and AAG promotes resistance, indicating that 3-meA is a lethal lesion in these cells. (b) Abasic sites resulting from 3-meA repair are cytotoxic and Ape1 promotes resistance to these derivative lesions. (c) A factor(s) associated with MGMT expression, other than repair of O(6)-meG, contributes to Me-lex resistance. (d) Me-lex may have clinical utility in the adjuvant therapy of gliomas. (e) AAG and Ape1 inhibitors may be useful in targeting alkylating agent resistance.
Insights
Methyl-lexitropsin (Me-lex) is cytotoxic to human glioma cells, with 3-methyladenine (3-meA) lesions being lethal. Alkyladenine DNA glycosylase (AAG) promotes resistance, while abasic sites also contribute to Me-lex cytotoxicity.
Area of Science:
- Molecular Biology
- Cancer Research
- DNA Damage and Repair
Background:
- Understanding DNA adduct cytotoxicity is crucial for brain tumor resistance mechanisms and developing antiresistance strategies.
- Methyl-lexitropsin (Me-lex) is a sequence-specific alkylating agent primarily forming 3-methyladenine (3-meA) DNA lesions.
Purpose of the Study:
- To characterize the cytotoxicity of Me-lex in human glioma cells.
- To investigate the roles of O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT), alkyladenine DNA glycosylase (AAG), and Ape1 in Me-lex resistance.
Main Methods:
- Quantified Me-lex cytotoxicity across 10 human glioma cell lines with varying MGMT and mismatch repair activity.
- Utilized antisense suppression of AAG and Ape1 to assess the contribution of 3-meA and abasic sites to Me-lex lethality.
- Measured abasic site formation.
Main Results:
- Me-lex exhibited wide-ranging cytotoxicity among glioma cell lines.
- MGMT-proficient lines showed unexpected resistance to Me-lex.
- Suppression of AAG enhanced Me-lex killing and reduced abasic sites, indicating 3-meA is a lethal lesion.
- Suppression of Ape1 increased Me-lex killing and abasic sites, suggesting cytotoxicity of these derivative lesions.
- MGMT ablation did not affect Me-lex cytotoxicity.
Conclusions:
- 3-methyladenine (3-meA) is a lethal DNA lesion in human glioma cells, and AAG promotes resistance.
- Abasic sites, arising from 3-meA repair, are also cytotoxic, with Ape1 contributing to resistance against these lesions.
- Factors associated with MGMT expression, beyond O(6)-meG repair, influence Me-lex resistance.
- Me-lex holds potential as an adjuvant therapy for gliomas.
- Inhibitors of AAG and Ape1 could be valuable in overcoming alkylating agent resistance.