student research day - Case Western Reserve University School of ...

Ben Tomlinson
The impact of base excision repair activity on the sensitivity to
temozolomide and the combination with methoxyamine in
melanoma cell lines in vitro and in xenograft tumors
Ben Tomlinson, Yanling Miao, Alina Bulgar, and Jon Donze and Lili Liu
Department of Oncology
University Hospitals and Case Western Reserve University
Methoxyamine (MX) blocks the base excision repair (BER) pathway by binding to abasic sites. These
sites are generated after glycosylases remove alkylated purines and pyrimidines that have been methylated by
the anti-cancer drug temozolomide (TMZ) that is an effective drug used in clinics for melanoma treatment. We
hypothesized that MX would sensitize melanoma cells to TMZ through its ability to specifically bind to abasic sites,
resulting in the blockage of BER pathway and accumulation of DNA damage. We tested our hypothesis in two
melanoma cell lines, A375 and WM164, by evaluating the expression of BER proteins and sensitivity to the TMZ
alone and in combination with MX in vitro and in xenograft setting. Results showed that A375 and WM164 cell
lines have normal MMR repair function and similar MGMT activity, which are well known TMZ resistant factors.
However, differential levels of BER proteins were detected in these two cell lines. In comparison with A375 cells,
the expression of BER proteins (including MNP, UDG, APE, and Pol ) were lower in WM164 cells. The sensitivity
to TMZ alone in these two cell lines was assessed by IC90 values generated by clonogenic survival assay,
showing 480 µM in A375 and 570 µM in WM164 cells. MX efficiently potentiated TMZ cytotoxicity in A375 but
failed to enhance the killing effect of TMZ in MW164 cells. Similar results were observed in nude mice carrying
human A375 or WM164 xenograft tumors that were treated with TMZ (80 mg/kg) or the combination of TMZ and
MX (2 mg/kg), ip injection daily for 5 days. The results suggest that the failure of TMZ-potentiation by MX in
WM164 cells was related to the low activity of BER proteins, particularly low levels DNA glycosylases (UDG,
MPG, OGG1). Deficiency of these BER proteins would decrease the formation of abasic sites, which are the
targets for MX action. Thus, results indicate that BER proteins may be differentially expressed in tumor cells that
impact the therapeutic effect of TMZ and the combination of TMZ and MX.
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