Michael G. Kemp, Ph.D. (Advisor); Jeffrey B. Travers, M.D., Ph.D. (Committee Member); Yong-jie Xu, M.D., Ph.D. (Committee Member)
Master of Science (MS)
The ATR protein kinase is activated in response to DNA damage and other forms of genotoxic stress caused by both environmental carcinogens and anti-cancer drugs. However, much of our understanding of ATR kinase function is limited to proliferating cells in which DNA replication stress is the primary signal for ATR activation and where the major regulatory targets of ATR signaling are proteins involved in DNA synthesis and cell cycle progression. Here we have used HaCaT keratinocytes maintained in a non-replicating, quiescent state in vitro to examine how cell killing by different genotoxic agents is impacted by cell growth status and by treatment with small molecule ATR kinase inhibitors. The genotoxins we examined included drugs from several classes of anti-cancer agents, including topoisomerase inhibitors (camptothecin, etoposide), alkylating agents (mitomycin C, temozolomide, and cisplatin), and compounds that interfere with RNA polymerase movement ((5-6-dichlorobenzimidazole 1-beta-D-ribofuranoside), actinomycin D). As expected, we find that quiescent cells are more resistant to the acute, lethal effects of these genotoxins than replicating cells. However, though we find that nearly all of these compounds led to the activation of ATR kinase signaling in the quiescent state, little-to-no effect of ATR kinase inhibitors was observed on quiescent cell viability. These results indicate that ATR can be activated in the absence of canonical replication stress and that its function does not significantly impact acute cell survival. To examine potential alternative functions for ATR signaling in quiescent cells, we then examined how ATR kinase inhibition impacted the activation of the translesion synthesis (TLS) pathway of DNA synthesis, which involves the use of specialized, potentially mutagenic DNA polymerases to fill in DNA repair gaps and complete DNA repair. Interestingly, we found that ATR kinase inhibition potentiated the activation of this pathway in response to mitomycin C and cisplatin but not following treatment with other genotoxins. Both mitomycin C and cisplatin induce the formation of DNA adducts that are repaired by the nucleotide excision repair system, and thus these results suggest that the ATR kinase may be required to limit the dependence on mutagenic TLS DNA polymerases in quiescent cells. Because ATR kinase inhibitors are currently being tested in clinical trials for use in anti-cancer therapies, this work provides valuable information on the positive and negative consequences of ATR kinase inhibition in quiescent or slowly growing cancer cells.
Year Degree Awarded
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