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Analysis of the Repair of
Topoisomerase II DNA Damage

By: Eric Goldstein | Mentor: Dr. Mark Muller


This project utilized reporter cassettes to analyze the repair of DNA damage induced by topoIIαand topoI poisons. Specific cassettes were employed that report repair of DSBs by either HR or NHEJ (Figure 4). The first cassette, in the case of RY-HR cultures, contains the topoIIα hot spot for the topoIIαpoison and/or an ISce-I restriction endonuclease cut site. The second cassette contains the template sequence to repair the induced DSB in the first cassette. Without drug treatment or pISce-I transfection, GFP is transcribed but not expressed due to mutations in the first sequence and a stop codon in the second sequence (Figure 4). The NHEJ pathway was specifically measured using the NHEJ reporter (Figure 4). With the NHEJ reporter, the formation of wild type GFP cannot proceed by HR due to the lack of a homologous donor sequence. The expression of GFP from the NHEJ reporter cassette can thus only result from a NHEJ event.

The RY-HR GFP reporter systems indicated that, with an increase in VP16 concentrations to 5 μM (Figure 10 - B), there is an increase in HR repair. A dose dependent decrease in the NHEJ pathway (Figure 10 – A) in response to VP16 was also observed. The RY-HR integrated cells showed a dose dependent decrease in HR treated with the topoI poison, CPT (Figure 10-B). The DSB initiated by VP16 appears to be repaired through the high fidelity, error free HR pathway. The high mitotic activity of cancer cells could make the cell cycle dependent HR repair pathway preferential compared to the non-cell cycle dependent NHEJ pathway. Most healthy cells are in the resting G1/G0 phase and, thus are not prone to HR DSB repair.

The data in Figure 9 define the toxicity profile of VP16. The highly specific topoI poison CPT had a higher toxicity than the VP16. HR may well be the preferred DSB repair pathway because the cancer cells are constantly dividing and HR is cell cycle specific. Moreover, HR proteins are associated with specific cell cycle checkpoints, whereas NHEJ is not [14]. Due to the high mitotic levels of most cancer cells, HR should be a prominent pathway for break repair. However, not all cancer cell types follow the HR pathway preferentially. For example, both normal and malignant urothelial cells upregulate the NHEJ pathway, while HR is more dominant in cervical cancer cells [15]. The data and literature suggest that DSB repair pathways are highly variable and may well be dependent on tumor tissue location. Most importantly, since healthy cells preferentially use NHEJ to repair DSBs, a selective agent for HR proteins could sensitize cells to anti-cancer treatments while leaving healthy cells relatively unharmed. One report demonstrated that NHEJ targeting sensitized tumor cells and caused normal cell damage [16]. The targeting of HR could lessen the malevolent impact of chemotherapy on healthy cells, and thereby target cancer cells selectively.

These data may illustrate that HeLa cells utilize the HR pathway over the NHEJ pathway to repair from the exposure of VP16, a topoIIα poison. Future work for this project includes analyzing, through the same reporter constructs, the effects of VP16 and CPT drug treatments on cell lines known for specific repair pathway upregulation. In addition, topoIIβ analysis can be performed based on the two pathways. TopoIIβ has been found to be a causative enzyme of secondary malignancies in cancer patients treated with topoII poisons. Finally, knockout or over expression of specific proteins upregulated in HR such as BRCA1 can be explored for its effects on both pathways via the GFP system.

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