<p>Base excision repair (BER) is a DNA repair mechanism that involves the removal of single damaged bases from DNA and their excision as free bases. Another DNA repair mechanism known as nucleotide excision repair (NER) involves the removal of bulky lesions from DNA. Previous published reports have suggested a role for certain NER proteins in BER. Methylene blue (MB) is a type II photosensitizer, which upon excitation by visible light (VL) produces singlet oxygen that reacts with DNA to form 8hydroxyguanine (8-oxoG) lesions. The BER mechanism has been shown to preferentially remove 8-oxoG lesions from DNA. In the current work, the role of NER proteins in the BER of [MB+VL]-induced DNA damage was examined using a reporter gene assay. AdMCMVlacZ and AdHCMVlacZ are non-replicating adenoviruses that express the bacterial β-galactosidase ( β-gal) reporter gene under the control of the murine or human cytomegalovirus immediate early promoter, respectively. Host cell reactivation (HCR) of β-gal activity for a [MB+VL]-treated AdMCMVlacZ was examined in two repair-proficient normal Chinese hamster ovary (CHO) cell lines, NER-deficient mutants from rodent complementation groups (RCGs) 1 through 6, and the HER-deficient cell line EM9. Results show a decreased HCR capacity of [MB+VL]-induced DNA damage in the UV61 (RCG-6; CSB) cell line when compared to the repair-proficient normal AA8. This suggests an involvement of CSB in BER of [MB+VL]-induced damage. In contrast, the XRCCl-deficient EM9 showed an increased HCR capacity when compared to the parental AA8. This suggests a beneficial role for an XRCCl deficiency or for the specific gain-of-function gene mutation in the XRCCl gene for the repair of [MB+VL]induced damage. Similarly, the ERCCl-deficient UV20 (RCG-1) showed an increased HCR capacity when compared to the parental AA8. In contrast, another ERCCJdeficient cell line, 30PV, and the ERCCJ knock-out cell line, CH0-7-27, showed no significant increase in β-gal activity when compared to the parental CHO-Kl. The ability to induce BER of [MB+VL]-induced DNA damage was also examined. HCR of β-gal activity for [MB+VL]-treated AdHCMVlacZ or AdMCMVlacZ was examined in several CHO cell lines that were either untreated or treated with low levels of UVC or MB plus VL. Pre-treatment of NER-deficient UV61 and repair-proficient AA8 cells with UVC resulted in an enhanced HCR for [MB+VL]-treated AdHCMVlacZ, although the results were only significant for UV61. Similarly, an enhanced HCR of [MB+VL]-treated AdMCMVlacZ was observed in AA8 cells, suggesting that the repair of the [MB+VL]treated reporter gene is inducible by UVC. A significant enhancement in HCR of [MB+VL]-treated AdMCMV lacZ was not observed in AA8 cells following pre-treatment with MB, VL, or both, indicating that the detection of any induction in the repair of DNA damage from MB plus VL could not be made with the conditions of the HCR assay employed. As investigations into the effects of MB plus VL on whole cells have been limited, clonogenic survival of BER-and NER-deficient CHO cell lines was also observed following treatment with MB alone or MB plus VL. Results showed that the sensitivities of the NER-deficient CHO cell lines from RCGs 2, 4 and 5 and the BERdeficient EM9 cell line to MB alone were within the range obtained for the two repairproficient CHO normal cell lines, AA8 and K 1. In contrast, the UV20 cell line was more sensitive to MB alone compared to the normal cell lines. Additionally, both UV24 (RCG-3; XPB) and UV61 showed a decreased sensitivity toMB alone when compared to the normal AA8 cell line. The sensitivity of cells to MB plus VL was greater in the UV24, UV135 (RCG-5; XPG) and the XRCCl-deficient EM9 cell lines compared to that of the range obtained for the two repair-proficient normal cell lines, AA8 and Kl. Taken together with the results from the HCR assays, these results suggest that [MB+VL]induced DNA damage to cells and its repair do not play a major role in the survival of cells following treatment with MB plus VL. It appears likely that damage to cellular components other than DNA, such as protein, lipid and biological membranes, play a more important role in cell killing by MB plus VL.</p> / Thesis / Master of Science (MSc)
Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21794 |
Date | 01 1900 |
Creators | Cowan, Robert |
Contributors | Rainbow, A.J., Biology |
Source Sets | McMaster University |
Language | en_US |
Detected Language | English |
Type | Thesis |
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