Abstract
BackgroundBreast cancer is the second leading cause of cancer deaths in women in the United States. Although the causes of this disease are incompletely understood, oxidative DNA damage is presumed to play a critical role in breast carcinogenesis. A common oxidatively induced DNA lesion is 8-hydroxyguanine (8-OH-Gua), which has been implicated in carcinogenesis. The aim of this study was to investigate the ability of HCC1937 and MCF-7 breast cancer cell lines to repair 8-OH-Gua relative to a nonmalignant human mammary epithelial cell line, AG11134.MethodsWe used oligonucleotide incision assay to analyze the ability of the two breast cancer cell lines to incise 8-OH-Gua relative to the control cell line. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the levels of 8-OH-Gua as its nucleoside, 8-OH-dG in the cell lines after exposure to H2O2 followed by 30 min repair period. Protein expression levels were determined by Western blot analysis, while the hOGG1 mRNA levels were analyzed by RT-PCR. Complementation of hOGG1 activity in HCC1937 cells was assessed by addition of the purified protein in the incision assay, and in vivo by transfection of pFlagCMV-4-hOGG1. Clonogenic survival assay was used to determine sensitivity after H2O2-mediated oxidative stress.ResultsWe show that the HCC1937 breast cancer cells have diminished ability to incise 8-OH-Gua and they accumulate higher levels of 8-OH-dG in the nuclear genome after H2O2 treatment despite a 30 min repair period when compared to the nonmalignant mammary cells. The defective incision of 8-OH-Gua was consistent with expression of undetectable amounts of hOGG1 in HCC1937 cells. The reduced incision activity was significantly stimulated by addition of purified hOGG1. Furthermore, transfection of pFlagCMV-4-hOGG1 in HCC1937 cells resulted in enhanced incision of 8-OH-Gua. HCC1937 cells are more sensitive to high levels of H2O2 and have up-regulated SOD1 and SOD2.ConclusionThis study provides evidence for inefficient repair of 8-OH-Gua in HCC1937 breast cancer cell line and directly implicates hOGG1 in this defect.
Highlights
Breast cancer is the second leading cause of cancer deaths in women in the United States
HCC1937 nuclear extracts but significantly overexpressed in MCF-7 nuclear extracts (Fig. 4A). These results suggest that the inefficient incision of 8-OH-Gua in HCC1937 cells could be due to undetectable levels of the hOGG1 in HCC1937
In order to determine whether the reduced expression level of hOGG1 in the two breast cancer cell lines correlated with transcriptional regulation, we examined the expression of hOGG1 1a mRNA by RT-PCR
Summary
Breast cancer is the second leading cause of cancer deaths in women in the United States. The aim of this study was to investigate the ability of HCC1937 and MCF-7 breast cancer cell lines to repair 8-OH-Gua relative to a nonmalignant human mammary epithelial cell line, AG11134. We show that HCC1937 breast cancer cells are deficient in the repair of 8-OH-Gua when compared to nonmalignant mammary epithelial cells. We further show that HCC1937 breast cancer cells express undetectable levels of hOGG1. These cells accumulate elevated levels of 8-OH-Gua after H2O2 challenge despite a cellular repair period at 37°C. This study directly implicates hOGG1 in the defective repair of 8-OH-Gua in HCC1937 breast cancer cells
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