Deficient repair of 8-hydroxyguanine in the BxPC-3 pancreatic cancer cell line

Abstract

Elevated levels of oxidatively induced DNA lesions have been reported in malignant pancreatic tissues relative to normal pancreatic tissues. However, the ability of the pancreatic cancer cells to remove these lesions has not previously been addressed. This study analyzed the effectiveness of the pancreatic cancer cell line, BxPC-3 to repair 8-hydroxyguanine (8-OH-Gua) relative to a nonmalignant cell line. We show that BxPC-3 cells repair 8-OH-Gua less effectively than the nonmalignant cells. This repair deficiency correlated with significant downregulation of the hOGG1 protein and the corresponding mRNA (30-fold lower than GAPDH) in BxPC-3 cell line. The repair defect was complemented in vivo by transient transfection of the hOGG1 gene and in vivo by recombinant hOGG1. These results are the first to show a deficiency of 8-OH-Gua repair in BxPC-3 cells, implicating this defect in the risk factor of pancreatic cancer.

Fig. 1

BER of 8-OH-Gua. Nuclear extracts (50 µg) were incubated at 37 °C with a duplex oligonucleotide containing a single 8-OH-Gua. After removing the aliquots the reactions were stopped and the products separated by electrophoresis. (A) Gel showing qualitative repair of 8-OH-Gua by AG11134, (B) gel showing qualitative repair of 8-OH-Gua by BxPC-3, and (C) quantified products of 8-OH-Gua repair by AG11134 (clear bars), and BxPC-3 (black bars). Lane 1: oligonucleotide size markers (8–32 bp), lanes 2–5: repair kinetics (0 h, 2 h, and 4 h, respectively), lane 6: hOGG1, and lane 7: undamaged duplex oligonucleotide plus extracts. At least three independent experiments were performed for each cell line and the error bars represent ±SEM. The asterisks denote a statistically significant difference (p < 0.05).

Fig. 2

Expression of hOGG1 protein and hOGG1 mRNA. (A) Western blot analysis of hOGG1 using 50 µg of nuclear extracts. Beta actin was used as a loading control. The gel shown is representative of at least three independent repeats. (B) Analysis of hOGG1 mRNA levels by quantitative real-time RT-PCR. The clear bars represent AG11134 cell line and the black bars represent BxPC-3 cell line. The first two bars represent GAPDH mRNAs while the last two represent hOGG1 mRNAs. The results represent at least six independent repeats and the error bars represent ±SEM. The asterisks denote a statistically significant difference (p < 0.05).

Fig. 3

Complementation of 8-OH-Gua-repair by transient transfection of the hOGG1. BxPC-3 cells were transfected with a plasmid carrying the hOGG1 gene or the vector alone. Nuclear extracts were subsequently prepared and the BER assay performed. (A) Expression of hOGG1, (B) repair of 8-OH-Gua by nuclear extracts from cells transfected with vector alone, (C) repair of 8-OH-Gua by nuclear extracts from cells transfected with plasmid carrying the hOGG1, and (D) quantified results. Lane 1: oligonucleotide size markers (8–32 bp), lanes 2–6: repair kinetics (0 h, 1 h, 2 h, 4 h, and 6 h, respectively), lane 7: hOGG1, and lane 8: undamaged duplex oligonucleotide plus extracts. The gray bars represent repair of 8-OH-Gua by extracts from hOGG1-transfected cells and black bars represent repair of 8-OH-Gua by extracts from empty vector-transfected cells. At least three independent experiments were performed and the error bars represent ±SEM. The asterisks denote a statistically significant difference (p < 0.05).

Fig. 4

Stimulation of 8-OH-Gua-repair by recombinant hOGG1. Recombinant hOGG1 protein was added to increasing amounts of BxPC-3 nuclear extracts and repair reactions performed. (A) Gel showing qualitative repair products, and (B) quantified results. Gray bars represent BxPC-3 reactions supplemented with recombinant hOGG1 and black bars represent BxPC-3 alone. At least three independent reactions were performed and the error bars represent ±SEM. The asterisks denote a statistically significant difference (p < 0.05).