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. 2015 Jan 1;11(2):144-55.
doi: 10.7150/ijbs.10692. eCollection 2015.

Clinical significance of a point mutation in DNA polymerase beta (POLB) gene in gastric cancer

Xiaohui Tan  1 , Hongyi Wang  2 , Guangbin Luo  3 , Shuyang Ren  4 , Wenmei Li  4 , Jiantao Cui  4 , Harindarpal S Gill  5 , Sidney W Fu  5 , Youyong Lu  4
Affiliations

Clinical significance of a point mutation in DNA polymerase beta (POLB) gene in gastric cancer

Xiaohui Tan et al. Int J Biol Sci. .

Abstract

Gastric cancer (GC) is a major cause of global cancer mortality. Genetic variations in DNA repair genes can modulate DNA repair capability and, consequently, have been associated with risk of developing cancer. We have previously identified a T to C point mutation at nucleotide 889 (T889C) in DNA polymerase beta (POLB) gene, a key enzyme involved in base excision repair in primary GCs. The purpose of this study was to evaluate the mutation and expression of POLB in a larger cohort and to identify possible prognostic roles of the POLB alterations in GC. Primary GC specimens and their matched normal adjacent tissues were collected at the time of surgery. DNA, RNA and protein samples were isolated from GC specimens and cell lines. Mutations were detected by PCR-RFLP/DHPLC and sequencing analysis. POLB gene expression was examined by RT-PCR, tissue microarray, Western blotting and immunofluorescence assays. The function of the mutation was evaluated by chemosensitivity, MTT, Transwell matrigel invasion and host cell reactivation assays. The T889C mutation was detected in 18 (10.17%) of 177 GC patients. And the T889C mutation was associated with POLB overexpression, lymph nodes metastases and poor tumor differentiation. In addition, patients with- the mutation had significantly shorter survival time than those without-, following postoperative chemotherapy. Furthermore, cell lines with T889C mutation in POLB gene were more resistant to the treatment of 5-fluorouracil, cisplatin and epirubicin than those with wild type POLB. Forced expression of POLB gene with T889C mutation resulted in enhanced cell proliferation, invasion and resistance to anticancer drugs, along with increased DNA repair capability. These results suggest that POLB gene with T889C mutation in surgically resected primary gastric tissues may be clinically useful for predicting responsiveness to chemotherapy in patients with GC. The POLB gene alteration may serve as a prognostic biomarker for GC.

Keywords: DNA Repair; DNA polymerase beta (POLB); chemotherapy.; gastric cancer; point mutation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Identification of the T889C mutation in primary GCs with PCR-RFLP, DHPLC and DNA sequencing. A. Restriction analysis of the POLB gene fragment amplified by PCR. RFLP patterns obtained after digestion of PCR products with Taq1 endonucleases. Primers were designed to amplify the region spanning 3' region of intron 12 to 5' region of exon 13 in POLB gene and length of the PCR products were 260bp. T889C mutation formed of a new Taq1 site from nucleotide 888 to 891 (TTGA to TCGA) of POLB (NM_002690.1) cDNA and therefore, the amplified product could be digested with Taq1. The molecular weights of digested fragments were 149bp and 111bp. B. The digested fragments were separated by 2.0% agarose gel electrophoresis. 100-bp ladder was used as a size marker. PCR products (260bp) amplified from BGC823, MGC803, SGC7901 and PAMC82 cell lines (carrying the T889C mutation) could be digested by Taq1, the molecular weights of digested fragments were 149 and 111bp while those from AGS, N87 and MKN45 cell lines couldn't be digested by Taq1. C. Restriction patterns obtained by Taq1 digestion of amplified product from GC tissues. The digested fragments were separated by 2.0% agarose gel electrophoresis and 100bp ladder was used as a size marker. PCR products (260bp) with T889C mutation could be digested by Taq1, the molecular weights of digested fragments were 149 and 111bp while the PCR products with wild type POLB couldn't be digested by Taq1. D. Chromatograph of DHPLC and sequencing analysis.
Figure 2
Figure 2
POLB expression on tissue array by immunohistochemistry, RT-PCR and Western blot analysis in GC patients. A, B and C. With T889C mutation; D and E. Without T889C mutation; F. Normal tissue. G. RT-PCR (top) and Western blot (bottom) analysis of POLB gene in GC tissues with-T889C mutation (lanes 1-3) and without- (lanes 5-6).
Figure 3
Figure 3
Correlation between T889C mutation and survival. Progression-free survival (top) and overall survival (bottom) for all 19 patients enrolled. The statistical significance of the difference was p = 0.027 and p = 0.026. Patients with T889C mutation had significantly shorter survival than did patients without the mutation after postoperative chemotherapy.
Figure 4
Figure 4
Correlation between T889C mutation and cytotoxicity of 5-fluorouracil, cisplatin and epirubicin. A. Phenotypic comparison of BGC823, SGC7901 and AGS cell lines before and after treatment. B. Sensitivity of cell lines to cisplatin, epirubicin, 5-fluorouracil and combination treatment with three drugs. Concentration of 5-fluorouracil, cisplatin, epirubicin, and was 5ug/ml, 0.5ug/ml and 0.1ug/ml respectively. The concentration of combination treatment of three drugs is cisplatin 0.5ug/ml, epirubicin 0.1ug/ml, and 5-fluorouracil 5ug/ml. Survival is expressed as the relative plating efficiency of treated cells to the control. The values shown in the figures are mean readings from six wells in each experiment and representative of at least three independent experiments. C. Protein expression of POLB in cell lines BGC823, SGC7901, AGS and N87.
Figure 5
Figure 5
The effect of POLB overexpression. AGS cells were transfected with empty pcDNA3.1, wild type POLB, T889C mutant and POLB siRNA. A. Expression of transfected POLB genes in human GC cell lines. Left panel, representative results of the QRT-PCR analysis of POLB mRNA levels in AGS cell lines. Each reaction was run twice in triplicates. POLB expression was normalized by GAPDH. Right panel, representative results of the Immunofluorescence for POLB protein expression. POLB protein of both the wild-type and mutant localized to the nucleus. B. The effect of POLB wild type, mutant and siRNA in AGS cell proliferation. MTT assays indicate cell proliferation rate was significantly higher in T889C overexpressors (left). The experiments were done three times in triplicates. Right panel shows phenotypic comparison of AGS cell lines before and after transfection of POLB wild type and mutant and siRNA. C. Matrigel analysis. Three fields of unit area on each membrane were counted for cell numbers, and the experiments were repeated twice. Data analysis was based on the average of parallel repeats. T889C mutation overexpressing cell lines exhibited very significantly higher invasiveness than wild type and empty vector control cell lines. D. Resistance assay. The cells were treated with or without 5-fluorouracil, cisplatin, and epirubicin for 24 h. The values are represented as a mean from three separate experiments; bars, ±SD. E. HCR assay showing increased post-UV DNA repair capability in T889C overexpressors. *p < 0.05.
Figure 6
Figure 6
A hypothetical model of the human POLB enzyme is shown in complex with DNA, and containing the L259S mutation that is found commonly in gastric cancer patients. The two known metal ions bound in the active site are shown as magenta spheres. A. The entire enzyme is shown revealing the relative location of the serine in proximity to the active site. B. A close-up view of additional hydrogen-bond networks is shown in a β-sheet that would lead to a predicted small distortion in the active site. The figure was made with the program, MOLSCRIPT .
See this image and copyright information in PMC

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