Dataset Information

Mutation of DNA polymerase beta in esophageal carcinoma of different regions.

ABSTRACT:

Aim

To observe the variation of DNA polymerase beta (polbeta) in esophageal carcinoma.

Methods

Thirty specimens containing adjacent normal epithelial tissues were collected from patients in Linzhou region (a high risk area for esophageal squamous carcinoma) and 25 specimens were from a non-high risk area. Total RNA was extracted from the samples and reverse transcription polymerase chain reaction (RT-PCR) was performed. PCR products were cloned and sequenced to investigate the polbeta gene with DNASIS and OMIGA. Statistical significance was evaluated using the chi(2) test.

Results

High-incidence area group: polbeta gene variation was detected in 13 of 30 esophageal carcinoma tissue specimens, and only one variation was found in 30 corresponding adjacent normal tissue specimens. Non high-incidence area group: polbeta gene variation was detected in 5 of 25 esophageal carcinoma tissue specimens, and no variation was found in 25 corresponding adjacent normal tissue specimens. The incidence of polbeta gene variation observed in the high-incidence area group was significantly higher than in the non-high incidence area group. Two mutation hot spots (454-466 and 648-670 nt) and a 58 bp deletion (177-234 nt) were found.

Conclusion

Variations of polbeta perform different functions between the high-incidence areas and the other areas, and may play a more important role in the high-incidence areas.

SUBMITTER: Zhao GQ

PROVIDER: S-EPMC4615399 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Publications

Mutation of DNA polymerase beta in esophageal carcinoma of different regions.

Zhao Guo-Qiang GQ Wang Tao T Zhao Qin Q Yang Hong-Yan HY Tan Xiao-Hui XH Dong Zi-Ming ZM

World journal of gastroenterology 20050801 30

<h4>Aim</h4>To observe the variation of DNA polymerase beta (polbeta) in esophageal carcinoma.<h4>Methods</h4>Thirty specimens containing adjacent normal epithelial tissues were collected from patients in Linzhou region (a high risk area for esophageal squamous carcinoma) and 25 specimens were from a non-high risk area. Total RNA was extracted from the samples and reverse transcription polymerase chain reaction (RT-PCR) was performed. PCR products were cloned and sequenced to investigate the pol ...[more]

PMID: 16094698

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Project description:DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca(2+) and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the "inactive" variant interferes with DNA repair.

Project description: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.

Dataset Information

Mutation of DNA polymerase beta in esophageal carcinoma of different regions.

Aim

Methods

Results

Conclusion

Publications

Mutation of DNA polymerase beta in esophageal carcinoma of different regions.

Similar Datasets

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