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Background: DNA polymerase β is one of the key enzymes for DNA repair and it was reported that about 30 percent of different types of cancers carried mutations in its coding gene Polb. However, it is still controversial whether it is true or false because of the small sample size in these studies. In current study, we performed genetic screening of promoter and coding regions of Polb gene in 69 Chinese lung cancer patients using Sanger sequencing method, so as to elucidate real mutation frequency of Polb mutations in Chinese Han population., Methods: Salting out extraction method was used to get the genome DNAs from tumor and normal matched tissues of 69 lung cancer patients. The promoter and 14 coding regions of Polb gene were then amplified using these DNAs as the template. After purification, amplicons were sequenced and aligned to the wild type Polb gene in NCBI database, in order to find out the mutated sites of Polb gene in Chinese lung cancer patients., Results: In this study, we totally found only 5 mutated sites in Polb gene. In detail, 3 mutations (-196G>T, -188_-187insCGCCC, -168C>A) were located in the promoter region; 2 mutations (587C>G, 612A>T) were found in coding regions. Specially, mutations of -188_-187insCGCCC and 587C>G (resulting to the amino acid substitution of Thr to Ser at position 196) had never been reported by other groups before. However, all these 5 mutated sites could be detected in both tumor and matched normal tissues, which inferred that they are not lung tumor specific mutations., Conclusions: No lung tumor specific mutations of Polb gene could be found in Chinese lung cancer patients and Polb gene mutation might not be a molecular marker for Chinese lung cancer patients.

Objective: To employ single nucleotide polymorphisms (SNP) microarray to detect copy number variations (CNVs) for the diagnosis of disease and molecular classification., Methods: For a patient with split-hand/split-foot malformation, genome-wide copy number variants SNP microarray was applied. Tiny copy number variations were verified by real-time fluorescent quantitative PCR., Results: The results of SNP microarray has revealed that the patient has carried a 0.39 Mb duplication in 10q24.31-24.32 (102 955 122-103 348 688), which has encompassed genes including LBX1, BTRC and POLL. By real-time fluorescent quantitative PCR, duplicate area encompassing the pathogenic genes have been verified. The results for LBX1, BTRC, POLL genes were all consistent with the SNP microarray test. Moreover, a duplication was detected in exon 9 of FBXW4 gene which is in nearby., Conclusion: SNP chips can efficiently identify tiny CNVs (< 1.0 Mb). In combination with real-time fluorescence quantitative PCR, this may provide valuable information for prenatal diagnosis.

Objective: To explore the mechanism of the hyper-expression of DNA polymerase beta (polbeta) in benzo[a]pyrene (BaP) induced malignant transformed cell (polbeta-T)., Methods: The mutation of polbeta gene exon and promoter were examined using reverse transcriptase-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) and gene sequencing. The expression of protein-arginine N-methyhransferase 6 (PRMT6) mRNA and protein in polbeta-T cell and control cell (polbeta cell) were investigated by RT-PCR and Western blot., Results: RT-PCR-SSCP and gene sequencing revealed that the hyper-expression of polbeta in polbeta-T cell was not associated with the mutation of polbeta gene exon while insert mutation (G) and point mutation (C-->A) were found located in the core region of polbeta gene promoter. Furthermore, the expression of PRMT6 mRNA and protein also increased in polbeta-T cell compared with control cell (P<0.05)., Conclusion: The enhancement of expression of polbeta in polbeta-T cell might be attributed to the mutations locating in polbeta gene promoter on transcription level of polbeta gene, and PRMT6 might also enhance the expression of polbeta in polbeta-T cell through relative epigenetic pathways.

Objective: To explore the relationship between the expression level of DNA polymerase beta (pol beta) and 60Co gamma-ray radiosensitivity and provide a basis on improving the efficiency of radiotherapy theoretically., Methods: pol beta wild-type cells (pol beta +/+), pol beta null cells (pol beta -/-) and pol beta overexpressed cells (polp beta oe) were applied as a model system. The radiosensitivity of 60Co gamma-ray on the cell was detected by MTT assay and clone formation assay. The DCFH-DA fluorescent probe was used to examine the cellular ROS after 60Co gamma-rays radiation., Results: MTT assay showed that after radiation by 60Co gamma-rays followed with 72 h incubation, the cell viabilities in the three kinds of cells decreased significantly with a dose-response relationship (r-/+ = -0.976, r-/- = -0.977, r(oe) = -0.982, P<0.05). In addition, the viability of pol beta -/- cell was lower than those of other two kinds of cells at the same dose (P<0.05). Likewise, the colony number and colony formation rate in all tested cells also decreased after exposure to 60Co gamma-rays. The ROS level in the three kinds of cells was enhanced after treatment with 60Co gamma-ray, and the ROS level in pol beta -/- cells was much higher than that in the other two kinds of cells (P<0.05)., Conclusion: Cell death caused by 60Co gamma-ray may associated with the DNA oxidative damage mediated by ROS; Overexpression of pol beta could protect against oxidative DNA damage, thus the cell apoptosis/death, thereby leading to reducing the radiosensitivity of 60Co gamma-rays, while null of DNA pol beta could increase radiosensitivity of 60Co gamma-rays by compromising the DNA repair.

Objective: To clarify the relationship between the expression of pol beta and DNA damage /repair induced by Benzo(a)pyrene (BaP)., Methods: pol beta wild-type cells (pol beta +/+), pol beta null cells (pol beta -/-) and wild-type pol beta overexpressed cells (pol beta oe) which had the same genetic background were studied. Firstly, RT-PCR and Western blot targeting to pol beta were carried out to measure the expression of pol beta mRNA and protein in above three kinds of cells, then MTT test and single cell gel electrophoresis (comet assay) were used to compare cell viability and DNA damage/repair of the three kinds of cells when exposed to BaP., Results: There was pol beta deletion in pol beta -/- cells and the level of pol beta mRNA and protein in pol beta oe cells was twice higher than that in pol beta +/+ cells. BaP could induce DNA damage and reduce cell viability, when compared with pol beta +/+ cells, IC50 of pol beta -/- cells was remarkably lower, DNA was prone to damage and more difficult to be repaired, on the other hand, IC50 of pol beta oe cells was obviously higher and the damage effect on DNA was weaker and prone to be repaired., Conclusion: Pol beta played an important role in the repair of DNA damage induced by BaP, deficiency of pol beta could decrease the DNA repair capability of cells, and overexpression of pol beta could help cells response to DNA damage and protect cells from death in a certain degree.

DNA polymerase beta (pol beta), a predominant enzyme involved in DNA base excision repair (BER), plays an important role in repair of DNA damage and maintaining stability and integrality of genome. Pol beta lacks 3' to 5' proofreading activity, which contributes to its low fidelity in DNA synthesis. There are different results about action rule of pol beta in genetic instability and mechanism of pol beta in tumorigenesis. The structure and function of pol beta, the relationship between pol beta and genome instability, and abnormal expression and mutation of pol beta in tumor were reviewed.

Objective: To study the influence of DNA polymerase beta (polbeta) gene silencing by small interfering RNA on biological behavior of human gastric cancer cell line BGC-823., Methods: The siRNA eukaryotic expression vectors targeting polbeta gene were constructed and transfected into BGC-823 cells by liposome. Stable cell lines were screened with G418. The expression levels of polbeta mRNA and protein were detected by real time PCR and Western blot in the cells of each group. The proliferation of each group was detected by flow cytometry and tumorigenicity was determined in nude mice., Results: The siRNA expression vector targeting polbeta gene was successfully constructed. The expression levels of polbeta mRNA and protein were significantly reduced in the experimental group transfected with siRNA expression vectors targeting polbeta, and the silencing effect of pRNAT-U6.1-sipolbeta2 (suppression degree was 83%) was stronger than that of pRNAT-U6.1-sipolbeta1 (depression degree is 56%). Compared with irrelevant siRNA control group, empty vector control group and untransfected group, the ratio of G0/G1 cells was increased, proportion of S phase cells and cell proliferation were decreased in the experimental group 1 cells transfected with pRNAT-U6.1-sipolbeta1 (P < 0.05). On the contrary, the ratio of G1/G0 was decreased, proportion of S phase cells and cell proliferation was increased in the experimental group 2 cells transfected with pRNAT-U6.1-sipolbeta2 (P < 0.05)., Conclusion: The siRNA expression vectors targeting DNA polymerase beta gene can significantly inhibit the expression of polbeta mRNA. Neither high nor extremely low expression of polbeta is beneficial to maintain the cellular physiological functions. The expression of polbeta silenced to a proper level by siRNA may play an important role in inhibiting tumorigenesis.

Objective: To knock down the expression of polymerase beta gene in human bronchial epithelial cells with technology of vector-mediated RNA interference (RNAi), to provide research tool for the study on the functions and mechanisms of polymerase beta in repairing of DNA damaged by environmental chemical pollutants (ECPs)., Methods: Technology of molecular clone was used to construct the recombination vector of "pEGFP-C1-U6-dsRNA" for the polymerase beta RNAi. The recombinants were transfected into human bronchial epithelial cells with kit of liperfectamine 2000. The control groups included normal human bronchial epithelial cells and human bronchial epithelial cells transfected with "pEGFP-Cl1. Cells were screened by G418, then technology of fluorescence microscopy imaging was used to observe the result of transrfection. The expresison level of polymerase beta was detected by Western blotting., Results: The expression level of polymerase beta in human bronchial epithelial cells transfected with the recombinant of "pEGFP-C1-U6-dsRNA" was about 17.3% of what in the normal cells., Conclusion: The RNAi of polymerase beta gene in human bronchial epithelial cells was successful.

Objective: To investigate the biological effects of overexpression of the human DNA polymerase (pol-beta) on cellular response to DNA damage., Methods: The cell strain HLFbeta from the stable overexpression of the human pol-beta was contaminated with methyl methanesulfonate (MMS) for investigating the effects of the pol-beta on the cellular responses to DNA damage on the aspects such as the DNA damage, the cell cycle and the induced mutation rate., Results: The cell HLFbeta from the stable overexpression of the human pol-beta was obtained through the screening. The cellular response to DNA damage of HLFbeta induced by the MMS in the intermediate and high dosage group (ranging from 0.5 to 0.8 mmol/L) was significantly lower than that in the control group. The analysis for the cell cycle distribution showed that both the two types of cells contaminated by MMS had retardation at G(2) phase. In the HLFbeta group, the cells had the obvious G(2) phase retardation and 49.0% of the cells were retarded at G(1) phase as well when the MMS was increased to 0.5 mmol/L while in the control, only 20.1% of the cells were retarded at the G(1) phase when the same dosage of MMS was administered. Moreover, the MMS-induced mutagenesis in HLFbeta was increased from 4.5 x 10(-6) to 8.2 x 10(-6), significantly higher than that in the control group (P < 0.05)., Conclusion: High Pol-beta level decreases cellular DNA damage induced by MMS. Nevertheless, the overexpression of Pol-beta can also increase error-prone DNA synthesis during DNA repair process.

Objective: To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs)., Methods: According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing., Results: The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment., Conclusion: The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.

Objective: To investigate whether DNA polymerase beta (POLB) gene mutations occur in human cervical cancer (CC)., Methods: To collect fresh specimens from 34 cervical cancer and examine the mutation of POLB gene using reverse transcription polymerase chain reaction (RT-PCR), single strand conformation polymorphism (SSCP) analysis and sequence analysis. According to their histological grading, the 34 cases of CC were divided into three groups: 9 cases of grade I (G(1)), 14 cases of grade II (G(2)), 11 cases of grade III (G(3))., Results: POLB mutations were detected in the tissues of CC. The mutation of POLB is related with the histological differentiated of the CC. The mutated rates in low differentiated cancer were significantly higher than those in moderate and high ones, and the difference was significant (P < 0.05). The sequencing of the PCR product showed an A to G chang at nucleotide 660, which resulted in a substitution of the amino acid from arginine to glycin at codon 182., Conclusions: There were POLB mutations in the tissues of CC, and this may be related with the development of the CC. The mutations impaired the catalytic activity of POLB. As a result, this may lead to observed accumulation of mutations in tumor cells.

Objective: To investigate whether DNA polymerase beta (POLB) gene mutations occur in esophageal cancer., Methods: Thirty specimens of esophageal squamous epithelial cancer were resected during operation. A piece of cancer tissue and a piece of juxtacancerous tissue were taken from each specimen. Fourteen specimens of preinvasive esophageal carcer were obtained by esophagoscopy. Reverse transcription polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) and sequence analysis were used to examine the DNA polymerase beta genes. DNASIS and OMIGA softwares were used for sequencing., Results: Obvious mutation was detected by SSCP in 13 of the 30 esophageal infiltrative cancer tissues (with a mutation rate of 43.3%) and in only 1 of the 30 juxtacancerous normal tissues from the preinvasive cancer group, obvious mutation was detected by SSCP in 5 of the 14 preinvasive esophageal cancer tissues (with the mutation rate of 35.7%). In addition, obvious mutation was also detected in one specimen of hyperplasia of squamous epithelium. Within 7 esophageal cancer specimens, 58 bp (177nt to 234nt) deleted mutation of POLB was found. There were 8 point mutation forms: (1) A --> G at 375nt (Ile --> Val) (2) T --> C at 454nt (Phe --> Ser) (3) G --> T at 462nt (Glu --> terminal code) (4) G --> A at 466nt (Gly --> Glu) (5) A --> T at 613nt (Lys --> Ile) (6) G --> C at 648nt (Gly --> Arg) (7) A --> G at 660nt (Arg --> Gly), and (8) A --> G at 670nt (Glu --> Gly)., Conclusion: DNA polymerase beta gene mutations are discovered in human esophageal carcinoma for the first time. It may be related to the development of esophageal cancer, suggesting that POLB activity may be changed.