Expression Of DNA Polymerase Beta Research Articles

Abstract 7129: DNA polymerase beta expression in head & neck cancer modulates the poly-ADP-ribose-mediated replication checkpoint

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) impose a significant health burden, necessitating innovative therapeutic strategies to enhance treatment efficacy. Current standard treatments, including surgery, radiation therapy, and chemotherapy, often exhibit limited effectiveness and cause substantial side effects. This underscores the critical need for novel therapies that selectively target cancer cells, sparing normal tissue. Survival from HNSCC is poor, and importantly, a racial disparity between African Americans (AfAm) and European Americans (EuAm) has been known for some time. Ancestry Informative Markers (AIMs) are associated with altered mRNA expression of Polβ where higher expression of Polβ was observed among AfAm patients in comparison to EuAm patients. Our research focuses on DNA polymerase beta (Polβ) and its role in replication stress and response to chemotherapy and DNA damage response modifiers. Elevated expression of Polβ is linked to increased DNA damage and genomic instability, contributing to treatment resistance. In this regard, our investigations reveal a regulatory role for base excision repair (BER) proteins, including Polβ, in the cellular response to inhibitors of poly(ADP-ribose) glycohydrolase (PARG), an enzyme involved in poly(ADP-ribose) (PAR) degradation. Inhibition of PARG triggers a robust intra-S phase response, activating ATR and CHK1 signaling. Exploiting the synthetic lethality between PARG inhibition and ATR/CHK1 inhibition, we have demonstrated the ability to overcome Polβ overexpression mediated treatment resistance in HNSCC cells. Specifically, our in vitro studies demonstrate that the response to a combination of an ATR inhibitor and PARG inhibitor is impacted by Polβ expression in HNSCC cells, with synergistic effects surpassing the efficacy of the individual inhibitors. Similarly, the combination of a CHK1 inhibitor and PARG inhibitor proves effective in cells with elevated Polβ expression, offering a potential dual therapeutic strategy. These combinations exhibit promising potential for HNSCC treatment when Polβ expression is elevated. In conclusion, our findings propose a novel therapeutic paradigm for HNSCC, employing combination therapy with ATR or CHK1 inhibitors and PARG inhibitors. This approach offers a targeted strategy, presenting a promising avenue for the development of more effective and less toxic treatment modalities in the management of HNSCC and may help overcome resistance from elevated expression of Polβ. Citation Format: Md Maruf Khan, Anusha Angajala, Denise Y. Gibbs, Jeffrey C. Liu, Camille Ragin, Robert W. Sobol. DNA polymerase beta expression in head & neck cancer modulates the poly-ADP-ribose-mediated replication checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7129.

Modulation of arsenic-induced epidermal growth factor receptor pathway signalling by resveratrol

Arsenic (As) is both a human carcinogen and an effective anticancer drug. These aspects of arsenic toxicity develop as a consequence of arsenic-induced oxidative stress and modifications to signal pathway activity which alter gene expression. Resveratrol (RVL) a food antioxidant found in grapes and other fruits, exhibits anti-carcinogenic properties by reducing oxidative stress and restoring signal pathway control. This study investigated the impact of RVL on arsenite [As(III)]-induced cell signalling in HaCaT keratinocytes by assaying phosphorylation status of epidermal growth factor receptor (EGFR) signalling intermediates and measuring changes in expression of Phase II and DNA repair biomarkers.As(III) exposure produced dose-dependent toxicity which was associated with increased activation of EGFR pathway intermediates, cSrc, Rac1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Arsenic-mediated ERK1/2 activation negatively regulated DNA polymerase beta expression and up regulated heme-oxygenase-1 at toxic concentrations. RVL treatment modulated As(III)-mediated ERK1/2 activation by shifting the balance of cSrc regulatory domain phosphorylation. These effects significantly altered the response of the EGFR pathway to growth factor-induced stimulation.Our research provides evidence that treatment with pharmacologically relevant doses of RVL influences cellular responses to As(III), largely due to RVL-mediated changes to Src and ERK1/2 activation.

Deregulated expression of DNA polymerase β is involved in the progression of genomic instability

Deregulated expression of DNA polymerase beta (pol β) has been implicated in genomic instability that leads to tumorigenesis, yet the mechanisms underlying the pol β-mediated genetic instability remain elusive. In this study, we investigated the roles of deregulated expression of pol β in spontaneous and xenobiotic-induced genetic instability using mouse embryonic fibroblasts (MEFs) that express distinct pol β levels (wild-type, null, and overexpression) as a model system. Three genetic instability endpoints, DNA strand breaks, chromosome breakage, and gene mutation, were examined under various expression levels of pol β by comet assay, micronuclei test, and hprt mutation assay. Our results demonstrate that neither pol β deficiency nor pol β overexpression is sufficient for accumulation of spontaneous DNA damage that promotes a hyperproliferation phenotype. However, pol β null cells exhibit increased sensitivity to exogenous DNA damaging agents with increased genomic instability compared with pol β wild-type and overexpression cells. This finding suggests that a pol β deficiency may underlie genomic instability induced by exogenous DNA damaging agents. Interestingly, pol β overexpression cells exhibit less chromosomal or DNA damage, but display a higher hprt mutation frequency upon methyl methanesulfonate exposure compared with the other two cell types. Our results therefore indicate that an excessive amount of pol β may promote genomic instability, presumably through an error-prone repair response, although it enhances overall BER capacity for induced DNA damage.

Ethanol-induced DNA damage and the expression of repair-related molecules in human gastric carcinoma AGS cells with use of an acid pump antagonist and proton pump inhibitor.

30 Background: Proton pump inhibitor (PPI) inhibits gastric acid secretion by strongly suppressing H + / K +- ATPase of parietal cells and is a major drug for peptic ulcers and gastroesophageal reflux. Recently it was reported that PPI has antioxidant and anti- inflammatory effects as well as a powerful acid inhibitory effect. To analyze the DNA repair capacity from the antioxidant effect of acid pump antagonist (Revanex-RN) and proton pump inhibitor (Omeprazole-OP), this study examined ethanol-induced DNA damage and the expression of repair related molecules in human gastric cancer AGS cells pretreated with an acid pump antagonist and a proton pump inhibitor. Methods: AGS, a human gastric cancer cell line was obtained from ATCC, was treated with ethanol (0, 2, 4, 6, 8, 10%) during 1hr. MTT and Comet assay were performed to evaluate ethanol-induced DNA damage. The optimum concentration of ethanol which caused DNA damage was established. And AGS cells were cultured in proper concentration of RN or OP overnight. Then, these cells were treated with 5% ethanol during 1hr. DNA damage and recovery were represented the value of Olive tail moment in comet assay. Western blot was also performed to detect expression of DNA polymerase beta, APE/ref-1, PCNA, and GADD45. Results: The DNA damage of AGS cell depended upon ethanol concentration distinctly. Damaged cells showed up recovered tendency in the group treated with low dose RN or OP. In addition, DNA repair enzymes including DNA polymerase beta and APE/ref-1 which were associated with mechanism of base excision repair (BER) were increased. A relevant protein such as GADD45 was also increased significantly. Conclusions: PPI has capacity to recover damaged DNA, which is associated with increased expression of GADD45 directly. Therefore, PPI offers cancer prevention by reducing the level of DNA damage in the gastric mucosa. GADD45 is also likely to suggest that it could be used a major factor on the injury mechanism and an indicator of mucosal damage in stomach. Moreover, RN, an acid pump antagonist is expected to have the higher safety compared to the existing PPIs and considered to be equal to PPIs in terms of anti-cancer effect. No significant financial relationships to disclose.

Links between DNA polymerase beta expression and sensitivity to bleomycin

Bleomycin (BLM), an important anti-tumor antibiotic, enables cell death through oxidative DNA damage mediated by reactive oxygen species (ROS). However, increasing cellular resistance has become a serious limitation to its clinical application. Base excision repair (BER), the major pathway for repairing oxidative bases, is involved in resistance of DNA-damaging anticancer drugs. DNA polymerase beta (pol β), a critical BER enzyme, has been reported to play a crucial role in combating BLM-induced oxidative DNA damage, as a result, pol β inhibition may increase the sensitivity to BLM. To test this hypothesis, we evaluated the sensitivity to BLM using mouse embryo fibroblasts (MEFs) with distinct pol β expression levels (wild-type, pol β deficiency) and explored the underlying mechanisms. The results showed that cell viability of pol β-deficient MEFs was significantly lower than that of isogenic wild type when treated with the same BLM dosage. In addition, increased ROS level, DNA single strand breaks, and chromosomal breakage were observed in pol β deficient cells, indicating impaired DNA repair and enhanced oxidative DNA damage under pol β deficiency. In agreement with the findings, an enhanced hprt gene mutation frequency was also detected in pol β null cells. In summary, this study demonstrated that BLM-induced DNA damage could be repaired through BER pathway and absence of pol β allows oxidative DNA/chromosome damage and gene mutation, which contributes to BLM hypersensitivity.

Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase β

BackgroundAltered expression of DNA polymerase β (Pol β) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol β over-expression has not yet been evaluated in a mouse model.Methodology/Principal FindingsWe have recently developed a novel transgenic mouse model that over-expresses Pol β. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner's gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol β over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol β expression. We observed elevated expression of Pol β in stomach adenomas and thyroid follicular carcinomas, but reduced Pol β expression in esophageal adenocarcinomas and squamous carcinomas.Conclusions/SignificanceThese data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.

Specific Up-Regulation of DNA Polymerase by Human Papillomavirus 16

To analyze how the infection of human papillomavirus 16 (HPV16) affects expression of DNA polymerase beta (DNA polB) with the aim of probing the mechanism of over-expression of DNA polB in human cancers. Four fragments of human DNA polB promoter were amplified and constructed into luciferase reporter vector pGL-Basic, generating pGL-BP, pGL-BMH, pGL-BMS, and pGL-BAT constructs respectively, and co-transfected with HPV16 or HPV6 into Hep2 cells. Luciferase activity was assayed 48 hours after transfection. Semi-quantitative RT-PCR was used to measure mRNA expression of endogenous DNA polB. Immunohistochemistry and in situ hybridization were used to analyze DNA polB expression and HPV16 or HPV6 infection in 38 cases of cervical lesions respectively. With co-transfection of HPV16 and DNA polB promoter-driving reporters into Hep2 cells, pGL-BP reporter in full-length DNA polB promoter presented markedly elevated luciferase activities (P < 0.05). However, the other three mutant reporters: pGL-BMH, pGL-BMS, and pGL-BAT, generated no reporting activities in the presence of HPV16 (P > 0.05). On the contrary, all of polB promoter reporters were little stimulated in co-transfection of HPV6 (P > 0.05). The transfection of HPV16 could enhance the endogenous polB mRNA expression compared with that of HPV6 (3.42 vs. 0.80, P < 0.05). The DNA polB expression was found in 8 of 10 HPV16-positive cervical intraepithelial neoplasia grade III (CIN III) cases, while was only found in 3 of 11 HPV6-positive condyloma accuminatum cases, but was negative in all chronic cervicitis cases. The correlation of DNA polB expression with HPV16 infection in cervical lesions was significant (P < 0.05). HPV16 is able to specifically stimulate the expression of DNA polB in human epithelial cells through interaction with the core upstream regulatory sequences of DNA polB promoter. Over-expression of DNA polB might be an explanation for the molecular mechanism underlying HPV-related human cancers.

Hairpin structure within the 3'UTR of DNA polymerase mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1

Aberrant expression of DNA polymerase β, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol β expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3′UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3′UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element—the anti-apoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA–protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol β mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol β.

Modulation of base excision repair by low density lipoprotein, oxidized low density lipoprotein and antioxidants in mouse monocytes.

In the present study, we found that oxidized low density lipoprotein, but not low density lipoprotein, down-regulated base excision repair activity in extracts of mouse monocyte cell line PU5-1.8. An enzyme required in this pathway, DNA polymerase beta, was also down-regulated. In contrast, treatment of monocytes with a combination of ascorbate and alpha-tocopherol up-regulated base excision repair activity and expression of DNA polymerase beta. Co-treatment of monocytes with antioxidants plus oxidized low density lipoprotein prevented down-regulation by oxidized low density lipoprotein. Oxidative DNA damage, as measured by 8-hydroxyguanine accumulation in genomic DNA, was found in cells treated with oxidized low density lipoprotein; 8-hydroxyguanine was not found in the cells treated with low density lipoprotein, antioxidants or oxidized low density lipoprotein plus antioxidants. These results establish a linkage between the DNA base excision repair pathway, oxidative DNA damage and oxidized low density lipoprotein treatment in mouse monocytes. Since oxidized low density lipoprotein is implicated in chronic disease conditions such as atherogenesis, these findings facilitate understanding of genetic toxicology mechanisms related to human health and disease.

Cisplatin resistance in human tumor cell lines is associated with DNA polymerase beta expression but not with thymidilate synthase expression

Cisplatin resistance in human tumor cell lines is associated with DNA polymerase beta expression but not with thymidilate synthase expression

Lack of mutations in DNA polymerase beta of estradiol-induced hamster kidney tumors: sequence of hamster DNA polymerase beta cDNA.

We examined the effects of estradiol (E2), the natural estrogenic hormone, on the structure and expression of DNA polymerase beta (DNA pol beta), a DNA repair gene, from E2-induced primary kidney tumors of twelve Syrian hamsters, their metastases, and from kidney tissues surrounding the tumors. We sequenced the coding region of the hamster DNA pol beta and found it to differ from that of the human by 11%. No mutations were detected in the entire coding region including the catalytic domain of the DNA pol beta from E2-induced primary kidney tumors, their metastases, or from kidney tissues surrounding the tumors. The expression of the DNA pol beta mRNA was also not significantly altered in E2-induced kidney tumors or in kidney tissues surrounding the tumors compared to that of control kidney tissues. These results suggest that mutations in the DNA pol beta gene may not be involved in the induction or malignant progression of hamster kidney tumors induced by E2. The nucleotide sequence of the hamster DNA pol beta described here will be useful for the study of the structure and expression of this gene.

SEMIQUANTITATIVE MEASUREMENT OF GENE-EXPRESSION BY RT-PCR - A CAUTIONARY TALE

The polymerase chain reaction (PCR) theoretically offers a very powerful means of quantifying gene expression in cells and tissues of different histological classes. From a technical viewpoint however, the use of RT-PCR to quantify gene expression can be demanding with poor reproducibility arising from several diverse sources. In this study, we describe and fully characterise an RT-PCR assay which generates highly reproducible estimates of DNA polymerase beta gene expression relative to the expression of beta-actin in a semi-quantitative manner. Particular emphasis has been placed on the efficiency of first strand cDNA synthesis and to aspects of primer design. In addition, various aspects associated with the quantification of gene expression required to generate reproducible results are discussed. Using the techniques described herein, the quantification of DNA polymerase beta expression in three independent experiments using human colon carcinoma cells was highly reproducible with ratios of target to internal standard gene expression of 3.68x10(-4) +/- 0.23x10(-4). Provided that careful consideration is given to key areas of the RT-PCR assay during experimental work up procedures, this assay can be used to provide an accurate measure of gene expression in cell lines.

Prenatal haloperidol alters the expression of DNA polymerases in brain regions of neonate rats

1. Previous studies have reported a marked reduction in the [3H]thymidine incorporation in forebrain after administration of a dopamine antagonist such as haloperidol. 2. We have investigated the possibility that the expression levels of genes related to DNA metabolism could be altered by haloperidol treatment. 3. By Northern blot analysis, we have studied the steady-state mRNA levels for genes involved in DNA metabolism, in neonate rat mesencephalon and forebrain, after chronic prenatal blockade of dopamine receptors with haloperidol. 4. We found that the expression levels for DNA polymerases alpha and beta were clearly reduced in forebrain by haloperidol treatment. On the contrary, the expression of DNA polymerase beta was increased in mesencephalon. 5. Our results suggest that dopamine receptors occupancy may be a critical factor in controlling cell proliferation during brain development, through a mechanism(s) involving changes in the expression of DNA polymerases.

In situ detection and characterization of DNA polymerase activities in the nucleus of eukaryotic cells.

We have developed a cytoenzymological method for localizing DNA polymerase activities in situ and for studying their responses to various chemical agents or environmental conditions. The incubation mixtures and the stimulatory or inhibitory agents added to these media were defined with reference to in vitro biochemical tests used to detect and to characterize DNA polymerases-alpha or -beta found in eukaryotic cells. This method has already been used to study DNA polymerase activities during cell differentiation or cell senescence. Apart from two exceptions found with lower organisms, the nuclear DNA polymerase activity was always higher under conditions which favoured the in vitro expression of DNA polymerase-beta rather than DNA polymerase-alpha. --In the various cell types studied, the cellular DNA polymerase activities were almost exclusively found in the nuclei. It is hoped that this methodology will be useful for obtaining more complete biochemical data on the intracellular localization of various DNA polymerases.