Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg^−/− mouse ES cells and human cancer cell lines. Parg^−/− mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg^−/− mouse ES cells. Parg^−/− ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.

Poly(ADP‐ribose) polymerase‐1 (Parp‐1) is activated by DNA strand breaks and functions in the maintenance of genomic integrity and cell death control. On the other hand, Parp‐1 is also involved in transcriptional regulation of various genes, and the relationship between Parp‐1 deficiency and susceptibility to tumorigenesis has not been fully elucidated. In the present study, Parp‐1⁻¹⁻ mice, harboring exon 1 disruption in Parp‐1, and Parp‐1^+l+ animals were administered azoxymethane (AOM) at a dose of 10 mg/kg body weight once a week for 6 weeks. At 30 weeks after the first carcinogen treatment, mice were sacrificed. The incidence of animals bearing either adenomas or adenocarcinomas in the colon and the average number of colon tumors per mouse were significantly higher in Parp‐1⁻¹⁻ mice than in Parp‐1^+l+ animals. β‐Catenin accumulation was observed in 43/44 of Parp‐1⁻¹⁻ tumors and 19/21 of the Parp‐1^+l+ tumors and was not statistically different between the genotypes. This suggests that most tumors developed through a pathway involving the alteration of Wnt‐β‐catenin signaling in both Parp‐1^−1‐ and Parp‐1^+l+ mice. In the liver, where AOM is primarily activated, the incidence of animals bearing nodules and the average number of nodules per section were significantly increased in Parp‐1^−1‐ mice compared with Parp‐1^+l+ mice. Therefore, the results indicate that susceptibility to AOM‐induced tumorigenesis in the colon and also in the liver is enhanced in Parp‐1^−1‐ mice, and Parp‐1 could have a substantial role in colon and liver tumorigenesis.

The autofluorescence visualization method (AVM) uses blue excitation light to assist in the diagnosis of epithelial dysplasia. It detects epithelial dysplasia as a black area, which is known as fluorescence visualization loss (FVL). In this study, we evaluated the detection accuracy for epithelial dysplasia of the tongue using the objective AVM and assessed its possible clinical utility. Seventy-nine tongue specimens clinically suspected to have leukoplakia or squamous cell carcinoma (SCC) were analyzed. First, the AVM was subjectively performed using the Visually Enhanced Lesion scope (VELscope), and the iodine-staining method was then performed. After biopsy, the histopathological results and the luminance ratio between the lesion and healthy tissue were compared, and a receiver operating characteristic curve was created. The cutoff value for the objective AVM was determined; the lesion was considered FVL-positive or -negative when the luminance ratio was higher or lower than the cutoff value, respectively. The histopathological diagnoses among the 79 specimens were SCC (n=30), leukoplakia with dysplasia (n=34), and leukoplakia without dysplasia (n=15). The cutoff value of the luminance ratio was 1.62, resulting in 66 FVL-positive and 13 FVL-negative specimens. The luminance ratio was significantly higher in the epithelial dysplasia-positive than -negative group (P<0.000 1). The objective AVM showed much higher consistency between histopathological results than did the two methods (kappa statistic=0.656). In conclusion, objective autofluorescence visualization has a potential as an auxiliary method for diagnosis of epithelial dysplasia.

Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg+/- and Parg-/-) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg-/- embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg+/+ ES cells. Parg-/- ES cells exhibited the same growth rate as Parg+/+ ES cells in culture. Sensitivity of Parg-/- ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and γ-irradiation was examined by clonogenic survival assay. Parg-/- ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and γ-irradiation compared with wildtype (Parg+/+) ES cells (p < 0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

Accumulated evidence suggests that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, cell-death induction, differentiation and tumorigenesis. Parp-1 deficiency also induces trophoblast differentiation from mouse embryonic stem cells during teratocarcinoma-like tumor formation. To understand the relationship of PARP-1 dysfunction and development of germ cell tumors, we conducted a genetic analysis of the PARP-1 gene in human germ cell tumors. Sixteen surgical specimens of germ cell tumors that developed in the brain and testes were used. Two known single nucleotide polymorphisms (SNPs) (Val762Ala and Lys940Arg), which are listed in the SNP database of the NCBI (National Center for Biotechnology Information), were detected. In both cases, cSNPs encoded amino acids located within peptide stretches in the catalytic domain, which are highly conserved among various animal species. Furthermore, another novel sequence alteration, a base change of ATG to ACG, was identified in a tumor specimen, which would result in the amino acid substitution, Met129Thr. This base change was observed in one allele of both tumor and normal tissues, suggesting that it is either a rare SNP or a germline mutation of the PARP-1 gene. Notably, the amino acid Met129 is located within the second zinc finger domain, which is essential for DNA binding and is conserved among animal species. One SNP in intron 2 and one in the upstream 5′-UTR (untranslated region) were also detected.