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.

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.

he polyADP-ribosylation reaction is catalyzed by poly(ADP-ribose) polymerase (Parp) family proteins using NAD as a substrate. Parp-1 is a 113-kDa nuclear enzyme which polyADP-ribosylates various nuclear proteins, including Parp-1 itself and histones, after activation by binding to DNA single and double strand breaks. Parp-1 −/− cells show delayed DNA rejoining after treatment with alkylating agents, and a role of Parp-1 in base excision repair and double strand break (DSB) repair has been suggested. 1) Parp-1 −/− cells also display enhanced genomic instability, including chromosome aberration and increased levels of micronuclei formation both in the absence of DNA damage and after DNA damage introduction. 2, 3)In addition, Parp-1 also has a leading role in cell death after DNA damage and Parp-1 overactivation. NAD depletion after DNA damage was abrogated in Parp-1 −/− cells and as a consequence, Parp-1 −/− cells became resistant to cell death induced by severe DNA damage. [4][5][6][7][8] This evidence suggests that loss of Parp-1 function could have a substantial contribution to tumorigenesis, through the introduction of genetic alterations.In fact, we previously demonstrated that Parp-1 −/− mice, harboring exon 1 disruption in Parp-1, showed increased incidences of liver and lung tumors after N-nitrosobis(2-hydroxypropyl)amine administration. Tong et al. 13) It is also known that pharmacological intakes of niacin augment NAD level and increase the latency of the ethylnitrosourea-induced liver carcinogenesis.14) Since Parp-1 is also involved in the transcriptional regulation of various genes, [15][16][17] the impact of Parp-1 deficiency may differ among various types of tumors and among various organs. Therefore, the effect of Parp-1 deficiency on tumorigenesis needs to be investigated in various models of tumorigenesis.In the present study, Parp-1 −/− mice, harboring exon 1 disruption of Parp-1, were treated with azoxymethane (AOM), a potent colon carcinogen. We observed that the Parp-1 −/− mice showed an enhanced incidence of tumor development in the colon and also in the liver. Colon tumors induced by AOM in rodents are frequently associated with alteration of the Wnt-β-catenin signaling cascade, and β-catenin accumulation is observed.18) Therefore, to examine whether the development of tumors in Parp-1 +/+ and Parp-1 −/− mice involves alteration of Wnt-β-catenin signaling, immunostaining of β-catenin was carried out. Materials and Methods Parp-1−/− mice generated by disrupting the Parp-1 exon 1 through the insertion of a neomycin resistance gene cassette 5) were used in this study. Mice were housed in plastic cages in an air-conditioned room with a 12 h light-dark cycle. Water and basal diet (CE-2, CLEA JAPAN, Tokyo) were available ad libitum. Parp-1 +/+ and Parp-1 −/− female mice (7-8 weeks old) of a mixed genetic background of ICR and 129Sv were produced by line-breeding. The mean body weights of Parp-1 + / + and Parp-1 −/− mice at the start of the experiment were 21.7±1.7 g and 21.4±2.9 g, respectively. P...

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.

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 gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (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.