Explanation In tumors found in ARH1 heterozygous mice and mouse embryonic fibroblasts (MEFs), mechanisms for inactivation of the active ARH1 gene included loss of heterozygosity (LOH) and ARH1 gene mutations. In some tumors from ARH1 heterozygous mice or from nude mice after subcutaneous injection of ARH1 heterozygous MEFs, an ARH1 protein band was observed by immunoblotting. In all likelihood, an ARH1 mutation in a small population of the heterozygous MEFs enabled them to proliferate more rapidly than did ARH1 heterozygous MEFs containing a normal allele, thus giving rise to colonies in soft agar and tumors in nude mice. Mutations in exons 2 and 3 of the ARH1 gene, which encode the catalytic site, were detected in lung adenocarcinoma isolated from ARH1 heterozygous mice and tumors in nude mice injected with ARH1 heterozygous MEFs. In all instances, mutations in the ARH1 gene were found in the tumor, but not in adjacent non-tumor tissue. Notably, no mutation was detected in cDNA from the ARH1 heterozygous MEFs that had been injected. Mutation types included missense mutations resulting from single-base substitutions (12 of 14, 85.7%), and deletion mutations with frame shifts, 2 of 14, 14.3%). The most frequent mutations of the coding strand were A>G (5 of 14, 35.7%), and T>C (4 of 14, 28.6%). To determine the effects of mutations on ARH1 enzymatic activity, the ARH1 mutant genes were expressed in ARH1KO MEFs. We generated stably ARH1KO MEFs transformed with ARH1 WT and mutant genes including mock (empty vector). Similar expression levels of protein were detected by Western blots. Surprisingly, when expressed in ARH1KO MEFs, the mutant proteins exhibited a wide variation of ARH1 catalytic activity (4 to 55% of WT activity). However, ARH1 activity was not detected in ARH1KO MEFs transformed with an ARH1 frame-shift or deletion mutant genes. These data suggested that effects on ARH1 enzymatic activity alone were not the sole basis for tumorigenesis. We reported previously that the proliferation of ARH1KO MEFs was faster than that of ARH1KO+wt and ARH1WT MEFs. To characterize the ARH1 mutations, their effects on rates of proliferation of ARH1KO MEFs transformed with empty vector (ARH1KO+Mock) were compared to ARH1KO MEFs transformed with an ARH1 wild-type gene that has 100% ARH1 activity (ARH1KO+WT1) and ARH1KO MEFs transformed with all ARH1 mutant genes (4-55% of WT activity). Interestingly, the proliferation rate of ARH1KO MEFs transformed with all mutant genes was significantly faster than that of ARH1KO+WT1 MEFs, and slower than that of ARH1KO Mock MEFs. In the case of ARH1 mutations leading to tumor development, the rate of proliferation of transformed ARH1KO MEFs depended upon the levels of ARH1 activity. These data also suggested that the proliferation assay and enzymatic activity were not good surrogates for tumorigenesis. Previously, we found that ARH1KO MEFs, but not ARH1WT and ARH1KO+WT MEFs formed colonies in soft-agar. The soft-agar colony formation assay is a common method to observe anchorage-independent growth, which correlates with tumorigenesis. All ARH1KO transformed with ARH1 mutant genes formed colonies in soft agar, whereas ARH1KO MEFs transformed with the ARH1 WT1 gene did not. Diameter of colonies with all mutant MEFs was significantly smaller than that of colonies formed by ARH1KO Mock MEFs. Diameters of colonies from MEFs transformed with the low catalytic activity group of ARH1 mutants were larger than that of colonies formed by the high activity group, but not larger than colonies formed by the intermediate ARH1 activity group. Number of colonies formed by MEFs transformed with any of the mutant MEFs was greater than that of colonies seen with ARH1KO+WT1 MEFs, but was fewer than those formed by ARH1 KO Mock MEFs. Data regarding diameter of colonies in soft-agar with the different groups of MEFs were similar to data related to the number of colonies. The numbers of colonies seen with the low ARH1 activity MEF group were greater than those seen with the intermediate ARH1 activity group and the high activity group. These data indicated that MEFs transformed with ARH1 mutant genes encoding proteins with residual ARH1 activity display anchorage-independent growth in soft agar with the number of colonies and diameters dependent on hydrolase activity. Growth of cells in athymic nude mice was used as a measure of tumorigenecity. It was observed previously that ARH1 genotype affected tumorigenesis; ARH1KO and ARH1 heterozygous MEFs, but not ARH1WT and ARH1KO+WT MEFs developed tumors in nude mice. Using ARH1KO MEFs transformed with ARH1 mutant genes, the effects of ARH1 mutant genes and activities of encoded proteins on subcutaneous tumor mass in athymic nude mice were determined. ARH1KO Mock MEFs formed tumors in nude mice, whereas ARH1KO MEFs transformed with ARH1 WT1 gene did not. Interestingly, all ARH1KO MEFs transformed with ARH1 mutant genes formed tumors in nude mice. In addition, ARH1KO MEFs transformed with a WT gene but expressing low levels of ARH1 protein and activity developed tumors, but they grew at a slower rate than ARH1KO MEFs transformed with ARH1 mutant genes having similar ARH1 activity. Interestingly, growth of tumors seen with ARH1KO MEFs transformed with ARH1 WT gene but expressing intermediate level of ARH1 protein and activity did not develop tumors in nude mice and were thus similar to ARH1KO+WT1 that was designated as 100% ARH1 activity. Thus, all ARH1KO MEFs transformed with ARH1 mutant genes developed tumors but the levels of expression of the WT and mutant gene were critical to tumor potential. Based on our tumorigenesis data, it appears that ARH1 deficiency and mutations were associated with development of lung adenocarcinoma and other cancers. Next, we asked whether human tumors might have ARH1 mutations and whether the mutations would preferentially occur in exons encoding the catalytic site, as was the case in the murine model. The human cancer database used to search for ARH1 mutation data was the Catalogue of Somatic Mutations in Cancer (COSMIC), Trust Sanger Institute, Genome Research Limited (England). Thirty-two ARH1 mutations in human cancers (e.g., lung, breast, colon) were found in the COSMIC database. ARH1 mutations were observed in human ARH1 exons 3 and 4, which are equivalent to mouse ARH1 exons 2 and 3, which comprise the active site. The ARH1 mutations in human cancer were mainly missense mutations with single-base substitution (71.2%, 23 out of 32) similar to the data seen with ARH1 heterozygous mice. The most frequent mutations of the coding strand were G>T (9 out of 30, 30%), G>A (6 out of 30, 20%) and C>T (6 out of 30, 20%). Human ARH1 gene mutations were more frequent in lung cancer (1.6%) than in cancers of other tissues. Some of the ARH1 sites mutated in the human gene were similar in location to those found in the mouse ARH1 gene. Also, the human ARH1 equivalent amino acid to mouse ARH1 D61, which was shown previously to be critical for ARH1 activity, is D56, and was mutated in human cancer. Since tumorigenesis was observed in both ARH1-deficient and heterozygous mice, ARH1 has properties of a tumor suppressor gene, and cancers follow a two-hit model. In agreement, we reported that six of 16 lung adenocarcinomas found in ARH1 heterozygous mice had LOH. We, therefore, also looked for LOH involving the human ARH1 gene as a potential mechanism for inactivation of ARH1 in human cancers. ARH1 LOH in human cancers was found in various types of tumors and tissues. In the human cancer database, percentage of LOH in lung (15.1%) and kidney (18.0%) cancers was greater than that observed in other tissues. Based on these data, it appears that ARH1 may participate in the pathogenesis of both human as well as murine cancer.

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Stevens, Linda A; Moss, Joel (2018) Mono-ADP-Ribosylation Catalyzed by Arginine-Specific ADP-Ribosyltransferases. Methods Mol Biol 1813:149-165
Pourfarjam, Yasin; Ventura, Jessica; Kurinov, Igor et al. (2018) Structure of human ADP-ribosyl-acceptor hydrolase 3 bound to ADP-ribose reveals a conformational switch that enables specific substrate recognition. J Biol Chem 293:12350-12359
Yahiro, Kinnosuke; Nagasawa, Sayaka; Ichimura, Kimitoshi et al. (2018) Mechanism of inhibition of Shiga-toxigenic Escherichia coli SubAB cytotoxicity by steroids and diacylglycerol analogues. Cell Death Discov 4:22
Mashimo, Masato; Moss, Joel (2018) ADP-Ribosyl-Acceptor Hydrolase Activities Catalyzed by the ARH Family of Proteins. Methods Mol Biol 1813:187-204
Bu, Xiangning; Kato, Jiro; Hong, Julie A et al. (2018) CD38 knockout suppresses tumorigenesis in mice and clonogenic growth of human lung cancer cells. Carcinogenesis 39:242-251
Abplanalp, Jeannette; Leutert, Mario; Frugier, Emilie et al. (2017) Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase. Nat Commun 8:2055
Sparrer, Konstantin M J; Gableske, Sebastian; Zurenski, Matthew A et al. (2017) TRIM23 mediates virus-induced autophagy via activation of TBK1. Nat Microbiol 2:1543-1557
Ida, Chieri; Yamashita, Sachiko; Tsukada, Masaki et al. (2016) An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells. Anal Biochem 494:76-81
Yamashita, Sachiko; Tanaka, Masakazu; Sato, Teruaki et al. (2016) Effect of mild temperature shift on poly(ADP-ribose) and ?H2AX levels in cultured cells. Biochem Biophys Res Commun 476:594-599
Chen, Pei-Wen; Jian, Xiaoying; Heissler, Sarah M et al. (2016) The Arf GTPase-activating Protein, ASAP1, Binds Nonmuscle Myosin 2A to Control Remodeling of the Actomyosin Network. J Biol Chem 291:7517-26

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