ADP-ribosylation Cycles

ADP-核糖基化循环

• 批准号: 9157310
• 负责人: Joel Moss
• 金额: $ 122.67万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157310
• 关键词: ADP Ribose Transferases; ADP ribosylation; ADP-Ribosylation Pathway; Active Sites; Adenosine Diphosphate Ribose; Affect; Agar; Agreement; Alleles; Amino Acids; Anchorage-Independent Growth; Anti-Bacterial Agents; Arginine; Bacterial Toxins; Biological; Biological Assay; Breast; Caliber; Carbamates; Carbon; Cataloging; Catalogs; Catalytic Domain; Cell Differentiation process; Cells; Cholera; Cholera Toxin; Cleaved cell; Code; Colon; Complementary DNA; Complex; DNA Repair; Data; Databases; Deacetylation; Defensins; Deletion Mutation; Development; Disease; Embryo; England; Epithelial Cells; Event; Exhibits; Exons; Family; Fibroblasts; Gene Mutation; Genes; Genome; Genotype; Goals; Human; Hydrolase; IL8 gene; Immunoblotting; Infection; Inflammation; Institutes; Interleukin-8; Kidney; Laboratory Study; Location; Loss of Heterozygosity; Lung; Lung Adenocarcinoma; Malignant Neoplasms; Malignant neoplasm of lung; Mammalian Cell; Measures; Methods; Missense Mutation; Modeling; Modification; Mus; Mutant Strains Mice; Mutate; Mutation; Niacinamide; Nude Mice; O-Acetyl-ADP-Ribose; Ornithine; Pathogenesis; Pathway interactions; Peptides; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Population; Post-Translational Protein Processing; Process; Proliferating; Property; Protein Family; Proteins; Reaction; Regulation; Reporting; Research; Residual state; Signal Transduction; Single base substitution; Site; Somatic Mutation; Subcutaneous Injections; Surface; Tissues; Transfection; Transforming Growth Factors; Trust; Tumor Suppressor Genes; Tumor Tissue; Variant; WT1 gene; Water; Western Blotting; alpha-Defensins; antimicrobial; base; biological systems; carcinogenesis; cell growth; cytotoxic; human neutrophil peptide 1; killings; loss of function; member; microorganism; mutant; neutrophil; poly ADP-ribose glycohydrolase; prevent; protein function; response; subcutaneous; tumor; tumor growth; tumorigenesis; tumorigenic; vector

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.

解释 在ARH1杂合小鼠和小鼠胚胎成纤维细胞（MEF）中发现的肿瘤中，活性ARH1基因失活的机制包括杂合性（LOH）和ARH1基因突变的丧失。在ARH1杂合小鼠或皮下注射ARH1杂合MEF后的一些肿瘤中，通过免疫印迹观察到ARH1蛋白带。在少量杂合MEF中，ARH1突变的可能性很可能使它们能够比含有正常等位基因的ARH1杂合MEF更快地增殖，从而引起裸鼠软琼脂和肿瘤的菌落。在从ARH1杂合小鼠中分离出的肺腺癌中检测到ARH1基因的外显子2和3中的突变，并在注射ARH1杂合子MEF的裸小鼠中检测到。在所有情况下，在肿瘤中发现了ARH1基因中的突变，但在相邻的非肿瘤组织中没有发现。值得注意的是，从注射的ARH1杂合MEF中未检测到突变。突变类型包括单基替换产生的错义突变（14个中的12个，85.7％）和带有框架移位的缺失突变，其中2个中的2个，14.3％）。编码链的最常见突变是A> g（14，35.7％）和T> C（14，28.6％）。 为了确定突变对ARH1酶活性的影响，在ARH1KO MEF中表达了ARH1突变基因。我们生成了稳定的ARH1KO MEFS，它用ARH1 WT和包括模拟（空矢量）的突变基因进行了转化。通过蛋白质印迹检测到相似的蛋白质表达水平。令人惊讶的是，当在ARH1KO MEF中表达时，突变蛋白表现出广泛的ARH1催化活性（4％至55％的WT活性）。然而，在用ARH1框架切换或缺失突变基因转化的ARH1KO MEF中未检测到ARH1活性。这些数据表明，仅对ARH1酶活性的影响并不是肿瘤发生的唯一基础。 我们以前报道说，ARH1KO MEF的扩散速度比ARH1KO+WT和ARH1WT MEF的速度快。为了表征ARH1突变，将其对用空载体（ARH1KO+模拟）转化的ARH1KO MEF的速率的影响与ARH1KO MEF进行了比较，该ARH1KO MEFs用ARH1野生型基因转变，其具有100％ARH1活性（ARH1KO+WT1）和ARH1KO MEF的野生型基因与ARH1KO MEFS的影响。有趣的是，用所有突变基因转变的ARH1KO MEF的增殖速率明显快于Arh1ko+WT1 MEF的速度，并且比Arh1ko Mock Mefs较慢。在导致肿瘤发展的ARH1突变的情况下，转化的ARH1KO MEF的增殖速率取决于ARH1活性的水平。这些数据还表明，增殖分析和酶促活性不是肿瘤发生的良好替代物。 以前，我们发现ARH1KO MEFS，但没有ARH1WT和ARH1KO+WT MEF在软性agar中形成殖民地。软性菌落形成测定是一种观察锚固非依赖生长的常见方法，与肿瘤发生相关。所有用ARH1突变基因转化的ARH1KO在软琼脂中形成菌落，而ARH1KO MEFs用ARH1 WT1基因转化。所有突变MEF的菌落直径明显小于Arh1ko Mock MEF形成的菌落。来自MEF的菌落的直径与低活性基团形成的菌落的催化活性基团转化的菌落大，但并不大于中间ARH1活性组形成的菌落。 由MEF形成的任何一个突变MEF形成的殖民地数量大于ARH1KO+WT1 MEF的殖民地的数量，但少于ARH1 KO MOCE MEF所形成的菌落。与不同基团的MEF组中有关软性菌落直径的数据类似于与菌落数量有关的数据。较低的ARH1活性MEF组看到的菌落数量大于中间ARH1活性组和高活性组的菌落数量。这些数据表明，MEF用编码蛋白质的ARH1突变基因转化，其残留ARH1活性在软琼脂中表现出锚定依赖性的生长，其菌落数量和直径取决于水解酶活性。 小鼠中细胞的生长被用作肿瘤性的量度。先前观察到ARH1基因型会影响肿瘤发生。 ARH1KO和ARH1杂合MEF，但没有ARH1WT和ARH1KO+WT MEF在裸鼠中出现肿瘤。使用用ARH1突变基因转化的ARH1KO MEF，确定了ARH1突变基因的作用以及编码蛋白的活性对无胸腺裸鼠皮下肿瘤质量的活性。 ARH1KO模拟MEF在裸鼠中形成肿瘤，而用ARH1 WT1基因转化的ARH1KO MEF没有。有趣的是，所有ARH1KO MEF都用ARH1突变基因转化为裸鼠中的肿瘤。此外，ARH1KO MEF用WT基因转化，但表达低水平的ARH1蛋白和活性会产生肿瘤，但它们的生长速度比ARH1KO MEF慢，而ARH1KO MEF被ARH1突变基因具有相似ARH1活性的ARH1突变基因。有趣的是，用ARH1 WT基因转化的ARH1KO MEF看到的肿瘤的生长，但表达ARH1蛋白和活性的中间水平并未在裸鼠中发展肿瘤，因此与ARH1KO+WT1相似，该ARH1KO+WT1被指定为100％ARH1活性。因此，用ARH1突变基因转化的所有ARH1KO MEF都会出现肿瘤，但是WT和突变基因的表达水平对于肿瘤潜力至关重要。 根据我们的肿瘤发生数据，ARH1缺乏症和突变似乎与肺腺癌和其他癌症的发展有关。接下来，我们询问人类肿瘤是否可能具有ARH1突变，以及该突变是否会像鼠模型中的催化位点一样优先发生在编码催化位点的外显子中。用于搜索ARH1突变数据的人类癌症数据库是癌症中体细胞突变的目录（宇宙），Trust Sanger Institute，Genome Research Limited（英国）。在宇宙数据库中发现了人类癌症（例如肺，乳房，结肠）中的32个ARH1突变。在人ARH1外显子3和4中观察到ARH1突变，这些突变等同于小鼠ARH1外显子2和3，其中包含活性位点。人类癌症中的ARH1突变主要是单基替代的错义突变（71.2％，32分中的23个），类似于ARH1杂合小鼠的数据。编码链的最常见突变是G> t（30％，30％），g> a（30％，20％中的6个）和c> t（30，20％中的6个）。与其他组织的癌症相比，人ARH1基因突变在肺癌中（1.6％）更为频繁。在人类基因中突变的某些ARH1位点的位置与小鼠ARH1基因中的位置相似。此外，人类ARH1等效氨基酸与小鼠ARH1 D61（以前证明对ARH1活性至关重要）是D56，并且在人类癌症中被突变。 由于在ARH1缺陷型和杂合小鼠中都观察到肿瘤发生，因此ARH1具有肿瘤抑制基因的特性，并且癌症遵循两次打击模型。总体而言，我们报道说，在ARH1杂合小鼠中发现的16个肺腺癌中有6个患有LOH。因此，我们还将涉及人类ARH1基因的LOH作为人类癌症中ARH1失活的潜在机制。在各种类型的肿瘤和组织中发现了人类癌症中的ARH1 LOH。在人类癌症数据库中，肺中LOH的百分比（15.1％）和肾脏（18.0％）的癌症大于其他组织中观察到的癌症。基于这些数据，似乎ARH1可以参与人类和鼠类癌的发病机理。