Masui T, Mann A M, Macatee T L, Okamura T, Garland E M, Fujii H, Pelling J C, Cohen S M
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-3135.
Cancer Res. 1991 Jul 1;51(13):3471-5.
Male F344 rats were fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazoly]formamide for 6 weeks and then fed 3% or 5% sodium saccharin, 5% sodium ascorbate, 3.12% calcium saccharin, 1.34% sodium chloride, 5.2% calcium saccharin plus 1.34% sodium chloride, or basal diet alone for 72 weeks. Protein and DNA were extracted from 89 bladder tumors [87 transitional cell carcinomas (TCC), 1 papilloma, and 1 sarcoma] from 86 rats p21 expression was examined by Western blotting using a monoclonal antibody against p21 (NCC-RAS-004). H-ras mutations in exons 1 and 2 were examined by direct sequencing of DNA amplified by polymerase chain reaction. Sequencing results demonstrated mutations at codon 61 (CAA to CGA in 15 TCCs; CAA to CTA in 2 TCCs), at codon 12 (GGA to TGG in 1 TCC), and at codon 13 (GGC to GTC in 3 TCCs). Mutations at codon 61 were confirmed by faster mobility of the p21 band in Western blots. The level of p21 expression varied among samples, but many TCCs appeared to express more p21 than controls. The overall incidence of H-ras mutations was 24.4% (21 of 86 rats). The type of chemical used for the promoting phase had essentially no effect on H-ras mutation, suggesting that the effects observed were related to FANFT administration. The frequency of H-ras mutation in each group was negatively related to the incidence of carcinoma (r = -0.85; P less than 0.01). Two groups of tumors (with or without the mutated ras gene) were compared for tumor size (reflected by the bladder weight), histological grading, and the presence of invasion. The size of tumors with mutated ras was significantly smaller than those without mutated ras. There was no difference in the histological grading between the two groups. Although not statistically significant, histological invasion was more frequently observed in tumors with mutated ras (14.3%) than in tumors without mutation (3.1%).
雄性F344大鼠先喂食0.2%的N-[4-(5-硝基-2-呋喃基)-2-噻唑基]甲酰胺6周，然后分别喂食3%或5%的糖精钠、5%的抗坏血酸钠、3.12%的糖精钙、1.34%的氯化钠、5.2%的糖精钙加1.34%的氯化钠，或仅喂食基础饲料72周。从86只大鼠的89个膀胱肿瘤（87个移行细胞癌、1个乳头状瘤和1个肉瘤）中提取蛋白质和DNA，使用抗p21的单克隆抗体（NCC-RAS-004）通过蛋白质印迹法检测p21表达。通过对聚合酶链反应扩增的DNA进行直接测序，检测第1和第2外显子中的H-ras突变。测序结果显示密码子61发生突变（15个移行细胞癌中CAA突变为CGA；2个移行细胞癌中CAA突变为CTA）、密码子12发生突变（1个移行细胞癌中GGA突变为TGG）以及密码子13发生突变（3个移行细胞癌中GGC突变为GTC）。蛋白质印迹中p21条带迁移速度加快证实了密码子61的突变。p21表达水平在不同样本间存在差异，但许多移行细胞癌似乎比对照组表达更多的p21。H-ras突变的总体发生率为24.4%（86只大鼠中有21只）。用于促癌阶段的化学物质类型对H-ras突变基本没有影响，这表明观察到的效应与给予FANFT有关。每组中H-ras突变频率与癌发生率呈负相关（r = -0.85；P小于0.01）。比较两组肿瘤（有或无ras基因突变）的肿瘤大小（以膀胱重量反映）、组织学分级和侵袭情况。有ras基因突变的肿瘤大小明显小于无ras基因突变的肿瘤。两组的组织学分级没有差异。虽然无统计学意义，但有ras基因突变的肿瘤（14.3%）比无突变的肿瘤（3.1%）更常观察到组织学侵袭。
