Division of Pathology, National Institute of Health Sciences, Kawasaki-ku, Kawasaki 210-9501, Japan.
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.
Toxicol Sci. 2022 Aug 25;189(1):51-61. doi: 10.1093/toxsci/kfac064.
We investigated γ-H2AX formation, a biomarker of DNA damage, and expression of stem cell markers (SCMs), including cytokeratin 14, aldehyde dehydrogenase 1A1 (ALDH1A1), and CD44, in the development of rat bladder tumors induced by short-term administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Histopathological examination showed that diffuse simple hyperplasia of the bladder urothelium induced by BBN recovered to the normal-appearing urothelium after withdrawal, whereas focal proliferative lesions were newly developed and subsequently progressed to benign papilloma and carcinoma. Immunohistochemical analysis revealed that BBN-induced γ-H2AX formation and ALDH1A1 and CD44 expression persisted at higher levels in the normal-appearing urothelium than those in the control group for long periods after withdrawal. Since persistent chronic inflammation was observed even after withdrawal, targeted gene expression analysis of inflammation-related factors revealed 101 genes, including Stat3 and Myc, that showed persistent high expression. Pathway analysis suggested that Stat3 and/or Myc activation may be associated with SCM expression. We focused on hepatocyte growth factor (Hgf), one of the genes predicted in relation to Stat3/Myc, and confirmed that HGF-positive cells increased by BBN persisted in the normal-appearing urothelium after withdrawal and colocalized with γ-H2AX and SCMs. These results suggested that the long-term persistence of γ-H2AX formation and SCM expression, which occurred during the early stages of bladder tumorigenesis, is not a transient response to exposure and might contribute to bladder tumorigenesis. Although further studies are needed, BBN-induced rat bladder tumors may originate from focal hyperplasia arising from SCM-positive cells via activation of the STAT3/MYC pathway after DNA damage involving γ-H2AX formation.
我们研究了 γ-H2AX 的形成，这是一种 DNA 损伤的生物标志物，以及干细胞标志物（SCMs）的表达，包括角蛋白 14、醛脱氢酶 1A1（ALDH1A1）和 CD44，这些标志物在短期给予 N-丁基-N-（4-羟丁基）亚硝胺（BBN）诱导的大鼠膀胱癌的发展过程中。组织病理学检查显示，BBN 诱导的膀胱尿路上皮弥漫性单纯性增生在停药后恢复为正常外观的尿路上皮，而局灶性增殖性病变则新出现并随后进展为良性乳头瘤和癌。免疫组织化学分析显示，BBN 诱导的 γ-H2AX 形成以及 ALDH1A1 和 CD44 的表达在停药后很长一段时间内仍持续高于对照组，在正常外观的尿路上皮中表达更高。由于即使在停药后仍观察到持续的慢性炎症，炎症相关因子的靶向基因表达分析显示，包括 Stat3 和 Myc 在内的 101 个基因持续高表达。通路分析表明，Stat3 和/或 Myc 激活可能与 SCM 表达有关。我们关注的是肝细胞生长因子（HGF），这是与 Stat3/Myc 相关的预测基因之一，并证实 BBN 诱导的 HGF 阳性细胞在停药后在正常外观的尿路上皮中持续存在，并与 γ-H2AX 和 SCM 共定位。这些结果表明，在膀胱癌发生的早期阶段，γ-H2AX 形成和 SCM 表达的长期持续存在不是对暴露的短暂反应，可能有助于膀胱癌的发生。尽管需要进一步研究，但 BBN 诱导的大鼠膀胱癌可能起源于 SCM 阳性细胞的局灶性增生，通过涉及 γ-H2AX 形成的 DNA 损伤后 STAT3/MYC 通路的激活而发生。
