Kyo Satoru, Nakamura Mitsuhiro, Kiyono Tohru, Maida Yoshiko, Kanaya Taro, Tanaka Masaaki, Yatabe Noriyuki, Inoue Masaki
Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Kanazawa, Japan.
Am J Pathol. 2003 Dec;163(6):2259-69. doi: 10.1016/S0002-9440(10)63583-3.
The human endometrium is a dynamic tissue, the proliferative activity of which dramatically changes throughout the menstrual cycle, with exquisite regulation by sex-steroid hormones. Primary endometrial epithelial cells fall into senescence within 2 weeks when cultured on plastic dishes, and more complete understanding of endometrial biology has been delayed because of, in part, a lack of an in vitro culture model for endometrial epithelial cells. Our goal was to establish immortalized human endometrial glandular cells that retain the normal functions and characteristics of the primary cells. Because the Rb/p16 and p53 pathways are known to be critical elements of epithelial senescence in early passages, we used human papillomavirus E6/E7 to target these pathways. The combination of human papillomavirus-16 E6/E7 expression and telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT) led to successful immortalization of the endometrial glandular cells. E6/E7 expression alone was sufficient to extend their life span more than 20 population doublings, but the telomerase activation was further required to enable the cells to pass through the subsequent replicative senescence at 40 population doublings. Isolated immortalized cells contained no chromosomal abnormalities or only nonclonal aberrations, retained responsiveness to sex-steroid hormones, exhibited glandular structure on three-dimensional culture, and lacked transformed phenotypes on soft agar or in nude mice. These findings support the notion that both Rb inactivation/p53 inactivation and telomerase activation are necessary to immortalize endometrial epithelial cells, but additional factors are required for endometrial carcinogenesis. Our established cell lines show great promise for investigation of hormone functions, endometrial biology, and endometrial carcinogenesis.
人子宫内膜是一种动态组织，其增殖活性在整个月经周期中会发生显著变化，并受到性甾体激素的精确调节。原代子宫内膜上皮细胞在塑料培养皿上培养时，2周内就会进入衰老状态，部分原因是缺乏子宫内膜上皮细胞的体外培养模型，这使得对子宫内膜生物学的更全面理解受到了延迟。我们的目标是建立永生化的人子宫内膜腺细胞，使其保留原代细胞的正常功能和特征。由于已知Rb/p16和p53信号通路是早期传代上皮细胞衰老的关键因素，我们使用人乳头瘤病毒E6/E7来靶向这些信号通路。人乳头瘤病毒16型E6/E7的表达与通过导入人端粒酶逆转录酶（hTERT）激活端粒酶相结合，成功实现了子宫内膜腺细胞的永生化。单独的E6/E7表达足以使它们的寿命延长超过20个群体倍增，但还需要进一步激活端粒酶，以使细胞能够在40个群体倍增时克服随后的复制性衰老。分离出的永生化细胞没有染色体异常或只有非克隆性畸变，保留了对性甾体激素的反应性，在三维培养中呈现出腺结构，并且在软琼脂上或裸鼠体内没有转化表型。这些发现支持了这样一种观点，即Rb失活/p53失活和端粒酶激活对于子宫内膜上皮细胞的永生化都是必要的，但子宫内膜癌发生还需要其他因素。我们建立的细胞系在激素功能、子宫内膜生物学和子宫内膜癌发生的研究方面显示出巨大的潜力。
