Successful Immortalization of Endometrial Glandular Cells with Normal Structural and Functional Characteristics

Abstract

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. The human endometrium is a unique tissue characterized by constant and rapid cell proliferation, differentiation, and breakdown during a menstrual cycle. This cyclic change in proliferation is exquisitely regulated by the cooperative actions of estrogen and progesterone, indicating that human endometrium is highly susceptive to sexsteroid hormones and that endometrial glandular epithelial cells may provide a good model with which to study hormone function and regulation. However, in classical tissue culture using plastic or glass dishes, epithelial cells loose their proliferative capacity during ongoing cultivation throughout several days, whereas stromal cells are more easily cultured in the longer term. Most investigators in this field have tried to develop endometrial cell cultures with a mixture of stromal cells and/or organotypic cultures, but both for short-term experiments only. [1][2][3] The lack of a stable in vitro culture system of endometrial cells also renders studying the molecular carcinogenesis of the endometrium difficult. Most endometrial cancers arise from endometrial glandular cells via the multistep accumulation of abnormalities in oncogenes and tumor suppressor genes, including PTEN, Ras, and p53. 4 These factors are frequently mutated or deregulated in endometrial cancers or even in its precursors. However, in vitro experiments to investigate the role of these factors in endometrial carcinogenesis have been impossible because of the extremely short life span of primary cultured endometrial epith...

HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes

“…To establish the in vitro model system for HPV16-mediated multistep carcinogenesis, these cells were infected with retroviral vectors expressing HPV16 E6 and E7 (HCK1T-E6E7), E6 alone (HCK1T-E6), E7 alone (HCK1T-E7), or control vector (HCK1T-vect). Functional expressions of these genes have been confirmed previously (Kiyono et al, 1998;Kyo et al, 2003). Decreased p53 expression levels in E6-infected cells indicated functional expression of the E6 protein ( Figure 1a) and expression of E7 was reconfirmed by Western blotting.…”

“…Vector construction and retroviral transfection Segments of HPV16 E6E7 (16E6E7), a splice donor sitemutant version of E6 (E6SD) (Kiyono et al, 1994) a series of E6 mutants, and hTERT were cloned and recombined into retroviral expression vectors to generate pCLXSN-16E6E7, -16E7, -16E6SD, -16E6 SAT, -16E6 D151, 16E6 L50G and pCLXSH-hTERT, as described previously (Kyo et al, 2003;Takeda et al, 2004). Construction of the destination vector, pDEST-CL-SI-MSCVpuro, for retroviral expression of shRNA, pCL-SI-MSCVpuro-ErbB2R-shRNA, -HPV16E6-shRNA, -p53-shRNA and -E6AP-shRNA was carried out by a previously described method (Sawada et al, 2004).…”

Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation

“…After 2-3 passages, the attached endometrial stromal cells were devoid of blood cells. Human EM-E6/E7/hTERT-2 cells, endometrium-derived progenitors, were obtained from surgical endometrial tissue samples and were immortalized by E6, E7, and hTERT (Kyo et al, 2003). C2C12 myoblast cells were supplied by RIKEN Cell Bank (The Institute of Physical and Chemical Research, Japan).…”

“…Implanted Endometrium-derived Cells Induce De Novo Myogenesis in Immunodeficient NOG Mice EM-E6/E7/hTERT-2 cells originate from the endometrial gland and are considered as endometrial progenitor cells or bipotential cells capable of differentiating into both glandular epithelial cells and endometrial stromal cells (Kyo et al, 2003). To determine whether EM-E6/E7/hTERT-2 cells and menstrual blood-derived cells generate complete endometrial structure in vivo, like endometriosis, the cells without any treatment or induction were injected into the right thigh muscle of immunodeficient NOG mice.…”

Immortalized cells as experimental models to study cancer

“…A telomere-independent growth arrest (M0) limits the growth of HMECs, but inactivation of the RB/ p16 INK4A pathway alleviates this proliferative block (Foster and Galloway 1996;Foster et al 1998). Once the RB/p16 INK4A pathway is experimentally inactivated, the constitutive ectopic expression of hTERT suffices to immortalize keratinocytes, HMECs and many other human cell types (Kiyono et al 1998;Lundberg et al 2002;Darimont et al 2003;Kyo et al 2003). These results suggest that the RB/p16 INK4A pathway plays an important role in the activation of senescence.…”