Extended life span of human endometrial stromal cells transfected with cloned origin-defective, temperature-sensitive simian virus 40

Abstract

Human endometrial stromal cells transfected with an origin-defective, temperature-sensitive simian virus 40 recombinant plasmid are dependent on T-antigen function for proliferation and at the permissive temperature have an extended life span in culture. Southern blot analysis indicates that the transfected gene is present in low copy number, possibly at a single integration site. Normal stromal cells are capable of 10 to 20 population doublings in culture. Transfected cultures have been carried at the permissive temperature to 80 population doublings before crisis. In the multistep model of malignant transformation of human cells, these cells represent one of the earliest stages: extended but finite life span. We have used these cells to investigate alterations in signal transduction that may be responsible for this early stage of transformation caused by the large T antigen. Temperature shift experiments indicate that the expression of ornithine decarboxylase (ODC) but not of c-fos is altered by the large T antigen. Induction of c-fos by serum or 12-O-tetradecanoylphorbol-13-acetate is independent of temperature. However, in the transfected cells, the induction of ODC by asparagine or serum is greatly enhanced at the permissive temperature. This result indicates that the large T antigen acts downstream of c-fos but upstream of ODC expression in the signal-transducing cascade.

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts

“…This result differs from those reported by others for polyomavirus-infected cells, in which T-antigendependent induction of c-fos was observed (20,64). On the other hand, conclusions similar to ours were recently drawn from studies on functions of SV40 T antigen (49). These studies and our findings may indicate that the viral proteins circumvent the c-fos-dependent signal transduction cascade.…”

Estrogen‐induced anchorage‐independence in human endometrial stromal cells

“…The limitations of finite lived cells, the changes that occur as normal human cells age in culture and the difficulties introduced by the inevitable variation between specimens made long-term and mechanistic studies with normal stromal cells difficult to pursue. Our efforts to circumvent these problems led first to the establishment of SV40-immortalized stromal cells (Rinehart et al, 1991(Rinehart et al, , 1993. The current studies were designed to determine the ability of estrogens to induce AIP in these SV40-immortalized cells and whether estrogen-induced changes in anchorage-independence increase in magnitude as a function of duration of treatment.…”

“…M4 cells were cloned subsequent to transfection of the primary cultures of stromal cells. Transfected M4 cells experienced an extension of life span (Rinehart et al, 1991). Following this phase of extended life span, M4 cells entered crisis.…”

Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen

“…Under the conditions used here, normal endometrial tissue specimens from 30 patients were successfully cultured as heterogeneous mixed cell populations, pure epithelial or stromal cell cultures for several weeks. The constituants of media were chosen using a synthetic approach already developed by us [2,9] and others [4,5,7,13,23]. It is known that EGF and low concentration of serum may facilitate epithelial cell growth [2,3], and that cholera toxin and cortisol may inhibit stromal cell growth [9].…”

“…Chemical carcinogens, human protooncogenes and viral immortalisation, using either SV 40 large T oncogene or adenovirus E1A oncogene have been widely used to immortalize or transform human cells, as prostatic, mammary [1,8] or other tissues [6]. Rinehart et al [23] reported immortalization with SV 40 large T of endometrial stromal cell immortalization using a similar protocol with liposomes. Our transfected endometrial cell lines have been cultured more than 10 months for HISEC and more than 11 months for HIEEC and respectively passaged more than 40 and 60 days, while the control cells could be maintained in culture for 37 (stromal) and 29 days (epithelial) only.…”