Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40

Rinehart, Clifford A.; Laundon, Caroline H.; Mayben, John P.; Lyn‐Cook, Beverly; Kaufman, David G.

doi:10.1093/carcin/14.5.993

Cited by 25 publications

(23 citation statements)

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“…5). We note that SV40 large T antigen, which binds and inactivates pRb as well as other cellular proteins, induces tetraploidy in certain cell types (Ornitz et al 1987;Kuhar and Lehman 1991;Rinehart et al 1992Rinehart et al , 1993. In addition, exocrine tissues of E2F1 -/ -mice contain cells with abnormally large nuclei and binucleated cells (Field et al 1996;Yamasaki et al 1996).…”

Section: Genes and Developmentmentioning

confidence: 86%

pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis.

Zacksenhaus

Jiang²,

Chung³

et al. 1996

Genes Dev.

282

254

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Mice deficient for the RB gene (RB-/-), prior to death at embryonic day 14.5, show increased cell death in all tissues that normally express RBI: the nervous system, liver, lens, and skeletal muscle precursor cells. We have generated transgenic mice (RBlox) that express low levels of pRb, driven by an RB1 minigene. RBIox/RB -/-mutant fetuses die at birth with specific skeletal muscle defects, including increased cell death prior to myoblast fusion, shorter myotubes with fewer myofibrils, reduced muscle fibers, accumulation of elongated nuclei that actively synthesized DNA within the myotubes, and reduction in expression of the late muscle-specific genes MCK and MRF4. Thus, insufficient pRb results in failure of myogenesis in vivo, manifest in two ways. First, the massive apoptosis of myoblasts implicates a role of pRb in cell survival. Second, surviving myotubes failed to develop normally and accumulated large polyploid nuclei, implicating pRb in permanent withdrawal from the cell cycle. These results demonstrate a role for pRb during terminal differentiation of skeletal muscles in vivo and place pRb at a nodal point that controls cell proliferation, differentiation, and death.[Key Words: Retinoblastoma gene; differentiation; myogenesis; cell cycle control; apoptosis] Received July 25, 1996; revised version accepted October 16, 1996.Terminal differentiation is a dynamic process coupled to cell cycle arrest that requires continuous active control (Blau 1993). The retinoblastoma gene (RB1) product (pRB) has been implicated in cell cycle exit and terminal differentiation. For example, SV40 large T antigen, which binds and inactivates pRb, can stimulate differentiated myotubes in culture to reenter the cell cycle (Gu et al. 1993). RB1 is a tumor suppressor gene, absence of which predisposes individuals to retinoblastoma in infancy and, to a lesser extent, osteosarcoma in the second decade of life, at times when these tissues normally undergo terminal differentiation (for review, see Zacksenhaus et al. 1993a). Inactivation of RB1 also contributes to the malignant progression of a wide spectrum of tumors including breast, prostate, lung, and bladder. Moreover, tumors with apparently normal RB1 frequently contain mutations in the pathway that regulates pRb function, 4These authors contributed equally to this work. 5Corresponding authors. resulting in inactivation of pRb (for review, see Weinberg 1995).pRb is a member of a family of proteins including p107 (Ewen et al. 1991) and p130 (Harmon et al. 1993;Li et al. 1993) that interact with transcription factors and viral oncoproteins through shared conserved domains. The RB family of proteins exerts a negative effect on cell proliferation by modulating the activity of certain transcription factors (Defeo-Jones et al.

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Section: Genes and Developmentmentioning

confidence: 86%

pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis.

Zacksenhaus

Jiang²,

Chung³

et al. 1996

Genes Dev.

282

254

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“…The tissue was digested in a 0.25% collagenase solution, and the stromal cells were separated from glands by a series of centrifugations. Immortalized ESF [10] were cultured in a 1:1 mixture of Ham's F12 and medium 199 containing a low concentration of phenol red (0.6 mg/mL) (GIBCO BRL, Gaithersburg, MD) supplemented with 1% charcoal-treated fetal bovine serum [37], 3% charcoaltreated bovine calf serum, 2 µg/mL insulin, and 4 mM glutamine in a humidified environment of 5% CO 2 at 33°C. For chronic treatment with DES, immortalized endometrial stromal cells were maintained in the same medium containing 0.1 or 1 nM DES.…”

Section: Cells and Tissue Culturementioning

confidence: 99%

“…These studies were difficult to pursue at a technical level because of the limited life span of ESF. To solve this problem, the long-term exposure portion of our study was performed with conditionally immortalized ESF [10]. Conditionally immortalized ESF are clonal in origin, aneuploid, and proliferate indefinitely at the permissive temperature.…”

Section: Introductionmentioning

confidence: 99%

Regulation of keratinocyte growth factor expression in human endometrium: Implications for hormonal carcinogenesis

Rinehart

1998

Mol. Carcinog.

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Estrogen is thought to be an important etiologic agent in endometrial and breast cancers. However, the mechanism or mechanisms by which estrogen acts as a hormonal carcinogen are not well understood. We hypothesize that in response to chronic exposure to estrogens, human endometrial stromal fibroblasts (ESF) produce factors that facilitate neoplastic transformation in epithelial cells. To test this hypothesis, we assessed the regulation of keratinocyte growth factor (KGF) mRNA and protein in ESF by interleukin-1 (IL-1) and diethylstilbestrol (DES). Short-term treatments with IL-1 but not with DES increased the abundance of KGF mRNA in ESF. However, chronic treatment with DES significantly increased KGF mRNA levels and protein production. KGF protein in medium conditioned by ESF chronically treated with 1 nM DES reached concentrations of approximately 100 ng/mL. At this concentration, KGF increased endometrial epithelial cell numbers fourfold and enhanced anchorage independence tenfold. These results suggest that KGF may play a role in hormonal carcinogenesis by mediating estrogen-induced changes in the interactions between stromal and epithelial cells. To address the potential role of nuclear transcription factor kappa B (NF-kappaB) in regulating KGF expression, we determined the effect of increased expression of its inhibitor, IkappaBalpha, on KGF mRNA and protein levels. Transfection with IkappaBalpha blocked induction of KGF expression by IL-1 but had no effect on the increase in KGF mRNA caused by chronic treatment with DES. These results suggest that IL-1 exerts its effects on KGF by an NF-kappaB-mediated pathway but that chronic treatment with DES stimulates KGF expression by some other mechanism.

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“…M4 cells were derived by transfection of NS cells with a tempera ture-sensitive mutant A209 of SV40 [12], M4 was cloned following transfection and initially experienced an extended lifespan followed by 'crisis'. M4 cells escaped from 'crisis' and appear to be immortal [ 13].…”

Section: Tissue Culturementioning

confidence: 99%

Expression of Tissue Kallikrein in Normal and SV40-Transfected Human Endometrial Stromal Cells

Xu²,

Jenzano

et al. 1993

Pathobiology

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The polymerase chain reaction with specific tissue kallikrein primers was utilized to demonstrate the presence of tissue kallikrein mRNA in human endometrial stromal cells. Enzymatic analysis measured with a specific tripeptide nitroanilide substrate demonstrated the presence of tissue kallikrein in the conditioned medium obtained from both normal stromal cells and stromal cells transfected with an origin-defective temperature-sensitive SV40 large T antigen. The transfected stromal cell supernatant exhibited approximately twice as much tissue kallikrein activity as normal stromal cells at 60-100% of cell confluence. The release of tisseu kallikrein from transfected stromal cells was confirmed by Western blot analysis and [³⁵S]-methionine incorporation into a 35-kD protein which retains tissue kallikrein activity. These results demonstrate for the first time the expression and secretion of tissue kallikrein in human endometrial stromal cells and provide evidence of possible involvement of tissue kallikrein in cell transformation.

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Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40

Cited by 25 publications

References 0 publications

pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis.

pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis.

Regulation of keratinocyte growth factor expression in human endometrium: Implications for hormonal carcinogenesis

Expression of Tissue Kallikrein in Normal and SV40-Transfected Human Endometrial Stromal Cells

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