NDLI: The isolation of an immortalized and tumorigenic cell line from p21 WAF1 null mouse bladders

Content Provider	Springer Nature Link
Author	McGarvey, Terence W. Nguyen, Trang B. Tomaszewski, John E. Malkowicz, S. Bruce
Copyright Year	2002
Abstract	Given a role for the deregulation of p21 WAF1 in the progression of bladder tumors, we examined the growth of cultured urothelial cells from wild-type and p21WAF1 null bladders. Bladders were excised, minced from euthanized p21 WAF1 and wild-type mice, treated overnight with dispase, and then placed into flasks coated with collagen type I in Dulbecco modified Eagle medium with 10% fetal calf serum. After an overnight incubation, the media was replaced with a serumfree media and a portion of explants were treated with 12-O-tetrade-canoylphorbol-13-acetate (TPA) on day 7 and continued for either 4 or 9 wk. The urothelial origin of any surviving epithelial cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) using uroplakin II-specific primers, and the expression of the cell cycle-related proteins, p16INK4 and p19ARF, was examined by semiquantitative RT-PCR and Western blotting. Isolated wild-type and serially passaged p21WAF1 null epithelial-like cells were then injected subcutaneously into nude mice. We found that phorbol ester treatment at two different concentrations significantly significantly enhanced uroepithelial colony formation from isolated wild-type mouse bladder tissue. On the other hand, significantly fewer urothelial colonies were derived from p21WAF1 null bladder cells treated with phorbol ester. Although there was apparent senescence and cell death of epithelial foci and stromal cells in phorbol ester-treated and-untreated p21WAF1 null cultures, after 3 mo there was an apparent subpopulation of epitheloid cells that overgrew each flask. There was a significant decrease in the number of these serially passaged cells in the G1 phase of the cell cycle when compared with initial explant wild-type or p21 WAF1 null cells. This subpopulation of epitheloid cells expressed the mouse uroplakin II gene, indicating a urothelial phenotype, but did also a high level of expression of the p53 protein and a significant decrease in the expression of the p19ARF transcript in both p21WAF1 null bladder and p21WAF1 null cells. These p21WAF1 null cells could be easily passaged and when injected subcutaneously into nude mice, large tumors developed. Therefore, it appears that a subpopulation of urothelial cells from the p21WAF1 null bladder can develop a tumorigenic phenotype in vitro.
Starting Page	394
Ending Page	400
Page Count	7
File Format	PDF
ISSN	10712690
Journal	In Vitro Cellular & Developmental Biology - Animal
Volume Number	38
Issue Number	7
e-ISSN	1543706X
Language	English
Publisher	Springer-Verlag
Publisher Date	2002-01-01
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	tumorigenesis immortalization p21WAF1 gene bladder Cell Biology Developmental Biology Animal Genetics and Genomics Animal Biochemistry Animal Physiology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Developmental Biology Medicine

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