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Increased p16 levels correlate with pRb alterations in human urothelial cells
T Yeager1, W Stadler, C Belair
1Department of Human Oncology, University of Wisconsin Medical School, Madison 53792.
Abstract:
The CDKN2 (MTS1) gene is located at 9p21; its product, p16, inhibits the cyclin D/CDK4 complex that phosphorylates pRb, thus negatively regulating cell cycle progression [M. Serrano et al., Nature (Lond.), 366: 704, 1994; A. Kamb et al., Science (Washington DC), 264: 436, 1994; T. Nobori et al., Nature (Lond.), 368: 753, 1994]. CDKN2 mutations are more common in cultured human uroepithelial cells (HUC) than in uncultured bladder cancers. We examined the status of CDKN2/p16 in early and late passage (P) cultures of HUC. HUC immortalization was not accompanied by p16 loss, even in cells with a hemizygous 9p21-pter deletion, but late passage cultures with a p16 loss showed decreased generation time. Thus, the data do not indicate that CDKN2 is a candidate for a chromosome 9 senescence gene but suggest that p16 loss may confer a growth advantage in vitro. Significant differences in p16 levels were observed among HUC cell lines, but no CDKN2 mutations were detected. However, an inverse correlation between elevated p16 and loss of pRb function was observed (P < 10(-4)). Ten samples with normal pRb showed low or undetectable p16 levels, while seven samples with known pRb alterations showed abundant p16 but nevertheless grew vigorously in culture. These results support the hypothesis that p16 mediated cell cycle inhibition, as well as p16 regulation, occurs via pRb dependent pathway(s).
Insights
Loss of the CDKN2 gene product, p16, may provide a growth advantage in human uroepithelial cells (HUC) in vitro, independent of senescence. This p16 loss correlates with altered pRb function, suggesting pRb-dependent pathways regulate p16.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The CDKN2 (MTS1) gene encodes p16, a cell cycle inhibitor that targets the cyclin D/CDK4 complex.
- p16 normally phosphorylates pRb, negatively regulating cell cycle progression.
- CDKN2 mutations are observed in cultured human uroepithelial cells (HUC).
Purpose of the Study:
- To investigate the status of CDKN2/p16 in early and late passage HUC cultures.
- To determine if CDKN2 is a senescence gene in HUC.
- To explore the relationship between p16 levels, pRb function, and cell growth in HUC.
Main Methods:
- Analysis of CDKN2/p16 status in early and late passage HUC cultures.
- Examination of HUC immortalization and senescence markers.
- Assessment of pRb function and its correlation with p16 levels.
Main Results:
- HUC immortalization was not associated with p16 loss, even with 9p21-pter deletion.
- Late passage HUC cultures with p16 loss exhibited decreased generation times, suggesting a growth advantage in vitro.
- No CDKN2 mutations were detected, but significant differences in p16 levels were observed.
- An inverse correlation was found between elevated p16 and loss of pRb function (P < 10(-4)).
- HUC with normal pRb had low p16, while those with pRb alterations had high p16 but grew vigorously.
Conclusions:
- CDKN2/p16 is not a candidate for a chromosome 9 senescence gene in HUC.
- Loss of p16 may confer a growth advantage in vitro for HUC.
- p16-mediated cell cycle inhibition and regulation likely occur via pRb-dependent pathways.