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Suppression of Ras-mediated NIH3T3 transformation by p19ARF does not involve alterations of cell growth properties
V Calabrò1, T Parisi, A Di Cristofano
1Department of Genetics, General and Molecular Biology, University of Naples Federico II, Italy.
Abstract:
The INK4a gene, one of the most frequently disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p19ARF, that both block cell proliferation. p16INK4a is a component of the Rb regulatory pathway, while p19ARF has been functionally related to p53. Moreover, p16INK4a is inactivated in many human tumors, while it has been very recently reported that p19ARF null mice develop tumors early in life. We show here that p19ARF is able to inhibit the formation of G418-resistant colonies when transfected into human and mouse cell lines expressing wild-type p53, regardless of p16 status. Moreover its amino terminal domain encoded by exon 1beta is still sufficient to obtain the same effect. We have analysed the ability of p19ARF to interfere with Ras-mediated cellular transformation in the NIH3T3 cell line. Cotransfection of p19ARF together with activated ras potently inhibited the formation of transformed foci in a dose-dependent manner. We have also isolated stable NIH3T3 transfectants expressing p19ARF and we have measured their growth properties as well as their efficiency of transformation by activated ras. Our results suggest that p19ARF can interfere with oncogene-mediated transformation, without significantly affecting NIH3T3 cell growth, at least at the levels of expression achieved in these experiments.
Insights
The p19ARF protein inhibits cell proliferation and oncogene-mediated transformation. This tumor suppressor function is mediated by its amino-terminal domain and is effective even without affecting cell growth.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- The INK4a gene produces two tumor suppressors: p16INK4a and p19ARF.
- p16INK4a acts in the Rb pathway, while p19ARF relates to p53.
- INK4a gene disruptions are common in human cancers, and p19ARF deficiency leads to early tumor development.
Purpose of the Study:
- To investigate the role of p19ARF in inhibiting cell proliferation and oncogene-induced transformation.
- To determine if p19ARF's amino-terminal domain is sufficient for its tumor-suppressive activity.
- To assess p19ARF's impact on cell growth and transformation mediated by Ras.
Main Methods:
- Transfection of human and mouse cell lines with p19ARF.
- Analysis of colony formation in G418-resistant cells.
- Cotransfection of NIH3T3 cells with p19ARF and activated Ras.
- Isolation of stable NIH3T3 transfectants expressing p19ARF.
Main Results:
- p19ARF inhibited colony formation in cells with wild-type p53, irrespective of p16 status.
- The amino-terminal domain of p19ARF was sufficient for this inhibitory effect.
- p19ARF potently inhibited Ras-mediated transformation in NIH3T3 cells in a dose-dependent manner.
- Stable p19ARF expression did not significantly affect NIH3T3 cell growth.
Conclusions:
- p19ARF functions as a tumor suppressor by inhibiting cell proliferation.
- p19ARF interferes with oncogene-induced cellular transformation.
- The amino-terminal domain of p19ARF is critical for its anti-transformation activity.