Abstract
The human Ink4a/Arf tumor suppressor locus encodes two distinct products: p16Ink4a which prevents phosphorylation and inactivation of the retinoblastoma protein and, p14Arf, a nucleolar protein which activates the function of the tumor suppressor p53 protein in the nucleoplasm in response to oncogenic stimulation through an as yet ill-defined mechanism. Here we show that the level of endogenous p14Arf and its balance between the nucleolus and the nucleoplasm in HeLa cells are exquisitely sensitive to changes in cell morphology and to short-lived perturbations in cell cycle and in nucleolar function such as those induced by the cyclin-dependent kinase inhibitor, roscovitine, and the casein kinase II and RNA synthesis inhibitor, DRB. Most remarkably, whereas p14Arf predominantly concentrates in the nucleolus of interphase cells and transiently disappears between metaphase and early G1 under normal growth conditions, it massively and reversibly accumulates in the nucleoplasm of postmitotic and S-phase cells upon short-term treatment with roscovitine and, at a lesser extent, DRB. In line with the fact that the nuclear level of p53 reaches a peak between mid-G1 and the G1/S border in p53-expressor cells which lack Arf expression, these results provide a clue that, in p53+/Arf+ cells, Arf proteins might serve both to speed and to amplify p53-mediated responses in conditions and cell cycle periods in which the mechanisms involved in p53 stabilization and activation are not fully operational. They further suggest that human endogenous p14Arf might activate p53 pathways in physiologic situations by acting inside the nucleoplasm, especially when normal cell cycle progression and nucleolar function are compromised.
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Abbreviations
- Cdk:
-
cyclin-dependent kinase
- CKII:
-
casein kinase II
- DRB:
-
5,6-dichloro-1-β-D-ribofuranosylbenzimidazole
- BrdUrd:
-
5-bromodeoxyuridine
- Dapi:
-
4′,6-diamidino-2-phenylindole
- MEFs:
-
mouse embryo fibroblasts
- NDFs:
-
nucleolus-derived foci
- PNBs:
-
prenucleolar bodies
- DMEM:
-
Dulbecco's modified Eagle's medium
- IPTG:
-
isopropyl β-D-thiogalactopyranoside
- FITC:
-
fluorescein isothiocyanate
- TRITC:
-
tetramethyl rhodamine isothiocyanate
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Acknowledgements
We are greatly indebted to Gordon Peters for his generosity in providing us with the 4c6/4 mouse monoclonal and JR14 rabbit polyclonal anti-p14Arf antibodies as well as with the NARF2 and NARF2/E6 cell lines and to Susana Llanos, for her valuable information and advices. We are thankful also to Tony Hunter for supplying the anti-cyclin A antiserum. Our acknowledgements extend to Frederic Coquelle for teaching us microscope imaging and to Dany Rouillard for her help with the flow cytometry experiments. The study was supported by the Centre National pour la Recherche Scientifique and the Curie Institute.
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David-Pfeuty, T., Nouvian-Dooghe, Y. Human p14Arf: an exquisite sensor of morphological changes and of short-lived perturbations in cell cycle and in nucleolar function. Oncogene 21, 6779–6790 (2002). https://doi.org/10.1038/sj.onc.1205871
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DOI: https://doi.org/10.1038/sj.onc.1205871
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