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International audience; Inhalation of radon is closely associated with an increased risk of lung cancers. While the involvement of Ink4a in lung tumor development has been widely described, the tumor suppressor gene has not been studied in radon-induced lung tumors. In this study, loss of heterozygosity (LOH) analysis of the Cdkn2a locus, common to the Ink4a and Arf genes, was performed on 33 radon-induced rat lung tumors and showed a DNA loss in 50% of cases. The analysis of p16(Ink4a) protein expression by immunohistochemistry revealed that 50% of the tumors were negative for this protein. Looking for the origin of this lack of expression, we observed a low frequency of homozygous deletion (6%), a lack of mutation, an absence of correlation between promoter methylation and Ink4a mRNA expression and no correlation between LOH and protein expression. However, a tendency for an inverse correlation between p16(Ink4a) and pRb protein expression was observed. The expressions of p19Arf, Mmd2 and Mdm4 were not deregulated and only 14% of the tumors were mutated for Tp53. These results indicated that Ink4a/Cdk4/Rb1 pathway deregulation, more than Arf/Mdm2/Tp53 pathway, has a major role in the development of these tumors through p16(Ink4a) deregulation. However, all known mechanisms of inactivation of the pathway do not play a recurrent role in these tumors and the actual origin of the lack of p16(Ink4a) protein expression remains to be established.

International audience; The nucleolar Arf protein has been shown to regulate cell cycle through both p53-dependent and -independent pathways. In addition to the well-characterized Arf-mdm2-p53 pathway, several partners of Arf have recently been described that could participate in alternative regulation process. Among those is the nucleolar protein B23/NPM, involved in the sequential maturation of rRNA. p19ARF can interact with B23/NPM in high molecular complexes and partially inhibit the cleavage of the 32S rRNA, whereas the human p14ARF protein has been shown to participate in the degradation of NPM/B23 by the proteasome. These data led to define Arf as a negative regulator of ribosomal RNA maturation. Our recent finding that the human p14ARF protein was able to specifically interact with the rRNA promoter in a p53-independent context, led us to analyse in vitro and in vivo the consequences of this interaction. Luciferase assay and pulse-chase experiments demonstrated that the rRNA transcription was strongly reduced upon p14ARF overexpression. Investigations on potential interactions between p14ARF and the transcription machinery proteins demonstrated that the upstream binding factor (UBF), required for the initiation of the transcriptional complex, was a new partner of the p14ARF protein. We next examined the phosphorylation status of UBF as UBF phosphorylation is required to recruit on the promoter factors involved in the transcriptional complex. Upon p14ARF overexpression, UBF was found hypophosphorylated, thus unable to efficiently recruit the transcription complex. Taken together, these data define a new p53-independent pathway that could regulate cell cycle through the negative control of rRNA transcription.

Evidence is accumulating about the role of individual AP-1 components in cell proliferation and transformation. Notably, Ras-mediated transformation is characterized by the upregulation of particular AP-1 members, such as c-Jun and Fra-1. The p14/p19ARF tumor suppressor gene is a key link between oncogenic Ras signaling and the p53 pathway. We explored the involvement of AP-1 dimers in the transcriptional regulation of the p14/p19ARF gene. We demonstrate that both the human and mouse ARF promoters are transcriptional targets of selective AP-1 dimers. The ARF promoter is regulated specifically by AP-1 heterodimers containing Fra-1. Overexpression of c-Jun approximately Fra-1 dimers in primary murine fibroblast cells led to the upregulation of the endogenous ARF protein and growth arrest. Conversely, inhibition of c-Jun or Fra-1 protein levels resulted in decreased ARF expression. In addition, we show that AP-1 dimers cooperate with oncogenic Ras in the transcriptional activation of the p14/p19ARF promoter. Thus, AP-1 heterodimers may contribute to the regulation of ARF expression upon oncogenic signaling.