The INK4a/ARF locus which is frequently inactivated in human tumours encodes two dierent tumour suppressive proteins, p16 INK4a and ARF. p16INK4a is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a ®nal eector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identi®ed. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance. Oncogene (2001) 20, 1033 ± 1041.

The ARF gene (p19 ARF in mouse and p14 ARF in man) has become a central actor of the cell cycle regulation process as it participates to the ARF-MDM2-p53 pathway and the Rb-E2F-1 pathway. By use of immunoprecipitation and Western blotting (IP/WB), we now show that ARF physically associates with Topoisomerase I (Topo I). ARF-Topo I immune complexes were detected in SF9 insect cells infected with recombinant baculoviruses encoding the two genes as well as in 293 cells that express endogeneously these proteins. Preparations of a GST ± ARF recombinant protein stimulated the DNA relaxation activity of Topo I but, in contrast, had no eect on the decatenation activity of Topo II. The Topo I stimulation was also detected in cell extracts of SF9 cells expressing both proteins. A confocal microscopy study indicated that part of ARF and Topo I colocalized in the granular component structure of the nucleolus. As a whole, our data indicate that Topo I is a new partner of ARF and suggest that ARF is involved in cell reactions that require Topo I. Oncogene (2001) 20, 836 ± 848.

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. p19 ARF can interact with B23/NPM in high molecular complexes and partially inhibit the cleavage of the 32S rRNA, whereas the human p14 ARF 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 p14 ARF 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 p14 ARF overexpression. Investigations on potential interactions between p14 ARF 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 p14 ARF 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 p14 ARF 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.

The tumor suppressor Arf (Alternative Reading Frame) protein (p14 ARF in human and p19 ARF in mouse) is mainly located in the nucleolus consistent with its subcellular localization, the protein has been shown to specifically interact with 5.8S rRNA and with B23/Nucleophosmin and to regulate ribosome biogenesis. Here, we show that the p14 ARF protein interacts with chromatin and is recovered by chromatin immunoprecipitation (ChIP) in a fraction that contains a DNA sequence of the rRNA gene promoter. In addition, topoisomerase I (Topo I) that has been shown to interact with p14 ARF coprecipitates with p14 ARF containing chromatin. These data, in view of the function for Topo I in rRNA transcription, are consistent with a role for the p14 ARF -Topo I complex in rRNA transcription and/or maturation.

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