Human P14arf: an Exquisite Sensor of Morphological Changes and of Short-Lived Perturbations in Cell Cycle and in Nucleolar Function

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Human p14Arf: an exquisite sensor of morphological changes and of short-lived perturbations in cell cycle and in nucleolar function

The´ re` se David-Pfeuty*,1 and Yolande Nouvian-Dooghe1

1UMR 146 du CNRS, Institut Curie-Recherche, Baˆtiment 110, Centre Universitaire, 91405 Orsay Ce´dex, France

The human Ink4a/Arf tumor suppressor locus encodes integrity of cell cycle checkpoint controls (Hartwell and two distinct products: p16Ink4a which prevents phosphor- Weinert, 1989). Defects in checkpoint controls can ylation and inactivation of the retinoblastoma protein trigger either cell death or survival of cells carrying and, p14Arf, a nucleolar protein which activates the genetic anomalies and prone to develop genomic function of the tumor suppressor p53 protein in the instability which is the hallmark of cancer cells nucleoplasm in response to oncogenic stimulation through (Hartwell, 1992). It has been realized for some time an as yet ill-defined mechanism. Here we show that the that the p53 tumor suppressor gene is a component of level of endogenous p14Arf and its balance between the DNA damage checkpoints in mammalian cells since its nucleolus and the nucleoplasm in HeLa cells are function is required to prevent traverse across the G1/S exquisitely sensitive to changes in cell morphology and and G2/M boundaries in the presence of many types of to short-lived perturbations in cell cycle and in nucleolar genotoxic stress (O’Connor, 1997; Giaccia and Kastan, function such as those induced by the cyclin-dependent 1998) via promoting cell cycle arrest or apoptosis. It kinase inhibitor, roscovitine, and the casein kinase II and has been realized for even longer that p53 is mutated or RNA synthesis inhibitor, DRB. Most remarkably, lost in more than 50% of human cancers (Hollstein et whereas p14Arf predominantly concentrates in the al., 1994) which provides a hint that it would also serve nucleolus of interphase cells and transiently disappears in vivo to bar cell proliferation in inappropriate between metaphase and early G1 under normal growth environmental settings, a conclusion which has been conditions, it massively and reversibly accumulates in the consolidated by all kinds of in vivo and in vitro nucleoplasm of postmitotic and S-phase cells upon short- investigations (Ko and Prives, 1996; Levine, 1997; term treatment with roscovitine and, at a lesser extent, Agarwal et al., 1998). The possibility that the function DRB. In line with the fact that the nuclear level of p53 of p53 in tumor suppression and in DNA damage reaches a peak between mid-G1 and the G1/S border in response might represent two distinct facets of its p53-expressor cells which lack Arf expression, these activity has emerged recently from the discovery that results provide a clue that, in p53+/Arf+ cells, Arf oncogenic stimulation can induce its activation via a proteins might serve both to speed and to amplify p53- mechanism quite different from that generally used by mediated responses in conditions and cell cycle periods in DNA damages. Whereas the latter utilizes protein which the mechanisms involved in p53 stabilization and kinases to phosphorylate p53 at various sites mostly activation are not fully operational. They further suggest within its N-terminal transactivation domain and to that human endogenous p14Arf might activate p53 disrupt its interaction with its target and negative pathways in physiologic situations by acting inside the regulator, Mdm2 (Prives, 1998), the first relies on the nucleoplasm, especially when normal cell cycle progres- expression of the Arf proteins (Quelle et al., 1995; sion and nucleolar function are compromised. Della Valle et al., 1997), encoded by the late-identified Oncogene (2002) 21, 6779 – 6790. doi:10.1038/sj.onc. Arf (alternate reading frame) transcript of the Ink4a 1205871 gene (Quelle et al., 1995; Duro et al., 1995; Stone et al., 1995; Mao et al., 1995). Human p14Arf, like mouse Keywords: p14Arf; cell cycle regulation; tumor suppres- p19Arf, stabilizes p53 owing to its capability to interact sion; cell morphology; cyclin-dependent kinase directly with Mdm2 (Pomerantz et al., 1998; Zhang et al., 1998; Kamijo et al., 1998; Stott et al., 1998) and, thereby, to inhibit the Mdm2-mediated nuclear export Introduction (Tao and Levine, 1999; Zhang and Xiong, 1999), ubiquitination and degradation of p53 (Honda and Faithful cell reproduction depends on the orderly Yasuda, 1999). Arf upregulation in primary cells completion of S and M phase which relies on the exposed to hyperproliferative signals such as myc (Zindy et al., 1998), E1A (de Stanchina et al., 1998), E2F1 (Bates et al., 1998; Dimri et al., 2000), oncogenic ras (Palmero et al., 1998; Lin and Lowe, 2001) and v- *Correspondence: T David-Pfeuty; E-mail: The´ re` [email protected] abl (Radfar et al., 1998; Cong et al., 1999) seems to be Received 20 February 2002; revised 8 July 2002; accepted 15 July directly involved in stabilizing p53, the physiologic 2002 outcome being cell cycle arrest or apoptosis and, p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6780 possibly, after diverting the p53 pathway, cell trans- response because it would permit to accumulate at the formation (Sherr, 1998; Sharpless and DePinho, 1999). same time high levels of Mdm2 ready to eliminate p53 A rationale for these observations is provided by the as soon as the triggering signal is switched off. This fact that Arf is a direct target gene of some hypothesis that Arf might work at extending the transcription factors of the E2F family (Degregori et duration of reversible p53-mediated cell cycle arrest is al., 1997; Bates et al., 1998) whose function is required already consistent with the report that NARF2 cells to promote S phase entry (Harbour and Dean, 2000; arrested by inducible expression of p14Arf resume Nevins, 2000). Altogether, these data have led to the normal cycling upon removal of the inducer after 2 concept that, via Arf, p53 would also participate in days (Stott et al., 1998). Whether this hypothesis is also oncogene checkpoints that guard cells against hyper- relevant to physiologic situations clearly deserves proliferative signals (Sherr, 1998). further investigation. The fact that the co-expression Yet, the mechanism how Arf proteins activate p53 is of p14Arf and p53 does not occur in tumor cells still unclear because the subcellular compartment in suggests, not only, that it is incompatible with in vitro which p53 and Mdm2 localize preferentially is the tumor cell growth, but also, that some important nucleoplasm and for Arf, it is the nucleolus. Based on aspects of the in vivo Arf regulation are preserved in the results of enforced co-expression experiments, one p53-negative tumor cell lines. Identifying parameters model proposes that Arf proteins function owing to involved in controlling the level of expression and their ability to sequester Mdm2 in the nucleolus and, subnuclear targeting of endogenous p14Arf in this type thence, to trap Mdm2-free p53 inside the nucleus (Tao of tumor cells might therefore be a source of and Levine, 1999; Weber et al., 1999, 2000; Sherr and information not to ignore more than those issued from Weber, 2000) or in PML-like nuclear bodies (Zhang the experiments of ectopic overexpression. and Xiong, 1999). It can not be excluded however that Here we report that both the level of expression and Arf proteins could utilize other tricks to activate p53 subnuclear targeting of endogenous p14Arf are highly given that the nucleolar localization of p14Arf is not variable among clonal variants of the HeLa cell line requested for its function in human cell lines (Llanos et which differ in their morphological appearance. Most al., 2001) and that Mdm2 does not relocalize to the remarkably also, we uncovered that endogenous, but nucleolus following p19Arf-dependent, ras-induced cell not ectopically-expressed, p14Arf disappears in meta- cycle arrest in primary mouse keratinocytes (Lin and phase when the nucleoli disintegrate and re- Lowe, 2001). The major caveat of in vitro studies accumulates in the newly-reformed nucleoli of the aiming at deciphering the mechanism of p53 activation daughter cells at the beginning of G1. Intriguingly by Arf proteins in physiologic situations comes from however, it massively and reversibly accumulates in the the fact that they all rely on the artifice of ectopic nucleoplasm of early G1 and S-phase cells upon short- overexpression because Arf is undetectable in untrans- term treatments with roscovitine and, at a lesser extent, formed cells and its co-expression with p53 does not DRB, that disrupt nucleolar organization. The implica- occur in tumor-derived ones (Pomerantz et al., 1998; tions of these results in relation to the possible in vivo Stott et al., 1998; Bates et al., 1998; Lindstro¨ m et al., functions of the Arf proteins are discussed. 2000). That is why a more conceptual approach to the problem should also be considered. In a recent report Results (David-Pfeuty et al., 2001), we have shown that a short-term treatment of human breast carcinoma Clonal variability in levels and subnuclear localization of MCF-7 cells and non-immortalized IMR-90 fibroblasts endogenous p14Arf with the Cdk inhibitor, roscovitine (Meijer et al., 1997), and the CKII and RNA synthesis inhibitor, DRB Human p14Arf has been reported to localize predomi- (Granick, 1975; Haaf and Ward, 1996), induces a nantly to the nucleolus (Pomerantz et al., 1998; Kamijo reversible activation of p53 that is coupled with a et al., 1998; Stott et al., 1998; Zhang and Xiong, 1999), reversible nucleolar fragmentation and blockade of 45S more precisely to its granular component (Lindstro¨ m et pre-rRNA processing and a reversible brake in G1/S al., 2000; Karayan et al., 2001), in a pattern very transit. In other words, we were able to shift human similar to that seen originally in mouse cells (Quelle et cultured cells into a reversible G0-like state through al., 1995). Occasionally, however, p14Arf has been simultaneously and reversibly activating the p53 path- found to display a speckled staining in the nucleus of way and repressing nucleolar functions. In vivo HeLa cells (Della Valle et al., 1997), or to form nuclear however, many cells can remain quiescent for long bodies with Mdm2 and p53 (Zhang and Xiong, 1999). times before they return to G1 while recovery from the The reports that the fraction of nucleolar p14Arf- roscovitine- or DRB-induced G0 state is hardly positive cells is highly variable from one human tumor possible past 8 h of treatment in cultured MCF-7 cells. cell line to another (Lindstro¨ m et al., 2000) and that Conceivably, normal cells in vivo might be equipped the protein is undetectable in hematopoietic human cell with devices allowing them to reversibly activate the lines that abundantly express its mRNAs (Della Valle p53 pathway and repress nucleolar activity over greatly et al., 1997) are intriguing too. extended periods of time. Now, the mechanism through Using the 4c6/4 monoclonal anti-p14Arf antibody which Arf apparently activates p53 would clearly be (Llanos et al., 2001), we also observed a great suitable to stabilize in a long-term a reversible p53 heterogeneity in the p14Arf distribution in the standard

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6781

Figure 1 (A) Double immunolabeling with mAb 4c6/4 anti-p14Arf and goat anti-B23 antibodies of the various HeLa cell lines and of NARF2/E6 cells (24 h after addition of 1 mM IPTG), and overlay image. Three main cell types, marked out by different types of arrows, can be discriminated according to their anti-p14Arf immunolabeling in HeLa M cells. In contrast p14Arf localizes predominantly to the nucleolus in interphase HeLa G and NARF2/E6 cells, and to nucleoplasmic dots, in HeLa S3 cells. The arrowheads point to mitotic cells that stain weakly for p14Arf and strongly for protein B23 in all HeLa cell lines, but not in NARF2/E6 cells. (B) Phase contrast images depicting the varying phenotypes of living HeLa M, G and S3 cells in culture

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6782 Hela M cell line (Figure 1A) whose representatives asynchronous Hela G cells whose intensity is 4 – 6 exhibit a characteristic epithelial-like phenotype and times higher than that from lysates of asynchronous have the propensity to form bidimensional multi- Hela M and HeLa S3 cells and from nocodazole- cellular aggregates, even at low plating densities arrested mitotic Hela G cells, but, roughly 1/3 of that (Figure 1B). Whereas protein B23 concentrates at a from lysates of NARF2/E6 cells treated for 24 h with relatively constant rate primarily in the nucleoli of all 1mM IPTG (Figure 2). Contrastingly, the 38 kDa interphase Hela M cells, three main cell types can be band corresponding to protein B23 is about of the discriminated according to their anti-p14Arf immuno- same strength in all lanes (Figure 2). labeling (Figure 1A): (i) the most individualized, well- spread cells (open arrows) that emerge from the Differential distribution of nucleolar proteins B23, p14Arf multicellular aggregates display intensely decorated and Ki-67 throughout mitosis in HeLa G cells nucleoli and a few bright nucleoplasmic speckles; (ii) in some isolated well-spread cells (thick arrows), Mammalian cell nucleoli disaggregate at the onset of however, the number of nucleoplasmic speckles gets M-phase into distinctive subcomponents that redis- prominent; (iii) cells embedded within the multicellular tribute to different cellular structures and in which are aggregates (not shown) and the less spread isolated selectively stored the dislocated parts of the ribosome ones (thin arrows) exhibit a variable density of factory during the course of mitosis (Dundr et al., nucleoplasmic speckles and a weak, if any, nucleolar 2000; Savino et al., 2001). There is good evidence also labeling. that nucleoli reconstruct in postmitotic cells owing in In view of these observations, we thought of part to conveyance of material from the mother cell. examining the p14Arf distribution in two morphologi- This is true in particular for nucleolar proteins cally distinct HeLa clones: HeLa G, whose involved in pre-rRNA processing (like protein B23) representatives present a more fibroblastoid, but well- which reenter the nucleolus via the PNB pathway spread phenotype and remain dispersed in sparse approximately at a stage where their function is cultures, and HeLa S3, whose representatives grow requested (Savino et al., 1999). As it is clearly depicted also dispersed but do not spread very well on the in Figure 3A, the dynamics of p14Arf during the cell substratum and appear refringent under the microscope cycle does not at all follow these rules, even in HeLa G (Figure 1B). Remarkably, p14Arf accumulates at an cells in which, yet, p14Arf distributes almost identically apparent level as high as that of protein B23 almost to protein B23 throughout the greatest part of exclusively in the nucleoli of most interphase HeLa G interphase. During late G2 phase (a) and prophase, cells while it is essentially found in nucleoplasmic p14Arf, similarly to protein B23, accumulates massively speckles in HeLa S3 cells (Figure 1A). Notably also inside large aggregates that encompass the still- and in striking contrast to protein B23, p14Arf is not compacted nucleoli and from which chromosomes are detectable above background level in most metaphase excluded (long arrows); at metaphase, however, the cells in anyone of the present HeLa cell lines (Figure p14Arf and B23 patterns obviously start diverging: 1A, arrowheads), no more than in two other p537/ Arf+ tumor-derived cell lines, Saos-2 and H1299. A staining pattern quite similar to that seen in the HeLa G clone has been detected in the HeLa Tet-on cell line commercialized by Clontech which, too, displays a fibroblastoı¨ d phenotype and in interphase NARF2 and NARF2/E6 cells in which p14Arf expression is strictly dependent upon the presence of IPTG (Stott et al., 1998). In the two latter cell lines, however, its distribution closely resembles that of protein B23 even in mitotic and early G1 cells (Figure 1A, lower panel, arrowheads) which demonstrates, first, that the 4c6/4 anti-p14Arf antibodies do interact with p14Arf when it is present in metaphase and, most importantly, that the mitosis-specific regulation of endogenous p14Arf is lost by the ectopically-expressed one. Neither the anti-B23 labeling (Figure 1A) nor the nucleoli (according to phase contrast microscopic observations; not shown) of the HeLa S3 cells differ grossly from those of the HeLa G and of the HeLa M cells and the anti-p14Arf- decorated nuclear speckles do not coı¨ ncide with any visible phase contrast (not shown) nor anti-B23- Figure 2 Western blot analysis of the expression of protein B23 decorated (Figure 1A) structures. Arf The JR14 rabbit polyclonal anti-p14Arf antiserum (or and p14 performed on equal amounts of whole-cell lysates from: NARF2/E6 (24 h IPTG, 1 mM); HeLa M; HeLa S3; HeLa the 4c6/4 mouse monoclonal antibody) readily detected Tet-On; HeLa G; HeLa G (8 h Roscovitine (Ro), 40 mM); HeLa a 14 kDa band on immunoblots of lysates from G (8 h DRB, 60 mM); HeLa G, mitotic

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6783

Figure 3 (A) Compared kinetics of redistribution of protein B23 and p14Arf throughout mitosis in HeLa G cells. (A) The two proteins keep colocalizing intimately in aggregated nucleolar structures from late G2 (long arrows; a) up to prophase; in metaphase (b) and anaphase (c), p14Arf is barely detectable in most cells in which protein B23 highly concentrates in perichromosomal areas and in the entire cell interior; in early telophase (d), many Arf-/B23+NDFs (thin arrows) and PNBs (arrowheads) can be detected; in late telophase (e), p14Arf weakly concentrates at sites where protein B23 already accumulates signiﬁcantly; at last, p14Arf rapidly collects in early G1 phase (f) up to reaching that of protein B23 in the newly reformed nucleoli. (B) Double immunolabeling of HeLa G cells with mAb 4c6/4 anti-p14Arf and goat anti-Ki-67 antibodies, overlay image and Dapi coloration. In prophase (a), Ki-67 deserts the nucleolar aggregates to which p14Arf remains associated (long arrows) and selectively decorates the condensing chromosomes, a location which it keeps in metaphase, anaphase and early telophase (b, c and d, respectively). (C) Double immunolabeling of HeLa G cells with mAb 4c6/4 anti-p14Arf and rabbit anti-H1Pi antibodies, overlay image and Dapi coloration. The anti-H1Pi antibodies always decorate intensely the p14Arf-negative metaphase (b) cells. They also concentrate, although at a much lesser extent, in the p14Arf-positive nuclear aggregates observed in late G2 and prophase (long arrows; a) and around the highly condensed chromosomes of early telophase cells (thin arrows, d)

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6784 p14Arf becomes barely detectable in many metaphase diffusely throughout the whole cell volume. This cells (75 – 80%; b cells) and in anaphase (c cell), at strongly suggested that p14Arf might be unstable in times when protein B23 highly concentrates in these cells when unbound to appropriate nucleolar perichromosomal areas and in the entire cell interior; structures and enforced to relocalize to the nucleo- in early telophase (d cells), numerous cytoplasmic plasm. We wondered then whether its nucleoplasmic NDFs (thin arrows) and nuclear PNBs (arrowheads) accumulation would not be prohibited also under which brightly stain for protein B23 but faintly for conditions outside mitosis in which the nucleoli p14Arf, re-emerge; in late telophase, a low level of disintegrate as occurs following treatment with roscov- p14Arf is sometimes seen at sites where protein B23 itine (David-Pfeuty, 1999) and DRB in a number of already accumulates significantly (e cells); finally, as the untransformed and tumor-derived cell lines (Granick, chromosomes keep decondensing in early G1 phase (f 1975; Haaf and Ward, 1996; David-Pfeuty et al., 2001). Arf cells), the p14 level rapidly rises up to reaching that A time of exposure of the cells to either 40 mM of protein B23 in the newly reformed nucleoli. roscovitine or 60 mM DRB as short as 1 h is sufficient Therefore, endogenous p14Arf apparently integrates to induce a full disintegration of the nucleolar postmitotic nucleoli later than protein B23 in HeLa G architecture into components which scatter through cells. Our observations also suggested that the slow the nucleus, as evidenced by phase contrast microscopy kinetics of nucleolar reappearance of p14Arf was (Figure 4A, C). Immunofluorescence observations, on probably consequent upon the fact that its stock is the other hand, allowed to distinguish three main cell dramatically depleted at metaphase and has therefore to types on the basis of their anti-p14Arf immunolabeling replenish in post-mitotic cells. We then questioned in roscovitine-treated cell populations (Figure 4A): (i) whether the nucleolar Ki-67 protein which, too, enters cells (marked with asterisks) in which both the anti- the nucleolus later than protein B23 (Savino et al., p14Arf and anti-B23 antibodies decorate mainly dark 1999), behaved like p14Arf during mitosis. As a matter nucleolar bodies (thin arrows) and, at a much lesser of fact, the pattern of expression of Ki-67 throughout extent, the nucleoplasm; (ii) others (large and short mitosis resembles neither that of p14Arf nor that of arrows) in which the nucleoplasmic concentration of protein B23, as shown in Figure 3B: in prophase (a cell), p14Arf is prominent and exceeds largely that of protein Ki-67 deserts the still-aggregated nucleolar structures to B23 which remains associated first with nucleolar which both protein B23 and p14Arf are intimately bodies (thin arrows); (iii) cells, identifiable as post- connected (long arrows); instead, it associates selectively mitotic, early G1 cells by phase contrast microscopy with the condensing chromosomes, a location which it which revealed the presence of a midbody and of keeps in metaphase, anaphase and early telophase (b, c numerous PNBs (open arrows and c region), in which and d cells); in late telophase and early G1 phase, its both the anti-p14Arf and anti-B23 antibodies strongly amount inside PNBs and newly forming nucleoli rises accumulate in a diffuse pattern throughout the whole more slowly than that of protein B23 (Savino et al., nuclear space in varying ratios from one cell-pair to 1999) but faster than that of p14Arf (not shown). another. Double immunolabeling experiments with Double immunolabeling experiments with anti-p14Arf anti-p14Arf and anti-cyclin A antibodies permitted in and anti-phospho-Histone H1 (H1Pi) antibodies were addition to show that the nucleoplasmic concentration performed to determine if there was a possible of p14Arf in roscovitine-treated cells varies in propor- correlation between the timing of disappearance of tion to that of cyclin A (Figure 4B), except in Arf p14 and that of the cyclin B1/Cdk1 kinase activity at postmitotic cells. It is noteworthy that 40 mM roscov- metaphase. As illustrated in Figure 3C, the level of itine is insufficient to visibly deplete the level of H1Pi is always high in the p14Arf-negative metaphase phospho-Histone H1 in mitotic cells, although it visibly cells present in unsynchronized populations (b cell) as but not totally reduces that in interphase cells, as well as in nocodazole-arrested mitotic cells, many of indicated by double immunofluorescence observations which lose p14Arf during the block (not shown). with anti-p14Arf and anti-H1Pi antibodies (not shown). Interestingly, concentrations of anti-H1Pi antibodies On the other side, 60 mM DRB generates effects higher than in the rest of the cell are often found in the somewhat similar to those of 40 mM roscovitine, apart p14Arf- and B23-positive aggregates present in late G2 from the fact that p14Arf accumulation in postmitotic and prophase (long arrows, a cell) and in association cells occurs in both the nucleus and the cytoplasm after with the highly condensed chromosomes of early DRB treatment (Figure 4C). Finally, the effects of both telophase cells (thin arrows, d cells). roscovitine and DRB on p14Arf distribution were fully reversible initially, as they are on nucleolar organization (David-Pfeuty et al., 2001), and p14Arf relocated A short-term treatment of HeLa G cells with roscovitine quasi-totally to the nucleoli as soon as these were fully or DRB induces reversible nucleolar fragmentation and reconstructed, that is, within 1 – 2 h after drug release nucleoplasmic accumulation of p14Arf (not shown). Thus, although endogenous p14Arf localizes predomi- Longer exposure (8 h) to either roscovitine or DRB nantly to the nucleolus of interphase HeLa G cells very resulted in an accumulation of cells in G1 and G2/M similarly to protein B23, it generally disappears at phase (5G1/S/G2-M4=54/33/13 and 58/30/12, metaphase when the nucleoli disassemble and protein respectively, versus 48/44/8 in controls) and beginning B23 redistributes in perichromosomal areas and of appearance of chromatin figures of apoptosis,

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6785

Arf Figure 4 (A) Double immunolabeling with mAb 4c6/4 anti-p14 and goat anti-B23 antibodies of HeLa G cells treated with 40 mM roscovitine for 1 h, overlay and magniﬁed phase contrast image of regions delineated on the overlays: (1) the asterisks designate cells which express low levels of both p14Arf and protein B23 mostly concentrated in dark nucleolar bodies (thin arrows); (ii) the large, short arrows point to a cell which displays a prominent nuclear anti-p14Arf immunostaining, largely exceeding that of protein B23 which is still associated primarily with nucleolar bodies (thin arrows); (iii) both p14Arf and protein B23 strongly accumulate in a diffuse pattern throughout the whole nuclear space in postmitotic cells identiﬁable by their midbody (open arrows and c region). Arf (B) Double immunolabeling of HeLa G cells treated with 40 mM roscovitine for 1 h with mAb 4c6/4 anti-p14 and rabbit anti- cyclin A antibodies, and Dapi coloration showing that the nucleoplasmic concentration of p14Arf in roscovitine-treated cells varies in proportion to that of cyclin A. (C) Double immunolabeling of HeLa G cells treated for 1 h with 40 mM roscovitine or 60 mM DRB, or untreated (Control), with mAb 4c6/4 anti-p14Arf and goat anti-B23 antibodies, overlay and phase contrast image (C Ph). Emphasis is laid here on postmitotic cells (whose midbody is marked out by an open arrow). In control cells, p14Arf barely accumulates in NDFs, PNBs (thin arrows) and small-sized nucleoli (open arrowheads). 1 h-treatment with either roscovitine or DRB prevents normal nucleolar reassembly in postmitotic HeLa G cells; however, it does not prevent accumulation of p14Arf inside the nucleoplasm in the presence of roscovitine and inside both the nucleus and the cytoplasm, in the presence of DRB

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6786 according to the Dapi coloration (not shown). Yet, exhibit a lower spreading capacity than the fibroblastoid survivor cells recovered, upon removal of the drugs, as derivatives, express a low level of p14Arf which resides fast as after 1 h-treatment. The level of expression of mostly in the nucleoplasm. Interestingly, clonal variants both p14Arf and protein B23 did not decline much by which we obtained from the Saos-2 (p53-/Rb-/Arf+) 8 h-treatment (Figure 2) and their subcellular distribu- cell line do not exhibit such a variability in morphology tion did not differ greatly from that seen after 1 h- and in p14Arf distribution and level of expression. Like treatment, except that postmitotic cells invaded with a most cervical carcinoma cells, HeLa cells harbor intact high diffuse immunolabeling for both p14Arf and p53 and Rb genes whose growth suppressor function is protein B23 (such as those pointed by open arrows in kept dormant by the HPV E6 and E7 oncoproteins, Figure 4) were not any longer visible. The whole cell respectively, but can be reactivated through repression populations eventually died upon continued exposure of E6/E7 transcription by HPV E2 proteins (Goodwin to roscovitine, somewhat faster for HeLa G than for and Dimaio, 2000). Perhaps, variation in the residual HeLa S3 and HeLa M cells – DRB is metabolized very level of activity of the p53 and Rb pathways might be fast in HeLa cells as observed earlier in MCF-7 cells responsible for the occurrence of this perplexing (David-Pfeuty et al., 2001) so that its action can not be phenomenon in HeLa cells. followed beyond 8 h. Another major finding of this work is that, although p14Arf and protein B23 distribute similarly through the nucleolus of interphase HeLa G cells, p14Arf, but not Discussion protein B23, suddenly disappears in metaphase, remind- ing of the behaviour of mitotic cyclins. Consistently, its The main debate as to how nucleolar Arf proteins level is still low in the B23-decorated NDFs and PNBs stabilize nucleoplasmic p53 and Mdm2 has focused up which re-emerge in late telophase. Afterwards, it rapidly to now on guessing in which subcellular compartment collects in the newly reformed nucleoli of early G1 cells, they actually manage to activate the p53 function. But, presumably when efficient translational activity is the issue when and in what particular context has restored. Importantly, this regulation of endogenous somewhat been neglected. Two recent reports have p14Arf is lost by ectopically-induced p14Arf. The challenged the earlier view that they would function by behaviour of endogenous p14Arf during mitosis sequestering Mdm2 in the nucleolus or in nuclear bodies contrasts also with that of many other hosts of the (Sherr and Weber, 2000): one, by showing that Mdm2 granular and dense fibrillar compartments of the does not relocalize to the nucleolus following p19Arf- nucleolus (including Ki-67 which enters nucleoli at the dependent, ras-induced cell cycle arrest in primary latest stage of nucleologenesis) which all remain present mouse keratinocytes (Lin and Lowe, 2001); the other, and redistribute to some subcellular structures during by demonstrating that nucleoplasmic forms of Arf mitosis (Gautier et al., 1992; Dundr and Olson, 1998; retain the capacity to stabilize Mdm2 and p53 in human this work). A possible explanation was that p14Arf is cells (Llanos et al., 2001). As emphasized by the latter unstable when unbound to fully assembled nucleoli. authors, when Arf is expressed at supraphysiological This yet would not be a general feature because its levels, it is always possible that the massive nucleolar nucleoplasmic level dramatically rises in early G1 cells accumulation induced would mask the presence of a upon 1 h-exposure to 40 mM roscovitine that is sufficient minor nucleoplasmic pool capable alone to execute the to induce a full nucleolar disintegration. Since roscov- physiologic response. Given the further observation that itine provokes a premature exit from mitosis in HeLa Arf expression in naturally-occurring systems appears cells (Sirri et al., 2000; not shown), it is conceivable that to be regulated at the level of translation and/or protein this could permit the birth of daughter cells that would stability and to be highly sensitive to environmental have inherited a maternal pool of p14Arf. This effect is parameters (Lindstro¨ m et al., 2000; Della Valle et al., specific for roscovitine because 1 h-DRB (60 mM) 1997), it is a matter of paramount importance to treatment (which also totally unravels the nucleoli) identify the parameters which, spatially and temporally, redistributes p14Arf in both the nucleoplasm and the control the level of expression and subnuclear targeting cytoplasm of early G1 cells. DRB, however, contrary to of the endogenous protein. roscovitine, does not provoke a premature exit from Here we show that both the level of expression and mitosis and also potently inhibits RNA polymerase II subnuclear targeting of p14Arf are highly variable (Haaf and Ward, 1996), so that its effects should be among clonal variants of the HeLa cell line which differ more complex. These striking observations provide a in their morphological appearance. In the original hint that Arf might be an essential component of the epithelial-like HeLa cells, there is a low level of p14Arf mitotic exit checkpoint and could account also for the which partitions among the nucleolus and the nucleo- reported implication of Arf proteins in the process of plasm in varying ratios from one cell to another in replicative senescence (Lundberg et al., 2000). It can be subconfluent cultures, but, which is exclusively nucleo- anticipated indeed that untimely exit of mitosis and, plasmic in confluent ones. Strikingly, two different more generally, exit of mitosis before total elimination fibroblastoı¨ d-like HeLa clones express much higher of the maternal pool of Arf, could induce in p53+/ levels of p14Arf which segregates almost exclusively in Arf+ cells a premature activation of the p53 pathway the nucleolus during interphase. Yet, HeLa S3 deriva- and, depending on concurrent signals, reversible or tives which also have lost epithelial-like features but irreversible cell cycle arrest or apoptosis. Interestingly,

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6787 reversible p53-dependent G1 arrest induced by micro- Our previous work (David-Pfeuty et al., 2001) tubule disruption (Khan and Wahl, 1998) depends on enabled us to propose that the capacity of the cyclin p19Arf in MEFs (Khan et al., 2000). The timing of E/Cdk2 complex activity to function as master inactivation of the cyclin B/Cdk1 kinase and that of regulator of restriction (R)-point transit (Pardee, p14Arf disappearance in metaphase HeLa G cells differ 1974; Vidal and Koff, 2000) takes root in the ability and should probably then be mediated by distinct of the complex to serve as a common upstream mechanisms. The first is known to rely on the anaphase- regulator of three distinct pathways involved in promoting complex (Morgan, 1999). As to p14Arf,we inducing cell cycle arrest, S-phase entry and cell failed to stabilize it in metaphase upon 7 h incubation in growth, whose major effectors are p53, E2F1 and the presence of 10 mM MG-132 proteasome inhibitor nucleolar activities, respectively. The complex should (not shown). What regulates Arf unstability in mitosis is therefore be able to perform a variety of tasks within presently under investigation. the three pathways which all should be readily A stable nucleoplasmic relocalization of p14Arf also reversible before R-point transit. This could be occurs beyond early G1 phase upon roscovitine- and achieved owing to devices which operate before the DRB-treatment, yet its apparent rate rises as cells R-point to minimize imbalance and/or interferences progress in the cell cycle, which is somewhat expected between the various cyclin E/Cdk2-activated pathways since Arf is a direct target gene of E2F. The effects of that would compromise their capacity of reversing, if roscovitine and DRB on both nucleolar organization necessary. Now, the Arf proteins can possibly interfere and p14Arf distribution are fully reversible initially but with each of these pathways. They could regulate some eventually become irreversible with time, the cells nucleolar activities since they carry nucleolar localiza- starting visibly to die past 8 h-treatment. This toxicity tion signals (Weber et al., 2000; Lohrum et al., 2000) is likely to be due to the fact that the nucleolar and can accumulate in the nucleolus. (Our preliminary unravelling induced by the two compounds is asso- observation that long-term (48 h) overexpression of ciated with an arrest of pre-rRNA processing in a p14Arf in NARF2 cells induces a full nucleolar variety of cells (David-Pfeuty et al., 2001; Sirri et al., disintegration in 530% of the cells supports indeed 2002), an effect that ultimately should end up in this hypothesis; T David-Pfeuty, unpublished data.) depleting the ribosome supply below a threshold They can also block reversibly entry in S phase by required for survival. activating a reversible p53 pathway (Stott et al., 1998). Together, our data show that short-lived perturba- Finally, human Arf has been shown to interact tions in cell cycle progression and in nucleolar physically with E2F1 and inhibit its transcriptional organization can trigger a swift, totally reversible and activity (Eymin et al., 2001). It is conceivable then that apparently harmless nucleoplasmic accumulation of the Arf proteins could participate in vivo in devices that endogenous Arf that is more extensive at the very are in charge to allow the various cyclin E/Cdk2- beginning of G1 phase and after G1/S transit in HeLa dependent pathways to evolve on an autonomous and cells in which both the p53 and Rb pathways are well-balanced mode for a certain period of time after dormant. Remembering that the p53 nuclear level peaks exit from G0. This in turn would permit G1 phase between mid-G1 and the G1/S border in p53-expressor events to occur reversibly until a critical no return cells in which Arf is either undetectable or not point, namely, the R-point. expressed (Takahashi and Suzuki, 1994; David-Pfeuty et al., 1996; David-Pfeuty, 1999), the presence of Arf in p53+/Arf+ cells could in principle work at broadening Materials and methods the cell cycle window during which the p53 pathway can be reversibly activated, and, thereby, contribute to Cell lines: growth conditions, synchronization and chemicals extend the sphere of activity of p53. For instance, it stands to reason that both speeding and amplifying the The parental ATCC CCL-2 epithelial-like HeLa cell line, p53 response in early G1 might help trigger a durable called afterwards HeLa M, and the Saos-2 osteosarcoma cell line were a gift from Evelyne May (CEA, Fontenay-aux- and reversible cell cycle arrest before the mitogen- Roses, France). The H1299 lung adenocarcinoma cell line stimulated pathways would have generated irreversible Arf and the HeLa S3 and HeLa G (Bosselut et al., 1990) clonal effects. Remind that inducible overexpression of p14 variants were provided by Thierry Soussi (Curie Institute, in NARF2 cells triggers a cell cycle arrest which can Paris, France), Tony Hunter (The Salk Institute, La Jolla, still be released after 2 days (Stott et al., 1998). CA, USA) and Jacques Ghysdael (Curie Institute, Orsay, Expectedly also, an increased nuclear level of Arf at, France), respectively. The HeLa Tet-On cell line was from and beyond G1/S transit should sensitize cells to p53 in Clontech (Palo Alto, CA, USA). All cells were grown in the presence of stress signals inefficient per se at DMEM supplemented with antibiotics and 10% fetal calf stabilizing p53 in the nucleus then. The physiologic serum, at 378C, in a 5% CO2 humidified atmosphere. The outcome should be a reinforcement of G1/S arrest if NARF2/E6 cell line was provided by Gordon Peters (ICRFL, London, UK). It has been obtained by transfection p21 induction is still permitted, but, cell death if it is of pBabepuro-E6 in NARF cells (derived from U2OS human not and if S phase entry occurs. Interestingly, altered osteosarcoma cells expressing inducibly p14Arf upon addition kinetics of p53 and p21 induction have been held of IPTG (Stott et al., 1998)). The NARF2/E6 cells are grown responsible for the lack of long-lasting G1 arrest in in DMEM+10% FCS containing 150 mg/ml hygromycin, DNA-damaged p19Arf-null MEFs (Khan et al., 2000). 300 mg/ml geneticin and 1.25 mg/ml puromycin.

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6788 Mitotic cells accumulating during a 8 h-exposure to carried out by electrophoresis on SDS/polyacrylamide gels 100 nM nocodazole (Sigma, l’Isle d’Abeau Chesnes, France) (16% acrylamide/0.33% bisacrylamide) after boiling the were detached by flushing and collected by centrifugation. samples for 3 min in 26SDS sample buffer. The fractio- The chemical inhibitors of Cdk, roscovitine, was purchased nated proteins are transferred to nitrocellulose at 600 mA from France Biochem (Meudon, France) and the CKII and for 3 h in cold room and, after blocking, the transfer RNA synthesis inhibitor, DRB, from Sigma. membranes are incubated in the presence of the appropriate antibody solution overnight at 48C. After several washes, they are exposed to horseradish peroxidase-conjugated Immunochemical, staining reagents and immunofluorescence antibody and detection of the immune complex is performed procedure using the Pierce ECL Western blotting SuperSignal system The 4c6/4 mouse monoclonal (Llanos et al., 2001) and JR14 (Interchim). rabbit polyclonal anti-p14Arf antibodies were kindly provided by Gordon Peters and the rabbit polyclonal anti-cyclin A Flow cytometry and Trypan blue assays antiserum, by Tony Hunter. The goat polyclonal anti-B23 (C- 19, sc-6013) and anti-Ki-67 (C20, sc-7844) IgGs were Prior to detection of BrdUrd-labeled cells as described purchased from Tebu (Le Perray-en-Yvelines, France) and (Wilson et al., 1985), the cells are pulse-labeled with 30 mM the rabbit polyclonal anti-phospho-Histone H1 (H1Pi) BrdUrd for 15 min, washed in PBS, trypsinized and collected antibodies (UBI), from Euromedex (Souffelweyersheim, by centrifugation. After fixation with 75% ethanol at 7208C France). Counterstaining of DNA was achieved using Dapi for 10 min, the nuclei are isolated following treatment with (1 mg/ml; Sigma). Pulse labeling of cells with BrdUrd (Sigma) 0.5% pepsin in 0.1 N HCl for 20 min and cellular DNA is was performed to identify cells undergoing DNA replication partially denatured with 2 N HCl for 20 min at 378C. After by flow cytometry. The rat monoclonal anti-BrdUrd antibody several washes, they are incubated at RT successively with rat (MAS 250b) was provided as a supernatant fluid from a anti-BrdUrd antibodies (1 h) and with FITC-conjugated goat hybridoma cell line (Interchim, Montluçon, France). Second- anti-rat IgG secondary antibody (1/2 h). They are then ary antibodies (Interchim, Montluçon, France) were Fab washed twice in PBS and stained with 25 mg/ml propidium fragments from goat or donkey IgGs conjugated either with iodide for 30 min at RT. Data are collected using a FACScan FITC or TRITC. flow cytometer (Becton Dickinson & Co., San Jose, CA, Double- or triple-fluorescence cell labeling is currently USA). The green fluorescence associated to BrdUrd-labeled performed after fixation with 3% paraformaldehyde and cells is measured at 520 nm and the red one of PI at subsequent permeabilization by treatment with 0.1% Triton 4620 nm. The G1/S/G2-M ratio is determined from the X-100 at room temperature. Immunolabeling with anti-cyclin bivariate BrdUrd vs DNA content plots by counting the A provides a reliable tool to determine cell cycle position number of dots in the appropriate area defined after during interphase as cyclin A first appears in the nucleus performing control experiments in which the incubation with shortly prior to the onset of S phase and then steadily BrdUrd has been omitted. Counting is made on 10 000 cells. accumulates during progression through the S and G2 phases, The rate of cell death as a function of the time of exposure reaching a maximal level in early prophase (Girard et al., to the different compounds is estimated by the Trypan blue 1991). Alternatively, immunolabeling with anti-phospho- exclusion assay. At the desired time, the medium containing Histone H1 (H1Pi) antibodies can be used for a similar floating cells is recolted and the adhering cells are detached purpose since histone H1 is a very good in vivo and in vitro by trypsinization and collected with the floating ones. Cell substrate for cyclin/Cdk complexes. The Dapi coloration is viability is determined after adding an equivalent volume of a particularly useful to identify a specific mitotic stage starting 0.125% Trypan blue solution to an aliquot of the whole from early prophase in which early chromosome condensation suspended cells and counting under the microscope the is reflected by the appearance of a characteristic punctate proportion of unstained versus total cells deposited in a pattern as well as to detect apoptotic cells which display hemacytometer. For each time point, an average value of cell different types of aberrant chromatin structures (Wyllie et al., viability is obtained from at least five counts over different 1980). Phase contrast microscopy is used to recognize fields of 100 cells. postmitotic cells which typically exhibit a low degree of spreading and remain connected through a visible midbody. This technique permits also to visualize nucleolar structures Abbreviations with some details. Fluorescence microscopy was performed as Cdk, cyclin-dependent kinase; CKII, casein kinase II; DRB, described previously (Savino et al., 2001) with a Leica DM 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole; BrdUrd, 5- RXA microscope equipped with a krypton/argon ion laser and bromodeoxyuridine; Dapi, 4’,6-diamidino-2-phenylindole; a UV filter (which allowed the detection of Dapi, fluorescein MEFs, mouse embryo fibroblasts; NDFs, nucleolus-derived and rhodamine at respectively 350, 492 and 550 nm) using foci; PNBs, prenucleolar bodies; DMEM, Dulbecco’s mod- 406 and 1006 PlanApo objectives. Images were acquired ified Eagle’s medium; IPTG, isopropyl b-D- using a MHz Micromax 1300Y interline CCD camera (Roper thiogalactopyranoside; FITC, fluorescein isothiocyanate; instruments) with the use of Metamorph Image Software TRITC, tetramethyl rhodamine isothiocyanate. (Universal Imaging Corp, West Chester, PA, USA).

Immunoblotting experiments Acknowledgments Cellular extracts for immunoblotting are prepared in lysis We are greatly indebted to Gordon Peters for his buﬀer containing 10 mM Tris-HCl (pH 7.2), 150 mM NaCl, generosity in providing us with the 4c6/4 mouse mono- 0.5% sodium deoxycholate, 1% Triton X-100, 5 mM clonal and JR14 rabbit polyclonal anti-p14Arf antibodies as EDTA, 5 mM dithiothreitol, a mixture of chymostatin, well as with the NARF2 and NARF2/E6 cell lines and to leupeptin, antipapaı¨ n and pepstatin (2 mg/ml each) and 100 Susana Llanos, for her valuable information and advices. kallikrein inactivator units of aprotinin. Protein separation is We are thankful also to Tony Hunter for supplying the

Oncogene p14Arf subnuclear localization and function T David-Pfeuty and Y Nouvian-Dooghe 6789 anti-cyclin A antiserum. Our acknowledgements extend to experiments. The study was supported by the Centre Frederic Coquelle for teaching us microscope imaging and National pour la Recherche Scientiﬁque and the Curie to Dany Rouillard for her help with the ﬂow cytometry Institute.

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Oncogene