The mouse mAb, mAb 327, that recognizes specifically both pp60v-src and pp60c-src in a wide variety of cells, has been used to determine precisely the various locations of pp60c-src in NIH c-src overexpresser cells, using the technique of immunofluorescence microscopy. In interphase cells, the protein exhibits two main distributions: one that appears uniform and in association with the cell surface and the other that is patchy and juxtanuclear and coincides with the centrosomes. The juxtanuclear aggregation of pp60c-src-containing patches depends on microtubules and does not seem to occur within the Golgi apparatus and the rough ER. At the G2-to-M-phase transition, a drastic change in the localization patterns of pp60c-src takes place. We also report experiments in which the NIH c-src overexpresser cells were exposed to Con A for various times to induce a redistribution of the cell surface Con A receptors. We show that, at each stage of the Con A-mediated endocytotic process, the Con A-receptor complexes redistribute into structures to which pp60c-src appears also to be associated: at first, into patches that form at the cell surface level and then, into a cap that stands at the cell center in a juxtanuclear position and that coincides with the Golgi apparatus. During this capping process, pp60c-src-containing vesicles continue to accumulate in a centriolar spot, as in interphase, Con A-untreated cells, from which Con A is excluded. The significance of the intracellular locations of pp60c-src to the possible functions of the protein is discussed. Also, the distribution patterns of the cellular protein in the NIH c-src overexpresser cells are compared with those of pp60v-src in RSV-transformed cells. The differences observed are discussed in relation with the differences in transforming capacities of the two proteins. Finally, the possible physiological significance of the association between pp60c-src and the structures generated after the binding of Con A to its surface receptors is addressed.

Two speci®c inhibitors of cyclin-dependent kinase 2 (Cdk2), roscovitine and olomoucine, have been shown recently to induce nuclear accumulation of wt p53 and nucleolar unravelling in interphase human untransformed IMR-90 and breast tumor-derived MCF-7 cells. Here, we show that the early response of MCF-7 cells to roscovitine is fully reversible since a rapid restoration of nucleolar organization followed by an induction of p21 WAF1/CIP1 , a downregulation of nuclear wt p53 and normal cell cycle resumption occurs if the compound is removed after 4 h. Interestingly, similar reversible e ects are also induced by the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole. Upon short-term treatment also, both compounds signi®cantly, but reversibly, reduce the level of 45S precursor ribosomal RNA. Cells exposed to the two types of protein kinase inhibitors for longer times keep exhibiting altered nucleolar and wt p53 features, yet they strikingly di erentiate in that most roscovitine-treated cells fail to ever accumulate high levels of p21 WAF1/CIP1 in contrast with DRB-treated ones. In both cases, however, the cells eventually fall into an irreversible state and die. Moreover, we found that constitutive overexpression of p21 WAF1/CIP1 alters the nucleolar unravelling process in the presence of DRB, but not of roscovitine, suggesting a role for this physiological Cdk inhibitor in the regulation of nucleolar function. Our data also support the notion that both roscovitine-and DRB-sensitive protein kinases, probably including Cdk2 and CKII, via their dual implication in the p53-Rb pathway and in ribosomal biogenesis, would participate in coupling cell growth with cell division. Oncogene (2001) 20, 5951 ± 5963.

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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 shortterm 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.