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The disruption of the normal function and nuclear localization of the promyelocytic leukemia-associated protein (PML) may play a major role in the pathogenesis of acute promyelocytic leukemia. PML, which is concentrated in nuclear bodies (PML bodies), has been shown to have growth- and transformation-suppressive properties. In this study, we have examined the intranuclear distribution of PML in a conditionally immortalized human cell line (IDH4) in which both proliferation and immortalization are dependent on the presence of SV40-encoded large T-antigen (SV40T). Expression of SV40T is controlled by a dexamethasone (Dex)-inducible promotor. Suppression of SV40DT (Dex removal) in IDH4 cells causes G1 arrest and expression of the senescent phenotype. This is accompanied by a redistribution of PML in most cells from the usual pattern containing only spherical bodies to a pattern, containing large doughnut-like or fiber-like structures in addition to the spherical bodies. This change in pattern is reversed when phenotypically senescent IDH4 cells are stimulated to proliferate again by SV40T-induction. Moreover, we find that there is a similar change in the PML pattern between young and senescent or serum-starved young IMR90 human fibroblasts, from which IDH4 cells are derived. However, fewer serum-starved cells contain large PML bodies than senescent cells. Our observations suggest senescence, although it may be partly related to growth arrest. Using three-dimensional fluorescence digital imaging microscopy, we have found that the apparently doughnut-like PML structures have a cylindrical or egg-shaped form and that PML is concentrated to the outer shell of the structure.

We have previously reported that EBNA-5, one of the Epstein-Barr virus-encoded proteins, accumulates in the nuclear bodies containing PML, the promyelocytic leukemia associated protein. In this study, we examine the intranuclear distribution of SV40 large T-antigen (SV40T), the p53 tumor suppressor protein (p53), and PML in a conditionally immortalized cell line, IDH4. In IDH4 cells, the expression of SV40T is regulated by a dexamethasone (Dex)-driven promoter. Withdrawal of Dex results in down-regulation of SV40T and growth arrest, whereas addition of Dex to the growth-arrested cells results in up-regulation of SV40T and proliferation. In proliferating IDH4 cells, SV40T is concentrated in nuclear dots that are also positive for p53. Many of these dots are juxtaposed to PML positive structures but do not colocalize with them. After removal of Dex, SV40T-p53 dots gradually disappear, while the PML structures remain. Induction of SV40T in nonproliferating IDH4 cells causes a coordinated redistribution of SV40T and p53. The immunostaining for SV40T and p53 is first weak, then strong with a homogeneous distribution, and 3-4 days later becomes dot-like again. This reappearance of SV40T-p53 dots coincides with the recovery of proliferation in restimulated IDH4 cells. Also, the p53 pattern correlates with the SV40T pattern with regard to both morphology and intensity during both suppression and induction of SV40T. Taken together, our data suggest that (i) the level of p53 is coregulated with the level of SV40T in a dose-dependent fashion; (ii) the formation of SV40T-p53 nuclear dots correlates with the transformed phenotype; (iii) the SV40T-p53 dots localize preferentially to the neighborhood of PML bodies which are already present in normal cells.

POM121 is an integral membrane protein specifically localized in the pore membrane domain of the nuclear envelope. We have investigated the intracellular distribution of rat POM121 heterologously overexpressed in monkey COS cells by immunofluorescence and fluorescence digital imaging microscopy. At low levels of expression overexpressed POM121 was distributed in the nuclear envelope in a punctate fashion, partially overlapping with the distribution of nuclear pores. At high levels of expression, however, the overexpressed protein accumulated in intranuclear bodies. These bodies represent a novel subnuclear structure, displaying a defined cylindrical structure and a distinct localization at or adjacent to the inner nuclear membrane. The C-terminal portion of POM121, which contains a pentapeptide repeat domain common to a subfamily of related nucleoporins, was sufficient to mediate targeting to the nuclear envelope as well as formation of intranuclear bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.