PML protein association with specific nucleolar structures differs in normal, tumor and senescent human cells Lenka Janderova ´-Rossmeislova ´, Zora Nova ´kova ´, Jana Vlasa ´kova ´, Vlada Philimonenko, Pavel Hoza ´k, Zdene ˇk Hodny ´ * Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vı ´den ˇ ska ´ 1083, 142 20 Prague 4, Czech Republic Department of Biology of the Cell Nucleus, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vı ´den ˇ ska ´ 1083, 142 20 Prague 4, Czech Republic Received 23 October 2006; received in revised form 9 February 2007; accepted 12 February 2007 Available online 12 March 2007 Abstract Promyelocytic leukemia protein (PML), a tumor suppressor, forms in most human cell types discrete multiprotein complexes termed PML nuclear bodies. Here, we have used indirect immunofluorescence and confocal microscopy to describe various forms of a novel nuclear PML compartment associated with nucleoli that is found under growth-permitting conditions in human mesenchymal stem cells (hMSC) and skin fibroblasts but not in several immortal cell lines with defects in the p53 and pRb pathways. In addition, we found that shut-off of rRNA synthesis induced by actinomycin D causes PML translocation to the surface of segregated nucleoli. This translocation is dynamic and reversible, following changes in nucleolar activity. Intriguingly, treatment causing premature senescence restores PML binding to nucleoli-derived structures and to the surface of segregated nucleoli in HeLa cells. These findings indicate that PML may be involved in nucleolar functions of normal non-transformed or senescent cells. The absence of nucleolar PML compartment in rapidly growing tumor-derived cells suggests that PML association with the nucleolus might be important for cell-cycle regulation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: PML; PML nuclear bodies; Nucleolus; Stem cells; Actinomycin D; Senescence 1. Introduction The nucleolus is the most prominent nuclear subdomain dedicated mainly to RNA polymerase I-dependent rRNA synthesis and pre-ribosome assembly (Olson, 2004). The synthesis of rRNA is the very first step in the ribosome bio- genesis as newly synthesized rRNA precursors provide binding sites for ribosomal proteins and are further pro- cessed to pre-40S and pre-60S ribosomal subunits (Fro- mont-Racine et al., 2003). Importantly, the number of functional ribosomes often limits the rate of overall protein synthesis. Therefore, the precise control of the nucleolar activity and consequently the number of ribosomes is one of the most effective and basic tools for the regulation of cellular growth and proliferation (Ruggero and Pandolfi, 2003; Grummt, 2003). Exact mechanisms of the regulation of rRNA synthesis and other nucleolar activities are under intense studies. It has been well-documented that the synthesis of rRNA is regulated by external growth conditions (Grummt, 1999). The molecular basis of this regulation is still not completely understood, however, the activation of mTOR signaling pathway by optimal growth conditions (i.e. accessibility of growth factors, essential aminoacids etc.) has been sug- gested to contribute to the activation of RNA polymerase I (Pol I) transcription machinery (Mayer et al., 2004). On the other hand, serum starvation, contact inhibition, stress and cellular senescence lead to inhibition of Pol I transcription (Grummt, 2003). 1047-8477/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2007.02.008 * Corresponding author. Fax: +420 241062289. E-mail address: hodny@biomed.cas.cz (Z. Hodny ´). www.elsevier.com/locate/yjsbi Journal of Structural Biology 159 (2007) 56–70 Journal of Structural Biology