Depletion of the Nucleolar Protein Nucleostemin Causes G1 Cell Cycle Arrest via the p53 Pathway

Abstract

Nucleostemin (NS) is a nucleolar protein expressed in adult and embryo-derived stem cells, transformed cell lines, and tumors. NS decreases when proliferating cells exit the cell cycle, but it is unknown how NS is controlled, and how it participates in cell growth regulation. Here, we show that NS is down-regulated by the tumor suppressor p14 ARF and that NS knockdown elevates the level of tumor suppressor p53. NS knockdown led to G1 cell cycle arrest in p53-positive cells but not in cells in which p53 was genetically deficient or depleted by small interfering RNA knockdown. These results demonstrate that, in the cells investigated, the level of NS is regulated by p14 ARF and the control of the G1/S transition by NS operates in a p53-dependent manner. INTRODUCTIONDestabilization or inactivation of the tumor suppressor p53 is typically required to maintain cell proliferation (Vogelstein et al., 2000). However, it is unclear which of many potential upstream regulators of p53 is responsible for cell cycle progression or exit. Nucleostemin (NS) is a nucleolar protein discovered in adult rat brain stem cells, and it is also preferentially expressed in embryo-derived stem cells, transformed cell lines, and human tumors (Tsai and McKay, 2002;Baddoo et al., 2003;Liu et al., 2004;Politz et al., 2005). NS dynamically shuttles between the nucleolus and nucleoplasm, and this exchange is based on its state of GTP binding (Tsai and McKay, 2005). Pull-down and coimmunoselection experiments reveal that NS interacts with p53 (Tsai and McKay, 2002), but whether NS controls cell proliferation in a p53-dependent manner remains unclear.In a previous investigation, we found that the NS is concentrated in regions of the nucleolus that are relatively devoid of nascent ribosomes (Politz et al., 2005). We subsequently asked whether any other cell cycle progression-related proteins might occupy this same intranucleolar territory, and we found that the tumor suppressor p14 ARF (alternate reading frame ͓ARF͔) precisely colocalizes with NS in these ribosome-sparse nucleolar regions (Supplemental Figure 1). Like NS, ARF is linked to p53, and yet NS and ARF play opposite roles in cell proliferation. These initial findings, therefore, motivated us to investigate whether ARF might regulate NS, and we found that it does. In turn, this led to the finding that NS regulates cell cycle progression via the p53 pathway. MATERIALS AND METHODS Cell Culture and TransfectionU2OS, Saos-2, and HeLa cells were cultured at 37°C in DMEM supplemented with 10% fetal bovine serum (FBS). The NARF6 cell line (Stott et al., 1998) was maintained in DMEM containing 10% FBS, 150 g/ml hygromycin, and 300 g/ml Geneticin (G-418; Invitrogen, Carlsbad, CA). For transient transfections, cells were plated in 35-mm dishes, and plasmid DNA was introduced using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. ARF expression in NARF6 cells was induced by the addition of isopropyl-␤-d-thiogalactopyranoside at 1 mM for 48 h. For analysis of cell cycle progr...

Differential Requirement for Nucleostemin in Embryonic Stem Cell and Neural Stem Cell Viability

“…Expression of the neural stem/progenitor cell marker genes Sox1, nestin, and Musashi, and bipotency (generation of both neurons and glial cells upon induction of differentiation) are maintained in the absence of nucleostemin. Our data also demonstrate that, as in cancer cells [16][17][18] and inner cell mass cells [12,13], nucleostemin is involved in cell cycle progression and prevention of apoptosis in ES cells and neural stem/progenitor cells.…”

“…High levels of nucleostemin expression are also detected in several types of cancer cells [16][17][18]. Although the role of nucleostemin in U2OS human osteosarcoma cells has been assessed extensively by siRNA-mediated knockdown experiments, the physiological function of nucleostemin in stem cells has been little characterized, with the exception of a few studies [15,45].…”

“…Thus, it is generally assumed that nucleostemin plays an important role in stem cell proliferation. However, with the exception of a few studies using stromal stem cells from adult human bone marrow [15], most studies investigating nucleostemin function have been performed using nonstem cells, such as U2OS osteosarcoma cells [16][17][18]. The role of nucleostemin in stem cells is therefore still unclear.…”

Distinct genome protective vs. ribosome synthetic functions of the paralogous nucleolar proteins nucleostemin and GNL3L

“…The subsequent identification of GNL3L, the mammalian paralog of nucleostemin, broadened the landscape of possible functions of these two nucleolar GTP-binding proteins. Subsequent work on both nucleostemin and GNL3L (Lin et al, 2010;Ma and Pederson, 2007;Meng et al, 2011a;Meng et al, 2011b;Meng et al, 2008;Zhu et al, 2009;Zhu et al, 2006) drew attention to the involvement of both proteins in cell proliferation, although the direct roles of the proteins were not rigorously proven. At the same time, evidence appeared in Drosophila linking the invertebrate protein, GNL3, to ribosome biosynthesis (Rosby et al, 2009), and another report implicated mammalian nucleostemin in ribosome biosynthesis (Romanova et al, 2009a).…”

“…Effects shown include a G1/S arrest (Dai et al, 2008;Ma and Pederson, 2007;Nikpour et al, 2009) 2009; Romanova et al, 2009a;Zhu et al, 2006). This discrepancy cannot be explained by the status of p53, Rb or p16 in the cell models used.…”