Inhibition of Ribosome Biogenesis by p19(ARF)
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Inhibition of Ribosome Biogenesis by p19(ARF)

CDKN2A (Cyclin Dependent Kinase Inhibitor-2A), which is also referred to as p16(INK4A) encodes ARFs (Alternative Reading Frames), or transcript variants. In mice the p16(INK4A) encodes a transcript variant known as p19(ARF), whereas in humans it encodes p14(ARF). The p19(ARF) acts a tumor suppressor but its action is distinct from the murine p16(INK4A). p19(ARF) acts on the p53 pathway by interacting with MDM2 (Mouse Double Minute-2), thereby blocking degradation of p53 and resulting in p53 stabilization and activation. This prevents shuttling of MDM2 between nucleus and cytoplasm (to where it must export p53 for degradation), thereby leaving p53 free within the nucleus to transactivate transcription of responsive genes. Interestingly, p19(ARF) is required for p53 activation by proliferative signals such as Myc (Myelocytomatosis Oncogene) but not for activation in response to ultraviolet or ionizing radiation. In addition p19(ARF) is also under the control of E2F1 (E2F Transcription Factor-1) (Ref.1). The 5.8S rRNA (5.8S (5.8 Svedberg Unit) Ribosomal RNA) forms a covalent linkage with tumor suppressor p53 protein and plays an important role in ribosome translocation. p53 binds specifically to a region upstream of the transcription start site for the human ribosomal gene cluster (rDNA or Ribosomal DNA). Implication of the p53-5.8S rRNA linkage is that p53 is involved in processing either a major or a minor portion of the rRNA transcript to produce 5.8S rRNA. 5.8S rRNA forms hydrogen bonds with 28S RNA in both the nuclear ribosomal precursor and in the cytoplasmic 60S ribosomal subunit. However, 5.8S rRNA also binds to the 40S ribosomal subunit, forms the ribosomal subunit interface and participates in the ribosomal-A (Aminoacyl) site. The p53 is associated with cytoplasmic ribosomes and is involved in translational control and ribosome biogenesis, under the influence of TFs (Transcription Factors) and RNA PolI (RNA Polymerase-I) (Ref.2).

MEFs (Mouse Embryo Fibroblasts) that survive Myc over-expression sustain p53 mutation or p19(ARF) loss during the process of establishment and become immortal. Therefore, p19(ARF) regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals. The physiologic signals that induce p19(ARF) remain unknown. Whether induced by enforced Myc or E1A (Mouse Adenovirus Early Region-1A) expression, chemical carcinogens, or by loss of p53 or p19(ARF) function, establishment and immortalization enable MEFs to be transformed into tumor cells by oncogenic Ras genes alone. Myc and E1A inactivate cellular responses that are normally required for Ras-mediated inhibition of cell proliferation, thereby converting Ras into a growth-promoting gene. Due to their apparent immortalizing functions, it seems paradoxical that Myc and E1A are also potent inducers of apoptosis. Moreover, p19(ARF), but not p16(INK4A), suppresses transformation by E1A plus Ras in a p53-dependent manner, consistent with the idea that p19(ARF) acts downstream of Rb (Retinoblastoma) (and E2F1) in countering oncogenic signaling (Ref.3).  p19(ARF) plays an important role in tumor suppression in the context of Rb inactivation. Inactivation of p19(ARF) acts more broadly than that of p53 in connecting abrogation of the Rb pathway to tumorigenesis (Ref.4). The E1A proteins regulate transcription of both viral and cellular genes in the infected cell. In the G0/G1 phase of the cell cycle, hypo-phosphorylated Rb complexes with transcription factor E2F1 preventing its ability to activate transcription. Expression of E1A overrides the normal cellular control of the Rb-E2F1 interaction by binding hypo-phosphorylated Rb and freeing E2F1. As only hypophosphorylated Rb complexes with and inactivates E2F, hyperphosphorylation results in the release of E2F1 and activation of E2F1 dependent transcription. The E1A oncogene also activates p53 through a signaling pathway involving the Rb protein and the tumor suppressor p19(ARF). Synergistic effects between the p19(ARF) and DNA damage pathways in inducing p53 contribute to E1As ability to enhance radio- and chemosensitivity. A variety of cellular stresses activate p53, including DNA damage, hypoxia and expression of mitogenic oncogenes. Following DNA damage, p53 becomes phosphorylated by kinases, leading to changes in p53 conformation and activity. In contrast, the E1A oncogene activates p53 through a fundamentally different mechanism, mediated largely by the tumor suppressor p19(ARF). Importantly, the DNA damage and E1A signaling pathways act in parallel: E1A does not produce p53 phosphorylation at serine-15 and DNA damage activates p53 independently of p19(ARF). Simultaneous activation of p53 by p19(ARF) and DNA damage synergizes to promote apoptosis in the presence of the E1A oncogene (Ref.5). The p19(ARF) forms homo-oligomers that are stabilized by disulphide links. p19(ARF) activity is linked to its oligomerization and is sensitive to the redox status of the cell. Oxidative stress affects p19(ARF) oligomerization (Ref.6). Tumor promotion is associated with an altered redox status, and it is known that cancer cells produce an excess of ROS (Reactive Oxygen Species). The oligomeric forms of p19(ARF)  are sensitive to reducing agents (Ref.7).

MDM2 degradation by p19(ARF) is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of p19(ARF)-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed p19(ARF) to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. The p53 protein is significantly destabilized by the over expression of MDM2, leading to the postulation that MDM2 plays a role in maintaining low levels of p53 in normal cells as well as in removing p53 during recovery from stress-induced arrest through a feedback regulatory loop. In the case of DNA double-strand breaks produced by Gamma-irradiation, p19(ARF) plays at most a redundant, if not a minor, role in mediating p53 accumulation (Ref.8). There are several regulators of the p19(ARF) promoter. Of these a number are most likely indirect regulators, such as Myc, Twist1 (Twist Homolog-1), Abl1 (v-Abl Abelson Murine Leukemia Viral Oncogene-1), etc. However, some of them potentially are direct regulators, like p53 and E2F1. Among several disease-implicated regulators of p19(ARF) includes the T-Box genes; TBx2 (T-Box-2) and TBx3. TBx2 potently down-regulate the p19(ARF) tumor suppressor, thereby causing efficient immortalization of primary fibroblasts (Ref.9). Twist1 plays multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis and interfering with the p53 tumor suppressor pathway. Loss of anchorage dependence is a hallmark of tumor cells, and the ability to promote anchorage-independent growth is a common property of oncogenes. Twist1 has the ability to promote anchorage-independent growth (Ref.10). The p19(ARF)-p53 tumor suppressor pathway plays a critical role in cell-cycle checkpoint control and apoptosis. The endogenous PI3K (Phosphatidylinositde-3 Kinase) and Rac1 (Ras-Related C3 Botulinum Toxin Substrate-1) activities are required for p19(ARF) and p53-regulated migration (Ref.11). Disruptions or mutations of the p19(ARF) have important implications for understanding the clinical behavior of several tumors (Ref.12).