Arrest of G₁-S Progression by the p53-Inducible Gene PC3 Is Rb Dependent and Relies on the Inhibition of Cyclin D1 Transcription

Abstract

The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G 1 arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G 1 arrest was Rb dependent. Furthermore, (i) the arrest of G 1 -S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 downregulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G 1 -S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.The control of the cell cycle plays an essential role in cell growth and in the activation of important cellular processes such as differentiation and apoptosis. pRb (retinoblastoma protein) and p53 are two molecules identified as key regulators of the cell cycle.pRb is a nuclear phosphoprotein whose phosphorylation state oscillates regularly during the cell cycle. Its underphosphorylated forms predominate in G 0 and G 1 , while highly phosphorylated forms exist in S, G 2 , and M phases (13,16,21). The primary biological function of underphosphorylated pRb is to inhibit progression toward S phase by controlling a checkpoint in late G 1 (for reviews, see references 8, 22, and 51). In fact, underphosphorylated pRb associates with members of the E2F family of transcription factors, impairing their activity and leading to a cell cycle block in G 1 . Conversely, the phosphorylation of pRb inactivates its growth suppression activity by freeing E2F molecules, thus enabling them to transactivate genes required for the progression of the cell into S phase and the remainder of the cell cycle (52, 97, 114).Cyclin-dependent kinases (CDKs) are the molecules responsible for pRb phosphorylation and its consequent inactivation (reviewed in references 70 and 102). Each CDK has its own functional specificity, based on the period of its activity during the cell cycle and on the specific cyclin partner. CDK4, CDK5, and CDK6 form complexes with D-type cyclins during the G 1 phase (65,69,116). CDK2, when bound to cyclin A or E, is instead essential for G 1 -to-S transition (28, 78), while the c...

Identification of fibroblast growth factor-8b target genes associated with early and late cell cycle events in breast cancer cells

“…Btg2 belongs to the BTG/Tob family which in mammals comprises six proteins that regulate cell cycle progression (Winkler, 2010). BTG2 controls G1 to S phase progression 18 negatively by direct inhibition of cyclin D1 transcription (Guardavaccaro et al, 2000).…”

Btg1 and Btg2 gene expression during early chick development

“…It has been suggested that Btg proteins exert cellular functions by interacting with transcriptional coactivators or corepressors, hence modulating their transcriptional activities (Matsuda et al, 2001;Tirone, 2001;Duriez et al, 2004;Lim, 2006). For example, it has been found that Btg2 impairs G1-S cell cyclephase progression by inhibiting cyclin D1 transcription (Guardavaccaro et al, 2000), that it can associate with HoxB9 (Prevot et al, 2000), and BMPregulated Smad1 and 8 (Park et al, 2004) to promote their transcriptional activity, and that it can enhance Math1 promoter activity (Canzoniere et al, 2004). Similarly, Btg1 has been shown to interact with and stimulate the activity of transcription factors that positively regulate myogenic processes, in particular myogenic factors such as MyoD, all-trans retinoic acid receptors, and the T3 receptor TR␣1 (Busson et al, 2005).…”

“…Studies in different model systems suggest that they act as antiproliferative genes, they can promote cell differentiation and also regulate apoptosis and cellular senescence (Matsuda et al, 2001;Tirone, 2001;Duriez et al, 2004;Lim, 2006). Btg/Tob genes encode for a group of structurally related proteins, characterized by the presence of two novel and highly conserved domains ("antiproliferative" boxes A and B) located in the first 120 residues of the protein (Guehenneux et al, 1997;Guardavaccaro et al, 2000). This gene family is conserved across phylogeny, with two members identified in invertebrates: the gene Fog-3 in C. elegans (Chen et al, 2000) and the mRNA with accession number AF177464 in D. melanogaster.…”

Blood-brain barrier genomics, proteomics, and new transporter discovery

“…The BBB can also act in an inhibitory mode by expression of osteonectin, a glycoprotein that has been shown to inhibit angiogenesis by binding vascular endothelial growth factor, 16 and PC3, an antiproliferative protein induced by the p53 protein. 17 The BBB is also rich in expression of molecular transporters and proteins involved in cellular trafficking. The differentially expressed transport systems that were identified in these studies facilitate organic anion transport (oatp2), sodium and potassium transport (Naϩ,Kϩ ATPase), energy substrate transport in the form of lactic acid and ketone bodies [monocarboxylate transporter (MCT1)], amino acid transport [cationic amino acid transporter (CAT1)], and protein transport [transferrin receptor (TfR)].…”