Distinct E2F-mediated transcriptional program regulates p14ARF gene expression

The tumor suppressor p14(ARF) gene is induced by ectopically expressed E2F, a positive regulator of the cell cycle. The gene is expressed at low levels in normally growing cells in contrast to high levels in varieties of tumors. How p14(ARF) gene is regulated by E2F in normally growing cells and tumor cells remains obscure. Here we show that regulation of p14(ARF) gene by E2F is distinct from that of classical E2F targets. It is directly mediated by E2F through a novel E2F-responsive element that varies from the typical E2F site. The element responds to E2F activity resulting from ectopic E2F1 expression, inactivation of pRb by adenovirus E1a or shRNA, but not to phosphorylation of pRb by serum stimulation or ectopic cyclin D1/cyclin-dependent kinase-4 expression in normal human fibroblasts. The element has activity in various tumor cells with defective pRb, but not in normally growing cells. These results indicate that the distinct regulation constitutes the basis of p14(ARF) function as a tumor suppressor, discriminating abnormal growth signals caused by defects in pRb function from normal growth signals.     

pRb inactivation in senescent cells leads to an E2F-dependent apoptosis requiring p73

Senescent cells in which pRb is inactivated undergo apoptosis on attempted reinitiation of DNA synthesis. To further explore the cell death resulting from loss of pRb function in senescent cells, we employed a temperature-sensitive pRb mutant protein (tspRb). We found that tspRb inactivation results in rapid E2F reactivation and subsequent S-phase reentry associated with the up-regulation of E2F target gene expression and cyclin E-dependent kinase activity. Total inhibition of cyclin-dependent kinase 2 activity results in a cell cycle arrest on pRb loss and a nearly complete suppression of apoptosis. Furthermore, blocking of E2F activity with a dominant-negative DP1 inhibits S-phase reentry and cell death following tspRb inactivation. Finally, inhibition of p73 activity abolishes apoptosis but not S-phase entry on pRb inactivation, suggesting that activation of E2F in senescent cells can result in the use of p73 as a cell death effector. Interestingly, senescent cells rescued from apoptosis maintain their altered shape and express senescence-associated beta-galactosidase despite loss of pRb function. Thus, maintenance of the terminal cell cycle arrest of senescent cells requires continuous pRb-mediated inactivation of E2F activity, the reappearance of which in these irrevocably altered cells triggers a cell death program instead of an inappropriate resumption of cell cycling.     

Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression

The activation of CDK2-cyclin E in late G1 phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G1-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G1 progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G1 progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or RbDeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to RbDeltaCDK renders it susceptible to inactivation in late G1, as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.     

4-Hydroxynonenal affects pRb/E2F pathway in HL-60 human leukemic cells

4-Hydroxynonenal (HNE), a highly reactive product of lipid peroxidation, has an antiproliferative effect in several tumor cell lines and provokes alteration of cell cycle progression in HL-60 cells. HNE down-regulates c-myc expression in K562, HL-60, and MEL cells. This prompted us to study the cascade of phenomena that, starting from the CKIs expression and the phosphorylation of pRb, arrives at the E2F binding to consensus sequence in the P2 promoter of the c-myc gene. Treatment of HL-60 cells with HNE (1 microM) causes a p53-independent increase of p21(WAF1/CIP1) expression, pRb dephosphorylation, a decrease of low molecular weight E2F complexes and an increase of high molecular weight E2F complexes bound to P2 c-myc promoter. E2F4 expression is reduced by HNE treatment as well as the amount of pRb/E2F4 complexes, whereas the amount of pRb/E2F1 complexes is increased. In conclusion, HNE can affect the pRb/E2F pathway by modifying the expression of several genes involved in the control of cell proliferation.     

Reduced Expression of FOXC2 and Brown Adipogenic Genes in Human Subjects with Insulin Resistance

Objective: We investigated subcutaneous adipose tissue expression of FOXC2 and selected genes involved in brown adipogenesis in adult human subjects in whom we have previously identified a reduced potential of precursor cell commitment to adipose‐lineage differentiation in relation to insulin resistance. Research Methods and Procedure: Gene expression was studied using quantitative real time polymerase chain reaction. The relation between the expression of brown adipogenic genes and the genes involved in progenitor cell commitment, adipose cell size, and insulin sensitivity in vivo was analyzed. Results: The expression of FOXC2, MASK, MAP3K5, retinoblastoma protein (pRb), peroxisome proliferator‐activated protein gamma (PPARγ), and retinoid X receptor gamma (RXRγ) was decreased in the insulin‐resistant compared with insulin‐sensitive subjects, whereas PPARγ‐2 and CCAAT/enhancer binding protein alpha (C/EBPα) showed no differential expression. The FOXC2 expression correlated with that of Notch and Wnt signaling genes, as well as of the genes studied participating in brown adipogenesis, including MASK, MAP3K5, PPARγ, pRb, RXRγ, and PGC‐1. A second‐level correlation between PPARγ and UCP‐1 was also significant. In addition, the expression of MASK, MAP3K5, pRb, RXRγ, and PGC‐1 inversely correlated with adipose cell mass and also correlated with the glucose disposal rate in vivo. Discussion: Our results suggest that a reduced brown adipose phenotype is associated with insulin resistance and that a basal brown adipose phenotype may be important for maintaining normal insulin sensitivity.