Expression Of E2F Target Genes Research Articles

G-Protein Coupled Receptor Kinase 5 Regulates Prostate Tumor Growth

The limited success of cancer therapeutics is largely attributable to the ability of cancer to become resistant to conventional cytotoxic chemotherapy. Thus, further identification of signaling molecules and pathways that influence tumorigenesis is needed to increase the overall therapeutic options. GRKs, originally recognized for their conserved role in GPCR signal control, have now emerged as regulators of additional biological molecules and functions. We used Western blot analysis to determine GRK expression in prostate cancer and RNA interference to establish the role of GRK5 in prostate cancer growth and progression through the cell cycle. GRK5 was expressed highly in the aggressive prostate cancer PC3 cell line and its silencing by RNA interference attenuated in vitro cell proliferation. PC3 cells that stably expressed lentiviral small hairpin RNA and targeted GRK5 evidence reduced xenograft tumor growth in mice. This was reversed by rescuing expression with wild-type but not with kinase inactive K215R GRK5, implying the need of GRK5 kinase activity for tumor growth. To investigate possible cellular mechanism(s) for GRK5 in cell growth regulation we tested whether kinase activity would impact cell cycle progression. Like forced over expression of kinase-inactive K215R GRK5, GRK5 knockdown led to G2/M arrest in the cell cycle. Also, evidence revealed that the loss of GRK5 activity resulted in decreased cyclin D1 expression, Rb protein phosphorylation and E2F target gene expression involved in cell cycle control. Results provide direct evidence that GRK5 has an immediate role in the regulation of prostate tumor growth.

Copy number defects of G1‐Cell cycle genes in neuroblastoma are frequent and correlate with high expression of E2F target genes and a poor prognosis

The tightly controlled network of cell cycle genes consists of a core of cyclin dependent kinases (CDKs) that are activated by periodically expressed cyclins. The activity of the cyclin-CDK complexes is regulated by cyclin dependent kinase inhibitors (CDKIs) and multiple signal transduction routes that converge on the cell cycle. Neuroblastoma are pediatric tumors that belong to the group of small round blue cell tumors, characterized by a fast proliferation. Here, we present high throughput analyses of cell cycle regulating genes in neuroblastoma. We analyzed a series of 82 neuroblastomas by comparative genomic hybridization arrays, single nucleotide polymorphism arrays, and Affymetrix expression arrays and analyzed the datasets in parallel with the R2 bioinformatic tool (http://r2.amc.nl). About 30% of the tumors had genomic amplifications, gains, or losses with shortest regions of overlap that suggested implication of a series of G1 cell cycle regulating genes. CCND1 (cyclin D1) and CDK4 were amplified or gained and the chromosomal regions containing the CDKN2 (INK4) group of CDKIs were frequently deleted. Cluster analysis showed that tumors with genomic aberrations in G1 regulating genes over-expressed E2F target genes, which regulate S and G2/M phase progression. These tumors have a poor prognosis. Our findings suggest that pharmacological inhibition of cell cycle genes might bear therapeutic promises for patients with high risk neuroblastoma.

Retinoblastoma‐independent regulation of cell proliferation and senescence by the p53–p21 axis in lamin A /C‐depleted cells

The expression of A-type lamin is downregulated in several cancers, and lamin defects are the cause of several diseases including a form of accelerated aging. We report that depletion of lamin A/C expression in normal human cells leads to a dramatic downregulation of the Rb family of tumor suppressors and a defect in cell proliferation. Lamin A/C-depleted cells exhibited a flat morphology and accumulated markers of cellular senescence. This senescent phenotype was accompanied by engagement of the p53 tumor suppressor and induction of the p53 target gene p21 and was prevented by small hairpin RNAs against p53, p21, or by the oncoprotein Mdm2. The expression of E2F target genes, normally required for cell cycle progression, was downregulated after lamin A/C depletion but restored after the inactivation of p53. A similar senescence response was observed in myoblasts from a patient with a lamin A mutation causing muscular dystrophy. We thus reveal a previously unnoticed mechanism of controlling cell cycle genes expression, which depends on p53 but does not require the retinoblastoma family of tumor suppressors and that can be relevant to understand the pathogenesis of laminopathies and perhaps aging.

ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein

The ADP-ribosylation factors (ARFs) 1 and 6 are small GTP-binding proteins, highly expressed and activated in several breast cancer cell lines and are associated with enhanced migration and invasiveness. In this study, we report that ARF1 has a critical role in cell proliferation. Depletion of this GTPase or expression of a dominant negative form, which both resulted in diminished ARF1 activity, led to sustained cell-growth arrest. This cellular response was associated with the induction of senescent markers in highly invasive breast cancer cells as well as in control mammary epithelial cells by a mechanism regulating retinoblastoma protein (pRB) function. When examining the role of ARF1, we found that this GTPase was highly activated in normal proliferative conditions, and that a limited amount could be found in the nucleus, associated with the chromatin of MDA-MB-231 cells. However, when cells were arrested in the G(0)/G(1) phase or transfected with a dominant negative form of ARF1, the total level of activated ARF1 was markedly reduced and the GTPase significantly enriched in the chromatin. Using biochemical approaches, we demonstrated that the GDP-bound form of ARF1 directly interacted with pRB, but not other members of this family of proteins. In addition, depletion of ARF1 or expression of ARF1T(31)N resulted in the constitutive association of pRB and E2F1, thereby stabilizing the interaction of E2F1 as well as pRB at endogenous sites of target gene promoters, preventing expression of E2F target genes, such as cyclin D1, Mcm6 and E2F1, important for cell-cycle progression. These novel findings provide direct physiological and molecular evidence for the role of ARF1 in controlling cell proliferation, dependent on its ability to regulate pRB/E2F1 activity and gene expression for enhanced proliferation and breast cancer progression.

MiR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor

Numerous microRNAs (miRNAs) are reported as differentially expressed in cancer, however the consequence of miRNA deregulation in cancer is unknown for many miRNAs. We report that two miRNAs located on chromosome 17p13, miR-132 and miR-212, are over-expressed in pancreatic adenocarcinoma (PDAC) tissues. Both miRNAs are predicted to target the retinoblastoma tumor suppressor, Rb1. Validation of this interaction was confirmed by luciferase reporter assay and western blot in a pancreatic cancer cell line transfected with pre-miR-212 and pre-miR-132 oligos. Cell proliferation was enhanced in Panc-1 cells transfected with pre-miR-132/-212 oligos. Conversely, antisense oligos to miR-132/-212 reduced cell proliferation and caused a G 2/M cell cycle arrest. The mRNA of a number of E2F transcriptional targets were increased in cells over expressing miR-132/-212. Exposing Panc-1 cells to the β2 adrenergic receptor agonist, terbutaline, increased the miR-132 and miR-212 expression by 2- to 4-fold. We report that over-expression of miR-132 and miR-212 result in reduced pRb protein in pancreatic cancer cells and that the increase in cell proliferation from over-expression of these miRNAs is likely due to increased expression of several E2F target genes. The β2 adrenergic pathway may play an important role in this novel mechanism.

The corepressor Alien as a novel tumor suppressor?

Alien has been characterized as a corepressor for nuclear hormone receptors that harbor a silencing domain such as the thyroid hormone receptor (TR), the vitamin D3 receptor (VDR) and DAX-1. In addition, the androgen receptor (AR), a steroid hormone receptor, interacts with Alien. Alien enhances gene silencing mediated by TR, VDR and DAX-1, whereas Alien inhibits AR-mediated transactivation. The inhibition of AR by Alien seems to be restricted to cases where AR is bound to AR antagonists. In line with this, Alien inhibits AR target gene expression and human prostate cancer cell proliferation in an antagonist-specific manner indicating that Alien has an inhibitory role for cell cycle progression. Alien mediates gene silencing by recruitment of histone deacetylase activity and interestingly through nucleo-some assembly activity. Hereby, Alien enhances nucleosome positioning mediated by nucleosome assembly protein 1, which suggests a novel molecular mechanism of corepressor function. Using a proteomic approach to identify Alien interacting partners, we detected the cell cycle factor E2F1 to bind to Alien in vivo. The E2F1-mediated transactivation and E2F target gene expression is inhibited by Alien, and in line with this Alien is observed to repress cell cycle progression.

1α,25‐dihydroxyvitamin D3 inhibits C4‐2 prostate cancer cell growth via a retinoblastoma protein (Rb)‐independent G1 arrest

The active metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D) reduces the growth of several prostate cancer cell lines, most commonly by inducing a cell-cycle arrest in G(1). This is mediated, in part, through down-regulation of c-Myc, a positive regulator of the transcription factor, E2F. There is evidence that prostate cancer cells lacking functional retinoblastoma protein (Rb), a negative regulator of E2F activity, are poorly responsive to 1,25D treatment. Since up to 60% of prostate cancers demonstrate a loss of heterozygosity for Rb, we sought to determine whether Rb is required for the growth inhibitory effects of 1,25D. Using siRNA, Rb was reduced in C4-2 prostate cancer cells, and the response of cells to 1,25D treatment or depletion of c-myc measured by [(3)H]-thymidine incorporation and flow cytometry. The effects of 1,25D treatment on E2F levels and activity, and E2F target gene expression were also measured. 1,25D treatment and c-Myc depletion both cause a G(1) arrest inhibiting C4-2 cell proliferation independently of Rb. 1,25D reduces c-Myc expression and causes a decrease in E2F and E2F target genes. Bcl-2, an E2F target and positive regulator of C4-2 cell growth, also is down-regulated by 1,25D independently of Rb. Redundant growth inhibitory pathways compensate for the loss of Rb, and tumors lacking functional Rb may be responsive to 1,25D.

Induction of Senescence by Doxorubicin Is Associated with Upregulated Mir-375 and Induction of Autophagy In CML Cell Line K562

AbstractAbstract 1843Senescence is a specialized form of growth arrest that it is generally irreversible and can be induced by telomere attrition, oxidative stress, oncogene expression and DNA damage signaling. Senescent cells display a typical upregulated senescence-associated (SA)-b-galactosidase activity and novel changes in chromatin architecture, the formation of SA heterochromatic foci (SAHF). Changes in gene expression, such as upregulated p16, p53, and p21 expression and silencing of E2F target genes, have been characterized to promote the establishment of senescence. Besides, by the actions of HP1g, HMGA, and DNMT proteins to produce a repressive chromatin environment, the transcription of proliferation-associated genes can be suppressed. Therefore, senescence has been suggested to functions as a natural brake to tumor development. In this study, we first sought to establish an in vitro senescence model using doxorubicin (DOX) and paclitaxel to treat CML cell line K562. We found that 50 nM DOX induced senescence, but did not induce apoptosis. In contrast, 10 nM and 100 nM paclitaxel induced apoptosis but did not induce senescence, and lower doses of paclitaxel (1 nM and 5 nM) had no effect on the cells. p53 and p16-pRb are the two major senescence pathways. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells is supposed to be established by a pathway independent of p53 and p16-pRb pathways. Indeed, the expression of the typical SA-premalignant cell markers (CDC6, Ki67, p19, p38, PU1, DNMT1, HMGA1, HP1g) did not change in the DOX-induced senescent K562 cells although the typical SA-b-galactosidase staining and SAHF were apparent. MicroRNA profiling revealed that miR-375 was upregulated in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor could rescue the proliferation ability suppressed by DOX (p < 0.05). The identification of miR-375 targets should help us to elucidate the substitution pathway that is responsible for the DOX-induced senescence in the absence of both p16 and p53 genes. With the observation that DOX treatment induced cells entering senescence but eventually lead to cell death, we also investigated if the alternative mode of cell death, autophagy, was involved. By examining the expression patterns of the 26 human autophagy-related (ATG) genes, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. Both ATG9 and ATG18 are crucial for the formation of autophagosome. Hence, in addition to the upregulation of miR-375, our results also demonstrated that senescence induced by DOX in K562 cells is associated with the initiation of autophagy. Updated results on the identification of miR-375 targets and the regulation of senescence and autophagy pathway will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

Abstract 36: Human ecdysoneless protein: Regulator of cholesterol homeostasis in pancreatic cancer

Abstract The human Ecdysoneless protein (hEcd) is a human orthologue of the D.melanogaster ecdysoneless protein, which is highly conserved from insects to mammals and identified as one of the factors regulating the synthesis of the steroid hormone ecdysone. Ecd is required autonomously throughout the body and its deficiency can lead to reproductive or developmental defects. hEcd, also known as human suppressor of GCR2 gene (hSGT) since it can rescue growth defects in S.cerevisiae mutants with a deletion of the growth control regulatory genes 2 (GCR2), a transcriptional activator of the glycolytic gene expression. Recent investigations revealed that hEcd interacts with murine double minute-2 and stabilizes p53 by inhibiting mdm-2 mediated degradation of p53. Another study done on MEFs generated from Ecd conditional knockout mice showed evidence that Ecd interacts with retinoblastoma protein at the pocket domain and competes with E2F for association with hypophosphorylated Rb, thereby regulating E2F target gene expression and cell cycle progression. In this study, we have analyzed the expression of Ecd in PanIN and Pancreatic adenocarcinoma tissues as well as pancreatic cancer cell lines. The studies show that Ecd is overexpressed in pancreatic cancer samples as compared to normal pancreas. Therefore based on its role as a cell cycle regulator and its increased expression in pancreatic tumors, we hypothesized that Ecd might act as a potential modulator contributing to the pathogenesis of pancreatic cancer cells. Stable knockdown clones of Ecd were established in CD/HPAF pancreatic cancer cell line using two different shRNA constructs, which led to reduced proliferation rate in vitro and decreased tumor weight in vivo. Further, microarray analysis of genes in Ecd knocked-down vs scrambled cells followed by bioinformatics analysis revealed that the major pathway affected due to Ecd downregulation is the cholesterol biosynthetic and regulatory pathway. The genes which were affected by more than two-fold in Ecd knocked-down cells, encoded key cholesterol biosynthetic pathway enzymes such as FDFT1, NSDHL, DHCR7 and CYP51A1 along with INSIG1, a protein which acts as a cholesterol level sensor in cells. This was further confirmed by Quantitative Real-Time PCR analysis. Flow-cytometric analysis for cholesterol-specific filipin staining showed increased cholesterol content in Ecd-kd cells vs the scrambled cells. Additionally, insulin stimulated GLUT-4 membrane translocation, one of the cholesterol-dependent pathways was also found to be affected with Ecd knockdown cells showing higher glucose uptake than the scrambled cells. Cholesterol being a major component of cells and a mediator of endocytic and cell signaling pathways, we conclude that Ecd contributes to pancreatic cancer pathogenesis by disrupting multiple cholesterol-dependent pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 36.

Abstract 1069: The role of mammalian Ecdysoneless in cell cycle regulation and cancer

Abstract Our long term research goal is to understand biochemical mechanisms of immortalization of human mammary epithelial cells (hMECs) that represents an early step in breast cancer progression. We have generated several hMEC immortalization models using viral oncogenes, mutated cancer genes as well as radiation treatment of cells. In one of such immortalization model, we identified Ecdysoneless (Ecd) protein as the human papilloma virus (HPV) E6 interacting protein. We have reported earlier that HPV16 E6 protein is highly efficient for hMEC immortalization. Here, we present evidence that Ecd, an evolutionarily conserved protein, is a novel regulator of the Rb/E2F pathway. We generated a conditional Ecd knockout mouse model and observed that complete deletion of Ecd leads to early embryonic lethality in mice. The experiments using conditional Ecd-null MEFs created from Ecdlox/lox revealed that lack of Ecd inhibits cellular proliferation by delaying G1-S cell cycle progression and down-regulates E2F target gene expression. Furthermore, Ecd directly binds to Rb at the pocket domain and disrupts the interaction of Rb with E2F, leading to impairment of repressive effect of Rb on E2F target promoters. Thus, we demonstrate that Ecd is an essential protein in embryonic development and a novel regulator of G1-S cell cycle transition by modulating Rb/E2F pathway. Consistent with these observations, overexpression of Ecd in hMECs hastens the exit of cells from G1 phase of the cell cycle. Significantly, immunohistochemical analyses showed Ecd is overexessed in ductal carcinoma in situ as well as in infiltrating ductal carcinoma of breast. In conclusion, Ecd plays an important role in cell cycle progression and its deregulation can lead to abnormal cell proliferation leading to breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1069.

Sequestration of Rb/E2F complex into PML-NBs provides a distinct mechanism to control the expression of E2F target genes

Sequestration of Rb/E2F complex into PML-NBs provides a distinct mechanism to control the expression of E2F target genes

Role of Mammalian Ecdysoneless in Cell Cycle Regulation

The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecd(lox/lox) mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecd(lox/lox) mouse embryonic fibroblasts led to a proliferative block due to a delay in G(1)-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb at the pocket domain and competed with E2F for binding to hypophosphorylated Rb. Our results demonstrate that mammalian Ecd plays a role in cell cycle progression via the Rb-E2F pathway.

Retinoblastoma/p107/p130 Pocket Proteins: PROTEIN DYNAMICS AND INTERACTIONS WITH TARGET GENE PROMOTERS

The retinoblastoma (RB) tumor suppressor and its family members, p107 and p130, function by repressing E2F transcription factor activity to limit the expression of genes required for cell cycle progression. Traditionally, it is thought that the RB family proteins repress E2F target gene expression through complexing with E2F at gene promoters. However, whereas chromatin immunoprecipitation experiments have demonstrated p107 and p130 at E2F-responsive promoters, RB chromatin association has not been reliably observed. Here we used green fluorescent protein-tagged proteins to rigorously explore the mechanism of RB-mediated transcriptional repression relative to its p107 and p130 family members. The use of live cell fluorescent imaging demonstrated that RB, p107, and p130 exhibit similar nuclear dynamics. Although these findings suggest a similar engagement with nuclear structures, chromatin immunoprecipitation approaches with multiple independent antibodies failed to detect the association of RB with target gene promoters. However, by employing antibodies directed against green fluorescent protein, we could utilize the same antibody to assess RB, p107, and p130 engagement. This approach demonstrated RB association with target gene promoters in a fashion analogous to p107 and p130. Extension of this technology demonstrated that direct RB phosphorylation disrupts promoter association to regulate transcription. Thus, RB is associated with promoters in a manner similar to p107/p130 and that association is modulated by phosphorylation during cell cycle progression.

E2f3a and E2f3b Contribute to the Control of Cell Proliferation and Mouse Development

The E2f3 locus encodes two Rb-binding gene products, E2F3a and E2F3b, which are differentially regulated during the cell cycle and are thought to be critical for cell cycle progression. We targeted the individual inactivation of E2f3a or E2f3b in mice and examined their contributions to cell proliferation and development. Chromatin immunoprecipitation and gene expression experiments using mouse embryo fibroblasts deficient in each isoform showed that E2F3a and E2F3b contribute to G(1)/S-specific gene expression and cell proliferation. Expression of E2f3a or E2f3b was sufficient to support E2F target gene expression and cell proliferation in the absence of other E2F activators, E2f1 and E2f2, suggesting that these isoforms have redundant functions. Consistent with this notion, E2f3a(-/-) and E2f3b(-/-) embryos developed normally, whereas embryos lacking both isoforms (E2f3(-/-)) died in utero. We also find that E2f3a and E2f3b have redundant and nonredundant roles in the context of Rb mutation. Analysis of double-knockout embryos suggests that the ectopic proliferation and apoptosis in Rb(-/-) embryos is mainly mediated by E2f3a in the placenta and nervous system and by both E2f3a and E2f3b in lens fiber cells. Together, we conclude that the contributions of E2F3a and E2F3b in cell proliferation and development are context dependent.

Sustained NF‐κB activation produces a short‐term cell proliferation block in conjunction with repressing effectors of cell cycle progression controlled by E2F or FoxM1

NF-kappaB transcription factors induce a host of genes involved in pro-inflammatory/stress-like responses; but the collateral effects and consequences of sustained NF-kappaB activation on other cellular gene expression programming remain less well understood. Here enforced expression of a constitutively active IKKbeta T-loop mutant (IKKbetaca) drove murine fibroblasts into transient growth arrest that subsided within 2-3 weeks of continuous culture. Proliferation arrest was associated with a G1/S phase block in immortalized and primary early passage MEFs. Molecular analysis in immortalized MEFs revealed that inhibition of cell proliferation in the initial 1-2 weeks after their IKKbetaca retroviral infection was linked to the transient, concerted repression of essential cell cycle effectors that are known targets of either E2F or FoxM1. Co-expression of a phosphorylation resistant IkappaBalpha super repressor and IKKbetaca abrogated growth arrest and cell cycle effector repression, thereby linking IKKbetaca's effects to canonical NF-kappaB activation. Transient growth arrest of IKKbetaca cells was associated with enhanced p21 (cyclin-dependent kinase inhibitor 1A) protein expression, due in part to transcriptional activation by NF-kappaB and also likely due to strong repression of Skp2 and Csk1, both of which are FoxM1 direct targets mediating proteasomal dependent p21 turnover. Ablation of p21 in immortalized MEFs reduced their IKKbetaca mediated growth suppression. Moreover, trichostatin A inhibition of HDACs alleviated the repression of E2F and FoxM1 targets induced by IKKbetaca, suggesting chromatin mediated gene silencing in IKKbetaca's short term repressive effects on E2F and FoxM1 target gene expression.

Activation of the retinoblastoma tumor suppressor mediates cell cycle inhibition and cell death in specific cervical cancer cell lines

High-risk human papilloma virus (HPV) encodes two oncoproteins, E6 and E7, which are vital to viral replication and contribute to the development of cervical cancer. HPV16 E7 can target over 20 cellular proteins, but is best known for inactivating the retinoblastoma (RB) tumor suppressor. RB functions by restraining cells from entering S-phase of the cell cycle, thus preventing aberrant proliferation. While it is well established that HPV16 E7 facilitates the degradation of the RB protein, the ability of the RB pathway to overcome E7 action is less well understood. In this study the RB-pathway was activated via the overexpression of the p16ink4a tumor suppressor or ectopic expression of an active allele of RB (PSM-RB). While p16ink4a had no influence on cell cycle progression, PSM-RB expression was sufficient to induce a cell cycle arrest in both SiHa and HeLa cells, HPV positive cervical cancer cell lines. Strikingly, this arrest led to the downregulation of E2F target gene expression, which was antagonized via enhanced HPV-E7 expression. Since downmodulation of E7 function is associated with chronic growth arrest and senescence, the effect of PSM-RB on proliferation and survival was evaluated. Surprisingly, sustained PSM-RB expression impeded the proliferation of SiHa cells, resulting in both cell cycle inhibition and cell death. From these studies we conclude that active RB expression can sensitize specific cervical cancer cells to cell cycle inhibition and cell death. Thus, targeted therapies involving activation of RB function may be effective in inducing cell death in cervical cancer.

RB Loss Promotes Aberrant Ploidy by Deregulating Levels and Activity of DNA Replication Factors

The retinoblastoma tumor suppressor (RB) is functionally inactivated in many human cancers. Classically, RB functions to repress E2F-mediated transcription and inhibit cell cycle progression. Consequently, RB ablation leads to loss of cell cycle control and aberrant expression of E2F target genes. Emerging evidence indicates a role for RB in maintenance of genomic stability. Here, mouse adult fibroblasts were utilized to demonstrate that aberrant DNA content in RB-deficient cells occurs concomitantly with an increase in levels and chromatin association of DNA replication factors. Furthermore, following exposure to nocodazole, RB-proficient cells arrest with 4 n DNA content, whereas RB-deficient cells bypass the mitotic block, continue DNA synthesis, and accumulate cells with higher ploidy and micronuclei. Under this condition, RB-deficient cells also retain high levels of tethered replication factors, MCM7 and PCNA, indicating that DNA replication occurs in these cells under nonpermissive conditions. Exogenous expression of replication factors Cdc6 or Cdt1 in RB-proficient cells does not recapitulate the RB-deficient cell phenotype. However, ectopic E2F expression in RB-proficient cells elevated ploidy and bypassed the response to nocodazole-induced cessation of DNA replication in a manner analogous to RB loss. Collectively, these results demonstrate that deregulated S phase control is a key mechanism by which RB-deficient cells acquire elevated ploidy.

E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1 , E2f2 , and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1- , E2f2- , and E2f3- deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21(CIP1). Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21(CIP1) and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

Control of the p53-p21CIP1 Axis by E2f1, E2f2, and E2f3 Is Essential for G1/S Progression and Cellular Transformation

The E2F family of transcription factors is believed to have an essential role in the control of cellular proliferation by regulating the transcription of genes involved in cell cycle progression. Previous work has demonstrated that the targeted inactivation of E2f1, E2f2, and E2f3 results in elevated p21(CIP1) protein levels, loss of E2F target gene expression, and cell cycle arrest at G1/S and G2/M, suggesting a strict requirement for these E2Fs in the control of normal cellular proliferation. We now demonstrate that E2f1, E2f2, and E2f3 are also required for oncogene-mediated transformation of mouse embryonic fibroblasts. Analysis of synchronized populations of mouse embryonic fibroblasts revealed that the inactivation of p21(CIP1) restores the ability of E2f1-3-deficient cells to enter and transit through G1/S (but not G2/M). In contrast, loss of p53 restored the ability of these cells to progress through both G1/S and mitosis, leading to their continued proliferation. The inactivation of p53 (but not p21(CIP1)) rendered E2f1-3-deficient cells sensitive to transformation and tumorigenesis. These results suggest that the negative regulation of the p53-p21(CIP1) axis by the E2F1-3 factors is critical for cell cycle progression and cellular transformation.