E2F DNA Binding Activity Research Articles

Abstract 209A: Involvement of pRb-E2F pathway in green tea extract-induced growth inhibition of human myeloid leukemia cells

Abstract It has long believed that green tea has many health benefits. However, the clinical research has not provided conclusive evidence on these benefits especially on cancer prevention and treatment. In basic research, there are also many inconsistent reports on the biological activity of green tea extract and its major chemical components (catechins) with both inhibitory and stimulatory effects of EGCG on various cancer cells being reported. The stimulatory or inhibitory activity is often linked to either activation or inactivation of receptor tyrosine kinase. In this study, we present evidence that green tea extract and its major chemical components, Epigallocatechin-3-gallate (EGCG), epigallocatechin (ECG), and epicatechin (EC) inhibit growth of two human myeloid leukemia cells (TF-1a and MV4-11) through the regulation of pRb synthesis and pRb-E2F complexes. Addition of green tea extract to the culture of the myeloid leukemia cells significantly inhibited their proliferation with a substantial portion of cell death and decreased their colony forming cells in soft-agar culture. EGCG, ECG, and EC showed a similar inhibitory effect on these cells with the inhibitory efficiency varied with each catechin and the dose applied. Green tea extract and EGCG had no significant effect on the expression of G1 CDKs and the CDK inhibitors but downregulated the formation of pRb-CDKs examined by Western blot and immunoprecipitation, respectively. Surprisingly, the expression of pRb was markedly upregulated while the phosphorylation of pRb downregulated. The upregulation of pRb was blocked by pre-treatment with cycloheximide, a protein synthesis inhibitor, suggesting a requirement of protein synthesis. In agreement with these results, pRb-E2F complexes were upregulated and E2F DNA binding activity decreased determined by EMSA assay. Since both TF-1a and MV4-11 cell are factor-independent cell lines, the upregulation of pRb-E2F complexes and downregulation of DNA binding activity by green tea extract is most likely through a receptor tyrosine kinase-independent pathway. In summary, this study reveals that green tea extract, EGCG, ECG, and EC inhibited growth in human myeloid leukemia cells via regulating pRb synthesis and the formation of pRb-E2F complexes, leading to the inhibition of the E2F DNA binding activity. In addition, we found that that the stem/progenitor cells derived from the leukemia cell lines tested are more sensitive to the inhibitory effect of green tea extract. We propose that as a beverage one should not consume high amounts of green tea (extract) due to its possible cytotoxic or apoptotic effect, and its significant inhibitory effect on stem/progenitor cells, although the biological effect of green tea extract in vivo could be quite different from in vitro. However, the concentrated green tea extract may have potential for clinical investigation as an inducer of cancer cell death. Citation Format: Darrell Henry, Sebastien Brumaire, Gabriela Alvarez, Beatriz Alvarez, Xiaotang Hu. Involvement of pRb-E2F pathway in green tea extract-induced growth inhibition of human myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 209A.

Involvement of pRb-E2F pathway in green tea extract-induced growth inhibition of human myeloid leukemia cells

Both inhibitory and stimulatory effect of EGCG on cancer cells have been reported, which often is linked to receptor tyrosine kinase signaling. In this study, we present evidence that green tea extract and its chemical component, Epigallocatechin-3-gallate (EGCG), inhibit growth of human myeloid leukemia cells through the regulation of pRb synthesis and formation of pRb-E2F complexes. Addition of green tea extract to the culture of TF-1a and MV4-11 myeloid leukemia cells significantly inhibited their proliferation with a substantial portion of cell death being observed. The green tea extract and EGCG had no significant effect on the expression of G1 CDKs and the CDK inhibitors but downregulated the formation of pRb-CDKs. Surprisingly, the expression of pRb was markedly upregulated while the phosphorylation of pRb downregulated. The upregulation of pRb was blocked by pre-treatment with cycloheximide, a protein synthesis inhibitor, suggesting a requirement of protein synthesis. In agreement with these results, pRb-E2F complexes were upregulated and E2F DNA binding activity decreased. Since both TF-1a and MV4-11 are factor-independent cell lines, the upregulation of pRb-E2F complexes and inhibition of DNA binding activity by green tea extract is most likely through a receptor tyrosine kinase-independent pathway. We also found that the stem/progenitor cells derived from these two leukemia cell lines are more sensitive to the inhibitory effect of green tea extract. Our result suggests that concentrated green tea extract and EGCG may have potential for clinical investigation as an inducer of cancer cell death.

The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans.

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.

E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome.

Arginine methylation controls growth regulation by E2F-1

E2F transcription factors are implicated in diverse cellular functions. The founding member, E2F-1, is endowed with contradictory activities, being able to promote cell-cycle progression and induce apoptosis. However, the mechanisms that underlie the opposing outcomes of E2F-1 activation remain largely unknown. We show here that E2F-1 is directly methylated by PRMT5 (protein arginine methyltransferase 5), and that arginine methylation is responsible for regulating its biochemical and functional properties, which impacts on E2F-1-dependent growth control. Thus, depleting PRMT5 causes increased E2F-1 protein levels, which coincides with decreased growth rate and associated apoptosis. Arginine methylation influences E2F-1 protein stability, and the enhanced transcription of a variety of downstream target genes reflects increased E2F-1 DNA-binding activity. Importantly, E2F-1 is methylated in tumour cells, and a reduced level of methylation is evident under DNA damage conditions that allow E2F-1 stabilization and give rise to apoptosis. Significantly, in a subgroup of colorectal cancer, high levels of PRMT5 frequently coincide with low levels of E2F-1 and reflect a poor clinical outcome. Our results establish that arginine methylation regulates the biological activity of E2F-1 activity, and raise the possibility that arginine methylation contributes to tumourigenesis by influencing the E2F pathway.

Impaired Hepatocellular Regeneration in Murine Sepsis Is Dependent on Regulatory Protein Levels

Sepsis is a poorly understood syndrome. Therefore, we examined the mechanisms underlying failed regeneration in sham-operated (SO), mildly septic (cecal ligation and single puncture [CLP]), and severely septic (cecal ligation with two punctures [2CLP]) C57Bl6 mice. Relative to no operation (T0) or SO, CLP, but not 2CLP, increased the number of cells staining for proliferating cell nuclear antigen, a marker for cell division. Levels of the retinoblastoma protein (pRb) were detected at T0 and after SO. CLP increased pRb abundance, whereas 2CLP decreased it. Changes in phosphorylated pRb were similar but more profound. The abundance of the transcription factor E2F was unaltered by SO, CLP, or 2CLP. However, E2F DNA binding activity, although unchanged after SO, increased after CLP and decreased after 2CLP. The abundance of cyclin D1 in nuclear fractions increased following CLP but decreased after 2CLP. Neither SO nor 2CLP altered the abundance of the cyclin-dependent kinase (cdk) 4. However, cdk-4 abundance increased after CLP. Finally, CLP increased the steady-state abundance of the mRNAs encoding thymidine kinase, DNA polymerase α, and dihydrofolate reductase, all required for DNA replication. No changes were noted after 2CLP. We conclude that 2CLP impaired hepatocyte proliferation following 2CLP in part via impaired cyclin D1/cdk-4-induced phosphorylation of pRb, maintaining the association between pRb and E2F and inhibited E2F transcriptional activity.

Par-4 gene silence inhibited hydrogen peroxide-induced apoptosis in alveolar epithelial cells

Objective To investigate the effects of Par-4 gene silence on hydrogen peroxide-induced apoptosis in alveolar epithelial cells. Method The alveolar epithelial cells A549 were cultured and exposed to hydrogen peroxide. The siRNA sequences targeted Par-4 gene was chemically synthesized and transfected to A549 cells with or without the exposure of hydrogen peroxide. The cells were divided into normal control groups, hydrogen peroxide-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide), hydrogen peroxide and Par-4-siRNA-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide after transfection of Par-4-siRNA), Non-specific DNA sequence transfection control group. The apoptosis of A549 cells was quantified by flow cytometry. The expression of Smac protein was detected by Western blot.Electrophoretic mobility shift assay was applied for evaluating the change of E2F1 DNA binding activity. Relative activity of Caspase-3 was detected by clolorimetric assay. Results The percent of apoptotic cells in hydrogen peroxide and Par-4-siRNA-treated group was (29.7 ± 2.3) %, which was significantly lower than that of hydrogen peroxide-treated group [(54.2 ± 4.1)%, q= 8.91, P ＜ 0.01)]. Par-4 siRNA could significantly suppress the increase of Smac protein, E2F1 DNA binding activity and caspase-3 activity induced by hydrogen peroxide in A549 cells. Conclusions Par-4 gene silence induced by siRNA might inhibit the apoptosis of alveolar epithelial cells, which might be resulted from suppression of the up-regulation of Smac gene expression, E2F1 DNA binding activity and caspase-3 activity. Key words: Par-4; Small RNA interference; Hydrogen peroxide; Alveolar epithelial cells; Apoptosis

Inhibition of Aldose Reductase Prevents Growth Factor–Induced G1-S Phase Transition through the AKT/Phosphoinositide 3-Kinase/E2F-1 Pathway in Human Colon Cancer Cells

Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH-dependent aldo-keto reductase, has been shown to be involved in growth factor-induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced HT29 cell proliferation by accumulating cells at G(1) phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat, prevented the EGF- and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF- and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G(1)-S transition regulatory proteins such as cyclin D1, cdk4, proliferating cell nuclear antigen, cyclin E, and c-myc. More importantly, inhibition of AR prevented the EGF- and bFGF-induced activation of phosphoinositide 3-kinase/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mouse xenografts. Collectively, these results show that AR mediates EGF- and bFGF-induced colon cancer cell proliferation by activating or expressing G(1)-S phase proteins such as E2F-1, cdks, and cyclins through the reactive oxygen species/phosphoinositide 3-kinase/AKT pathway, indicating the use of AR inhibitors in the prevention of colon carcinogenesis. Mol Cancer Ther; 9(4); 813-24. (c)2010 AACR.

Interaction of E2F–Rb family members with corepressors binding to the adjacent E2F site

Cell cycle-dependent transcriptional repression of the E2F1 and B-myb promoters is mediated through E2F-binding sites and adjacent corepressor site (cell cycle gene homology region (CHR)/downstream repression site (DRS)). Here, we show that a factor binding to the B-myb CHR is co-purified with E2F DNA-binding activity, and coimmunoprecipitated with components of E2F/Rb-family repressor complexes, E2F4 and retinoblastoma (Rb) family proteins. In spite of structural and functional similarities, however, the E2F1 and B-myb CHRs exhibited distinct factor-binding specificities. Furthermore, substitution of E2F1 CHR with the B-myb CHR in the E2F1 promoter revealed that the B-myb CHR was unable to repress the E2F1 promoter completely in the G0 phase. These results suggest that transcriptional repression of the E2F1 and B-myb promoters is mediated by physical interaction of E2F/Rb-family repressor complexes with promoter-specific corepressors.

The candidate tumor suppressor BTG3 is a transcriptional target of p53 that inhibits E2F1

Proper regulation of cell cycle progression is pivotal for maintaining genome stability. In a search for DNA damage-inducible, CHK1-modulated genes, we have identified BTG3 (B-cell translocation gene 3) as a direct p53 target. The p53 transcription factor binds to a consensus sequence located in intron 2 of the gene both in vitro and in vivo, and depletion of p53 by small interfering RNA (siRNA) abolishes DNA damage-induced expression of the gene. Furthermore, ablation of BTG3 by siRNA in cancer cells results in accelerated exit from the DNA damage-induced G2/M block. In vitro, BTG3 binds to and inhibits E2F1 through an N-terminal domain including the conserved box A. Deletion of the interaction domain in BTG3 abrogates not only its growth suppression activity, but also its repression on E2F1-mediated transactivation. We also present evidence that by disrupting the DNA binding activity of E2F1, BTG3 participates in the regulation of E2F1 target gene expression. Therefore, our studies have revealed a previously unidentified pathway through which the activity of E2F1 may be guarded by activated p53.

Anti-HER2 Antibody Trastuzumab Inhibits CDK2-Mediated NPAT and Histone H4 Expression via PI3K Pathway

Anti-HER2 antibody trastuzumab (Herceptin®) has been used effectively to treat patients with breast cancer that overexpresses HER2. We have demonstrated that p27Kip1 upregulation is one of the key events that cause G1 arrest upon trastuzumab treatment. Here, we have examined the effect of trastuzumab on CDK2, Rb, E2F1, NPAT, histone H4, and their associated signaling pathway. Results showed that trastuzumab was able to dramatically inhibit the kinase activity and expression of CDK2, whereas the kinase activity and expression of CDK4 were not affected by trastuzumab treatment. Unlike the p27Kip1 upregulation that occurs primarily through post-translational mechanisms, CDK2 was mainly downregulated at a transcriptional level as shown by Northern blotting and real-time RT-PCR analyses. Trastuzumab decreased the kinase activity of cyclin E (because of CDK2 inhibition) although it had little effect on cyclin E protein level. Overexpression of wild-type cyclin E or its lower molecular weight forms did not influence the response to trastuzumab. Levels and activities of CDK6, cyclin A, and cyclin D1 were all suppressed by trastuzumab. As a result, Rb phosphorylations that associates with CDK2, cyclin E, CDK6, cyclin A, or cyclin D1 were substantially inhibited by trastuzumab. Trastuzumab decreased the DNA-binding activity of E2F1. Trastuzumab decreased the level of NPAT protein. Trastuzumab decreased the level of histone H4 mRNA. Blockade of PI3K pathway with LY294002 produced similar effects to trastuzumab treatment. Taken together, these results demonstrated that anti-HER2antibody trastuzumab inhibits CDK2, Rb phosphorylation, E2F1 activity, NPAT, and histone H4 via PI3K signaling pathway, which are essential to both DNA and histone synthesis during the progression of G1 phase to S phase of the cell cycle.

FAILED COMPENSATORY HEPATIC REGENERATION PARALLELS E2F-1 ACTIVATION IN LATE SEPSIS

Objective: E2F-1 is a well-known transcription factor responsible for regulating cell proliferation. Inturn, E2F is regulated by the retinoblastoma protein (pRB). Previous work in our lab suggests that failed compensatory regeneration parallels mortality in a fulminant murine sepsis model. Since hepatocellular regeneration in sepsis has not been well defined, we hypothesize that dysfunctional phosphophorylated pRB (PpRB)/ E2F-1 signaling may be responsible for the sepsis-induced impairment of compensatory regeneration. Methods: 6-8 w/o C57 B1/6 mice underwent cecal ligation and double puncture (2CLP). Sham operated (SO) and unoperated mice (T0) served as controls. Livers were harvested at 3,6,16,24,48 and 72 hours after SO/2CLP and the nuclear protein fractions were isolated. Electromobility shift assays (EMSA) and immunoblots were used to evaluate E2F-1 binding activity and nuclear abundance of E2F-1 and P-pRb. Results: P-pRb and E2F-1 nuclear protein levels were markedly decreased at 48 and 72 hours after 2CLP compared to T0 and SO controls. Although there was an increase in E2F-1 DNA binding activity at 48 hours post 2 CLP binding was undetectable at 72 hours. Conclusions: Although the pathologic significance of failed hepatic regeneration remains unknown, it may be important in the recovery from sepsis. Our findings suggest that failed E2F-1 activation may limit compensatory regeneration in fulminant sepsis.

E2F–HDAC complexes negatively regulate the tumor suppressor gene ARHI in breast cancer

ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.

Cilostazol Inhibits Vascular Smooth Muscle Cell Growth by Downregulation of the Transcription Factor E2F

Neointimal formation, the leading cause of restenosis, is caused by proliferation of vascular smooth muscle cells (VSMCs). Patients with diabetes mellitus have higher restenosis rates after coronary angioplasty than nondiabetic patients. Cilostazol, a selective type 3 phosphodiesterase inhibitor, is currently used to treat patients with diabetic vascular complications. Cilostazol is a potent antiplatelet agent that inhibits VSMC proliferation. In the present study, we examine whether the antiproliferative effect of cilostazol on VSMCs is mediated by inhibition of an important cell cycle transcription factor, E2F. Cilostazol inhibited the proliferation of human VSMCs in response to high glucose in vitro and virtually abolished neointimal formation in rats subjected to carotid artery injury in vivo. Moreover, the compound suppressed high-glucose-induced E2F-DNA binding activity, and the expression of E2F1, E2F2, cyclin A, and PCNA proteins. These data suggest that the beneficial effects of cilostazol on high-glucose-stimulated proliferation of VSMCs are mediated by the downregulation of E2F activity and expression of its downstream target genes, including E2F1, E2F2, cyclin A, and PCNA.

δ-Opioid agonist induced regulation of E2F1 DNA binding activity in NG108-15 cells

Activation of opioid receptors have been implicated in the modulation of cell proliferation and the E2F family of transcription factors may play a role in opioid inhibition of DNA synthesis. Gel shift assays and Western blotting of nuclear extracts from NG108-15 cells revealed increased E2F1 DNA binding activity and higher levels of E2F1 following activation of δ-opioid receptors. It is suggested that DADLE-induced regulation of E2F DNA binding activity involves ERKs.

The Adenovirus E4-6/7 Protein Directs Nuclear Localization of E2F-4 via an Arginine-Rich Motif

E2F transcription factors are key participants in the regulation of proliferation, apoptosis, and differentiation in mammalian cells. E2Fs are negatively regulated by members of the retinoblastoma protein (pRb) family. During adenovirus (Ad) infection, viral proteins that displace pRb family members from E2Fs and recruit E2F complexes to viral and cellular promoter regions are expressed. This recruitment of E2F involves the induction of stable E2F binding to inverted E2F binding sites in the Ad E2a and cellular E2F-1 promoters and induces both viral and cellular gene expression. E2F-4 has abundant E2F activity within cells, and the majority of E2F-4 in asynchronous cells is found in the cytoplasm. Upon expression of the adenovirus E4-6/7 protein, a significant portion of E2F-4 is translocated to the nucleus, and its activity constitutes the majority of Ad-induced nuclear E2F DNA binding activity. This redirection of E2F-4 from cytoplasm to the nucleus requires an N-terminal arginine-rich nuclear localization sequence within E4-6/7. The directed targeting of E4-6/7 to the nucleus is important for the function of this protein in the context of viral infection. This function of E4-6/7 has a redundant component as well as nonredundant components in cooperation with the adenovirus E1A oncoproteins to deregulate and usurp host cell E2F function.

Altered transcriptional regulators in response to serum in immortalized lymphocytes from Alzheimer's disease patients

Cell cycle disturbances may precede neuronal death in Alzheimer's disease (AD). We described alterations, in lymphocytes from AD patients, on the activity of two transcription factors, E2F and NF-κB, involved in cell proliferation and survival regulation, demonstrating that cell cycle dysfunction also occurs in peripheral cells. The analysis of E2F–DNA binding activity revealed lower signal intensity of protein–DNA complexes in AD cells, which correlated with increased phosphorylation of retinoblastoma (pRb) related proteins and enhanced proliferation. The calmodulin (CaM) antagonist calmidazolium (CMZ) abrogated the increased activity of AD cells by partially dephosphorylating pRb and p130. The NF-κB–DNA binding activity increased as cell progress through the cell cycle. The reduced NF-κB activation observed in AD cells appears not to be related to the increased phosphorylation of the pRb family proteins nor with the enhanced proliferative activity of AD cells, but seems to protect them from death induced by the loss of trophic support. Ca 2+/CaM antagonists rescue NF-κB–DNA binding activity and sensitize AD cells to serum withdrawal. These observations suggest that disruption of Ca 2+/CaM signaling pathway could be linked mechanistically to its pro cell survival actions, promoting enhanced proliferation or decreased cell death depending on the presence of growth-stimulatory signals.

Role of E2F and ERK1/2 in STI571-mediated smooth muscle cell growth arrest and cyclin A transcriptional repression

Platelet-derived growth factor (PDGF) ligand and receptors (PDGF-R) activate smooth muscle cell (SMC) proliferation, a key event during vascular obstructive disease. The PDGF-R tyrosine kinase inhibitor STI571 attenuates SMC proliferation and experimental neointimal thickening. Here, we investigated the molecular mechanisms underlying STI571-dependent SMC growth arrest. STI571 abrogates PDGF-BB-dependent cyclin D1 and cyclin A protein expression and inhibits transcriptional activation of reporter genes driven by the human cyclin A gene promoter. Repression of cyclin A promoter activity by STI571 requires a functional E2F-binding site, and forced expression of E2F overrides this inhibitory effect. Moreover, STI571 inhibits E2F DNA-binding activity in SMCs. We also found that STI571 abrogates PDGF-BB-dependent activation of extracellular-regulated kinase 1 and 2 (ERK1/2), and forced activation of these factors impaired STI571-dependent inhibition of both cyclin A promoter activity and SMC proliferation. Thus, E2F and ERK1/2 play an important role in STI571-mediated SMC growth arrest and cyclin A transcriptional repression. These findings may have importance in the development of novel therapeutic strategies for the treatment of neointimal hyperplasia.

Inhibition of E2F Abrogates the Development of Cardiac Myocyte Hypertrophy

Growth of the post-natal mammalian heart occurs primarily by cardiac myocyte hypertrophy. Previously, we and others have shown that a partial re-activation of the cell cycle machinery occurs in myocytes undergoing hypertrophy such that cells progress through the G1/S transition. In this study, we have examined the regulation of the E2F family of transcription factors that are crucial for the G1/S phase transition during normal cardiac development and the development of myocyte hypertrophy in the rat. Thus, mRNA and protein levels of E2F-1, 3, and 4 and DP-1 and DP-2 were down-regulated during development to undetectable levels in adult myocytes. Interestingly, E2F-5 protein levels were substantially up-regulated during development. In contrast, an induction of E2F-1, 3, and 4 and the DP-1 protein was observed during the development of myocyte hypertrophy in neonatal myocytes treated with serum or phenylephrine, whereas the protein levels of E2F-5 were decreased with serum stimulation. E2F activity, as measured by a cyclin E promoter luciferase assay and E2F-DNA binding activity, increased significantly during the development of hypertrophy with serum and phenylephrine compared with non-stimulated cells. Inhibiting E2F activity with a specific peptide that blocks E2F-DP heterodimerization prevented the induction of hypertrophic markers (atrial natriuretic factor and brain natriuretic peptide) in response to serum and phenylephrine, reduced the increase in myocyte size, and inhibited protein synthesis in stimulated cells. Thus, we have shown that the inhibition of E2F function prevents the development of hypertrophy. Targeting E2F function might be a useful approach for treating diseases that cause pathophysiological hypertrophic growth.

Cleavage of E2F-1-regulating proteins and activation of E2F-1 during CD95-induced death of thymocytes.

The CD95 death receptor activates caspases that cleave a variety of intracellular substrates, including cell cycle control proteins. However, the significance of this cleavage for the induction of apoptosis is unclear. In this study, CD95-induced cleavage of the G1/S checkpoint regulator proteins, retinoblastoma protein (pRb) and murine-double-minute-2 (mdm-2), was associated with an increased protein concentration of a key transcription factor, E2F-1, which is regulated by both of them. Furthermore, DNA-binding activity to E2F sites is increased. In thymocytes, CD95-induced apoptosis was associated with increased E2F-1 DNA-binding activity, while thymocytes that lacked E2F-1 were less susceptible to CD95-induced apoptosis. We conclude that the G1/S checkpoint is an important target of CD95 signalling. CD95-activated caspases cleave regulator proteins to increase E2F-1 activity, and inappropriate activation of E2F-1 is part of the mechanism of CD95-induced apoptosis.