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Biomedical subjects

W Douglas Cress

Publications and source records attributed to W Douglas Cress.

18 recordsLinked to original sources
PubMed2006

Interactions between E2F1 and SirT1 regulate apoptotic response to DNA damage.

The nicotinamide adenine dinucleotide (NAD)-dependent deacetylase Sir2 (silent information regulator 2) regulates gene silencing in yeast and promotes lifespan extension during caloric restriction. The mammalian homologue of Sir2 (SirT1) regulates p53, NF-kappaB and Forkhead transcription factors, and is implicated in stress response. This report shows that the cell-cycle and apoptosis regulator E2F1 induces SirT1 expression at the transcriptional level. Furthermore, SirT1 binds to E2F1 and inhibits E2F1 activities, forming a negative feedback loop. Knockdown of SirT1 by small interference RNA (siRNA) increases E2F1 transcriptional and apoptotic functions. DNA damage by etoposide causes E2F1-dependent induction of SirT1 expression and knockdown of SirT1 increases sensitivity to etoposide. These results reveal a mutual regulation between E2F1 and SirT1 that affects cellular sensitivity to DNA damage.

Apoptosis↗
PubMed2006

Bok, Bcl-2-related Ovarian Killer, Is Cell Cycle-regulated and Sensitizes to Stress-induced Apoptosis.

Bok/Mtd (Bcl-2-related ovarian killer/Matador) is considered a pro-apoptotic member of the Bcl-2 family. Although identified in 1997, little is known about its biological role. We have previously demonstrated that Bok mRNA is up-regulated following E2F1 overexpression. In the current work, we demonstrate that Bok RNA is low in quiescent cells and rises upon serum stimulation. To determine the mechanism underlying this regulation, we cloned and characterized the mouse Bok promoter. We find that the mouse promoter contains a conserved E2F binding site (-43 to -49) and that a Bok promoter-driven luciferase reporter is activated by serum stimulation dependent on this site. Chromatin immunoprecipitation assays demonstrate that endogenous E2F1 and E2F3 associate with the Bok promoter in vivo. Surprisingly, we find that H1299 cells can stably express high levels of exogenous Bok protein. However, these cells are highly sensitive to chemotherapeutic drug treatment. Taken together these results demonstrate that Bok represents a cell cycle-regulated pro-apoptotic member of the Bcl-2 family, which may predispose growing cells to chemotherapeutic treatment.

Animals↗
PubMed2005

Activation of p27Kip1 Expression by E2F1. A negative feedback mechanism.

The E2F1 transcription factor is a critical regulator of cell cycle due to its ability to promote S phase entry. However, E2F1 overexpression also sensitizes cells to apoptosis and E2F1-null mice are predisposed to tumor development, suggesting that it also has properties of a growth suppressor. E2F1 transcription function is regulated by interaction with hypophosphorylated pRb. Cdk inhibitors such as p16INK4a and p27Kip1 inhibit pRb phosphorylation by the cyclin D/Cdk4 and cyclin E/Cdk2 complexes, thus keeping E2F1 in an inactive state. We found that E2F1 binds to the p27 promoter in vivo and activates p27 mRNA and protein expression. Depletion of endogenous E2F1 by siRNA causes a reduction in basal p27 expression level. Inhibition of endogenous p27 expression by siRNA increases E2F1 transcriptional activity and permits accelerated cell cycle progression by exogenous E2F1. These observations suggest that induction of p27 acts as a negative feedback mechanism for E2F1 and may also contribute to other functions of E2F1.

Animals↗
PubMed2005

Ribonucleotide reductase M1 gene promoter activity, polymorphisms, population frequencies, and clinical relevance.

RRM1 is a gene crucial for determination of the tumor phenotype. It encodes the regulatory subunit of ribonucleotide reductase, and it is a molecular target of gemcitabine. In addition, RRM1 induces PTEN expression and inhibits cell migration, invasion, and metastasis formation. In patients with resected lung cancers, increased levels of RRM1 are highly associated with long survival. In contrast, patients on gemcitabine and cisplatin therapy for advanced disease have a poor survival if RRM1 expression is high presumably because of decreased efficacy of chemotherapy. We analyzed the RRM1 promoter for polymorphisms in an effort to develop a practical and inexpensive assay for RRM1 expression. Two single nucleotide polymorphisms, RR37 and RR524, were discovered. These polymorphisms impacted promoter activity in vitro and had different frequencies in various populations. Promoter allelotypes were highly associated with overall (P = 0.06) and disease-free (P = 0.03) patient survival. The allelotype with the highest predicted activity was associated with the best patient outcome. However, we did not find an association between allelotype and tumoral RRM1 expression. This is likely a result of the limited impact of the described promoter polymorphisms on overall in vivo gene expression. We conclude that clinical studies using RR37 and RR524 for decisions on chemotherapy are premature and that further functional studies on the RRM1 promoter are required to fully elucidate factors controlling RRM1 expression.

Biological Assay↗
PubMed2005

Role of Stat3 in regulating p53 expression and function.

Loss of p53 function by mutation is common in cancer. However, most natural p53 mutations occur at a late stage in tumor development, and many clinically detectable cancers have reduced p53 expression but no p53 mutations. It remains to be fully determined what mechanisms disable p53 during malignant initiation and in cancers without mutations that directly affect p53. We show here that oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Site-specific mutation of a Stat3 DNA-binding site in the p53 promoter partially abrogates Stat3-induced inhibition. Stat3 activity also influences p53 response genes and affects UV-induced cell growth arrest in normal cells. Furthermore, blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. As a point of convergence for many oncogenic signaling pathways, Stat3 is constitutively activated at high frequency in a wide diversity of cancers and is a promising molecular target for cancer therapy. Thus, repression of p53 expression by Stat3 is likely to have an important role in development of tumors, and targeting Stat3 represents a novel therapeutic approach for p53 reactivation in many cancers lacking p53 mutations.

Animals↗
PubMed2004

E2F4 deficiency promotes drug-induced apoptosis.

E2F1 and E2F4 are known to have opposing roles in cell cycle control. In the present work, we examine the role of both E2F1 and E2F4 in apoptosis induced by three cyclin-dependent kinase inhibitors (roscovitine, BMS-387032, and flavopiridol) as well as by three established chemotherapeutic drugs (VP16, cisplatin and paclitaxel). We find that E2F4 levels are diminished following treatment with cyclin dependent kinase inhibitors (flavopiridol, roscovitine and BMS-387032) or with DNA damaging drugs (cisplatin and VP16). In contrast, each of these drugs induced E2F1. We find that mouse fibroblasts nullizygous for the E2F4 gene are more sensitive to apoptosis induced by roscovitine, flavopiridol, cisplatin, and VP16, whereas E2F1-deficient fibroblasts are less sensitive. Likewise, we find that RNAi-mediated reductions in E2F4 in human cancer cells results in increased drug sensitivity. Taken together, these results support a model in which E2F1 and E2F4 play opposing roles during drug-induced apoptosis.

Animals↗
PubMed2004

Deregulated expression of LRBA facilitates cancer cell growth.

LRBA expression is induced by mitogens in lymphoid and myeloid cells. The Drosophila LRBA orthologue rugose/DAKAP550 is involved in Notch, Ras and EGFR pathways. These findings suggest that LRBA could play a role in cell types that have increased proliferative and survival capacity. Here, we show by microarray and real-time PCR analyses that LRBA is overexpressed in several different cancers relative to their normal tissue controls. We also show that LRBA promoter activity and endogenous LRBA mRNA levels are reduced by p53 and increased by E2F1, indicating that mutations in the tumor suppressors p53 and Rb could contribute to the deregulation of LRBA. Furthermore, inhibition of LRBA expression by RNA interference, or inhibition of its function by a dominant-negative mutant, leads to significant growth inhibition of cancer cells, demonstrating that deregulated expression of LRBA contributes to the altered growth properties of a cancer cell. Finally, we show that the phosphorylation of EGFR is affected by the dominant-negative mutant, suggesting LRBA plays a role in the mammalian EGFR pathway. These findings demonstrate that LRBA facilitates cancer cell growth and thus LRBA may represent a novel molecular target for cancer therapy.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Antiapoptotic signaling pathways in non-small-cell lung cancer: biology and therapeutic strategies.

One of the hallmarks of lung cancer is the deregulation of apoptotic or programmed cell death mechanisms usually found in normal cells that allow for corrupted cells to undergo cellular suicide. This includes mechanisms that attenuate proapoptotic pathways and/or amplify antiapoptotic pathways. Increasing evidence suggests that lung cancer cells use multiple and perhaps redundant pathways to maintain survival. Increasing knowledge of these pathways offers a better understanding of the biology of lung cancer as well as novel therapeutic strategies that can enhance lung cancer cell death. This review discusses the apoptotic machinery and signal transduction pathways that regulate apoptosis, methods of identifying the presence of activated survival signaling pathways in human lung cancers, and the clinical significance and relevance for therapy for patients with lung cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

AG490 inhibits G1-S traverse in BALB/c-3T3 cells following either mitogenic stimulation or exogenous expression of E2F-1.

AG490, a member of the tryphostin family of protein kinase inhibitors, repressed G(0)-G(1) traverse in BALB/c-3T3 cells. While the early induction of STAT activity was repressed by AG490, extracellular signal-regulated kinase (ERK) activation was unaffected and a pattern of gene expression suggested that cells exited G(0) in the presence of the inhibitor. Although AG490 did not alter the induction of cyclin D1 protein, neither cyclin D1- nor cyclin D3-associated kinase activity was observed in growth-inhibited cells. Surprisingly, p130 was partially phosphorylated, and E2F3A protein was expressed in mitogen-stimulated AG490-treated cells despite the lack of cyclin D-associated kinase activity. These data suggest that AG490 inhibits a cellular pathway required for mid-G(0)-G(1) traverse that is located after the induction of early processes potentially mediated by E2F (although independent of cyclin D-associated kinase activity) but before the late G(1) increase in E2F-dependent transcription. Infection of AG490-treated cells with an E2F-1 adenovirus caused the induction of cyclin A, but could not overcome the drug-induced cell cycle arrest that was coincident with the repression of cyclin-dependent kinase 2 (cdk2)-associated kinase activation. We conclude that cdk2-associated kinase activity is modulated by a cellular process repressed by AG490. Furthermore, this cdk2-associated kinase activity is required for G(0)-G(1) traverse in some role other than the regulation of E2F-dependent transcription.

Animals↗
PubMed2004

Efficient down-regulation of cyclin A-associated activity and expression in suspended primary keratinocytes requires p21(Cip1).

When suspended in methylcellulose, primary mouse keratinocytes cease proliferation and differentiate. Suspension also reduces the activity of the cyclin-dependent kinase cdk2, an important cell cycle regulatory enzyme. To determine how suspension modulates these events, we examined its effects on wild-type keratinocytes and keratinocytes nullizygous for the cdk2 inhibitor p21(Cip1). After suspension of cycling cells, amounts of cyclin A (a cdk2 partner), cyclin A mRNA, and cyclin A-associated activity decreased much more rapidly in the presence than in the absence of p21(Cip1). Neither suspension nor p21(Cip1) status affected the stability of cyclin A mRNA. Loss of p21(Cip1) reduced the capacity of suspended cells to growth arrest, differentiate, and accumulate p27(Kip1) (a second cdk2 inhibitor) and affected the composition of E2F DNA binding complexes. Cyclin A-cdk2 complexes in suspended p21(+/+) cells contained p21(Cip1) or p27(Kip1), whereas most of the cyclin A-cdk2 complexes in p21(-/-) cells lacked p27(Kip1). Ectopic expression of p21(Cip1) allowed p21(-/-) keratinocytes to efficiently down-regulate cyclin A and differentiate when placed in suspension. These findings show that p21(Cip1) mediates the effects of suspension on numerous processes in primary keratinocytes including cdk2 activity, cyclin A expression, cell cycle progression, and differentiation.

Animals↗
PubMed2003

Regulation of the cyclin D3 promoter by E2F1.

We have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G(1) progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G(1) cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides -143 and -135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation.

Cell Cycle Proteins↗
PubMed2003

Flavopiridol-induced apoptosis is mediated through up-regulation of E2F1 and repression of Mcl-1.

Flavopiridol treatment can lead to apoptosis via a mechanism that has been associated with down-regulation of Mcl-1. Likewise, recent studies from our laboratory demonstrated that E2F1 leads to transcriptional repression of Mcl-1 and subsequently apoptosis. Given the ability of cyclin/cyclin-dependent kinase 2 antagonists to kill transformed cells, we surmised that flavopiridol may stabilize E2F1 and enhance apoptosis via repression of Mcl-1. Here we demonstrate that flavopiridol is associated with a dose-dependent increase in E2F1 protein levels, a corresponding reduction in Mcl-1, and apoptosis in H1299 lung carcinoma cells. Treatment of H1299 cells with 200 nM flavopiridol resulted in the rapid elevation of E2F1 and reduction in Mcl-1 levels within 12 h of treatment. The elevation of E2F1 and reduction in Mcl-1 clearly preceded the induction of apoptosis. Both H1299 and NIH3T3 fibroblast cell lines that constitutively express Mcl-1 under the control of the cytomegalovirus promoter have no reductions in Mcl-1 levels with flavopiridol treatment and are resistant to apoptosis induced by flavopiridol. H1299 cells that have E2F1 deleted through RNAi vector targeting are less sensitive to flavopiridol-induced cell death, and likewise, mouse embryo fibroblast cell lines deficient in E2F1 are less susceptible to apoptosis induced by flavopiridol compared with wild-type control fibroblasts. These data suggest that apoptosis induced by flavopiridol is dependent on the enhancement of E2F1 levels and the repression of Mcl-1.

3T3 Cells↗
PubMed2002

Histone deacetylase 3 binds to and regulates the multifunctional transcription factor TFII-I.

Histone deacetylase 3 (HDAC3) is one of four members of the human class I histone deacetylases that are implicated in transcriptional repression through deacetylation of acetyllysines in amino-terminal tails of core histones. In an immunoaffinity purification using anti-HDAC3, transcription factor TFII-I copurified with HDAC3. Specificity of the HDAC3-TFII-I interaction was confirmed by coimmunoprecipitation of epitope-tagged proteins, GST pull-down assays, and protein colocalization with indirect immunofluorescence. An anti-TFII-I immunoprecipitate contained histone deacetylase enzymatic activity. Mutational analyses revealed that the carboxyl-terminal of HDAC3 (residues 373-401) and residues 363-606 of TFII-I were required for the HDAC3-TFII-I interaction. Transcriptional activation by TFII-I was severely reduced by overexpression of HDAC3. These results suggest that HDAC3 modulates some of the functions of TFII-I and provides a link between histone deacetylase and a multifunctional transcriptional activator.

Amino Acid Sequence↗
PubMed2002

E2F-3B is a physiological target of cyclin A.

The E2F family of transcription factors controls the expression of numerous genes that are required for the G(1)/S transition. Among the mechanisms that modulate the activity of the E2F proteins, cyclin A has been found to be important for the down-regulation of E2F-1, -2, and -3A activity after cells have progressed through G(1)/S. Specifically, phosphorylation of these E2F proteins by cyclin A/Cdk2 ultimately results in their necessary degradation as cells progress through S phase. E2F-3B was recently identified as an alternatively spliced form of E2F-3A that was predicted to lack a functional cyclin A binding domain. In this paper, we present considerable evidence that contradicts this prediction. First, we demonstrate binding of cyclin A to E2F-3B as bacterially expressed proteins in vitro. Second, we demonstrate binding of cyclin A to E2F-3B in mammalian cells in vivo. Third, we show that co-expression of cyclin A with E2F-3B significantly reduces E2F-3B-mediated transcriptional activity. Finally, in synchronized cells, we observe down-regulation of E2F-3B protein expression coincident with the up-regulation of cyclin A. We conclude that E2F-3B is a physiological target of cyclin A.

3T3 Cells↗
PubMed2002

Identification of novel E2F1-regulated genes by microarray.

The E2F pathway has been proposed to regulate genes involved in the transition from quiescence into DNA synthesis. However, this hypothesis has not been rigorously tested on a genomic scale. Toward this end, we have infected quiescent mouse fibroblasts, which do not express E2F1, with an E2F1-expressing adenovirus and examined the expression of more than 6000 genes using high-density microarrays. Microarray results clearly support the current paradigm; however, they suggest that E2F1 may also regulate unanticipated cellular functions including pathways involved in apoptosis, signal transduction, transcriptional control, and membrane biology. Most surprisingly, we identified a number of genes that are repressed by E2F1 expression, suggesting that E2F1 may have the potential to repress transcription of numerous genes through an unknown mechanism.

3T3 Cells↗
PubMed2002

Differences in DNA binding properties between E2F1 and E2F4 specify repression of the Mcl-1 promoter.

E2F1 is a potent inducer of apoptosis whereas its relative, E2F4, generally does not promote cell death. Other work from our laboratory has demonstrated that E2F1 can directly bind and represss the Mcl-1 promoter - contributing to E2F1-mediated apoptosis. Here we show that while E2F1 can repress the Mcl-1 promoter, other members of the E2F family (such as E2F4) cannot. Characterization of the Mcl-1 promoter demonstrates that the -143/+10 region is critical for E2F1-mediated downregulation. We demonstrate that the ability of E2F1 to repress the Mcl-1 promoter correlates with its ability to bind within the required -143/+10 region of this promoter. In contrast, E2F4 is unable to bind to the -143/+10 region of the Mcl-1 promoter. We propose that E2F4 is unable to repress the Mcl-1 promoter primarily as a result of insufficient binding to the essential regulatory region. This is the first evidence of DNA binding specificity among E2F family members that results in differential regulation of a naturally occurring promoter.

3T3 Cells↗
PubMed2002

Direct repression of the Mcl-1 promoter by E2F1.

E2F1 induces apoptosis via both p53-dependent and p53-independent mechanisms. The direct targets in the p53-independent pathway remain enigmatic; however, the induction of this pathway does not require the transactivation domain of E2F1. Using cells that are defective in p53 activation, we show that E2F1 potently represses the expression of Mcl-1--an anti-apoptotic Bcl-2 family member whose depletion results in apoptosis. We also show that this transcriptional repression is direct and dependent upon E2F1's DNA-binding domain, but does not require the transactivation domain of E2F1. Consistent with this DNA binding requirement of E2F1 to repress Mcl-1, we show that E2F1 binds to the Mcl-1 promoter both in vitro and in vivo, and have identified the DNA element (-143/-117) within this promoter that is required for E2F1 binding and repression. Additionally, cell lines constitutively expressing Mcl-1 are resistant to E2F1-mediated apoptosis--suggesting that Mcl-1 downregulation is a necessary event in the p53-independent apoptotic process. Thus, we identify a p53 family-independent mechanism of E2F1-induced apoptosis in which E2F1 directly represses Mcl-1 expression.

3T3 Cells↗