Biomedical subjects

Doron Ginsberg

Publications and source records attributed to Doron Ginsberg.

14 recordsLinked to original sources

PubMed2006

E2F1 modulates p38 MAPK phosphorylation via transcriptional regulation of ASK1 and Wip1.

The E2F family of transcription factors regulates a diverse array of cellular functions, including cell proliferation, cell differentiation, and apoptosis. Recent studies indicate that E2F can also regulate transcription of upstream components of signal transduction pathways. We show here that E2F1 modulates the activity of the p38 MAPK pathway via E2F1-induced transient up-regulation of p38 MAPK phosphorylation. The mechanism by which E2F1 modulates p38 MAPK phosphorylation involves transcriptional induction of the kinase ASK1, a member of the MAPKKK family that phosphorylates p38 MKKs. Subsequent E2F-dependent down-regulation of the p38 signaling pathway is achieved through E2F-induced up-regulation of Wip1, a phosphatase that dephosphorylates and inactivates p38. Both ASK1 and Wip1 are essential mediators of the E2F-p38 connection: knock down of ASK1 inhibits E2F1-induced phosphorylation of p38, whereas knock down of Wip1 prolongs E2F1-induced p38 phosphorylation. Furthermore, Wip1 knock down enhances E2F1-induced apoptosis. Therefore, our data reveal a novel link between a central signaling pathway and the transcription factor E2F and identify Wip1 as a modulator of E2F1-induced apoptosis.

Apoptosis↗

PubMed2005

Transcriptional programs following genetic alterations in p53, INK4A, and H-Ras genes along defined stages of malignant transformation.

The difficulty to dissect a complex phenotype of established malignant cells to several critical transcriptional programs greatly impedes our understanding of the malignant transformation. The genetic elements required to transform some primary human cells to a tumorigenic state were described in several recent studies. We took the advantage of the global genomic profiling approach and tried to go one step further in the dissection of the transformation network. We sought to identify the genetic signatures and key target genes, which underlie the genetic alterations in p53, Ras, INK4A locus, and telomerase, introduced in a stepwise manner into primary human fibroblasts. Here, we show that these are the minimally required genetic alterations for sarcomagenesis in vivo. A genome-wide expression profiling identified distinct genetic signatures corresponding to the genetic alterations listed above. Most importantly, unique transformation hallmarks, such as differentiation block, aberrant mitotic progression, increased angiogenesis, and invasiveness, were identified and coupled with genetic signatures assigned for the genetic alterations in the p53, INK4A locus, and H-Ras, respectively. Furthermore, a transcriptional program that defines the cellular response to p53 inactivation was an excellent predictor of metastasis development and bad prognosis in breast cancer patients. Deciphering these transformation fingerprints, which are affected by the most common oncogenic mutations, provides considerable insight into regulatory circuits controlling malignant transformation and will hopefully open new avenues for rational therapeutic decisions.

Cell Differentiation↗

PubMed2005

E2F and signal transduction pathways.

The E2F family of transcription factors is most well known for its ability to regulate the expression of genes required for DNA replication and cell cycle progression. However, recent studies indicate that E2F can also regulate transcription of upstream components of signal transduction pathways. Here we discuss the effects of E2F activity on signal transduction pathways and its potential biological consequences.

Animals↗

PubMed2004

Transcriptional regulation of AKT activation by E2F.

The pRB-E2F pathway is a downstream target of mitogenic signaling pathways. The E2F family of transcription factors has a pivotal role in regulating cell proliferation since it controls the timely expression of many genes that are required for cell cycle progression. Moreover, at least one member of this family, E2F1, can mediate apoptotic cell death. We show here that E2F also modulates the activity of a major signal transduction pathway: we demonstrate that E2F upregulates AKT activity through a transcription-dependent mechanism. We identify the adaptor protein Grb2 associated binder 2 (Gab2) as a direct E2F target gene and an essential effector of E2F-dependent AKT activation. AKT activation was shown to inhibit E2F1induced apoptosis. Therefore, our results suggest the existence of a negative feedback loop involving E2F and AKT.

Adaptor Proteins, Signal Transducing↗

PubMed2004

p53-dependent down-regulation of telomerase is mediated by p21waf1.

Inactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominant-negative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F.

Base Sequence↗

PubMed2004

E2F3-a novel repressor of the ARF/p53 pathway.

The Arf tumor suppressor is a key component of the p53 tumor surveillance pathway, and its expression is activated by abnormal proliferation signals. In a recent paper, Lees and coworkers investigate the regulation of Arf expression by E2Fs and demonstrate that in normal cells E2F3 is a pivotal repressor of Arf.

Animals↗

PubMed2003

Up-regulation of Bcl-2 homology 3 (BH3)-only proteins by E2F1 mediates apoptosis.

The E2F1 transcription factor is a critical downstream target of the tumor suppressor pRB. The retinoblastoma (RB) pathway is often inactivated in human tumors, resulting in deregulated E2F activity that can induce both proliferation and apoptosis. Bcl-2 homology 3 (BH3)-only proteins are pro-apoptotic members of the Bcl-2 protein family that trigger apoptosis in response to diverse stimuli. We show here that E2F1 up-regulates the expression of the pro-apoptotic BH3-only proteins PUMA, Noxa, Bim, and Hrk/DP5 through a direct transcriptional mechanism. Expression of the E7 protein of HPV16, which disrupts RB/E2F complexes, also up-regulates the expression of these four BH3-only proteins, implicating endogenous E2F in this phenomenon. Indeed, endogenous E2F1 binds the promoters of these four genes. Furthermore, inhibition of E2F1-induced expression of either Noxa or PUMA results in a significant reduction in E2F1-induced apoptosis, indicating that increased Noxa and PUMA levels mediate this E2F1-induced apoptosis. Importantly, inhibition of E2F activity abolishes DNA damage-induced elevation of PUMA levels, implicating E2F in the physiological regulation of PUMA expression. These data provide a novel direct link between E2F and the apoptotic machinery and may explain the increased sensitivity of cells with a defective RB/E2F pathway to chemotherapy.

Animals↗

PubMed2003

Prolonged culture of telomerase-immortalized human fibroblasts leads to a premalignant phenotype.

Telomere shortening in primary human fibroblasts results in replicative senescence, which can be overcome by telomerase (hTERT) overexpression. However, because immortalization is one of the hallmarks of malignant transformation, careful analysis of hTERT-immortalized cells is of crucial importance for understanding both processes. To this end, we infected WI-38 fibroblasts with a retrovirus carrying the hTERT cDNA and analyzed their proliferative behavior during 600 days [ approximately 500 population doublings (PDLs)] of continuous culture. Growth of three independent mass cultures was uniform for approximately 150 PDLs after telomerase infection, followed by a progressive acceleration of growth in two of three cultures. Expression of p16(INK4A) was significantly elevated in the immortalized cells but gradually disappeared during the accelerated growth phase. This alteration correlated with loss of the contact inhibition response and conferred the cells with sensitivity to H-Ras-induced transformation. In contrast, the p53- and pRb-mediated checkpoints such as the DNA damage response, chromosomal stability and entry into quiescence remained intact, irrespective of INK4A locus expression. Importantly, detailed examination of one of the WI-38/hTERT cultures during the accelerated growth phase revealed overexpression of the c-myc and Bmi-1 oncogenes, as well as loss of p14(ARF) expression. Collectively, our results indicate that although hTERT-immortalized cells behave similarly to primary cells during the first 150 PDLs, long-term growth in culture may favor the appearance of clones carrying potentially malignant alterations.

Cell Division↗

PubMed2003

ATM is a target for positive regulation by E2F-1.

The E2F-1 transcription factor is a critical downstream target of the tumor suppressor, RB. When activated, E2F-1 induces cell proliferation. In addition, deregulation of E2F-1 constitutes an oncogenic stress that can induce apoptosis. The protein kinase ATM is a pivotal mediator of the response to another type of stress, genotoxic stress. In response to ionizing radiation, ATM activates the tumor suppressor p53, a key player in the control of cell growth and viability. We show here that E2F-1 elevates ATM promoter activity and induces an increase in ATM mRNA and protein levels. This is accompanied by an E2F-induced increase in p53 phosphorylation. Expression of the E7 protein of HPV16, which dissociates RB/E2F complexes, also induces the elevation of ATM levels and p53 phosphorylation, implicating endogenous E2F in these phenomena. These data demonstrate that ATM is transcriptionally regulated by E2F-1 and suggest that ATM serves as a novel, ARF-independent functional link between the RB/E2F pathway and p53.

3T3 Cells↗

PubMed2002

E2F mediates sustained G2 arrest and down-regulation of Stathmin and AIM-1 expression in response to genotoxic stress.

Exposure of cells to genotoxic agents results in activation of checkpoint pathways leading to cell cycle arrest. These arrest pathways allow repair of damaged DNA before its replication and segregation, thus preventing accumulation of mutations. The tumor suppressor retinoblastoma (RB) is required for the G(1)/S checkpoint function. In addition, regulation of the G(2) checkpoint by the tumor suppressor p53 is RB-dependent. However, the molecular mechanism underlying the involvement of RB and its related proteins p107 and p130 in the G(2) checkpoint is not fully understood. We show here that sustained G(2)/M arrest induced by the genotoxic agent doxorubicin is E2F-dependent and involves a decrease in expression of two mitotic regulators, Stathmin and AIM-1. Abrogation of E2F function by dominant negative E2F abolishes the doxorubicin-induced down-regulation of Stathmin and AIM-1 and leads to premature exit from G(2). Expression of the E7 papilloma virus protein, which dissociates complexes containing E2F and RB family members, also prevents the down-regulation of these mitotic genes and leads to premature exit from G(2) after genotoxic stress. Furthermore, genotoxic stress increases the levels of nuclear E2F-4 and p130 as well as their in vivo binding to the Stathmin promoter. Thus, functional complexes containing E2F and RB family members appear to be essential for repressing expression of critical mitotic regulators and maintaining the G(2)/M checkpoint.

3T3 Cells↗

PubMed2002

E2F1 pathways to apoptosis.

The E2F family of transcription factors plays a pivotal role in the regulation of cell proliferation and their activity is often deregulated in human tumors. Recent studies demonstrate that E2F1 can induce both proliferation and apoptosis. E2F1-induced apoptosis occurs via multiple pathways, some of which induce stabilization and activation of the tumor suppressor p53. The pro-apoptotic activity of E2F1 suggests that its deregulation constitutes an oncogenic stress that may target pre-malignant cells to undergo apoptosis, thus preventing tumor development.

Animals↗

PubMed2002

Induction of chondrocyte growth arrest by FGF: transcriptional and cytoskeletal alterations.

The effect of fibroblast growth factor (FGF) on mature chondrocytes, the cells responsible for axial skeletal development, is growth attenuation rather than stimulation. This singular response has been linked to signaling via FGF receptor 3 (FGFR3), partly because mutations causing chronic FGFR3 activation lead to various human disorders of bone growth. In order to study how FGF inhibits growth, we analyzed its effect on a rat chondrocyte-derived cell line. We show that the FGF-induced growth arrest occurs at the G1 phase, accompanied by profound changes in gene expression and cytoskeletal organization. Within minutes of binding, FGF induces tyrosine kinase activity in the focal substrate adhesions where it colocalizes with vinculin. Upon FGF stimulation, FGFR3 is selectively removed from the focal adhesions, which is followed by their disassembly and disruption of the organized cytoskeleton. Multiple genes are induced following FGF stimulation in chondrocytes, which has been shown by DNA array screening and confirmed for some by immunoblotting. These genes include regulators of cell differentiation and proliferation such as c-jun, JunD, cyclin-D1, NFkappaB1 and of plasma-membrane microdomain morphology, such as ezrin. The transcription factor Id1 is downregulated, consistent with the cells' exit from the mitotic cycle. Moreover, following FGF stimulation, levels of FGFR3 mRNA and protein decline, as does downstream signaling through the MAPK pathway. The importance of this FGFR3-mediated on-off control is illustrated in transgenic mice expressing mutant, hyperactive FGFR3, where abnormally high levels of NFkappaB are expressed throughout their bone growth-plates. A working model is presented of the signaling network involved in regulating FGF-induced chondrocyte differentiation and receptor downregulation.

Animals↗

PubMed2002

E2Fs up-regulate expression of genes involved in DNA replication, DNA repair and mitosis.

The E2F family of transcription factors plays a pivotal role in the regulation of cell proliferation in higher eukaryotes. We used DNA microarrays and cell lines containing either inducible E2F-1 or inducible E2F-3 to identify novel E2F target genes. Our data indicate that E2F up-regulates the expression of genes not previously described as E2F target genes. A number of these E2F-regulated genes are involved in DNA replication, DNA repair and mitosis. These results suggest that E2F affects cell cycle progression both at S phase and during mitosis. Furthermore, our findings indicate that E2F-dependent gene activation may contribute to the cellular response to DNA damage.

Animals↗

PubMedDate not supplied

E2F and Ras synergize in transcriptionally activating p14ARF expression.

The INK4a/ARF locus, which is frequently inactivated in human tumors, encodes two distinct tumor suppressive proteins, ARF and p16INK4a. ARF stabilizes and activates p53 by negating the effects of mdm2 on p53. Furthermore, its function is not restricted to the p53 pathway and it also inhibits cell proliferation in cells lacking p53. Expression of ARF is up-regulated in response to a number of oncogenic stimuli including E2F1. We show here that while oncogenic Ras does not significantly affect p1(4AR)F expression in normal human cells it activates p1(4AR)F in cells containing deregulated E2F. Moreover, oncogenic Ras and E2F1 synergize in activating p1(4AR)F expression. Activation of p1(4AR)F promoter by E2F1 persists in the absence of the consensus E2F-binding sites in this promoter, indicating that this activation also occurs through non- canonical binding sites. The activation by oncogenic Ras requires both E2F and Sp-1 activity, demonstrating the complex regulation of p14(ARF) in response to oncogenic stimuli.

Animals↗