Publications
132 results found
Humbert N, Navaratnam N, Augert A, et al., 2010, Regulation of ploidy and senescence by the AMPK-related kinase NUAK1, EMBO J, Vol: 29, Pages: 376-386
Senescence is an irreversible cell-cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss-of-function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant-negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy. The EMBO Journal (2010) 29, 376-386. doi: 10.1038/emboj.2009.342; Published online 19 November 2009
Banito A, Rashid S, Acosta JC, et al., 2009, Senescence impairs successful reprogramming to pluripotent stem cells, Genes Dev
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16(INK4a), and p21(CIP1). Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.
Maertens GN, Messaoudi-Aubert SE, Racek T, et al., 2009, Several distinct polycomb complexes regulate and co-localize on the INK4a tumor suppressor locus, PLOS One, Vol: 4, ISSN: 1932-6203
Misexpression of Polycomb repressive complex 1 (PRC1) components in human cells profoundly influences the onset of cellular senescence by modulating transcription of the INK4a tumor suppressor gene. Using tandem affinity purification, we find that CBX7 and CBX8, two Polycomb (Pc) homologs that repress INK4a, both participate in PRC1-like complexes with at least two Posterior sex combs (Psc) proteins, MEL18 and BMI1. Each complex contains a single representative of the Pc and Psc families. In primary human fibroblasts, CBX7, CBX8, MEL18 and BMI1 are present at the INK4a locus and shRNA-mediated knockdown of any one of these components results in de-repression of INK4a and proliferative arrest. Sequential chromatin immunoprecipitation (ChIP) reveals that CBX7 and CBX8 bind simultaneously to the same region of chromatin and knockdown of one of the Pc or Psc proteins results in release of the other, suggesting that the binding of PRC1 complexes is interdependent. Our findings provide the first evidence that a single gene can be regulated by several distinct PRC1 complexes and raise important questions about their configuration and relative functions.
Humbert N, Martien S, Augert A, et al., 2009, A Genetic Screen Identifies Topoisomerase 1 as a Regulator of Senescence, Cancer Res
Normal cell growth can be permanently blocked when cells enter a state known as senescence. This phenomenon can be triggered by various stresses, such as replicative exhaustion, oncogenic stimulation, or oxidative stress. Senescence prevents transmission of aberrant signals to daughter cells and thus prevents irreversible damage that could favor cancer development. To identify new genetic events controlling senescence, we have performed a loss-of-function genetic screen on normal human cells. We report that knockdown of topoisomerase I (Top1) results in an increased replicative potential associated with a decrease in senescence markers and a diminished DNA damage response. In addition, Top1 depletion also favors a bypass of oncogene-induced senescence. Conversely, Top1 constitutive expression induces growth arrest, the appearance of a senescence marker, and an activation of the DNA damage response. Altogether, these results reveal an unanticipated function of Top1 in regulating senescence. [Cancer Res 2009;69(10):4101-6].
Barradas M, Anderton E, Acosta JC, et al., 2009, Histone demethylase JMJD3 contributes to epigenetic control of INK4a/ARF by oncogenic RAS, Genes Dev, Vol: 23, Pages: 1177-82
The INK4a/ARF tumor suppressor locus, a key executor of cellular senescence, is regulated by members of the Polycomb group (PcG) of transcriptional repressors. Here we show that signaling from oncogenic RAS overrides PcG-mediated repression of INK4a by activating the H3K27 demethylase JMJD3 and down-regulating the methyltransferase EZH2. In human fibroblasts, JMJD3 activates INK4a, but not ARF, and causes p16(INK4a)-dependent arrest. In mouse embryo fibroblasts, Jmjd3 activates both Ink4a and Arf and elicits a p53-dependent arrest, echoing the effects of RAS in this system. Our findings directly implicate JMJD3 in the regulation of INK4a/ARF during oncogene-induced senescence and suggest that JMJD3 has the capacity to act as a tumor suppressor.
Acosta JC, Gil J, 2009, A Role for CXCR2 in Senescence, but What about in Cancer?, Cancer Res
Senescence is an irreversible arrest triggered by stresses such as telomere shortening, DNA damage, or oncogenic signaling. Oncogene-induced senescence occurs in preneoplastic lesions, but it is absent from full-blown malignancies suggesting a tumor suppressor function. We recently found that depletion of the receptor CXCR2 [which binds to chemokines such as interleukin (IL)-8 or GROalpha] delays both replicative senescence and impairs the senescence response to oncogenic signals. Our findings suggest that signaling by IL-8 and GROalpha might limit tumor growth by reinforcing senescence early in tumorigenesis. The challenge remains in how to integrate this with the well-known tumor promoting effects of IL-8 and GROalpha. [Cancer Res 2009;69(6):2167-70].
Augert A, Payré C, Launoit YD, et al., 2009, The M-type receptor PLA2R regulates senescence through the p53 pathway, EMBO Rep
Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss-of-function genetic screen in primary human fibroblasts. We report that knockdown of the M-type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress-induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species-DNA damage-p53-dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.
Acosta JC, Loghlen AO, Banito A, et al., 2008, Control of senescence by CXCR2 and its ligands, Cell Cycle, Vol: 7, Pages: 2956-9
Senescence is an irreversible growth arrest with important physiological implications as it contributes to tumour suppression and may have a role in aging. During senescence, cells suffer profound phenotypic changes affecting amongst others cell morphology and chromatin structure. Senescent cells also undergo significant transcriptional changes, such as the increased production of a plethora of different secreted factors, which are the basis of the so-called senescence-associated secretory phenotype. While some of these factors have been previously shown to possess different pro-tumorigenic activities, we recently demonstrated that the secretion of CXCR2-binding chemokines (such as IL-8 or GROalpha) by senescent cells contribute to reinforce senescence via activation of the p53 pathway. Importantly, our data adds to that presented by several groups suggesting that also other factors secreted during senescence (such as PAI-1, IGFBP-7 or IL-6) contribute to the senescent response. Here, we discuss our findings in the context of the emerging role for secreted factors in regulating senescence through paracrine and/or autocrine mechanisms.
Acosta JC, Loghlen AO, Banito A, et al., 2008, Chemokine signaling via the CXCR2 receptor reinforces senescence, Cell, Vol: 133, Pages: 1006-18
Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.
Kondoh H, Lleonart ME, Nakashima Y, et al., 2007, A High Glycolytic Flux Supports the Proliferative Potential of Murine Embryonic Stem Cells, Antioxidants & Redox Signaling, Vol: 9, Pages: 293-299
Scott CL, Gil J, Hernando E, et al., 2007, Role of the chromobox protein CBX7 in lymphomagenesis, Proceedings of the National Academy of Sciences, Vol: 104, Pages: 5389-5394
Kondoh H, Lleonart ME, Bernard D, et al., 2007, Protection from oxidative stress by enhanced glycolysis; a possible mechanism of cellular immortalization, Histology and Histopathology, Vol: 22, Pages: 85-90, ISSN: 0213-3911
Reactive oxygen species (ROS) play a crucial role not only in the physiological signal transduction but also in the pathogenesis of several human diseases such as atherosclerosis, neurodegenerative diseases, metabolic disorders, aging or cancer amongst others. Oxidative stress is also responsible for cellular and organism senescence, in accordance with what Harman initially proposed in the free radical theory of aging. Recent findings support the notion that protection from oxidative stress can increase life span significantly. We reported that enhanced glycolysis could modulate cellular life span with reduction of oxidative stress. Moreover, the tumor suppressor gene p53 controls post-transcriptionally the level of the glycolytic enzyme, phosphoglycerate mutase (PGM). As enhanced glycolysis is a distinctive and prominent feature of cancer cells (termed the Warburg effect), our findings disclosed a novel aspect of the Warburg effect: the connection between senescence and oxidative stress.
Kondoh H, Lleonart ME, Bernard D, et al., 2007, Protection from oxidative stress by enhanced glycolysis; a possible mechanism of cellular immortalization, Histol Histopathol, Vol: 22, Pages: 85-90
Reactive oxygen species (ROS) play a crucial role not only in the physiological signal transduction but also in the pathogenesis of several human diseases such as atherosclerosis, neuro-degenerative diseases, metabolic disorders, aging or cancer amongst others. Oxidative stress is also responsible for cellular and organism senescence, in accordance with what Harman initially proposed in the free radical theory of aging. Recent findings support the notion that protection from oxidative stress can increase life span significantly. We reported that enhanced glycolysis could modulate cellular life span with reduction of oxidative stress. Moreover, the tumor suppressor gene p53 controls post-transcriptionally the level of the glycolytic enzyme, phosphoglycerate mutase (PGM). As enhanced glycolysis is a distinctive and prominent feature of cancer cells (termed the Warburg effect), our findings disclosed a novel aspect of the Warburg effect: the connection between senescence and oxidative stress.
García MA, Gil J, Ventoso I, et al., 2006, Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action, Microbiol Mol Biol Rev, Vol: 70, Pages: 1032-60
The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by interferons. Not only is PKR an effector molecule on the cellular response to double-stranded RNA, but it also integrates signals in response to Toll-like receptor activation, growth factors, and diverse cellular stresses. In this review, we provide a detailed picture on how signaling downstream of PKR unfolds and what are the ultimate consequences for the cell fate. PKR activation affects both transcription and translation. PKR phosphorylation of the alpha subunit of eukaryotic initiation factor 2 results in a blockade on translation initiation. However, PKR cannot avoid the translation of some cellular and viral mRNAs bearing special features in their 5’ untranslated regions. In addition, PKR affects diverse transcriptional factors such as interferon regulatory factor 1, STATs, p53, activating transcription factor 3, and NF-kappaB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IkappaB and NF-kappaB nuclear translocation has been intensively studied. At the cellular and organism levels PKR exerts antiproliferative effects, and it is a key antiviral agent. A point of convergence in both effects is that PKR activation results in apoptosis induction. The extent and strength of the antiviral action of PKR are clearly understood by the findings that unrelated viral proteins of animal viruses have evolved to inhibit PKR action by using diverse strategies. The case for the pathological consequences of the antiproliferative action of PKR is less understood, but therapeutic strategies aimed at targeting PKR are beginning to offer promising results.
Gil J, Peters G, 2006, Regulation of the INK4b–ARF–INK4a tumour suppressor locus: all for one or one for all, Nat Rev Mol Cell Biol, Vol: 7, Pages: 667-677
Bernstein E, Duncan EM, Masui O, et al., 2006, Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin, Mol Cell Biol, Vol: 26, Pages: 2560-9
The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.
Bernard D, Gil J, Dumont P, et al., 2006, The methyl-CpG-binding protein MECP2 is required for prostate cancer cell growth, Oncogene, Vol: 25, Pages: 1358-1366
Bernard D, Martinez-Leal JF, Rizzo S, et al., 2005, CBX7 controls the growth of normal and tumor-derived prostate cells by repressing the Ink4a/Arf locus, Oncogene, Vol: 24, Pages: 5543-51
Control of cell proliferation by Polycomb group proteins (PcG) is an important facet of cellular homeostasis and its disruption can promote tumorigenesis. We recently described CBX7 as a novel PcG protein controlling the growth of normal cells. In an attempt to identify a putative role of CBX7 in tumorigenesis, we analysed CBX7 expression in a panel of cancer cell lines and primary tissues. CBX7 was highly expressed in three different prostate cancer cell lines and present at elevated levels in normal prostate. Ablation of CBX7 expression using short hairpin RNAs (shRNA) resulted in upregulation of p16Ink4a and p14Arf in both LNCaP and PC-3 prostate cell lines. CBX7 knockdown caused an impairment of cell growth that was dependent on the status of the p14Arf/p53 and p16Ink4a/Rb pathways in both normal and cancer prostate cells. CBX7 overexpression in LNCaP cells resulted in a slight growth advantage in both androgen-dependent and -independent conditions. Moreover, CBX7 expression cooperated with c-Myc in rendering LNCaP cells insensitive to growth arrest by androgen receptor inhibition. Together, these data suggest that CBX7 represses p16Ink4a and p14Arf expression in normal and tumor-derived prostate cells, affecting their growth depending on the status of the p16Ink4a/Rb and the p14Arf/p53 pathways.
Collado M, Gil J, Efeyan A, et al., 2005, Tumour biology: senescence in premalignant tumours, Nature, Vol: 436, Pages: 642-642
Oncogene-induced senescence is a cellular response that may be crucial for protection against cancer development, but its investigation has so far been restricted to cultured cells that have been manipulated to overexpress an oncogene. Here we analyse tumours initiated by an endogenous oncogene, ras, and show that senescent cells exist in premalignant tumours but not in malignant ones. Senescence is therefore a defining feature of premalignant tumours that could prove valuable in the diagnosis and prognosis of cancer.
Gil J, Kerai P, Lleonart M, et al., 2005, Immortalization of primary human prostate epithelial cells by c-Myc, Cancer Res, Vol: 65, Pages: 2179-85
A significant percentage of prostate tumors have amplifications of the c-Myc gene, but the precise role of c-Myc in prostate cancer is not fully understood. Immortalization of human epithelial cells involves both inactivation of the Rb/p16INK4a pathway and telomere maintenance, and it has been recapitulated in culture by expression of the catalytic subunit of telomerase, hTERT, in combination with viral oncoproteins. Here, we show the immortalization of human prostate epithelial cells (HPrEC) by a single genetic event, the expression of the c-Myc oncogene. Myc stabilizes telomere length in HPrEC through up-regulation of hTERT expression and overrides the accumulation of cell cycle inhibitory proteins, such as p16INK4a. Overall, HPrECs expressing c-Myc retain many characteristics of normal cells, such as the induction of a senescence-like growth arrest in response to oncogenic Ras, an intact p53 response, and an absence of gross karyotypic abnormalities. However, HPrECs expressing c-Myc lack a Rb/p16INK4a checkpoint and can be transformed without the need for additional genetic lesions in that pathway. These results give a partial explanation for the physiologic role of c-Myc overexpression in prostate cancer.
Gil J, Bernard D, Peters G, 2005, Role of polycomb group proteins in stem cell self-renewal and cancer, DNA Cell Biol, Vol: 24, Pages: 117-25
Polycomb group proteins (PcG) form part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. The mechanism relies on epigenetic modifications on specific histone tails that are inherited through cell divisions, thus behaving de facto as a cellular memory. This cellular memory governs key events in organismal development as well as contributing to the control of normal cell growth and differentiation. Consequently, the dysregulation of PcG genes, such as Bmi1, Pc2, Cbx7, and EZH2 has been linked with the aberrant proliferation of cancer cells. Furthermore, at least three PcG genes, Bmi1, Rae28, and Mel18, appear to regulate self-renewal of specific stem cell types suggesting a link between the maintenance of cellular homeostasis and tumorigenesis. In this review, we will briefly summarize current views on PcG function and the evidence linking specific PcG proteins with the behavior of stem cells and cancer cells.
Kondoh H, Lleonart M, Gil J, et al., 2005, Glycolysis and cellular immortalization, Drug Discovery Today: Disease Mechanisms, Vol: 2, Pages: 263-267
Kondoh H, Lleonart ME, Gil J, et al., 2005, Glycolytic enzymes can modulate cellular life span, Cancer Res, Vol: 65, Pages: 177-85
An unbiased screen for genes that can immortalize mouse embryonic fibroblasts identified the glycolytic enzyme phosphoglycerate mutase (PGM). A 2-fold increase in PGM activity enhances glycolytic flux, allows indefinite proliferation, and renders cells resistant to ras-induced arrest. Glucosephosphate isomerase, another glycolytic enzyme, displays similar activity and, conversely, depletion of PGM or glucosephosphate isomerase with short interfering RNA triggers premature senescence. Immortalized mouse embryonic fibroblasts and mouse embryonic stem cells display higher glycolytic flux and more resistance to oxidative damage than senescent cells. Because wild-type p53 down-regulates PGM, mutation of p53 can facilitate immortalization via effects on PGM levels and glycolysis.
Gil J, Esteban M, 2004, Vaccinia virus recombinants as a model system to analyze interferon-induced pathways, JOURNAL OF INTERFERON AND CYTOKINE RESEARCH, Vol: 24, Pages: 637-646, ISSN: 1079-9907
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Gil J, Esteban M, 2004, Vaccinia virus recombinants as a model system to analyze interferon-induced pathways, J Interferon Cytokine Res, Vol: 24, Pages: 637-46
The interferons (IFNs) are a family of cytokines with broad antiviral activities that also control cell proliferation and modulate immune responses. IFNs exert their pleiotropic actions through the regulation of multiple pathways that have been subjected to extensive study using diverse approaches. The scope of this review is to show how we can take advantage of vaccinia virus (VV) to study IFN-related pathways. We summarize and present the different VV models available for studying IFN function and the possibilities that they offer to analyze IFN-induced pathways, IFN modulators, and the biologic effects at the molecular and cellular levels. Emphasis is given to studies of dsRNA-activated signaling with VV lacking E3L (VV DeltaE3L) and in RNA-activated protein kinase (PKR)-related pathways, through the use of VV recombinants (VVr) with inducible PKR (VV PKR). The latest system is versatile, as expression of PKR can be regulated and induced at different times; similarly, VVr can be generated expressing other PKR modulators. As an example of the utility of VVr, we describe how this model has been used to analyze the antiviral and proapoptotic functions of PKR, the impact of PKR on translation, and the PKR-induced activation of the nuclear factor-kappaB (NF-kappaB) pathway.
Gil J, García MA, Gomez-Puertas P, et al., 2004, TRAF family proteins link PKR with NF-kappa B activation, Mol Cell Biol, Vol: 24, Pages: 4502-12
The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-kappa B via the I kappa B kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-kappa B activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-kappa B by the dsRNA-dependent protein kinase.
Gil J, Bernard D, Martínez D, et al., 2004, Polycomb CBX7 has a unifying role in cellular lifespan, Nat Cell Biol, Vol: 6, Pages: 67-72
In contrast to cancer cells and embryonic stem cells, the lifespan of primary human cells is finite. After a defined number of population doublings, cells enter in an irreversible growth-arrested state termed replicative senescence. Mutations of genes involved in immortalization can contribute to cancer. In a genetic screen for cDNAs bypassing replicative senescence of normal human prostate epithelial cells (HPrEC), we identified CBX7, a gene that encodes a Polycomb protein, as shown by sequence homology, its interaction with Ring1 and its localization to nuclear Polycomb bodies. CBX7 extends the lifespan of a wide range of normal human cells and immortalizes mouse fibroblasts by downregulating expression of the Ink4a/Arf locus. CBX7 does not inter-function or colocalize with Bmi1, and both can exert their actions independently of each other as shown by reverse genetics. CBX7 expression is downregulated during replicative senescence and its ablation by short-hairpin RNA (shRNA) treatment inhibited growth of normal cells though induction of the Ink4a/Arf locus. Taken together, these data show that CBX7 controls cellular lifespan through regulation of both the p16(Ink4a)/Rb and the Arf/p53 pathways.
Bernard D, Pourtier-Manzanedo A, Gil J, et al., 2003, Myc confers androgen-independent prostate cancer cell growth, Journal of Clinical Investigation, Vol: 112, Pages: 1724-31
Prostate cancer is one of the most diagnosed and mortal cancers in western countries. A major clinical problem is the development of androgen-independent prostate cancer (AIPC) during antihormonal treatment. The molecular mechanisms underlying the change from androgen dependence to independence of these tumors are poorly understood and represent a challenge to develop new therapies. Based on genetic data showing amplification of the c-myc gene in AIPC, we studied the ability of c-myc to confer AIPC cell growth. Human androgen-dependent prostate cancer cells overexpressing c-myc grew independently of androgens and presented tumorigenic properties in androgen-depleted conditions. Analysis of signalling pathways by pharmacological inhibitors of the androgen receptor (AR) or by RNA interference directed against AR or c-myc showed that c-myc acted downstream of AR through multiple growth effectors. Thus c-myc is required for androgen-dependent growth and following ectopic expression can induce androgen-independent growth. Moreover, RNA interference directed against c-myc showed that growth of human AIPC cells, AR-positive or -negative, required c-myc expression. Furthermore, we showed that c-myc-overexpressing cells retain a functional p53 pathway and thus respond to etoposide.
García MA, Guerra S, Gil J, et al., 2002, Anti-apoptotic and oncogenic properties of the dsRNA-binding protein of vaccinia virus, E3L, Oncogene, Vol: 21, Pages: 8379-87
The vaccinia virus (VV) E3L gene encodes a dsRNA binding protein that inhibits activation of the IFN-induced, dsRNA-dependent protein kinase, (PKR), the 2-5A synthetases/RNase L system and other dsRNA dependent pathways, thus leading to efficient VV replication. To analyse E3L effects over cellular metabolism in a virus-free system, we have generated stable mouse 3T3 cell lines expressing E3L. Expression of E3L in NIH3T3 cells results in inhibition of eIF-2alpha phosphorylation and Ikappa(B)alpha degradation in response to dsRNA. Antiviral responses induced by IFN-alpha/beta were partially impaired in 3T3-E3L cells, as determined by a viability assay upon VSV infection. E3L expression also confers resistance to dsRNA-triggered apoptosis. Interestingly, cells expressing E3L grew faster than control cells, and showed increased expression of cyclin A and decreased levels of p27(Kip1). E3L cooperated with H-ras in a focus formation assay, and NIH3T3 E3L cells formed solid tumors when injected in nude mice. Overall, our findings reveal that interference of E3L protein with several cellular pathways, results in promotion of cellular growth, impairment of antiviral activity and resistance to apoptosis.
Gil J, García MA, Esteban M, 2002, Caspase 9 activation by the dsRNA-dependent protein kinase, PKR: molecular mechanism and relevance, FEBS Lett, Vol: 529, Pages: 249-55
The double-stranded RNA-dependent protein kinase (PKR) induces apoptosis by activation of the FADD/caspase 8 pathway. Here we show that upon PKR expression, caspase 9 is processed and activated, correlating with the translocation of cytochrome c to the cytoplasm and breakdown of mitochondrial potential upon Bax insertion. However, treatment of cells with an inhibitor of caspase 9 could not prevent PKR-induced apoptosis. During PKR-induced apoptosis, caspase 9 is activated downstream of caspase 8. Our findings revealed that caspase 9, although dispensable, is a mediator of PKR-induced cell death.
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