RESUMO
BACKGROUND: Non-invasive biomarkers of disease progression in mice with cancer are lacking making it challenging to implement appropriate humane end points. We investigated whether body temperature, food and water consumption could be used to predict tumour burden. METHODS: Thirty-six male, wild-type C57Bl/6 mice were implanted with subcutaneous RFID temperature sensors and inoculated with Eµ-myc tumours that infiltrate lymphoid tissue. RESULTS: Decrease in body temperature over the course of the study positively predicted post-mortem lymph node tumour burden (R(2)=0.68, F(1,22)=44.8, P<0.001). At experimental and humane end points, all mice that had a mean decrease in body temperature of 0.7 °C or greater had lymph nodes heavier than 0.5 g (100% sensitivity), whereas a mean decrease in body temperature <0.7 °C always predicted lymph nodes lighter than 0.5 g (100% specificity). The mean decrease in food consumption in each cage also predicted mean post-mortem lymph node tumour burden at 3 weeks (R(2)=0.89, F(1,3)=23.2, P=0.017). CONCLUSION: Temperature, food and water consumption were useful biomarkers of disease progression in mice with lymphoma and could potentially be used more widely to monitor mice with other forms of cancer.
Assuntos
Temperatura Corporal , Ingestão de Líquidos , Ingestão de Alimentos , Linfoma/patologia , Carga Tumoral , Animais , Biomarcadores Tumorais , Progressão da Doença , Linfoma/diagnóstico , Linfoma/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-myc/genética , Dispositivo de Identificação por Radiofrequência , Tecnologia de Sensoriamento RemotoRESUMO
The p21 gene encodes a cyclin-dependent kinase inhibitor that affects cell-cycle progression, but the potential of this gene product to serve as a tumour suppressor in vivo has not been established. In this report, we show that the growth of malignant cells in vitro and in vivo is inhibited by expression of p21. Expression of p21 resulted in an accumulation of cells in G0/G1, altered morphology, and cell differentiation, but apoptosis was not induced. Introduction of p21 with adenoviral vectors into malignant cells completely suppressed their growth in vivo and also reduced the growth of established pre-existing tumours. Gene transfer of p21 may provide a molecular genetic approach to arresting cancer cell growth by committing malignant cells irreversibly to a pathway of terminal differentiation.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/genética , Ciclinas/metabolismo , Genes Supressores de Tumor , Inibidores do Crescimento/metabolismo , Células 3T3 , Animais , Ciclo Celular , Diferenciação Celular , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/farmacologia , Técnicas de Transferência de Genes , Humanos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais CultivadasRESUMO
The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacted with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibited p300-associated cyclin E-Cdk2 activity, stimulated kappaB-dependent gene expression, which was also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Ativação Transcricional , Ciclo Celular , Linhagem Celular , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Genes Reporter , Humanos , Células Jurkat , NF-kappa B/genética , Proteínas Nucleares/genética , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição RelA , Fatores de Transcrição/genética , TransfecçãoRESUMO
The members of the Rel/NF-kappa B family of transcription factors form one of the first lines of defense against infectious diseases and cellular stress. These proteins initiate a highly coordinated response in multiple cell types that effectively counteracts the threat to the health of the organism. Conversely, disruption of the regulatory mechanisms that control the specificity and extent of this response, which results in aberrant activation of NF-kappa B, can be one of the primary causes of a wide range of human diseases. Thus, targeting NF-kappa B might lead to the development of new pharmaceutical reagents that could provide novel treatments for many inflammatory diseases and cancer.
Assuntos
Proteínas I-kappa B/fisiologia , NF-kappa B/fisiologia , Animais , Apoptose , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Proteínas I-kappa B/química , Proteínas I-kappa B/genética , Inflamação/metabolismo , Modelos Biológicos , Família Multigênica , NF-kappa B/química , NF-kappa B/genética , Neoplasias , Transcrição GênicaRESUMO
The forkhead associated (FHA) domain-containing protein Smad nuclear interacting protein 1 (SNIP1) has multiple cellular functions, including the ability to interact with DNA-binding transcription factors and transcriptional coactivators. Moreover, we have demonstrated previously that SNIP1 regulates cyclin D1 expression and promoter activity. Here, we identify a new function for SNIP1 as a regulator of ATR checkpoint kinase-dependent pathways in human U-2 OS osteosarcoma cells: SNIP1 is required for p53 induction in response to ultraviolet light treatment and selectively regulates the phosphorylation of known ATR target proteins, including p53, Chk1 and the histone variant H2AX. These activities are independent of its ability to regulate cyclin D1 expression. Significantly, SNIP1 is also required for ATR-dependent functions of the human p14(ARF) tumour suppressor, including its ability to modulate the activity of the RelA(p65) NF-kappaB subunit. This, together with its other described functions, suggests that SNIP1 could have an important role during tumorigenesis and cancer therapy.
Assuntos
Proteínas de Ciclo Celular/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Osteossarcoma/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Quinase 1 do Ponto de Checagem , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Raios UltravioletaRESUMO
The respective pro- and antiapoptotic functions of the transcription factors p53 and nuclear factor kappaB (NF-kappaB), and their potential impact on tumorigenesis and response to tumor therapy are well recognized. The capacity of the RelA(p65) subunit of NF-kappaB to specify a pro-apoptotic outcome in response to some stimuli is less well recognized, but needs to be understood if rational manipulation of the NF-kappaB pathway is to be deployed in cancer therapy. In this report, we provide evidence that the growth-responsive nuclear protein, proenkephalin (Penk), is required, in part, for apoptosis induction, in response to activation or overexpression of p53 and RelA(p65). We describe UV-C-inducible physical associations between endogenous Penk and p53 and RelA(p65) in mammalian cell lines. Depletion of Penk by RNA interference (RNAi) substantially preserves viable cell number following exposure to UV-C irradiation or hydrogen peroxide and confers transient protection in cells exposed to the genotoxin etoposide. In virally transformed and human tumor cell lines, overexpression of nuclear Penk with overabundant or activated p53, or RelA(p65) even in the absence of p53, enhances apoptosis to the point of synergy. We have further shown that Penk depletion by RNAi substantially derepresses transcription of a range of antiapoptotic gene targets previously implicated in repression-mediated apoptosis induction by NF-kappaB and p53. Physical association of endogenous Penk with the transcriptional co-repressor histone deacetylase suggests that it may be a component of a transcriptional repression complex that contributes to a pro-apoptotic outcome, following activation of the NF-kappaB and p53 pathways, and could therefore help to facilitate an antitumor response to a broad range of agents.
Assuntos
Apoptose , Encefalinas/metabolismo , NF-kappa B/metabolismo , Precursores de Proteínas/metabolismo , Fator de Transcrição RelA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Encefalinas/genética , Etoposídeo/farmacologia , Humanos , Precursores de Proteínas/genética , Interferência de RNA , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
The diverse cellular and biological functions of the nuclear factor kappa B (NF-kappaB) pathway, together with the catastrophic consequences of its aberrant regulation, demand specific and highly regulated control of its activity. As described in this review, regulation of the NF-kappaB pathway is brought about through multiple post-translational modifications that control the activity of the core components of NF-kappaB signaling: the IkappaB kinase (IKK) complex, the IkappaB proteins and the NF-kappaB subunits themselves. These regulatory modifications, which include phosphorylation, ubiquitination, acetylation, sumoylation and nitrosylation, can vary, depending on the nature of the NF-kappaB-inducing stimulus. Moreover, they frequently have distinct, sometimes antagonistic, functional consequences and the same modification can have different effects depending on the context. Given the important role of NF-kappaB in human health and disease, understanding these pathways will not only provide valuable insights into mechanism and function, but could also lead to new drug targets and the development of diagnostic and prognostic biomarkers for many pathological conditions.
Assuntos
NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ativação Enzimática , Humanos , Hidrólise , Quinase I-kappa B/metabolismoRESUMO
Complexes formed from the nuclear factor kappaB (NF-kappaB) family of transcription factors are ubiquitously expressed and are induced by a diverse array of stimuli. This results in their becoming activated in a wide variety of different settings. While the functions of NF-kappaB in many of these contexts have been the subject of intense research and are now well established, it is also clear that there is great diversity in the effects and consequences of NF-kappaB activation. NF-kappaB subunits do not necessarily regulate the same genes, in an identical manner, in all of the different circumstances in which they are induced. This review will discuss the different functions of NF-kappaB, the pathways that modulate NF-kappaB subunit activity and, in contrast to its more commonly thought of role as a promoter of cancer cell growth and survival, the ability of NF-kappaB, under some circumstances, to behave as a tumor suppressor.
Assuntos
Modelos Biológicos , NF-kappa B/metabolismo , Neoplasias/terapia , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Carcinógenos/metabolismo , Humanos , Quinase I-kappa B/metabolismo , NF-kappa B/fisiologiaRESUMO
Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF-kappaB. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF-kappaB has been shown to promote resistance to programmed cell death. These observations suggest that a regulatory mechanism must exist to integrate these opposing outcomes and coordinate this critical cellular decision-making event. Here we show that both p53 and NF-kappaB inhibit each other's ability to stimulate gene expression and that this process is controlled by the relative levels of each transcription factor. Expression of either wild-type p53 or the RelA(p65) NF-kappaB subunit suppresses stimulation of transcription by the other factor from a reporter plasmid in vivo. Moreover, endogenous, tumor necrosis factor alpha-activated NF-kappaB will inhibit endogenous wild-type p53 transactivation. Following exposure to UV light, however, the converse is observed, with p53 downregulating NF-kappaB-mediated transcriptional activation. Both p53 and RelA(p65) interact with the transcriptional coactivator proteins p300 and CREB-binding protein (CBP), and we demonstrate that these results are consistent with competition for a limiting pool of p300/CBP complexes in vivo. These observations have many implications for regulation of the transcriptional decision-making mechanisms that govern cellular processes such as apoptosis. Furthermore, they suggest a previously unrealized mechanism through which dysregulated NF-kappaB can contribute to tumorigenesis and disease.
Assuntos
NF-kappa B/genética , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genética , Apoptose/genética , Ligação Competitiva , Linhagem Celular , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Humanos , Mutação/genética , Proteínas Repressoras/genética , Transativadores/genética , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/genética , Raios UltravioletaRESUMO
Adhesive interactions between cells are essential for the organization and function of differentiated tissues and organs and are mediated by inducible cell surface glycoproteins. In normal tissues, cell adhesion molecules contribute to immune regulation, inflammation, and embryogenesis. Additionally, they play an important role in a variety of pathogenic processes. Cell adhesion molecule expression can be induced by stimuli known to activate NF-kappa B, a ubiquitous transcription factor found in a variety of cell types. To investigate the role of NF-kappa B in cell adhesion molecule expression, we treated HL-60 cells with a double-stranded oligonucleotide which specifically inhibits NF-kappa B-mediated transcription. This treatment resulted in the inhibition of phorbol 12-myristate 13-acetate (PMA)-induced cellular adhesion, morphological changes, and the expression of leukocyte integrin CD11b. In a similar fashion, expression of intercellular adhesion molecule 1 on human endothelial cells induced by PMA was specifically inhibited by the NF-kappa B antagonist. We suggest that NF-kappa B activation is a necessary event for the PMA-induced differentiation of HL-60 cells and the expression of certain activation is a necessary event for the PMA-induced differentiation of HL-60 cells and the expression of certain adhesion molecules. Furthermore, the inhibition of transcription factor functions by this generally applicable mechanism can be used to define their role in cellular differentiation and function.
Assuntos
Adesão Celular , NF-kappa B/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Sequência de Bases , Ligação Competitiva , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/biossíntese , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , DNA , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Citometria de Fluxo , Humanos , Molécula 1 de Adesão Intercelular , Dados de Sequência Molecular , NF-kappa B/antagonistas & inibidores , Oligonucleotídeos/metabolismo , Tionucleotídeos/metabolismo , Células Tumorais CultivadasRESUMO
Induction of human immunodeficiency virus type 1 (HIV-1) gene expression in stimulated T cells has been attributed to the activation of the transcription factor NF-kappa B. The twice-repeated kappa B sites within the HIV-1 long terminal repeat are in close proximity to three binding sites for Sp1. We have previously shown that a cooperative interaction of NF-kappa B with Sp1 is required for the efficient stimulation of HIV-1 transcription. In this report, we define the domains of each protein responsible for this effect. Although the transactivation domains seemed likely to mediate this interaction, we find, surprisingly, that this interaction occurs through the putative DNA-binding domains of both proteins. Sp1 specifically interacted with the amino-terminal region of RelA(p65). Similarly, RelA bound directly to the zinc finger region of Sp1. This interaction was specific and resulted in cooperative DNA binding to the kappa B and Sp1 sites in the HIV-1 long terminal repeat. Furthermore, the amino-terminal region of RelA did not associate with several other transcription factors, including MyoD, E12, or Kox15, another zinc finger protein. These findings suggest that the juxtaposition of DNA-binding sites promotes a specific protein interaction between the DNA-binding regions of these transcription factors. This interaction is required for HIV transcriptional activation and may provide a mechanism to allow for selective activation of kappa B-regulated genes.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/genética , NF-kappa B/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Células Cultivadas , DNA/metabolismo , Análise Mutacional de DNA , Drosophila/citologia , HIV-1/crescimento & desenvolvimento , Humanos , Dados de Sequência Molecular , Ligação Proteica , Deleção de Sequência , Fator de Transcrição Sp1/genética , Linfócitos T/microbiologia , Fator de Transcrição RelA , Ativação Transcricional , Dedos de ZincoRESUMO
The transcriptional coactivators p300 and CREB binding protein (CBP) are important regulators of the cell cycle, differentiation, and tumorigenesis. Both p300 and CBP are targeted by viral oncoproteins, are mutated in certain forms of cancer, are phosphorylated in a cell cycle-dependent manner, interact with transcription factors such as p53 and E2F, and can be found complexed with cyclinE-Cdk2 in vivo. Moreover, p300-deficient cells show defects in proliferation. Here we demonstrate that transcriptional activation by both p300 and CBP is stimulated by coexpression of the cyclin-dependent kinase inhibitor p21(WAF/CIP1). Significantly this stimulation is independent of both the inherent histone acetyltransferase (HAT) activity of p300 and CBP and of the previously reported carboxyl-terminal binding site for cyclinE-Cdk2. Rather, we describe a previously uncharacterized transcriptional repression domain (CRD1) within p300. p300 transactivation is stimulated through derepression of CRD1 by p21. Significantly p21 regulation of CRD1 is dependent on the nature of the core promoter. We suggest that CRD1 provides a novel mechanism through which p300 and CBP can switch activities between the promoters of genes that stimulate growth and those that enhance cell cycle arrest.
Assuntos
Ciclinas/genética , Proteínas Nucleares/genética , Transativadores/genética , Ativação Transcricional , Animais , Proteína de Ligação a CREB , Ciclo Celular/genética , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , HumanosRESUMO
Transforming mutants of the p53 tumor suppressor gene can positively regulate transcription from several promoters that do not contain known p53 binding sites. Here, we report the identification of a novel p53 binding site in the human immunodeficiency virus long terminal repeat that specifically mediates mutant p53 transactivation. This DNA element was bound by endogenous Jurkat p53 when these cells were stimulated by tumor necrosis factor. Mutation of this sequence inhibited p53 transactivation and tumor necrosis factor inducibility of the human immunodeficiency virus type 1 long terminal repeat. In addition, this DNA element was found to be sufficient to confer mutant p53 responsiveness on a heterologous minimal promoter. It has been hypothesized that transforming mutants of p53 represent a proliferative conformational stage that can be adopted by the native protein under stimulation by growth factors. The data presented suggest that proliferative and antiproliferative p53 conformations recognize different DNA binding sites in order to mediate distinct biological functions. Thus, transforming mutants of p53 that fold into the proliferative conformation would favor proliferative over antiproliferative functions.
Assuntos
HIV-1/genética , Sequências Repetitivas de Ácido Nucleico/efeitos dos fármacos , Ativação Transcricional , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/genética , Sequência de Bases , Sítios de Ligação/genética , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Sequências Repetitivas de Ácido Nucleico/genética , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Ampliador HIV/genética , HIV/genética , Proteínas I-kappa B , Transcrição Gênica , Animais , Linfócitos B/citologia , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/análise , Sondas de DNA , Proteínas de Ligação a DNA/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , NF-kappa B/farmacologia , Conformação Proteica , Proteínas Recombinantes/metabolismo , Fator de Transcrição RelA , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Post-translational modifications of nuclear factor (NF)-κB subunits provide a mechanism to differentially regulate their activity in response to the many stimuli that induce this pathway. However, the physiological significance of these modifications is largely unknown, and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines, but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride (CCl4) treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine model of hepatocellular carcinoma. These data reveal a critical pathway controlling NF-κB function in the liver that acts to suppress the tumour-promoting activities of RelA.
Assuntos
Apoptose/genética , Neoplasias Hepáticas/genética , Regeneração Hepática/genética , NF-kappa B/genética , Fator de Transcrição RelA/genética , Animais , Tetracloreto de Carbono/toxicidade , Proliferação de Células/efeitos dos fármacos , Técnicas de Introdução de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Mutação/genética , Fosforilação/genéticaRESUMO
The REL gene, encoding the NF-κB subunit c-Rel, is frequently amplified in B-cell lymphoma and functions as a tumour-promoting transcription factor. Here we report the surprising result that c-rel-/- mice display significantly earlier lymphomagenesis in the c-Myc driven, Eµ-Myc model of B-cell lymphoma. c-Rel loss also led to earlier onset of disease in a separate TCL1-Tg-driven lymphoma model. Tumour reimplantation experiments indicated that this is an effect intrinsic to the Eµ-Myc lymphoma cells but, counterintuitively, c-rel-/- Eµ-Myc lymphoma cells were more sensitive to apoptotic stimuli. To learn more about why loss of c-Rel led to earlier onset of disease, microarray gene expression analysis was performed on B cells from 4-week-old, wild-type and c-rel-/- Eµ-Myc mice. Extensive changes in gene expression were not seen at this age, but among those transcripts significantly downregulated by the loss of c-Rel was the B-cell tumour suppressor BTB and CNC homology 2 (Bach2). Quantitative PCR and western blot analysis confirmed loss of Bach2 in c-Rel mutant Eµ-Myc tumours at both 4 weeks and the terminal stages of disease. Moreover, Bach2 expression was also downregulated in c-rel-/- TCL1-Tg mice and RelA Thr505Ala mutant Eµ-Myc mice. Analysis of wild-type Eµ-Myc mice demonstrated that the population expressing low levels of Bach2 exhibited the earlier onset of lymphoma seen in c-rel-/- mice. Confirming the relevance of these findings to human disease, analysis of chromatin immunoprecipitation sequencing data revealed that Bach2 is a c-Rel and NF-κB target gene in transformed human B cells, whereas treatment of Burkitt's lymphoma cells with inhibitors of the NF-κB/IκB kinase pathway or deletion of c-Rel or RelA resulted in loss of Bach2 expression. These data reveal a surprising tumour suppressor role for c-Rel in lymphoma development explained by regulation of Bach2 expression, underlining the context-dependent complexity of NF-κB signalling in cancer.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-rel/genética , Animais , Apoptose/genética , Linfócitos B/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Western Blotting , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Humanos , Linfoma de Células B/metabolismo , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-rel/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Nuclear Factor-Kappa B (NF-kappa B) was first identified by Sen and Baltimore (1986, Cell 46, 705-716) as a constitutively active transcription factor binding the kappa light chain immunoglobulin enhancer in B cells. Shortly afterwards, the same researchers found NF-kappa B to be present in other cell types in an inactive cytoplasmic form which upon cellular stimulation could be induced to translocate to the nucleus and bind DNA. Subsequently, it has been demonstrated that NF-kappa B performs a critical role as a regulator of the immune system, the response to stress, apoptosis, viral replication and is involved in many diseases, leading to it becoming one of the most intensively studied transcription factors of the last decade. The pivotal role played by NF-kappa B is illustrated not only by the great diversity of genes that it regulates, but also by the large variety of stimuli leading to its activation. This article will address how NF-kappa B, a ubiquitously expressed transcription factor composed of dimers formed from five subunits, differentially regulates the expression of such a diverse array of genes with different functions, in different cell types and at different times. Recent research indicates that this behavioral diversity arises from a delicately balanced network of protein: protein interactions: NF-kappa B activity is determined not only through its regulated nuclear localization but is also dependent on the cellular context in which it is found.
Assuntos
Regulação da Expressão Gênica/fisiologia , NF-kappa B/fisiologia , Transcrição Gênica/fisiologia , Animais , HumanosRESUMO
To respond to changes in its environment, the cell utilizes mechanisms that integrate extracellular signals with specific changes in gene expression. To better understand these critical regulatory mechanisms, research has focused, for the most part, on the identification of sequence-specific DNA-binding proteins, such as the nuclear factor kappaB (NF-kappaB) or activator protein 1 (AP-1) families of transcription factors, that interact with the promoter and enhancer elements of genes induced or repressed during cellular activation. More recently, however, it has become apparent that non-DNA-binding transcriptional coactivators, such as p300 and CREB binding protein (CBP), previously thought to function primarily as "bridging" proteins between DNA-bound transcription factors and the basal transcription complex, play a critical regulatory role as integrators of diverse signalling pathways with the selective induction of gene expression. In this commentary, we shall discuss the implications of a particular aspect of this growing and expanding field: how cell cycle regulation of p300 and CBP impacts our understanding of cellular differentiation, the response to DNA damage, and oncogenesis.
Assuntos
Ciclo Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Transcrição Gênica/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Understanding the mechanisms that promote aberrant tumour cell survival is critical for the determination of novel strategies to combat colorectal cancer (CRC). We have recently shown that the anti-apoptotic protein BAG-1, highly expressed in pre-malignant and CRC tissue, can potentiate cell survival through regulating NF-κB transcriptional activity. In this study, we identify a novel complex between BAG-1 and the p50-p50 NF-κB homodimers, implicating BAG-1 as a co-regulator of an atypical NF-κB pathway. Importantly, the BAG-1-p50 complex was detected at gene regulatory sequences including the epidermal growth factor receptor (EGFR) and COX-2 (PTGS2) genes. Suppression of BAG-1 expression using small interfering RNA was shown to increase EGFR and suppress COX-2 expression in CRC cells. Furthermore, mouse embryonic fibroblasts derived from the NF-κB1 (p105/p50) knock-out mouse were used to demonstrate that p50 expression was required for BAG-1 to suppress EGFR expression. This was shown to be functionally relevant as attenuation of BAG-1 expression increased ligand activated phosphorylation of EGFR in CRC cells. In summary, this paper identifies a novel role for BAG-1 in modulating gene expression through interaction with the p50-p50 NF-κB complexes. Data presented led us to propose that BAG-1 can act as a selective regulator of p50-p50 NF-κB responsive genes in colorectal tumour cells, potentially important for the promotion of cell survival in the context of the fluctuating tumour microenvironment. As BAG-1 expression is increased in the developing adenoma through to metastatic lesions, understanding the function of the BAG-1-p50 NF-κB complexes may aid in identifying strategies for both the prevention and treatment of CRC.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Subunidade p50 de NF-kappa B/metabolismo , NF-kappa B/fisiologia , Fatores de Transcrição/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Neoplasias Colorretais/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos , Receptores ErbB/genética , Fibroblastos , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Luciferases/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/genética , Regiões Promotoras Genéticas , Multimerização Proteica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , TransfecçãoRESUMO
The RelA(p65) NF-kappaB (nuclear factor kappaB) subunit is typically thought of as being antiapoptotic and tumour-promoting. However, in our laboratory, we have discovered that RelA can inhibit, rather than induce, antiapoptotic gene expression when activated by certain chemotherapeutic drugs, UV light or through the action of the ARF tumour suppressor. These observations explain why RelA can sometimes facilitate rather than inhibit apoptosis and also exhibits tumour-suppressor characteristics in vivo. A better understanding of these processes and an ability to analyse RelA function in tumours could lead to improved cancer diagnosis, choice of therapy and, ultimately, development of new drugs.