RESUMO
BACKGROUND: Limited data are available regarding the ability of biomarkers to predict complete pathological response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Complete response translates to better patient survival. DEK is a transcription factor involved not only in development and progression of different types of cancer, but is also associated with treatment response. This study aims to analyze the role of DEK in complete pathological response following chemoradiotherapy for locally advanced rectal cancer. METHODS: Pre-treated tumour samples from 74 locally advanced rectal-cancer patients who received chemoradiation therapy prior to total mesorectal excision were recruited for construction of a tissue microarray. DEK immunoreactivity from all samples was quantified by immunohistochemistry. Then, association between positive stained tumour cells and pathologic response to neoadjuvant treatment was measured to determine optimal predictive power. RESULTS: DEK expression was limited to tumour cells located in the rectum. Interestingly, high percentage of tumour cells with DEK positiveness was statistically associated with complete pathological response to neoadjuvant treatment based on radiotherapy and fluoropyrimidine-based chemotherapy and a marked trend toward significance between DEK positiveness and absence of treatment toxicity. Further analysis revealed an association between DEK and the pro-apoptotic factor P38 in the pre-treated rectal cancer biopsies. CONCLUSIONS: These data suggest DEK as a potential biomarker of complete pathological response to treatment in locally advanced rectal cancer.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas Cromossômicas não Histona/biossíntese , Proteínas Oncogênicas/biossíntese , Proteínas de Ligação a Poli-ADP-Ribose/biossíntese , Neoplasias Retais/metabolismo , Neoplasias Retais/terapia , Idoso , Idoso de 80 Anos ou mais , Quimiorradioterapia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Valor Preditivo dos Testes , Prognóstico , Neoplasias Retais/patologia , Resultado do TratamentoRESUMO
One of the defining features of aggressive melanomas is their complexity. Hundreds of mutations and an ever increasing list of changes in the transcriptome and proteome distinguish normal from malignant melanocytic cells. Yet, despite this altered genetic background, a long-known attribute of melanomas is a relatively low rate of mutations in the p53 gene. However, it is unclear whether p53 is maintained in melanoma cells because it is required for their survival, or because it is functionally disabled. More pressing from a translational perspective, is to define whether there is a tumor cell-selective wiring of p53 that offers a window for therapeutic intervention. Here, we provide genetic and pharmacological evidence demonstrating that p53 represents a liability to melanoma cells, which they thwart by assuming an oncogenic dependency on the E3 ligase murine double minute-2 (MDM2). Specifically, we used a combination of RNA interference and two structurally independent small molecule inhibitors of the p53-MDM2 interaction to assess the relative requirement of both proteins for the viability of normal melanocytes and a broad panel of melanoma cell lines. We demonstrated in vitro and in vivo that MDM2 is selectively required to blunt latent pro-senescence signals in melanoma cells. Notably, the outcome of MDM2 inactivation depends not only on the mutational status of p53, but also on its ability to signal to the transcription factor E2F1. These data support MDM2 as a drug target in melanoma cells, and identify E2F1 as a biomarker to consider when stratifying putative candidates for clinical studies of p53-MDM2 inhibitors.
Assuntos
Fator de Transcrição E2F1/metabolismo , Melanócitos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Divisão Celular , Linhagem Celular Tumoral , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator de Transcrição E2F1/genética , Fase G2 , Humanos , Imidazóis/farmacologia , Immunoblotting , Imuno-Histoquímica , Melanócitos/citologia , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oncoprotein C-MYC is overexpressed in human metastatic melanomas and melanoma-derived cells where it is required for the suppression of oncogene-induced senescence (OIS). The genetic events that maintain high levels of C-MYC in melanoma cells and their role in OIS are unknown. Here we report that C-MYC in cells from several randomly chosen melanoma lines was upregulated at the protein level, and largely because of the increased protein stability. Of all known regulators of C-MYC stability, levels of B56α subunit of the PP2A tumor suppressor complex were substantially suppressed in all human melanoma cells compared with normal melanocytes. Accordingly, immunohistochemical analysis revealed that the lowest and the highest amounts of PP2A-B56α were predominantly detected in metastatic melanoma tissues and in primary melanomas from patients with good clinical outcome, respectively. Importantly, PP2A-B56α overexpression suppressed C-MYC in melanoma cells and induced OIS, whereas depletion of PP2A-B56α in normal human melanocytes upregulated C-MYC protein levels and suppressed BRAF(V600E)- and, less efficiently, NRAS(Q61R)-induced senescence. Our data reveal a mechanism of C-MYC overexpression in melanoma cells and identify a functional role for PP2A-B56α in OIS of melanocytic cells.
Assuntos
Genes myc , Melanoma/genética , Proteína Fosfatase 2/metabolismo , Linhagem Celular Tumoral , Senescência Celular , Humanos , Melanócitos/metabolismo , Melanoma/secundário , Estabilidade Proteica , Regulação para CimaRESUMO
Malignant melanomas often harbor activating mutations in BRAF (V600E) or, less frequently, in NRAS (Q61R). Intriguingly, the same mutations have been detected at higher incidences in benign nevi, which are largely composed of senescent melanocytes. Overexpression of BRAF(V600E) or NRAS(Q61R) in human melanocytes in vitro has been shown to induce senescence, although via different mechanisms. How oncogene-induced senescence is overcome during melanoma progression remains unclear. Here, we report that in the majority of analysed BRAF(V600E)- or NRAS(Q61R)-expressing melanoma cells, C-MYC depletion induced different yet overlapping sets of senescence phenotypes that are characteristic of normal melanocytes undergoing senescence due to overexpression of BRAF(V600E) or NRAS(Q61R), respectively. These senescence phenotypes were p16(INK4A)- or p53-independent, however, several of them were suppressed by genetic or pharmacological inhibition of BRAF(V600E) or phosphoinositide 3-kinase pathways, including rapamycin-mediated inhibition of mTOR-raptor in NRAS(Q61R)-expressing melanoma cells. Reciprocally, overexpression of C-MYC in normal melanocytes suppressed BRAF(V600E)-induced senescence more efficiently than NRAS(Q61R)-induced senescence, which agrees with the generally higher rates of activating mutations in BRAF than NRAS gene in human cutaneous melanomas. Our data suggest that one of the major functions of C-MYC overexpression in melanoma progression is to continuous suppress BRAF(V600E)- or NRAS(Q61R)-dependent senescence programs.
Assuntos
Senescência Celular , Expressão Gênica , Melanoma/genética , Melanoma/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Progressão da Doença , Deleção de Genes , Humanos , Melanócitos/metabolismo , Melanoma/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
A major role for c-Myc in the proliferation of normal cells is attributed to its ability to promote progression through G(1) and into S phase of the cell cycle. The absolute requirement of c-Myc for cell cycle progression in human tumor cells has not been comprehensively addressed. In the present work, we used a lentiviral-based short hairpin RNA (shRNA) expression vector to stably reduce c-Myc expression in a large number of human tumor cell lines and in three different types of normal human cells. In all cases, cell proliferation was severely inhibited, with normal cells ultimately undergoing G(0)/G(1) growth arrest. In contrast, tumor cells demonstrated a much more variable cell cycle response with cells from several lines accumulating in S or G(2)/M phases. Moreover, in some tumor lines, the phase of cell cycle arrest caused by inhibition of c-Myc could be altered by depleting tumor suppressor protein p53 or its transcriptional target p21(CIP/WAF). Our data suggest that, as in the case of normal cells, c-Myc is essential for sustaining proliferation of human tumor cells. However its rate-limiting role in cell cycle control is variable and is reliant upon the status of other cell cycle regulators.
Assuntos
Ciclo Celular/fisiologia , Proliferação de Células , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/deficiência , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Queratinócitos/citologia , Queratinócitos/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Expression of Bcl-x(L) correlates with the clinical outcomes of patients with cancer. While the role of Bcl-2 in angiogenesis is becoming increasingly evident, the function of Bcl-x(L) in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-x(L) expression in primary endothelial cells. Bcl-x(L)-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-x(L)), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-x(L) induced expression of the pro-angiogenic chemokines CXC ligand-1 (CXCL1) and CXC ligand-8 (CXCL8), and that blockade of CXC receptor-2 (CXCR2) signaling inhibited spontaneous sprouting of HDMEC-Bcl-x(L). Bcl-x(L) led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-x(L), suggesting the existence of a unidirectional crosstalk from Bcl-x(L) to Bcl-2. EGF and Bcl-x(L) activate the mitogen-activated protein kinase/ERK pathway resulting in upregulation of vascular endothelial growth factor (VEGF), a known inducer of Bcl-2 in endothelial cells. Inhibition of VEGF receptor signaling in HDMEC-Bcl-x(L) prevented Bcl-2 upregulation and demonstrated the function of a VEGF-mediated autocrine loop. Bcl-2 downregulation by RNAi blocked CXCL1 and CXCL8 expression downstream of Bcl-x(L), and markedly decreased angiogenesis in vivo. We conclude that Bcl-x(L) functions as a pro-angiogenic signaling molecule controlling Bcl-2 and VEGF expression. These results emphasize a complex interplay between Bcl-2 family members beyond their classical roles in apoptosis.
Assuntos
Células Endoteliais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína bcl-X/metabolismo , Animais , Apoptose/fisiologia , Quimiocina CXCL1/metabolismo , Células Endoteliais/citologia , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos SCID , Transdução GenéticaRESUMO
Melanoma cells depend on sustained proteasomal function for survival. However, bortezomib, the first proteasome inhibitor in clinical use, is not sufficient to improve the poor prognosis of metastatic melanoma patients. Since the proteasome is also expressed in all normal cell compartments, it is unclear how to enhance the efficacy of bortezomib without exacerbating secondary toxicities. Here, we present pharmacological and genetic analyses of mechanisms of resistance to proteasome inhibition. We focused on Bcl-2, Bcl-x(L) and Mcl-1 as main antiapoptotic factors associated with melanoma progression. Despite an efficient blockage of the proteasome, bortezomib could not counteract the intrinsically high levels of Bcl-2 and Bcl-x(L) in melanoma cells. Moreover, Mcl-1 was only downregulated at late time points after treatment. Based on these results, a combination treatment including (-)-gossypol, an inhibitor of Mcl-1/Bcl-2/Bcl-x(L), was designed and proven effective in vivo. Using a specific RNA interference approach, the survival of bortezomib-treated melanoma cells was found to rely primarily on Mcl-1, and to a lesser extent on Bcl-x(L) (but not on Bcl-2). Importantly, neither Mcl-1 nor Bcl-x(L) inactivation affected the viability of normal melanocytes. This hierarchical requirement of Bcl-2 family members for the maintenance of normal and malignant cells offers a therapeutic window to overcome melanoma chemoresistance in a tumor cell-selective manner.
Assuntos
Ácidos Borônicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/farmacologia , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/fisiologia , Ácidos Borônicos/metabolismo , Bortezomib , Caspases/metabolismo , Linhagem Celular Tumoral , Gossipol/metabolismo , Gossipol/farmacologia , Humanos , Melanoma Experimental/imunologia , Camundongos , Transplante de Neoplasias , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/metabolismo , Transplante HeterólogoRESUMO
Malignant melanoma is the most aggressive form of skin cancer and has proven to be highly resistant to conventional chemotherapy. Intriguingly, the p53 tumor suppressor, a main mediator of chemoresistance in other tumor types, is rarely mutated in melanoma. However, we have previously shown that anti-apoptotic isoforms of p73 (deltaTA-p73), another member of the p53 family, are overexpressed in metastatic melanomas. DeltaTA-p73 can oppose the pro-apoptotic functions of p53 and full length p73, and thus it could contribute to melanoma chemoresistance. In this study, we use an efficient adenoviral-based gene transfer approach to introduce a transcriptionally active form of p73 (TA-p73beta) in melanoma cells, with the objective of overcoming drug resistance. Interestingly, TA-p73beta significantly sensitized 5 out of 7 aggressive melanoma cell lines to the standard therapeutic agents adriamycin and cisplatin. More importantly, TA-p73beta displayed a synergistic effect in vivo allowing adriamycin or cisplatin to block melanoma cell growth in mouse xenograft models (p < 0.05). In summary, our data show that Ad-mediated TA-p73beta gene expression can markedly sensitize a subset of melanoma cell lines to adriamycin and cisplatin in vitro and in vivo, suggesting a new chemosensitization strategy for malignant melanomas.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Ligação a DNA/genética , Técnicas de Transferência de Genes , Terapia Genética , Melanoma/tratamento farmacológico , Proteínas Nucleares/genética , Adenoviridae/genética , Animais , Antibióticos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/uso terapêutico , Sinergismo Farmacológico , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Nus , Proteínas Nucleares/metabolismo , Proteína Tumoral p73 , Proteínas Supressoras de Tumor , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , Proteínas/genética , Apoptose , Fator Apoptótico 1 Ativador de Proteases , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/metabolismo , Proteínas/metabolismo , Tamanho da Amostra , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Mistletoe extracts are used as alternative cancer treatment in addition to standard chemotherapy and radiation treatment and have an immunostimulatory and pain-relieving effect. A direct antitumour effect of mistletoe extracts against tumour cells of lymphoid origin has been linked to the D-galactoside-specific mistletoe lectin I. In this study, we investigated the cellular effect of bacterially expressed, recombinant mistletoe lectin alone or in combination with ionising radiation in a genetically defined p53-wild-type and p53-deficient E1A/ras-transformed murine tumour cells system. Downregulation of the proliferative activity and cell killing by recombinant mistletoe lectin occurred in a clear dose response (0.1-1 ng ml(-1)). Induction of apoptosis was p53-independent, but apoptosis-associated factor-1-dependent. Cellular treatment with lectin in combination with ionising radiation resulted in both p53-wild-type and p53-deficient tumour cells in an at least additive, antiproliferative effect and enhanced activation of caspase-3. Combined treatment with ionising radiation and lectin revealed a similar cytotoxic effect in human, p53-mutated adenocarcinoma cells. Thus, recombinant mistletoe lectin alone and in combination with ionising radiation bypasses often prevalent apoptotic deficiencies in treatment-resistant tumour cells.
Assuntos
Apoptose/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Preparações de Plantas/farmacologia , Proteínas de Plantas , Toxinas Biológicas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Anexina A5/metabolismo , Fator Apoptótico 1 Ativador de Proteases , Western Blotting , Caspase 3 , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fibroblastos/metabolismo , Genes ras , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Camundongos , Erva-de-Passarinho/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Mutação/genética , Proteínas/genética , Proteínas/metabolismo , Radiação Ionizante , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2 , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.
Assuntos
Apoptose , Melanoma/metabolismo , Proteínas/metabolismo , Antineoplásicos/farmacologia , Fator Apoptótico 1 Ativador de Proteases , Caspase 9 , Caspases/metabolismo , Cromossomos Humanos Par 12 , Clonagem Molecular , Metilação de DNA , DNA de Neoplasias/metabolismo , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Genes p53 , Humanos , Perda de Heterozigosidade , Melanoma/patologia , Melanoma/secundário , Mutação , Proteínas/genética , Células Tumorais CultivadasAssuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Dano ao DNA , Fatores de Transcrição E2F , Genes Supressores de Tumor , Genes p53 , Humanos , Camundongos , Camundongos Knockout , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
Caspases are a family of cysteine proteases that constitute the apoptotic cell death machinery. We report the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by the protein kinase C (PKC) inhibitors staurosporine (STP) and PKC-412. In our genetically defined tumor cells, treatment with low doses of STP or the conventional PKC-specific inhibitor PKC-412 in combination with irradiation (5 Gy) potently reduced viability, enhanced mitochondrial cytochrome c release into the cytosol, and specifically stimulated the initiator caspase-9. Whereas treatment with each agent alone had a minimal effect, combined treatment resulted in enhanced caspase-3 activation. This was prevented by broad-range and specific caspase-9 inhibitors and absent in caspase-9-deficient cells. The tumor suppressor p53 was required for apoptosis induction by combined treatment but was dispensable for dose-dependent STP-induced caspase activation. These results demonstrate the requirement for an intact caspase-9 pathway for apoptosis-based radiosensitization by PKC inhibitors and show that STP induces apoptosis independent of p53.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Caspases/efeitos dos fármacos , Caspases/efeitos da radiação , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/efeitos da radiação , Proteína Quinase C/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Estaurosporina/análogos & derivados , Animais , Apoptose/fisiologia , Caspase 9 , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/radioterapia , Proteína Quinase C/metabolismo , Tolerância a Radiação/fisiologia , Estaurosporina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/efeitos da radiação , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 were found to be essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, were resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substituted for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development.
Assuntos
Apoptose , Caspases/fisiologia , Genes p53 , Neoplasias Experimentais/patologia , Proteínas/fisiologia , Animais , Fator Apoptótico 1 Ativador de Proteases , Caspase 9 , Caspases/genética , Divisão Celular , Transformação Celular Neoplásica , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Genes myc , Genes ras , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Proteínas/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. Caspase processing is inhibited in Casp9-/- ES cells but not in thymocytes or splenocytes. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of at least four different apoptotic pathways in mammalian cells.
Assuntos
Apoptose/genética , Caspases , Cisteína Endopeptidases/fisiologia , Transdução de Sinais/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Caspase 9 , Linhagem Celular , Córtex Cerebral/anormalidades , Cisteína Endopeptidases/genética , Grupo dos Citocromos c/metabolismo , Dexametasona/farmacologia , Embrião de Mamíferos , Ativação Enzimática/genética , Fibroblastos/citologia , Fibroblastos/enzimologia , Raios gama , Regulação da Expressão Gênica no Desenvolvimento , Ativação Linfocitária , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Mitocôndrias/enzimologia , Especificidade de Órgãos/genética , Prosencéfalo/anormalidades , Baço/citologia , Baço/imunologia , Baço/efeitos da radiação , Células-Tronco , Timo/citologia , Timo/enzimologiaRESUMO
Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.
Assuntos
Apoptose/fisiologia , Caspases , Núcleo Celular/metabolismo , Cisteína Endopeptidases/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linfócitos B/citologia , Linfócitos B/fisiologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Complexo CD3/farmacologia , Caspase 3 , Morte Celular/fisiologia , Divisão Celular/fisiologia , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal , Feminino , Expressão Gênica/genética , Expressão Gênica/fisiologia , Longevidade/genética , Longevidade/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Mutação/genética , Mutação/fisiologia , Neutrófilos/fisiologia , Pressão Osmótica , Células-Tronco/efeitos da radiação , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/fisiologia , Células Tumorais Cultivadas/efeitos da radiação , Raios Ultravioleta , Receptor fas/farmacologiaRESUMO
The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage phi29 is absolutely required for viral DNA replication in vivo. About approximately 95% of the intrinsic tyrosine fluorescence of phi29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the phi29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. phi29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the phi29 SSB fluorescence emission (phiF = 0.067 +/- 0.010), (ii) the fast motions of the tyrosine side chains (phi(short) = 0.14 +/- 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I- indicates that phi29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and omega (cooperativity) defining phi29 SSB-poly(dT) interaction, the effective DNA binding constant, K(eff) = K omega, being largely independent of temperature. Altogether, the fluorescent properties of phi29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA.
Assuntos
Fagos Bacilares/química , Proteínas de Ligação a DNA/química , Tirosina/química , Proteínas Virais/química , Replicação do DNA , Concentração de Íons de Hidrogênio , Solubilidade , Solventes , Espectrometria de Fluorescência , Temperatura , Termodinâmica , Replicação ViralRESUMO
The strand-displacement mechanism of Bacillus subtilis phage phi29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, phi29 SSB, to be replicated in vivo. To understand the characteristics of phi29 SSB-ssDNA complex that could explain the requirement of phi29 SSB, we have (i) determined the hydrodynamic behavior of phi29 SSB in solution and (ii) monitored the effect of complex formation on phi29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 +/- 0.1) x 10(8) gs(-1), and its rotational correlation time, 7.0 +/- 0.5 ns, phi29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that phi29 SSB is highly organized within a wide range of temperatures (15 to 50 degrees C), being mainly constituted by beta-sheet elements (approximately 50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of phi29 SSB.
Assuntos
Fagos Bacilares/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Conformação Proteica , Temperatura , Proteínas Virais/químicaRESUMO
The single-stranded DNA (ssDNA)-binding protein (SSB) of bacteriophage phi 29 is one of the virus-encoded proteins required for viral DNA replication. We have found that phi 29 SSB has helix-destabilizing activity since it removes secondary structure of the ssDNA in phi 29 replicative intermediates, as revealed by electron microscopy, and displaces oligonucleotides annealed to M13 ssDNA. To investigate the mechanism of the SSB-dependent stimulation of phi 29 DNA replication we have characterized the helix-destabilizing activity of phi 29 SSB and measured its effect on the DNA elongation rate by phi 29 DNA polymerase, which does not require an accessory helicase. The use of replication reactions where strand displacement is either required (phi 29 DNA replication) or not (conversion of primed M13 ssDNA into double-stranded DNA (dsDNA)) has allowed us to find that (1) strand displacement DNA replication was affected by lowering the temperature or by increasing the salt concentration, since the DNA elongation rate on the phi 29 template was three to fourfold slower than on primed M13 ssDNA, (2) under those conditions, addition of phi 29 SSB stimulated to different extents the DNA elongation rate during phi 29 DNA replication, whereas it had a marginal effect on primed M13 ssDNA replication, and (3) phi 29 SSB increased four to sixfold the phi 29 DNA elongation rate by phi 29 DNA polymerase strand displacement mutants, reaching approximately 50% the rate of the wild-type enzyme. The implications of the helix-destabilizing properties of the phi 29 SSB under conditions in which DNA opening is impaired are discussed.
Assuntos
Fagos Bacilares/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Replicação Viral , Fagos Bacilares/enzimologia , Sequência de Bases , DNA Helicases/genética , DNA de Cadeia Simples/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , TemperaturaRESUMO
phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.