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1.
N Engl J Med ; 384(5): 440-451, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33471974

RESUMO

BACKGROUND: Population-based estimates of the risk of breast cancer associated with germline pathogenic variants in cancer-predisposition genes are critically needed for risk assessment and management in women with inherited pathogenic variants. METHODS: In a population-based case-control study, we performed sequencing using a custom multigene amplicon-based panel to identify germline pathogenic variants in 28 cancer-predisposition genes among 32,247 women with breast cancer (case patients) and 32,544 unaffected women (controls) from population-based studies in the Cancer Risk Estimates Related to Susceptibility (CARRIERS) consortium. Associations between pathogenic variants in each gene and the risk of breast cancer were assessed. RESULTS: Pathogenic variants in 12 established breast cancer-predisposition genes were detected in 5.03% of case patients and in 1.63% of controls. Pathogenic variants in BRCA1 and BRCA2 were associated with a high risk of breast cancer, with odds ratios of 7.62 (95% confidence interval [CI], 5.33 to 11.27) and 5.23 (95% CI, 4.09 to 6.77), respectively. Pathogenic variants in PALB2 were associated with a moderate risk (odds ratio, 3.83; 95% CI, 2.68 to 5.63). Pathogenic variants in BARD1, RAD51C, and RAD51D were associated with increased risks of estrogen receptor-negative breast cancer and triple-negative breast cancer, whereas pathogenic variants in ATM, CDH1, and CHEK2 were associated with an increased risk of estrogen receptor-positive breast cancer. Pathogenic variants in 16 candidate breast cancer-predisposition genes, including the c.657_661del5 founder pathogenic variant in NBN, were not associated with an increased risk of breast cancer. CONCLUSIONS: This study provides estimates of the prevalence and risk of breast cancer associated with pathogenic variants in known breast cancer-predisposition genes in the U.S. population. These estimates can inform cancer testing and screening and improve clinical management strategies for women in the general population with inherited pathogenic variants in these genes. (Funded by the National Institutes of Health and the Breast Cancer Research Foundation.).


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Variação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Razão de Chances , Risco , Análise de Sequência de DNA , Adulto Jovem
2.
JCO Precis Oncol ; 4: 32-43, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32832836

RESUMO

PURPOSE: In studies of men of European ancestry, rare pathogenic variants in DNA repair pathway genes have been shown to be associated with risk of aggressive prostate cancer. The contribution of rare coding variation to prostate cancer risk in men of African ancestry has not been established. METHODS: We sequenced a panel of 19 DNA repair and cancer predisposition genes in 2,453 African American and 1,151 Ugandan prostate cancer cases and controls. Rare variants were classified as pathogenic or putatively functionally disruptive and examined in association with prostate cancer risk and disease aggressiveness in gene and pathway-level association analyses. RESULTS: Pathogenic variants were found in 75 out of 2,098 cases (3.6%) and 31 out of 1,481 controls (2.1%) (OR=1.82, 95% CI=1.19 to 2.79, P=0.0044) with the association being stronger for more aggressive disease phenotypes (OR=3.10, 95% CI=1.54 to 6.23, P=0.0022). The highest risks for aggressive disease were observed with pathogenic variants in the ATM, BRCA2, PALB2 and NBN genes, with odds ratios ranging from ~4 to 15 in the combined study sample of African American and Ugandan men. Rare, non-pathogenic, non-synonymous variants did not have a major impact on risk of overall prostate cancer or disease aggressiveness. CONCLUSIONS: Rare pathogenic variants in DNA repair genes have appreciable effects on risk of aggressive prostate cancer in men of African ancestry. These findings have potential implications for panel testing and risk stratification in this high-risk population.

3.
JAMA ; 319(23): 2401-2409, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29922827

RESUMO

Importance: Individuals genetically predisposed to pancreatic cancer may benefit from early detection. Genes that predispose to pancreatic cancer and the risks of pancreatic cancer associated with mutations in these genes are not well defined. Objective: To determine whether inherited germline mutations in cancer predisposition genes are associated with increased risks of pancreatic cancer. Design, Setting, and Participants: Case-control analysis to identify pancreatic cancer predisposition genes; longitudinal analysis of patients with pancreatic cancer for prognosis. The study included 3030 adults diagnosed as having pancreatic cancer and enrolled in a Mayo Clinic registry between October 12, 2000, and March 31, 2016, with last follow-up on June 22, 2017. Reference controls were 123 136 individuals with exome sequence data in the public Genome Aggregation Database and 53 105 in the Exome Aggregation Consortium database. Exposures: Individuals were classified based on carrying a deleterious mutation in cancer predisposition genes and having a personal or family history of cancer. Main Outcomes and Measures: Germline mutations in coding regions of 21 cancer predisposition genes were identified by sequencing of products from a custom multiplex polymerase chain reaction-based panel; associations of genes with pancreatic cancer were assessed by comparing frequency of mutations in genes of pancreatic cancer patients with those of reference controls. Results: Comparing 3030 case patients with pancreatic cancer (43.2% female; 95.6% non-Hispanic white; mean age at diagnosis, 65.3 [SD, 10.7] years) with reference controls, significant associations were observed between pancreatic cancer and mutations in CDKN2A (0.3% of cases and 0.02% of controls; odds ratio [OR], 12.33; 95% CI, 5.43-25.61); TP53 (0.2% of cases and 0.02% of controls; OR, 6.70; 95% CI, 2.52-14.95); MLH1 (0.13% of cases and 0.02% of controls; OR, 6.66; 95% CI, 1.94-17.53); BRCA2 (1.9% of cases and 0.3% of controls; OR, 6.20; 95% CI, 4.62-8.17); ATM (2.3% of cases and 0.37% of controls; OR, 5.71; 95% CI, 4.38-7.33); and BRCA1 (0.6% of cases and 0.2% of controls; OR, 2.58; 95% CI, 1.54-4.05). Conclusions and Relevance: In this case-control study, mutations in 6 genes associated with pancreatic cancer were found in 5.5% of all pancreatic cancer patients, including 7.9% of patients with a family history of pancreatic cancer and 5.2% of patients without a family history of pancreatic cancer. Further research is needed for replication in other populations.


Assuntos
Carcinoma Ductal Pancreático/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas/genética , Idoso , Estudos de Casos e Controles , DNA de Neoplasias/análise , Bases de Dados Genéticas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Sistema de Registros , Risco , Análise de Sequência de DNA , Análise de Sobrevida
4.
Cancer Res ; 77(8): 1892-1904, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28108509

RESUMO

To modulate T-cell function for cancer therapy, one challenge is to selectively attenuate regulatory but not conventional CD4+ T-cell subsets [regulatory T cell (Treg) and conventional T cell (Tconv)]. In this study, we show how a functional dichotomy in Class IA PI3K isoforms in these two subsets of CD4+ T cells can be exploited to target Treg while leaving Tconv intact. Studies employing isoform-specific PI3K inhibitors and a PI3Kδ-deficient mouse strain revealed that PI3Kα and PI3Kß were functionally redundant with PI3Kδ in Tconv. Conversely, PI3Kδ was functionally critical in Treg, acting there to control T-cell receptor signaling, cell proliferation, and survival. Notably, in a murine model of lung cancer, coadministration of a PI3Kδ-specific inhibitor with a tumor-specific vaccine decreased numbers of suppressive Treg and increased numbers of vaccine-induced CD8 T cells within the tumor microenvironment, eliciting potent antitumor efficacy. Overall, our results offer a mechanistic rationale to employ PI3Kδ inhibitors to selectively target Treg and improve cancer immunotherapy. Cancer Res; 77(8); 1892-904. ©2017 AACR.


Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Neoplasias Experimentais/terapia , Fosfatidilinositol 3-Quinases/imunologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Feminino , Imunoterapia/métodos , Isoenzimas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/imunologia , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia
5.
Int J Legal Med ; 130(4): 905-914, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26932869

RESUMO

Within forensic genetics, there is still a need for supplementary DNA marker typing in order to increase the power to solve cases for both identity testing and complex kinship issues. One major disadvantage with current capillary electrophoresis (CE) methods is the limitation in DNA marker multiplex capability. By utilizing massive parallel sequencing (MPS) technology, this capability can, however, be increased. We have designed a customized GeneRead DNASeq SNP panel (Qiagen) of 140 previously published autosomal forensically relevant identity SNPs for analysis using MPS. One single amplification step was followed by library preparation using the GeneRead Library Prep workflow (Qiagen). The sequencing was performed on a MiSeq System (Illumina), and the bioinformatic analyses were done using the software Biomedical Genomics Workbench (CLC Bio, Qiagen). Forty-nine individuals from a Swedish population were genotyped in order to establish genotype frequencies and to evaluate the performance of the assay. The analyses showed to have a balanced coverage among the included loci, and the heterozygous balance showed to have less than 0.5 % outliers. Analyses of dilution series of the 2800M Control DNA gave reproducible results down to 0.2 ng DNA input. In addition, typing of FTA samples and bone samples was performed with promising results. Further studies and optimizations are, however, required for a more detailed evaluation of the performance of degraded and PCR-inhibited forensic samples. In summary, the assay offers a straightforward sample-to-genotype workflow and could be useful to gain information in forensic casework, for both identity testing and in order to solve complex kinship issues.


Assuntos
Impressões Digitais de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Linhagem , Polimorfismo de Nucleotídeo Único , Frequência do Gene , Genótipo , Heterozigoto , Humanos , Reação em Cadeia da Polimerase , Suécia
6.
Cancer Immunol Res ; 2(11): 1080-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25080445

RESUMO

Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses are still needed. The temporary nature of the therapy-induced immune response can be attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, regulatory T cells (Treg). Although the depletion of Tregs has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. We found that PI3K-Akt pathway inhibitors selectively inhibit Tregs with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Tregs in comparison with Tconvs when treated with different Akt and PI3K inhibitors. This effect has been observed in both human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Tregs in both naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors led to a significant therapeutic antitumor effect, which was shown to be Treg dependent. Here, we report the use of PI3K-Akt pathway inhibitors as potent agents for the selective depletion of suppressive Tregs. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg depletion.


Assuntos
Ativação Linfocitária/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
7.
Eur J Immunol ; 41(10): 2977-86, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21710477

RESUMO

Programmed death-1 receptor (PD-1) is expressed on T cells following TCR activation. Binding of this receptor to its cognate ligands, programmed death ligand (PDL)-1 and PDL-2, down-regulates signals by the TCR, promoting T-cell anergy and apoptosis, thus leading to immune suppression. Here, we find that using an anti-PD-1 antibody (CT-011) with Treg-cell depletion by low-dose cyclophosphamide (CPM), combined with a tumor vaccine, induces synergistic antigen-specific immune responses and reveals novel activities of each agent in this combination. This strategy led to complete regression of established tumors in a significant percentage of treated animals, with survival prolongation. We show for the first time that combining CT-011 and CPM significantly increases the number of vaccine-induced tumor-infiltrating CD8(+) T cells, with simultaneous decrease in infiltrating Treg cells. Interestingly, we find that CT-011 prolongs Treg-cell inhibition induced by CPM, leading to a sustainable significant synergistic decrease of splenic and tumor-infiltrated Treg cells. Surprisingly, we find that the anti-tumor effect elicited by the combination of CT-011 and CPM is dependent on both CD8(+) and CD4(+) T-cell responses, although the antigen we used is a class I MHC-restricted peptide. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Antígeno B7-H1/imunologia , Ciclofosfamida/farmacologia , Neoplasias Experimentais/terapia , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Apoptose , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Ciclofosfamida/administração & dosagem , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Proteínas E7 de Papillomavirus/administração & dosagem , Proteínas E7 de Papillomavirus/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo
8.
Curr Mol Med ; 9(6): 766-73, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19689303

RESUMO

HPV has been linked to many human malignancies and, as such, represents a major public health crisis. The understanding of HPV biology, however, has helped tremendously in developing prophylactic vaccines, which should help in decreasing mortality due to HPV infections. Understanding HPV biology has allowed researchers to use the virus as a model for the development of not only prophylactic vaccines, but also therapeutic ones. The advantages of HPV as a model stem from the limited number of proteins encoded by the HPV genome that can be targeted by vaccines, and also from the restricted expression of certain viral proteins during different stages of infection. In this review, we discuss how HPV can be used as a model for the development of both prophylactic and therapeutic vaccines.


Assuntos
Vacinas Anticâncer/uso terapêutico , Modelos Imunológicos , Papillomaviridae/imunologia , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/terapia , Humanos , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Vacinas contra Papillomavirus/uso terapêutico , Proteínas Virais/imunologia
9.
Cancer Res ; 68(3): 909-17, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18245494

RESUMO

As human colorectal cancer (CRC) cells metastasize to distant sites, they are susceptible to detachment-induced cell death or anoikis - a form of apoptosis that occurs when anchorage-dependent CRC cells go into suspension. Our goal was to identify whether tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL) receptors mediate anoikis in human CRC cells. First, we assessed whether caspases of the extrinsic (caspase-8) or intrinsic (caspase-9) death pathways were involved. Caspase-8 was cleaved during exposure to suspension culture in four CRC lines, and cell death was inhibited by caspase-3 and caspase-8 inhibitors but not by a caspase-9 inhibitor. Gene transcripts in macrophage inflammatory protein-101 (MIP-110), a weakly metastatic human CRC, were increased at least 2-fold for TRAIL-R2 (DR5) and TRAIL after 24 h of suspension culture compared with cells in monolayer culture. The increased expression of DR5 was confirmed at the protein level at 24 h, and exposure of MIP-101 cells to an antagonistic antibody to DR5 decreased caspase-8 activation. The antagonistic antibody to DR5 inhibited anoikis in four human CRC lines. Treatment with an antagonistic DR4 antibody or a neutralizing antibody to TRAIL ligand did not reduce anoikis consistently. Knockdown of DR5 or TRAIL also inhibited anoikis, whereas exogenous TRAIL or FasL did not consistently increase anoikis. In summary, DR5 receptor mediates death signals for anoikis in human CRC cells through the extrinsic apoptotic pathway.


Assuntos
Anoikis/fisiologia , Neoplasias Colorretais/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ativação Enzimática , Proteína Ligante Fas/farmacologia , Proteína Ligante Fas/fisiologia , Humanos , RNA Interferente Pequeno/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Transfecção
10.
Cancer Res ; 67(10): 4774-82, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17510406

RESUMO

Carcinoembryonic antigen (CEA) is a tumor marker that is associated with metastasis, poor response to chemotherapy of colorectal cancer (CRC), and anoikis, a form of apoptosis caused by cell detachment from matrix that is dependent on TRAIL-R2 (DR5) and caspase-8 activation in CRC. Although CEA is a homophilic binding protein that may provide survival signals through homotypical cell aggregation, we now report that CEA binds TRAIL-R2 (DR5) directly in two-hybrid assays to decrease anoikis through the extrinsic pathway. Deletion of the PELPK sequence (delPELPK) of CEA (delPELPK CEA) restores sensitivity to anoikis while it maintains its cell aggregation function. Wild-type (WT) CEA also increases experimental hepatic metastasis, whereas the delPELPK CEA does not. Thus, membrane CEA interacts with DR5 to inhibit anoikis and increase metastatic potential in CRC.


Assuntos
Anoikis/fisiologia , Antígeno Carcinoembrionário/metabolismo , Neoplasias Colorretais/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/genética , Caspase 8/metabolismo , Inibidores de Caspase , Adesão Celular/fisiologia , Neoplasias Colorretais/metabolismo , Células HT29 , Humanos , Integrinas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Transdução de Sinais/fisiologia
11.
Int J Cancer ; 111(3): 332-7, 2004 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15221959

RESUMO

Tumor cells cause ischemia/reperfusion (I/R) injury as they arrest within the hepatic microvasculature with the production of nitric oxide (NO) and reactive oxygen species (ROS) that kill both host liver and implanting tumor cells. Carcinoembryonic antigen (CEA) both facilitates the survival of experimental metastasis to nude mouse liver by weakly metastatic human colorectal carcinomas (CRCs) and induces the release of the proinflammatory cytokine IL-6. We hypothesized that CEA also stimulates the release of the antiinflammatory cytokine IL-10 causing inhibition of the toxicity of hepatic I/R injury and indirect stimulation of tumor cell colonization of the liver. Intravenous injection of CEA produced more than 1 ng/ml of IL-10 in the systemic circulation within 1 hr which subsided by 8 hr. The IL-10 response is specific to CEA since the pentapeptide sequence in CEA that binds to the CEA receptor stimulated isolated Kupffer cells to produce IL-10. IL-10, but not IL-6, increased the survival of weakly metastatic CRC cocultured with ischemic-reoxygenated liver fragments but did not affect the survival of CRC exposed to oxidative stress in the absence of any host cells. CEA, IL-6 and IL-10 pretreatment reduced expression of iNOS but only CEA and IL-10 strongly inhibited NO and total reactive species production by ischemic-rexoygenated liver. IL-6 was toxic to CRC exposed to oxidative stress while IL-10 did not have a direct effect on CRC. Thus, CEA stimulates production of IL-10 that may enhance metastasis by promoting the ability of circulating CRC cells to survive the I/R injury of implantation.


Assuntos
Antígeno Carcinoembrionário/farmacologia , Neoplasias Colorretais/patologia , Interleucina-10/farmacologia , Interleucina-6/farmacologia , Circulação Hepática/efeitos dos fármacos , Neoplasias Hepáticas/secundário , Traumatismo por Reperfusão/prevenção & controle , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Interleucina-10/fisiologia , Interleucina-6/fisiologia , Cinética , Camundongos , Camundongos Nus , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transplante Heterólogo
12.
Cancer Res ; 64(8): 2643-8, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15087371

RESUMO

Malignant cells undergo anoikis as they encounter fluid shear stress during transit to a metastatic site. We postulated that intracellular nitric oxide (NO) contributes to this cell death by comparing the growth of human colorectal carcinoma cells in low fluid shear stress rotated three-dimensional (Rotated 3-D) cultures with growth in static three-dimensional (Static 3-D) cultures on nonadherent surfaces and with two-dimensional monolayer (Monolayer 2-D) cultures. NO, loss of microtubules, and apoptosis increased significantly in Rotated 3-D cultures within 10 min and persisted at 24 h, whereas inhibition of NO synthase decreased apoptosis and intracellular NO and prevented tubulin degradation. Thus, fluid shear stress and three-dimensional growth increases NO synthase and NO to cause tubulin breakdown and induce anoikis. Intracellular NO in malignant cells entering the circulation may be a novel target for metastasis by colorectal carcinoma.


Assuntos
Apoptose/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Microtúbulos/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Antioxidantes/farmacologia , Reatores Biológicos , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Espécies Reativas de Oxigênio/metabolismo , ômega-N-Metilarginina/farmacologia
13.
Oncogene ; 22(52): 8460-71, 2003 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-14627987

RESUMO

The transcription factor E2F-1 is implicated in the activation of S-phase genes as well as induction of apoptosis, and is regulated by interactions with Rb and by cell cycle-dependent alterations in E2F-1 abundance. We earlier demonstrated a pivotal role for poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of E2F-1 expression and promoter activity during S-phase re-entry when quiescent cells re-enter the cell cycle. We now investigate the putative mechanism(s) by which PARP-1 may upregulate E2F-1 promoter activity during S-phase re-entry. DNase-1 footprint assays with purified PARP-1 showed that PARP-1 did not directly bind the E2F-1 promoter in a sequence-specific manner. In contrast to p53, a positive acceptor in poly(ADP-ribosyl)ation reactions, E2F-1 was not poly(ADP-ribosyl)ated by wild-type PARP-1 in vitro, indicating that PARP-1 does not exert a dual effect on E2F-1 transcriptional activation. Protein-binding reactions and coimmunoprecipitation experiments with purified PARP-1 and E2F-1, however, revealed that PARP-1 binds to E2F-1 in vitro. More significantly, physical association of PARP-1 and E2F-1 in vivo also occurred in wild-type fibroblasts 5 h after re-entry into S phase, coincident with the increase in E2F-1 promoter activity and expression of E2F-1-responsive S-phase genes cyclin A and c-Myc. Mapping of the interaction domains revealed that full-length PARP-1 as well as PARP-1 mutants lacking either the catalytic active site or the DNA-binding domain equally bind E2F-1, whereas a PARP-1 mutant lacking the automodification domain does not, suggesting that the protein interaction site is located in this central domain. Finally, gel shift analysis with end-blocked E2F-1 promoter sequence probes verified that the binding of PARP-1 to E2F-1 enhances binding to the E2F-1 promoter, indicating that PARP-1 acts as a positive cofactor of E2F-1-mediated transcription.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Genética , Animais , Sítios de Ligação , Ciclina A/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Genes myc/fisiologia , Humanos , Testes de Precipitina
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