RESUMO
BACKGROUND & AIMS: Genomic instability promotes colon carcinogenesis by inducing genetic mutations, but not all genes affected by this process have been identified. We investigated whether genomic instability in human colorectal cancer (CRC) cells produces mutations in the hepatocyte growth factor (HGF) gene. METHODS: We genotyped human colon tumor tissues and adjacent nontumor tissues collected from 78 patients University of Pittsburgh Health Sciences and Veterans Hospital, along with 40 human CRC and adjacent nontumor tissues in a commercial microarray. We used cellular, biochemical, and molecular biological techniques to investigate the factors that alter HGF signaling in colon cancer cells and its effects on cell proliferation and survival. RESULTS: All tested human CRC tissues and cell lines that had microsatellite instability contained truncations in the regulatory deoxyadenosine tract element (DATE) of the HGF gene promoter. The DATE was unstable in 14% (11 of 78) of CRC samples; DATE truncation was also polymorphic and detected in 18% (13 of 78) of CRC tissues without microsatellite instability. In CRC cell lines, truncation of DATE activated expression of HGF, resulting in its autocrine signaling via MET. This promoted cell proliferation and resistance to necroptosis. HGF signaling via MET reduced levels of the receptor-interacting serine-threonine kinase 1, a mediator of necroptosis, in CRC cells. High levels of HGF protein in tumor tissues correlated with lower levels of receptor-interacting serine-threonine kinase 1 and shorter survival times of patients. CONCLUSIONS: Thirty-one percent of CRC samples contain alterations in the DATE of the HGF promoter. Disruption of the DATE increased HGF signaling via MET and reduced levels of receptor-interacting serine-threonine kinase 1 and CRC cell necroptosis. DATE alteration might be used as a prognostic factor or to select patients for therapies that target HGF-MET signaling.
Assuntos
Adenocarcinoma/genética , Apoptose , Neoplasias do Colo/genética , Instabilidade Genômica , Fator de Crescimento de Hepatócito/genética , Ativação Transcricional , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Comunicação Autócrina , Proliferação de Células , Sobrevivência Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Reparo de Erro de Pareamento de DNA , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Células HCT116 , Células HT29 , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Necrose , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Prognóstico , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
Our previous study showed a characteristic p53 mutational spectrum in lung tumors from lung cancer patients in the Western Pennsylvania region. To further understand the involvement of p53 mutations in lung tumor development, in this study we compared p53 mutational spectra and distribution between tumor cells taken from lung tumor tissue and histologically normal cells taken from tumor-surrounding tissue obtained from 122 lung cancer patients [67 adenocarcinomas (ACs) and 55 squamous cell carcinomas (SCCs)]. Overall, mutations were detected in exons 5-8 of the p53 gene in cell samples from 39.3% (48/122) of the patients. Twenty-four mutations were found among the ACs (35.8%, 24/67) and consisted mostly of G to T transversions at codon 248 in either only the tumor tissue (12 cases, 50%), or only the histologically normal tissue (2 cases, 8.3%), or both tissue types (10 cases, 41.7%). Among the SCCs, 24 mutations of both transition and transversion types were detected at multiple codons in either only the tumor tissue (17 cases, 70.8%), or only the histologically normal tissue (3 cases, 12.5%), or both tissues (4 cases, 16.7%). Overall, the distribution of mutations among the tumor tissue and histologically normal tissue was not significantly different between the ACs and SCCs (P > 0.05). In both groups, the mutations in the histologically normal tissue may be identical to or different from those in the tumor tissue. Therefore, p53 mutations are frequent in tumor-surrounding histologically normal tissue, and some of them might be involved in lung carcinogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genes p53 , Neoplasias Pulmonares/genética , Pulmão/metabolismo , Mutação , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We sought to determine the clinical and immunohistopathological prognostic factors for overall survival (OS) in adult patients with post-transplant lymphoproliferative disorders (PTLDs). Eighty-four patients diagnosed with PTLDs between 1980 and 2004 at the University of Pittsburgh Medical Center were identified. Immunohistochemical staining was performed on tumor tissue at the time of diagnosis for the following proteins: Bcl-2, Bcl-6, c-myc and p53. The median survival for all patients was 20.8 months, 95% CI: (7.4-77.6). On univariate analysis for OS, the following poor prognostic factors were identified: age at transplant >60 years (p = 0.024), multiorgan transplant (p = 0.019), ECOG > 2 (p < 0.0001), grafted organ involvement (p < 0.0001), extranodal disease (p = 0.011), early (<1 year) PTLDs (p < 0.0001), stage IV (p = 0.0017), EBV positive (p = 0.012) and elevated white blood count (p = 0.010). Good prognostic factors included ECOG<2 (p < 0.0001), late (>1 year) PTLDs (p = 0.002), early stage at diagnosis (stages I and II, p = 0.005), nodal disease (p = 0.0053), monomorphic disease (0.0034), initial immunosuppression reduction (p = 0.0015) and use of rituximab (p = 0.045). Bcl-2 but not Bcl-6, c-myc, or p53 correlated with poor survival, p = 0.0036. This study identifies new clinical and pathological markers for poor survival in PTLDs.
Assuntos
Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/mortalidade , Transplante de Órgãos/efeitos adversos , Biomarcadores/análise , Transtornos Linfoproliferativos/etiologia , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-6/análise , Proteínas Proto-Oncogênicas c-myc/análise , Fatores de Risco , Análise de Sobrevida , Proteína Supressora de Tumor p53/análiseRESUMO
Little is known about the biology of post-transplant lymphoproliferative disorders (PTLDs). The objective of this study was to determine the molecular alterations that occur at the protein level in patients with PTLDs. Six tumor samples from adult patients with PTLD and four benign lymph nodes were studied using protein microarray technique. Proteins that were dysregulated included proteins in the PI3K/mTOR, NFkB and HSP90 pathways. Inhibitors of these proteins induced cytotoxicity and apoptosis in EBV+ve and -ve cell lines. These results provide insight into pathways that are dysregulated in PTLD and can be targeted in future clinical trials.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Regulação da Expressão Gênica , Herpesvirus Humano 4 , Transtornos Linfoproliferativos/metabolismo , Transplante de Órgãos , Transplante de Células-Tronco , Infecções por Vírus Epstein-Barr/virologia , Feminino , Perfilação da Expressão Gênica/métodos , Proteínas de Choque Térmico HSP90/biossíntese , Humanos , Transtornos Linfoproliferativos/virologia , Masculino , NF-kappa B/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Quinases/biossíntese , Proteômica/métodos , Serina-Treonina Quinases TOR , Transplante HomólogoRESUMO
Our previous studies indicate that prolonged caffeine consumption exacerbates renal failure in nephropathy associated with the metabolic syndrome. Reduced activity of the antioxidant defense system and beneficial effects of antioxidant therapy have been reported in diabetic rats and humans. The purpose of this study was to examine the early renal effects of caffeine consumption and the effects of concomitant antioxidant therapy in young obese, diabetic ZSF1 rats. Eleven-week-old male ZSF1 rats were randomized to drink tap water, caffeine (0.1%), tempol (1 mmol/L), or a solution containing caffeine and tempol for nine weeks. Caffeine significantly reduced body weight and glycosuria (weeks 2-9), improved glucose tolerance (week 9), had no effect on elevated plasma triglycerides, plasma cholesterol (week 9) and blood pressure (week 9), and significantly increased plasma cholesterol level (weeks 5 and 9). Yet, as early as after two weeks, caffeine greatly augmented proteinuria and increased renal vascular resistance (RVR) and heart rate (HR: week 9). Tempol had no effects on metabolic status and development of proteinuria, did not alter caffeine-induced metabolic changes and early proteinuria, and attenuated caffeine-induced increase in HR and RVR. Immunohistochemical analysis revealed significant glomerular and interstitial inflammation, proliferation, and fibrosis in control animals. Caffeine augmented the influx of glomerular and interstitial macrophages (ED1+ cells) influx, glomerular and tubular proliferative response, and glomerular collagen IV content. Tempol abolished the exacerbation of renal inflammation, proliferation, and fibrosis induced by caffeine. In conclusion, in nephropathy associated with the metabolic syndrome, caffeine--most likely through the interaction with adenosine receptors and interference with anti-inflammatory and/or glomerular hemodynamic effects of adenosine--augments proteinuria and stimulates some of the key proliferative mechanisms involved in glomerular remodeling and sclerosis. Tempol does not prevent early renal injury (i.e., proteinuria) induced by caffeine, yet abolishes late renal inflammatory, proliferative, and fibrotic change induced by chronic caffeine consumption in obese ZSF1 rats.
Assuntos
Cafeína/toxicidade , Nefropatias Diabéticas/induzido quimicamente , Rim/efeitos dos fármacos , Obesidade/complicações , Animais , Peso Corporal/efeitos dos fármacos , Colágeno Tipo IV/análise , Óxidos N-Cíclicos/farmacologia , Hemodinâmica/efeitos dos fármacos , Rim/patologia , Lipídeos/sangue , Masculino , Antígeno Nuclear de Célula em Proliferação/análise , Ratos , Receptor A2A de Adenosina/efeitos dos fármacos , Receptor A2A de Adenosina/fisiologia , Receptor A2B de Adenosina/efeitos dos fármacos , Receptor A2B de Adenosina/fisiologia , Renina/sangue , Marcadores de SpinRESUMO
Indices of renal injury and oxidative stress were examined in mice with deficiency of cytosolic Cu(2+)/Zn(2+) superoxide dismutase (SOD1-/-, KO) and their wild-type (WT) littermates with streptozotocin-induced diabetes. After 5 weeks of diabetes, KO diabetic (D) but not WT-D mice developed marked albuminuria, increases in glomerular content of transforming growth factor beta, collagen alpha1(IV), and nitrotyrosine, and higher glomerular superoxide compared with corresponding values in nondiabetics. After 5 months of diabetes, increases in these parameters, mesangial matrix expansion, renal cortical malondialdehyde content, and severity of tubulointerstitial injury were all significantly greater, whereas cortical glutathione was lower, in KO-D than in WT-D. In contrast to WT-D, after 4 weeks of diabetes, KO-D mice did not develop the increase in inulin clearance (C(In)) characteristic of early diabetes. The nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methylester suppressed C(In) in WT-D, but had no effect on C(In) in KO-D. Treatment of KO-D with the SOD mimetic tempol for 4 weeks suppressed albuminuria, increases in glomerular transforming growth factor beta, collagen alpha1(IV), nitrotyrosine, and glomerular superoxide, and concurrently increased C(In). The latter action of tempol in KO-D was blocked by the N(omega)-nitro-l-arginine methylester. The findings provide support for a role for superoxide and its metabolism by SOD1 in the pathogenesis of renal injury in diabetes in vivo, and implicate increased interaction of superoxide with nitric oxide as a pathogenetic factor.
Assuntos
Óxidos N-Cíclicos/farmacologia , Óxidos N-Cíclicos/uso terapêutico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Superóxido Dismutase/genética , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Progressão da Doença , Hemoglobinas Glicadas/análise , Inulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/fisiologia , Marcadores de Spin , Estreptozocina , Superóxido Dismutase-1 , Fatores de TempoRESUMO
Human parvovirus B19 is a single-stranded DNA virus with a predilection for infecting rapidly dividing cell lines, such as bone marrow erythroid progenitor cells. People with defective cell-mediated immunity (eg, severe combined immunodeficiency syndrome; acquired immunodeficiency syndrome; and patients receiving immunosuppressive therapy, ie, post organ transplant) can develop pure red cell aplasia, in which suppression of erythroid precursors is permanent. Identification of parvovirus inclusions in marrow biopsies and subsequent confirmation of infection by in situ hybridization is important in the assessment of anemia in immunodeficient patients. Our objective is to provide a general overview of the parvovirus B19 infection and its characteristics in immunocompromised patients and to summarize updated information regarding the clinicopathologic features, pathobiology, and laboratory diagnosis of this subject. The pathologist should be aware of the wide spectrum of manifestations of parvovirus B19 infection depending on the patient's hematologic and immunologic status.
Assuntos
Hospedeiro Imunocomprometido , Infecções por Parvoviridae/imunologia , Humanos , Parvovirus B19 HumanoRESUMO
To better understand the molecular changes that occur in Waldenstrom macroglobulinemia (WM), we employed antibody-based protein microarrays to compare patterns of protein expression between untreated WM and normal bone marrow controls. Protein expression was defined as a >2-fold or 1.3-fold change in at least 67% of the tumor samples. Proteins up-regulated by >2-fold included Ras family proteins, such as Rab-4 and p62DOK, and Rho family proteins, such as CDC42GAP and ROKalpha. Other proteins up-regulated by >1.3-fold included cyclin-dependent kinases, apoptosis regulators, and histone deacetylases (HDAC). We then compared the samples of patients with symptomatic and asymptomatic WM and showed similar protein expression signatures, indicating that the dysregulation of signaling pathways occurs early in the disease course. Three proteins were different by >2-fold in symptomatic versus asymptomatic, including the heat shock protein HSP90. Elevated protein expression was confirmed by immunohistochemistry and immunoblotting. Functional significance was validated by the induction of apoptosis and inhibition of proliferation using specific HDAC and HSP90 inhibitors. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated in WM, which both enhance our understanding of disease pathogenesis and represent targets of novel therapeutics.
Assuntos
Macroglobulinemia de Waldenstrom/metabolismo , Idoso , Apoptose/fisiologia , Benzoquinonas/farmacologia , Células da Medula Óssea/metabolismo , Processos de Crescimento Celular/fisiologia , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Lactamas Macrocíclicas/farmacologia , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Plasmócitos/metabolismo , Proteômica , Reprodutibilidade dos Testes , Macroglobulinemia de Waldenstrom/patologiaRESUMO
We developed methods for prolonged (12 h), sterile, normothermic perfusion of rat kidneys and screened compounds for renal preservation including: mitochondrial transition pore inhibitor (decylubiquinone); caspase inhibitor (Z-VAD); peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists (gemfibrozil, WY-14643); antioxidants (trolox, luteolin, quercetin); growth factors (HGF, PDGF, EGF, IGF-1, VEGF, transferrin); calpain inhibitor (Z-Val-Phe-CHO); calmodulin inhibitor (W7); K(ATP) opener (minoxidil, minoxidil sulfate); PARP inhibitor (3-aminobenzamide); calcium channel blocker (verapamil); V(2) agonist (DDAVP); diuretics (acetazolamide, hydrochlorothiazide, furosemide, mannitol); peroxisome proliferator-activated receptor-beta agonist (L-165041); dopamine agonist (dopamine); essential fatty acid (linolenic acid); beta-NAD; urea; uric acid; and aldosterone. In pilot studies, only PPARalpha agonists and mannitol provided promising results. Accordingly, these agents were investigated further. Fifteen rat kidneys were perfused for 12 h with L-15 media at 37 degrees C in the absence or presence of mannitol, gemfibrozil, gemfibrozil + mannitol or WY-14643. Chronic perfusion in untreated kidneys caused destruction of glomerular and tubular architecture (light and electron microscopy), disappearance of Na(+)-K(+)-ATPase-alpha(1) (Western blotting), and apoptosis (Apoptag staining). Gemfibrozil and WY-14643 marginally improved some biomarkers of renal preservation. However, the combination of gemfibrozil with mannitol markedly improved all parameters of renal preservation. We conclude that PPARalpha agonists, particularly when combined with mannitol, protect organs from normothermic, perfusion-induced damage.
Assuntos
Genfibrozila/farmacologia , Rim/fisiologia , Manitol/farmacologia , Preservação de Órgãos/métodos , PPAR alfa/agonistas , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Interações Medicamentosas , Técnicas In Vitro , Rim/efeitos dos fármacos , Rim/patologia , Rim/ultraestrutura , Masculino , Microscopia Eletrônica , Perfusão , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/análiseRESUMO
BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) plays a key role in the recruitment and activation of monocytes during inflammation. Increased MCP-1 serum levels in patients with various cancers were correlated with advanced stage. Here, we evaluated the role of MCP-1 on prostate cancer (CaP) cell proliferation and invasion. METHODS: Expression of MCP-1 in tissue specimens was analyzed by immunohistochemical staining. MCP-1 production was determined by ELISA in conditioned media collected from primary prostate epithelia (PrEC), LNCaP, C4-2B, PC3 cells, and hFOB. Cell proliferation and invasion were assayed by MTS assay and invasion chambers. RESULTS: All CaP cells, as well as hFOB, produced high amount of MCP-1 compared to PrEC cells. MCP-1 expression levels were associated with advanced pathologic stage. MCP-1 induced proliferation and invasion of CaP cells and this was abolished partially either by CCR2 antagonist or PI3 Kinase inhibitor. CONCLUSION: MCP-1 acts as a paracrine and autocrine factor for CaP growth and invasion.
Assuntos
Proliferação de Células , Quimiocina CCL2/fisiologia , Invasividade Neoplásica/fisiopatologia , Neoplasias da Próstata/fisiopatologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Cromonas/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Morfolinas/farmacologia , Invasividade Neoplásica/patologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Próstata/metabolismo , Receptores CCR2 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Pulmonary arterial hypertension (PH) is a deadly disease characterized by pulmonary arterial vasoconstriction and hypertension, pulmonary vasculature remodeling, and right ventricular hypertrophy. Our previous in vivo studies, performed in several models of cardiac, vascular, and/or renal injury, suggest that the metabolites of 17beta-estradiol may inhibit vascular and cardiac remodeling. The goal of this study was to determine whether 2-methoxyestradiol (2ME), major non-estrogenic estradiol metabolite, prevents the development and/or retards the progression of monocrotaline (MCT)-induced PH. First, a total of 27 male Sprague Dawley rats were injected with distillated water (Cont, n=6) or monocrotaline (MCT; 60 mg/kg, i.p.; n=21). Subsets of MCT animals (n=7 per group) received 2ME or its metabolic precursor 2-hydroxyestradiol (2HE; 10 microg/kg/h via osmotic minipumps) for 21 days. Next, an additional set (n=24) of control and MCT rats was monitored for 28 days, before right ventricular peak systolic pressure (RVPSP) was measured. Some pulmonary hypertensive animals (n=8) were treated with 2ME (10 microg/kg/h) beginning from day 14 after MCT administration. MCT caused pulmonary hypertension (ie, increased right ventricle/left ventricle+septum [RV/LV+S] ratio and wall thickness of small-sized pulmonary arteries, and elevated RVPSP) and produced high and late (days 22 to 27) mortality. Pulmonary hypertension was associated with strong proliferative response (PCNA staining) and marked inflammation (ED1+cells) in lungs. Both metabolites significantly attenuated the RV/LV+S ratio and pulmonary arteries media hypertrophy and reduced proliferative and inflammatory responses in the lungs. Furthermore, in diseased animals, 2ME (given from day 14 to 28) significantly decreased RVPSP, RV/LV+S ratio and wall thickness, and reduced mortality by 80% (mortality rate: 62.5% vs. 12.5%, MCT vs. MCT+2ME day 14 to 28). This study provides the first evidence that 2ME, a major non-estrogenic, non-carcinogenic metabolite of estradiol, prevents the development and retards the progression of monocrotaline-induced pulmonary hypertension. Further evaluation of 2ME for management of pulmonary arterial hypertension is warranted.
Assuntos
Estradiol/análogos & derivados , Hipertensão Pulmonar/prevenção & controle , 2-Metoxiestradiol , Animais , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Estradiol/farmacologia , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/prevenção & controle , Imuno-Histoquímica , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Masculino , Monocrotalina , Antígeno Nuclear de Célula em Proliferação/análise , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Moduladores de Tubulina/farmacologiaRESUMO
Our previous studies in rodent models of nephropathy demonstrate that 2-hydroxyestradiol (2HE), an estradiol metabolite with little estrogenic activity, exerts renoprotective effects. In vivo, 2HE is readily converted to 2-methoxyestradiol (2ME), a major estradiol metabolite with no estrogenic activity. The goal of this study was to determine whether 2ME has renal and cardiovascular protective effects in vivo. First, the acute (90 minutes) and chronic (14 days) effects of 2ME (10 microg/kg/h) on blood pressure and renal function were examined in normotensive and spontaneously hypertensive rats (SHR). Second, a rat model of cardiovascular and renal injury induced by chronic nitric oxide synthase inhibition (N-nitro-L-arginine; 40 mg/kg/d; LNNA group) was used to examine the protective effects of estradiol metabolites. Subsets of LNNA-treated rats were administered either 2HE or 2ME (10 microg/kg/h via osmotic minipump; LNNA+2ME and LNNA+2HE groups, respectively. 2-Methoxyestradiol had no acute or chronic effects on blood pressure or renal function in normotensive animals or on hypertension in SHR. Prolonged, 5-week NOS inhibition induced severe cardiovascular and renal disease and high mortality (75%, LNNA group). 2ME, but not 2HE, significantly decreased elevated blood pressure and attenuated the reduction in GFR. 2HE delayed the onset of proteinuria, whereas no proteinuria was detected in the 2-ME group. 2HE and 2ME reduced mortality rate by 66% and 83%, respectively (P < 0.001). In the kidney, 2HE and 2ME abolished LNNA-induced interstitial and glomerular inflammation, attenuated glomerular collagen IV synthesis, and inhibited glomerular and tubular cell proliferation. In the heart, 2HE and 2ME markedly reduced vascular and interstitial inflammation and reduced collagen synthesis and vascular/interstitial cell proliferation. This study provides the first evidence that, in a model of severe cardiovascular and renal injury, 2-methoxyestradiol (a major nonestrogenic estradiol metabolite) exerts renal and cardiovascular protective effects and reduces mortality.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Estradiol/análogos & derivados , Nefropatias/tratamento farmacológico , Óxido Nítrico Sintase/antagonistas & inibidores , 2-Metoxiestradiol , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Peso Corporal/efeitos dos fármacos , Doenças Cardiovasculares/induzido quimicamente , Creatinina/sangue , Inibidores Enzimáticos/farmacologia , Estradiol/administração & dosagem , Estradiol/metabolismo , Estradiol/uso terapêutico , Taxa de Filtração Glomerular/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Infusões Intravenosas , Nefropatias/induzido quimicamente , Masculino , Nitroarginina/farmacologia , Proteinúria/sangue , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Circulação Renal/efeitos dos fármacos , Fatores de TempoRESUMO
Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19(+) purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.
Assuntos
Linfoma de Célula do Manto/química , Proteínas de Neoplasias/análise , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Linfócitos B , Biópsia , Análise por Conglomerados , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfonodos/patologia , Linfoma de Célula do Manto/patologia , Masculino , Pessoa de Meia-Idade , Análise Serial de ProteínasRESUMO
Lung cancer mortality rates in the Xuan Wei County population are among the highest in China and are associated with exposure to indoor emissions from the burning of smoky coal. Previous studies of lung tumors from both non-smoking women and smoking men in this region showed high frequencies of mutations, consisting mostly of G-->T transversions in the p53 tumor suppressor gene and K-ras oncogene, suggesting that these mutations were caused primarily by polycyclic aromatic hydrocarbons. In this study sputum samples from 92 individuals with no evidence of lung cancer from Xuan Wei County were screened for p53 and K-ras mutations. Sputum cells were collected on glass slides by sputum cytocentrifugation, stained and cytopathologically analyzed. Cytologically non-malignant epithelial cells were taken from each sputum sample using a laser capture microdissection microscope and molecularly analyzed. Cells taken from the sputum of 15 (16.3%) individuals were mutation positive, including 13 (14.1%) individuals each with a p53 mutation, 1 (1.1%) individual with a K-ras mutation and 1 (1.1%) individual with a p53 and a K-ras mutation. p53 mutations were found in both the sputum of individuals with evidence of chronic bronchitis (3 of 46 or 6.5%) and those without evidence of this disease (11 of 46 or 23.9%). Therefore, mutations in the p53 gene and, to a lesser extent, the K-ras gene were frequent in non-malignant epithelial cells taken from the sputum of individuals without evidence of lung cancer who were exposed to smoky coal emissions in Xuan Wei County and were at a high risk for developing the disease.
Assuntos
Poluição do Ar em Ambientes Fechados/efeitos adversos , Genes p53/genética , Genes ras/genética , Neoplasias Pulmonares/genética , Escarro/citologia , Bronquite Crônica/genética , Bronquite Crônica/patologia , China , Carvão Mineral/efeitos adversos , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Microdissecção , Mutação/genéticaRESUMO
Mutations in the p53 tumor suppressor gene are frequent in breast tumors but the implication of p53 mutations in breast cancer development remains poorly understood. In this study, we applied laser capture microdissection (LCM) microscope to histologically review and sample cells from paraffin-embedded breast tissue sections obtained from six cases of ductal carcinoma in situ (DCIS) and ten cases of atypical ductal hyperplasia (ADH). p53 mutations were detected, using single stranded conformational polymorphism (SSCP) and sequencing, in cell samples of three cases with DCIS and five cases with ADH. p53 mutations are therefore present in DCIS and ADH of the breast, considered as pre-malignant precursors to breast cancer, and some of them may represent early events in breast cancer development.
Assuntos
Neoplasias da Mama/genética , Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Análise Mutacional de DNA , Genes p53 , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Transformação Celular Neoplásica , Feminino , Humanos , Hiperplasia , Microdissecção , Inclusão em Parafina , Polimorfismo Conformacional de Fita Simples , Lesões Pré-CancerosasRESUMO
The effects of overexpression of Cu(2+)/Zn(2+) superoxide dismutase-1 (SOD-1) on indexes of renal injury were compared in 5-month-old nontransgenic (NTg) db/db mice and db/db mice hemizygous for the human SOD-1 transgene (SOD-Tg). Both diabetic groups exhibited similar hyperglycemia and weight gain. However, in NTg-db/db mice, albuminuria, glomerular accumulation of immunoreactive transforming growth factor-beta, collagen alpha1(IV), nitrotyrosine, and mesangial matrix were all significantly increased compared with either nondiabetic mice or SOD-Tg-db/db. SOD-1 activity and reduced glutathione levels were higher, whereas malondialdehyde content was lower, in the renal cortex of SOD-Tg-db/db compared with NTg-db/db mice, consistent with a renal antioxidant effect in the transgenic mice. Inulin clearance (C(IN)) and urinary excretion of guanosine 3',5'-cyclic monophosphate (U(cGMP)) were increased in SOD-Tg-db/db mice compared with corresponding values in nondiabetic mice or NTg-db/db mice. C(IN) and U(cGMP) were suppressed by Nomega-nitro-L-arginine methyl ester in SOD-Tg-db/db but not in NTg-db/db mice, implying nitric oxide (NO) dependence of these increases and enhanced renal NO bioactivity in SOD-Tg-db/db. Studies of NO-responsive cGMP in isolated glomeruli supported greater quenching of NO in glomeruli from NTg-db/db compared with SOD-Tg-db/db mice. Evidence of increased NO responsiveness and the suppression of glomerular nitrotyrosine may both reflect reduced NO-superoxide interaction in SOD-Tg-db/db mice. The results implicate superoxide in the pathogenesis of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/prevenção & controle , Rim/patologia , Óxido Nítrico/fisiologia , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Animais , Sequência de Bases , GMP Cíclico/metabolismo , Primers do DNA , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Humanos , Imuno-Histoquímica , Inulina/urina , Glomérulos Renais/enzimologia , Glomérulos Renais/patologia , Camundongos , Camundongos Transgênicos , NG-Nitroarginina Metil Éster/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mutations in the p53 tumor suppressor gene and the K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and other patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cytocentrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) for p53 mutations and by PCR and denaturing gradient gel electrophoresis (DGGE) for K-ras mutations. This method was used to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with p53 mutations, 1 patient with a K-ras mutation, and 1 patient with K-ras and p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low-fraction mutations occurring throughout the p53 and K-ras genes in sputum of lung cancer patients. This method may be applicable to the analysis of epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.
Assuntos
DNA de Neoplasias/análise , Genes p53 , Genes ras , Lasers , Neoplasias Pulmonares/diagnóstico , Microdissecção , Escarro/metabolismo , Biomarcadores Tumorais/análise , Lavagem Broncoalveolar , Análise Mutacional de DNA , DNA de Neoplasias/genética , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Microdissecção/métodos , Mutação/genética , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Escarro/citologiaRESUMO
Nonsteroidal anti-inflammatory drugs decrease sporadic colorectal carcinoma and adenomas in patients with familial adenomatous polyposis and in rodent models of sporadic colon cancer and familial adenomatous polyposis. Similarly, selective cyclooxygenase 2 inhibitors decrease adenomas in humans and rodents. However, their effects on chronic colitis and colitis-associated neoplasia are unknown. Interleukin 10-/- mice (C57/B6) were fed regular chow (n = 20) or chow with celecoxib (1,500 ppm, n = 18) or rofecoxib (75 ppm, n = 20) for 12 weeks. Twenty-eight percent of the celecoxib group died versus 5% of the control and rofecoxib groups (p < 0.05 compared with control). Celecoxib and rofecoxib increased the incidence of colitis (26% vs. 92% and 68%, p < 0.01), colitis score (0.4 +/- 0.2 vs. 2.5 +/- 0.3 and 2 +/- 0.4, p < 0.01), aberrant crypt foci (0.5 +/- 0.3 vs. 3.7 +/- 2.6 and 2.8 +/- 0.7, p < 0.01), aberrant crypts per mouse (4.11 +/- 2.1 vs. 41.2 +/- 9.7 and 27.1 +/- 7.5, p < 0.01) and dysplasia (11% vs. 54% and 42%, p < 0.01). Similarly, indomethacin (9 ppm, n = 15) increased colitis score, aberrant crypt foci, and dysplasia after 27 days of treatment. Two selective cyclooxygenase 2 inhibitors exacerbate colitis and premalignant changes in the interleukin 10-/- mouse model of chronic colitis and colitis-associated colon carcinoma.
Assuntos
Colite/induzido quimicamente , Inibidores de Ciclo-Oxigenase/toxicidade , Interleucina-10/farmacologia , Lactonas/toxicidade , Lesões Pré-Cancerosas/induzido quimicamente , Sulfonamidas/toxicidade , Animais , Celecoxib , Colite/patologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Lesões Pré-Cancerosas/patologia , Pirazóis , SulfonasRESUMO
Lung cancer, a disease related mostly to tobacco smoke exposure and a leading cause of cancer-related death in industrialized countries, is frequently associated with mutations in the p53 tumor suppressor gene. Genetic differences resulting in inter-individual variation in DNA repair capacity may in part account for susceptibility of a cell to genotoxic agents leading to somatic mutations, including p53 mutations, and eventual transformation of a normal cell into a malignant phenotype. The objective of this study is to investigate the relationship between the polymorphisms of two DNA repair genes, the nucleotide excision repair xeroderma pigmentosum group D (XPD) gene (codons 312 and 751) and the base excision repair X-ray repair cross-complementing group 1 (XRCC1) gene (codon 399), and p53 mutations among lung cancer patients. Lung tumors from 204 smokers with non-small cell lung cancer (NSCLC) were analyzed for mutations in exons 5-8 of the p53 gene and genotypes of XPD and XRCC1. p53 mutations were found in 20% (40/204) of the patients. Patients with the XPD codon 312 Asn allele were less likely to have p53 mutations (13.8%) than XPD 312 Asp/Asp (27.3%) [odds ratio (OR) 0.43, 95% confidence interval (CI) 0.20-0.89, P = 0.023]. No association was found between p53 mutations and either XPD Lys751Gln or XRCC1 Arg399Gln. However, the p53 mutation frequency increased with the increased number of the combined genotypes among XPD 312WT (Asp/Asp), XPD 751VT (Lys/Gln or Gln/Gln) or XRCC1 399VT (Arg/Gln or Gln/Gln) (P = 0.01, trend test). These results suggest that individuals who smoke and have the XPD codon 312 Asp/Asp genotype may be at a greater risk of p53 mutations, especially if combined with other polymorphisms that may result in deficient DNA repair.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA Helicases , Genes p53 , Neoplasias Pulmonares/genética , Proteínas/genética , Fumar/efeitos adversos , Fatores de Transcrição , Alelos , Carcinoma Pulmonar de Células não Pequenas/etiologia , Análise Mutacional de DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Genótipo , Humanos , Neoplasias Pulmonares/etiologia , Masculino , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Genético , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteína Grupo D do Xeroderma PigmentosoRESUMO
The p53 gene is frequently mutated in lung tumors, and mutations may be caused by both polycyclic aromatic hydrocarbons (PAHs) and nitrosamines found in tobacco smoke. The two major forms of lung cancer, adenocarcinoma (AC) and squamous cell carcinoma (SCC), are known to differ in the proportion of tumors exhibiting p53 mutation, and may also differ in the mutational spectra produced. Previous studies comparing p53 mutational spectra between AC and SCC of the lung have been limited by small sample size. We examined p53 mutations in exons 5-8 in 202 cases of AC and 82 cases of SCC from smoking lung cancer patients in the Western Pennsylvania region. The percent of cases with p53 mutation was significantly lower in ACs (40/202, 20%) compared to SCCs (29/82, 35%, P=0.006). The proportion of mutations present that were G to T transversions was not significantly different between the two tumor types (52% of p53 mutations in AC compared to 32% in SCC). G to A transitions either did not differ in frequency in the two types of lung cancer (20% of mutations in AC and 24% of mutations in SCC). A distinct spectrum was observed, however, in the p53 mutation pattern in the two types of lung cancer. ACs showed a strong preference for a mutational hotspot at codons 248 and 249, while squamous cell tumors showed mutational events spread throughout exons 5-8, with a preference for codon 267. Mutations at codon 267 in SCC were all C to T transitions that occurred at CpG sites. Both tumor types demonstrated preferential mutation of the non-transcribed strand (100% of all G to T transversions and 55% of the G to A transitions). These results suggest that p53 mutations in both types of lung tumors may arise from adduction by both PAHs and nitrosamines. Mutations arising in ACs appear selectively in regions of p53 that produce more rigid proteins, suggesting a drastic change in p53 function is needed to result in ACs, while less constrained changes in p53 function can result in SCCs. Mutation in p53 was not found to be related to patient survival in this group of patients, while tumor size and degree of differentiation were poor survival predictors.