RESUMO
Lack of detectable expression of p27kip1 cyclin dependent kinase inhibitor has previously been correlated with high degree of malignancy in human breast, colorectal, gastric and small cell lung carcinomas. Here we demonstrate that an inverse correlation between p27kip1 expression and tumour malignancy also exists in most types of human B cell lymphomas examined. A clear exception was Burkitt's lymphoma (BL), a highly malignant tumour which often expresses high levels of p27kip1. Analysis of p27kip1 derived from Burkitt's lymphoma cell lines expressing high levels of p27kip1, BL40 and BL41, in a cyclin E/cdk2 kinase inhibition assay demonstrated that p27kip1 is not permanently inactivated since heat treatment can restore the inhibitory activity of p27kip1. However, p27kip1 expressed in these two cell lines is largely sequestered in inactive complexes and we have no evidence that c-myc or Epstein-Barr virus are responsible for the sequestration of p27kip1 in these two cell lines although c-myc and EBV are two oncogenic agents often associated with Burkitt's lymphomas. Interestingly, we observed that high level p27kip1 expression often correlated with cyclin D3 overexpression both in vivo and in BL cell lines. The majority of p27kip1 in BL40 cells was complexed with cyclin D3 indicating that overexpressed cyclin D3 may at least be part of the sequestering activity for the inhibitory function of p27kip1. Furthermore, cyclinD3/cdk4 complex could sequester p27kip1 in a cyclin E/cdk2 kinase assay in vitro. Finally, we show that cyclin D3 transfected into an inducible p27kip1 cell line could overcome the G1 arrest mediated by p27kip1. These results argue that in addition to down-regulation of p27kip1 expression, some tumour cells can sequester and tolerate the antiproliferative function of p27kip1. They also suggest a novel role for the overexpression of D-type cyclins as one pathway allowing tumour cells to overcome the antiproliferative function of p27kip1.
Assuntos
Linfócitos B/metabolismo , Linfoma de Burkitt/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Linfócitos B/patologia , Linfoma de Burkitt/patologia , Carcinoma/patologia , Ciclo Celular , Ciclina D3 , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Temperatura Alta , Humanos , Neoplasias Pulmonares/patologia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prognóstico , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína do Retinoblastoma/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The purpose of the current study was to evaluate myocardial creatinine kinase (CK) and lactate dehydrogenase (LDH) systems in a model of epinephrine-induced cardiomyopathy in rabbits. Eight rabbits received four repetitive epinephrine infusions (300 mg/kg/60 min, i.v.) in 12-day intervals and eight untreated rabbits served as controls (CTRL). Echocardiography demonstrated a significant deterioration of LV function as well as increased LV-diameter and -mass index in catecholamine-induced cardiomyopathy. Histological examination revealed that repetitive catecholamine infusion resulted in LV fibrous areas with collagenous content and an increase in myocyte width (16.9+/-0.8 microm vs. CTRL 12.9+/-0.9; P<0.05). LV dysfunction was associated with a decreased total LV lactate dehydrogenase activity (LDH; 0.43+/-0.03 IU/mg protein vs. CTRL 0.52+/-0.04; P<0.05) whereas total creatinine kinase activity was unchanged (CK; 7.30+/-0.63 IU/mg protein vs. CTRL 9.20+/-0.49, n.s.). Furthermore, myocardial LDH isoenzymes were shifted with a decrease in LDH(1) and an increase in LDH2 and LDH3 (LDH(1): 84.90+/-2.60% vs. CTRL 94.50+/-0.40; LDH2: 7.30+/-1.20% vs. 1.50+/-0.13; LDH3: 5.40+/-0.90% vs. 3.20+/-0.25; all P<0.05). Foetal B-CK isoenzymes were significantly increased (CK-MB 5.30+/-0.66 vs. 2.20+/-0.35%; P<0.05). The current study demonstrates changes in cardiac energy metabolism including an impaired LDH activity with a shift towards anaerobic isoenzymes as well as a more efficient CK system in a model of catecholamine-induced LV dysfunction.
Assuntos
Creatina Quinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Disfunção Ventricular Esquerda/enzimologia , Animais , Modelos Animais de Doenças , Ecocardiografia , Epinefrina , Isoenzimas/metabolismo , Masculino , Miocárdio/enzimologia , Coelhos , Estatísticas não Paramétricas , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/patologiaRESUMO
Congestive heart failure (CHF) is often associated with atrial fibrillation. The safety of many antiarrhythmic drugs in CHF is limited by proarrhythmic effects. We aimed to assess the safety of a novel atrial-selective K(+)-channel blocker AVE0118 in CHF compared to a selective (dofetilide) and a non-selective IKr blocker (terfenadine). For the induction of CHF, rabbits (n = 12) underwent rapid right ventricular pacing (330-380 bpm for 30 days). AVE0118 (1 mg/kg) dofetilide (0.02 mg/kg) and terfenadine (2 mg/kg) were administered in baseline (BL) and CHF. A six-lead ECG was continuously recorded digitally for 30 min after each drug administration. At BL, dofetilide and terfenadine significantly prolonged QTc interval (218 +/- 30 ms vs 155 +/- 8 ms, p = 0.001 and 178 +/- 23 ms vs. 153 +/- 12 ms, p = 0.01, respectively) while QTc intervals were constant after administration of AVE0118 (p = n.s.). In CHF, dofetilide and terfenadine caused torsades de pointes and symptomatic bradycardia, respectively, and prolonged QTc interval (178 +/- 30 ms vs. 153 +/- 14 ms, p = 0.02 and 157 +/- 7 ms vs. 147 +/- 10 ms, p = 0.02, respectively) even at reduced dosages, whereas no QTc-prolongation or arrhythmia was observed after full-dose administration of AVE0118. In conclusion, atrial-selective K(+)-channel blockade by AVE0118 appears safe in experimental CHF.
Assuntos
Compostos de Bifenilo/efeitos adversos , Átrios do Coração/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Bloqueadores dos Canais de Potássio/efeitos adversos , Canais de Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/induzido quimicamente , Compostos de Bifenilo/administração & dosagem , Compostos de Bifenilo/uso terapêutico , Modelos Animais de Doenças , Eletrocardiografia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Sistema de Condução Cardíaco/efeitos dos fármacos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/terapia , Frequência Cardíaca/efeitos dos fármacos , Infusões Intravenosas , Marca-Passo Artificial , Bloqueadores dos Canais de Potássio/administração & dosagem , Bloqueadores dos Canais de Potássio/uso terapêutico , CoelhosRESUMO
BACKGROUND: Aldosterone is an important mediator of cardiovascular and renal remodeling. Type II diabetes mellitus leads to renal and cardiac end organ damage. We investigated the renin-angiotensin-aldosterone system in a model of type 2 diabetes mellitus with known diabetic nephropathy and cardiac remodeling, the Zucker Diabetic Fatty rat with and without ACE-inhibition (ZDF and ZDF+ACE-I) and its control, the Zucker Lean (ZDL) rat. METHODS: Male animals were studied from an age of 7-24 weeks. At ages 7, 14, 17, 20, and 23 weeks, urinary excretion of aldosterone-glucuronide and potassium was assessed. ACE-inhibition with ramipril was started orally at week 13 (1 mg/kg/d). At the end of the study rats were sacrificed and plasma aldosterone concentration and plasma renin activity were measured. Aldosterone synthase (CYP11B2) mRNA expression in the adrenals, kidney, heart and adipose tissue was assessed by real-time PCR. Urinary albumin excretion as marker for diabetic nephropathy was measured in metabolic cages and correlated to aldosterone. RESULTS: Plasma aldosterone concentration and aldosterone-glucuronide was significantly elevated in ZDF rats, and significantly reduced by ACE-inhibiton. In contrast, plasma renin activity was significantly reduced in ZDF rats and normalized by ACE-inhibition. The urinary aldosterone correlated significantly to albuminuria. Adrenal CYP11B2 expression was not significantly higher in ZDF rats. CYP11B2 mRNA was not detected in the kidney, heart and adipose tissue. CONCLUSION: In ZDF rats, urinary and plasma aldosterone levels were elevated despite reduced plasma renin activity. The reversible effect of ACE-inhibition shows that the up-regulation of aldosterone must be dependent of the renin-angiotensin-system in this type II diabetes model. The correlation between aldosterone and diabetic nephropathy suggests a clinical relevance of this observation.
Assuntos
Aldosterona/sangue , Diabetes Mellitus Tipo 2/sangue , Actinas/genética , Albuminúria , Aldosterona/análogos & derivados , Aldosterona/urina , Animais , Pressão Sanguínea , Citocromo P-450 CYP11B2/genética , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/urina , Modelos Animais de Doenças , Frequência Cardíaca , Masculino , RNA Mensageiro/genética , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The aim of our study was to determine whether planimetry of the anatomic regurgitant orifice (ARO) in patients with aortic regurgitation (AR) by magnetic resonance imaging (MRI) is feasible and whether ARO by MRI correlates with the severity of AR. METHODS AND RESULTS: Planimetry of ARO by MRI was performed on a clinical magnetic resonance system (1.5 T Sonata, Siemens Medical Solutions) in 45 patients and correlated with the regurgitant fraction (RgF) and regurgitant volume (RgV) determined by MRI phase velocity mapping (PVM; MRI-RgF, MRI-RgV, n = 45) and with invasively quantified AR by supravalvular aortography (n = 32) and RgF upon cardiac catheterisation (CATH-RgF, n = 15). Determination of ARO was possible in 98% (44/45) of the patients with adequate image quality. MRI-RgF and CATH-RgF were modestly correlated (n = 15, r = 0.71, p<0.01). ARO was closely correlated with MRI-RgF (n = 44, r = 0.88, p<0.001) and was modestly correlated with CATH-RgF (n = 14, r = 0.66, p = 0.01). Sensitivity and specificity of ARO to detect moderately severe and severe aortic regurgitation (defined as MRI-RgF > or =40%) were 96% and 95% at a threshold of 0.28 cm2 (AUC = 0.99). Of note, sensitivity and specificity of ARO to detect moderately severe and severe AR at catheterisation (defined as CATH-RgF > or =40% or supravalvular aortography > or =3+) were 90% and 91% at a similar threshold of 0.28 cm2 (AUC = 0.95). Lastly, sensitivity and specificity of ARO to detect severe aortic regurgitation (defined as MRI-RgF > or =50% and/or regurgitant volume > or =60 ml) were 83% and 97% at a threshold of 0.48 cm2 (AUC = 0.97). CONCLUSIONS: Visualisation and planimetry of the ARO in patients with AR are feasible by MRI. There is a strong correlation of ARO with RgV and RgF assessed by PVM and with invasively graded AR at catheterisation. Therefore, determination of ARO by MRI is a new non-invasive measure for assessing the severity of AR.
Assuntos
Insuficiência da Valva Aórtica/diagnóstico , Angiografia por Ressonância Magnética/métodos , Adulto , Idoso , Cateterismo Cardíaco/métodos , Métodos Epidemiológicos , Feminino , Humanos , Angiografia por Ressonância Magnética/normas , Masculino , Pessoa de Meia-IdadeRESUMO
E2F1 overexpression has been shown to induce apoptosis in cooperation with p53. Using Saos-2 cells, which are null for p53 and lack functional Rb, we have demonstrated that E2F1 overexpression can also induce apoptosis in the absence of p53 and retinoblastoma protein (Rb). E2F1-induced apoptosis can be specifically inhibited by Rb but not mdm2, which is known for its ability to inhibit p53-induced apoptosis. Through the study of the apoptotic function of a set of E2F1 mutants, it was clear that the transactivation and the apoptotic function of E2F1 are uncoupled. The transactivation-defective E2F1 mutants E2F1(1-374), E2F1(390-1)DF(delta mdm2), and E2F1(406-415)(delta Rb) can induce apoptosis as effectively as wild-type E2F1. In contrast to E2F1 transactivation, the DNA-binding activity of E2F1 was proven to be essential for its apoptotic function, as the DNA-binding-defective mutants E2F1(132) and E2F1(132)(1-374) failed to induce apoptosis. Therefore Rb may inhibit E2F1-induced apoptosis by mechanisms other than the suppression of the transactivation of E2F1. This hypothesis was supported by our observation that although Rb overexpression can specifically repress the apoptosis induced by wild-type E2F1 and a Rb-binding-competent E2F1 mutant E2F1(390-1)DF(delta mdm2), it failed to inhibit the apoptosis induced by mutants E2F1(1-374) and E2F1(delta 406-415)(delta Rb), which are defective or reduced in Rb binding and transactivation. All of these points argue for a novel function for E2F1 and Rb in controlling apoptosis. The results also indicate that transcriptional repression rather than the transactivation function of E2F1 may be involved in its apoptotic function. The results presented here may provide us some physiological implication of the repression function of the Rb-E2F1 complex.
Assuntos
Apoptose , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Linhagem Celular , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Proteína 1 de Ligação ao Retinoblastoma , Fatores de Transcrição/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
As a universal inhibitor of cyclin-dependent kinases and one of the target genes of the tumor suppresser p53, p21Waf1/Cip1 can act as a tumor suppresser through its ability to control cell cycle progression. To study the function of p21Waf1/Cip1 protein and to investigate its tissue distribution, a panel of anti-p21Waf1/Cip1 monoclonal antibodies was generated. These anti-p21Waf1/Cip1 monoclonal antibodies were initially raised against a GST-p21Waf1/Cip1 fusion protein produced in bacteria. Detailed characterization of the antibodies showed that they can specifically detect p21Waf1/Cip1 by immunoblotting, immunoprecipitation, and immunostaining. The specific induction of p21Waf1/Cip1 expression in response to gamma-radiation in cells containing p53 was also detected by these antibodies. The ability to detect p21Waf1/Cip1 expression in conventionally fixed tissue sections allowed us to investigate the distribution of p21Waf1/Cip1 in 23 different types of normal human tissues, and p21Waf1/Cip1 expression was found in most tissues. A close inverse relationship between p21Waf1/Cip1 expression and proliferation was seen in some tissues, including gastrointestinal tract. However, such association is not universal. In tissues such as lung, kidney, thyroid, pancreatic ducts and acini, and liver, despite the fact that most of the cells are quiescent, expression of p21Waf1/Cip1 was detected only in occasional epithelial cells. All these suggest that the expression of p21Waf1/Cip1 varies among different human tissues. Finally, epitope mapping of the anti-p21Waf1/Cip1 antibodies using a peptide library covering the entire p21Waf1/Cip1 protein sequence indicates that two of the antibodies recognize a region of p21Waf1/Cip1 close to that bound by proliferating cell nuclear antigen. These two monoclonal antibodies will therefore be additionally useful in further understanding the functions of p21Waf1/Cip1 both in vitro and in vivo.
Assuntos
Ciclinas/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Inibidor de Quinase Dependente de Ciclina p21 , Regulação da Expressão Gênica , Humanos , Immunoblotting , Imunoquímica/métodos , Dados de Sequência Molecular , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Testes de Precipitina , Proteínas Recombinantes , Valores de Referência , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
Left ventricular hypertrophy (LVH) is accompanied by progressive accumulations of extracellular matrix proteins. They are produced predominantly by cardiac fibroblasts that surround the cardiac myocytes. The aim of this study was to emphasize the role of a combined approach using both in vivo and in vitro studies to elucidate the effects of carvedilol on cardiac remodeling. We therefore used an established model of supravalvular aortic banding and cardiac fibroblasts. LVH was induced by banding of the ascending aorta. Male Wistar rats were allocated to four groups: sham-operated, sham+carvedilol, aortic stenosis (AS), and AS+carvedilol. Treatment time was four weeks. Fibroblasts were isolated from the entire left ventricle of sham and AS rats. Carvedilol/metoprolol/prazosin were added (0.1, 1.0 and 10 microM; 24 h). In addition, interferon- gamma was applied for 24 h (10, 100 and 1000 IU). AS rats revealed increased LV weights (+27%) and cardiomyocyte widths as compared to sham-operated rats (1.6-fold, P<0.01). Carvedilol reduced LVH by 20%. This finding was accompanied by a decrease of laminin, fibronectin, collagen I and III in vivo. Collagen I/III and fibronectin were increased in fibroblasts of AS v sham rats (P<0.0001, each). Carvedilol reduced collagen I, III and fibronectin by 40/60/35% (0.1 microM; P<0.001) irrespective of LVH. Carvedilol had no effects on collagen IV and laminin. Carvedilol dose-dependently reduced the proliferation rate by 20% at 0.1 microM(P<0.0001). Metoprolol and prazosin had no effect on the expression of extracellular matrix proteins and on the proliferation of the cells of either origin. Interferon- gamma blunted the proliferation rate of cultured fibroblasts and lead to a significant decrease in extracellular matrix deposits. These results indicate that the effects of carvedilol may be due to the antiproliferative or antioxidative properties of this unselective beta-adrenergic receptor antagonist. These changes of the extracellular matrix represent a new mechanism of carvedilol that may contribute to the observed beneficial effects in congestive heart failure.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1 , Antagonistas Adrenérgicos beta/metabolismo , Anti-Hipertensivos/metabolismo , Carbazóis/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Ventrículos do Coração/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Propanolaminas/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Western Blotting/métodos , Carbazóis/farmacologia , Carvedilol , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Ventrículos do Coração/citologia , Hemodinâmica , Interferon gama/metabolismo , Interferon gama/farmacologia , Masculino , Metoprolol/metabolismo , Metoprolol/farmacologia , Miocárdio/metabolismo , Prazosina/metabolismo , Prazosina/farmacologia , Propanolaminas/farmacologia , Ratos , Ratos Wistar , Pressão VentricularRESUMO
The expression of cyclin-dependent kinase inhibitor p27(kip1) in human tumors and normal tissues was investigated using a panel of novel anti-p27(kip1) mAbs. An inverse correlation between expression of p27(kip1) and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27(kip1) expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27(kip1) and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27(kip1) and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27(kip1) may be a useful prognostic marker in these cancers.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclinas/biossíntese , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Oncogênicas/biossíntese , Proteínas Supressoras de Tumor , Biomarcadores Tumorais , Ciclo Celular , Divisão Celular , Ciclina D1 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/análise , Feminino , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Cinética , Masculino , Proteínas Associadas aos Microtúbulos/análise , Proteínas Oncogênicas/análise , Especificidade de Órgãos , Prognóstico , Valores de Referência , Células Tumorais CultivadasRESUMO
BACKGROUND: Published data regarding effects of growth hormone (GH) on the renin system are controversial. The aim of this study therefore was to evaluate the effects of GH on the renin system in normal rats and rats with myocardial infarction (MI). METHODS: Normal rats received 2, 5, or 10 IU GH/kg/day or vehicle subcutaneously for 4 weeks. Furthermore rats with MI were randomized to receive 2 IU GH/kg/day or vehicle for 4 weeks. Subdivision into MI groups (mild, moderate, and large) was by histological determination of infarct size. Renal renin gene expression was assessed by RNAase protection assay and plasma renin activity by radioimmunoassay. In addition, isolated mouse juxtaglomerular cells were exposed to GH for 20 h, and renin secretion rates were assessed. RESULTS: GH treatment in normal rats for 4 weeks increased body weight, and kidney weight to body weight ratio, but did not affect renin secretion and renal renin gene expression. In rats with large MI, renal renin gene expression increased about fourfold, but was unchanged in rats with small and moderate MI as compared to normal rats. In rats with MI, body weight decreased and this decrease was partially reversed by GH treatment. GH treatment did not change renal renin gene expression, and renin secretion in rats with MI. Renin secretion of isolated juxtaglomerular cells was unaffected by GH. CONCLUSIONS: Our study demonstrates that GH treatment has no significant effect on renin secretion and on renal renin gene expression in normal rats and in rats with stimulated renin system due to MI in vivo. In isolated juxtaglomerular cells in vitro, renin secretion was also unaffected by GH.