RESUMO
The epidermal growth factor (EFG) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antigen of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggressive transformed cells. Dual staining of EGFR and AR was observed in 50-60% of NS2T2A1 cells, while 30-40% cells expressed AR only. To explore the potential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientation was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to < 5%). The anchorage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4-6.8% relative to the control cell line transfected with the vector alone. The clones expressing AR antisense RNA showed a reversion of the malignant phenotype when injected in nude mice, since a significant reduction of tumor intake was observed coincident with a significant tumor mass reduction (> 96%). Moreover, intra-tumoral vascularization decreased significantly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). These in vitro and in vivo data reveal the oncogenic nature of AR in transformed breast epithelial cells and imply a role for AR in tumor angiogenesis.
Assuntos
Mama/citologia , Transformação Celular Neoplásica/genética , Glicoproteínas/fisiologia , Substâncias de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , RNA Antissenso/genética , Anfirregulina , Animais , Divisão Celular/genética , Linhagem Celular Transformada , Família de Proteínas EGF , Células Epiteliais/citologia , Receptores ErbB/genética , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/genética , Fenótipo , Receptores Androgênicos/genéticaRESUMO
In patients with idiopathic hemochromatosis, retinopathy was investigated by ophthalmoscopy and fluorescein angiography and was present in eight out of 23; this prevalence is similar to that reported in patients with diabetes aged between 30 and 60 years at onset of diabetes and with the same duration of the disease; in these eight patients retinopathy was of mild degree, with no impairment of visual acuity, fewer than 10 microaneurysms in each fundus, and no other retinal abnormatic islets directly. These studies confirm that anticholinergic drugs may be useful adjuvants in treating these patients.
Assuntos
Retinopatia Diabética/sangue , Hormônio do Crescimento/sangue , Hemocromatose/sangue , Adulto , Idoso , Arginina , Glicemia/metabolismo , Retinopatia Diabética/complicações , Feminino , Hemocromatose/complicações , Humanos , Insulina , Testes de Função Hepática , Masculino , Pessoa de Meia-IdadeRESUMO
The expression of the P-glycoprotein which is associated with the development of multidrug resistance in various cell lines was investigated in 87 fresh acute leukaemia and multiple myeloma samples using the specific mouse monoclonal antibody MRK16 in an indirect immunofluorescence assay. Considering a 10% positive cell cut-off value, a heterogeneous expression of P-glycoprotein was observed in 5/22 (22.7%) de novo acute leukaemias, 7/22 (31.8%) relapse or secondary acute leukaemias, 14/27 (51.8%) acute transformation of myeloproliferative or myelodysplastic syndromes and 5/16 (31.2%) multiple myelomas. This expression was not associated with specific cytogenetic abnormalities, especially alterations of chromosome 7q. Verapamil, a calcium channel blocker, has been demonstrated to circumvent the multidrug resistance in cell lines, possibly by interfering with P-glycoprotein function. Using the microculture tetrazolium assay, verapamil was demonstrated to increase the sensitivity of fresh leukaemic or myeloma cells to doxorubicin in 19/43 (43.1%) samples. The doxorubicin IC50 level and the capacity of verapamil to increase the sensitivity of blast cells to doxorubicin in vitro did not correlate with the expression of P-glycoprotein. We conclude that high non-cytotoxic concentrations of verapamil were able to increase the in vitro doxorubicin sensitivity of fresh acute leukaemia and myeloma cells without detectable expression of the P-glycoprotein.
Assuntos
Doxorrubicina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Verapamil/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Resistência a Medicamentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoRESUMO
Parathyroid hormone-related protein (PTHrP) is the main mediator of humoral hypercalcemia of malignancy (HHM) and it is produced by many tumors, including breast cancers. Breast epithelial cells as well as breast cancer tumors and cell lines have been reported as expressing PTHrP and the PTH/PTHrP receptor, suggesting that PTHrP may act as an autocrine factor influencing proliferation or differentiation of these cell types. We investigated PTHrP gene expression, PTH/PTHrP receptor signaling, and PTHrP-induced mitogenesis in three immortalized human mammary epithelial cell lines that exhibit differential tumorigenicity. The most tumorigenic cells expressed the highest levels of PTHrP messenger RNA (mRNA) and protein. We used reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting to detect the PTH/PTHrP receptor transcripts and proteins in all of the three cell lines. Treatment with human PTHrP(1-34) [hPTHrP(1-34)] and hPTH(1-34) increased intracellular cyclic adenosine monophosphate (cAMP) but not free Ca2+ in the nontumorigenic line. These agonists increased both cAMP and free Ca2+ levels in the moderately tumorigenic line, but only increased free Ca2+ in the highly tumorigenic line. Application of the PTH/PTHrP receptor antagonist [Asn10,Leu11,D Trp12]PTHrP(7-34) or PTHrP antibodies reduced [3H]thymidine incorporation in a dose-dependent fashion in the highly tumorigenic cell line but did not affect the other lines. Thus, treatment with a PTH/PTHrP receptor antagonist reduced cell proliferation, suggesting that PTHrP signaling mediated by the phospholipase C (PLC) pathway stimulates proliferation of a highly tumorigenic immortalized breast epithelial cell line.
Assuntos
Linhagem Celular Transformada/patologia , Epitélio/patologia , Proteínas/metabolismo , Animais , Sequência de Bases , Mama/patologia , Testes de Carcinogenicidade , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/virologia , AMP Cíclico/metabolismo , Epitélio/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/farmacologia , Proteínas/genética , Proteínas/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/antagonistas & inibidores , Receptores de Hormônios Paratireóideos/genética , Receptores de Hormônios Paratireóideos/metabolismo , Transdução de Sinais , Vírus 40 dos SímiosRESUMO
The purpose of the work was to assess the predictive value of biologic factors on the efficacy of highly active antiretroviral therapy alone or combined with chemotherapy on AIDS-associated Kaposi's sarcoma. Twenty-six AIDS-Kaposi's sarcoma patients who started therapy with protease inhibitors were investigated. No baseline chemotherapy was associated with less severe initial clinical status. Median follow-up was 652 d. The main outcome measures were as follows: best Kaposi's sarcoma clinical response; Kaposi's-sarcoma-associated herpesviral load in peripheral blood mononuclear cells using real-time quantitative polymerase chain reaction (non-detectable if less than 100 copies per microg); human immunodeficiency viral charge in plasma (non-detectable if less than 200 copies per ml); and CD4 lymphocyte count. Time to undetectable Kaposi's-sarcoma-associated herpesviral load, time to undetectable human immunodeficiency viral charge, and time to CD4 >or= 150 per microl were also recorded over time, from 2 mo measurements. Patients were staged according to the AIDS Clinical Trials Group-based tumor, immune, systemic staging system criteria. At baseline, Kaposi's sarcoma was progressive for 25 (96%) of the 26 enrolled patients. Complete or partial response to highly active antiretroviral therapy alone or combined with chemotherapy was achieved in 22 patients (85%). Median time to clinical response was estimated at 251 d. Clinical response was faster in patients without chemotherapy at baseline (p = 0.003) as well as in patients not previously treated with reverse transcriptase inhibitors (p = 0.0012). Using univariable analyses, predictive factors of clinical response were undetectable Kaposi's-sarcoma-associated herpesviremia (p = 0.013), undetectable human immunodeficiency viremia (p = 0.03), and relative variation of CD4 lymphocytes (p = 0.004). Using multivariable analysis, undetectable Kaposi's-sarcoma-associated herpesviremia (p = 0.009) and relative variation of CD4 (p = 0.005) were independently selected as having a predictive value for clinical response. Occurrence of nondetection of either Kaposi's-sarcoma-associated herpesvirus or human immunodeficiency virus was not associated with baseline CD4 value. Kaposi's-sarcoma-associated herpesvirus quantitative viral charge is an independent predictive factor of the efficacy of highly active antiretroviral therapy on AIDS-Kaposi's sarcoma. Our results support immune reconstitution as a mechanism of response of Kaposi's sarcoma to highly active antiretroviral therapy.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Terapia Antirretroviral de Alta Atividade , Proteínas de Bactérias , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/etiologia , Síndrome da Imunodeficiência Adquirida/virologia , Antígenos CD4/análise , Seguimentos , Proteínas de Choque Térmico/sangue , Infecções por Herpesviridae/etiologia , Infecções por Herpesviridae/virologia , Humanos , Monócitos/metabolismo , Monócitos/virologia , Prognóstico , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/virologia , Carga Viral , Viremia/virologiaRESUMO
We investigated somatostatin receptors (SSTRs) in surgical specimens of prostate cancer and benign prostate hyperplasia (BPH), a normal immortalized epithelial cell line (PNT1), epithelial cancer cell lines, and stromal cells in short-term culture derived from normal and BPH biopsies. Cross-linking studies with 125I-Tyr11-SRIF-14 (125I-SRIF) and the SRIF analog 125I-BIM-23104 identified one major 57-kDa band both in surgical specimens and in epithelial and stromal cells cultures. In membrane-enriched fractions and whole stromal cells from a normal prostate and from one BPH, a single type of SSTR was characterized (Kd = 6.10(-9) and 10(-8) M, respectively, Bmax = 1.6 pmol per mg of proteins). mRNA for SSTR1 was detected in all epithelial and stromal cells tested except for PNT1, while SSTR2 mRNA was detected in one BPH stromal cell culture. BIM-23104 had no effect on the in vitro growth of the epithelial cells tested. Conversely, 10(-10) M BIM-23104 induced >50% growth inhibition of stromal cells after 6 days in culture. These results may have implications for therapeutic strategies using SRIF analogs in BPH and prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Peptídeos Cíclicos/farmacologia , Próstata/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Sequência de Aminoácidos , Linhagem Celular , Células Cultivadas , Reagentes de Ligações Cruzadas , Epitélio/metabolismo , Expressão Gênica , Humanos , Radioisótopos do Iodo , Masculino , Dados de Sequência Molecular , Próstata/patologia , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Somatostatina/genética , Somatostatina/metabolismo , Somatostatina/farmacologia , Células Estromais/metabolismo , Células Tumorais CultivadasRESUMO
The human histiocytic lymphoma line U-937 consists of cells having characters of immature monocytes. We have demonstrated that these cells possess highly specific insulin receptors with binding properties similar to that found for mature human blood monocytes. 125I-insulin binding increased progressively with time to reach a maximum at 90 min at 22 degrees C and was proportional to the number of cells in the incubation medium. Insulin degradation as assessed by TCA precipitation was negligible. Scatchard analysis of the binding data was curvilinear and the total number of insulin binding sites was around 13,500. The average affinity profile gave an 'unoccupied site' affinity constant of 1.34 nM-1. When the U-937 cells were induced to differentiate into morphologically and functionally monocyte-like cells, after incubation with retinoic acid, the total number of binding sites decreased significantly with no change in the affinity of the hormone for its receptor.
Assuntos
Insulina/metabolismo , Leucemia Mieloide/metabolismo , Ligação Competitiva , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Glucagon/metabolismo , Humanos , Receptor de Insulina/metabolismo , Somatostatina/metabolismo , Tretinoína/farmacologiaRESUMO
The effect of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the active form of vitamin D3, on cell growth, clonogenicity, and cyclic adenosine monophosphate (cAMP) production was examined in human breast cancer cell line T47D. 1,25(OH)2D3 markedly inhibited proliferation of T47D cells in a time- and concentration-dependent manner. 1,25(OH)2D3 5 X 10(-7) reduced to 70% [3H]thymidine incorporation into DNA. Specific high affinity nuclear receptors for 1,25(OH)2D3 were present in this cell line. The cAMP produced by T47D cells was measured during 10 min stimulation by effectors (prostaglandin E1 or forskolin). Without effector, T47D cells produced similar amounts of cAMP in control and 1,25(OH)2D3-treated cells. After 3 days in the presence of 1,25(OH)2D3, cAMP production was significantly increased compared to control cells when stimulated by 10(-4) M prostaglandin E1 or 5 X 10(-7) M forskolin (3.2- and 2.4-fold increase, respectively). This cAMP increase was concentration dependent within the same range that inhibited cell growth and clonogenicity. These results suggest that 1,25(OH)2D3 may indirectly affect cAMP production by modulating the target cell response to stimulatory agents of cAMP production.
Assuntos
Alprostadil/farmacologia , Calcitriol/farmacologia , Colforsina/farmacologia , AMP Cíclico/biossíntese , Células Tumorais Cultivadas/metabolismo , Neoplasias da Mama/metabolismo , Calcitriol/metabolismo , Divisão Celular/efeitos dos fármacos , Humanos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The effects of recombinant gamma interferon (IFN gamma) on proliferation, estrogen-receptor (ER) content, mRNA level and protein secretion of a breast cancer estrogen-induced protein pS2/BCEI were investigated in two human breast cancer cell lines, ZR75-1 and T47D. Both cell lines have estrogen and progesterone receptors and previously showed HLA class I and class II responses to IFN gamma (Int J Cancer 1990; 45: 1169). An antiproliferative effect of IFN gamma (1000-5000 U/ml) was observed in serum containing medium on ZR75-1 but not on T47D cells. Noninhibitory concentration of IFN gamma (100 U/ml) had sensitising antiproliferative effect with antiestrogens on ZR75-1 cells whereas IFN gamma did not modify the growth inhibition observed in T47D cells with antiestrogens. In serum-free, estradiol-free, phenol-red-free chemically defined medium (Cancer Res 1984; 44: 4553), IFN gamma abolished in ZR75-1 but not in T47D the 30% growth stimulation induced by estradiol. In ZR75-1 cells, IFN gamma induced a transitory 30-50% increase of ER content, as measured by ER-enzymoimmunoassay, at day 2 of culture, and reduced mRNA level and secretion of pS2/BCEI. In T47D cells, a 30-50% decrease of ER content was observed but only when cells were long term cultured (30 weeks) with IFN gamma. In this cell line, no transcription of pS2/BCEI was observed. Antiproliferative action of IFN gamma on ZR75-1 cells is associated with an inhibition of estradiol effects and a reduction of pS2/BCEI mRNA level and protein secretion.
Assuntos
Neoplasias da Mama/terapia , Interferon gama/farmacologia , Proteínas , Receptores de Estradiol/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores de Estradiol/metabolismo , Proteínas Recombinantes , Serina/metabolismo , Fator Trefoil-1 , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Proteínas Supressoras de Tumor , Uridina/metabolismoRESUMO
Insulin release is coupled with a calcium entry into the pancreatic B cells. The use of calcium-antagonists may eventually alter glucose homeostasis. To evaluate this possibility, nicardipine action was tested both in vitro and in vivo: 1. on insulin release and vascular resistances from isolated perfused rat pancreases; 2. on 7 hypertensive patients with an established glucose intolerance, during two oral glucose tolerance tests (OGTT) performed successively under placebo and nicardipine (90 mg daily) at a two-week interval. In vitro, the basal insulin release from isolated perfused rat pancreases (86 +/- 15 ng.min-1; n = 27; M +/- SE) was inhibited according to the nicardipine dose by the 5 th min. of infusion: 7.2 +/- 1.5 p.100 of the initial output at 10(-4) M (n = 6; p less than %.001); 33.4 +/- 2.7 p.100 at 10(-6) M (n = 6; p less than 0.001); 87.5 +/- 14.8 p.100 at 10(-8) M (n = 6; ns). The pancreatic vascular resistances declined significantly for the 3 doses, but no dose-response could be registered. In vivo, the mean arterial blood pressure was significantly reduced by nicardipine from 114 +/- 3 mmHg to 95 +/- 3 mmHg (p less than 0.001) without any significant alteration of either glucose tolerance (glycaemias at the 120 th min of OGTT: 9.2 +/- 1.1 mmol.l-1 vs 8.9 +/- 1.2 mmol.-1) or insulin peak: 70 +/- 20 micrograms U.ml-1 vs 67 +/- 22 microU.ml-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Insulina/metabolismo , Nifedipino/análogos & derivados , Resistência Vascular/efeitos dos fármacos , Adulto , Idoso , Animais , Feminino , Humanos , Secreção de Insulina , Masculino , Pessoa de Meia-Idade , Nicardipino , Nifedipino/farmacologia , Ratos , Ratos EndogâmicosRESUMO
The effect of somatostatin on plasma C-peptide immunoreactivity was studied in seven normal men following an intravenous 25 g glucose load. No elevation in plasma insulin was observed and similarly no rise in plasma C-peptide immunoreactivity occurred, indicating that somatostatin inhibits glucose-induced C-peptide release as well as insulin release.
Assuntos
Peptídeo C/sangue , Peptídeos/sangue , Somatostatina , Glicemia/metabolismo , Peso Corporal , Peptídeo C/imunologia , Glucose , Humanos , Insulina/sangue , Masculino , Pessoa de Meia-Idade , RadioimunoensaioRESUMO
The antihypertensive effect of 2,000 mg of acebutolol investigated with an acute 48 hr test in 60 diabetic and 60 non-diabetic in-patients with essential hypertension. In hypertensive diabetic patients, acebutolol was induced a significant fall in blood pressure similar to that observed in non-diabetics. The acute antihypertensive effect of acebutolol was not uniform in hypertensive subjects: a significant decrease of blood pressure was observed in 34 diabetics and 31 non-diabetic patients. Fifteen out of the 34 diabetic responders to the 48 hr test were treated by acebutolol alone for six months; a highly significant correlation between the acute and the chronic antihypertensive effect of the beta-blocker was observed. As long-term results paralleled those of the short-term experiment, acute acebutolol administration appears to be a rapid means to select hypertensive diabetics sensitive or resistant to betablockers. Plasma renin activity was not found to give, in hypertensive diabetics, a reliable predictive index of the response to acute administration of acebutolol.
Assuntos
Acebutolol/uso terapêutico , Complicações do Diabetes , Hipertensão/diagnóstico , Pressão Sanguínea/efeitos dos fármacos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Renina/sangueRESUMO
Six normal subjects and six normotensive insulin-dependent diabetics underwent two insulin hypoglycaemia tests after administration for three days of either a placebo or of acebutolol--a cardioselective beta-blocker--at a dose of 400 mg per day. The order in which the tests were performed was decided by random selection. Acebutolol suppressed the tachycardia which occurred as a reaction to hypoglycaemia but did not interfere with other warning symptoms and signs. In both normal subjects and diabetics, acebutolol neither worsened the initial hypoglycaemia nor did it delay a return to normal values. The increase in lactate levels following hypoglycaemia was not reduced by acebutolol but free fatty acid rebound was suppressed. Hormonal responses (glucagon, cortisol, growth hormone) were unaffected by the beta-blocker. If they are confirmed by long term studies, these results would suggest that acebutolol is safer to use than non-cardioselective beta-blockers in the treatment of coronary insufficiency and of hypertension in diabetics exposed to the risk of hypoglycaemia.
Assuntos
Acebutolol/farmacologia , Diabetes Mellitus/sangue , Hipoglicemia/sangue , Doença Aguda , Adulto , Glicemia/metabolismo , Ácidos Graxos não Esterificados/sangue , Glucagon/sangue , Hormônio do Crescimento/sangue , Humanos , Hidrocortisona/sangue , Insulina/farmacologia , Lactatos/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
The aim of this prospective, randomized, double-blind, placebo controlled study was to investigate the effect of nifedipine on carbohydrate metabolism in diabetic patients after a 3-day and a 3-month course of treatment. Sixteen non obese, well controlled non-insulin dependent diabetics, (HbA1 less than 10%), with moderate untreated hypertension were divided in two groups: nifedipine (group N, 8 patients) and placebo (group P, 8 patients). An oral glucose tolerance test (OGTT, 75 g glucose) and an arginine infusion were performed before, after a 3-day, and a 3-month course, either of nifedipine 30 mg/D or placebo. Blood samples obtained during OGTT were assayed for glucose and insulin, and during arginine infusion for insulin, glucagon and growth hormone. The differences between basal and peak values during tests were compared between both groups before and after treatment using Wilcoxon's rank sum test. Neither acute nor chronic administration of nifedipine or placebo modified the glucose tolerance. However, basal insulin levels were reduced by 3 month-administration of nifedipine (from 19 +/- 2 micromicrons/ml to 10 +/- 1 micromicrons/ml, p = 0,01). Otherwise the basal and peak hormonal values during tests were not significantly affected by nifedipine either at the start of after 3 months of treatment. These results suggest that nifedipine, when given in standard dosage for 3 months, has minor effects on carbohydrate metabolism in non-insulin dependent diabetic patients.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Nifedipino/uso terapêutico , Adulto , Idoso , Arginina , Ensaios Clínicos como Assunto , Diabetes Mellitus Tipo 2/sangue , Método Duplo-Cego , Glucagon/sangue , Teste de Tolerância a Glucose , Hormônio do Crescimento/sangue , Humanos , Insulina/sangue , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Distribuição AleatóriaRESUMO
Nuclear magnetic resonance spectroscopy was performed on breast cancer cell lines MCF-7, MDA-MB231 and T47D. Proton spectra showed discrepancies of lipid quantity in the different lines. The high resolution lines of lipids were not as intense in the membrane preparations. These results show the potential of NMR spectroscopy to study the involvement of membrane lipids in proliferation, metastasis or drug resistance process.
Assuntos
Adenocarcinoma/química , Neoplasias da Mama/química , Lipídeos de Membrana/análise , Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Divisão Celular , Linhagem Celular Transformada , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
The anti-hypertensive effect of clonidine is closely related to its central alpha-agonist action. In acute administration, the drug provokes a significant increase in the plasma concentrations of growth hormone (GH). In chronic administration, the effects of clonidine on GH secretion are not well documented. Clonidine was administered at a daily dose of 0,15 to 0,30 mg to 10 non-obese diabetic hypertensive male subjects for at least 3 months. Blood glucose and GH plasma concentrations were determined 15 times during the 24-hour cycle. Blood glucose and GH values recorded before the intake and after stopping the drug, in the basal state, after meals, after measured muscular exercise and during the first stage of sleep could be superimposed when on and off clonidine. The effects of prolonged administration of clonidine on GH secretion thus differ markedly from the effects of acute administration as in non diabetic subjects. The long-term use of clonidine does not induce a chronic increase of GH plasma concentrations which might worsen the evolution of the diabetic microangiopathy.
Assuntos
Clonidina/uso terapêutico , Diabetes Mellitus/fisiopatologia , Hormônio do Crescimento/metabolismo , Hipertensão/complicações , Adulto , Glicemia/análise , Complicações do Diabetes , Diabetes Mellitus/sangue , Humanos , Hipertensão/tratamento farmacológico , Masculino , Pessoa de Meia-IdadeRESUMO
The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.
Assuntos
Bombesina/análise , Neoplasias da Mama/análise , Mama/análise , Peptídeos/análise , Receptores de Neurotransmissores/análise , Northern Blotting , Bombesina/metabolismo , Bombesina/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular , Membrana Celular/análise , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas/análise , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Epitélio/análise , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Peptídeo Liberador de Gastrina , Humanos , Peptídeos/metabolismo , Peptídeos/farmacologia , Ensaio Radioligante , Receptores da Bombesina , Receptores de Neurotransmissores/efeitos dos fármacos , Receptores de Neurotransmissores/metabolismo , Células Tumorais Cultivadas/análise , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
The mRNA levels of the ras-related human rhoA, rhoB and rhoC genes were studied in human breast-cancer cell lines (HBCal), and in normal and immortalized mammary epithelial cells (HMEC) by Northern blot analysis and in situ hybridization. In contrast to the ubiquitous rhoA and rhoC gene expression, dramatic variations in the mRNA level of the rhoB gene were evidenced. The rhoB mRNA level appeared to be inversely correlated to the amounts of the epidermal-growth-factor(EGF) receptors in these cells. The rhoB transcripts were detected at high levels in ZR75-1, MCF7, HSL 53, HSL 59, HSL 90, T47D and SKBR3 HBCal, at hardly detectable levels in BT 20, MDA-MB 231 and H466B HBCal and at intermediate levels in normal and immortalized breast epithelial cells. Rapid and transient induction of the rhoB transcription was observed after EGF treatment in serum-deprived MDA-MB231, T47D and immortalized epithelial cells. In contrast, no modulation of rhoB expression by EGF could be objectified in the MCF7 and ZR75-1 cell lines. Yet a normal function of EGF receptors was evidenced, since the immediate early gene c-fos was rapidly induced, suggesting a constitutive expression of rhoB in these cell lines bypassing the regulation by EGF. In human mammary epithelial cells, rhoB mRNA is rapidly and transiently induced with EGF concentrations known to stimulate cell proliferation. This suggests that the rhoB product might be involved in a cascade that initiates or promotes cell proliferation, and plays an important role in EGF-stimulated growth of breast normal and cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Epidérmico/farmacologia , GTP Fosfo-Hidrolases/biossíntese , Proteínas de Ligação ao GTP/biossíntese , Regulação Neoplásica da Expressão Gênica/imunologia , Proteínas de Membrana/biossíntese , Northern Blotting , Feminino , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Humanos , Hibridização In Situ , Proteínas de Membrana/genética , Sondas de Ácido Nucleico , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Células Tumorais Cultivadas , Proteína rhoB de Ligação ao GTPRESUMO
The lipid composition of five human breast cancer cell lines (MCF-7, T47D, ZR-75-1, SKBR3 and MDA-MB231) was assessed by proton magnetic resonance spectroscopy (MRS) in whole cells and membrane-enriched fractions. The proportions of the three main lipid resonances in 1D spectra were different for each cell line. These resonances included mobile methyl and methylene functions from fatty acids of triglycerides and phospholipids and N-trimethyl from choline of phospholipids. T47D and ZR-75-1 cells presented a high methylene/methyl ratio (6.02 +/- 0.35 and 6.28 +/- 0.90). This ratio was significantly lower for SKBR3, MCF-7 and MDA-MB231 cells (2.76 +/- 0.22, 2.27 +/- 0.57 and 1.39 +/- 0.39). The N-trimethyl/methyl ratio was high for MDA-MB231 and SKBR3 cells (1.38 +/- 0.54 and 0.86 +/- 0.32), but lower for MCF-7, T47D and ZR-75-1 cells (0.49 +/- 0.11, 0.16 +/- 0.07 and 0.07 +/- 0.03). 2D COSY spectra confirmed these different proportions in mobile lipids. From 1D spectra obtained on membrane preparations, T47D and ZR-75-1 were the only cell lines to retain a signal from mobile methylene functions. These differences might be related to the heterogeneity found for several parameters of these cells (tumorigenicity, growth rate, hormone receptors); an extended number of cases from fresh samples might enable clinical correlations.
Assuntos
Neoplasias da Mama/química , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/análise , Sequência de Carboidratos , Feminino , Humanos , Lipídeos de Membrana/química , Dados de Sequência Molecular , Prótons , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Tumorais CultivadasRESUMO
H466-B and T47-D breast carcinoma cell lines were treated with recombinant gamma interferon (r gamma IFN) to study major histocompatibility complex (MHC) class I and class II antigen responses. Untreated H466-B cells released B2 microglobulin (B2M) into the culture medium and expressed B2M and class I heavy chain on 100% of the cells. The expression of class II antigens (DR) was limited to 8 +/- 4% of the cells. This subpopulation was isolated by cell sorting and labelled with 35S methionine. Protein extracts were immunoprecipitated with anti-DR antibody and subjected to two dimensional non-equilibrium pH gradient electrophoresis (2D-PAGE). A normal pattern of expression of invariant, alpha and beta chains was shown. The MHC antigenic expression of H466-B parental cell line was not modified by interferon treatment. Untreated T47-D cells did not release B2M into the culture medium, expressed B2M and class I heavy chain on 100% of the cells but did not express class II molecules using radio-immunoassay or 2D-PAGE. As early as 24 h after r gamma IFN addition, T47-D cells released B2M into the medium, B2M and class I heavy chain were significantly greater than that of untreated cells, and class II antigenic expression was found, all these in a dose dependent manner. 2D-PAGE analysis of class II antigens revealed the profile of human DR molecules but this expression seemed incomplete since only single alpha and beta spots were detected suggesting a possible defect in the sialilation of DR molecules. These results show a heterogeneity in MHC antigenic responses to r gamma IFN and suggest that synthetized class II molecules may be incompletely processed.