RESUMO
BACKGROUND: Clinical outcome of high-risk melanoma patients is not reliably predicted from histopathological analyses of primary tumours and is often adjusted during disease progression. Our study aimed at extending our previous findings in skin metastases to evaluate the prognostic value of tyrosinase-related protein 1 (TYRP1) in lymph node metastases of stages III and IV melanoma patients. METHODS: TYRP1 mRNA expression in 104 lymph node metastases was quantified by real-time PCR and normalised to S100 calcium-binding protein B (S100B) mRNA expression to correct for tumour load. TYRP1/S100B ratios were calculated and median was used as cutoff value. TYRP1/S100B mRNA values were correlated to clinical follow-up and histopathological characteristics of the primary lesion. RESULTS: A high TYRP1/S100B mRNA ratio significantly correlated with reduced disease-free (DFS) and overall survival (OS; Cox regression analysis, P=0.005 and 0.01, respectively), increased Breslow thickness (Spearman's rho test, P<0.001) and the presence of ulceration (Mann-Whitney test, P=0.02) of the primaries. Moreover, high TYRP1/S100B was of better prognostic value (lower P-value) for OS than Breslow thickness and ulceration. Finally, it was well conserved during disease progression with respect to high/low TYRP1 groups. CONCLUSION: High TYRP1/S100B mRNA expression in lymph node metastases from melanoma patients is associated with unfavourable clinical outcome. Its evaluation in lymph node metastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lesions and may allow different management of the two groups of patients.
Assuntos
Linfonodos/metabolismo , Melanoma/genética , Glicoproteínas de Membrana/genética , Oxirredutases/genética , RNA Mensageiro/biossíntese , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Intervalo Livre de Doença , Feminino , Humanos , Linfonodos/patologia , Metástase Linfática , Masculino , Melanoma/enzimologia , Melanoma/patologia , Glicoproteínas de Membrana/biossíntese , Pessoa de Meia-Idade , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/genética , Oxirredutases/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/biossíntese , Proteínas S100/genética , Taxa de Sobrevida , Adulto JovemRESUMO
BACKGROUND: Clinical outcome of patients with high-risk melanoma cannot be reliably predicted on the basis of classical histopathological examination. Our study aimed to determine in melanoma metastases a gene expression profile associated with patient survival, and to identify and validate marker(s) of poor clinical outcome. METHODS: Skin and lymph node metastases from melanoma patients (training population) were used to identify candidate prognostic marker(s) based on DNA microarray analysis. Additional skin metastases (validation population) were used to assess the prognostic value of the first ranked gene by real-time PCR. RESULTS: We performed microarray analysis in the training population and generated a list of 278 probe sets associated with a shorter survival. We used the first ranked gene, tyrosinase-related protein 1 (TYRP1), further measured its expression in the validation population by real-time PCR and found it to be significantly correlated with distant metastasis-free survival (DMFS), overall survival (OS) and Breslow thickness. We also found that it was fairly well conserved in the course of the disease regardless of the delay to metastasis occurrence. Finally, although Tyrp1 protein (immunohistochemistry (IHC)) was only detected in about half of the samples, we showed that its expression also correlated with Breslow thickness. CONCLUSION: Our data indicate that TYRP1 mRNA expression level, at least in skin metastases, is a prognostic marker for melanoma, and is particularly useful when prognostic pathology parameters at the primary lesion are lacking. Its conserved expression further supports its use as a target for therapy.
Assuntos
Melanoma/genética , Glicoproteínas de Membrana/genética , Oxirredutases/genética , RNA Mensageiro/biossíntese , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Testes Genéticos/métodos , Humanos , Metástase Linfática , Masculino , Melanoma/enzimologia , Melanoma/patologia , Melanoma/secundário , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oxirredutases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/secundário , Resultado do Tratamento , Adulto JovemRESUMO
Clara cell secretory protein (CC16, CC10, or CCSP), the major secretory protein of the Clara cell, presents several biologic properties, suggesting that it may play a protective role against intrapulmonary inflammatory processes. The aim of the present study was to investigate the changes of CC16 concentrations in the lung, bronchoalveolar lavage fluid (BALF), and serum of rats with acute lung injury induced by lipopolysaccharide (LPS). These changes were compared with Clara cell density, CC16 mRNA level in the lung and classic indices of inflammation in BALF. Injected at doses of 10, 100, or 200 microgram/100 g body weight, LPS induced an acute lung inflammation as estimated by an increased influx of cells and albumin in the BALF. This inflammatory response was associated with a marked reduction of CC16 concentrations in BALF and lung homogenate as well as of the CC16 mRNA levels in the lung. At the highest dose of LPS, the CC16-positive cell density in the bronchiolar epithelium was also decreased. In serum, by contrast, the concentration of CC16 was elevated as a consequence of increased airway permeability. Pretreating rats intraperitoneally with dexamethasone (2 mg/kg) significantly lowered the leukocyte influx and attenuated the albumin increase in BALF. Dexamethasone, however, failed to prevent the increased airway permeability to CC16, suggesting that during inflammation different mechanisms regulate the leakage of proteins across the alveolocapillary barrier depending on the direction of passage and/or the size of the protein. Our results show a marked decrease of the secretion and synthesis of CC16 during LPS-induced acute lung inflammation.
Assuntos
Proteínas/análise , Síndrome do Desconforto Respiratório/metabolismo , Uteroglobina , Actinas/análise , Animais , Anti-Inflamatórios/farmacologia , Northern Blotting , Brônquios/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Lipopolissacarídeos/administração & dosagem , Pulmão/química , Pulmão/patologia , Masculino , Proteínas/genética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/patologiaRESUMO
The 16 kDa Clara cell protein (CC16), an abundant component of airway secretions, has recently been proposed in humans as a pulmonary marker measurable not only in bronchoalveolar lavage fluid (BALF) but also in serum. The aim of the present study was to investigate the changes and determinants of CC16 concentrations in these fluids in normal rats and rats with lung injury. Female Sprague-Dawley rats were given a single i.p. injection of arachis oil (n=20) or chemicals in arachis oil (n=10) that mainly damage Clara cells (4-ipomeanol (IPO) 8 mg x kg(-1) and methylcyclopentadienyl manganese tricarbonyl (MMT) 5 mg x kg(-1)) or endothelial cells (alpha-naphthylthiourea (ANTU) 5 mg x kg(-1)). CC16 concentration (mean+/-sD in microg x L(-1)), measured by a sensitive latex immunoassay, was significantly reduced in BALF of all treated groups (IPO 380+/-100; MMT 730+/-200; ANTU 1,070+/-200; controls 1,700+/-470). The same pattern of decrease was observed in the labelling of Clara cells with an anti-CC16 antiserum as well as in the CC16 messenger ribonucleic acid levels assessed by Northern enzyme-linked immunosorbent assay. In serum, by contrast, CC16 was significantly increased in all treated groups (IPO 31+/-7; MMT 22+/-12; ANTU 52+/-24; controls 15+/-6). This rise of CC16 in serum was associated with an elevation of albumin in BALF which is an index of increased bronchoalveolar/blood barrier permeability. In conclusion, lung injury induces a decrease of the 16 kDa Clara cell protein in bronchoalveolar lavage fluid owing to a reduced production by damaged Clara cells, and an increase in serum protein levels resulting from its enhanced leakage across the bronchoalveolar/blood barrier. This study provides new insights into the understanding of the changes of lung secretory proteins in bronchoalveolar lavage fluid and serum.
Assuntos
Inibidores Enzimáticos/metabolismo , Fosfolipases A/antagonistas & inibidores , Proteínas/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Uteroglobina , Animais , Barreira Alveolocapilar/fisiologia , Líquido da Lavagem Broncoalveolar/química , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Feminino , Pulmão/efeitos dos fármacos , Pulmão/patologia , Ratos , Ratos Sprague-Dawley , Síndrome do Desconforto Respiratório/patologiaRESUMO
Clara cell protein is a 16-17 kDa protein (CC16) secreted by Clara cells in the bronchiolar lining fluid of the lung. In order to investigate the potential of this protein as a pulmonary marker in animals, CC16 was isolated from rat bronchoalveolar lavage fluid (BALF) and a sensitive latex immunoassay applicable to both rat and mouse CC16 was developed. The pattern of CC16 concentrations in rat biological fluids determined by the immunoassay was consistent with the hypothesis of a passive diffusion of the protein across the bronchoalveolar/blood barriers showing a difference of more than 5,000 fold between the concentration in the epithelial lining fluid (mean, 140 mg x L(-1)) and that in serum (20 microg x L(-1)) or urine (3 microg x L(-1)). In BALF, the CC16 concentration averaged 5,500 microg x L(-1) and was of the same magnitude as that determined on lung and trachea homogenates. CC16 was also detectable in amniotic fluid with a mean value of 800 microg x L(-1) before delivery. Damage of Clara cells produced by methylcyclopentadienyl manganese tricarbonyl resulted in a significant decrease of CC16 in BALF but did not affect the serum levels of the protein. The nephrotoxicant sodium chromate by contrast had no influence on the CC16 content of BALF but markedly increased CC16 levels in both serum and urine as a result of impaired glomerular filtration and tubular reabsorption, respectively. In conclusion, mouse or rat Clara cell protein of 16-17 kDa can easily be quantified, not only in bronchoalveolar lavage fluid, but also in extrapulmonary fluids such as serum or urine. Thus, in rodents, Clara cell protein of 16-17 kDa follows the same metabolic pathway as in humans, diffusing from the respiratory tract into serum where it is eliminated by the kidneys. This serum Clara cell protein of 16-17 kDa may be useful as a peripheral marker of events taking place in the respiratory tract.
Assuntos
Inibidores Enzimáticos/análise , Fosfolipases A/antagonistas & inibidores , Proteínas/análise , Uteroglobina , Animais , Western Blotting , Líquidos Corporais/química , Líquido da Lavagem Broncoalveolar/química , Inibidores Enzimáticos/metabolismo , Feminino , Imunoensaio/métodos , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Gravidez , Proteínas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Human placental lactogen B (hCS-B) promoter activity is strongly stimulated by triiodothyronine (T3) in pituitary GC cells through interaction between the thyroid receptor and a thyroid receptor-binding element (TBE) spanning coordinates -67 to -41. This TBE is adjacent to the binding site for pituitary factor GHF1 (-95 to -68) which seems necessary for T3 stimulation of hCS-B promoter activity (M. L. Voz, B. Peers, A. Belayew, and J. A. Martial, J. Biol. Chem. 266:13397-13404, 1991). We here demonstrate actual synergy between the thyroid receptor and GHF1. Indeed, in placental JEG-3 cells devoid of factor GHF1, hCS promoter activity is barely stimulated by T3, while a strong response is observed in pituitary GC cells. In the latter, furthermore, neither the TBE nor the GHF1-binding site alone is sufficient to render the thymidine kinase promoter responsive to T3, while in combination they promote strong T3 stimulation. Close proximity between these sites is required for optimal synergy: T3 stimulation globally decreases with increased spacing. Furthermore, synergy occurs not only with a GHF1-binding site but also with all other factor recognition sequences tested (Sp1, NF1, CP1, Oct1, and CACCC boxes) and even with two other copies of the TBE. Nor is it specific to hCS TBE, since the palindromic sequence TCAGGTCA TGACCTGA (TREpal) also exhibits cooperativity.