RESUMO
The BCR/ABL hybrid gene plays a central role in the pathogenesis of the chronic phase of chronic myeloid leukemia (CML). We used a very sensitive quantitative reverse transcriptase-polymerase chain reaction to investigate the levels of hybrid BCR/ABL mRNA in bone marrow cells of 20 patients with Philadelphia positive (Ph(+)) CML treated with interferon-alpha (IFN-alpha) as a single agent. Bone marrow samples were collected at diagnosis and at hematologic remission induced by IFN-alpha, or by hydroxyurea in case of resistance to IFN-alpha. The mean levels of BCR/ABL transcripts in bone marrow mononuclear cells of patients who showed a complete hematologic response to IFN-alpha were significantly reduced with respect to those at diagnosis (48 x 10(3) v 168 x 10(3); P <.001), whereas no difference was detected between the values at diagnosis and at hematologic remission in patients resistant to IFN-alpha. In cell culture experiments, IFN-alpha priming significantly reduced the levels of BCR/ABL hybrid transcripts in a dose-dependent manner in Ph+ bone marrow precursors obtained at diagnosis from patients who subsequently responded to IFN-alpha treatment (P < .005). No downmodulation was observed in bone marrow precursors from patients who subsequently proved to be IFN-resistant. These results indicate that downmodulation of BCR/ABL gene expression could be one of the mechanisms involved in the response of CML patients to IFN-alpha treatment.
Assuntos
Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Transcrição Gênica , Antineoplásicos/uso terapêutico , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Hidroxiureia/uso terapêutico , Contagem de Leucócitos , Monócitos/metabolismo , Monócitos/patologia , Cromossomo Filadélfia , Contagem de Plaquetas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fas-R is expressed constitutively in CD34(+) cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that alpha-interferon (IFN-alpha) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-alpha can prime CML cells for the effects of Fas, the response to IFN-alpha in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34(+) CML cells after Fas-R triggering and the clinical response to IFN-alpha. After priming with IFN-alpha, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-alpha; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-alpha. In responders to IFN-alpha, Fas-R agonist induced dose-dependent apoptosis of CD34(+) cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-alpha, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and no bcr/abl downmodulation was observed. Finally, we measured bcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/abl protein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210 bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-alpha treatment.
Assuntos
Apoptose/fisiologia , Biomarcadores Tumorais/biossíntese , Proteínas de Fusão bcr-abl/biossíntese , Interferon-alfa/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide de Fase Crônica/patologia , Receptor fas/fisiologia , Adulto , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Relação Dose-Resposta a Droga , Proteína Ligante Fas , Feminino , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia Mieloide de Fase Crônica/terapia , Masculino , Glicoproteínas de Membrana/farmacologia , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Células Tumorais Cultivadas , Receptor fas/biossíntese , Receptor fas/genéticaAssuntos
Neoplasias/química , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , Sequência de Bases , Biópsia , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia/genética , Leucemia/patologia , Fígado/química , Fígado/enzimologia , Fígado/patologia , Linfócitos/química , Linfócitos/enzimologia , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias/patologia , RNA Mensageiro/síntese química , RNA Neoplásico/análise , DNA Polimerase Dirigida por RNA , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Homologia de Sequência do Ácido Nucleico , Superóxido Dismutase/genéticaRESUMO
No laboratory test completely distinguishes malignant ascites (MA) from ascites associated with cirrhosis and (or) hepatocellular carcinoma (A/C-HC). Ascitic cytology is highly specific but has a diagnostic sensitivity of only 40-60%. We determined 11 ascitic analytes and cytology in 58 patients with cirrhosis, 15 with hepatocellular carcinoma, and 21 with MA (10 ovarian cancers, 4 mesotheliomas, 6 gastrointestinal neoplasias, 1 leukemia). Ascitic total protein, cholesterol, pseudouridine, and lactate dehydrogenase (LD), and the ascitic:serum ratios of total protein and of LD showed the most significant differences between the two groups of patients. Stepwise multiple linear discriminant analysis (applying the Wilks' lambda criterion) of several variables, corroborated by the "jack-knife" reallocation procedure, showed that the ascitic cholesterol and ascitic LD association correctly identified 100% of MA and A/C-HC; cytology had a diagnostic specificity of 100%, but identified only 48% of MA. This association may represent a primary tool for the discrimination of ascites of unknown origin, particularly in the presence of negative cytology findings.
