Asunto(s)
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Estabilidad de Enzimas , Magnesio/metabolismo , Manganeso/metabolismo , Virus de la Leucemia Murina de Moloney/enzimología , Virus de la Leucemia Murina de Moloney/genética , Ingeniería de Proteínas , ARN Helicasas/genética , ARN Helicasas/metabolismo , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y Especificidad , Thermococcus/enzimología , Thermococcus/genéticaRESUMEN
The SSX family proteins have been considered new members of the cancer/testis antigens because of the restricted expression in testis among normal tissues and the activation in a wide range of cancers. Thus, they would be potential molecular targets for immunotherapeutic strategies. We have developed a competitive nucleic acid sequence-based amplification (NASBA) assay to analyze SSX mRNA expression in 211 bone and soft tissue tumors. The copy numbers of SSX mRNA per mug of total RNA in tumor tissues were widely distributed, ranging logarithmically from 0.6 to 6.6. We found that malignant tumors showed significantly higher expression of SSX mRNA than benign tumors (P < 0.0001). Further, SSX mRNA expression in stage III tumors was significantly higher than that in stage I or II tumors (P < 0.005). This NASBA assay was also more sensitive compared to immunohistochemistry using newly affinity-purified polyclonal antibody against SSX. Collectively, these results suggest that the SSX quantitative NASBA assay could provide useful information to select eligible patients for SSX-specific cancer vaccines.
Asunto(s)
Neoplasias Óseas/genética , Proteínas de Neoplasias/genética , ARN Mensajero/análisis , Proteínas Represoras/genética , Replicación de Secuencia Autosostenida , Neoplasias de los Tejidos Blandos/genética , Adolescente , Adulto , Anciano , Animales , Neoplasias Óseas/inmunología , Células COS , Niño , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , ARN Mensajero/metabolismo , ARN Neoplásico/análisis , ARN Neoplásico/metabolismo , Proteínas Represoras/análisis , Neoplasias de los Tejidos Blandos/inmunologíaRESUMEN
Accurate quantification of multidrug resistance-1 gene (MDR1) expression in target cells would be of important therapeutic value in predicting cellular response to anticancer drugs. Because certain normal cells in peripheral blood physiologically express MDR1, increasing the sensitivity of the detection methods might result in confounding low-degree expression in tumor cells with physiologic expression in normal cells. The purpose of this study was to determine MDR1 mRNA expression levels in peripheral blood leukocytes obtained from healthy adult volunteers using a competitive nucleic acid sequence-based amplification (NASBA) assay. We determined the reference intervals of MDR1 mRNA expression in peripheral blood obtained from 98 healthy adults by measuring its expression with the quantitative NASBA assay between 5.50 x 10(4) copies/microg RNA and 6.76 x 10(5) copies/microg RNA. The new reference intervals were evaluated using a number of sensitive or resistant cell lines as control; positive or negative MDR1 expression was clearly demonstrated. We also reevaluated MDR1 expression levels in leukemia cells obtained from patient peripheral blood; 18 of 31 samples (58%) exceeded the newly established upper reference limit. The cutoff value established could be used to distinguish significant MDR1 expression in tumor cells from physiologic expression in certain normal cells coexistent in peripheral blood.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Leucocitos Mononucleares/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adulto , Estudios de Casos y Controles , Expresión Génica , Humanos , ARN Mensajero/análisis , Replicación de Secuencia Autosostenida/métodos , Células Tumorales CultivadasRESUMEN
BACKGROUND: Because clinical drug resistance is caused by low-grade expression of a responsible gene, highly sensitive methods are desirable for its detection in clinical settings. We developed a quantitative nucleic acid sequence-based amplification (NASBA) assay for multidrug resistance-1 (MDR1) transcripts, and applied it to clinical samples. METHOD: MDR1 transcripts were amplified using the NASBA technique combined with sandwich hybridization of amplified MDR1 mRNA followed by chemiluminescence detection on an automated analyzer. Quantification of MDR1 mRNA was achieved through competitive coamplification of in vitro-generated RNA, which acts as an internal control. RESULTS: The competitive NASBA assay exhibited higher sensitivity (reliable detection limit was 100 copies of MDR1 mRNA) and linearity over a broader dynamic range (7 logarithmic orders) than the competitive reverse transcription-polymerase chain reaction assay. All 33 clinical samples obtained from patients with leukemia were successfully assayed, demonstrating its feasibility. MDR1 expression-compensated with beta-actin expression-ranged from 1.4 x 10(2) to 2.5 x 10(6) (median 4.8 x 10(5)) copies/microg RNA, while the range of MDR1 expression in peripheral blood samples from 15 healthy adults was from 8.9 x 10(4) to 5.2 x 10(5) (median 2.2 x 10(5)) copies/microg RNA. MDR1 expression in 8 of 33 clinical samples exceeded the median of healthy adult samples. CONCLUSIONS: The competitive NASBA assay is applicable to MDR1 mRNA quantification in clinical samples and would contribute to detection of clinical multidrug resistance.
