RESUMEN
Melanoma is heterogeneous for its biological properties and melanoma-associated antigens (MAAs). This diversity is partially observed in the expression of the MAAs involved with the melanin synthesis pathway. We therefore developed a sensitive multimarker reverse transcription-PCR plus Southern blot assay using five MAAs as molecular markers to detect primary and metastatic melanoma cells. Melanoma cell lines, melanocytes (cultured), primary and metastatic malignant melanoma tissues, and blood from patients with American Joint Committee on Cancer stage I-IV melanoma were assessed for tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17, and MART-1/Melan-A. All of the MAA mRNA markers were expressed in 100% of melanoma cell lines and cultured melanocytes, 74% of primary and metastatic tumors (excluding tumor-draining lymph nodes), 43% of tumor-involved lymph nodes, and 43% of patients' bloods. Hypomelanotic melanoma tissues expressed a lower frequency of individual mRNA markers. Overall, at least one mRNA marker was expressed in more than 86% of specimens assayed. Normal tissue specimens from patients and blood from normal volunteer donors were negative for MAA mRNA expression. The multimarker MAA reverse transcription-PCR plus Southern blot analysis was more reliable and sensitive than a single-molecular marker assay for the detection of melanoma cells. This molecular assay can also provide information on MAA mRNA expression of metastatic melanoma cells that may assist in monitoring the therapeutic efficacy of active specific immunotherapy toward specific MAA-bearing melanomas.
Asunto(s)
Antígenos de Neoplasias , Biomarcadores de Tumor/análisis , Oxidorreductasas Intramoleculares , Melanoma/diagnóstico , Sondas ARN , ARN Mensajero/análisis , Neoplasias Cutáneas/diagnóstico , Southern Blotting , Humanos , Interferón gamma/sangre , Interferón gamma/genética , Isomerasas/sangre , Isomerasas/genética , Antígeno MART-1 , Melanoma/genética , Melanoma/inmunología , Glicoproteínas de Membrana , Monofenol Monooxigenasa/sangre , Monofenol Monooxigenasa/metabolismo , Proteínas de Neoplasias/sangre , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , Proteínas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Células Tumorales Cultivadas , Antígeno gp100 del MelanomaRESUMEN
Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277). rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E. coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro. Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates. Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic. Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP. These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.
Asunto(s)
Cisteína/análogos & derivados , Inmunotoxinas/química , Proteínas de Plantas/química , Ribosomas/efectos de los fármacos , Toxinas Biológicas , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cisteína/química , Cisteína/inmunología , Pruebas Inmunológicas de Citotoxicidad , Disulfuros/química , Estabilidad de Medicamentos , Escherichia coli/metabolismo , Humanos , Inmunotoxinas/inmunología , Inmunotoxinas/farmacología , Ratones , Datos de Secuencia Molecular , N-Glicosil Hidrolasas , Proteínas de Neoplasias/biosíntesis , Proteínas de Plantas/inmunología , Proteínas de Plantas/farmacología , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
A cDNA copy of the gel gene, encoding gelonin (Gel), has been cloned from the seeds of the Asian plant, Gelonium multiflorum. Gel is a type-I ribosome-inactivating protein which has been produced in Escherichia coli as a secreted protein under the transcriptional control of the Salmonella typhimurium araB promoter and linked to the pectate lyase (pelB) leader sequence from Erwinia carotovora. Recombinant, soluble Gel (re-Gel) can be recovered from the E. coli culture supernatant at a yield of greater than 1 mg/ml, and it inhibits protein synthesis in vitro to the same extent as the native protein isolated from plant seeds.