RESUMEN
Downregulation of HLA class I antigen expression has been reported in a significant proportion of primary breast carcinomas suggesting an escape mechanism from CTL mediated lysis leading to tumor dissemination and metastasis. We have previously reported the biochemical and immunohistochemical analysis of HLA total class I (W6/32 mAb), alpha-chain (Q1/28,TP25.99 mAbs) and beta(2)-microglobulin (Namb-1 mAb) subunits expression in 25 primary breast carcinomas. This study at protein level resulted in the observation of three different HLA class I expression patterns by both techniques: high, low, and absent downregulation patterns. To better characterize the HLA class I antigens downregulation we extended such analysis also at RNA level by RT-PCR using HLA-A, HLA-B, HLA-C, and beta(2)-microglobulin specific primers either in breast cancer or normal tissues derived from the same patient. None (100%) of the alpha-chain genes analyzed in patient tumor tissues showed significant reduction of expression. In 10 patients out of 25 (40%) the beta(2)-microglobulin gene showed complete loss of expression compared with the corresponding normal tissue counterpart, which showed a constitutive expression, whereas in 2 patients (12.5%) its expression was comparable with the normal counterpart. Sequence analysis at genomic level revealed no defects affecting beta(2)-microglobulin gene in those patients showing lack of expression. Also TAP1 and TAP2 genes expression were investigated in order to confirm or exclude involvement of the MHC class I molecules assembling machinery. The RT-PCR approach mainly confirmed our beta(2)-microglobulin biochemical analysis indicating that in breast cancer specimens it is possible to address the HLA class I gene downregulation as a phenomenon occurring at post-transcriptional level mainly affecting the beta(2)-microglobulin gene expression.
Asunto(s)
Neoplasias de la Mama/inmunología , Regulación hacia Abajo/inmunología , Antígenos HLA/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Actinas/genética , Anticuerpos Monoclonales/análisis , Western Blotting , Neoplasias de la Mama/química , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Antígenos HLA/genética , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunohistoquímica , Células K562 , Microglobulina beta-2/genética , Microglobulina beta-2/inmunologíaRESUMEN
The expression of HLA class I alpha-chain and beta(2)-m subunits was studied at the protein level by a semiquantitative Western blot (WB) approach, in 25 primary breast tumors. The results indicated three pathways of alterations defined comparing the tumor WB gel band with the corresponding PBL gel band: (i) high downregulation pattern (the tumor WB gel band was < or =50% relative to the PBL band), which was found in 44% and 36% of tumors for alpha-chain and beta(2)-m, respectively; (ii) low downregulation pattern (the tumor gel band was between 51% and 75%), which was found in 24% and 20% of tumors for alpha-chain and beta(2)75%), which was found in 32% and 44% of tumors for alpha-chain and beta(2)-m, respectively. The concordance rate with immunohistochemistry (IHC) performed on the same tissue samples was 72% for alpha-chain and 64% for beta(2)-m. This study shows that the use of a semiquantitative WB technique can well define the levels of HLA class I antigens in an autologous setting allowing the biochemical analysis of HLA class I downregulation directly in solid tumor tissues. In addition, the WB technique can be a valuable tool to objectively support the IHC method.
