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Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid

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DNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells. Methods Cell lines C33A (HPV-), CaSki (HPV-16+) and MS751 (HPV-18+) were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 μM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid. Results Valproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma. Conclusion These results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines is used.
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Journal of Translational Medicine Bio Med  Central
Research Open Access Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cerv ical cancer cells by the demethylating agent hydralazine and the histone de acetylase inhibitor valproic acid María de Lourdes Mora-García 1 , Alfonso Duenas-González 2 , Jorge Hernández-Montes 1,7 , Erick De la Cruz-Hernández 2 , Enrique Pérez-Cárdenas 2 , Benny Weiss-Steider 1 , Edelmiro Santiago-Osorio 3 , Vianney Francisco Ortíz-Navarrete 4 , Víctor Hugo Rosales 4 , David Cantú 2 , Marcela Lizano-Soberón 2 , Martha Patricia Rojo-Aguilar 5 and Alberto Monroy-García* 6
Address: 1 Laboratorio de Inmunobiología, Unidad de Investigación en Diferenciación Celu lar y Cáncer. FES-Zaragoza, UNAM, México, 2 Unidad de Investigación Biomédica en Cáncer, Institut o de Investigaciones Biomédicas, UNAM, Inst ituto Nacional de Cancerología, México, 3 Laboratorio de Biología Molecular del Cáncer, Unidad de Investigación en Dife renciación Celular y Cáncer, FES-Zaragoza, UNAM, México, 4 Departamento de Biomedicina, CINVESTAV, IPN, México, 5 Unidad de Reumatologia, IMSS, CMN SXXI, México, 6 Unidad de Investigación Médica en Enfermedades Oncológicas. IMSS, CMN SXXI, México and 7 Alumno del Doctorado en Ciencias Biológicas UNAM, México Email: María de Lourdes Mora-García - mogl@s ervidor.unam.mx; _duenasg@yahoo.com; J g z-Alfonso Duenas-González - alfonso or e Hernánde Montes - jhzms@yahoo.com; Erick De la Cruz-H ernández - qcerick2000@yahoo.com; Enrique Pérez- Cárdenas - zperez@salud.gob.mx; Benny Weiss-Steider - bennyweiss@hotmail.com; Edelmiro Santiago-O sorio - edelmiros@yahoo.com.mx; Vianney Francisco Ortíz-Navarrete - vortiz@mail.cinvestav.mx; Víctor Hugo Rosales - vícto r@yahoo.com; David Cantú - dcanru3 @excite.com; Marcela Lizano-Soberón - mlsoberon@yahoo.com.mx; Martha Patricia Rojo-Aguilar - mroj oa@cis.gob.mx; Alberto Monroy-García* - albertomon@yahoo.com * Corresponding author
Published: 27 December 2006 Received: 30 June 2006 Journal of Translational Medicine 2006, 4 :55 doi:10.1186/1479-5876-4-55 Accepted: 27 December 2006 This article is available from: http://www .translational-medicine.com/content/4/1/55 © 2006 Mora-García et al; licensee BioMed Central Ltd. This is an Open Access article distribute d under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/2.0), which per-mits unrestricted use, distribution, and reproduction in any medium, provided the orig inal work is properly cited.
Abstract Background: DNA hypermethylation and histone deacetylation are ep igenetic events that contribute to the absence or downregulated expression of different comp onents of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on th e expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells. Methods: Cell lines C33A (HPV-), CaSki (HPV-16+) and MS751 (HPV -18+) were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Pr imary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 µ M of the HPV peptides. Cytotoxicity of stimulated CTL's was as sayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid. Results: Valproic acid and hydralazine/valproic acid up-regulated th e constitutive HLA class-I expr ession as evaluated by flow cytometry and RT-PCR despite constitutive promoter demeth ylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetyla ted histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma. Conclusion: These results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines is used.
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