Vincristine regulates the phosphorylation of the antiapoptotic protein HSP27 in breast cancer cells Pedro Casado, Pedro Zuazua-Villar, Eva del Valle, Carlos Martı ´nez-Campa 1 Pedro S. Lazo, Sofı ´a Ramos * Departamento de Bioquimica y Biologı´a Molecular, Instituto Universitario de Oncologı´a del Principado de Asturias (I.U.O.P.A), Universidad de Oviedo, 33071 Oviedo, Spain Received 7 April 2006; received in revised form 4 May 2006; accepted 9 May 2006 Abstract Vincristine is an antitumor drug that inhibits microtubule polymerization, causes G2/M arrest and induces apoptosis. 2D-PAGE and MALDI-TOF-MS analysis of vincristine effects on MCF7 cells, revealed a vincristine upregulated form and a vincristine downregulated form of the antiapoptotic protein HSP27. These findings linked to the lack of vincristine effect over HSP27 mRNA, suggest a protein post-translational modification. Further assays indicated the presence of a phosphorylated peptide, containing serine 82, only in the vincristine upregulated form. Serine 82 phosphorylation was con- firmed using specific antibodies. Thus, phosphorylation of HSP27 may play a role in the cellular response to vincristine. Ó 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: 2D-PAGE; Breast cancer; HSP27; MALDI-TOF-MS; Phosphorylation; Vincristine 1. Introduction Microtubule dynamics and stability have a relevant role in many cellular events, particularly in cell divi- sion where vivid changes in microtubule architecture occur. This cell division regulation makes microtu- bules an important target for cancer chemotherapy [1]. Vinca alkaloids are a group of antitumor drugs that includes natural compounds such as vincristine or vinblastine [2] and semisynthetic compounds such as vinorelvine [3] and vindesine [4]. This micro- tubule-interfering agents introduce a wedge at the interface of two tubulin molecules that prevents the addition of heterodimers onto growing microtu- bules [5]. As a consequence, the mitotic spindle of dividing cells cannot be formed, leading to c-Jun NH2-terminal Kinase (JNK) activation [6], Bcl-2 phosphorylation [7], G2/M arrest and apoptosis [6]. Resistance development to chemotherapy agents means a major clinical problem. Cancer cells turn resistant to vinca alkaloids by mutations in tubulin [8], expression of alternative tubulin classes [8,9] and other cytoskeletal alterations [10]. Another mechanism of resistance is the decrease of drug con- centration inside the cell caused by overexpression 0304-3835/$ - see front matter Ó 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.05.005 * Corresponding author. Tel.: +34 985102764; fax: +34 985104213. E-mail address: sramos@uniovi.es (S. Ramos). 1 Present address: Departamento de Fisiologı ´a y Farmacologı ´a, Facultad de Medicina, Universidad de Cantabria, 39011 Sant- ander, Spain Cancer Letters 247 (2007) 273–282 www.elsevier.com/locate/canlet