title: Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells.
abstract: We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O6-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.
authors.affiliation: Oncology Laboratory, IMBECU-CCT, CONICET, National Research Council, Av. Dr. Ruiz Leal s/n, Parque General San Martín, CP 5500, Mendoza, Argentina.
authors.forename: Gisela Natalia
authors.initials: GN
authors.lastname: Castro
authors.forename: Niubys
authors.initials: N
authors.lastname: Cayado-Gutiérrez
authors.forename: Felipe Carlos Martín
authors.initials: FC
authors.lastname: Zoppino
authors.forename: Mariel Andrea
authors.initials: MA
authors.lastname: Fanelli
authors.forename: Fernando Darío
authors.initials: FD
authors.lastname: Cuello-Carrión
authors.forename: Mayra
authors.initials: M
authors.lastname: Sottile
authors.forename: Silvina Beatriz
authors.initials: SB
authors.lastname: Nadin
authors.forename: Daniel Ramón
authors.initials: DR
authors.lastname: Ciocca
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000187:drug effects; D045643:DNA Repair Enzymes / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005910:Glioma / Q000473:pathology; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018832:Molecular Chaperones; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D000077204:Temozolomide
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018906:Antineoplastic Agents, Alkylating; D050883:HSC70 Heat-Shock Proteins; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C490786:HSPA1A protein, human; C494371:HSPA8 protein, human; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; C501486:MLH1 protein, human; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D045643:DNA Repair Enzymes; D000077204:Temozolomide