Research Communication Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro Marcel Benadiba, Juliano Andreoli Miyake, and Alison Colquhoun Department of Cell and Developmental Biology, University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Summary Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 lM GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous stud- ies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA’s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronu- cleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. Ó 2009 IUBMB IUBMB Life, 61(3): 244–251, 2009 Keywords gamma-linolenic acid; Ku80; E2F1; glioma; cell cycle; apoptosis. INTRODUCTION Gliomas are the most common form of central nervous sys- tem tumor, and high-grade gliomas (WHO III-IV) are notori- ously difficult to treat. Although standard treatment involves surgical resection and radio/chemotherapy, the incidence of re- currence is very high because of the diffusing nature of these tumors. Previous studies have shown that polyunsaturated fatty acids such as gamma-linolenic acid (GLA) and eicosapentaenoic acid are cytotoxic to tumor cells both in vitro and in vivo (1–4). GLA can alter tumor cell metabolism and proliferation through the production of lipid peroxides and reactive oxygen species, changes in mitochondrial membrane lipid composition, loss of mitochondrial membrane potential, induction of apoptosis, and cell cycle arrest (3–5). Several PUFAs including GLA have been reported to alter bcl2 expression thereby inducing apopto- sis (6). GLA also has synergistic effects on both radiotherapy and chemotherapy (7). The difficulty in treating patients with gliomas has led to the proposed use of GLA as an adjuvant therapy (7, 8). However, the mechanisms by which GLA causes its antitumor effects remain largely unknown in gliomas (5, 7). At subcytotoxic concentrations, GLA reduces both rates of glioma cell proliferation and migratory capacity (9), without inducing the apoptosis typically seen with higher concentrations of GLA (2, 5, 8). This study aimed to identify the possible mo- lecular targets of GLA treatment to provide a better understand- ing of the mechanisms behind these previous findings in glio- mas. Because GLA may alter DNA cell cycle, control and repair mechanisms was analyzed as lipid peroxides can cause substantial DNA and protein damage (6). The expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 and the effects of the natural antioxidant vitamin E on expression were analyzed. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation (10). The fact that GLA exposure is known to enhance the effi- cacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other mole- cules such as roscovitine (10) and flavopiridol (11). Address correspondence to: Alison Colquhoun, Department of Cell and Developmental Biology, University of Sa ˜o Paulo, Sa ˜o Paulo, CEP 05508-900, SP, Brazil. Tel.: 155-11-3091-7261. Fax: 155-11-3091- 7402. E-mail: alison@usp.br Received 23 July 2008; accepted 18 October 2008 ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.154 IUBMB Life, 61(3): 244–251, March 2009