RESUMO
Rb2/p130 tumor suppressor protein regulates cell cycle progression primarily through interactions with members of the E2F family of transcription factors and repression of the transactivation of E2F target genes. In ATRA sensitive ovarian carcinoma CA-OV3 cells, a dramatic increase in Rb2/p130 protein mediates growth arrest at G0/G1. However, although Rb2/p130 is expressed at high levels in SK-OV3 cells, they fail to growth arrest in response to ATRA treatment. We show that the functional activity of Rb2/p130 in SK-OV3 cells is reduced when compared to CA-OV3 cells. To determine the basis for the reduced functional activity, we characterized the Rb2/p130 protein in SK-OV3 cells and investigated the possible role of alterations to this molecule in mediating resistance to ATRA growth suppression. Direct sequencing of Rb2/p130 cDNA cloned from SK-OV3 cells revealed the presence of two homozygous missense mutations (T178C and C259G) which result in amino acid changes Ser60 to Pro60 and Pro87 to Ala87 respectively. Unfortunately the same missense mutations were observed in Rb2/p130 cDNA cloned from ATRA sensitive CA-OV3 cells. We next investigated differences in Rb2/p130 protein subcellular localization. While Rb2/p130 was localized in the nucleus in both cell lines, we observed regions of intense staining within the nucleus of SK-OV3 cells. This is suggestive of aggregation and/or subnuclear sequestration of the Rb2/p130 protein. Finally, the PAGE migration pattern of Rb2/p130 suggested that a hyperphosphorylated form of Rb2/p130 accumulated in SK-OV3 cells but not in CA-OV3 cells. It is possible that this hyperphosphorylated form can be responsible for the decreased Rb2/p130 functional activity observed in SK-OV3 cells and may contribute to the resistance of these cells to ATRA mediated growth suppression.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Ovarianas/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Tretinoína/metabolismo , Adenocarcinoma/tratamento farmacológico , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Mutação de Sentido Incorreto , Neoplasias Ovarianas/tratamento farmacológico , Fosforilação , RNA Mensageiro/análise , Proteína p130 Retinoblastoma-Like/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
More than 80 years ago, Wolbach and Howe provided the first evidence suggesting a link between alterations within human cells that lead to malignancies and vitamin A deficiencies (Wolbach and Howe 1925 Nutr. Rev. 36: 16-19). Since that time, epidemiological, preclinical and clinical studies have established a causative relationship between vitamin A deficiency and cancer. Laboratory research has provided insight into the intracellular targets, various signaling cascades and physiological effects of the biologically-active natural and synthetic derivatives of vitamin A, known as retinoids. Collectively, this body of research supports the concept of retinoids as chemopreventive and chemotherapeutic agents that can prevent epithelial cell tumorigenesis by directing the cells to either differentiate, growth arrest, or undergo apoptosis, thus preventing or reversing neoplasia. Continued refinement of the retinoid signaling pathway is essential to establishing their use as effective therapeutics for tumor subtypes whose oncogenic intracellular signaling pathways can be blocked or reversed by treatment with retinoids.
