RESUMO
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Tumor dissemination to the extra-hepatic region of the portal vein, lymph nodes, lungs or bones contributes to the high mortality seen in HCC; yet, the molecular mechanisms responsible for HCC metastasis remain unclear. Prior studies have suggested a potential link between accumulated cytoplasm-localized p16 and tumor progression. Here we report that p16 enhances metastasis-associated phenotypes in HCC cells - ectopic p16 expression increased cell migration in vitro, and lung colonization after intravenous injection, whereas knockdown of endogenous p16 reduced cell migration. Interestingly, analysis of p16 mutants indicated that the Cdk4 interaction domain is required for stimulation of HCC cell migration; however, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. Intriguingly, we found that in p16-positive HCC samples, p16 protein is predominantly localized in the cytoplasm. In addition, we identified a potential role for nuclear-cytoplasmic shuttling in p16-stimulated migration, consistent with the predominantly cytoplasmic localization of p16 in IHC-positive HCC samples. Finally, we determined that p16-stimulated cell migration requires the Cdc42 GTPase. Our results demonstrate for the first time a pro-migratory role for p16, and suggest a potential mechanism for the observed association between cytoplasmic p16 and tumor progression in diverse tumor types.
Assuntos
Carcinoma Hepatocelular/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Ion exchange chromatography, one of the major procedures for protein purification, seldom provides single-step purification due to a lack of specific affinity. In this work, a novel and simple method called "back flush" (i.e., reversing the flow direction of elution relative to that of sample loading) was developed to achieve single-step purification on an ion exchanger. Tips for the conditions and operation by back flush are presented. Our study demonstrates, for the first time, the feasibility and dramatic improvement for protein purification by the back-flush method.
