RESUMO
Human securin, also known as human pituitary tumor-transforming gene 1 (pttg1), plays a key role in cell-cycle regulation. Two homologous genes, pttg2 and pttg3, have been identified although very little is known about their physiological function. In this study, we aimed at the characterization of these two pttg1 homologs. Real-time PCR analysis using specific probes demonstrated that Pttg2 is expressed at very low levels in various cell lines and tissues whereas Pttg3 was largely undetectable. We focused on the study of Pttg2 and found that, unlike PTTG1, PTTG2 lacks transactivation activity and does not bind to separase, making improbable a role in the control of sister chromatids separation. To further investigate the biological role of pttg2, we used short hairpin RNA inhibition of Pttg2 and found that cells with reduced Pttg2 levels assumed a rounded morphology compatible with a defect in cell adhesion and died by apoptosis in a p53- and p21-dependent manner. Using microarray technology, we generated a gene expression profile of Pttg2-depleted cells versus wild-type cells and found that knockdown of PTTG2 results in concomitant downregulation of E-cadherin and elevated vimentin levels, consistent with EMT induction. The observation of aberrant cellular behaviors in Pttg2-silenced cells reveals functions for pttg2 in cell adhesion and provides insights into a potential role in cell invasion.
Assuntos
Apoptose , Transição Epitelial-Mesenquimal , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Caderinas/metabolismo , Adesão Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Células HCT116 , Humanos , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Securina , Tubulina (Proteína)/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Vimentina/metabolismoRESUMO
The faithful repair of DNA damage, especially chromosomal double-strand breaks (DSBs), is crucial for genomic integrity. We have previously shown that securin interacts with the Ku70/80 heterodimer of the DSB non-homologous DNA end-joining (NHEJ) repair machinery. Here we demonstrate that securin deficiency compromises cell survival and proliferation, but only after genotoxic stress. Securin(-/-) cells show a significant increase in gross chromosomal rearrangements and chromatid breaks after DNA damage, and also reveal an altered pattern of end resection in an NHEJ assay in comparison with securin(+/+) cells. These data suggest that securin has a key role in the maintenance of genomic stability after DNA damage, thereby providing a previously unknown mechanism for regulating tumour progression.
Assuntos
Proliferação de Células , Dano ao DNA , Reparo do DNA , Proteínas de Neoplasias/metabolismo , RNA Interferente Pequeno/metabolismo , Sequência de Bases , Camptotecina/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Aberrações Cromossômicas , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Doxorrubicina/farmacologia , Instabilidade Genômica , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , SecurinaRESUMO
We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Divisão Celular , Núcleo Celular/metabolismo , Cromossomos Humanos Par 3 , Clonagem Molecular , Dano ao DNA , Proteínas de Ligação a DNA/química , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA , Ratos , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Regulação para Cima , Dedos de ZincoRESUMO
The activity of AV200, a synthetic ardeemin derivative, in reversing the multidrug resistance phenotype has been investigated. At non-toxic doses, AV200 was able to completely restore vincristine and paclitaxel toxicities and partially restore that of doxorubicin in multidrug-resistant cells. The potency of AV200 as a modulator of the resistance to doxorubicin, vincristine and paclitaxel resulted to be seven-, 59 and 12-fold, respectively, higher than that of verapamil. In vitro measurements of rhodamine 123 accumulation in human resistant cells suggest that AV200 reverses multidrug resistance by directly inhibiting the P-glycoprotein-mediated drug efflux. This work underscores the possibility of utilizing ardeemin derivatives as a source of non-toxic modulators of the multidrug resistance phenotype.
