RESUMO
Prostate cancer is one of the most frequent malignancies in the Western world. The identification of additional molecular markers is needed to refine the diagnosis of prostate cancer and to develop more effective therapies. In order to identify molecular abnormalities involved in prostate cancer progression, we performed gene expression analysis of prostate cancer samples compared to matched normal tissue from the same patient using a cancer-related microarray. Amplified RNA was hybridized to a cDNA microarray containing 6386 genes and tissue microarrays were used to study protein expression levels. Using significance analysis of microarrays, we identified >1300 genes differentially expressed in prostate cancer compared to normal tissue. Forty-two of these genes were highly upregulated in prostate cancer while 169 were highly repressed. We found that the gene coding for tspan13 was upregulated >2-fold in 75% of the samples analyzed. Immunohistochemistry analysis of prostate cancer tissue microarrays showed that tspan13 is overexpressed in 80% of prostate cancer samples analyzed. We found that tspan13 expression inversely correlates with Gleason score (p=0.01) and PSA preoperative levels (p=0.11) and directly correlates with presence of prostatic intraepithelial neoplasia in tumor tissue (p=0.04). Moreover, we detected tspan13 expression in low-grade prostatic intraepithelial neoplasia. Thus, our results show that tspan13 is overexpressed in prostate cancer and its expression correlates with factors of favourable outcome. Therefore we suggest that tspan13 may have an important role in the progression of prostate cancer.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Neoplasias da Próstata/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Humanos , Imuno-Histoquímica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraspaninas , Resultado do Tratamento , Regulação para CimaRESUMO
For several DNA-dependent DNA polymerases it has been shown that their synthetic and degradative activities are organized in two separated modules. The functional coordination required between them to accomplish successfully the replication process is provided by important contacts with the substrate contributed by residues coming from both modules. These domains are connected by a central "linker" region adjacent to the "YxGG/A" motif, the putative limit of the polymerization domain. We describe here the mutational analysis of phi29 DNA polymerase in several residues of this region, connecting the N- and C-terminal domains and conserved in DNA polymerases able to start replication by protein-priming. The mutant polymerases with the less conservative changes showed reduced DNA binding activity. Additionally, their TP binding capacity was reduced, affecting the TP-deoxynucleotidylation in the absence of template. Interestingly, the role of the residues studied here in DNA binding seems to be especially important to start replication, when the polymerase enters from the closed binary into the ternary complex. These results allow us to propose that this interdomain region of phi29 DNA polymerase is playing an important role for substrate binding including both DNA and TP.
Assuntos
Fagos Bacilares/enzimologia , DNA Polimerase Dirigida por DNA/genética , DNA/metabolismo , Exonucleases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Sequência Conservada/genética , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Polímeros/metabolismo , Ligação Proteica/genética , Homologia de Sequência de AminoácidosRESUMO
Doxazosin is a quinazoline-based compound acting as an alpha-1-adrenergic inhibitor shown to induce apoptosis in prostate cancer cell lines via an alpha-1-adrenergic receptor-independent mechanism. To better understand the mechanism of doxazosin-induced apoptosis in prostate cancer, we performed cDNA microarray to analyze gene expression changes produced by doxazosin in the androgen-dependent human prostate cancer cell line, LNCaP. We found that 70 and 92 genes were deregulated after 8 and 24 h of doxazosin treatment, respectively. These genes are involved in several cellular processes such as cell-cycle regulation, cell adhesion and signal transduction pathways. Strikingly, we found that doxazosin induces deregulation of genes implicated in DNA replication and repair, such as GADD45A, XRCC5 and PRKDC. These facts, together with the demonstration of the ability of doxazosin to bind DNA, allowed us to propose a novel mechanism of action for doxazosin in prostate cancer cells that implies DNA-damage mediated apoptosis by down-regulation of XRCC5 and PRKDC genes.