RESUMO
DRA is a gene that is down-regulated in colon adenomas and adenocarcinomas in humans. We have previously shown that DRA proteins are found as various forms in tissue due to differential glycosylation. This study has focused on the function of DRA related to its subcellular localization. We used the baculovirus expression system and overexpressed a nearly full-length DRA driven by a polyhedrin promoter in Sf9 insect cells. DRA protein expressed in this cell was underglycosylated relative to normal colon mucosa, but uniformly targeted to the cell membrane. It also appears to undergo posttranslational cleavage, removing about 100 amino acids from its amino terminus. This membrane localization is similar to what we observed in the colon mucosa. An ion transport assay demonstrated that DRA functions as a sulfate transporter. When DRA was expressed, sulfate import was increased more than threefold compared to the control. Sulfate import was inhibitable by the anion transporter inhibitor, DIDS, in a dose-dependent fashion. Given that (1) DRA has high similarity to other identified sulfate transporters and the proposed structure of DRA polypeptide is characteristic of those transporters, (2) DRA localization is limited to the cell membrane, and (3) DRA expression correlates with intestinal differentiation in mouse, we suggest that DRA represents a tissue-specific member of the sulfate transporter family.
Assuntos
Antiporters , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Sulfatos/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/metabolismo , Antiportadores de Cloreto-Bicarbonato , Colo/metabolismo , DNA Complementar/genética , Expressão Gênica , Glicosilação , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Spodoptera , Transportadores de SulfatoRESUMO
The human S29 ribosomal protein (S29 rp) cDNA has been isolated from differential hybridization screening of a colon carcinoma cDNA library. Northern blot analysis showed that the level of S29 rp mRNA was higher in undifferentiated HT29 human colon carcinoma cells than in a morphologically differentiated subclone under the same growth condition. Furthermore, the level of S29 rp mRNA was downregulated in rapidly proliferating HT29 cells, as compared to the contact inhibited cells. Interestingly, the amount of Krev-1 mRNA was inversely correlated with respect to the amount of S29 rp mRNA in these cells. To examine a functional link between S29 rp and Krev-1 protein, we co-transfected the expression vectors containing wild-type or mutant S29 rp and mutationally activated Krev-1(63E) cDNAs into the v-Ki-ras-transformed NIH3T3 (DT) cells, and observed the induction of flat revertants. Krev-1(63E) induced a certain amount of flat colonies, while S29 rp alone also induced flat colonies at low frequencies. Interestingly, revertant-inducing activity of Krev-1(63E) was significantly enhanced by S29 rp. We have also demonstrated that a zinc finger-like domain of S29 rp indeed has a zinc binding activity and a derivative, S29 rp(ms), which was unable to bind zinc ion but still retained revertant inducing activity by itself, could not functionally interact with Krev-1(63E) protein.
Assuntos
Proteínas de Ligação ao GTP/genética , Genes Supressores de Tumor , Proteína Oncogênica p21(ras)/fisiologia , Proteínas Ribossômicas/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Transformação Celular Viral , Neoplasias do Colo/genética , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Dedos de Zinco , Proteínas rap de Ligação ao GTPAssuntos
Sequência de Bases , DNA Complementar , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismo , RNA , Animais , Biotina , Primers do DNA/síntese química , DNA Complementar/síntese química , Biblioteca Gênica , Indicadores e Reagentes , Dados de Sequência Molecular , RNA Mensageiro/isolamento & purificação , Mapeamento por RestriçãoRESUMO
Three complementary DNA encoding S19 ribosomal protein (S19), laminin-binding protein (LBP), and HLA class I (HLA-I) genes were isolated from a colon tumor-enriched subtraction library. To evaluate this mRNA expression, surgically removed colon tumors as well as matched normal tissue and human colon carcinoma cell lines showing various differentiation states, anchorage dependence, and proliferation states were examined by Northern blot analysis. The mRNA level of S19 mRNA (0.6 kilobase) was higher in primary colon carcinoma tissue than in matched normal colon tissue in 5 of 6 cases. In 2 of 4 cases, the expression of LBP mRNA (1.2 kilobases) was higher in carcinoma than in normal tissue. In 12 human colon cell lines, the level of LBP mRNA was higher in poorly differentiated cells. On the other hand, HLA-I mRNA (1.7 kilobases) was higher in well-differentiated cells. Although the S19 mRNA was expressed in both well- and poorly differentiated cells, a concomitant increase with tumor progression was observed in two pairs of cell lines derived from the same patients (SW480 and SW620; COLO201 and COLO205). Anchorage dependence of butyrate-treated HT29 colon carcinoma cells was correlated with lower levels of S19 and LBP mRNAs and higher levels of HLA-I mRNA expression compared with untreated cells. While the expression of S19 and LBP mRNAs was not changed due to cell growth states, HLA-I mRNA levels were found to be low in proliferating HT29 cells but highly induced in contact-inhibited cells. In summary, therefore, high expression of S19 and LBP combined with low expression of HLA-I were well correlated with colon carcinoma cells of higher malignant potential.