RESUMEN
Using proviral tagging in combination with in vitro selection for invasiveness, we have identified a gene, designated Tiam-1, that affects invasion. In the selected invasive T lymphoma variants, proviral insertions were found within coding exons of the Tiam-1 gene, resulting in both truncated 5'-end and 3'-end transcripts that give rise to N- and C-terminal Tiam-1 protein fragments. In one invasive variant, amplification of the Tiam-1 locus was observed with concomitant increase in the amount of normal Tiam-1 protein. Cell clones that were invasive in vitro produced experimental metastases in nude mice, and transfection of truncated Tiam-1 cDNAs into noninvasive cells made these cells invasive. The predicted Tiam-1 protein harbors a Dbl- and Pleckstrin-homologous domain, which it shares with GDP-GTP exchangers for Rho-like proteins that have been implicated in cytoskeletal organization.
Asunto(s)
Linfoma de Células T/genética , Invasividad Neoplásica/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/análisis , ADN de Neoplasias/análisis , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido , Metástasis Linfática , Linfoma de Células T/microbiología , Linfoma de Células T/patología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/genética , Mutagénesis Insercional , Proteínas Oncogénicas/genética , Proteínas/química , Proteínas Proto-Oncogénicas/genética , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Células Tumorales Cultivadas , Integración Viral/genética , Proteínas de Unión al GTP rapRESUMEN
Non-invasive, non-metastatic mouse BW5147 T-lymphoma cells were treated with non-mutagenic concentrations of the hypomethylating agent 5-azacytidine (5-aza-C). Subsequently, invasive variants were selected on monolayers of rat embryo fibroblasts. The estimated frequency of induction of invasive variants was smaller than 1 in 10(6) cells. We obtained several independent clones that were stable in the expression of the invasive phenotype. In contrast to the parental cell line, the highly invasive clones produced widespread metastases upon tail vein injection in all the syngeneic AKR mice tested, whereas clones with an intermediate level of invasiveness formed metastases only in part of the mice tested. DNA analysis using the methylation-sensitive and insensitive restriction enzymes, Hpa-II and Msp-I, respectively, showed that the DNA of the invasive variants remained hypomethylated, up to 6 months after 5-aza-C treatment. 5-aza-C is thus able to induce invasive and metastatic potential in the BW5147 T-lymphoma cells, similar to the activated human c-Ha-ras oncogene or human chromosome 7, as studied previously. The acquisition of invasive and metastatic potential is presumably caused by DNA hypomethylation and thus activation of one or more silent invasion controlling genes.