RESUMO
Using an expression cDNA cloning approach, we examined human tumor cell lines for novel oncogenes that might evade detection by conventional techniques. We isolated a transforming sequence that was highly efficient in transforming NIH 3T3 mouse fibroblasts. DNA sequence analysis identified the gene as the human homolog of a recently cloned alpha subunit of mouse GTP-binding protein G alpha 12. NIH 3T3 cells transfected with G alpha 12 cDNA grew in soft agar and were tumorigenic in nude mice. There were no apparent mutations in the cloned cDNA in comparison with a G alpha 12 cDNA clone isolated from a normal human epithelial cell library, implying that overexpression alone was sufficient to cause NIH 3T3 cell transformation. The observed altered growth properties mediated by G alpha 12 showed a certain degree of dependency on serum factors, and its mitogenic potential was also potently inhibited by suramin treatment.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação ao GTP/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , DNA/biossíntese , DNA/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/fisiologia , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Oncogenes , Homologia de Sequência de Aminoácidos , Suramina/farmacologia , Células Tumorais CultivadasRESUMO
By ectopic expression of cDNAs derived from a Ewing sarcoma cell line in NIH3T3 cells, we isolated a transforming gene (est). Sequence analysis revealed homology to the cot oncogene, which encodes a novel serine kinase. Whereas the cot product was truncated at its carboxy-terminal end as a result of gene rearrangement during transfection, est encodes the normal cot product. Thus, this gene can be activated as an oncogene by overexpression as well as by gene rearrangement. NIH3T3 cells transfected with est formed progressively growing colonies in soft agar and were tumorigenic in nude mice. The 3.2-kb est transcript was expressed at low level in both human fibroblasts and epithelial cells. Addition of the tumor promoter, okadaic acid (OA), or cytokine, interleukin 1 (IL-1), but not serum or platelet-derived growth factor (PDGF), induced increased expression of the est transcript. Using fluorescence in situ hybridization, we localized the est gene to the short arm of human chromosome 10 at band p11.2.
Assuntos
Clonagem Molecular , DNA/genética , Oncogenes , Proteínas Serina-Treonina Quinases/genética , Sarcoma de Ewing/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 10 , DNA/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Proto-Oncogenes , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
We generated a cDNA expression library from a human mammary epithelial cell line for detection of novel oncogenes by focus formation assay in NIH3T3 cells. A morphologically unique focus was identified and the transforming plasmid was isolated. The transforming gene, designated TIM, encoded a predicted protein species of 60 kDa containing a Dbl-Homology (DH) motif. This motif is also present in other growth regulatory molecules including Bcr, Cdc24, Vav, Ras-grf, and Ect2 which have been implicated as regulators of small GTP-binding proteins. NIH3T3 cells transfected with TIM expression plasmid showed altered growth properties in vitro and were tumorigenic when injected into nude mice. The 6.5 kilobasepair (kb) transcript of the TIM gene was mainly expressed in kidney, liver, pancreas, lung, and placenta. By analysing a panel of human-hamster somatic cell hybrids, we localized the TIM gene to human chromosome 7.