RESUMO
Cyclin D1, a putative G1 cyclin, has been implicated in cell cycle control. The human cyclin D1 gene is located on chromosome 11q13 where DNA rearrangement and amplification have been detected in several types of human cancer. Previous studies demonstrated that the cyclin D1 gene is not only rearranged or amplified but also overexpressed in some of these human tumors and tumor-derived cell lines. To further address the roles of cyclin D1 in cell cycle control and tumorigenesis, we have stably overexpressed the human cyclin D1 cDNA in Rat6 embryo fibroblasts by using retrovirus mediated transduction. The cyclin D1 protein was overproduced about 10-fold and was localized predominately in the nucleus. Cyclin D1 overexpressing cells displayed a decrease in the duration of the G1 phase, decreased cell size, and induced tumors when injected into athymic (nude) mice. In addition, overexpression of cyclin D1 in Rat6 cells perturbed the expression of several cellular growth-related genes including c-myc, c-jun, and cyclin A, but not cyclin D3. Taken together, these results indicate that deregulated expression of the cyclin D1 gene can cause disturbances in cell cycle control and gene expression and also enhance tumorigenesis.
Assuntos
Ciclinas/genética , Ciclinas/fisiologia , Fibroblastos/citologia , Expressão Gênica/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/fisiologia , Animais , Northern Blotting , Western Blotting , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Núcleo Celular/química , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Ciclina D1 , Ciclinas/análise , DNA/análise , DNA/genética , Embrião de Mamíferos/química , Embrião de Mamíferos/citologia , Fibroblastos/química , Fibroblastos/metabolismo , Citometria de Fluxo , Fase G1 , Amplificação de Genes , Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Proteínas Oncogênicas/análise , Proteínas Proto-Oncogênicas c-jun/análise , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-myc/análise , Proteínas Proto-Oncogênicas c-myc/genética , RatosRESUMO
Many cell types contain a subset of long-lived, 'stable' microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca(2+)-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150(Glued) and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.