Related Experiment Videos
Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor
Abstract:
Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a "competence" function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus, transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.
Insights
Oncogenes like c-myc, H-ras, and E1a immortalized rat kidney cells, with H-ras conferring a "competence" function. Cell transformation required high H-ras expression, not just the presence of multiple oncogenes.
Area of Science:
- Cell Biology
- Oncogenesis
- Molecular Biology
Background:
- Cellular senescence is a barrier to uncontrolled proliferation.
- Oncogenes can override senescence and promote immortalization.
- Different oncogenes may confer distinct cellular properties.
Purpose of the Study:
- To investigate the immortalization and transformation potential of specific oncogenes in rat kidney cells.
- To characterize the growth factor responses of oncogene-immortalized cell lines.
- To determine the role of oncogene expression levels in cellular transformation.
Main Methods:
- Immortalization of early-passage rat kidney cells using viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a.
- Quantitation of oncogene RNA expression levels.
- Short-term growth factor response assays.
- Secondary transfection experiments to assess transformation.
Main Results:
- Viral promoter-driven c-myc, H-ras (Val-12), and E1a oncogenes immortalized rat kidney cells, while normal c-myc and H-ras (Gly-12) did not.
- Immortalized cell lines exhibited distinct growth factor dependencies.
- H-ras-immortalized cells showed high expression of H-ras and deregulated c-myc, suggesting a "competence" function.
- Morphological transformation required high levels of H-ras expression, not co-expression with c-myc.
Conclusions:
- Specific oncogenes can immortalize primary cells and confer distinct phenotypes.
- High-level expression of the H-ras oncogene is critical for morphological transformation.
- Cellular transformation is dependent on the dosage of activated oncogenes.