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Expression and phenotypic alterations caused by an inducible transforming ras oncogene introduced into rat liver
1Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Abstract:
Although transforming ras oncogenes have been implicated as causative factors in liver cell transformation, the exact function and phenotypic alterations generated by the expression of such transforming genes in liver epithelial cells has yet to be defined. We have utilized a retroviral vector system to deliver an inducible transforming ras gene into normal, anchorage dependent rat liver epithelial cells. The Moloney murine sarcoma virus based vector is composed of a dominant selectable marker, Neo, which is transcriptionally driven from the 5' proviral long terminal repeat (LTR) and a transforming Ha-ras gene under the transcriptional control of a glucocorticoid inducible LTR of the mouse mammary tumor virus. Subsequent to infection, G418 resistant, tumorigenic cell lines were isolated and one particular cell line, designated REL-Ras3, was extensively characterized. Single copies of a full length as well as a truncated provirus were integrated into REL-Ras3 cells. The integrated ras gene was transcribed into poly(A+) RNA with dexamethasone treatment increasing both the steady state level of ras mRNA as well as transcription initiated from the MMTV LTR. Western blot analysis confirmed the presence of P21 containing a transforming mutation in position 12. Phenotypic alterations associated with ras expression in REL-Ras3 cells include: gross morphological alterations; loss of contact inhibition of growth; becoming lethally tumorigenic and anchorage independent; alterations in growth kinetics involving a diminished lag phase of the growth curve; and increases in glucose transport. Differences in growth kinetics and glucose transport could be directly correlated with the levels of ras expression.
Insights
Introducing a transforming ras oncogene into rat liver epithelial cells induced significant phenotypic changes. These included loss of growth control, increased tumorigenicity, and altered glucose transport, directly correlating with ras expression levels.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Transforming ras oncogenes are implicated in liver cell transformation.
- The precise function and phenotypic alterations of ras oncogenes in liver epithelial cells remain unclear.
Purpose of the Study:
- To define the function and phenotypic alterations of a transforming ras gene in rat liver epithelial cells.
- To investigate the effects of inducible ras expression on cell behavior and characteristics.
Main Methods:
- Utilized a retroviral vector system to deliver an inducible transforming Ha-ras gene into normal rat liver epithelial cells.
- Isolated and characterized a tumorigenic cell line (REL-Ras3) expressing the ras gene.
- Analyzed ras gene transcription, protein expression (P21), and phenotypic changes via molecular and cellular assays.
Main Results:
- Dexamethasone treatment increased ras mRNA and transcription from the MMTV LTR in REL-Ras3 cells.
- Confirmed the presence of transforming P21 protein with a mutation at position 12.
- Observed morphological alterations, loss of contact inhibition, anchorage independence, increased tumorigenicity, altered growth kinetics, and elevated glucose transport, all correlated with ras expression levels.
Conclusions:
- Inducible expression of a transforming ras oncogene in rat liver epithelial cells causes significant phenotypic alterations.
- Ras-induced changes include loss of growth control, acquisition of tumorigenic properties, and metabolic shifts like increased glucose transport.
- These findings elucidate the role of ras oncogenes in liver cell transformation and provide a model for studying associated phenotypic changes.