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p21ras-mediated decrease of the retinoblastoma protein in fibroblasts occurs through growth factor-dependent
L Kivinen1, E Tiihonen, T Haapajärvi
1Haartman Institute, Department of Virology, University of Helsinki, Finland.
Abstract:
Stable coexpression of the human retinoblastoma protein (pRB) cDNA and EJ c-Ha-ras oncogene in murine fibroblasts leads to loss of pRB expression with concomitant transformation of the cells (1). We show here that conditional expression of p21ras in mouse fibroblasts expressing human pRB leads to a rapid decrease of pRB expression at both protein and mRNA levels. The decrease of pRB mRNA is blocked by cycloheximide, suggesting the requirement of ongoing protein synthesis. p21ras expression leads also to decreases of c-myc and tissue metalloproteinase inhibitor-2 mRNAs, whereas cyclin-dependent kinase 4, cyclin D1, E2F-1, and ornithine decarboxylase are unaffected. The decrease in pRB is accompanied by progressive morphological transformation of the cells. The effect of p21ras on pRB expression was serum and growth factor dependent. A shift of the cells to low serum (0.2% FCS) abolished the effects of p21ras on pRB, but this effect was reconstituted by the addition of growth factors epidermal growth factor, fibroblast growth factor-2, transforming growth factor beta 1, and platelet-derived growth factor to the cells. The results suggest a complex interaction between p21ras, pRB, and growth factors in the control of cell growth. p21ras appears to drive the cell cycle by deregulation of key cell cycle regulators, the functions of which in low serum become redundant or require the presence of growth factors positively driving the cell cycle.
Insights
Ras oncogene expression rapidly decreases retinoblastoma protein (pRB) levels in mouse fibroblasts, leading to cell transformation. This effect is dependent on growth factors and protein synthesis, highlighting complex cell cycle regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The retinoblastoma protein (pRB) is a key tumor suppressor regulating cell cycle progression.
- Oncogenic mutations, such as in the Ras pathway, are frequently observed in human cancers.
- Understanding the interplay between oncogenes and tumor suppressors is crucial for cancer research.
Purpose of the Study:
- To investigate the effect of oncogenic Ras (p21ras) expression on pRB levels and cellular transformation in murine fibroblasts.
- To elucidate the molecular mechanisms underlying Ras-induced pRB downregulation.
- To determine the role of growth factors and protein synthesis in this process.
Main Methods:
- Stable coexpression of human pRB cDNA and EJ c-Ha-ras oncogene in murine fibroblasts.
- Conditional expression of p21ras.
- Analysis of pRB, c-myc, and other cell cycle regulator mRNA and protein levels.
- Treatment with cycloheximide, varying serum concentrations, and addition of specific growth factors.
Main Results:
- p21ras expression led to a rapid decrease in both pRB protein and mRNA levels.
- The decrease in pRB mRNA was dependent on ongoing protein synthesis.
- p21ras also reduced c-myc and tissue metalloproteinase inhibitor-2 mRNA but did not affect CDK4, cyclin D1, E2F-1, or ODC.
- Cellular transformation, including morphological changes, accompanied the decrease in pRB.
- The effect of p21ras on pRB was serum and growth factor dependent, being rescued by specific growth factors.
Conclusions:
- p21ras oncogene expression induces a significant reduction in pRB levels, promoting cellular transformation.
- Cell cycle regulation involves a complex interplay between p21ras, pRB, and growth factors.
- p21ras may override normal cell cycle control mechanisms, potentially by altering the dependency on growth factors for cell cycle progression.