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The effects of p21N-ras expression in NIH-3T3 cells upon cyclic AMP metabolism
S A Davies1, M D Houslay, M J Wakelam
1Department of Biochemistry, University of Glasgow, U.K.
Abstract:
The effects of overexpression of p21N-ras upon cyclic AMP metabolism have been examined in the inducible T15 cell line. In cells overexpressing the N-ras gene product, beta-adrenergic stimulation of cyclic AMP generation was reduced. The reduction was more pronounced the longer the ras gene was expressed and in chronically transformed cells a reduction in forskolin-stimulated cyclic AMP generation was also observed. The transformed cells exhibited a reduction in beta-adrenergic binding sites, but no change in the apparent EC50 for agonist induced cyclic AMP generation. Treatment of the cells with dibutyryl cyclic AMP induced a dose-dependent inhibition of proliferation, with the transformed cells being more sensitive than the control cells.
Insights
Overexpressing the N-ras gene in T15 cells reduced cyclic AMP (cAMP) generation and inhibited proliferation. Transformed cells showed increased sensitivity to dibutyryl cAMP, impacting cell growth.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Ras proteins are key regulators of cellular signaling pathways.
- Aberrant Ras signaling is implicated in various cancers.
- Cyclic AMP (cAMP) is a crucial second messenger involved in diverse cellular processes.
Purpose of the Study:
- To investigate the impact of N-ras gene overexpression on cyclic AMP (cAMP) metabolism in T15 cells.
- To determine how N-ras-induced cellular transformation affects beta-adrenergic and forskolin-stimulated cAMP generation.
- To assess the role of N-ras in regulating cell proliferation in response to cAMP.
Main Methods:
- Utilized an inducible T15 cell line for controlled N-ras gene expression.
- Measured beta-adrenergic and forskolin-stimulated cyclic AMP (cAMP) generation.
- Quantified beta-adrenergic binding sites and EC50 for agonist-induced cAMP.
- Assessed cell proliferation inhibition using dibutyryl cyclic AMP treatment.
Main Results:
- N-ras overexpression led to reduced beta-adrenergic stimulation of cAMP generation, with effects intensifying over time.
- Chronically transformed cells showed diminished forskolin-stimulated cAMP generation.
- Transformed cells displayed fewer beta-adrenergic binding sites but unchanged EC50 for cAMP induction.
- Dibutyryl cAMP dose-dependently inhibited proliferation, with transformed cells exhibiting higher sensitivity.
Conclusions:
- N-ras overexpression significantly disrupts cAMP metabolism and signaling.
- Ras-induced transformation alters cellular responses to cAMP, affecting both second messenger generation and proliferation.
- These findings highlight a critical role for N-ras in regulating cellular homeostasis and suggest potential therapeutic targets.