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Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth
J J Yang1, J S Kang, R S Krauss
1Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Abstract:
Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.
Insights
Ras oncoprotein signals through multiple pathways to overcome cell adhesion requirements for cell cycle progression. Combinations of Ras effector mutants synergistically promote anchorage-independent growth and cell cycle activity.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Cell cycle progression in normal cells requires adhesion to a substrate.
- The Ras oncoprotein can induce anchorage-independent growth by overriding this adhesion requirement.
- Ras signaling involves multiple downstream effectors, potentially acting synergistically.
Purpose of the Study:
- To investigate cell cycle regulation of anchorage-independent growth using Ras effector loop mutants.
- To determine how Ras signaling pathways contribute to overcoming cell adhesion dependence.
- To explore the synergistic interactions between Ras effector mutants in promoting anchorage-independent growth and cell cycle progression.
Main Methods:
- Stable expression of activated H-Ras (12V) and its effector loop mutants in NIH 3T3 and Rat 6 cells.
- Assay for soft agar colony formation to measure anchorage-independent growth.
- Analysis of cell cycle regulatory events, including pRB phosphorylation, cyclin E-dependent kinase activity, and cyclin A expression.
- Ectopic expression of cyclins D1, E, and A to assess their synergistic effects with Ras mutants.
Main Results:
- Individual Ras effector loop mutants were defective in inducing anchorage-independent growth.
- Pairwise combinations of effector loop mutants synergized to induce anchorage-independent growth in NIH 3T3 cells, with specific combinations effective in Rat 6 cells.
- Mutants partially relieved adhesion dependence of cell cycle activities (pRB phosphorylation, cyclin E-kinase, cyclin A expression) in NIH 3T3 cells.
- Synergistic combinations of mutants in Rat 6 cells also synergistically abrogated adhesion requirements for cell cycle activities.
- Ectopic expression of cyclins D1, E, or A, particularly cyclin A, synergized with individual mutants to promote soft agar colony formation.
Conclusions:
- Ras utilizes multiple signaling pathways to regulate the cell cycle machinery.
- These pathways synergize to override the adhesion requirements for specific cell cycle events, leading to anchorage-independent growth.
- The findings provide insights into the complex mechanisms by which Ras transformation occurs and suggest potential therapeutic targets.