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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.

Molecular and Cellular Biology
|May 5, 1998
PubMed

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.

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