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Transfection of human mutated K-ras in mouse NIH-3T3 cells is associated with increased cloning efficiency and DNA

S Nigro1, E Geido, E Infusini

  • 1Laboratory of Biophysics and Cytometry, National Institute for Cancer Research (I.S.T.), Genoa, Italy.

International Journal of Cancer
|September 17, 1996
PubMed

Insights

A mutated human K-ras oncogene in mouse cells caused abnormal cell division and increased the rate of sub-clone development. These K-ras-driven sub-clones showed higher cloning efficiency and DNA aneuploidy, indicating genomic instability.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • The K-ras oncogene plays a critical role in cell signaling pathways.
  • Mutations in K-ras are frequently observed in various human cancers.
  • Understanding the downstream effects of mutated K-ras is crucial for cancer research.

Purpose of the Study:

  • To investigate if a mutated human K-ras protein induces mitotic abnormalities and sub-clone development.
  • To determine if these sub-clones exhibit changes in DNA ploidy and proliferation.
  • To assess the impact of activated K-ras on genomic stability.

Main Methods:

  • NIH-3T3 mouse cells were transfected with a mutated human K-ras oncogene (codon 12 G-C).
  • Abnormal mitoses were quantified by observing chromosome behavior during cell division.
  • Sub-clone generation was assessed using limiting-dilution experiments and cloning efficiency.
  • DNA content and cell cycle phase distribution were analyzed by flow cytometry.

Main Results:

  • Transfected cells showed a significantly higher frequency of abnormal mitoses, including lagging chromosomes.
  • K-ras-transfected cells exhibited a markedly increased cloning efficiency (41%) compared to control cells (22%).
  • A higher proportion of DNA aneuploid sub-clones (9%) were generated from K-ras-transfected cells versus control cells (1%).

Conclusions:

  • High expression of the activated human K-ras protein in NIH-3T3 cells is linked to mitotic abnormalities.
  • Mutated K-ras promotes increased cloning efficiency and contributes to DNA aneuploidization.
  • These findings suggest that activated K-ras can drive genomic instability and sub-clone evolution.

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