Replicative activity and actinomycin binding in mouse diploid fibroblasts (in vitro ageing)

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Embryonic mouse fibroblasts exhibit limited proliferation in vitro, undergoing approximately 10 doublings before growth cessation.
• In vitro aging involves progressive cellular changes affecting DNA properties.

Purpose of the Study:

• To investigate alterations in DNA properties during the in vitro aging of mouse fibroblasts.
• To understand the molecular mechanisms underlying cellular senescence.

Main Methods:

• Serial subcultivation of mouse fibroblasts.
• Pulse labeling with [3H]thymidine to assess DNA synthesis.
• Quantification of [3H]actinomycin binding to cellular nuclei.
• Feulgen cytophotometry for DNA content analysis.
• Induction of quiescence via serum deprivation.

Main Results:

• Progressive decrease in [3H]thymidine labeling with serial subcultivation, with a slower decline after five passages.
• Up to 50% of cells in the final passage incorporated [3H]thymidine after prolonged labeling.
• Decreased binding of [3H]actinomycin to nuclei during serial subcultivation.
• Feulgen cytophotometry revealed a wide spectrum of DNA content in terminal cultures, with 38% of cells having increased DNA.
• 40% of senescent fibroblasts showed reduced actinomycin binding compared to early passage cells.

Conclusions:

• Age-dependent changes in chromatin condensation, rather than DNA amount or cell cycle variations, are implicated in the observed alterations.
• In vitro aging of fibroblasts is associated with significant modifications in DNA accessibility and potentially DNA content regulation.