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Evidence for a CDK4-dependent checkpoint in a conditional model of cellular senescence

Sharon Brookes1, Sladjana Gagrica, Elaine Sanij

  • 1a Cancer Research-UK London Research Institute ; London , UK.

Cell Cycle (Georgetown, Tex.)
|February 20, 2015
PubMed

Insights

Cellular senescence involves cell cycle arrest and tumor suppression. This study reveals cyclin-dependent kinase 4 (CDK4) activity is crucial for a G2 checkpoint, contributing to the senescent phenotype.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Cancer Research

Background:

  • Cellular senescence is a stable cell cycle arrest mechanism vital for tumor suppression.
  • Key regulators like pRb, p53, p16(INK4a), and p21(CIP1) are known, but details of senescence implementation remain unclear.
  • SV40 large T-antigen (T-Ag) in human fibroblasts provides a model for studying conditional senescence.

Purpose of the Study:

  • To investigate the role of CDK4 in cellular senescence.
  • To elucidate the function of cyclin D-CDK4 complexes during senescence.
  • To understand CDK4's involvement in cell cycle checkpoints during senescence.

Main Methods:

  • Used human fibroblasts expressing temperature-sensitive SV40 large T-antigen (T-Ag).
  • Manipulated CDK4 activity using shRNA knockdown and chemical inhibition.
  • Analyzed cell cycle progression, cell size, and protein complex formation (cyclin D-CDK4-p21).

Main Results:

  • At non-permissive temperatures, p21(CIP1) facilitates cyclin D-CDK4 reassembly, leading to hypo-phosphorylated pRb and cell cycle arrest.
  • CDK4 inhibition or knockdown prevented cell enlargement characteristic of senescence.
  • Blocking CDK4 activity shifted cell arrest from G2/M to G1, indicating a role in the G2 checkpoint.

Conclusions:

  • CDK4 kinase activity plays a significant role in a G2 checkpoint that contributes to the establishment of cellular senescence.
  • The findings uncover a previously unrecognized function for CDK4 in the senescence pathway.
  • Targeting CDK4 may offer new strategies for cancer therapy by modulating senescence.
Keywords:
BrdU, bromodeoxyuridineCDK, cyclin dependent kinaseCDK4FACS, fluorescence actvated cell sortingHFs, human fibroblastsPI, propidium iodideSA-βgal, senescence-associated β-galactosidase activitySV40 T-antigenSV40, simian virus 40TERT, telomerase reverse transcriptasehuman fibroblastsp16INK4ap21CIP1p53pRb, retinoblastoma proteinretinoblastoma proteinsenescenceshRNA, short-hairpin RNAts, temperature sensitive

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