Metabolic changes during cell growth inhibition by p27 overexpression

  • Carvalhal A
  • Marcelino I
  • Carrondo M
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Abstract

The overexpression of p27, a cyclin-dependent kinase ({CDK}) inhibitor, has been shown to effectively inhibit cell growth at the G1-phase of different cell lines, potentiating a valid genetic strategy for cell proliferation control. In order to characterize the energy requirements after p27 overexpression in {CHO} cells expressing {SEAP} (secreted form of the human alkaline phosphatase enzyme), key metabolic parameters were evaluated. Cell growth inhibition led to a significant increase in cell size concomitant with a 2-fold increase in cell protein content. The simultaneous increase of the intracellular proteolytic activity with protein content suggests higher protein synthesis. A general 2-fold increase in oxygen, glutamine and glucose consumption rates, coupled with an increase in lactate and ammonia production was observed. p27 overexpression led to a significant increase in the intracellular pool of {AMP} (8.5-fold), {ADP} (6-fold) and, more uncommonly, {ATP} (4.5-fold). Nevertheless, cells were able to maintain the equilibrium among the three adenine nucleotides since both the {ATP}/{ADP} ratio and the energy charge values remained similar to those observed with non-growth inhibited cells. This work shows that the observed 4-fold increase in {SEAP} specific productivity after cell growth inhibition by p27, occurred concomitantly with a higher expenditure of cell energy. This characterization of cell metabolism becomes important in demonstrating the applicability of growth inhibition systems.

Author-supplied keywords

  • Adenine Nucleotides
  • Alkaline Phosphatase
  • Animals
  • Cell Cycle Proteins
  • Cell Survival
  • Cricetinae
  • Culture Media
  • Cyclin-Dependent Kinase Inhibitor p27
  • Energy Metabolism
  • G1 Phase
  • Humans
  • Proteins
  • Tumor Suppressor Proteins
  • Up-Regulation
  • {CHO} Cells

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Authors

  • A V Carvalhal

  • I Marcelino

  • M J T Carrondo

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