Metabolic changes during cell growth inhibition by p27 overexpression.
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.
Carvalhal
A V
AV
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal.
Marcelino
I
I
Carrondo
M J T
MJ
D000227:Adenine Nucleotides / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects*; D006224:Cricetinae; D003470:Culture Media; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004734:Energy Metabolism / Q000187:drug effects*; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D011506:Proteins / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism* / Q000494:pharmacology; D015854:Up-Regulation
D000227:Adenine Nucleotides; D018797:Cell Cycle Proteins; D003470:Culture Media; D011506:Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000469:Alkaline Phosphatase