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.
Similar content being viewed by others
References
Atkinson DE (1969) Regulation of enzyme function. Annu Rev Microbiol 23:47–68
Bax BE, Bloxam DL (1997) Energy metabolism and glycolysis in human placental trophoblast cells during differentiation. Biochim Biophys Acta 1319:283–292
Carvalhal AV, Coroadinha AS, Alves PM, Moreira JL, Hauser H, Carrondo MJT (2002) Metabolic changes during cell growth inhibition by the IRF-1 system. Enzyme Microb Technol 30:95–109
Carvalhal AV, Sá Santos S, Calado J, Haury M, Carrondo MJT (2003) Cell growth arrest by nucleotides, nucleosides and bases as a tool for improved production of recombinant proteins. Biotechnol Prog 19:69–83
Cazin M, Paluszezak D, Bianchi A, Cazin JC, Aerts C, Voisin C (1990) Effects of anaerobiosis upon morphology and energy metabolism of alveolar macrophages cultured in gas phase. Eur Respir J 3:1015–1022
Chapman JD, Webb RG, Borsa J (1971) ATP pools levels in synchronously growing chinese hamster cells. J Cell Biol 49:229–233
Cruz PE, Martins PC, Alves PM, Peixoto CC, Santos H, Moreira JL, Carrondo MJT (1999a) Proteolytic activity in infected and noninfected insect cells: degradadtion of HIV-1 Pr55gag particles. Biotechnol Bioeng 65:133–143
Cruz HJ, Ferreira AS, Freitas CM, Moreira JL, Carrondo MJT (1999b) Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture. Appl Microbiol Biotechnol 51:579–585
Doyle A, Griffiths JB, Newell DG (1996) Cell and tissue culture: laboratory procedures. Wiley, Chichester
Freudenberg H, Mager J (1971) Studies on the mechanism of the inhibition of protein synthesis induced by intracellular ATP depletion. Biochim Biophys Acta 232:537–555
Fussenegger M (2001) The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies. Biotechnol Prog 17:1–51
Fussenegger M, Mazur X, Bailey JE (1997) A novel cytostatic process enhances the productivity of Chinese hamster ovary cells. Biotechnol Bioeng 55:927–939
Glaken MW, Adema E, Sin Skey AJ (1988) Mathematical descriptions of hybridoma culture kinetics I. Initial metabolic rates. Biotechnol Bioeng 32:491–506
Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551
Grummt F, Grummt PD (1977) Regulation of ATP pools, rRNA and DNA synthesis in 3T3 cells in response to serum or hypoxanthine. Eur J Biochem 76:7–12
Hasenson M, Hartley-Asp B, Kihlfors C, Lundin A, Gustafsson JA, Pousette A (1985) Effect of hormones on growth and ATP content of a human prostatic carcinoma cell line, LNCaP-r. Prostate 7:183–194
Hengst L, Reed SI (1996) Translational control of p27Kip1 accumulation during the cell cycle. Science 271:1861–1864
Kato JY, Matsuoka M, Polyak K, Massague J, Sherr CJ (1994) Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell 79:487–496
Lundin A, Hasenson M, Persson J, Pousette A (1986) Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol 133:27–42
Mazur X, Fussenegger M, Renner WA, Bailey JE (1998) Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27. Biotechnol Prog 14:705–713
Neermann J, Wagner R (1996) Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells. J Cell Physiol 166:152–169
Ngo J, Orlando RA, Ibsen KH (1986) Pyruvate kinase and total protein are regulated differently during growth of P-815 mastocytoma cells. Arch Biochem Biophys 274:171–182
Nguyen H, Gitig DM, Koff A (1999) Cell-free degradation of p27(kip1), a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome. Mol Cell Biol 19:1190–1201
Nourse J, Firpo E, Flanagan WM, Coats S, Polyak K, Lee M-H, Massague J, Crabtree GR, Roberts JM (1994) Interleukin-2-mediated elimination of the p27kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372:570–573
Ryll T, Wagner R (1991) Improved ion-pair high-performance liquid chromatographic method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells. J Chromatogr 570:77–88
Santarén JF, Bravo R (1986) A basic cytoplasmic protein (p27) induced by serum in growth-arrested 3T3 cells but constitutively expressed in primary fibroblasts. EMBO J 5:877–882
Shankland SJ, Pippin J, Flanagan M, Coats SR, Nangaku M, Gordon KL, Roberts JM, Couser WG, Johnson RJ (1997) Mesangial cell proliferation mediated by PDGF and bFGF is determined by levels of the cyclin kinase inhibitor p27Kip1. Kidney Int 51:1088–1099
Siems W, Muller M, Dubiel W, Dumdey R, Rapoport S (1986) ATP production and consumption of rabbit reticulocytes increase in an amino-acid-enriched medium. Biomed Biochim Acta 45:585–591
Skog S, Tribukait B (1985) Discontinuous RNA and protein synthesis and accumulation during cell cycle of Ehrlich ascites tumour cells. Exp Cell Res 159:510–518
Spanier AM, Clark WA, Zak R (1984) Replacement perfusion of cultured eucaryotic cells: a method for the accurate measurement of the rates of growth, protein synthesis, and protein turnover. J Cell Biochem 26:47–64
Stein GS, Baserga R, Giordano A, Denhardt DT (1999) The molecular basis of cell cycle and growth control. Wiley-Liss, N.Y.
Toyoshima H, Hunter T (1994) p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78:67–74
Twining SS (1984) Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal Biochem 143:30–34
Vlach J, Hennecke S, Amati B (1997) Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27. EMBO J 16:5334–5344
Wagner R, Ryll T, Krafft H, Lehmann J (1988) Variation of amino acid concentration in the medium of HU β-IFN and HU IL-2 producing cell lines. Cytotechnology 1:145–150
Acknowledgements
The authors are grateful to Prof. James Bailey (deceased, 2001), Dr. Martin Fussenegger and Dr. Xenia Mazur of the ETH, Zurich, for the CHO cells clones. The authors are grateful to Maria do Rosário Clemente from IBET/ITQB for technical support. The authors acknowledge and appreciate the financial support received from the European Commission (BIO4-CT95–0291) and Fundação para a Ciência e Tecnologia—Portugal (PRAXIS XXI/BD/13344/97).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Carvalhal, A.V., Marcelino, I. & Carrondo, M.J.T. Metabolic changes during cell growth inhibition by p27 overexpression. Appl Microbiol Biotechnol 63, 164–173 (2003). https://doi.org/10.1007/s00253-003-1385-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-003-1385-5