Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Dec 1;376(Pt 2):403–411. doi: 10.1042/BJ20030816

Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence.

Werner Zwerschke 1, Sybille Mazurek 1, Petra Stöckl 1, Eveline Hütter 1, Erich Eigenbrodt 1, Pidder Jansen-Dürr 1
PMCID: PMC1223775  PMID: 12943534

Abstract

Cellular senescence is considered a major tumour-suppressor mechanism in mammals, and many oncogenic insults, such as the activation of the ras proto-oncogene, trigger initiation of the senescence programme. Although it was shown that activation of the senescence programme involves the up-regulation of cell-cycle regulators such as the inhibitors of cyclin-dependent kinases p16INK4A and p21CIP-1, the mechanisms underlying the senescence response remain to be resolved. In the case of stress-induced premature senescence, reactive oxygen species are considered important intermediates contributing to the phenotype. Moreover, distinct alterations of the cellular carbohydrate metabolism are known to contribute to oncogenic transformation, as is best documented for the phenomenon of aerobic glycolysis. These findings suggest that metabolic alterations are involved in tumourigenesis and tumour suppression; however, little is known about the metabolic pathways that contribute to these processes. Using the human fibroblast model of in vitro senescence, we analysed age-dependent changes in the cellular carbohydrate metabolism. Here we show that senescent fibroblasts enter into a metabolic imbalance, associated with a strong reduction in the levels of ribonucleotide triphosphates, including ATP, which are required for nucleotide biosynthesis and hence proliferation. ATP depletion in senescent fibroblasts is due to dysregulation of glycolytic enzymes, and finally leads to a drastic increase in cellular AMP, which is shown here to induce premature senescence. These results suggest that metabolic regulation plays an important role during cellular senescence and hence tumour suppression.

Full Text

The Full Text of this article is available as a PDF (217.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alcorta D. A., Xiong Y., Phelps D., Hannon G., Beach D., Barrett J. C. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13742–13747. doi: 10.1073/pnas.93.24.13742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boros Laszlo G., Lee Wai-Nang Paul, Go Vay Liang W. A metabolic hypothesis of cell growth and death in pancreatic cancer. Pancreas. 2002 Jan;24(1):26–33. doi: 10.1097/00006676-200201000-00004. [DOI] [PubMed] [Google Scholar]
  3. Bürkle A. Poly(ADP-ribosyl)ation, genomic instability, and longevity. Ann N Y Acad Sci. 2000 Jun;908:126–132. doi: 10.1111/j.1749-6632.2000.tb06641.x. [DOI] [PubMed] [Google Scholar]
  4. Collavin L., Lazarevic D., Utrera R., Marzinotto S., Monte M., Schneider C. wt p53 dependent expression of a membrane-associated isoform of adenylate kinase. Oncogene. 1999 Oct 21;18(43):5879–5888. doi: 10.1038/sj.onc.1202970. [DOI] [PubMed] [Google Scholar]
  5. Crook T., Morgenstern J. P., Crawford L., Banks L. Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras. EMBO J. 1989 Feb;8(2):513–519. doi: 10.1002/j.1460-2075.1989.tb03405.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dimri G. P., Lee X., Basile G., Acosta M., Scott G., Roskelley C., Medrano E. E., Linskens M., Rubelj I., Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9363–9367. doi: 10.1073/pnas.92.20.9363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Domingo M., Einig C., Eigenbrodt E., Reinacher M. Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies. J Histochem Cytochem. 1992 May;40(5):665–673. doi: 10.1177/40.5.1374093. [DOI] [PubMed] [Google Scholar]
  8. Dürst M., Dzarlieva-Petrusevska R. T., Boukamp P., Fusenig N. E., Gissmann L. Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene. 1987;1(3):251–256. [PubMed] [Google Scholar]
  9. Eigenbrodt E., Basenau D., Holthusen S., Mazurek S., Fischer G. Quantification of tumor type M2 pyruvate kinase (Tu M2-PK) in human carcinomas. Anticancer Res. 1997 Jul-Aug;17(4B):3153–3156. [PubMed] [Google Scholar]
  10. Ethier M. F., Hickler R. B., Dobson J. G., Jr Aging increases adenosine and inosine release by human fibroblast cultures. Mech Ageing Dev. 1989 Nov;50(2):159–168. doi: 10.1016/0047-6374(89)90011-0. [DOI] [PubMed] [Google Scholar]
  11. Glaser G., Giloh H., Kasir J., Gross M., Mager J. On the mechanism of the glucose-induced ATP catabolism in ascites tumour cells and its reversal by pyruvate. Biochem J. 1980 Dec 15;192(3):793–800. doi: 10.1042/bj1920793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gutierrez G. Cellular energy metabolism during hypoxia. Crit Care Med. 1991 May;19(5):619–626. doi: 10.1097/00003246-199105000-00008. [DOI] [PubMed] [Google Scholar]
  13. Hara E., Smith R., Parry D., Tahara H., Stone S., Peters G. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol Cell Biol. 1996 Mar;16(3):859–867. doi: 10.1128/mcb.16.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harley C. B. Human ageing and telomeres. Ciba Found Symp. 1997;211:129–144. doi: 10.1002/9780470515433.ch9. [DOI] [PubMed] [Google Scholar]
  15. Hayflick L. Aging, longevity, and immortality in vitro. Exp Gerontol. 1992 Jul-Aug;27(4):363–368. doi: 10.1016/0531-5565(92)90066-9. [DOI] [PubMed] [Google Scholar]
  16. Heldt H. W., Schwalbach K. The participation of GTP-AMP-P transferase in substrate level phosphate transfer of rat liver mitochondria. Eur J Biochem. 1967 Apr;1(2):199–206. doi: 10.1007/978-3-662-25813-2_31. [DOI] [PubMed] [Google Scholar]
  17. Henderson J. F., Scott F. W. Inhibition of animal and invertebrate cell growth by naturally occurring purine bases and ribonucleosides. Pharmacol Ther. 1980;8(3):539–571. doi: 10.1016/0163-7258(80)90076-5. [DOI] [PubMed] [Google Scholar]
  18. Hugo F., Mazurek S., Zander U., Eigenbrodt E. In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation. J Cell Physiol. 1992 Dec;153(3):539–549. doi: 10.1002/jcp.1041530315. [DOI] [PubMed] [Google Scholar]
  19. Höckel M., Vaupel P. Biological consequences of tumor hypoxia. Semin Oncol. 2001 Apr;28(2 Suppl 8):36–41. [PubMed] [Google Scholar]
  20. Inouye S., Yamada Y., Miura K., Suzuki H., Kawata K., Shinoda K., Nakazawa A. Distribution and developmental changes of adenylate kinase isozymes in the rat brain: localization of adenylate kinase 1 in the olfactory bulb. Biochem Biophys Res Commun. 1999 Jan 27;254(3):618–622. doi: 10.1006/bbrc.1998.0116. [DOI] [PubMed] [Google Scholar]
  21. Jazwinski S. M. Metabolic control and ageing. Trends Genet. 2000 Nov;16(11):506–511. doi: 10.1016/s0168-9525(00)02119-3. [DOI] [PubMed] [Google Scholar]
  22. Lane M. A., Black A., Handy A., Tilmont E. M., Ingram D. K., Roth G. S. Caloric restriction in primates. Ann N Y Acad Sci. 2001 Apr;928:287–295. doi: 10.1111/j.1749-6632.2001.tb05658.x. [DOI] [PubMed] [Google Scholar]
  23. Lee H. M., Greeley G. H., Jr, Englander E. W. Age-associated changes in gene expression patterns in the duodenum and colon of rats. Mech Ageing Dev. 2001 Apr 15;122(4):355–371. doi: 10.1016/s0047-6374(00)00254-2. [DOI] [PubMed] [Google Scholar]
  24. Lin S. J., Defossez P. A., Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science. 2000 Sep 22;289(5487):2126–2128. doi: 10.1126/science.289.5487.2126. [DOI] [PubMed] [Google Scholar]
  25. Lin S. S., Manchester J. K., Gordon J. I. Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae. J Biol Chem. 2001 Jul 18;276(38):36000–36007. doi: 10.1074/jbc.M103509200. [DOI] [PubMed] [Google Scholar]
  26. Longnus Sarah L., Wambolt Richard B., Parsons Hannah L., Brownsey Roger W., Allard Michael F. 5-Aminoimidazole-4-carboxamide 1-beta -D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms. Am J Physiol Regul Integr Comp Physiol. 2003 Apr;284(4):R936–R944. doi: 10.1152/ajpregu.00319.2002. [DOI] [PubMed] [Google Scholar]
  27. Marsin Anne-Sophie, Bouzin Caroline, Bertrand Luc, Hue Louis. The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase. J Biol Chem. 2002 Jun 13;277(34):30778–30783. doi: 10.1074/jbc.M205213200. [DOI] [PubMed] [Google Scholar]
  28. Mathupala S. P., Rempel A., Pedersen P. L. Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase. J Bioenerg Biomembr. 1997 Aug;29(4):339–343. doi: 10.1023/a:1022494613613. [DOI] [PubMed] [Google Scholar]
  29. Mazurek S., Hugo F., Failing K., Eigenbrodt E. Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: role of P36. J Cell Physiol. 1996 May;167(2):238–250. doi: 10.1002/(SICI)1097-4652(199605)167:2<238::AID-JCP7>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  30. Mazurek S., Michel A., Eigenbrodt E. Effect of extracellular AMP on cell proliferation and metabolism of breast cancer cell lines with high and low glycolytic rates. J Biol Chem. 1997 Feb 21;272(8):4941–4952. doi: 10.1074/jbc.272.8.4941. [DOI] [PubMed] [Google Scholar]
  31. Mazurek S., Weisse G., Wüst G., Schäfer-Schwebel A., Eigenbrodt E., Friis R. R. Energy metabolism in the involuting mammary gland. In Vivo. 1999 Nov-Dec;13(6):467–477. [PubMed] [Google Scholar]
  32. Mazurek S., Zwerschke W., Jansen-Dürr P., Eigenbrodt E. Metabolic cooperation between different oncogenes during cell transformation: interaction between activated ras and HPV-16 E7. Oncogene. 2001 Oct 18;20(47):6891–6898. doi: 10.1038/sj.onc.1204792. [DOI] [PubMed] [Google Scholar]
  33. Nestelbacher R., Laun P., Vondráková D., Pichová A., Schüller C., Breitenbach M. The influence of oxygen toxicity on yeast mother cell-specific aging. Exp Gerontol. 2000 Feb;35(1):63–70. doi: 10.1016/s0531-5565(99)00087-x. [DOI] [PubMed] [Google Scholar]
  34. Raghunathan R., Russell J. D., Arinze I. J. Pyruvate carboxylase and phosphoenopyruvate carboxykinase in cultured human fibroblasts. J Cell Physiol. 1977 Aug;92(2):285–292. doi: 10.1002/jcp.1040920217. [DOI] [PubMed] [Google Scholar]
  35. Rapaport E. Experimental cancer therapy in mice by adenine nucleotides. Eur J Cancer Clin Oncol. 1988 Sep;24(9):1491–1497. doi: 10.1016/0277-5379(88)90340-9. [DOI] [PubMed] [Google Scholar]
  36. Rivera-Nieves J., Thompson W. C., Levine R. L., Moss J. Thiols mediate superoxide-dependent NADH modification of glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem. 1999 Jul 9;274(28):19525–19531. doi: 10.1074/jbc.274.28.19525. [DOI] [PubMed] [Google Scholar]
  37. Ronen S. M., DiStefano F., McCoy C. L., Robertson D., Smith T. A., Al-Saffar N. M., Titley J., Cunningham D. C., Griffiths J. R., Leach M. O. Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis. Br J Cancer. 1999 Jun;80(7):1035–1041. doi: 10.1038/sj.bjc.6690459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schoonen W. G., Wanamarta A. H., van der Klei-van Moorsel J. M., Jakobs C., Joenje H. Characterization of oxygen-resistant Chinese hamster ovary cells. III. Relative resistance of succinate and alpha-ketoglutarate dehydrogenases to hyperoxic inactivation. Free Radic Biol Med. 1991;10(2):111–118. doi: 10.1016/0891-5849(91)90004-m. [DOI] [PubMed] [Google Scholar]
  39. Schoonen W. G., Wanamarta A. H., van der Klei-van Moorsel J. M., Jakobs C., Joenje H. Respiratory failure and stimulation of glycolysis in Chinese hamster ovary cells exposed to normobaric hyperoxia. J Biol Chem. 1990 Jul 5;265(19):1118–1124. [PubMed] [Google Scholar]
  40. Serrano M., Blasco M. A. Putting the stress on senescence. Curr Opin Cell Biol. 2001 Dec;13(6):748–753. doi: 10.1016/s0955-0674(00)00278-7. [DOI] [PubMed] [Google Scholar]
  41. Serrano M., Lin A. W., McCurrach M. E., Beach D., Lowe S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell. 1997 Mar 7;88(5):593–602. doi: 10.1016/s0092-8674(00)81902-9. [DOI] [PubMed] [Google Scholar]
  42. Sirover M. A. New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta. 1999 Jul 13;1432(2):159–184. doi: 10.1016/s0167-4838(99)00119-3. [DOI] [PubMed] [Google Scholar]
  43. Susor W. A., Rutter W. J. Method for the detection of pyruvate kinase, aldolase, and other pyridine nucleotide linked enzyme activities after electrophoresis. Anal Biochem. 1971 Sep;43(1):147–155. doi: 10.1016/0003-2697(71)90119-9. [DOI] [PubMed] [Google Scholar]
  44. Tissenbaum Heidi A., Guarente Leonard. Model organisms as a guide to mammalian aging. Dev Cell. 2002 Jan;2(1):9–19. doi: 10.1016/s1534-5807(01)00098-3. [DOI] [PubMed] [Google Scholar]
  45. Van Rompay A. R., Johansson M., Karlsson A. Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases. Pharmacol Ther. 2000 Aug-Sep;87(2-3):189–198. doi: 10.1016/s0163-7258(00)00048-6. [DOI] [PubMed] [Google Scholar]
  46. Wagner M., Hampel B., Bernhard D., Hala M., Zwerschke W., Jansen-Dürr P. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploidization and senescence-associated apoptosis. Exp Gerontol. 2001 Aug;36(8):1327–1347. doi: 10.1016/s0531-5565(01)00105-x. [DOI] [PubMed] [Google Scholar]
  47. Wagner M., Hampel B., Hütter E., Pfister G., Krek W., Zwerschke W., Jansen-Dürr P. Metabolic stabilization of p27 in senescent fibroblasts correlates with reduced expression of the F-box protein Skp2. Exp Gerontol. 2001 Dec;37(1):41–55. doi: 10.1016/s0531-5565(01)00165-6. [DOI] [PubMed] [Google Scholar]
  48. Wang Wengong, Yang Xiaoling, López de Silanes Isabel, Carling David, Gorospe Myriam. Increased AMP:ATP ratio and AMP-activated protein kinase activity during cellular senescence linked to reduced HuR function. J Biol Chem. 2003 May 1;278(29):27016–27023. doi: 10.1074/jbc.M300318200. [DOI] [PubMed] [Google Scholar]
  49. Weisman G. A., Lustig K. D., Lane E., Huang N. N., Belzer I., Friedberg I. Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine. J Biol Chem. 1988 Sep 5;263(25):12367–12372. [PubMed] [Google Scholar]
  50. Yamada K., Noguchi T. Regulation of pyruvate kinase M gene expression. Biochem Biophys Res Commun. 1999 Mar 16;256(2):257–262. doi: 10.1006/bbrc.1999.0228. [DOI] [PubMed] [Google Scholar]
  51. Yu Seong-Woon, Wang Hongmin, Poitras Marc F., Coombs Carmen, Bowers William J., Federoff Howard J., Poirier Guy G., Dawson Ted M., Dawson Valina L. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science. 2002 Jul 12;297(5579):259–263. doi: 10.1126/science.1072221. [DOI] [PubMed] [Google Scholar]
  52. Zerfass K., Schulze A., Spitkovsky D., Friedman V., Henglein B., Jansen-Dürr P. Sequential activation of cyclin E and cyclin A gene expression by human papillomavirus type 16 E7 through sequences necessary for transformation. J Virol. 1995 Oct;69(10):6389–6399. doi: 10.1128/jvi.69.10.6389-6399.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zwerschke W., Mazurek S., Massimi P., Banks L., Eigenbrodt E., Jansen-Dürr P. Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein. Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1291–1296. doi: 10.1073/pnas.96.4.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. ter Kuile B. H., Westerhoff H. V. Transcriptome meets metabolome: hierarchical and metabolic regulation of the glycolytic pathway. FEBS Lett. 2001 Jul 6;500(3):169–171. doi: 10.1016/s0014-5793(01)02613-8. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES