Frequent Sampling Reveals Dynamic Responses by the Transcriptome to Routine Media Replacement in HepG2 Cells

Abstract

Cultured cell lines are employed extensively for biological research. Large-scale differential gene expression (LSDGE) is being used to study mechanisms of toxicity in such cultures. `Normal' gene expression dynamics could have a major impact on the design and interpretation of these studies. In order to provide understanding of such dynamics, we investigated LSDGE responses to media replacement in human hepatoblastoma cells (HepG2) using 5-minute sampling frequencies for 6 hours post routine media replacement. Each mRNA transcript was found to exhibit a characteristic `operating range' based on signal intensity. Following media replacement, which replenishes nutrients (eg, glucose and glutamate) and removes excretory products (eg, lactate), a complex set of gene expression changes was observed. Some transcripts appeared to switch on from a quiescent state to a very active one (eg, CYP1A1), others exhibited `clocklike' oscillations (eg, asparagine synthetase), or a synchronous burst (chirp) of expression up regulation (eg, timeless). Mathematical analysis (Fourier Transform, Singular Value Decomposition, Wavelets, Phase Analysis) of oscillating expression patterns identified cycle lengths ranging from 11.8 to 210 minutes. There were prominent 36.5- and 17.4-minute cycles, for subsets of genes, and transcript-specific differences in phase angle with respect to these cycles. The functional consequences of these novel observations remain to be determined. It is clear that dense time-course studies provide a valuable approach to the investigation of physiological responses to nutrients, toxicants, and other environmental variables. This research also highlights the need for an understanding of biological dynamics when using cell culture systems.

An Excel data file representing individual transcripts from the respective Clontech cDNA arrays referred to in this article is available at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. Rows represent data for individual transcripts and columns represent the time-points from 0 to 360 minutes. To access this file, click on the issue link for 31(4), then select this article. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Keywords

Transcriptome dynamics media replacement HepG2 liver human CYP1A1 asparagine synthetase.

References

Alter O. , Brown P. , Botstein D. (2000). Singular value decomposition for genome-wide expression data processing and modeling. Proc Natl Acal Sci USA 97: 10101—10106.

Barbosa-Tessmann IP , Pineda VL , Nick HS , Schuster SM , Kilberg MS (1999). Transcriptional regulation of the human asparagine synthetase gene by carbohydrate availability. Biochem J 339: 151—158.

Berg H. (1993). Random Walks in Biology. Princeton University Press, Princeton, New Jersey, pp 1—152.

Chenchik A. (1997). Atlas human cDNA expression array 1. Clontechniques 12(2): 1—8.

Clayton JD , Kyriacou CP , Reppert SM (2001). Keeping time with the human genome. Nature 409: 829—831.

Collins FS (1999). Overview of DNA chip technology. Nature Gen. 21: 1—60.

Crosby LM , Benavides G. , Yoon L. , Hyder K. , DeAngelo AB , Morgan KT (2000). Morphologic analysis correlates with gene expression changes in cultured F344 rat mesothelial cells. Toxicol Appl Pharmacol 169: 205—221.

Eisen MB , Spellman PT , Brown PO , Botstein D. (1998). Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95: 14863—14868.

Elowitz MB , Leibler S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature 403: 335—338.

10.

Goldbeter A. (1997). Biochemical Oscillations and Cellular Rhythms, The Molecular Bases of Periodic and Chaotic Behaviour. Cambridge University Press, Cambridge, UK, pp 1—605.

11.

Goldman LR , Harnly M. , Flattery J. , Patterson DGJ , Needham LL (2000). Serum polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans among people eating contaminated home-produced eggs and beef . Environ Health Perspect 108(1): 13—19.

12.

Holland MJ (2002). Transcript abundance in yeast varies over six orders of magnitude. J Biol Chem 277(17): 14363—14366.

13.

Holter NS , Mitra M. , Maritan A. , Cieplak M. , Banavar JR , Fedoroff NV (2000). Fundamental patterns underlying gene expression profiles: simplicity from complexity. Proc Natl Acad Sci USA 97(15): 8409— 8414.

14.

Hubbard BB (1998). The World According to Wavelets; The Story of a Mathematical Technique in the Making. 2nd ed, AK Peters, Ltd, Natick, Massachusetts .

15.

Iyer VR , Eisen MB , Ross DT , Schuler G. , Moore T. , Lee JCF , Trent JM , Staudt LM , Hudson JJ , Boguski MS , Lashkari D. , Shalon D. , Botstein D. , Brown PO (1999). The transcriptional program in the response of human fibroblasts to serum. Science 283: 83.

16.

Kepler TB , Elston TC (2001). Stochasticity in transcriptional regulation: Origins, consequences, and mathematical representations. Biophys J 81: 3116—3136.

17.

Klassen CD ed (2001). Casarett and Doull's Toxicology, The Basic Science of Poisons. 6th ed. McGraw-Hill Medical Publishing Division , New York.

18.

Klevecz RR , Ruddle FH (1968). Cyclic changes in synchronized mammalian cell cultures. Science 159(3815): 634—636.

19.

Klevecz RR (1969). Temporal order in mammalian cells: 1. The periodic synthesis of lactate dehydrogenase in the cell cycle. J Biol Chem 43(2): 207—219.

20.

Klevecz RR , Bowse HB (2000). Tuning in the transcriptome: Basins of attraction in the yeast cell cycle. Cell Prolif 33: 209—218.

21.

Klevecz RR , Murray JD (2001). Genome wide oscillations in expression: Wavelet analysis of time series data from yeast expression arrays uncovers the dynamic architecture of phenotype. Mol Biol Rep 28: 73—82.

22.

Mathews MB , Sonenberg N. , Hershey JWB (2000). Origins and principles of translational control . In: Translational Control of Gene Expression, Sonenberg N , Hershey JWB , Mathews MB (eds). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 1—31.

23.

McAdams HH , Arkin A. (1997). Stochastic mechanisms in gene expression. Proc Natl Acad Sci USA 94: 814—819.

24.

Merrill C. , Ni H. , Yoon L. , Tirmenstein MA , Narayanan P. , Benavides G. , Easton M. , Creech D. , Hu CX , Thomas HC , Morgan KT (2002). Etomoxirinduced oxidative stress detected by differential gene expression is confirmed biochemically. Toxicol Sci 68: 93—101.

25.

Morel Y. , Barouki R. (1998). Down-regulation of cytochrome P4501A1 gene promotor by oxidative stress. J Biol Chem 273(41): 26969—26976.

26.

Morgan KT , Ni H. , Brown HR , Yoon L. , Qualls CWJ , Crosby LM , Reynolds R. , Gaskill B. , Anderson SP , Kepler TB , Brainard T. , Liv N. , Easton M. , Merrill C. , Creech D. , Sprenger D. , Conner G. , Johnson PR , Fox T. , Sartor M. , Richard E. , Kuruvilla S. , Casey W. , Benavides G. (2002). Application of cDNA microarray technology to in vitro toxicology and the selection of genes for a real time RT-PCR-based screen for oxidative stress in Hep-G2 cells. Toxicol Pathol 30(4): 435—451.

27.

Murray JD (1993). Mathematical biology. In: Biomathematics , Vol 19, 2nd ed, Levin SA (ed). Springer , New York, pp 1—767.

28.

Potter VR , Watanabe M. , Pitot HC , Morris HP (1969). Systematic oscillations in metabolic activity in rat liver and hepatomas. Survey of normal dipoid and other hepatoma lines . Cancer Res 29: 55—78.

29.

Puri PL , Maclachlan TK , Levrero M. , Giordano A. (1999). The intrinsic cell cycle: from yeast to mammals . In: The Molecular Basis of Cell Cycle and Growth Control , Stein GS , Bajerga R , Giordano A , Denhardt DT (eds). Wiley-Liss, New York, pp 15—79.

30.

Rier SE (2002). The potential role of exposure to environmental toxicants in the pathophysiology of endometriosis. Ann NY Acad Sci 955: 201—212.

31.

Shymko RM , Klevecz RR , Kauffman SA (1984). The cell cycle as an oscillatory system. In: Cell Cycle Clocks, Edmunds L (Ed). Marcel Dekker, Inc, New York, pp 273—293.

32.

Solé R. , Goodwin B. (2000). Signs of Life, How ComplexityPervadesBiology. Basic Books, New York.

33.

Strogatz SH (1994). Nonlinear Dynamics and Chaos. Perseus Book Publishing, New York, pp 1—498.

34.

Wager-Smith K. , Kay SA (2000). Circadian rhythm genetics: From flies to mice to humans. Nature Gen 26: 23—27.