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

Kevin T. Morgan

GlaxoSmithKline, Research Triangle Park, North Carolina 27709, kevin.morgan{at}aventis.com, Aventis Pharmaceuticals, Route 202-206, PO Box 6800, Bridgewater, New Jersey 08807, Mailing Address, 5020 F Edwards Mill Road, Raleigh, North Carolina 27612

Warren Casey

GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Marilyn Easton

GlaxoSmithKline, Research Triangle Park, North Carolina 27709, PresentAddress 121 Windward Court, Cary, North Carolina 27513

Don Creech

GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Hong Ni

GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Lawrence Yoon

GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Steve Anderson

GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Charles W. Qualls, JR

GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Lynn M. Crosby

Mailing Address, 5020 F Edwards Mill Road, Raleigh, North Carolina 27612

Alistair Macpherson

Department of Mathematics, LeHigh University, Bethlehem, Pennsylvania, 18015

Peter Bloomfield

Department of Statistics, North Carolina State University (NCSU), Raleigh, North Carolina 27695

Timothy C. Elston

Department of Mathematics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3250

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.

Key Words: Transcriptome • dynamics • media replacement • HepG2 • liver • human • CYP1A1 • asparagine synthetase.

Toxicologic Pathology, Vol. 31, No. 4, 448-461 (2003)
DOI: 10.1080/01926230390213784
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