Cell Transformation Assays

Max Costa1, Jessica E. Sutherland1

1 New York University School of Medicine, New York, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.4
DOI:  10.1002/0471140856.tx0304s01
Online Posting Date:  May, 2001
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Morphological transformation of mammalian cells following exposure to a chemical agent is frequently used as a preliminary screen for the carcinogenic potential of the agent. This unit describes a protocol using Syrian hamster embryo (SHE) cells to assay transformation.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Pregnant Golden Syrian hamsters
  • Chinese hamster ovary (CHO; ATCC)
  • Disinfectant solution: e.g., 2% (v/v) Orsyl
  • 95% (v/v) ethanol
  • Serum‐free Dulbecco's minimal essential medium (DMEM)
  • 0.5% (w/v) trypsin solution in recipenormal saline A
  • Fetal bovine serum (FBS; tested in cell culture)
  • Complete DMEM supplemented with 10% (v/v) FBS
  • Normal saline A (see recipe)
  • Dimethyl sulfoxide (DMSO) or glycerol
  • Complete DMEM supplemented with 20% (v/v) FBS
  • F‐12 medium supplemented with 10% (v/v) FBS
  • Test agent
  • 0.5% (w/v) crystal violet solution in 95% ethanol
  • Positive and negative control agents
  • Serum‐free F‐12 medium
  • Absorbant cloth, sterile
  • Laminar flow hood
  • Dissecting instruments
  • 100‐mm tissue culture plates, sterile
  • 10‐ml pipets, sterile
  • Freezing vials for storing cells
  • 150‐ml beakers
  • Inverted tissue culture microscope
  • Additional reagents and equipment for sacrificing animals ( appendix 3A), for counting cells with a Coulter counter or hemacytometer ( appendix 3A), and for trypan blue exclusion ( appendix 3A)
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Literature Cited

Literature Cited
   Berwald, Y. and Sachs, L. 1965. In vitro transformation of normal cells to tumor cells by carcinogenic hydrocarbons. J. Natl. Cancer Inst. 35:641‐661.
   Costa, M. 1980. Metal carcinogenesis testing. Principles and in vitro methods. Humana Press, Clifton, N.J.
   Dunkel, V.C., Rogers, C., Swierenga, S.H.H., Brillinger, R.L., Gilman, J.P.W., and Nestman, E.R. 1991. Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: III. Cell transformation in C3H/10T1/2 mouse embryo cell, BALB/c3T3 mouse fibroblast and Syrian hamster embryo cell cultures. Mutat. Res. 246:285‐300.
   Evans, C.H. and DiPaulo, J.A. 1981. In vitro mammalian cell transformation for identification of carcinogens, cocarcinogens, and anticarcinogens. ,In Short‐Term Tests for Chemical Carcinogens (H.F. Stich and R.H.C. San, eds.) 306‐322. Springer‐Verlag, New York.
   IARC/NCI/EPA Working Group. 1985. Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for the prediction of carcinogenic chemicals: Overview and recommended protocols. Cancer Res. 45:2395‐2399.
   Kakunaga, T. 1973. A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3. Intl. J. Cancer. 12:463‐473.
   Kerckaert, G.A., Brauninger, R., LeBoeuf, R.A., and Isfort, R.J. 1996a. Use of the Syrian hamster embryo cell transformation assay for carcinogenicity prediction of chemicals currently being tested by the National Toxicology Program in rodent bioassays Environ. Health Perspect. 104:1075‐1084.
   Kerckaert, G.A., Isfort, R.J., Carr, G.J., Aardema, R.A., and LeBoeuf, R.A. 1996b. A comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70. Mutat. Res. 356:65‐84.
   Reznikoff, C.A., Bertram, J.S., Brankow, D.W., and Heidelberger, C. 1973. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res. 33:3239‐3249.
   Rundell, J.O. 1984. In vitro cell transformation: An overview In Carcinogenesis and Mutagenesis Testing (J.F.. Douglas, ed.) pp. 39‐62. Humana Press. Clifton, N.J.
   Trott, D.A., Cuthbert, A.P., Overell, R.W., Russo, I., and Newbold, R.F. 1995. Mechanisms involved in the immortalization of mammalian cells by ionizing radiation and chemical carcinogens. Carcinogenesis 16:193‐204.
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