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Two-Dimensional Gel Electrophoretic Analysis of Cytoplasmic Proteins from Friend Erythroleukemia Cells Chemically Induced to Undergo Terminal Erythroid Differentiation¹

Biology Division [B. K. M.] and Engineering Physics and Mathematics Division [R. C. M.], Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 and National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [J. K. S.]

The proerythroblastoid Friend erythroleukemia cell (FELC) line, clone TR 19-9, was treated with 4 mM hexamethylene bisacetamide (HMBA) over a 6-day period. Greater than 94% of the FELC reacted positively to the benzidine assay for hemoglobin by Day 4 of treatment. Protein accumulation during the final 4 days of treatment (from Days 2 to 6) was monitored by labeling for 24-h periods with a ¹⁴C-labeled amino acid mixture. At the end of each radiolabeling time point, cells were harvested and cytoplasmic proteins were isolated and subjected to two-dimensional gel electrophoresis in triplicate. Short-term fluorographic exposures were made in the linear X-ray film response range to monitor those polypeptides which were most rapidly accumulated. Fluorographs were digitized for computer image analysis and gel data comparison rationales were used to combine the polypeptides contained on the replicate fluorographs into a single cytoplasmic polypeptide profile or Master Image for each of the two experimental conditions, control and HMBA-treated FELC. These two images were merged into a single Master Composite Image containing a total of 211 polypeptides so that those polypeptides common to both and/or unique to each of the experimental conditions could be viewed graphically in the same plane. A total of 98 polypeptides in HMBA-treated FELC were shown to have large accumulation rate differences from the control FELC; 32 of these polypeptides were present in the HMBA Master Image which were not detected in the Control Master Image and 66 polypeptides were present in the Control Master Image but not detected in the HMBA Master Image. Five polypeptides, found in both Master Images, were shown to vary quantitatively in the HMBA-treated FELC from the corresponding polypeptides in the control. These quantitative data measurements on the rates of accumulation of various common polypeptides offer a mode for simultaneously monitoring the kinetics of induction and repression of many gene products throughout an experimental time course.

¹ Research sponsored by the Office of Health and Environmental Research, United States Department of Energy, under contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc. A preliminary report of this work has been presented at the 78th Annual Meeting of the American Association for Cancer Research, Atlanta, GA, May 20–23, 1987 (1). This work was conducted for the National Toxicology Program under the National Institute of Environmental Health Sciences, Interagency Agreement No. Y01-ES-60151, DOE No. 40-1669-85.

² To whom requests for reprints should be addressed, at Carcinogenesis and Toxicology Evaluation Branch, National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709.

Received 8/19/87. Revised 11/19/87. Accepted 11/30/87.