Microelectrophoresis in Agarose Gel of Soluble Rat Liver Proteins During Aminoazo Dye Carcinogenesis

Agarose-Gel Electrophoresis of Soluble Proteins from Bronchial Mucosa and Bronchogenic Carcinoma

“…In part, these alterations may represent an altered pattern of gene expression in the tumor. There is also some evidence that in the early stages of carcinogenesis, some proteins present in the soluble fractions were originally derived from the region of the cell membrane [9], Thus, dislodgement or leakage of proteins from cell organelles under the influence of carcinogenic agents, as suggested by Sorof [14], might contribute to the altered soluble protein profile.…”

“…Individual tracheal and bronchial mucosae and nonnecrotic tumors were dissected free of surrounding tissues, rinsed in homogenizing medium and homogenized in ice-cold 0.08 m sodium phosphate buffer, pH 7.8, containing 0.075 m NaCl (approximately 10 ml), as de scribed previously [8]. Soluble proteins were obtained by centrifuging the homogenates for 60 min at 100,000 g at 4'C in a Spinco model-L ultracentrifuge and the supernatants collect ed with a Pasteur pipette.…”

“…The soluble proteins from the tracheal mucosae and some tumors were dialyzed against homogenizing medium containing Carbowax 6000 (Union Carbide, Melbourne, Australia) or, in the case of some later samples, Dextran T40 (Pharmacia, Uppsala, Sweden) in an Oxford multiple dialyzer to a final protein concentration of 1-5 mg/ml, as determined by the biuret method [4], The soluble proteins were electrophoresed immediately or after storage at -20 'C. Elec trophoresis was performed at pH 8.6 in 0.9% agarose gel, as described previously [8], ex cept that the samples were applied to strips of Whatman No. 1 filter paper (1 mm x 12 mm), which were placed on the gel 2 cm from the cathode end of the slide.…”

“…Electrophoretograms were fixed, stained with Ponceau S (G.T. Gurr, England) and dried as described previously [8] and then scanned, us ing a Joyce-Loebl Chromoscan recording and integrating densitometer. Four or more elec trophoretograms were prepared from each tissue sample and the proportion of protein pres ent in each band was calculated from the mean of the values obtained from these densito meter scans.…”

“…This tumor was selected for investigation, first, because various chemical carcinogens present in cigarette smoke have been implicated as etiological agents [11]; secondly, because of the tumor's increasing incidence [13]. Agarose gel electrophoresis was employed in this study, as in a previous report [8], it was shown to be sufficiently sensitive to confirm the alterations which take place in liver soluble proteins during azo dye-induced hepatocarcinogenesis in the rat.…”