The Effects of Tween 80 on the in J―itroMetabolism of Cells of the Ehrlich-Lettré Ascites Carcinoma1 E. R. M. KAY (Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, Rochester, New York) SUMMARY Marked alterations in cell permeability were brought about by the presence of the detergent Tween 80 in preparations of Ehrlich-Lettré ascites cells. The growth of these cells was normal in host mice after treatment and thus they were viable as transplants. The permeability of the cells increased greatly, as shown by the uptake of Lissamine green. Soluble nucleotides and amino acids were removed by the Tween treatment. Oxygen uptake was reduced by 50 %. The incorporation of formate into protein and deoxyribonucleic acid (DNA) decreased, but it increased into nuclear ribonucleic acid (RNA). Incorporation of @Pinto the phospholipids increased greatly and was found to be localized in phosphatidyl serine. The interpretation of the alterations brought about by Tween 80 are discussed in relation to the special properties of the tumor cells. It has been recognized for some time that the peculiar The tumor was used directly or was treated with Tween properties of tumor cells which permit their invasiveness, 80. After removal of the ascites fluid from tumor-bearing and adhesiveness are dependent to a large extent on the mice the fluid was centrifuged in 3 15-mi centrifuge tubes properties of the cell membrane (1, 3, 24). The surface to give equal volumes of packed cells for the experiment. properties of cells are, in turn, dependent on the composi The supematant serum from the centrifugation was tion of the membrane, which includes both proteins and saved separately for later use. To 1 tube containing phospholipids. Alteration of the composition of the packed cells, 10 ml of saline were added. To a 2nd tube plasma membrane or of the endoplasmic reticulum and were added 10 ml of 0.25 Msucrose, and to a 3rd tube were mitochondria would be expected to alter markedly the added 10 ml of 0.25 M sucrose containing 1 % Tween 80. metabolic properties of the cells in question. In this The tubes were stoppered and mixed thoroughly to disperse paper are presented the results of a study in which altera the cells and were allowed to stand at room temperature for tions were made in the membrane structure of cells of the 15 or 30 mm. The tubes were then centrifuged to pack Ehrlich-Lettré ascites carcinoma using the detergent the cells (5 mm at 3000 rpm in an International clinical cen Tween 80. This study has been carried out to show the trifuge) and the supernatants were saved for further analy changes in metabolic activity resulting from the action of sis. The packed cells were washed and centrifuged twice, this surface active agent. The experiments have included using 0.9 % NaCl as a wash solution, and the supematants observations on changes in permeability, respiration, and were discarded. Finally, the original serum samples were incorporation of inorganic @Pand ‘@Cformateinto the added to the packed cells and, after shaking, the cell sus nucleic acids, proteins, and phospholipids of this tumor pensions were ready for use either for reinoculation experi under in vitro conditions. A preliminary report of this ments or for in vitro metabolic experiments. work has been published (13). Extraction of acid soluble nucleotides was carried out by adding ice cold 4 % HCJO4 to an aliquot of the suspension, MATERIALS AND METHODS after centrifugation to pack the cells, and reading of the optical density of the acid extract at 260 nip using a Beck The Ehrlich-Lettré ascites carcinoma used in this study was carried as a stock tumor in mice of the CFW strain man model DU spectrophotometer. The amino acids were extracted from a similar aliquot of the suspension, obtained from Carworth Farms. The tumor was main tained by transplanting 0.2 ml of the ascites fluid at 7- to which was then centrifuged; 95 % ethanol (5 ml) was used 10-day intervals. Fluid containing tumor cells to be used as the extracting agent. The alcoholic supematant ob in in vitro experiments was drained from host mice, pooled, tamed was dried and the residue dissolved in a small and used without treatment as a control tumor prepara volume of 70 % ethanol and applied to sheets of Whatman tion, or treated with Tween 80 as described below. No. 1 chromatograph paper for 2-dimensional chromatog raphy using the solvent system of Hardy (6). 1 Supported by grant No. CA 05172 from the National Cancer Institute, USPHS. Respiration of the control and treated cells was measured Received for publication October 28, 1964. by the appropriate procedures using a Bronwill-Warburg 764 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1965 American Association for Cancer Research. KAY—Effects of Tween 80 on Ehrlich-Lettré Ascites Cells 765 respirometer. Metabolic experiments employing ‘4C sample of the solution was plated on a stainless steel formats or 32@carrier-free inorganic phosphate were carried planchet for counting and a 0.1-mi aliquot was diluted to out using a Dubnoff metabolic shaking incubator. 4ml;theopticaldensitywasdeterminedatanappropriate For incubation experiments an aliquot of the above wave length using the Beckman model DU spectropho suspensions was added to the flasks as follows: 2.0 ml cell tometer (25). suspension (containing 0.5 ml packed cells); 1.0 ml 0.25 M sucrose—0.05 M phosphate buffer, pH 7.4; 0.8 ml 0.25 RESULTS AND DISCUSSION M sucrose—O.1 M glucose; 0.2 ml H2O containing the In preliminary studies treatment of the tumor cells with isotope (8 pc ‘@Cformate, 20 pC @2P)(12). Glutamine was 0.25 i@sucrose or with 0.25 Msucrose containing 1 % Tween added to the incubation mixture before adding the cells, 80, appeared to effect no loss in viability of the cells when so that the final concentration of glutamine was 100 pg/mi. they were injected into host mice and the tumor growth The incubations were carried out for periods of 1 hr. After was measured by weight increase. This finding is in this time, the flask contents were centrifuged and the agreement with the findings of Morgan (23) and others. packed cells were frozen prior to preparation of the cell When the cells were examined with the dye Lissamine fractions by a modification of a previously published green, marked permeability changes were noticed after method (22). incubation with Tween 80. This dye is a negatively The nuclear and cytoplasmic fractions were extracted charged nontoxic triphenylmethane dye and has been 1st with ice cold 10 % trichloroacetic acid (TCA) to remove used extensively by a number of workers as an indicator acid soluble components. Lipids were removed from the of cell viability. It has been studied extensively by acid extracted fraction by using acetone, ethanol, ethanol Holmberg (9) as an indicator of cell death. According to ether, and ether extractions from the nuclear and cyto this author the cells become freely permeable when ir plasmic fractions of the incubation experiment. These combined extracts were dried, dissolved in chloroform TABLE 1 methanol (1 : 1), and applied to silicic impregnated paper EFFECT OF TWEEN 80 TREATMENT ON CELLS OF EHRLICH-LETTR@ for separation of the phospholipids according to the ASCITES CARCINOMA methods already outlined (19). The lipid-free residues Incubation conditions: 2.0 ml cells (containing 0.5 ml packed were hydrolyzed in 0.5 ml of 0.3 N KOH at 37°Cfor18 hr. cells) ; 1.0 ml sucrose (0.25 M)-phosphate buffer (0.05 M) pH 7.4; After this time the alkaline solutions were cooled and 0.8 ml sucrose (0.25 M) + 0.1 M glucose; 0.2 ml H,O (or isotope acidified with 1 drop of 70 % HC1O4. The RNA in the solution). acidified extract was decanted after centrifugation and diluted to 5 ml. An aliquot of 0.5 ml was plated on a nIp)Controla 02Extractable (O.D. at 260 stainless steel planchet and counted in a gas flow Nuclear Chicago counter. Aliquots were analyzed in duplicate for phosphorus by the Allen (2) procedure. All corrections Sucrose 5.5 1.020 were made for half life and background as required, when Sucrose + 1% Tween 806.2 3.31.000 0.550 32@ was used. The specific activity was recorded as cpm/ 100 pg phosphorus. a Control, sucrose, and sucrose + 1% Tween 80 refer to treat ment of the cells prior to incubation. The DNA was extracted from the nuclear residues, after RNA hydrolysis as above, by using 4 % HC1O4at 90°Cfor 15 min. The DNA activity was measured by diluting the supematant extract to 5 ml, plating a 0.5-mi aliquot as above, and measuring the phosphorus of aliquots, in duplicate, by the Allen (2) method. When ‘4Cformate was used as labeled precurser the various RNA and DNA samples were hydrolyzed to their constituent purines and pyrimidines. For this procedure the total extracts of these nucleic acids, obtained as above, were dried in a vacuum desiccator and hydrolyzed with 70 % HC1O4by adding 1 drop of the concentrated acid to the dry residue. The hydrolysis was carried out at 100°C for 1 hr. After this process the samples were diluted to about 0.3 mi and the aliquots were placed at 1 end (10 It cm) of a 50 by 3.8 cm strip of Whatman No. 3 MM paper; descending chromatography was carried out overnight ORIGIN using the solvent system of Wyatt (25), consisting of CHART 1.—Amino acids of Ehrlich-Lettré ascites carcinoma.