The Effects of Tween 80 on the in Jв€•Itrometabolism of Cells of The

The Effects of Tween 80 on the in Jâ€•itroMetabolism of Cells of the Ehrlich-LettrÃ© 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-LettrÃ© 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-LettrÃ© 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 â€˜@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-LettrÃ© 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. KAYâ€”Effects of Tween 80 on Ehrlich-LettrÃ© Ascites Cells 765 respirometer. Metabolic experiments employing â€˜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 sucroseâ€”0.05 M phosphate buffer, pH 7.4; 0.8 ml 0.25 RESULTS AND DISCUSSION M sucroseâ€”O.1 M glucose; 0.2 ml H2O containing the In preliminary studies treatment of the tumor cells with isotope (8 pc â€˜@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 37Â°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 90Â°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 â€˜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 100Â°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.â€”Amino acids of Ehrlich-LettrÃ© ascites carcinoma. isopropanol, 65 ml; concentrated HC1, 16.5 ml; and enough Free amino acid pattern of control Ehrlich ascites cells. Amino water to make 100 ml. The bases were identified by their acids identifiedfrom solvent amino acid maps prepared using the R@values, eluted, and lyophiized ; 0.3 mi of appropriate solvents : I = ethanol, 1-butanol, water, propionic acid (10:10:5:2); II = 1-butanol, acetone, water, dicyclohexylamine concentrations of acid was added to these dry residues. (10:10:5:2). ASP, aspartic acid; GLY, glycine; GLT, glutamine; With the purine, adenine and the thymine, 0.1 N HC1 was SER, serine; GLU, glutomic acid; PR, proline; ALA, alanine; TY, used; with guanine, 1.6 N HC1 was employed. A 0.1-mi tyrosine; VAL, valine; LEU, leucine.

tion with adenylic and guanylic acid nucleotides reduced to 0 the greatest extent. Since this reduction in free nucleo tides occurred during the Tween treatment it would be 0 reflected in the lower Qo, found for these cells during subsequent incubation. In view of the loss of acid soluble materials during Tween treatment, it was of interest to find a corresponding loss in amino acids from the cells. In Charts 1 and 2 are chromatographs of the ethanol extractable materials of the cells. It can be seen readily that there are marked quanti tative differences in the Tween 80 treated cells. The 2 Q chromatographs were prepared in identical fashion from 14t the same volume of packed cells and thus the effects of the Tween 80 treatment are obvious. This is a further indica ORIGIN tion of the effects of the Tween on cell permeability. In the Tween 80 treated preparation there is a marked CHART 2.â€”Free amino acid pattern of Tween 80 treated Ehr lich-LettrÃ© ascites cells. Absicca and ordinate refer to solvent diminution of aspartic acid, glycine, glutamic acid, and systems as in Chart 1. alanine. Glutamine was reduced considerably below the normally low levels found in these cells. A very low level TABLE 2 of extractable leucine and tyrosine was observed in the EFFECT OF TWEEN 80 TREATMENT ON INCORPORATION OF 14C treated cells. The level of cellular serine was not reduced FORMATE BY ASCITES CELLS noticeably by the Tween treatment. An examination of Specific activity = cpm/JLMbase or = cpm/mg protein. the amino acid pattern has confirmed the fact that Tween treatment has decided effects on the permeability of these cells.

FRACTIONRNA@DNACytoplasm The use of 14(@formate as a label for metabolic experi ments permits the determination of purine synthesis in TweenControlTweenAdenineControl TweenNuclearControl nuclear and cytoplasmic RNA, and in the purines and thymine of DNA. It also permits the determination of protein synthesis in the cells because the glycine-serine Guanine 2451 1558 15,615 19,285 2033 615 conversion system is known to be present in many tissues Thymine â€” â€” â€” â€” 3853 1715 (15), including tumor cells (14). There is also evidence Protein557229562911169134,0781,28851,2502113000â€”1045 â€” that tumors depend to a marked degree on glycine and a RNA, ribonucleic acid; DNA, deoxyribonucleic acid. serine (17, 20, 21). The effects of Tween 80 treatment on 14Cformate in corporation are shown in Table 2. It can be seen that reversible damage has occurred, presumably because of a there is a reduction in this incorporation into protein in severe metabolic disturbance associated with changes in nembrane activity. Holmberg claimed that penetration both nuclear and cytoplasmic fractions. Since it was of the Lissamine green was an indication of irreversibly damaged and, therefore, â€œdeadâ€•cells.A normal prepara TABLE 3 tion of ascites tumor cells (Fig. 1) and a preparation of EFFECT OF TWEEN 80 TREATMENT ON INCORPORATION OF 32@ tumor cells (Fig. 2) were treated with Tween 80 under the INTO Ascrris CELLS @ above experimental conditions. It can be seen readily Specific activity = cpm/100 P.

that after Tween treatment about 90 % of the cells took up ACIDControl the Lissamine green. Figs. 3 and 4 are comparison FRACTIONRIBONUCLEIC photomicrographs of control and treated cells under high changeCytoplasm Tween % power. The penetration of the dye can be seen in the nuclei and cytoplasm of the cells after Tween 80 treatment. 16,586 +29 In view of the findings of Holmberg (9), with 90 % of the Nuclei11,614 +12TABLE20,438 22,818 cells taking up the dye after Tween 80 treatment, it might be assumed that the preparation was metabolically in 4 active. However, in the work outlined in this paper, the uptake of this dye indicates only that the cells are more EFFECT OF TWEEN 80 TREATMENT ON INCORPORATION OF 32@ INTO ASCITES CELLS permeable, without necessarily implying irreversibility of SpecificP.FRACTIONPHOSPUOLIPID@â€” activity = cpm/100 pg metabolic activity. In Table 1 it is seen that the Qo, of the preparation after Tween treatment was reduced to half that of the control value. In an examination of the acid soluble fraction, using the optical density at 260 mp changeCytoplasm Control Tween % as a measure, it was found that the Qo, was reduced to about 60 % of the control cells. The constituent nucleo 9343 +134 tides of this fraction were selectively reduced in concentra Nuclei3990 2353 9354 +297

CYTOPLASM NUCLEI results obtained in other experiments (19) have demon strated that the cells of the Ehrlich-LettrÃ© ascites car @@@ â€˜â€”.._, PHOS cinoma can incorporate â€˜@Cformatsinto several lipids. Chromatographic separation of the phospholipids showed CC the presence of radioactivity in phosphatidyl serine, phosphatidyl-ethanolamine, lecithin, sphingomyelin, and some unidentified lipids. The findings confirmed that, in @e4J \ this tumor, the conversion of formate to serine takes place via the glycine-serine conversion system (14, 15). The experiments reported in this paper which show very rapid incorporation of 32@into the phosphatidyl serine after @ 65::;:@ â€˜P Tween 80 treatment, suggest a reconstitution of cell mem brane material, in view of the other observations of normal @ c::@ growth of the reinoculated tumor in host mice. The CONTROL TWEEN80 CONTROL TWEEN80 complex phospholipids have been implicated in membrane activity (7, 8) in relation to cation transport. Recent CHART 3. Effect of Tween 80 treatment on incorporation of â€œPinto phospholipids of Ehrlich-LettrÃ© ascites cells. Chromat studies on â€œinducedâ€•pinocytosis by Karnofsky et al. (11) ograph of 32@labeledphosphatides. Crosshatchedspotsrepresent have also shown that phosphatidyl serine is markedly and only radioactivity detected on radioautograph. NON-PHOS, consistently labeled under these conditions. Thus the nonphosphatides; PGP, polyglycerolphosphatides; PE, phos evidence presented here would support the conclusion that phatidylethanolamine; PS, phosphatidylserine; LEC, lecithin; SPH, sphingomyelin; IP, inositol phosphatides; UNID, un Tween 80 treatment disrupts membrane structure, and identified. that these membrane components are reconstituted during the course of recovery. shown above that there is a loss in amino acids and nucleo The fact that nuclear RNA synthesis is stimulated by tides from cells during their contact with the detergent it Tween 80 treatment is of interest, in view of the recent can be assumed that both substrate level and energy findings of LaCour (16) that when Tween was used in a supply are affected. Of interest, however, was the finding nuclear isolation technic (4) the nucleoli became invisible that despite these reductions in activity of protein there but were shown to retain their rapidly labeled RNA. The was observed a stimulation of purine formation in the nuclear RNA may then be implicated indirectly in the nuclear RNA from â€˜@Cformats. In contrast to this effect reconstitution mechanisms seen in this study. If the was an observed reduction in the specific activity of the observed stimulation of RNA indicated an increased purines of the cytoplasmic RNA and of DNA at this 1-hr synthesis of â€œmessengerâ€•responsible for protein synthesis incubation time. The specific activity of the thymine of of the altered membranes, then the evidence for the DNA was also reduced during incubation in the presence of stimulation of the phosphatidyl serine might be evidence â€˜@Cformate. for the formation of the new lipid component of this mem The results obtained when using @Pcarrier-free phos brane system. phate as a label for nucleic acid synthesis are shown in In further examination of the phospholipid fractions of Table 3. Although the above mentioned increase in the treated cells it was observed that there was an absence nuclear RNA synthesis occurred after Tween treatment, of the cardiolipins of the treated cells (Chart 3). This has the activity of cytoplasmic RNA was also stimulated. indicated that the effect of Tween 80 may not be solely at The RNA was measured in these @Pexperiments without the level of the plasma membrane. The cardiolipins are separation of the individual nucleotides; hence the activity considered essentially mitochondrial components (5, 18). of the RNA fraction includes concomitants of nonnucleo The loss of cardiolipins from the treated cells would tide phosphorus (10) which might elevate the observed indicate changes in mitochondrial structure. This would specific activities, especially those of the cytoplasmic also indicate the possible loss of mitochondrial constit fraction. uents. It has already been shown that the acid extracta The incorporation of @Pintothe phospholipid fractions ble nucleotides are considerably reduced in the Tween 80 took place very rapidly. The results (Table 4) indicate an treated cells. The reduction in observed Q@,2isa reflec extraordinary increase in the labeling of this fraction after tion of this loss. These facts might argue in favor of a Tween 80 treatment of the cells. The observed increase specific action of Tween 80 on these cell organelles. In in the nuclear fraction amounted to almost 300 %. The further studies of the action of this detergent on ascites phospholipid fraction was subjected to siicic acid-im cells the various cytoplasmic components, including the pregnated paper chromatography (Chart 3); the separation mitochondria, are being separated from the cytoplasm of the various phospholipids showed that at 1 hr the only fraction labeled, as seen in a radioautograph of the fraction and studied separately. This work will be re chromatograph, was phosphatidyl serine. The identifica ported elsewhere. tion of this component was made according to RF values REFERENCES and the ninhydrin reaction. The high specific activity of 1. Abercrombie, M., and Ambrose, E. J. The Surface Properties the phospholipid fraction, as observed above, is thus due of Cancer Cells. A Review. Cancer Res., 22: 525-48, 1962. entirely to the active phosphatidyl serine found in both 2. Allen, R. J. L. The Estimation of Phosphorus. Biochem. J., nuclear and cytoplasmic fractions of these cells. The 34:858â€”65,1940.

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Fxo. 1.â€”Normalcontrol preparation of cells of Ehrlich-LettrÃ© ascites carcinoma stained with Lissamine green. Fia. 2.â€”Tween 80 treated preparation of cells of Ehrlich-LettrÃ© ascites carcinoma stained with Lissamine green. Fio. 3.â€”Normalcontrol cells of Ehrlich-LettrÃ©a8cites car cinoma stained with Lissamine green, and under high magnifica tion. X 562. Fio. 4.â€”Tween 80 treated cells of Ehrlich-LettrÃ© ascites car cinoma stained with Lissamine green, and under high magnifica tion. X 562.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1965 American Association for Cancer Research. The Effects of Tween 80 on the in Vitro Metabolism of Cells of the Ehrlich-Lettré Ascites Carcinoma

E. R. M. Kay

Cancer Res 1965;25:764-769.

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