It has been recognized for some time that the peculiarproperties of tumor cells which permit their invasiveness,and adhesiveness are dependent to a large extent on theproperties of the cell membrane (1, 3, 24). The surfaceproperties of cells are, in turn, dependent on the composition of the membrane, which includes both proteins andphospholipids. Alteration of the composition of theplasma membrane or of the endoplasmic reticulum andmitochondria would be expected to alter markedly themetabolic properties of the cells in question. In thispaper are presented the results of a study in which alterations were made in the membrane structure of cells of theEhrlich-Lettré ascites carcinoma using the detergentTween 80. This study has been carried out to show thechanges in metabolic activity resulting from the action ofthis surface active agent. The experiments have includedobservations on changes in permeability, respiration, andincorporation of inorganic @Pand ‘@Cformate into thenucleic acids, proteins, and phospholipids of this tumorunder in vitro conditions. A preliminary report of thiswork has been published (13).

MATERIALS AND METHODS

The Ehrlich-Lettré ascites carcinoma used in this studywas carried as a stock tumor in mice of the CFW strainobtained from Carworth Farms. The tumor was maintained by transplanting 0.2 ml of the ascites fluid at 7- to10-day intervals. Fluid containing tumor cells to be usedin in vitro experiments was drained from host mice, pooled,and used without treatment as a control tumor preparation, or treated with Tween 80 as described below.

1 Supported by grant No. CA 05172 from the National Cancer

Institute, USPHS.Received for publication October 28, 1964.

The tumor was used directly or was treated with Tween80. After removal of the ascites fluid from tumor-bearingmice the fluid was centrifuged in 3 15-mi centrifuge tubesto give equal volumes of packed cells for the experiment.The supematant serum from the centrifugation wassaved separately for later use. To 1 tube containingpacked cells, 10 ml of saline were added. To a 2nd tubewere added 10 ml of 0.25 Msucrose, and to a 3rd tube wereadded 10 ml of 0.25 M sucrose containing 1 % Tween 80.The tubes were stoppered and mixed thoroughly to dispersethe cells and were allowed to stand at room temperature for15 or 30 mm. The tubes were then centrifuged to packthe cells (5 mm at 3000 rpm in an International clinical centrifuge) and the supernatants were saved for further analysis. The packed cells were washed and centrifuged twice,using 0.9 % NaCl as a wash solution, and the supematantswere discarded. Finally, the original serum samples wereadded to the packed cells and, after shaking, the cell suspensions were ready for use either for reinoculation experiments or for in vitro metabolic experiments.

Extraction of acid soluble nucleotides was carried out byadding ice cold 4 % HCJO4 to an aliquot of the suspension,after centrifugation to pack the cells, and reading of theoptical density of the acid extract at 260 nip using a Beckman model DU spectrophotometer. The amino acidswere extracted from a similar aliquot of the suspension,which was then centrifuged; 95 % ethanol (5 ml) was usedas the extracting agent. The alcoholic supematant obtamed was dried and the residue dissolved in a smallvolume of 70 % ethanol and applied to sheets of WhatmanNo. 1 chromatograph paper for 2-dimensional chromatography using the solvent system of Hardy (6).

Respiration of the control and treated cells was measured

by the appropriate procedures using a Bronwill-Warburg764

The Effects of Tween 80 on the in J―itroMetabolism of Cells ofthe 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 thedetergent Tween 80 in preparations of Ehrlich-Lettré ascites cells. The growth ofthese cells was normal in host mice after treatment and thus they were viable astransplants. The permeability of the cells increased greatly, as shown by the uptakeof Lissamine green. Soluble nucleotides and amino acids were removed by the Tweentreatment. Oxygen uptake was reduced by 50 %. The incorporation of formate intoprotein and deoxyribonucleic acid (DNA) decreased, but it increased into nuclearribonucleic acid (RNA). Incorporation of @Pinto the phospholipids increasedgreatly and was found to be localized in phosphatidyl serine. The interpretation ofthe alterations brought about by Tween 80 are discussed in relation to the specialproperties of the tumor cells.

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02Extractable (O.D. at 260nIp)Controla

SucroseSucrose + 1% Tween 806.2

5.53.31.000

1.0200.550

KAY—Effects of Tween 80 on Ehrlich-Lettré Ascites Cells 765

respirometer. Metabolic experiments employing ‘4Cformats or 32@carrier-free inorganic phosphate were carriedout using a Dubnoff metabolic shaking incubator.

For incubation experiments an aliquot of the abovesuspensions was added to the flasks as follows: 2.0 ml cellsuspension (containing 0.5 ml packed cells); 1.0 ml 0.25M sucrose—0.05 M phosphate buffer, pH 7.4; 0.8 ml 0.25

M sucrose—O.1 M glucose; 0.2 ml H2O containing the

isotope (8 pc ‘@Cformate, 20 pC @2P)(12). Glutamine wasadded to the incubation mixture before adding the cells,so that the final concentration of glutamine was 100 pg/mi.The incubations were carried out for periods of 1 hr. Afterthis time, the flask contents were centrifuged and thepacked cells were frozen prior to preparation of the cellfractions by a modification of a previously publishedmethod (22).

The nuclear and cytoplasmic fractions were extracted1st with ice cold 10 % trichloroacetic acid (TCA) to removeacid soluble components. Lipids were removed from theacid extracted fraction by using acetone, ethanol, ethanolether, and ether extractions from the nuclear and cytoplasmic fractions of the incubation experiment. Thesecombined extracts were dried, dissolved in chloroformmethanol (1 : 1), and applied to silicic impregnated paperfor separation of the phospholipids according to themethods already outlined (19). The lipid-free residueswere hydrolyzed in 0.5 ml of 0.3 N KOH at 37°Cfor 18 hr.After this time the alkaline solutions were cooled andacidified with 1 drop of 70 % HC1O4. The RNA in theacidified extract was decanted after centrifugation anddiluted to 5 ml. An aliquot of 0.5 ml was plated on astainless steel planchet and counted in a gas flow NuclearChicago counter. Aliquots were analyzed in duplicatefor phosphorus by the Allen (2) procedure. All correctionswere made for half life and background as required, when32@ was used. The specific activity was recorded as cpm/

100 pg phosphorus.The DNA was extracted from the nuclear residues, after

RNA hydrolysis as above, by using 4 % HC1O4at 90°Cfor15 min. The DNA activity was measured by diluting thesupematant extract to 5 ml, plating a 0.5-mi aliquot asabove, and measuring the phosphorus of aliquots, induplicate, by the Allen (2) method.

When ‘4Cformate was used as labeled precurser thevarious RNA and DNA samples were hydrolyzed to theirconstituent purines and pyrimidines. For this procedurethe total extracts of these nucleic acids, obtained as above,were dried in a vacuum desiccator and hydrolyzed with70 % HC1O4by adding 1 drop of the concentrated acid tothe dry residue. The hydrolysis was carried out at 100°Cfor 1 hr. After this process the samples were diluted toabout 0.3 mi and the aliquots were placed at 1 end (10cm) of a 50 by 3.8 cm strip of Whatman No. 3 MM paper;descending chromatography was carried out overnightusing the solvent system of Wyatt (25), consisting ofisopropanol, 65 ml; concentrated HC1, 16.5 ml; and enoughwater to make 100 ml. The bases were identified by theirR@values, eluted, and lyophiized ; 0.3 mi of appropriateconcentrations of acid was added to these dry residues.With the purine, adenine and the thymine, 0.1 N HC1 wasused; with guanine, 1.6 N HC1 was employed. A 0.1-mi

sample of the solution was plated on a stainless steelplanchet for counting and a 0.1-mi aliquot was diluted to4ml;theopticaldensitywasdeterminedatanappropriatewave length using the Beckman model DU spectrophotometer (25).

RESULTS AND DISCUSSION

In preliminary studies treatment of the tumor cells with0.25 i@sucrose or with 0.25 Msucrose containing 1 % Tween80, appeared to effect no loss in viability of the cells whenthey were injected into host mice and the tumor growthwas measured by weight increase. This finding is inagreement with the findings of Morgan (23) and others.

When the cells were examined with the dye Lissaminegreen, marked permeability changes were noticed afterincubation with Tween 80. This dye is a negativelycharged nontoxic triphenylmethane dye and has beenused extensively by a number of workers as an indicatorof cell viability. It has been studied extensively byHolmberg (9) as an indicator of cell death. According tothis author the cells become freely permeable when ir

TABLE 1EFFECT OF TWEEN 80 TREATMENT ON CELLS OF EHRLICH-LETTR@

ASCITES CARCINOMA

Incubation conditions: 2.0 ml cells (containing 0.5 ml packedcells) ; 1.0 ml sucrose (0.25 M)-phosphate buffer (0.05 M) pH 7.4;0.8 ml sucrose (0.25 M) + 0.1 M glucose; 0.2 ml H,O (or isotopesolution).

a Control, sucrose, and sucrose + 1% Tween 80 refer to treatment of the cells prior to incubation.

CHART 1.—Amino acids of Ehrlich-Lettré ascites carcinoma.Free amino acid pattern of control Ehrlich ascites cells. Aminoacids identifiedfrom solvent amino acid maps prepared using thesolvents : I = ethanol, 1-butanol, water, propionic acid(10:10:5:2); II = 1-butanol, acetone, water, dicyclohexylamine(10:10:5:2). ASP, aspartic acid; GLY, glycine; GLT, glutamine;SER, serine; GLU, glutomic acid; PR, proline; ALA, alanine; TY,tyrosine; VAL, valine; LEU, leucine.

It

ORIGIN

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FRACTIONRNA@DNACytoplasm

Control TweenNuclearControlTweenControlTweenAdenine

GuanineThymineProtein5572

2451—

29562911

1558—

169134,078

15,615—

1,28851,250

19,285—

2113000

20333853

—1045

6151715

—

FRACTIONRIBONUCLEIC

ACIDControl

Tween %changeCytoplasm

Nuclei11,61416,586 +29

20,438 22,818+12TABLE

4

EFFECT OF TWEEN 80 TREATMENT ON INCORPORATION OF 32@

INTO ASCITES CELLS

Specific activity = cpm/100 pgP.FRACTIONPHOSPUOLIPID@—

Control Tween %changeCytoplasm

Nuclei39909343 +134

2353 9354 +297

766 Cancer Research Vol. 25, June 1965

tion with adenylic and guanylic acid nucleotides reduced tothe greatest extent. Since this reduction in free nucleotides occurred during the Tween treatment it would bereflected in the lower Qo, found for these cells duringsubsequent incubation.

In view of the loss of acid soluble materials duringTween treatment, it was of interest to find a correspondingloss in amino acids from the cells. In Charts 1 and 2 arechromatographs of the ethanol extractable materials of thecells. It can be seen readily that there are marked quantitative differences in the Tween 80 treated cells. The 2chromatographs were prepared in identical fashion fromthe same volume of packed cells and thus the effects of theTween 80 treatment are obvious. This is a further indication of the effects of the Tween on cell permeability. Inthe Tween 80 treated preparation there is a markeddiminution of aspartic acid, glycine, glutamic acid, andalanine. Glutamine was reduced considerably below thenormally low levels found in these cells. A very low levelof extractable leucine and tyrosine was observed in thetreated cells. The level of cellular serine was not reducednoticeably by the Tween treatment. An examination ofthe amino acid pattern has confirmed the fact that Tweentreatment has decided effects on the permeability of thesecells.

The use of 14(@formate as a label for metabolic experiments permits the determination of purine synthesis innuclear and cytoplasmic RNA, and in the purines andthymine of DNA. It also permits the determination ofprotein synthesis in the cells because the glycine-serineconversion system is known to be present in many tissues(15), including tumor cells (14). There is also evidencethat tumors depend to a marked degree on glycine andserine (17, 20, 21).

The effects of Tween 80 treatment on 14Cformate incorporation are shown in Table 2. It can be seen thatthere is a reduction in this incorporation into protein inboth nuclear and cytoplasmic fractions. Since it was

TABLE 3

EFFECT OF TWEEN 80 TREATMENT ON INCORPORATION OF 32@

INTO Ascrris CELLS

Specific activity = cpm/100@ P.

0

0

CHART 2.—Free amino acid pattern of Tween 80 treated Ehrlich-Lettré ascites cells. Absicca and ordinate refer to solventsystems as in Chart 1.

TABLE 2EFFECT OF TWEEN 80 TREATMENT ON INCORPORATION OF 14C

FORMATE BY ASCITES CELLS

Specific activity = cpm/JLMbase or = cpm/mg protein.

14t

ORIGIN

a RNA, ribonucleic acid; DNA, deoxyribonucleic acid.

reversible damage has occurred, presumably because of asevere metabolic disturbance associated with changes innembrane activity. Holmberg claimed that penetration

of the Lissamine green was an indication of irreversiblydamaged and, therefore, “dead―cells. A normal preparation of ascites tumor cells (Fig. 1) and a preparation oftumor cells (Fig. 2) were treated with Tween 80 under theabove experimental conditions. It can be seen readilythat after Tween treatment about 90 % of the cells took upthe Lissamine green. Figs. 3 and 4 are comparisonphotomicrographs of control and treated cells under highpower. The penetration of the dye can be seen in thenuclei and cytoplasm of the cells after Tween 80 treatment.

In view of the findings of Holmberg (9), with 90 % of thecells taking up the dye after Tween 80 treatment, it mightbe assumed that the preparation was metabolically inactive. However, in the work outlined in this paper, theuptake of this dye indicates only that the cells are morepermeable, without necessarily implying irreversibility ofmetabolic activity. In Table 1 it is seen that the Qo, ofthe preparation after Tween treatment was reduced tohalf that of the control value. In an examination of theacid soluble fraction, using the optical density at 260 mpas a measure, it was found that the Qo, was reduced toabout 60 % of the control cells. The constituent nucleotides of this fraction were selectively reduced in concentra

Q

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Ki@@—Effectsof Tween 80 on Ehrlich-Lettré Ascites Cells 767

results obtained in other experiments (19) have demonstrated that the cells of the Ehrlich-Lettré ascites carcinoma can incorporate ‘@Cformats into several lipids.Chromatographic separation of the phospholipids showedthe presence of radioactivity in phosphatidyl serine,phosphatidyl-ethanolamine, lecithin, sphingomyelin, andsome unidentified lipids. The findings confirmed that, inthis tumor, the conversion of formate to serine takes placevia the glycine-serine conversion system (14, 15). Theexperiments reported in this paper which show very rapidincorporation of 32@into the phosphatidyl serine afterTween 80 treatment, suggest a reconstitution of cell membrane material, in view of the other observations of normalgrowth of the reinoculated tumor in host mice. Thecomplex phospholipids have been implicated in membraneactivity (7, 8) in relation to cation transport. Recentstudies on “induced―pinocytosis by Karnofsky et al. (11)have also shown that phosphatidyl serine is markedly andconsistently labeled under these conditions. Thus theevidence presented here would support the conclusion thatTween 80 treatment disrupts membrane structure, andthat these membrane components are reconstituted duringthe course of recovery.

The fact that nuclear RNA synthesis is stimulated byTween 80 treatment is of interest, in view of the recentfindings of LaCour (16) that when Tween was used in anuclear isolation technic (4) the nucleoli became invisiblebut were shown to retain their rapidly labeled RNA. Thenuclear RNA may then be implicated indirectly in thereconstitution mechanisms seen in this study. If theobserved stimulation of RNA indicated an increasedsynthesis of “messenger―responsible for protein synthesisof the altered membranes, then the evidence for thestimulation of the phosphatidyl serine might be evidencefor the formation of the new lipid component of this membrane system.

In further examination of the phospholipid fractions ofthe treated cells it was observed that there was an absenceof the cardiolipins of the treated cells (Chart 3). This hasindicated that the effect of Tween 80 may not be solely atthe level of the plasma membrane. The cardiolipins areconsidered essentially mitochondrial components (5, 18).

The loss of cardiolipins from the treated cells wouldindicate changes in mitochondrial structure. This wouldalso indicate the possible loss of mitochondrial constituents. It has already been shown that the acid extracta

ble nucleotides are considerably reduced in the Tween 80treated cells. The reduction in observed Q@,2is a reflection of this loss. These facts might argue in favor of aspecific action of Tween 80 on these cell organelles. Infurther studies of the action of this detergent on ascitescells the various cytoplasmic components, including themitochondria, are being separated from the cytoplasmfraction and studied separately. This work will be reported elsewhere.

REFERENCES

1. Abercrombie, M., and Ambrose, E. J. The Surface Propertiesof Cancer Cells. A Review. Cancer Res., 22: 525-48, 1962.

2. Allen, R. J. L. The Estimation of Phosphorus. Biochem. J.,34:858—65,1940.

CONTROL TWEEN80 CONTROL TWEEN80

CHART 3. Effect of Tween 80 treatment on incorporation of“Pinto phospholipids of Ehrlich-Lettré ascites cells. Chromatograph of 32@labeled phosphatides. Crosshatchedspotsrepresentonly radioactivity detected on radioautograph. NON-PHOS,nonphosphatides; PGP, polyglycerolphosphatides; PE, phosphatidylethanolamine; PS, phosphatidylserine; LEC, lecithin;SPH, sphingomyelin; IP, inositol phosphatides; UNID, unidentified.

shown above that there is a loss in amino acids and nucleotides from cells during their contact with the detergent itcan be assumed that both substrate level and energysupply are affected. Of interest, however, was the findingthat despite these reductions in activity of protein therewas observed a stimulation of purine formation in thenuclear RNA from ‘@Cformats. In contrast to this effectwas an observed reduction in the specific activity of thepurines of the cytoplasmic RNA and of DNA at this 1-hrincubation time. The specific activity of the thymine ofDNA was also reduced during incubation in the presence of‘@Cformate.

The results obtained when using @Pcarrier-free phosphate as a label for nucleic acid synthesis are shown inTable 3. Although the above mentioned increase innuclear RNA synthesis occurred after Tween treatment,the activity of cytoplasmic RNA was also stimulated.The RNA was measured in these @Pexperiments withoutseparation of the individual nucleotides; hence the activityof the RNA fraction includes concomitants of nonnucleotide phosphorus (10) which might elevate the observedspecific activities, especially those of the cytoplasmicfraction.

The incorporation of @Pinto the phospholipid fractionstook place very rapidly. The results (Table 4) indicate anextraordinary increase in the labeling of this fraction afterTween 80 treatment of the cells. The observed increasein the nuclear fraction amounted to almost 300 %. Thephospholipid fraction was subjected to siicic acid-impregnated paper chromatography (Chart 3); the separationof the various phospholipids showed that at 1 hr the onlyfraction labeled, as seen in a radioautograph of thechromatograph, was phosphatidyl serine. The identification of this component was made according to RF valuesand the ninhydrin reaction. The high specific activity ofthe phospholipid fraction, as observed above, is thus dueentirely to the active phosphatidyl serine found in bothnuclear and cytoplasmic fractions of these cells. The

CYTOPLASM NUCLEI

‘—.._,@@@ PHOS

C C

@e4J \

6@ 5::;:@ ‘P

c::@@

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768 Cancer Research

3. COMAN, D. R. Mechanisms Responsible for the Origin andDistribution of Blood-borne Tumor Metastases : A I@eview.Cancer Res., 13: 397—404,1953.

4. Fisher, H. W., and Harris, H. The Isolation of Nuclei fromAnimal Cells in Culture. Proc. Roy. Soc. (London) Ser. B,156: 521—23,1962.

5. Getz, G. S., and Bartley, W. A Cardiolipin-like Compound inRat Liver Mitochondria. Na2ure, 184: 1229—30,1959.

6. Hardy, T. L. One-Phase Solvent Mixtures for the Separationof Amino Acids. Anal. Chem., 57: 971—74,1955.

7. Hokin, L. E., and Hokin, M. R. Studies on the Carrier Function of Phosphatidic Acid in Sodium Transport. I. The Turnover of Phosphatidic Acid and Phosphoinositide in the AvianSalt Gland on Stimulation of Secretion. J. Gen. Physiol.,44: 61—85,1960.

8. . Phosphatidic Acid Metabolism and Active Transportof Sodium. Federation Proc., 55: 8—18,1963.

9. Holmberg, B. On the Permeability to Lissamine Green andOther Dyes in the Courseof Cell Injury and CellDeath. Exptl.Cell Res., 55: 406—14,1961.

10. Hutchison, W. C., Crosbie, G. W., Mendes, C. B., McLndoe,W. M., Childs, M., and Davidson, J. N. Protein Bound Cornpounds of Phosphorus and Inositol. Biochim. Biophys. Acta,51: 44—58,1956.

11. Karnovsky, M. L., Shafer, A. W., Saito, K., and Glass, E. A.Pinocytosis and Phagocytosis—SorneCommon BiochemicalFeatures. Abstracts of 6th International Congress of Biochemistry, New York, 1964,p. 655.

12. Kay, E. R. M. Incorporation of Nucleic Acid and ProteinPrecursors into Ascites Cells. Federation Proc., 19: 393, 1960.

13. . The Effects of Tween 80 on the Metabolism of Cellsof the Ehrlich Ascites Carcinoma. Proc. Can. Fed. Biol. Soc.,7: 6, 1964.

14. Kit, S. The Biosynthesis of Free Glycine and Serine by Tumors. Cancer Res., 15: 715—18,1955.

15. Koshland, D. E., and Erwin, M. J., Enzyme Catalysis andEnzyme Specificity Combination of Amino Acids at theActive Site of Phosphoglucomotase. J. Am. Chem. Soc., 79:2657—58, 1957.

16. LaCour, L. F. Behaviour of Nucleoli in Isolated Nuclei.Exptl. Cell Res., 54: 239—42,1964.

17. Lockart, R. Z., and Eagle, H. Requirements for Growth ofSingle Human Cells. Science, 129: 252—54,1959.

18. Marinetti, G. V., Erbland, J., Albrecht, M., and Stotz, E.The in Vitro Incorporation of p22 labelled Orthophosphateinto the Phosphatides of Isolated Rat Liver Mitochondria.Biochim. Biophys. Acta, 56: 130-43, 1957.

19. Marinetti, G. V., and Kay, E. R. M. The Incorporation of C1@Formate into Ehrlich Ascites Tumor Lipids. Thid., 70: 168-75,1963.

20. McCoy, T. A., Maxwell, M., and Kruse, P. Amino Acid Requirements of the Novikoff Hepatoma in Vitro. Proc. Soc.Exptl. Biol. Med., 100: 115—18,1959.

21. McCoy, T. A., and Neuman, R. E. The Cultivation of WalkerCarcinosarcoma 256 in Vitro from Cell Suspensions. J. Natl.Cancer Inst., 16: 1221—27,1956.

22. Smellie, R. M. S., Humphrey, G. F., Kay, E. R. M., andDavidson, J. N. The Incorporation of Radioactive Phosphorusinto the Nucleic Acids of Different Rabbit Tissues. Biochern.J., 60: 177—85,1955.

23. Tolnai, S., and Morgan, J. F. Studies on the in Vitro antitumor activity of Fatty Acids. V. Unsaturated Acids. Can.J. Biochem. Physiol., 40: 869-75, 1962.

24. Weiss, P. Cell Contact. Intern. Rev. CytoL, 7: 391—423,1958.25. Wyatt, G. R. The Separation of Nucleic Acid Components by

Chromatography on Filter Paper. In: The Nucleic Acids,E. Chargaff and J. N. Davidson (eds.) Vol. 1. New York:AcademicPress, Inc., 1955.

Fxo. 1.—Normalcontrol preparation of cells of Ehrlich-Lettréascites carcinoma stained with Lissamine green.

Fia. 2.—Tween 80 treated preparation of cells of Ehrlich-Lettréascites carcinoma stained with Lissamine green.

Fio. 3.—Normalcontrol cells of Ehrlich-Lettréa8cites carcinoma stained with Lissamine green, and under high magnification. X 562.

Fio. 4.—Tween 80 treated cells of Ehrlich-Lettré ascites carcinoma stained with Lissamine green, and under high magnification. X 562.

Vol. 25, June 1965

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1965;25:764-769. Cancer Res E. R. M. Kay the Ehrlich-Lettré Ascites Carcinoma

Metabolism of Cells ofin VitroThe Effects of Tween 80 on the

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