The action of synthetic surfactants on membranes of tumor cells

Experimenlnl

Cell

Research

24, 429-439

(1961)

129

THE ACTION OF SYNTHETIC SURFACTANTS MEMBRANES OF TUMOR CELLS I. MORPHOLOGICAL C. G. PALMER, Department

of Medicine

OBSERVATIONS

M. E. HODES

and A. K. WARREIC

and Department of Biochemistry, Indianapolis, Indiana, Received

ON

December

Indiana U.S.A.

Unicersity

Medical

Center.

12. 1960

THEstudy

reported here originated in attempts by the authors to find a surfactant which might facilitate the isolation of nuclei from tumor tissues 19:. Some tumor cells, notably the V-2 rabbit carcinoma, were difficult to fragment into constituent parts by classical biochemical techniques. Phase microscope observations of the results of such separations revealed that “clean” nuclei were a rarity. “Phase” observations during titration with surfactant was initially used as a criterion of usefulness of a surfactant for such a procedure. Later, staining methods were attempted. Sigrosin uptake i 10 ] proved the most satisfactory indicator of activity of surfactant on Ehrlich ascites (E&4) cells [Sl. These cells were chosen instead of V-2 carcinoma since manipulation necessary for preparation of the cell suspension from the solid tumor in itself resulted in damage sufficient to make further evaluation of surface active agents difficult. More recent work on surface active agents in this laboratory has been directed toward assay of release of cellular constituents (i.e., cholesterol, phosphorus, nitrogen) during treatment with low concentrations of surfactants capable of initiating nigrosin uptake [l, 6ij.

MATERIALS

AND

METHODS

Cells.----Sevenday old Ehrlich ascites cells grown in Swissmice were used throughout. The cells were washed twice in normal saline. Red blood cells present in hemorrhagic ascites could be removed for the most part by centrifugation and careful 1 This work was supported by grants from the Damon Runyon Foundation (Grant So. 4’LtiA) and L’.S. Public Health Service (Grant No. 3475-C2). The authors wish to thank the Riley Foundation for continued personal support. Electron microscopy was performed with the cooperation of Robert M. Smith. The electron microscope which was used was given to the Indiana University Medical Center by the Indiana Elks Association, to which grateful acknowledgement is made. Experimental

Cell Research

24

430

C. G. Palmer,

M. E. Nodes

and A. K. Warren

pipetting. The LLC-He-l cells are from a tissue culture line originating in human embryonic connective tissue and carried, for these experiments, in stirrer cu1tures.l The cells were centrifuged, washed free of medium in normal saline and resuspended in that diluent to a concentration of lo6 cells per ml. Titration.-Dropwise addition of surfactant from a burette to a 5 ml aliquot of suspension slowly agitated by a mechanical stirrer was carried out at room temperature or in a constant temperature water bath. Surfactants used are described in detail in a previous publication [S]. Aliquots of cells were removed for phase microscope observation or nigrosin staining (0.75 per cent nigrosin in normal saline). Phase observation was made with dark M phase objectives (N.A. 0.66 or N.A. 1.25). Cells for electron microscopy were fixed after treatment by adding an equal volume of 2 per cent osmic acid (buffered at pH 7.4) to the suspension. After 15 min preliminary fixation the cells were centrifuged and the fixative was replaced with fresh 1 per cent buffered osmic acid for an additional 20 min. The material was dehydrated in ethyl alcohol and embedded in butyl-methyl methacrylate (4: 1). RESULTS

The Action of SLS on Ehrlich Ascites Cells and Cells of Cell Line LLC-He-l The action of sodium lauryl sulfate (SLS) on Ehrlich ascites cells was followed by phase microscopy as aliquots of detergent were added to a normal saline suspension of washed cells. Initially the suspended cells were observed to be spherical and highly refractile (Fig. 1). Rare cells had blebs or blisters appearing on the surface of the cells. Addition of SLS (cont. 0.13 m&Z) resulted in a great increase in blebbing and blistering of the cell surface. Such blebs might be small and discrete or encompass large areas of the surface. The cytoplasm appeared diffuse, flattened and most of the refractility was lost. Often the cytoplasm and nucleus appeared to be side by side in the cell within the bleb (expanded membrane). The nucleus eventually swelled to a size larger than the original intact cell. At higher concentrations (0.4-0.6 mM) only wisps of cytoplasm appeared to be attached to the nucleus (Fig. 4). Finally, free nuclei were observed (concentration 0.6-0.7 mM). These appeared to have intact nucleoli. Continued action of the SLS resulted in swelling and lysis of the nucleus. Breaks in the nuclear membrane occurred and the contents streamed out of the nucleus (1.3 mM). Frequently one encountered fragments of nuclear membrane (ghosts) in the preparation. Jlacroscopically, the suspension became highly viscid as nuclei acid extraction occurred. In our titrations, and in the comparisons of the ability of different surfactants to produce cell lysis, we have used the designation “cells” for those 1 These

cultures

i3xperimental

Cdl

were Research

kindly 24

provided

by Mr.

P. Simpson

and

Dr.

I. Johnson

of Eli Lilly

Co.

Surfactants;

The

sequence

of morphological

changes

cell morphology

after

surfactant

-131

treatment

of cells.

Note: This is a summary of effects of surfactants on cells using a number of detergents scvcral cell lines. Sot all detergents produce the complete sequence (set text ). Fig.

I .--Ehrlich

Fig.

2. -Rlebbing

Fig. 3.---Sucleus Irralmtwt Phase

ascites

(FX)

suspension

of I,LC-He-l and cytoplasm 500, enlarged

I.‘@. 4. -- Fragments 0.1 111‘11 SI‘S. Phase

of cytoplasm 1 100.

cells after

in normal

saline.

SLS treatment.

side by side within i 2. attached

to nuclei,

Phase

1100.

I’hase

1 lO(l.

expanded “tabs”.

membrane after

of l.l.(:-He-l

trcatnrent

of I<.\

ant1

after

SI.5

cells

with

cells \vhich appear to have cytoplasm completely surrounding the nuclens, “tabs” for those cells or portions of cells with granules or wisps of cytoplasm attached to a nucleus but not necessarily surrounding the nucleus, anti have rcwrvcct lhc trrm “nucleus" for those nuclei appearing stripped 01’ cytoplasm.

C. G. Palmer,

Fig. 5.-Nucleus with SLS. Phase

of LLGHe-1 x 1100.

111. E. Hades and A. K. Warren

and fragments

of nuclear

Fig. 6.-Electron photomicrograph of untreated EA enlarged x 2. Note microvilli, mitochondria, nucleus. Fig. 7.-Electron photomicrograph loss of mitochondrial structure. Experimental

Cell

Research

24

of SLS-treated x 4400, enlarged

membrane cell from

normal

(lower

right)

saline

(0.35 m&i’) EA cells. Note x 2.

after

suspension. raised

treatment x 4400

cell membrane,

Surfactants;

cell morphology

433

Phase microscope observation of SLS lysis of a number of tissue culture lines which had been grown as single cells in stirrer cultures \vere also carried out. In LLC-He-l, a sequence analogous to that described for PI.4 was observed (Figs. 2, 3, and 5). Observations made with the phase microscope were supplemented t)~ light microscope observation of aliquots removed during titrations and diluted (1 : 20) in a 0.075 per cent solution of nigrosin in normal saline, in \\‘I<(: counting pipettes [ 101. Nigrosin staining of SLS-treated EA cells hriell> preceded lysis of the cells (0.13-0.17 mM). (see Table \., 17 1). Fresh suspensions of ISA cells in normal saline arc made up largely ol’ cells mistained hy nigrosin. Addition of SLS results in uptake of stain by $1 portion of the cells. This staining appears first in the nuc*lcus and later in the cytoplasm. Finally, large, swollen, darkly stained nuclei arc ohscrwd (,Fig. 10). ISlwtron microscope pictures of the action of lo\\. concentrations of’ Sl,S on FAA cells showed that the blebs or blisters observed with phase microscop\ v.crc artuall~ extensions of the cell membrane. Fig. 6 is an rlwtron microphotograph of an untreated I<.4 cell from the suspension in normal saline sho\\.ing the microvilli, mitochondria and nucleus. ATier addition of SlA (final concentration 0.35 mM) to a suspension of li.4 cells (I .2 - 106 cells pel ml) all of the ~~~11sstained with nigrosin. An aliquot of the SI,S-treated I<;\ cells \vas proccssctl for electron microscopy \vith the above control. ‘I’hc el(scbon micrographs (Fig. 7) showed a raised cell mr~mbranc and tiisrul,lion ot’ Ihc mitochontiria. \\‘e feel that the raised areas of’ the ccl1 memhranck probably c~orrw~~ond to the blebs or blisters ohscrvcd \\-ith I)hasc rnicroscol)!~. Similar t~lr~tron-mi~ros~~)~~e observations ha\-c hen made 1)~ I,ohtich an(l IAanciscliutz -1 0 after treatment of ISA cells \\dth hc,terologous antist~ra. The Action

of Lauryl

Pyridinium

Chloride

(LPC)

on Ehrlich

.4srites

Cells

I,auryl p,vridinium chloride, a cationic detergent, also produced hlehhing of the ~11 membrane anti enabled the penetration of the dye, nigosin, into thr II.4 wits. So cytotysis was observed nor \verc fret, nuclei l~roducrti. I’hc raising or htebbing of the membrane is observed at all concrntrations l’ronl 0.2 to 1 .% mM. At times there appear to he larger blcbs \vith smaller OIIW lvithin, hut at the highest concentrations there was no cdolyis or exutraction of nucleic acid. I3y the time 2.2 mM was reached, clumping of the cells anti clearing of the suspension occurred. LI’C did produce alterations in the cell mcmhrane \\hic*h resulted in an uptake of nigrosin in 50 per scant of the cells

C. C. Palmer,

434

M. E. E-lodes and A. K. Warren

at 0.21 mM and 100 per cent of the cells at about 0.27 mM. Fig. 8 is a photomicrograph of nigrosin-stained EA cells after treatment with LPC to show stained and unstained cells at the lower concentrations. Here, too, the nucleus stained first and the cytoplasm later. The Action

of Igepal

DM-710

The action of this non-ionic detergent was also followed by phase microscopy. This surfactant also induces blebbing, cytolysis and the production of free nuclei. The nuclear yield is low, however, in the case of the EA cells and higher with LLC-He-1 cells (see Table VII, [7]). Blebbing after treatment wih this agent may be in the form of small discrete blebs or larger blebs similar to those observed with the other surfactants. Higher concentrations result in some free nuclei (LIP to 35 per cent with LLC-He-1 cells) and large quantities of cytoplasmic debris in the surrounding medium. Finally, gross clumping occurred before a high yield of free nuclei could be obtained. So viscous solution occurred with this surfactant. The agglutinated mass as observed with phase appeared to consist of nuclei, tabs, and large globules of cytoplasm in the surrounding medium. No fibers were observed. Nigrosin staining after treatment of EA cells with non-ionic detergents is shown in Fig. 9. In this figure, a lightly and a darkly stained cell (after treatment with 0.034 mM 11%710) may be observed. The blebbed cells appear to be the only ones to have stained darkly with nigrosin. The penetration of stain seemed to occur at about the same time as the blebbing of the cell membrane or to slightly precede the blebbing. The lightly stained cell (i.e., nucleus) is not blebbed, but the cell bearing a darkly stained nucleus shows blebbing of the membrane. Eventually all of the cells are stained with nigrosin after treatment with 0.98 mM DM-710.

The Eflect of IO-Hydroxy-

AZ-decenoic

Acid

on Ehrlich

Ascites

Cells

Reports of the anti-tumor action of lo-hydroxy-AZ-decenoic acid (HDA) [17 ], coupled with our observed action of lytic action of another long-chain Fig. Note

8.-EA staining

cells after treatment with LPC (9.21 mBf) and staining with nigrosin. of only a portion of the cells and that nuclear staining precedes cytoplasmic

Fig. light

9.--EA staining

cells after of nuclei.

Fig. lO.-Nuclei after stained with nigrosin. Experimental

Cell

treatment Darkly

with stained

SLS treatment x 1250.

Research

24

DM710 (0.34 cell blebbed. of EA

cells.

mM) and staining x 1250. Note

that

the nuclei

with

nigrosin.

are swollen

x 1250. staining. Dark and

and darkly

Surjacfanfs;

cell morphology

1%

436

C. G. Palmer,

M. E. Hodes and A. K. Warren

fatty acid, lauric acid, suggested that HDA might be acting in a similar manner to lauric acid and SLS. Therefore, the morphological changes following treatment of EA cells with HDA were observed, This agent did not produce blebbing, and cytolysis occurred only at high concentration (4.6-6.2 mM). The cells became shrunken, and the surface appeared roughened and crenulated. After 20 min at this concentration, above the effective chemotherapeutic dose [17], the cells were all stained with nigrosin but no increase in the number of nuclei or cell fragments was observed.

The E$ect of pH

Changes on EA Cells

The original suspensions of EA cells in normal saline were at a pH of 6-6.4. Increases in pH produced by addition of 0.01 N NaOH to the suspension, resulted in cytolysis and extraction of nucleic acids at pH 10.5. In this case, however, the lysis did not follow in the stepwise pattern observed with the detergents but individual cells became greatly swollen and lysed almost immediately. Few “tabs” were produced and any nuclei which were present were greatly swollen. Some extractions apparently occurred at lower pH, since micro-fibers were observed at pH 7.6-9.4. No increase in nigrosin uptake was observed until pH 10.5 at which time 100 per cent of the remaining cells were stained (gross extraction had occurred). Decreases in pH produced by addition of 0.01 il; HCl to EA cells resulted in only slight increases in nigrosin uptake and cytolysis did not occur. The cells became quite shrunken and resembled these cells treated with HDA at pH values from 2-2.6. A few cells with blebs were observed. Between pH 0.9 and 1.8 the cells were swollen, the greater amount of swelling occurring at the lower pH, but even here only 7 per cent of the cells were stained with nigrosin. DISCUSSION

The morphological changes initiated by a series of surface active agents have been described. These include the production of “blebs” or blisters on the cell membrane, the uptake of a dye, nigrosin, the lysis of the cell and the production of free nuclei. The phenomenon of blebbing of the cell membrane is not limited to situations where surface active agents are involved. Various studies seem to indicate that it is an index of cell membrane damage. Zollinger [18], who observed this phenomenon in considerable detail in a variety of “physiological” solutions, considers it to be a special Experimental

Cell Research

24

Surfactants;

cell morphology

13;

type of fluid uptake by living cells occurring particularly in unnatural c-onditons (potocytosis). Freed, Engle et al. [4] observed blebbing in E,4 cells which had been damaged by ultraviolet light. When a portion of a cell was subjected to C.V. irradiation, only that portion exhibited blebbing. Bennet and Connon !a] observed blebbing as a result of the action of fatty acids, bile acids and steroids on EA cells. Streptococcal hemolysins likewise initiate blebs, “pseupodia”, on EA cells and facilitate the uptake of trypan blue I’:5:. S-rays will also produce blebbing of EA cells. In their studies of cell death King et nl. [12, 13, 14, 151 found blebbing to be one of the earliest changes morphologically relevant to cellular death. The cellular death resulting from surfactant lysis does not strictly correspond to their sequence of morphological and biochemical events leading to cell death. IJptakc ot stain in our experiments occurs simultaneously \vith blebbing or may slightly precede it, while blebbing in King’s experiments occurred several hours prior lo trypan blue staining (i.e., death of the cells). In our experiments gross extraction of DNA occurred after lysis of the cell, xvhile King et rrl. found that lIS.4 loss (as measured microspectrophotometricallp) preceded blebbing, loss of enzyme activity, and cell death. It must he remembered that the surfactants probably act primarily on the lipids and lipoproteins of the cell membrane, which would account for the read\- access of nigrosin into the cell in our experiments. The fact that S-ray damage of cells occurs primarily in the nucleus might account for the early release of NA by S-ray. Similarly, the efTect of Salygran, a sulfhpdryl inhibitor, in initiating earl\- nucleic acid release might also be related to the larger number of sulfhydrvl linkages found within the nucleus. In addition, the experiments of King c>f (11. encompassed a number of hours, while the reactions of cells \vith surfactants were telescoped into a relatively short period (less than an hour). Nonetheless, w-e are currently investigating the biochemical sequence of lysis prior to cellular uptake of nigrosin [l I. Unpublished experiments with I,. Higgs suggest losses of protein, nitrogen and LT.\‘.-absorbing materials (26X--28C) m/l) on treatment with sub-lytic concentration of detergents. Electron microscope studies of sections of SLS-treated ISA cells indicate that the blehs correspond to raised areas of the cell membrane. Loblich and Landschutz [ 16 ] have described similar “Abhebung tier %ellmembran” after treatment of 13 cells with heterologous antisera. They concluded that the “blisters” arose from the “alteration of microvilli into surface vesicles”. The hlisters thus appear in situations where surface injury has occurred. We hare observed such blistering following temperature shock (removing cells from an iced solution to room temperature) or prolonged storage (6 to

C. G. Palmer,

438

M. E. Hodes and A. K. Warren

8 hr in normal saline) of IL4 cells. Similar by King et al. [12-l 51. h’igrosin, an anionic dye, containing indulin a black color [3], has been used in a manner etc. as an indicator of cell death [ll]. In our to parallel cellular death unequivocally [lo]. an indicator of changes in permeability of the large indulin molecule. TABLE

I. The effectiveness

observations

plus a yellow dye to impart similar to eosin, trypan blue, hands it has not been found It appears to serve well as cell membrane relative to the

of different classes of surfactants nuclear membrane. Cell membrane

Surfactant Cationic Anionic Non-ionic

have been made

class (LX) (SLS) (DM-710)

Dye

uptake x x x

Nuclear

membrane

Lysis

Lysis

-

x x

on cell and

x -

The different classes of detergents produce difyerent responses in cell and nuclear membrane in both EL4 and tissue culture suspensions (Table I). Cationic detergents can initiate surface changes in the cell membranes but lysis of neither the cell nor nuclear ( i.e., blebbing and nigrosin uptake), membrane occurs. Anionic detergents can lyse both nuclear and cell membranes and non-ionics produce cellular lysis, but nuclear lysis does not appear to occur. The possible modes of action of detergents on the cell membrane are discussed more fully in the accompanying paper [7]. Dye uptake and blebbing occurs with all three classes of surfactant. Xigrosin stains the nucleus preferentially, since it is an acid dye (and combines with the basic proteins of the nucleus). This does not imply that initial action of surfactant also occurs on the nuclear membrane, since the nuclear membrane is known to be freely permeable to large molecules (DNA, RNA) and morphologically to have a pore structure compatible with such permeability. It does appear that the nuclear membrane and cell membrane differ in their chemical structure, as evidenced by their response to surfactants. The nuclear membrane would seem to have more protein and less lipid than the cell membrane. Final decisions on composition must await actual analysis of the components.

Experimental

Cell

Research

24

Surfactants;

cell morphology

139

SUMMARY The morphological effects of surfactants on ISA cells precede sequentialI! and include: I, hlehbing of cell membrane (coincident to tiyc uptake by the cells); ?, gradual loss of cytoplasmic fragments; 3, the appearance of free nuclei followed by nuclear lysis and 4, nucleic acid extraction. The surfactants differ in effectiveness of producing the above changes on PI.4 cells: Anionic (SLS) -’ nonionic (DM-710) > cationic (LPC) surfactants. REFERENCES 1. BARHETT,

I-. and

HODES,

ikl. E.,

Ezptl.

Cell

Heseurch

21, 209 (1960).

2. BENNET, L. R. and CONNEN, F. E., J. Natl. Cancer Inst. 19, 999 (195i). 3. CO.UN,J. J., Biological Stains, Biotech. Publications, New York, 1953. 1. FREEI,, J. J., ENGLE. J. L., RUDKIS, G. T. and SCHULTZ, .J.. Riophys. Is'iochem. (1959). 5. GINSBURG, I., &if. ,I. Expfl. 6. HIGGS, I-., Ilnpublished.

5. Honm,

M. E.,

P.~LMER,

X. HODES,

XI. E..

PALMEH,

Pafhol.

C:ylol.

5. 205

40, 417 (1959).

C. G. and IAVEKGOOD,

D., Expff. Cell Reseurch. 24, 29X (1961). C;. Cr. and N~;ARREN, A. K., Ercpfl. Cell Xeseorch 21. 164 (1960). 9. Hoom, M. E., PALMER, C. G. and WILLIAMS, I). S., Sature 182, ,529 (1958). IO. HODES, bl. E., W.ARREX, A. K. and PALMER, C. G., Scrfure 188, 157 (1960). 11. K.~LTENBACH, .J. P., KALTEXBACH, XI. H. and Luors, W. H., E.cpfl. Cc/l Keseurch 15,

(1958). 12. KIYC.

I>. \V.,

P~I~LSOS,

S.

R., HASNAFORD,

N. C. and

KREIS.

A. T.. .t171. .I. I'rtlhol.

35,

112 36:)

(1959).

13. ~' - ibid. 35, 575 (1959). 14. KIYG, I). W.,PAULSOK, S. R., PUCRETT,

N. I,. and KREDS,

h. 'I‘., ibirl.

15. ~-- ihid. 35, 1067 (1959). 16. I.OHLICH, .J. .J. and I,.~NIX,C~UTZ, C., Z. Krebsforsch. 63, 3% (1960). li. TOWXSEND, G. I-.. MORGAN, .J. G. and HAZLETT, R., A’afurr 183, 1350 1X.

%OI,LI~GER,

H.

IT., .-lm.

J. Pathof.

35,

X35 (19%)).

(lO.i!b).

24, 545 (1948).

B.zperimerlfni

Cdl

I~rswrrc~h

24