The action of synthetic surfactants on membranes of tumor cells

THE ACTION OF SYNTHETIC SURFACTANTS MEMBRANES OF TUMOR CELLS II.

TITRATION

M. E. HODES, Department

of Medicine

ON

EXPERIMENTS’

C. G. PALMER

and D. LIVENGOOD

and Department of Biochemistry, Indiana Indianapolis, Indiana, U.S.A.

University

Medical

Center,

Received December 12, 1960

THE lysis

of tumor cells by surfactants proceeds in stages [g]. The plasma membrane is probably affected first, followed by the various contents of the cell. I’he initial attack is at the submicroscopic level, and has been followed by observing the entrance of a vital dye into [S] and the exit of cholesterol from [ 1 ] Ehrlich ascites tumor cells. Such observations are relatively simple when the cells are free and can be harvested with little damage. This is true of ascites tumors, but not the case with most solid tumors. Here, initial preparation of the cell suspensions, while leaving the cells morphologically intact, causes damage sufficient to allow penetration .of dye into many presumably still viable cells [S]. These cells must be studied by observation of morphological alterations following treatment with surfactant. In the present study we have observed the effects produced by a number of surfactants on a variety of cells, and attempted to correlate the morphological changes with the entrance of a dye, nigrosin, into the cell. The preceding paper [ 151 outlines the sequential changes in morphology in detail. MATERIALS

AND

METHODS

The surfactants were described in a previous publication [8]. lo-Hydroxy-AZdecenoic acid (HDA) was a gift from Prof. Butenandt, of Munich. These materials were made up to 13.9 mM in physiological saline solution. The Ehrlich ascites (EA) cells used are described in the preceding paper [15]. The V-2 carcinoma was carried by serial transplantation in New Zealand white rabbits.

Following --

sacrifice by air injection

the tumors were harvested

and used fresh or

1 This work was supported by grants from the Damon Runyon Foundation (Grant No. 4a6A), U.S. Public Health Service (Grant Ko. 3475-C2), and the Indiana Elks. Several surface active agents were gifts from E. I. DuPont de Nemours and Company, American Alcolac, Antara Chemicals and Hooker Chemical Company. Experimental

Cell Research 24

Action

of synthetic

surfactants

on memhranrs.

II

a99

frozen and stored at -- 30”. Fresh tumor was minced fine with scissors and frozen tissue ground in a porcelain mortar in about 5 volumes of cold (I.13 AU NaCl. The crude suspensions were then filtered through No. 80 gauze and passed through X0 and 120 mesh wire screens. The cells were washed once or twice in saline and harvested by low-speed (600 y) centrifugation in the cold. ‘l’he cells were resuspended in saline for use. derived from human embryonic COW Tissue culture preparations of LLC-HE,, ncctivc tissue, and .J-!K, originally derived from the peripheral blood of a patient with monoqtic leukemia, were given to us by Mr. Pat Simpson and Dr. Irving Johnson of Eli Lilly and Co. These were grown in Xl99 medium with 5 per cent horse serum in stirrer culture. They were maintained in single cell suspension. Aliquots were removed for use, the cells collected at the centrifuge, washed and rt’suspended in 0.15 53 NaCl. The final dilution provided 10” cells per ml. Cell counts were performed in a standard hemacgtometer chamber with the aid 01’ a leukocyte pipette. In all experiments, recoveries ranged from about X0 to 100 per cent. Observations of cell morphology were made with the aid of phase contrast while nigrosin (final concentration, O.‘iT, microscopy of uustained preparations, per cent) staining was observed in the hemacytometer chamber using light microscopy. In general, note was made of the degree of nigrosin staining, fraction of cells stained, appearance of the cells in unstained preparations, and the point at which clumping or gross extraction of nucleic acid occurred. The latter was obvious front the change in viscosity of the suspension, and microscopic alteration of the nuclei.

RESULTS \‘-‘L c,trr~inoll,cr.-Our initial attempts were tfirectect to a method !‘!)r the isolation of nuclei from this tumor [9]. Because ol’ the paucity of unstaint>cl cells in suspensions prepared 1)~ our method ;:,I, no attempts \vere matic IO correlate morphological changes in the cells \\-ith dye uptake, hut the rclativc was judged by the ease A\-ith \vhich tumor nuclei efficar? of the surfactants

\\-ere liherateti,

and bp the number

liberated.

M. E. Hodes, C. G. Palmer

and D. Livengood

Results obtained when a freshly excised tumor was titrated are presented in Table I. Table II outlines a comparison of the action of a series of homologous alkyl sulfates. The C 12 and C 14 sulfates are roughly comparable in action. It has been possible to prepare nuclei by grinding frozen tumor in a porcelain mortar and pestle in the presence of 2.5 mM sodium lauryl sulfate (SLS) in 0.15 A4 NaCl at about 0”. One such preparation contained 02 per cent nuclei, 44 per cent “tabs” and 24 per cent cells. Because of the large number of cells remaining, and the variability of results as compared to the titration procedure, we have not pursued this possibility further. We

TABLE

II.

Comparison

of the action of homologous carcinoma cells.

alkyl

Differential Alkyl sulfate

Concentration @Ml

octy1 Decyl Lauryl’ Myristylb

Nuclei %

13.9 3.0 2.7 2.0

13 22 40 57

sulfates

on V-2

counts

“Tabs” %

Cells %

23 22 35 38

63 56 25 25

a See Table III. ’ Fresh tumor cells.

III.

TABLE

Effect of CaCl, on lysis of V-2 carcinoma Differential

SLS cont. (mW 0 1.9 2.3 2.4 2.7 2.3

CaCI, cont. (mW

Suclei ?A

0 1.8 0

1.8

a 27°C. The experiments were performed * Includes four ghost nuclei. Experimental

Cell Research 24

20 15 4ob 45

counts

“Tabs” %

20 “5 35 40

on separate aliquots

CPllS

x

cells by SIS. Recovery of nucleated fragments %

100 100 50 50

100 100

so 25 15 of the same tumor.

84

301

Action of synthetic surfactants on membranes. 11

have, however, used such crude preparations as starting material for the preparation of tumor deoxyribonucleic acid. In several procedures designed for their isolation, calcium chloride has nuclei [-I, 111. Addition of CaCl, in varying roncenbeen used to “harden”

‘I‘aHr,l: I\‘.

Efect

o/ increasing concentrations lysis of V-2 cells.a Differential

Ka laurate cont. (1ll~lZ)

of sodiam Irrrzrrrfe 011

counts

“Tabs” Y”

Nuclei Y”

Cells ‘)a

0

1 00

0.3

100

1.3

1

6

93

3.1

9

18

73

4.0

14

15

il

.4.5

21

24 nuclei-clumping

Mostly

-4.75

-5.i

a Fresh tumor.

\‘. Comparison

‘I’.\n~t-

y-2

(kmc. range ( 111M )

0

O.(l(i-0.07 0.13-0.1 i l~.21-0.:34 0.~44-0.46 0.51-0.5:~ 0.6-0.50 O.S8 1.:3ti-1 .:x3 l.!)‘) 2.32

II

Stained 96

5 14 37

J-96

13

16 51 48 21 Extraction

Nuclei“Tabs” 0’ :b 10

Stained 0’ ,13

0

13

0

6

10 16

14 36

3

57

81

46

65 51

21 79 71 02

2 0 4

80

1 3 70 31

93

62

38

100

1

85

100

:+

96

Extraction

29

of

1.1X-HE,

Nuclei “Tabs” 1” “b

0

30 :j

of SLS on the nlembrnnes sever01 cell lines.

Ehrlich

Nuclei”Tabs” ‘I,> “C,

4

of the action

15

Stained (‘0

Nuclei”Tabs” 0.” (II,

41

1

41

-4%

i

21

80 100

12 10 Clumping

71 SI

302

M. E. Hodes, C. G. Palmer

nnd D. Linengood

trations has not materially improved the recovery of cell fragments containing nuclei, nor appreciably changed the differential count (Table 111). Results similar to those obtained with SLS \vere seen when V-2 cells \verc titrated with sodium laurate (Table IV), although higher concentrations \vere required.

Fig.

1.

Ehrlich nscites rind tissue culture cells.-Preliminary experiments on the effect of surfactants in promoting dye penetration [8] indicated that SLS was one of the most effective as well as the anionic agent easiest to handle. It was therefore used as the prototype in experiments designed to compare morphological and tinctorial changes. The results of the individual experiments with SLS are summarized in Table \‘, together with a titration of V-2 cells for comparison. Staining slightly precedes, then roughly parallels Iysis of the cell membrane (lysed cells = nuclei + “ tabs”), and the suspectibility of the three types of single cells is roughly similar. These results are depicted graphically in Fig. 1. The curve for staining is always above that for cell membrane rupture. Retn-een SLS concentration of 0.3 to 0.4 mM there is a rather abrupt change in staining properties. The cells are almost all light gray when examined immediately, and shortly after being placed in the counting chamber, a majority of them assume a rather dark hue. When a concentration of about 0.4 mM is exceeded, the cells all reach the final color immediately. Lauryl pyridinium chloride (LX) was used as an example of the cationic agents. Results are tabulated in Table VI. While the EA and *J-Y6 cells take up stain in the presence of this agent, only very few of the cells lyse. The LLC-HE1 cells, on the other hand, undergo both staining and lysis. Experimental

Cell Research 24

;Icfion

of synthetic

surfacfanfs

:303

on membrtrnes. Ii

‘l’hc nonionic Igepal I)M-710 (Table VII) is similar to the anionic SLS in action. The yield of free nuclei is lower lvith li.4, and higher \vith LIX-HK,. 1O-H~drox~-A~-dccenoic, acid has heen show-n to have antitumor propertiw 21 . l{ecausc trf its chain length, it might hchavc similarly to sodium laurattb and SI,S in the system described above. Ho~vcver, as she\\-11 in ‘I’ahlc \‘III, this material is a relatively inef’f’ective rytolytic agrnt against PX, although it might hc cxpccted to “kill” ahout one-half of the cells. ;\ comparison of thtb Icthal anal lytic properties of this suhstancc has hwn undcrtalicn v10 -.

Ehrlich (hnc. range (m.12)

Stained (‘<,

Suclei “0

.I-
“Tabs” 00

Stained 0;)

I.l.(:-HI‘,

Suclei “0

“Tabs” “/,

Stainctl 0

Suclci ‘Jo

‘“l‘ab~” (‘4,

(I (1.0; 0.17-0.21

0.21 0.27~-0.40 0.31-0.51 o.li7-o.i:l O.!)r;-1.1,; ‘,.th-2.M

‘1’.~111.1<\‘II.

Compwison

of’ the rrction

o/ Igepul

two cell lines. Ilhrlich C:onc. range (nl.11)

Nuclei

0,
“Tabs” “<>

lj.ll-‘710

011 ihr

IJW~J?~~IYIIIP,S o/

M. E. Hodes, C. G. Palmer TABLE VIII.

and D. Livengood

Effect of increasing concentration of HDA on nigrosin and lysis (Ehrlich ascites).a

alptuke

Differential Concentration MM)

Unstained

0 0.12 0.24 0.36 0.42 0.47 0.59 0.75 1.58 4.62 6.23

100 95 95 83 76 85 78 79 81 81 75

0.52

63

a Room temperature: 2.8 x 106.

Cells x

cells

pH 5.5 (citrate

buffer;

“Tabs” %

Suclei %

93 94 94 82 48 59 68 66 -

5 6 6 16 48 38 26 28 -

2 0 0 2 4 3 6 6

54 53

34 30

12 17

ionic strength

0.3); cell count initially

2.4, finally

IX. Effect of cell concentration on nigrosin uptake and lysis at various times after SLS treatment (Ehrlich ascites).”

TABLE

Differential Total cells ( x 10-6)

Time (min)

Concentration WW

Unstained cells

Cells %

“Tabs” %

8

Nuclei %

1.2 1.3

0 9

0 0.15

98 86

92 84

16

0 0

2.0 2.0 1.8

0 9 40

0 0.15 0.15

94 82 74

92 82 85

8 18 15

0 0 0

3.3 2.7

0 9

0 0.15

95 77

90 75

10 25

0 0

39

0

0

97

86

14

0

49 28 30

9 38 66

0.15 0.15 0.15

81 73 82

80 75 73

20 25 22

0 0 0

a Room temperature. Experimental

SLS added immediately

Cell Research 24

after control

determinations.

30.13

Action of synthetic surfactants on membranes. II

The interaction of proteins with surfactants seems to be more dependent on the protein:detergent ratio than on the absolute detergent concentration [18j. The effect of varying the concentration of cells, and hence the protein, in the suspension was tested, using a single, moderately effective concentration

_'

80

8 0 70 : 'a :: 60 : 2 50 8 i

40

0:

I! 0

1

2

I

I

3

4

Fig. 2.--O - ~-0, NaCl, 0.15 M; O-O, * -~ A, SLS, 0.05 m,Vf, in 0.15 M NaCl.

‘r.\l3r.l-

S.

Rffecl

1: 5 6 Time in hours

Tyrodes

I: 7

8

(no CaCl,);

of 1.8 mM CaCl, on nigrosin unrioris time interuds (Ehrlich

I

I:

9

10

CaCl, 0

(1

Time (min)

0 0.24 0.24

-

0 0.24 0.24

-

0

trscites).” 1)ifferential “l’af,s” “
47

97 0 0

96 50 11

-

95

94 x9 06

II 33

x9 x4 x9

11 1(i 11

0.07

9

87

0.07

47

86 94

0.05

0

97

0.07

47

98

temperature.

Cells o” 96 59 2

9

(with CaCI,): and

uptnke rind lysis by SLS rti

98 9 0

9 47

0

a Iioom

Vnstained cells

24

.P) - -3, Tyrodes

SI,S Concentration (ndf)

I 21

Ii

Suclri 1’1,

M. E. Nodes, C. G. Palmer

and D. Livengood

of SLS (Table IS). Within the range 1 to 30 x 106, at low surfactant conccntration, EA cell number is without appreciable efIect on the action of SLS. As time might be expected to have an influence on the uptake of dye, several concentrations of EA cells and surfactants were brought into contact for various periods. These results are also she\\-n in Table IS. The effect of the surfactant is usually complete in 9 or 10 min, and the major part of the action seems to take place almost instantly. The slow increase of staining which occurs in the absence of surfactant is depicted in Fig. 2. TABLE

PH

XI.

Effect of pH on niyrosin Total cells ( x 10-y

Unstained cells

A. 6.4 3.3 2.6 2.0 1.5 0.9

2.1 2.1 2.1 3.2 1.5 1.7

6.0 6.9 7.6 9.4 10.5

1.1 1.4 1.9 1.1 0

Titration 100 100 98 90 92 86

R. Titration

a Room temperature. b Four cells found in chamber,

93 97 95 97 100b

uptake and lysis (Ehrlich CCllS

%

“Tabs” %

ascites).’ Nuclei 96

wifh acid. 90 100 98 99 95 100

10 0 2 1 5 0

with base. 100 0 55 15 82 18 a5 5 Gross extraction

0 0 0 10

all unstained.

The effect of CaCl, on staining and lysis was also assayed on the EA cell. The results are shown in Table X. As EA cells in glucose-free solutions may continue to metabolize for some time, then either die of starvation or as the result of accumulated acidic products of that metabolism, the effect of varying the external pH by addition of acid or alkali was tested. The results of these experiments are shown in Table XI. (The final pH of the reagent cell mixtures in our experiments was always 6.5 to 7.4, except in the case of lo-hydroxy-AZ-decenoic acid, when a citrate bufIer of pH 5.5 was employed). Esperimental

Cell Research 24

&lrtion

of synthetic

surfactants

on membranes.

II

3Oi

DISCUSSION

I’rs\v experiments on tysis of tumor cells have been reported, and inosl \\-orkcrs have used experimental designs dift’erent from those reported hrrc. t%cnnctt and Connon 121 studied the efTects of a number of’ tytic agents on the plasma mcmhrancs of tL4 cells. Morphological observations after beatmrnt \vith fixed concentrations of lysin for 1 hour at 37‘ in their tlantls gavcb rt~ults similar to those reported here, with maximum tytic activity for fatt\ avicls centering ahout the C 12 (tauric) acid. ‘t’hc mcthott cmptoycd was purposely designed lo detect only lytic action. .\ similar approach, utilizing changes in morphology atonc~, \\-as nwessar! in evaluating the rtfects of surfactants on the solid V-2 carcinoma. ‘t’tio wsutts of the experiments reported here, as welt as other unpuhtishect data, indicate that a large variety of surfactants can producv some degree of tysis of V-2 ~~~11s.Ltosl agents leave the cell itself more or less intact. after breaking the plasma mrmhranc. This may he due to the nature 01’ rabbit (*(~I1cytot)tasm, \vhivh swms to cling tenaciously to the nucleus. ‘I’hc approach chosen with the EA and tissue cutturc cells ~-3s designed to which could IX evaluated nncctuivocally. ‘t’hcw inc*luctv t)roduce ~tlangc~s uptake of dye, tysis of the plasma memhranc anct adherence of cytoptasmic fragments (“labs”), production of free nuctci, anti clumping or cxslraction of the “tabs” and nuclei. Incubation methods generally lead lo changes in Ihe morphology of the cell as a result of the passage of time ant1 m(>Iahotit vhangcs within the cell itself. These changes are sometimes hard to ditl’rrentiatv from those produced by the surfactants (sw pwcding patwr), nncl it \\-a~ for this rcason that Ihe rapid titration mrthocl w-as c~t~oscn for this stutiy. ‘t’hc uptake of vital dye as a more precise tool for the study ot’ the actions 01 surfactants sull’crs from h\-o shortcomings. One ot’ Ihew is Ihc I’:I~~IPI apriipt transition from little to almost complete staining oyvr a n:irro\\ range of conc.rntration; a transition Ivhich is roughly rcproducihle. Similar “all-or-none” cll’wts have heen ohserveci by \\‘attiaus anct dc I)u;-c ‘)‘) __ in their studies on riipturc of tysosomes h? nonionic surfavtants. Its t)ossit)lc rclationshit) to a transition from protectiw lo lytic ac*tion of tht> surfactant as has been observed in the case of erythrocytes [7, 11 merits I’urU~cr PXt)loration. ‘1’11~second difficulty concerns the tack of correlation with plasma mc~mhranc lysis in some y&ems (Table VI). This lack of correlation \vith staining is also ohserved in Ihe loss of reprottwti~e ability produvwt \vhc,n surt’actants avl on tC.4 cells [lo:, and indicates a dilf’errnt motic> of aclion ot

308

M. E. Hodes, C. G. Palmer and D. Livengood

the various agents, as well as failure of the system to measure the same types of phenomena. Eaton, Scala and Jewel1 [5], among others, have noted that loss of reproduction of EA cells treated with noxious agents precedes staining and loss of other functions. Despite the abruptness of staining, reproducibility seems quite good. In general, only 100 cells were counted, as both staining and morphology tend to change while cells are on the microscope slide. This is due to heat from the lamp and evaporation around the edges of the coverslip. Repeat experiments indicate that these phenomena are not responsible for the difference in staining and lysis reported above, as both processes proceed in the same direction. After due consideration of the difficulties involved, some conclusions are possible. If several cell types are treated with any single surfactant (Tables V, VI and VIl), comparison of the susceptibilities of the cells can be made. Thus, LLC-HE, cells are susceptible to lysis by lauryl pyridinium chloride, whereas the other cell lines tested are resistant. This line originated from human embryonic connective tissue; the other materials are all of malignant origin. The similar susceptibility of all cell lines tested to cell membrane rupture by SLS would suggest, in view of the experiments of Pethica and Schulman [17] on penetration of surfactants into cholesterol monolayers, similar proportions of cholesterol in the lipid complex of the membrane. Further data here would be helpful, as anionic agents could also complex with protein in the membrane. The relative independence of lysis and cell concentration (Table 1X) would suggest that protein is not the determining factor at the surfactant concentrations employed. Greisman’s data [6] on erythrocyte hemolysis by sodium oleate indicate that lysis is rapid and independent of cell concentration above approximately 0.1 mM surfactant. Comparative data using SLS (Table IX) would suggest roughly comparable critical concentrations for the EL4 cell line reported here. The absence of marked time dependence for the reaction with SLS is perhaps explicable by the concentration of surfactant chosen, but the small number of cells stained even after 40-66 min, seems to indicate an equilibrium reaction between cell and surfactant. Attempts to determine the mode of action of the diverse chemical entities producing lysis or dye uptake are under way. Preliminary studies [ 1 ] indicate that staining and cholesterol release proceed hand in hand, in the case of EA cells, thus indicating action on the lipid portion of the cell membrane. It is interesting to note that surfactants promote hyperlipermia in animals [ 161, and cause a slight decrease in adrenal cholesterol. How much, if any, of this Experimental

Cell Research 24

Action of synthetic surfactants on membranes. II

309

action is due to action on cell membranes is unknown. Lovelock [13, 11, has noted a correlation between release of cholesterol and phospholipid and lysis of erythrocptes by nonionic surfactants. In his work, as \vell as in our own, hydrocarbon chain length is the structure best correlated with activity. The nature of some possible membrane alterations allo\ving dye penctration has been discussed above. Variations in the lipid layer could C;~LISV distortions in the chemical of the structure membrane, followed by increased permeability. Hotchkiss [12] has noted permeability increases in surfactanttrcatrd bacteria. That some surfactants act directly on the membranes is rather apparent from the rapid lytic effects noted in the tables (differential columns). It has been suggested that dye uptake follows interference \vith metabolism, and death of the cell, as decreased uptake of O2 has been noted to prccectc staining [.ji. That staining without respiratory depression can be produced hy alteration of the membrane directly is indicated in cxpcriments by thcsr authors rising lecithinase (Cl. welchii toxin) at 1 : 16 dilution. Some respiration remained cvm when all cells had become permeable to cosin. Similar elfrcls might be produced by long chain fatty acids, some of \vhich are kno\vn to affect respiration [lo, 20:. SUMMARY A comparison has been made of the effect of increasing concentrations 01 a number of nonionic, anionic, and cationic surfactants on the cell mctnbrancs of the V-2 carcinoma, the Ehrlich ascitcs carcinoma and tissue culture preparations of human embryonic connective tissue and peripheral blood The concentrations necessary to cells from human monocvtic leukemia. produce rupture of the cell membrane have been compared \vith those x\ hich promote uptake of the vital dye, nigrosin. The action of the agents in promoting staining and lysis has heen roughly similar with the following cucct)tions: the V-2 cell membrane is more resistant to thr action of SLS than the other cell lines, lanryl pyridinium chloride promotes staining l)ut not Iysis of the malignant and ascites lines, while the LLC-HE, human conncc’tivc tissue line is both stained and lysed in the presence of this surfactant. \\‘itliin the range of 1 to 30 million cells, cell concentration is \vitliout much ttTect on the action of sodium lauryl sulfate against Ehrlich ascites cells. The presence of calcium chloride likewise does not c’ausc much change in the action of this surfactant. Altering the pH from 1 to 9.1 causes littlc change in the uptake of dye by the cells. In general, \vhen lysis does occur, it is preceded slightly hy nigrosin staining.

310

M. E. Hodes, C. G. Palmer and U. Liuengood REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

BARRETT, L. K. and HODES, M. E., E~ptl. Cell Research 21, 209 (1960). BEXNETT, L. R. and CONNON, F. E., J. Null. Cancer Inst. 19, 999 (1957). DIXON, F., PALMER, C. G. and HODES, IN E., Transplantation Bull. 7, 91 (1960). DOUNCE, A. L., in The Nucleic Acids (E. CHARGAFF and J. N. D~~IUSON, eds.) Acadelnic Press, New York, 1955. EATON, M. D., SCALA, A. R. and .JEWELL, M., Cancer Research 19, 945 (1959). GREISMAN, S. E., Proc. Sot. Exptl. Biol. Med. 101, 117 (1959). HERZ, A., Z. Rio!. 111, 139 (1959). HODES, M. E., PALMER, C. G. and WARREN, A., Exptl. Cell Research 21, 164 (1960). Hooq M. E., PALMER; C. G. and WILLIAMS, D. S.; Nature 182, 529 (1958). HODES. M. E.. WARREN, A. K. and PALMER, C. G.. Nafure 188, 157 (1960). Ho~E&Io?.,, G. H., SCI&IDER, W. C. and STRIE&CH, M. J., J. Bioi Chem. 196, 111 (1952). HOTCHKISS, R. D., Ann. N. Y. Acad. Sci. 46, 479 (1946). LOVELOCK, J. E., Nature 173, 659 (1954). LOVELOCK, J. E. and REES, R. J. W., Nature 175, 161 (1955). PAL~IER, C. G., HODES, M. E. and WARREN, A. K., Exptl. Cell Research (In Press). PATNODE, R. A., HUDGINS, P. C. and JANICKI, B. W., J. Exptl. Med. 107, 33 (1955). PETHICA, B. A. and SCHULMAN, J. H., Biochem. .I. 53, 177 (1953). PUTNA~~, F. IV., Advunces in Protein Chem. 4, 79 (1948). SCHOLEFIELD, P. G.. Cancer Research 18. 1026 (1958): C. A. 53, 1528 (1958). SCHOLEFIELD; P. G.; Can. J. Biochem. Physiol. ‘34, li27 (1956). ’ TOWNSEND, G. I;., MORGAN, J. F. and HAZLETT, B., Nature 183, 1270 (1959). WATTI~UX, R. and DE DUVE, C., Riochem. .I. 63, 606 (1956).

Experimenlal

Cell Research 24