I

Surv. immunol. Res. 2:122-128 (1983) �9 1983 S. Karger AG, Basel

0252-9564/83/0022--012252.75/0

Synthesis of Lipids or Lipid-Containing Macromolecules in Tumor Cells Relevance to Host Defense

Sarkis H. Ohanian

Laboratory of Immunobiology, National Cancer Institute, National Institutes of Health, Bethesda, Md., USA

The relative success of tumor cell growth in a host may be a reflection of the ability of the cells to respond in some manner to negate or nullify the mechanism(s) of the host's cel- lular and humoral immune attack. The re- sponse(s) which are available to influence the outcome of immune attack would include the ability to repair or reseal damaged plasma and cellular membranes and/or the ability of the cell to modify the chemical-physical properties of the cellular membranes. The success of the cells to survive potentially cy- totoxic damage would also depend upon the rate and extent with which the cell is able to respond to the attack mechanism. In this paper I will focus on the humoral immune system and the properties of tumor cells which appear to influence the effectiveness of immune attack.

A major component of the humoral im- mune mechanism is complement (C). The complement system is composed of at least 11 distinct protein and/or glycoprotein com- ponents and several inhibitors. The system has been extensively characterized in man [ 1 ] and guinea pigs [2] and has been detected in the sera of many mammalian species and lower vertebrates [3]. The C system can be

divided into two pathways of activation (classical and alternative or properdin path- ways) and a common membrane damaging pathway (fig. 1).

The classical pathway is initiated by the interaction of the Clq subunit of C1 with the Fc receptor of IgG or IgM in an antigen-anti- body complex (fig. 1). The subsequent events are a complex series of actions and interac- tions of the initial components (C4, C2 and C3) and the late acting C component (C5, C6, C7, C8 or C9) with one another and the cell membrane. The generation of the membrane attack complex C5b-9 and the nature of the damage-causing steps is being investigated intensively and at the present time appears to involve the formation of transmembrane channels [4] or the fixation of C-protein- membrane phospholipid micelles [5] which lead to the loss of semipermeable properties of the membrane. Additional studies on the kinetics and extent of lysis of target erythro- cytes by antibody plus C led to the one-hit mechanism of immune hemolysis or cytoly- sis [6]. The one-hit theory states the interac- tion of an antigen-antibody complex with C components which leads to the generation of a single lesion is sufficient to lyse or kill a cell.

Lipid Synthesis in Tumor Cells and Host Defense 123

~;}~: : ~i �84

C3b

~ B.P C3b. 8

C3b n B, P

C \ \ C5b U

/ ~ C6, 7,8,9 d

C5b-9

Fig. 1. General schematic of reaction sequence of classical and alternative complement pathway. In the classical pathway Clq interacts with antibody of antigen-antibody complexes (AgAb) which leads to activation of Clr and Cls subunits (Clq~). The activated C~r and Cls enzymaticatly cleave C4 and C2 to generate on the cell surface the enzymaticatly active complex C42 (C3 convertase). C3, activated by C42, binds to the cell surface and leads to the generation of the C5 cleaving enzyme C 1---4~. The alternative or properdin pathway is initiated by the interaction of naturally occurring substance and certain classes ofimmunoglobulins with native C3. The C3b which is generated complexes with factor B. The C3b,B complex, in turn activated by factor D, results in the enzymatic cleavage of additional C3. The enzymatical]y active complex C3b,B is converted to a C5 cleaving enzyme following the uptake of additional activated C3 or activated C3 plus properdin (P). The membrane attack complex of C which is composed of C5b,6,7,8 and 9 can be generated by the C5 cleaving enzymes C1,4,2,3, C3b,~.13,P or C3b.,B.

124 Ohanian

Thus, the time to reach an endpoint in killing of nucleated cells or release of hemoglobin from red cells is dependent upon the number of cytotoxic hits per target cell. The rate of killing of cells with one lesion would be less than that of cells with more than one le- sion.

In contrast to the classical pathway in which the later-acting C components (C5 to C9) are activated by C1, C4, C2, and C3, an alternative or properdin pathway exists by which the later C components can be activated by naturally occurring polysaccharides, by en- dotoxin, polyanions and some classes of im- munoglobulins (fig. 1). While most of the ac- tivity occurs in the fluid phase, the interme- diates also can bind to target cell membrane. This pathway has been reviewed [7-9]. Vari- ants of this pathway of C activation and activ- ity have been described and include reactive lysis [ 10-12] and deviated lysis [ 13, 14].

At the present time the classical pathway of C action appears to be the major mecha- nism of humoral immune killing of nucleated cells. However, there are reports that under selected conditions, the alternative pathway of C activity can also be effective [15-19].

In nucleated cells it has been shown that changes in physical properties [20-25] and inhibition of respiration, glycolysis and pro- tein synthesis can occur within minutes after the addition of C [23, 24, 26-28]. Stimula- tion of phospholipid synthesis as measured by 32p incorporation has also been detected in HeLa [29, 30] and mouse L cells [31]. In additional studies with L cells under appro- priate conditions addition of C to antibody- sensitized cells can stimulate nucleoside up- take and cell growth [32]. These results sug- gest that metabolic changes do occur in different nucleated cells under attack by anti- body plus C.

In studies with guinea pig hepatoma cell lines, evidence has been obtained which sug- gests that the relative resistance of the cells to humoral immune attack is correlated with the metabolic properties of the cells [for re- view see ref. 33]. In contrast to other reports, however, no changes in the synthesis of DNA, RNA, protein or complex carbohy- drate was observed in antibody-sensitized cells within 1 rain to 3 h after the addition of GPC (a source of C not effective in killing the cells) or HuC (a source effective in killing the cells). However, lipid synthesis (cardiolipin, cholesteryl ester and phosphatidylcholine), as measured by the incorporation of acetate, glycerol or fatty acid into the lipid pool of the cells and the release oflipids (cardiolipin, tri- glycerides and phosphatidylcholine), was markedly enhanced within 3-5 min after ad- dition of the GPC. On the other hand, no increase in the net synthesis of lipid was observed with ceils treated with HuC, al- though increased release of lipids was ob- served (cholesteryl ester and triglyceride). Al- though cells treated with HuC demonstrated no net increase in lipid synthesis, less cardio- lipin and more phosphatidylcholine was syn- thesized by these cells as compared to cell controls. The observation that the synthesis and/or release of cardiolipin, a major intra- cellular lipid, is affected by C action suggests that intracellular events as well as those at the cell surface may be associated with the ability of the cells to resist humoral immune at- tack.

Other investigators have also found that C-mediated attack of different types of target cells is accompanied by the release of phos- pholipids and/or cholesterol from the mem- brane [34-37]. The phospholipids include phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin.

Lipid Synthesis in Tumor Cells and Host Defense 125

These observations have led in part to the present concept of the mechanism of C- induced cytolysis, i.e. that the terminal C component (C5 to C9) form a group of am- phiphilic proteins the terminus of which is hydrophobic and inserted into the lipid bi- layer of the membrane. Release of some of the lipids from the bilayer could, therefore, be a primary step in loss of the cells osmotic barrier and finally death or lysis.

The observation that lipids or lipid-con- taining macromolecules may enable a cell to resist C-mediated attack is further supported by observations that treatment of cells with chemical or physical agents (chemothera- peutic drugs, X-ray, Atromid S) which render cells more sensitive than untreated control cells reversibly interfered with the synthesis of specific lipids [38, 39]. In addition treat- ment of cells with selected hormones renders the cells more resistant than untreated cells reversibly enhanced the synthesis of selected lipids [33, 40]. Analysis of the fatty acid com- position of the lipids of the cells modified by treatment with the selected agents showed that resistant cells had a higher content of sat- urated fatty acids while sensitive cells had a higher content of unsaturated fatty acids.

Many cell properties such as membrane fluidity and mobility, cell adhesion and cell permeability were influenced by the arrange- ment and content of lipids or fatty acids in the cell membrane [41-46]. It has been re- ported that the ability of cells to maintain membrane integrity in the face of changes in osmotic pressure or attack by antibody and C is dependent on membrane lipid fluidity [33]. Alterations of the fatty acid content or com- position of EL-4 mouse leukemia cells by inducing the cells to incorporate C19:0 and C18:3 fatty acid was shown to affect capping of certain antigenic membrane components

[47]. However, the susceptibility of the cells to cell-mediated or antibody-C-mediated killing was not affected by such treatments [48]. Unfortunately, fluidity measurements of the cell membrane were not performed. In studies with bacterial cells, however, it was shown that a threshold amount ofrnembrane fluidity in the cells was required for C-me- diated killing to occur [49].

Preliminary results using fluorescent po- larization techniques have shown that mem- branes of guinea pig hepatoma cells rendered susceptible to humoral immune attack fol- lowing treatment with chemotherapeutic drugs were more fluid than control cells. In contrast, membranes of cells rendered resis- tant following treatment with hormones were less fluid [Ohanian, unpublo observations]. In additional studies in which the lipid and fatty acid composition were manipulated by chemical and physical means, it was shown that when the phosphatidylcholine/sphingo- myelin mole ratio of the hepatoma cells was increased 5- to 8-fold the cells were more sus- ceptible than control cells [50]. In contrast, when the content of unsaturated fatty acids was decreased following homogenous cata- lyzed hydrogenation the cells were more resis- tant than control cells. It has been reported that phosphatidylcholine/sphingomyelin ratio is directly proportional to membrane fluid prop- erties [43] whereas high unsaturated fatty acid content should result in decreased membrane fluidity [50]. While there may be some corre- lation between increased fluidity and in- creased sensitivity to killing by C with the guinea pig hepatoma cells, there was no de- tectable correlation with human cells [51]. Similar results were noted with P815 mouse mastocytoma cells [Ohanian, in preparation].

The use ofl iposomes or BLM has made it possible to determine the effects of fluidity of

126 Ohanian

the target surface on the efficiency of C- mediated killing. These studies have shown that increased membrane fluidity can lead to enhanced [52, 53] or decreased efficiency [54] of the C. These results would confirm those of nucleated cells that membrane fluidity may not play a major role in the outcome of C attack. One drawback in the available methods for the measure of fluidity is that the analysis is by nature an average of the actual values for the individual probes in the membrane. Since C appears to act at discrete sites on the membrane, subtle changes in the physical and chemical interaction of mem- brane components at selected sites in the membrane may not be detected. At the present time the role of membrane physical and chemical properties in susceptibility of the cell to C killing has not been unequivo- cally answered.

The humoral attack mechanism is a com- plex series of events involving the interaction of antigen, antibody and C with one another and the cell membrane. The observations that binding of many thousands of C compo- nents and development of ultrastructural changes occurring on or within the cell mem- brane without cell killing suggests that prop- erties of the target cell can greatly influence the outcome of the attack mechanism [25, 38]. The present information leads one to conclude that synthesis and composit ion lipid or lipid containing macromolecules are of primary importance in the maintenance of cell integrity since DNA, RNA, protein and complex carbohydrate synthesis can be in- hibited without leading to immediate suscep- tibility of the cell to killing [25]. These obser- vations imply the existence of a system(s) which is relatively independent of direct ge- netic control. Such a system(s) would have an obvious advantage in that an immediate re-

sponse to repair potentially cytoxic events occurs at the cell membrane or even at intra- cellular sites. The response mechanisms, at the present time, appear to be associated with the synthesis and incorporation of lipids or lipid-containing macromolecules into the surface or intracellular membranes . Thus, the effectiveness of cell killing by complement may be the net result of the qualitative and quantitative aspects of the attack mechanism versus the rate and extent of the cell response to nullify the cytotoxic activity generated.

R e f e r e n c e s

1 M~iller-Eberhard, H.J.: Chemistry and reaction mechanisms of complement. Adv. Immunol. 8: 1- 80 (1968).

2 Nelson, R.A.; Jensen, J.; Gigli, I.; Tamura, R.: Methods for the separation, purification and mea- surement of nine components of hemolytic com- plement in guinea pig serum. Immunochemistry 3: 111-135 (1966).

3 Ballow, M.: Phylogenetics and ontogenetics of the complement system; in Day, Good, Comprehen- sive immunology. Biological and amplification systems in immunology, vol. 2, pp. 183-204 (Ple- num Press, New York 1977).

4 Mayer, M.M." Membrane damage by complement. Johns Hopkins med. J. 148:243-258 (1981).

5 Podack, E.R.; M/iller-Eberhard, H.J.: Binding of desoxycholate, phosphatidylcholine vesicles, lipo- protein and the S-protein to complexes of the ter- minal complement components. J. Immun. 121: 1025-1030 (1978).

6 Mayer, M.M.: Development of the one-hit theory of immune hemolysis; in Heidelberger, Plescia, Immunochemical approaches to its problems in microbiology, pp. 268-279 (Rutgers University Press, New Brunswick 1961).

7 G6tze, O.; M/iller-Eberhard, H.J.: Lysis oferythro- cytes by complement in the absence of antibody. J. exp. Med. 132:898-915 (1970).

8 Medicus, R.G.; Schreiber, R.D.; G6tze, O.; Mfil- ler-Eberhard, H.J.: A molecular concept of the properdin pathway. Proc. natn. Acad. Sci. USA 73:

612-616 (1976).

Lipid Synthesis in Tumor Cells and Host Defense 127

9 Gewurz, H.; Lint, T.: Alternative modes and path- ways of complement activation; in Day, Good, Comprehensive immunology. Biological and am- plification systems in immunology, vol. 2, pp. 17- 45 (Plenum Press, New York 1977).

10 Thompson, R.A.; Rowe, D.S.: Reactive hemolysis. A distinctive form of red cell lysis. Immunology 14:745-762 (1968).

11 Thompson, R.A.; Lachman, P.J.: Reactive lysis. The complement mediated lysis of unsensitized cells. I. The characterization of the indicator factor and its identification as C7. J. exp. Med. 131: 629- 642 (1970).

12 Lachman, P.J.; Thompson, R.A.: Reactive lysis. The complement mediated lysis of unsensitized cells. II. The characterization of activated reactor as C56 and the participation of C8 and C9. J. exp. Med. 131:643-657 (1970).

13 Rother, U.; H/insh, G.; Menzel, J.; Rother, K.: Deviated lysis. Transfer of complement lytic activ- ity to unsensitized cells. I. Generation of the trans- ferable activity on the surface of complement resis- tant bacteria. Z. ImmunForsch. 148:172 -186 (1974).

14 Rother, U.; H/insh, G.; Rother, K.: Deviated lysis. Transfer of complement lytic activity to unsensi- tized cells. II. Generation by inulin and by antigen- antibody complexes. Z. ImmunForsch. 151: 442- 451 (1976).

15 Ferrone, S.; Cooper, N.R.; Pellegrino, M.A.; Reis- feld, R.A.: Activation of human complement by human lymphoid cells sensitized with histocom- patibility (HL-A) alloantisera. Proc. natn. Acad. Sci. USA 70:3665-3669 (1973).

16 Perrin, L.H.; Joseph, B.S.; Cooper, N.R.; Old- stone, M.B.A.: Mechanism of injury of virus- infected cells by antiviral antibody and comple- ment. Participation of IgG, F(ab')z, and the alter- native complement pathway. J. exp. Med. 143: 1027-1043 (1976).

17 Joseph, B.S.; Cooper, N.R.; Oldstone, M.B.A.: Im- munologic injury of cultured cells infected with measles virus. I. Role of IgG antibody and the alternative complement pathway. J. exp. Med. 141:761-774 (1975).

18 Theophilopoulos, A.N.; Perrin, L.C.: Lysis of hu- man cultured lymphoblastoid cells by cell-me- diated activation of the properdin pathway. Science 195." 878-880 (1977).

19 Baker, P.J.; Lint, T.; Mortensen, R.T.; Gewurz,

H.J.: C56--7 initiated cytolysis of lymphoid cells. Description of the phenomenon and studies on its control by C56--7 inhibitors. J. Immun. 118: 198- 202 (1977).

20 Latta, H.; Kutsakis, A.: Cytotoxic effects of spe- cific antiserum and 17-hydroxycorticosterone on cells in tissue culture. Lab. Invest. 6." 12-17 (1957).

21 Miller, D.G.; Hsu, T.G.: The action of cytotoxic antisera on the HeLa strain in human carcinoma. Cancer Res. 16." 306-312 (1956).

22 Ellam, K.A.O.: Studies on the mechanism of the cytotoxic action of antisera. Aust. J. Sci. 20." 116- 117 (1957).

23 Bickis, l.J.; Quastel, J.H.; Vas, S.I.: Effect of Ehr- lich ascites antisera on the biochemical activities of Ehrlich ascites carcinoma cells in vitro. Cancer Res. 19." 602-607 (1959).

24 Green, H.; Goldberg, B.: The action of antibody and complement on mammalian cells. Ann. N.Y. Acad. Sci. 87.' 352-362 (1960).

25 Ohanian, S.H.; Schlager, S.I.; Borsos, T.: Molecu- lar interactions of cells with antibody and comple- ment. Influence of metabolic and physical proper- ties of the target on the outcome of humoral immune attack; in Reisfeld, Inman, Contemporary topics in molecular immunology, vol. 7, pp. 153- 180 (Plenum Press, New York 1977).

26 Colter, J.J.; Kritchevsky, D.; Bird, H.A.; McCand- less, R.F.: In vitro studies with antisera against tumor cell protein fractions. Cancer Res. 17." 272- 276 (1957).

27 Flax, M.A.: The action of anti-Ehrlich ascites tu- mor antibody. Cancer Res. 16." 774-783 (1956).

28 Ellem, K.A.O.: Some aspects of the ascites tumor response to a heterologous antisera. Cancer Res. 18. 1179-1185 (1958).

29 Guttler, F.; Clausen, J.: Changes in lipid pattern of HeLa cells exposed to immunoglobulin G and complement. Biochem. J. 115." 959-968 (1969).

30 Guttler, F.: Phospholipid synthesis in HeLa cells exposed to immunoglobulin G and complement. Biochem. J. 128." 953-960 (1972).

31 Shearer, W.T.; Crouch, J.A.: Humoral immuno- stimulation. VIII. Increased incorporation of phosphate and turnover of phosphatidylinositol in cells treated with antibody. J. Immun. 119." 911- 917 (1977).

32 Shearer, W.T.; Atkinson, J.P.; Frank, M.M.; Par- ker, C.W.: Humorat immunostimulation. IV. Role

128 Ohanian

of complement. J. exp. Med. 1 4 1 : 7 3 6 - 7 5 2 (1971).

33 Ohanian, S.H.; Schlager, S.I.: Humoral immune killing of nucleated cells. Mechanism of comple- ment-mediated attack and target cell defense; in Atassi, CRC reviews in immunology, vol. 1, pp. 165-209 (CRC Press, Florida 1981).

34 Smith, J.K.; Beeker, E.L.: Serum complement and the enzymatic degradation of erythrocyte phos- pholipid. J. Immun. 100." 419-474 (1968).

35 Kinsky, S.C.; Haxby, J.A.; Zopf, D.A.; Airing, C.R.; Kinsky, C.B.: Complement dependent dam- age to lysosomes prepared from pure lipids and Forssman antigen. Biochemistry 8:4149--4159 (1969).

36 Inoue, K.; Kinoshita, T.; Okada, M.; Akiyama, Y.: Release of phospholipids for complement-me- diated lesions on the surface structure of Escheri- chia coll. J. Immun. 119:65-72 (1977).

37 Giavedoni, E.B.; Dalmasso, A.P.: The induction by complement of a change in KSCN-dissociable red cell membrane lipid. J. Immun. 116:1163- 1169 (1971).

38 Ohanian, S.H.; Borsos, T.: Killing of nucleated cells by antibody and complement; in Day, Good, Comprehensive immunology, vol. 2, pp. 115-135 (Plenum Press, New York 1977).

39 Schlager, S.I.; Ohanian, S.H.: Correlation between lipid synthesis in tumor cells and their sensitivity to humoral immune attack. Science 197. 773-776 (1977).

40 Schlager, S.I.; Ohanian, S.H.: Tumor cell lipid composition and sensitivity to humoral immune killing. I. Modification of cellular lipids and fatty acid content by metabolic inhibitors and hor- mones. J. Immun. 124:626-634 (1980).

41 Baldassare, J.J.; Rhinehardt, K.B.; Silbert, D.F.: Modification of membrane lipid. Physical proper- ties in relation to fatty acid structure. Biochemistry 15:2986-2994 (1976).

42 Baldassare, J.J.; Brenckle, G.M.: Modification of membrane lipid. Functional properties of mem- brane relative to fatty acid structure. Fed. Proc. 36: 640 (1977).

43 Borochou, H.; Zahler, P.; Wilbrandt, W.; Shinitz- ky, M.: The effect ofphosphatidylcholine to sphin- gomyelin mole ratio in the dynamic properties of sheep erythroeyte membrane. Biochim. biophys. Acta 170:382-399 (1977).

44 Cherry, R.J.: Protein and lipid mobility in biologi-

cal and model membranes; in Chapman, Wallach, Biological membranes, vol. 3, pp. 47-90 (Aca- demic Press, New York 1976).

45 Selkirk, J.K.; Elwood, J.C.; Morris, H.P.: Study on the proposed role of phospholipid in tumor cell membrane. Cancer Res. 31:27-31 (1971).

46 Goldman, S.S.: Cold resistance of the brain during hibernation. III. Evidence of a lipid adaptation. Am. J. Physiol. 228:834-838 (1975).

47 Mandel, G.; Shimza, S.; Gill, R.; Clark, W.: Alter- ation of the fatty acid composition of membrane phospholipids in mouse lymphoid cells. J. Immun. 120:1631-1636 (1978).

48 Mandel, G.; Clark, W.: Functional properties of EL-4 tumor cell lipid altered membranes. J. Im- mun. 120:1637-1643 (1978)o

49 Kato, K.; Bito, Y.: Relationship between bacteri- cidal action of complement and fluidity of cellular membranes. Infect. Immunity 19:12-21 (1978).

50 Schlager, S.I.; Ohanian, S.H.: Modulation of tu- mor cell susceptibility to humoral immune killing through chemical and physical manipulation of cellular lipid and fatty acid composition. J. Im- mun. 125:1196-1200 (1980).

51 Ohanian, S.H.: Physical and chemical properties of nucleated cells which vary in their sensitivity to antibody-complement-mediated killing. Fed. Proc. 40:358 (1981).

52 Hesketh, T.R.; Payne, S.N.; Humphrey, J.H.: Complement and phospholipase C lysis of lipid membranes. Immunology 23:705-714 (1972).

53 Kitasawa, T.; Inoue, K.: Effect of temperature on immune damage of lysosomes prepared in the presence or absence of cholesterol. Nature, Lond. 254:254-256 (1975).

54 Humphries, G.M.K.; McConnell, H.M.: Antigen mobility in membranes and complement mediated attack. Proc. hath. Acad. Sci. USA 72:2483-2491 (1971).

Sarkis H. Ohanian, Laboratory. of Immunobiology, National Cancer Institute, National Institutes of Health, Building 37. Room 2B15, Bethesda, MD 20205 (USA)