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Membrane fluidity in malignancy Adversative and recuperative
Biochimica et Biophysica Acta, 738 (1984) 251-261
251
Elsevier
BBA 87123
M E M B R A N E FLUIDITY IN M A L I G N A N C Y ADVERSATIVE A N D RECUPERATIVE MEIR SHINITZKY
Department of Membrane Research, The Weizmann Institute of Science, Rehovot 76100 (lsrael) (Received S e p t e m b e r 14th, 1984)
Contents I.
Introduction
.............................................................................
II.
Physiological d e t e r m i n a n t s of m e m b r a n e microviscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Degree of u n s a t u r a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. S p h i n g o m y e l i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................................... D. P h o s p h a t i d y l e t h a n o l a m i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The p r o t e i n c o n t e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Physical effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
252 253 253 254 254 254 254
III. M e m b r a n e microviscosity of m a l i g n a n t t r a n s f o r m e d cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. H o m e o s t a s i s of m e m b r a n e fluidity - is it i m p a i r e d in t u m o u r cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. L e u k e m i a a n d l y m p h o m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. H e p a t o m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
254 255 255 256
IV. I m m u n o t h e r a p y aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The effect of lipid fluidity on m e m b r a n e functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. T u m o u r vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cholesteryl h e m i s u c c i n a t e - t r e a t e d cells (V l) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pressurized cells (Vn) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Shed i m m u n o g e n s (Viu) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Variabilities in vaccine p o t e n c y with t u m o u r type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Rectification of i m m u n e c o m p e t e n c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
256 256 257 257 258 258 259 260
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. Introduction Biological processes are confined by membranes to separate compartments in order to miniA b b r e v i a t i o n s : TAA, t u m o u r associated antigens; M H C , m a j o r h i s t o c o m p a t i b i l i t y complex.
0 3 0 4 - 4 1 9 X / 8 4 / $ 0 3 . 0 0 © 1984 Elsevier Science Publishers B.V.
251
mize random dissipation of energy and information. In membrane-sealed organelles, reactions are conducted by specialized macromolecules, while small substrates and products diffuse selectively through the membrane boundary. In addition, many reactions and signal transductions take place on the membrane surface itself where diffusion
252 processes in this two-dimensional array are nonrandom. In all these processes the membrane lipid fluidity plays a key regulatory role and is therefore of prime physiological significance [1]. This complex parameter, which combines both the order and the diffusion aspects of the lipid constituents [2,3], is readily altered by various metabolic pathways or by passive exchange with the surroundings. Virtually any cellular transformation is associated with some changes in lipid constituents and in almost all types of tumour cells changes in membrane lipid fluidity have in fact been reported. Yet, except for a few selected cases, the physiological implications of the changes in membrane fluidity are only poorly understood. The main objective of the following article is to present a general overview on the involvement of membrane fluidity in malignancy and its management, rather than reviewing the vast, and somewhat confusing, literature on this important issue. Updated summaries on the various physiological aspects of membrane fluidity can be found in a recent book [4], which also includes a comprehensive review on alteration of membrane lipid fluidity in tumour cells [5].
II. Physiological determinants of membrane microviscosity Data on lipid fluidity can, in principle, belong to either one of three distinct levels of resolution: microscopic, macroscopic or submacroscopic [3]. the microscopic level provides information on individual atoms or molecular segments at various depths of the lipid layer. The macroscopic level deals with lipid domains in bulk thermodynamic terms. The intermediate submacroscopic level provides low molecular resolution of lipid fluidity, generally expressed in macroscopic terms, and is evaluated with the aid of spectral probes. Parameters of lipid fluidity obtained by fluorescence depolarization, excimer formation and electron spin resonance belong to this category. Inasmuch as the physiological relevance of a certain dynamic parameter relates to its effect on the overt activity, the submacroscopic level of resolution of membrane fluidity complies well with the majority of membrane processes. Fluorescence polarization of a well-defined probe, such as DPH [6], may re-
solve some physiologically relevant parameters of membrane fluidity. According to the Fluid Mosaic Model [7] and the hydrodynamic elaboration of that model [8], a biological membrane is a two-dimensional fluid where the motions of individual molecules (lipids and proteins) are hampered by a viscous drag which should be expressible as a 'microviscosity'. However, in recent years it has become clear that there are several parameters relevant for a proper description of membrane fluidity or the physical state of the lipid bilayer [2,9,10]. The measured steady-state fluorescence polarization (P value) has been shown to contain two of these parameters: the angular range and the rate-contribution of the hindering of wobbling motion of the lipid chains [2,9]. The range parameter, which is the dominant one in biological membranes, can be translated in a lipid order parameter. The term 'microviscosity' is used here to express the combination of both the angular range (order) and the rotational rate of lipid-chain motion. Since it is not yet established how the actual lateral viscosity (relevant for translational mobility of membrane proteins) can be expressed in the present 'microviscosity', a quantification in macroscopic viscosity units (poises) is not always appropriate. Therefore 'microviscosity' is here expressed in the measured P values (0 < P < 0.5 being the limits for extremely 'fluid' and extremely ' rigid', respectively). The natural modulators of lipid fluidity can be divided into chemical modulators and physical effectors. The main modulators are (a) the cholesterol level - presented as cholesterol/ phospholipids, (b) the degree of unsaturation of the phospholipid acyl chains, (c) the level of s p h i n g o m y e l i n - generally presented as sphingomyelin/lecithin, and (d) the level of membrane proteins - presented as protein/lipid. The stationary levels of these modulators in a biological membrane can change in response to a regulatory signal or stress. The change can be effected either by passive exchange of one of the chemical modulators with the external medium (e.g., serum) or by membrane biogenesis, both of which processes reach a new steady-state within hours or days. The physical effectors of lipid fluidity are temperature, pressure, pH, membrane potential and Ca z+, and their effect is practically instantaneous.
253 For most physiological functions, the submacroscopic parameters of lipid fluidity are most relevant. This is mostly reflected in the fact that modulation of a membrane function by alterations of lipid composition corresponds to similar changes in microviscosity, irrespective of the type of lipid change, and with only little dependence on its specific microscopic details. Physiological restoration of an impaired membrane microviscosity proceeds mostly by changes in either cholesterol level or the composition of phospholipid acyl chains, with the possibility of mutual interchange [11,12]. The physiological relevance of the submacroscopic approach to membrane lipid fluidity is further demonstrated by the fact that a proper physiological function is maintained at a certain range of microviscosity, while this can - in principle - be attained by any combination of chemical or physical factors. For example, in response to changes in temperature (e.g., in poikilotherms or during hybernation) the level of cholesterol or the degree of phospholipid unsaturation changes to the levels required for restoration of the apparent lipid microviscosity [13-15]. Thus, the change in temperature is compensated for by changes in chemical composition, which are obviously different from the microscopic point of view. Similar compensatory mechanisms on a submacroscopic level operate when lipid fluidization induced by narcotics (e.g., alcohol) is reversed by hydrostatic pressure to restore normal function [16]. Also, when the biosynthesis of one of the chemical modulators (e.g., cholesterol) is impaired, it can be compensated for by another chemical modulator (degree of unsaturation) [11]. In some specific membrane functions, however, microscopic details play, nevertheless, an important role. This is especially pertinent to cases where the functional unit, e.g., a membrane-associated enzyme, is activated by a specific interaction with a lipid, which itself can also act as a fluidity modulator. In such cases, the effect on lipid fluidity induced by this lipid is only partially relevant. The important chemical and physical modulators of membrane lipid fluidity are discussed in some detail in the following.
11.4. Cholesterol Under physiological conditions, cholesterol is the main membrane rigidifier in eukaryotes [1]. Its effect is to increase the microviscosity, which simultaneously increases the degree of order in the lipid domain. Up to a certain level, cholesterol is more or less evenly distributed between the phospholipids and the mole index of cholesterol/ phospholipids may serve as a good qualitative parameter for correlation with the overt lipid microviscosity [17]. At abnormally high cholesterol levels (cholesterol/phospholipid > 2) a sharp transition in cholesterol organization, presumably to segregated domains, takes place, where further addition of cholesterol only slightly affects the apparent membrane microviscosity [18]. The weak and nonspecific association of cholesterol with phospholipids enables it to freely exchange between various phospholipid pools. This process of cholesterol translocation is largely determined by the cholesterol/phospholipid ratio of the participating pools and is especially pertinent to cholesterol exchange between the membranes of blood cells and the serum lipoproteins [17-19]. Metabolic changes in cholesterol level operate, on the one hand, via intraceUular degradation or synthesis and, on the other hand, by the formation or hydrolysis of cholesterol esters. Esterification and de-esterification of cholesterol provide an efficient preservation of a constant cholesterol/ phospholipid ratio, which in turn helps in maintaining a constant level of membrane fluidity [20].
liB. Degree of unsaturation When a c& double bond is introduced into a saturated phospholipid acyl chain, it induces a marked increase in the specific volume which causes membrane fluidization. A single double bond has the greatest effect on membrane fluidization, while a second bond has a much less fluidizing effect and, at higher degrees of unsaturation, the effect becomes progressively less pronounced [21]. Special intracellular enzymes ('desaturases') can regulate the number of double bonds and length of the phospholipid acyl chains [22]. Exchange processes, either spontaneous or with the aid of
254 special protein carriers [23], can also induce a net change in the overall degree of unsaturation.
HC. Sphingomyelin. The two most abundant phosphorylcholine phospholipids, phosphatidylcholine and sphingomyelin, are highly dissimilar in physicochemical properties [24]. Natural sphingomyelin and phosphatidylcholine are at about the extreme edges of contribution to rigidification (sphingomyelin) or fluidization (phosphatidylcholine). Besides imparting rigidity on lipid layers, sphingomyelin can also act as a coupler of the two lipid monolayers [25], and can form separate domains [24], Changes in lipid fluidity due to changes in sphingomyelin content can be presented in terms of changes in phosphatidylcholine-to-sphingomyelin mole ratio.
liD. Phosphatidylethanolamine The primary amine headgroup of phosphatidylethanolamine renders the ability to form hydrogen bonds with an adjacent phosphate group which imparts an increase in lipid microviscosity [1]. The methylating enzymes which reside in biological membranes and can sequentially methylate phosphatidylethanolamine to phosphatidylcholine, with a net increase in lipid fluidity, presumably play an important role in lipid fluidization which follows signal transduction by hormones, neurotransmitters or mitogens [26].
HE. The protein content Proteins, in general, are incompressible and in membranes they act as bulky rigid isles installed in a fluid lipid matrix. In essence, the effect of membrane proteins on lipid dynamics is similar to that of cholesterol [27]. Both rigidify and increase the order in fluid lipid domains and act conversely below the lipid phase transition.
IIF. Physical effectors Among the physical parameters which can affect the membrane lipid fluidity, temperature and hydrostatic pressure are of direct physiological and practical relevance. Temperature, which is the
most fundamental, and most versatile tool in thermodynamics, simultaneously affects all components of any dynamic system. In a biological membrane, changes in temperature will affect not only the lipid microviscosity but also the tertiary and quaternary structure of the membrane proteins, the structure and dynamics of the boundary water layer as well as the kinetic constants of any physical or chemical process. The overt change in a certain function with temperature is therefore a complex resultant of a series of dynamic changes that can even counteract each other. Focussing the effect of temperature on a selected process can be done only if the process has a particularly high energy of activation (e.g., lipid phase transition). Temperature, despite being so easy to handle, is therefore a physical parameter which with most complex systems can yield only qualitative information. Unlike temperature, pressure is highly selective, since it operates exclusively through changes in free volume. Therefore only compressible components yield to pressure changes and, in biological tissues subjected to pressure below 1500 atmospheres, these are the membrane lipid layer and the quaternary structure of protein assemblies. Lipid regions in the fluid phase have a relatively high free volume and are highly compressible. Upon imposition of pressure the free volume is reduced, the lipids are condensed and the microviscosity and order increase [28,29]. The change in volume is predominantly due to change in the free area per lipid (reviewed in Ref. 10). Under extreme pressures the lipids can even be transformed into the solid phase [30]. Assemblies of protein subunits tend to dissociate under pressure, since the aggregated form includes 'dead spaces' and therefore has a higher net volume than the sum of the separated units [31]. In isolated membranes the effect of hydrostatic pressure is almost exclusively confined to an increase in the lipid density (microviscosity). In intact cells the disintegration of microtubules and microfilaments also contributes to the overt effect of pressure [32]. II1. Membrane microviscosity of malignant transformed cells Normal cells at their stationary state possess a defined membrane fluidity which maintains proper
255 function [1,33]. The membrane microviscosity of proliferative cells, on the other hand, can be markedly affected by the cell density, the cell cycle, the lipid metabolism, as well as by the external tissue or fluid [34]. Nevertheless, the general important trend within a certain cell type is that the proliferative activity (i.e., tumourigenic virulence) increases with membrane fluidity. The actual correspondence between membrane fluidity and tumourigenicity is not clear, though intuitive ideas can be readily offered. Because of the above reasons it is difficult to clearly evaluate the inherent membrane fluidity of most malignant cells as their environmental parameters and physiological status should be taken into account. This is presumably the main cause for the frequent disagreements in the literature as to the actual microviscosity values of tumour cells [5]. From the data in the literature [5] it appears that, with a few exceptions, tumour cells from solid tissues (e.g., hepatoma) have a lower membrane fluidity than their normal analogues, while tumours of flowing cells (e.g., leukemia) have a higher membrane fluidity than their normal analogues. The membrane microviscosity of leukemia, lymphoma and hepatoma are currently the best characterized of these and are briefly discussed below. I l i A . Homeostasis of membrane fluidity impaired in tumour cells?
is it
The continuous encounters of normal tissue cells with a variety of nutrients and chemicals which can affect the membrane fluidity is buffered by a very effective homeostasis mechanism of membrane fluidity. This mechanism (termed a 'homeoviscous adaptation'; Ref. 13) operates mostly via alterations in the degree of unsaturation of the membrane phospholipids and in the level of cholesterol [12-15]. As a result, normal cells at their natural locus maintain their specific membrane lipid fluidity irrespective of the composition of the various chemical constituents in the external fluid. This fact re-emphasizes the relevance of the submacroscopic membrane fluidity as the principal physiological scale for the overt membrane activities. Malignant cells, despite being of clonal origin,
tend to acquire a typical membrane fluidity depending on the organ where they settle [4,34]. This fact indicates a greater susceptibility than normal cells to effects of external fluidity modulators. The origin of this trend is not clear and could be due to the fast cycle of the malignant cells which can override the response of the relatively slow fluidity homeostasis [14]. Alternatively, it is conceivable that malignant cells are impaired in their fluidity homeostasis enzymes, compared to normal cells, or even lack them altogether. These possibilities, however, have not yet been verified experimentally. Impaired resistance to incorporated fluidity modulators (e.g., from the serum) may be another reason for the disagreements appearing in the literature for the actual membrane microviscosity of various malignant cells. It might be that defined conditions and media, both in vitro and in vivo, could settle some of the ambiguities concerning the inherent values of microviscosity parameters of malignant cells. IIIB. Leukemia and lymphoma
Since the original observation by Shinitzky and Inbar in 1974 that the membrane of leukemic cells is more fluid than that of normal lymphocytes due to a lower cholesterol/phospholipid [35], many studies have followed in order to verify this important assertion [5]. Though, in general, most studies confirm it, there are a few disagreements which originate from the fact that data on membrane fluidity and cholesterol level which are obtained with intact cells can be significantly different from those obtained with isolated plasma membranes, and that normal lymphocytes from different organs also differ in their membrane fluidity [5]. In addition, disseminated malignant cells tend to change their membrane fluidity in compliance with the host organ (section IliA), and therefore lymphoma cells grown in ascites, for example, can adopt a more fluid membrane than in their original lymphoid gland. A proper comparison between membrane fluidity of normal and malignant lymphocytes should therefore be determined with plasma membranes isolated from cells of the relevant tissue. Taking these into account, the data presented in the literature [5]
256
indicate the following trends: leukemia is indeed associated with increase in plasma membrane lipid fluidity, mostly due to decrease in cholesterol/ phospholipid and to a lesser extent due to other factors. The virulence of the leukemic cells is reflected in the magnitude of the increase in membrane fluidity or decrease in cholesterol/ phospholipid. Lymphoma cells, unlike leukemic cells, have a similar or slightly lower membrane fluidity than normal gland lymphocytes, similar to the trend of tumours in solid tissue. Table I presents a summary of data accumulated by Van Blitterswijk [5] for plasma membranes of human leukemic cells. Except for the hairy cell leukemia, the figures agree well with the above conclusions.
IIIC. Hepatoma Along the malignancies of solid tissue hepatoma is currently the best-characterized for membrane composition and fluidity. Compared to normal hepatocytes, hepatoma cells, both slowly and rapidly dividing types, possess a lower plasmamembrane fluidity due to an increase in cholesterol/phospholipid [36-40]. It is interesting TABLE I M E M B R A N E MICROVISCOSITY (EXPRESSED AS T H E D E G R E E OF F L U O R E S C E N C E P O L A R I Z A T I O N OF T H E PROBE DPH AT 25°C) AND CHOLESTEROL/ P H O S P H O L I P I D ( C / P L ) O F ISOLATED PLASMA MEMBRANES F R O M N O R M A L A N D L E U K E M I C H U M A N LYMPHOCYTES Values were taken from data accumulated in Ref. 5 and a r e presented a s m e a n s + S . D . n . d . , not determined. The microviscosity (P0ph) is approx. 2 P / ( 0 . 4 6 - P ) [3,6].
Normal peripheral blood lymphocytes Chronic lymphatic leukemia Acute lymphoblastic leukemia Cronic myeloid leukemia Acute myeloid leukemia Hairy cell leukemia a Measured in whole cells.
Pdph at 25°C
C/PL
0.295 + 0.017
0.61
0.277 + 0.020
0.43
0.274 + 0.010
0.39
n.d.
0.46 a
n.d. 0.305 + 0.020
0.31 a 0.66
that the intracellular membranes of hepatoma cells are also of higher microviscosity and cholesterol/ phospholipid [36]. The reason for that lies, presumably, in the aberration in the intracellular cholesterol metabolism which is inherently of high turnover in hepatocytes.
IV. Immunotherapy aspects IVA. The effect of lipid fluidity on membrane functions Membrane processes can be grossly divided into those driven by metabolic energy (active processes) and those carried out through diffusion (passive processes). The latter are spontaneous and comply with the thermodynamics of protein diffusion and position where the membrane lipid fluidity is a critical determinant. The overt effect of the lipid fluidity on a passive membrane process is mainly mediated through the degree of accessibility of the functional sites and their rates of rotational and lateral diffusions [33,39]. Increase in the lipid microviscosity decreases the lipid free volume and in turn decreases the solubilization of the protein in the hydrocarbon core [40]. In parallel, the energy of interactions between the protein residues and the lipid chains decreases [41,42] and the net effect is a shift in the equilibrium position towards the aqueous domain on either side of the membrane (' vertical displacement') [40]. Changes in lipid microviscosity can alternatively be compensated for by protein-protein association through 'lateral displacement' which, in extreme cases, can create segregated domains of lipids and proteins [43,44]. Changes in lipid fluidity can thus expose or mask antigenic determinants [45,46], receptors [47,48] or transport carriers [39]. On the dynamic level the lipid fluidity operates as a frictional force which opposes lateral and rotational mobility of membrane proteins and thus determines their rate of collisional coupling to form active units, e.g., microaggregation of receptors, association between receptors and their second messenger or association between antigens with the major histocompatibility complex (MHC). In relation to tumour growth two important parameters may be associated with the membrane lipid fluidity: the expression of tumour associated
257
antigens (TAA) and the anti-tumour activity of the host's leukocytes. By in vitro or in vivo manipulation of the membrane lipid fluidity it is now possible to augment the expression of TAA and to increase the activity of immune leukocytes. This may open novel approaches to cancer immunotherapy, as discussed in the following.
IVB. Tumour vaccines It is now well established that animal tumour cells bear specific neo-antigens [49] and can thus provide a source for tumour vaccine preparations. However, untreated tumour cells are, in general, only weakly immunogenic and for most tumours it is impractical to use untreated-irradiated tumour cells or their m e m b r a n e s for active immunotherapy. Attempts to increase the immunogenicity of tumour cells by chemical coupling with strong antigens [50,51] or administration together with adjuvant [52,53] indicated only a moderate increase in efficacy. The reason why the immunogenicity of TAA in untreated cells is lower than expected is still unclear. It is plausible that the projection of TAA to the immune system is masked by other membrane proteins or that the TAA entities are separated from each other or from the MHC. These types of structural impairment are believed to reduce considerably the immunogenicity of membrane antigens [54]. If these are indeed the inherent reasons for the low immunogenicity of TAA on tumour cells, increase in the lipid microviscosity could provide an intriguing route for augmenting the expression of TAA. This possibility is based on the notion that upon increase in microviscosity the equilibrium position of diffusible membrane proteins is shifted vertically or laterally (' vertical displacement' or 'lateral displacement') [40-48]. The implications of this approach are far-reaching and open up a new avenue for cancer immunotherapy in human patients. The current state of the art of tumour vaccines prepared by this method is summarized below. The most effective and convenient means for increasing the membrane microviscosity of tumour cells are incorporation of cholesteryl hemisuccinate [39,55] or application of hydrostatic pressure [46]. The duration of the treatment by these
means is relatively short (about 1 h) and for most cells it does not affect viability. A schematic representation of these treatments and their putative effect on the expression of membrane-associated antigens is shown in Fig. 1. It indicates three possible types of vaccine products: cholesteryl hemisuccinate-treated cells (Vx), pressurized cells (VII) and the material shed from pressurized cells (V.I).
IVB1. Cholesteryl hemisuccinate-treated cells (1/1) Incorporation of cholesterol into tumour cell membranes requires relatively long treatment and somewhat unfavourable conditions. Alternatively, one can use charged natural cholesterol esters which can intercalate into the membrane lipid layer relatively fast and induce an increase in lipid microviscosity, similar, though not identical, to that obtained with cholesterol [39]. Among these, cholesteryl hemisuccinate, a natural fatty-acid-like cholesterol ester, is most suitable for manipulation of cell plasma membranes [39-47]. Marked aug-
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Fig. 1. Three types of tumour vaccine obtained by treatment of tumour cells with cholesteryl hemisuccinate, pressure or their combination and their proposed effect on antigen expression. Vl, cholesteryl hemisuccinate-treated cells, TAA are more exposed. Vn, pressure-treated cells, TAA are more exposed and aggregated, presumably around MHC. Vm, shed tumour immunogens, including soluble and aggregated TAA and other membrane proteins.
258
mentations in expression of membrane receptors [47-48], antigens [46] or transport channels [39] were accomplished by incorporation of cholesteryl hemisuccinate. In relation to augmentation of tumour immunogenicity, cholesteryl hemisuccinate presumably affects TAA similarly to other membrane antigens. The unequivocal immunogenic potency of cholesteryl hemisuccinate-treated irradiated tumour cells was demonstrated by prophylactic immunization against subsequent challenge with viable untreated tumour cells in a series of mouse and hamster syngeneic tumours [55-59]. Moreover, immunization with vaccine V I after tumour implantation, which is more relevant to human cancer, was also found to be very effective [56,57]. The results of immunotherapy with V~ of mice bearing Lewis lung carcinoma (3LL) are shown in Fig. 2 (From Ref. 57). These results indicate the great potency of V~ in eliminating lung metastasis of this tumour. The immunotherapeutic potential of V~ in human cancer has been tested by delayed-type hypersensitivity (skin reaction) with autologous tumour cells isolated from solid tumours immediately after
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Fig. 2. Suppression of pulmonary metastases in mice infected with 3LL tumour. The primary growth in the footpad was excised at a stage of metastatic dissemination and immunotherapy consisting of 107 cholesteryl hemisuccinate-enriched irradiated 3LL cells was given by intraperitoneal injection at the day of excision and after 6 days. In control animals untreated 3LL cells were used for immunotherapy. Metastasis was scored by lung weight, [125I]UdR incorporation and number of metastatic nodules, all indicating a substantial reduction of metastatic mass (from Ref. 57, by permission).
surgery, and then enriched with cholesteryl hemisuccinate and irradiated [59,60]. In about 70% of the cases the cholesteryl hemisuccinate-treated cells elicited a marked skin reaction, while untreated cells or irrelevant cells (e.g., lymphocytes) treated with cholesteryl hemisuccinate displayed almost no reaction [59,60]. Most of the patients who did not respond to V~ were in an advanced stage of the disease and were practically anergic [59,60]. IVB2. Pressurized cells (VIi) Hydrostatic pressure has a unique potential in manipulating membrane structure (see section II). In practice, pressures of up to about 1500 atmospheres can be well tolerated by most cells provided that the system is air-free. When subjected to a pressure of such a magnitude the membrane lipids are condensed to a level of about twice the microviscosity [46] and the microfilaments and microtubules are practically dissociated [32]. Under such conditions, where the lipid layer is rigidifled and diffusion barriers are released, most membrane proteins are freely displaced vertically or laterally, and some of them are even shed off [46]. The ensuing reshuffling of organization enables proteins with some affinity (e.g., TAA to TAA or TAA to MHC) to associate with each other and to form more-or-less stable complexes. Upon slow release of pressure the lipid microviscosity returns to its initial level and most of the cytoskeletal elements reassemble [32]. However, the initial organization of the membrane proteins is only partially recovered, partly because of the loss of shed material and partly because of the reimposed diffusional restrictions by the membrane cytoskeleton. These may bear important implications for the expression of TAA. Preliminary results from our laboratory [61] indicate that pressurized and then irradiated tumour cells of specific lines become strong tumour vaccines (VH) with a potency which can exceed that of Vl (see Table II). IVB3. Shed tumour immunogens (Vzzz) In line with vertical displacement notion, the increase in lipid microviscosity can exceed a critical level where proteins are shed off to the external medium [46]. Excessive rigidification can be brought about by massive incorporation of cholesteryl hemisuccinate [43] or by high hydro-
259 T A B L E II P O T E N C Y O F T H R E E T U M O R VACCINES (VI, V H A N D VxH) IN I N C R E A S I N G SURVIVAL IN F O U R H A M S T E R A N D MOUSE SYNGENEIC TUMOURS Animals were first treated twice (107 irradiated cells or their equivalent) and then challenged with viable tumour cells. The potency of immunization is qualitatively presented as mean day of 50% survival (D-50) and quantitatively by the relative immunity parameter. RIP = ((D-50 with treatment)/(D-50 without treatment))- 1. RIP > 0 indicates acquired immunity [55]. CHS, cholesteryl hemisuccinate. T u m o r cells
Hamster pancreatic carcinoma (CBP) Hamster pancreatic carcinoma (LSP-1) Mouse mastocytoma (P-815) Mouse T-lymphoma (EL-4)
No treatment
Untreated cells
CHS-treated cells (Vj)a
Pressurized cells (Vrl)
Supernatant of VII (Vil 0
D-50
RIP
D-50
RIP
D-50
D-50
D-50
RIP
45
0
44
= 0
78
n.d.
51
0.1
58
35
0
36
-- 0
38
n.d.
70
1.0
58
14-16
0
14-16
-- 0
30
1.0
n.d.
n.d.
16
0
20
22
0.38
36
0.25
RIP
0.7
--- 0
RIP
1.25
21
Ref.
57
0.31
61
a Other examples can be found in Ref. 55.
static pressure, or better by a combination of moderate cholesteryl hemisuccinate incorporation followed by pressure application [46]. The shed material is a mixture of single proteins which stay in solution through the support of bound phospholipids which are shed off concomitantly, and aggregated membrane proteins or membrane fragments. These can be separated and isolated by conventional centrifugation or gel filtration methods. Shedding of active membrane proteins by lipid 'hyperrigidification' surpasses the main hurdle in isolation of functional membrane proteins the obligatory use of detergents. Shed tumour immunogens (Vln) may be void of the M H C components and therefore could be specific to the kind of tumour from which they were prepared but not restricted for autologous use only. This bears some practical advantage over V~ or V n. In the current state of the art the actual potency of V m is hard to evaluate, as it has been tested in only a few cases (see Table II) and in a few skin tests in humans.
IVC. Variabilities in oaccine potency with tumour type
Inasmuch as the tumour vaccines described above are relatively new in the field of cancer research the comparison between them should await more data. From the few cases where such a comparison could be made (see Table II) it may only be concluded that each cell line may possess a specific type of potent vaccine. Indeed, for practical reasons it may be advisable to use a mixture of the three vaccines which may also evoke some profitable synergistic effects. Another attractive variation which has not yet been tested is vaccine preparation made of allogeneic cells. Here the tissue markers of the vaccine could act as additional adjuvant and may thus augment the potency of the vaccine. If this route proves successful, tumour vaccines could be prepared from banks of human tumour cells grown in tissue culture which offers obvious advantage over the use of autologous cells.
260
IVD. Rectification of immune competence by lipid treatment 6
At the advanced stages of cancer, when most body functions deteriorate, the immune competence declines and the patient approaches a state of anergy. At such a stage the immune reaction against tumour development is negligible and the application of tumour vaccine for skin test or therapy becomes futile. It is not yet clear what is the etiological factor responsible for the decline in responsiveness of the leukocytes in cancer patients. One possibility is that it relates to improper lipid composition of the cell plasma membranes, notably increase in cholesterol level. Such a situation prevails in aged individuals whose leukocytes are of high cholesterol/phospholipid and their immune reactivity is markedly suppressed [62]. The excess cholesterol in leukocytes from aged mice or men can be extraced by phosphatidylcholine [63] or better with a mixture designated 'Active Lipid', consisting of phosphatidylcholine, phosphatidylethanolamine and neutral lipids, all from egg yolk [64,65]. This treatment may be applied in vivo by hyperalimentation and was found to be effective even when given per os [64,65]. Old mice and men who received Active Lipid demonstrated a substantial recovery of their immune responsiveness [64,65]. It remains to be tested whether indeed Active Lipid can enhance leukocyte reactivity to specific, cancer-associated antigens and thus rectify the immune competence of cancer patients. This may add an essential therapeutic modality to cancer immunotherapy of advanced cases. When combined with tumour vaccine or other immunotherapeutic agents the overall beneficial effect of the integrated regimen could be synergistic, and may, one hopes, lead to tumour regression. References 1 Shinitzky, M. and Henkart, P. (1979) Int. Rev. Cytol. 60, 121-147 2 Van Blitterswijk, W.J., Van Hoeven, R.P. and Van der Meer, B.W. (1981) Biochim. Biophys. Acta 644, 323-332 3 Shinitzky, M. and Yuli, I. (1982) Chem. Phys. Lipids 30, 261-282 4 Shinitzky, M. (ed.) (1984) in Physiology of Membrane Fluidity, Vols. I and II, CRC Press, Boca Raton, FL 5 Van Blitterswijk, W.J. (1984) in Physiology Membrane
7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Fluidity (Shinitzky, M., ed.), Vol. 1I, pp. 53-84, CRC Press, Boca Raton, FL Shinitzky, M. and Barenholz, Y. (1978) Biochim. Biophys. Acta 515, 367-394 Singer, S.J. and Nicolson, G.L. (1972) Science 175, 720-731 Saffman, P.G. and Delbruch, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111-3113 Stubbs, C.D. and Smith, A.D. (1984) Biochim. Biophys. Acta 779, 89-137 Van der Meer, W. (1984) in Physiology of Membrane Fluidity (Shinitzky, M., ed.), Vol. I, pp. 53-72, CRC Press, Boca Raton, FL Freter, C.E., Laderson, R.C. and Silbert, D.V. (1979) J. Biol. Chem. 254, 6909-6916 Sinensky, M. (1980) J. Cell Biol. 85, 166-169 Sinensky, M. (1974) Proc. Natl. Acad. Sci. USA 71,522-525 Hazel, J.R. and Prosser, C.L. (1974) Physiol. Rev. 54, 620-677 Cossins, A.R. (1977) Biochim. Biophys. Acta 470, 395-411 Johnson, S.W. and Miller, K.W. (1970) Nature 228, 75-76 Cooper, R.A. (1978) J. Supramol. Struct. 8, 413-430 Cooper, R.A., Leslie, M.H., Fischkoff, S., Shinitzky, M. and Shattil, S. (1978) Biochemistry 17, 327-334 Hagerman, J.S. and Gould, R.G. (1951) Proc. Soc. Exp. Biol. Med. 78, 329-332 Arbogast, L.Y., Rothblat, G.H., Leslie, M.H. and Cooper, R.A. (1976) Proc. Natl. Acad. Sci. USA 73, 3680-3684 Hubbell, W.L. and McConnell, H.M. (1971) J. Am. Chem. Soc. 93, 314-326 Dickens, B.F. and Thompson, G.A., Jr. (1982) Biochemistry 21, 3604-3611 Wirtz, K.W.A. and Zilversmit, D.B. (1969) Biochim. Biophys. Acta 193, 105-116 Barenholz, Y. and Thompson, T.E. (1980) Biochim. Biophys. Acta 604, 129-158 Schmidt, C.F., Barenholz, Y., Huang, C. and Thompson, T.E. (1978) Nature 271, 775-777 Hirata, F. and Axelrod, J. (1980) Science 209, 1082-1090 Shinitzky, M. and Inbar, M. (1976) Biochim. Biophys. Acta 433, 133-149 Chong, P.L.G. and Cossins, A.R. (1983) Biochemistry 22, 409 -415 Flamm, M., Okubo, T., Turro, W.J. and Schachter, D. (1982) Biochim. Biophys. Acta 687, 101-104 Liu, N. and Kay, R.L. (1977) Biochemistry 16, 3484-3486 Paladini, A.A., Jr. and Weber, G. (1981) Biochemistry 20, 2587-2593 Salmon, E.D. (1975) J. Cell Biol. 65, 114-127 Shinitzky, M. (1979) Dev. Cell Biol. 4, 173-181 Rivnay, B., Gorelik, E., Segal, S. and Shinitzky, M. (1981) Invasion Metastasis 1, 99-110 Shinitzky, M. and Inbar, M. (1974) J. Mol. Biol. 85,603-615 Van Hoeven, R.P. and Emmelot, P. (1972) J. Membrane Biol. 9, 105-120 Emmelot, P., Bos, C., Van Hoeven, R.P. and Van Blitterswijk, W.J. (1974) Methods Enzymol. 31, 75-90 Van Hoeven, R.P., Van Blitterswijk, W.J. and Emmelot, P. (1979) Biochim. Biophys. Acta 551, 44-54
261 39 Yuli, I., Wilbrandt, W. and Shinitzky, M. (1981) Biochemistry 20, 4250-4256 40 Borochov, H. and Shinitzky, M. (1976) Proc. Natl. Acad. Sci. USA 73, 4526-4530 41 Gerson, D.F. (1982) Biophys. J. 37, 145-147 42 Maroudas, N.G. (1984) J. Theor. Biol. 107, 243-248 43 Wunderlich, F., Ronai, V., Speth, J., Seelig, J. and Blumen, A. (1975) Biochemistry 18, 251-255 44 Cherry, R.J., Muller, U., Holenstein, C. and Heyn, M.P. (1980) Biochim. Biophys. Acta 596, 145-151 45 Shinitzky, M. and Souroujon, M. (1979) Proc. Natl. Acad. Sci. USA 76, 4438-4440 46 Muller, C.P. and Shinitzky, M. (1981) Exp. Cell Res. 136, 53-62 47 Heron, D.S., Shinitzky, M., Hershkowitz, M. and Samuel, D. (1980) Proc. Natl. Acad. Sci. USA 77, 7463-74 48 Heron, D.S., Israeli, M. Hershkowitz, M., Samuel, D. and Shinitzky, M. (1981) Eur. J. Pharmacol. 72, 361-364 49 Shiku, H., Takahashi, T., Oettgen, H.F. and Old, L.J. (1976) J. Exp. Med. 144, 873-881 50 Lackmann, O.J. and Sikora, K. (1978) Nature 271,463 51 Austin, F. and Boone, C.W. (1979) Adv. Cancer Res. 30, 301-340 52 Goodnight, J.A., Jr. and Morton, D.L. (1978) Annu. Rev. Med. 29, 231-283 53 Baldwin, R.W. and Pimm, M.V. (1978) Adv. Cancer Res. 28. 91-147
54 Zinkernagel, R.M. and Doharty, P.C. (1979) Adv. Immunol. 27, 52-80 55 Shinitzky, M., Skornick, Y. and Haran-Ghera, N. (1979) Proc. Natl. Acad. Sci USA 76, 5313-5316 56 Shinitzky, M. and Skornick, Y. (1982) Dev. Cancer Res. 7, 61-68 57 Skornick, Y., Gorelik, E. and Sindelar, W.F. (1982) Surgical Forum 33, 396-398 58 Skornick, Y., Kurman, C.C. and Sindelar, W.F. (1984) Cancer Res. 44, 946-946 59 Skornick, Y., Danciger, E., Rozin, R.R. and Shinitzky, M. (1981) Cancer Immunol. Immunother. 11, 93-96 60 Skornick, Y., Dresdale, A.R. and Sindelar, W.F. (1983) J. Natl. Cancer Inst. 70, 465-467 61 Richert, L., Or, A., Klausner, J. and Shinitzky, M. submitted for publication 62 Rivnay, B., Bergman, B., Shinitzky, M. and Globerson, A. (1980) Mech. Age. Dev. 12, 119-126 63 Rivnay, B., Orbital-Harel, T., Shinitzky, M. and Globerson, A. (1983) Mech. Age. Dev. 23, 329-336 64 Lyte, M., Rabinovich, H., Klajman, A. and Globerson, A. (1982) lmmunobiology 163, 258 (abstract) 65 Shinitzky, M., Lyte, M., Heron, D.S. and Samuel, D. (1983) in Intervention in the Aging Process (Regelson, W., ed.), Vol. 3, pp. 175-187, Alan Liss Inc., New York
251
Elsevier
BBA 87123
M E M B R A N E FLUIDITY IN M A L I G N A N C Y ADVERSATIVE A N D RECUPERATIVE MEIR SHINITZKY
Department of Membrane Research, The Weizmann Institute of Science, Rehovot 76100 (lsrael) (Received S e p t e m b e r 14th, 1984)
Contents I.
Introduction
.............................................................................
II.
Physiological d e t e r m i n a n t s of m e m b r a n e microviscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Degree of u n s a t u r a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. S p h i n g o m y e l i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................................... D. P h o s p h a t i d y l e t h a n o l a m i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The p r o t e i n c o n t e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Physical effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
252 253 253 254 254 254 254
III. M e m b r a n e microviscosity of m a l i g n a n t t r a n s f o r m e d cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. H o m e o s t a s i s of m e m b r a n e fluidity - is it i m p a i r e d in t u m o u r cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. L e u k e m i a a n d l y m p h o m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. H e p a t o m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
254 255 255 256
IV. I m m u n o t h e r a p y aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The effect of lipid fluidity on m e m b r a n e functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. T u m o u r vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cholesteryl h e m i s u c c i n a t e - t r e a t e d cells (V l) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pressurized cells (Vn) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Shed i m m u n o g e n s (Viu) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Variabilities in vaccine p o t e n c y with t u m o u r type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Rectification of i m m u n e c o m p e t e n c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
256 256 257 257 258 258 259 260
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
260
I. Introduction Biological processes are confined by membranes to separate compartments in order to miniA b b r e v i a t i o n s : TAA, t u m o u r associated antigens; M H C , m a j o r h i s t o c o m p a t i b i l i t y complex.
0 3 0 4 - 4 1 9 X / 8 4 / $ 0 3 . 0 0 © 1984 Elsevier Science Publishers B.V.
251
mize random dissipation of energy and information. In membrane-sealed organelles, reactions are conducted by specialized macromolecules, while small substrates and products diffuse selectively through the membrane boundary. In addition, many reactions and signal transductions take place on the membrane surface itself where diffusion
252 processes in this two-dimensional array are nonrandom. In all these processes the membrane lipid fluidity plays a key regulatory role and is therefore of prime physiological significance [1]. This complex parameter, which combines both the order and the diffusion aspects of the lipid constituents [2,3], is readily altered by various metabolic pathways or by passive exchange with the surroundings. Virtually any cellular transformation is associated with some changes in lipid constituents and in almost all types of tumour cells changes in membrane lipid fluidity have in fact been reported. Yet, except for a few selected cases, the physiological implications of the changes in membrane fluidity are only poorly understood. The main objective of the following article is to present a general overview on the involvement of membrane fluidity in malignancy and its management, rather than reviewing the vast, and somewhat confusing, literature on this important issue. Updated summaries on the various physiological aspects of membrane fluidity can be found in a recent book [4], which also includes a comprehensive review on alteration of membrane lipid fluidity in tumour cells [5].
II. Physiological determinants of membrane microviscosity Data on lipid fluidity can, in principle, belong to either one of three distinct levels of resolution: microscopic, macroscopic or submacroscopic [3]. the microscopic level provides information on individual atoms or molecular segments at various depths of the lipid layer. The macroscopic level deals with lipid domains in bulk thermodynamic terms. The intermediate submacroscopic level provides low molecular resolution of lipid fluidity, generally expressed in macroscopic terms, and is evaluated with the aid of spectral probes. Parameters of lipid fluidity obtained by fluorescence depolarization, excimer formation and electron spin resonance belong to this category. Inasmuch as the physiological relevance of a certain dynamic parameter relates to its effect on the overt activity, the submacroscopic level of resolution of membrane fluidity complies well with the majority of membrane processes. Fluorescence polarization of a well-defined probe, such as DPH [6], may re-
solve some physiologically relevant parameters of membrane fluidity. According to the Fluid Mosaic Model [7] and the hydrodynamic elaboration of that model [8], a biological membrane is a two-dimensional fluid where the motions of individual molecules (lipids and proteins) are hampered by a viscous drag which should be expressible as a 'microviscosity'. However, in recent years it has become clear that there are several parameters relevant for a proper description of membrane fluidity or the physical state of the lipid bilayer [2,9,10]. The measured steady-state fluorescence polarization (P value) has been shown to contain two of these parameters: the angular range and the rate-contribution of the hindering of wobbling motion of the lipid chains [2,9]. The range parameter, which is the dominant one in biological membranes, can be translated in a lipid order parameter. The term 'microviscosity' is used here to express the combination of both the angular range (order) and the rotational rate of lipid-chain motion. Since it is not yet established how the actual lateral viscosity (relevant for translational mobility of membrane proteins) can be expressed in the present 'microviscosity', a quantification in macroscopic viscosity units (poises) is not always appropriate. Therefore 'microviscosity' is here expressed in the measured P values (0 < P < 0.5 being the limits for extremely 'fluid' and extremely ' rigid', respectively). The natural modulators of lipid fluidity can be divided into chemical modulators and physical effectors. The main modulators are (a) the cholesterol level - presented as cholesterol/ phospholipids, (b) the degree of unsaturation of the phospholipid acyl chains, (c) the level of s p h i n g o m y e l i n - generally presented as sphingomyelin/lecithin, and (d) the level of membrane proteins - presented as protein/lipid. The stationary levels of these modulators in a biological membrane can change in response to a regulatory signal or stress. The change can be effected either by passive exchange of one of the chemical modulators with the external medium (e.g., serum) or by membrane biogenesis, both of which processes reach a new steady-state within hours or days. The physical effectors of lipid fluidity are temperature, pressure, pH, membrane potential and Ca z+, and their effect is practically instantaneous.
253 For most physiological functions, the submacroscopic parameters of lipid fluidity are most relevant. This is mostly reflected in the fact that modulation of a membrane function by alterations of lipid composition corresponds to similar changes in microviscosity, irrespective of the type of lipid change, and with only little dependence on its specific microscopic details. Physiological restoration of an impaired membrane microviscosity proceeds mostly by changes in either cholesterol level or the composition of phospholipid acyl chains, with the possibility of mutual interchange [11,12]. The physiological relevance of the submacroscopic approach to membrane lipid fluidity is further demonstrated by the fact that a proper physiological function is maintained at a certain range of microviscosity, while this can - in principle - be attained by any combination of chemical or physical factors. For example, in response to changes in temperature (e.g., in poikilotherms or during hybernation) the level of cholesterol or the degree of phospholipid unsaturation changes to the levels required for restoration of the apparent lipid microviscosity [13-15]. Thus, the change in temperature is compensated for by changes in chemical composition, which are obviously different from the microscopic point of view. Similar compensatory mechanisms on a submacroscopic level operate when lipid fluidization induced by narcotics (e.g., alcohol) is reversed by hydrostatic pressure to restore normal function [16]. Also, when the biosynthesis of one of the chemical modulators (e.g., cholesterol) is impaired, it can be compensated for by another chemical modulator (degree of unsaturation) [11]. In some specific membrane functions, however, microscopic details play, nevertheless, an important role. This is especially pertinent to cases where the functional unit, e.g., a membrane-associated enzyme, is activated by a specific interaction with a lipid, which itself can also act as a fluidity modulator. In such cases, the effect on lipid fluidity induced by this lipid is only partially relevant. The important chemical and physical modulators of membrane lipid fluidity are discussed in some detail in the following.
11.4. Cholesterol Under physiological conditions, cholesterol is the main membrane rigidifier in eukaryotes [1]. Its effect is to increase the microviscosity, which simultaneously increases the degree of order in the lipid domain. Up to a certain level, cholesterol is more or less evenly distributed between the phospholipids and the mole index of cholesterol/ phospholipids may serve as a good qualitative parameter for correlation with the overt lipid microviscosity [17]. At abnormally high cholesterol levels (cholesterol/phospholipid > 2) a sharp transition in cholesterol organization, presumably to segregated domains, takes place, where further addition of cholesterol only slightly affects the apparent membrane microviscosity [18]. The weak and nonspecific association of cholesterol with phospholipids enables it to freely exchange between various phospholipid pools. This process of cholesterol translocation is largely determined by the cholesterol/phospholipid ratio of the participating pools and is especially pertinent to cholesterol exchange between the membranes of blood cells and the serum lipoproteins [17-19]. Metabolic changes in cholesterol level operate, on the one hand, via intraceUular degradation or synthesis and, on the other hand, by the formation or hydrolysis of cholesterol esters. Esterification and de-esterification of cholesterol provide an efficient preservation of a constant cholesterol/ phospholipid ratio, which in turn helps in maintaining a constant level of membrane fluidity [20].
liB. Degree of unsaturation When a c& double bond is introduced into a saturated phospholipid acyl chain, it induces a marked increase in the specific volume which causes membrane fluidization. A single double bond has the greatest effect on membrane fluidization, while a second bond has a much less fluidizing effect and, at higher degrees of unsaturation, the effect becomes progressively less pronounced [21]. Special intracellular enzymes ('desaturases') can regulate the number of double bonds and length of the phospholipid acyl chains [22]. Exchange processes, either spontaneous or with the aid of
254 special protein carriers [23], can also induce a net change in the overall degree of unsaturation.
HC. Sphingomyelin. The two most abundant phosphorylcholine phospholipids, phosphatidylcholine and sphingomyelin, are highly dissimilar in physicochemical properties [24]. Natural sphingomyelin and phosphatidylcholine are at about the extreme edges of contribution to rigidification (sphingomyelin) or fluidization (phosphatidylcholine). Besides imparting rigidity on lipid layers, sphingomyelin can also act as a coupler of the two lipid monolayers [25], and can form separate domains [24], Changes in lipid fluidity due to changes in sphingomyelin content can be presented in terms of changes in phosphatidylcholine-to-sphingomyelin mole ratio.
liD. Phosphatidylethanolamine The primary amine headgroup of phosphatidylethanolamine renders the ability to form hydrogen bonds with an adjacent phosphate group which imparts an increase in lipid microviscosity [1]. The methylating enzymes which reside in biological membranes and can sequentially methylate phosphatidylethanolamine to phosphatidylcholine, with a net increase in lipid fluidity, presumably play an important role in lipid fluidization which follows signal transduction by hormones, neurotransmitters or mitogens [26].
HE. The protein content Proteins, in general, are incompressible and in membranes they act as bulky rigid isles installed in a fluid lipid matrix. In essence, the effect of membrane proteins on lipid dynamics is similar to that of cholesterol [27]. Both rigidify and increase the order in fluid lipid domains and act conversely below the lipid phase transition.
IIF. Physical effectors Among the physical parameters which can affect the membrane lipid fluidity, temperature and hydrostatic pressure are of direct physiological and practical relevance. Temperature, which is the
most fundamental, and most versatile tool in thermodynamics, simultaneously affects all components of any dynamic system. In a biological membrane, changes in temperature will affect not only the lipid microviscosity but also the tertiary and quaternary structure of the membrane proteins, the structure and dynamics of the boundary water layer as well as the kinetic constants of any physical or chemical process. The overt change in a certain function with temperature is therefore a complex resultant of a series of dynamic changes that can even counteract each other. Focussing the effect of temperature on a selected process can be done only if the process has a particularly high energy of activation (e.g., lipid phase transition). Temperature, despite being so easy to handle, is therefore a physical parameter which with most complex systems can yield only qualitative information. Unlike temperature, pressure is highly selective, since it operates exclusively through changes in free volume. Therefore only compressible components yield to pressure changes and, in biological tissues subjected to pressure below 1500 atmospheres, these are the membrane lipid layer and the quaternary structure of protein assemblies. Lipid regions in the fluid phase have a relatively high free volume and are highly compressible. Upon imposition of pressure the free volume is reduced, the lipids are condensed and the microviscosity and order increase [28,29]. The change in volume is predominantly due to change in the free area per lipid (reviewed in Ref. 10). Under extreme pressures the lipids can even be transformed into the solid phase [30]. Assemblies of protein subunits tend to dissociate under pressure, since the aggregated form includes 'dead spaces' and therefore has a higher net volume than the sum of the separated units [31]. In isolated membranes the effect of hydrostatic pressure is almost exclusively confined to an increase in the lipid density (microviscosity). In intact cells the disintegration of microtubules and microfilaments also contributes to the overt effect of pressure [32]. II1. Membrane microviscosity of malignant transformed cells Normal cells at their stationary state possess a defined membrane fluidity which maintains proper
255 function [1,33]. The membrane microviscosity of proliferative cells, on the other hand, can be markedly affected by the cell density, the cell cycle, the lipid metabolism, as well as by the external tissue or fluid [34]. Nevertheless, the general important trend within a certain cell type is that the proliferative activity (i.e., tumourigenic virulence) increases with membrane fluidity. The actual correspondence between membrane fluidity and tumourigenicity is not clear, though intuitive ideas can be readily offered. Because of the above reasons it is difficult to clearly evaluate the inherent membrane fluidity of most malignant cells as their environmental parameters and physiological status should be taken into account. This is presumably the main cause for the frequent disagreements in the literature as to the actual microviscosity values of tumour cells [5]. From the data in the literature [5] it appears that, with a few exceptions, tumour cells from solid tissues (e.g., hepatoma) have a lower membrane fluidity than their normal analogues, while tumours of flowing cells (e.g., leukemia) have a higher membrane fluidity than their normal analogues. The membrane microviscosity of leukemia, lymphoma and hepatoma are currently the best characterized of these and are briefly discussed below. I l i A . Homeostasis of membrane fluidity impaired in tumour cells?
is it
The continuous encounters of normal tissue cells with a variety of nutrients and chemicals which can affect the membrane fluidity is buffered by a very effective homeostasis mechanism of membrane fluidity. This mechanism (termed a 'homeoviscous adaptation'; Ref. 13) operates mostly via alterations in the degree of unsaturation of the membrane phospholipids and in the level of cholesterol [12-15]. As a result, normal cells at their natural locus maintain their specific membrane lipid fluidity irrespective of the composition of the various chemical constituents in the external fluid. This fact re-emphasizes the relevance of the submacroscopic membrane fluidity as the principal physiological scale for the overt membrane activities. Malignant cells, despite being of clonal origin,
tend to acquire a typical membrane fluidity depending on the organ where they settle [4,34]. This fact indicates a greater susceptibility than normal cells to effects of external fluidity modulators. The origin of this trend is not clear and could be due to the fast cycle of the malignant cells which can override the response of the relatively slow fluidity homeostasis [14]. Alternatively, it is conceivable that malignant cells are impaired in their fluidity homeostasis enzymes, compared to normal cells, or even lack them altogether. These possibilities, however, have not yet been verified experimentally. Impaired resistance to incorporated fluidity modulators (e.g., from the serum) may be another reason for the disagreements appearing in the literature for the actual membrane microviscosity of various malignant cells. It might be that defined conditions and media, both in vitro and in vivo, could settle some of the ambiguities concerning the inherent values of microviscosity parameters of malignant cells. IIIB. Leukemia and lymphoma
Since the original observation by Shinitzky and Inbar in 1974 that the membrane of leukemic cells is more fluid than that of normal lymphocytes due to a lower cholesterol/phospholipid [35], many studies have followed in order to verify this important assertion [5]. Though, in general, most studies confirm it, there are a few disagreements which originate from the fact that data on membrane fluidity and cholesterol level which are obtained with intact cells can be significantly different from those obtained with isolated plasma membranes, and that normal lymphocytes from different organs also differ in their membrane fluidity [5]. In addition, disseminated malignant cells tend to change their membrane fluidity in compliance with the host organ (section IliA), and therefore lymphoma cells grown in ascites, for example, can adopt a more fluid membrane than in their original lymphoid gland. A proper comparison between membrane fluidity of normal and malignant lymphocytes should therefore be determined with plasma membranes isolated from cells of the relevant tissue. Taking these into account, the data presented in the literature [5]
256
indicate the following trends: leukemia is indeed associated with increase in plasma membrane lipid fluidity, mostly due to decrease in cholesterol/ phospholipid and to a lesser extent due to other factors. The virulence of the leukemic cells is reflected in the magnitude of the increase in membrane fluidity or decrease in cholesterol/ phospholipid. Lymphoma cells, unlike leukemic cells, have a similar or slightly lower membrane fluidity than normal gland lymphocytes, similar to the trend of tumours in solid tissue. Table I presents a summary of data accumulated by Van Blitterswijk [5] for plasma membranes of human leukemic cells. Except for the hairy cell leukemia, the figures agree well with the above conclusions.
IIIC. Hepatoma Along the malignancies of solid tissue hepatoma is currently the best-characterized for membrane composition and fluidity. Compared to normal hepatocytes, hepatoma cells, both slowly and rapidly dividing types, possess a lower plasmamembrane fluidity due to an increase in cholesterol/phospholipid [36-40]. It is interesting TABLE I M E M B R A N E MICROVISCOSITY (EXPRESSED AS T H E D E G R E E OF F L U O R E S C E N C E P O L A R I Z A T I O N OF T H E PROBE DPH AT 25°C) AND CHOLESTEROL/ P H O S P H O L I P I D ( C / P L ) O F ISOLATED PLASMA MEMBRANES F R O M N O R M A L A N D L E U K E M I C H U M A N LYMPHOCYTES Values were taken from data accumulated in Ref. 5 and a r e presented a s m e a n s + S . D . n . d . , not determined. The microviscosity (P0ph) is approx. 2 P / ( 0 . 4 6 - P ) [3,6].
Normal peripheral blood lymphocytes Chronic lymphatic leukemia Acute lymphoblastic leukemia Cronic myeloid leukemia Acute myeloid leukemia Hairy cell leukemia a Measured in whole cells.
Pdph at 25°C
C/PL
0.295 + 0.017
0.61
0.277 + 0.020
0.43
0.274 + 0.010
0.39
n.d.
0.46 a
n.d. 0.305 + 0.020
0.31 a 0.66
that the intracellular membranes of hepatoma cells are also of higher microviscosity and cholesterol/ phospholipid [36]. The reason for that lies, presumably, in the aberration in the intracellular cholesterol metabolism which is inherently of high turnover in hepatocytes.
IV. Immunotherapy aspects IVA. The effect of lipid fluidity on membrane functions Membrane processes can be grossly divided into those driven by metabolic energy (active processes) and those carried out through diffusion (passive processes). The latter are spontaneous and comply with the thermodynamics of protein diffusion and position where the membrane lipid fluidity is a critical determinant. The overt effect of the lipid fluidity on a passive membrane process is mainly mediated through the degree of accessibility of the functional sites and their rates of rotational and lateral diffusions [33,39]. Increase in the lipid microviscosity decreases the lipid free volume and in turn decreases the solubilization of the protein in the hydrocarbon core [40]. In parallel, the energy of interactions between the protein residues and the lipid chains decreases [41,42] and the net effect is a shift in the equilibrium position towards the aqueous domain on either side of the membrane (' vertical displacement') [40]. Changes in lipid microviscosity can alternatively be compensated for by protein-protein association through 'lateral displacement' which, in extreme cases, can create segregated domains of lipids and proteins [43,44]. Changes in lipid fluidity can thus expose or mask antigenic determinants [45,46], receptors [47,48] or transport carriers [39]. On the dynamic level the lipid fluidity operates as a frictional force which opposes lateral and rotational mobility of membrane proteins and thus determines their rate of collisional coupling to form active units, e.g., microaggregation of receptors, association between receptors and their second messenger or association between antigens with the major histocompatibility complex (MHC). In relation to tumour growth two important parameters may be associated with the membrane lipid fluidity: the expression of tumour associated
257
antigens (TAA) and the anti-tumour activity of the host's leukocytes. By in vitro or in vivo manipulation of the membrane lipid fluidity it is now possible to augment the expression of TAA and to increase the activity of immune leukocytes. This may open novel approaches to cancer immunotherapy, as discussed in the following.
IVB. Tumour vaccines It is now well established that animal tumour cells bear specific neo-antigens [49] and can thus provide a source for tumour vaccine preparations. However, untreated tumour cells are, in general, only weakly immunogenic and for most tumours it is impractical to use untreated-irradiated tumour cells or their m e m b r a n e s for active immunotherapy. Attempts to increase the immunogenicity of tumour cells by chemical coupling with strong antigens [50,51] or administration together with adjuvant [52,53] indicated only a moderate increase in efficacy. The reason why the immunogenicity of TAA in untreated cells is lower than expected is still unclear. It is plausible that the projection of TAA to the immune system is masked by other membrane proteins or that the TAA entities are separated from each other or from the MHC. These types of structural impairment are believed to reduce considerably the immunogenicity of membrane antigens [54]. If these are indeed the inherent reasons for the low immunogenicity of TAA on tumour cells, increase in the lipid microviscosity could provide an intriguing route for augmenting the expression of TAA. This possibility is based on the notion that upon increase in microviscosity the equilibrium position of diffusible membrane proteins is shifted vertically or laterally (' vertical displacement' or 'lateral displacement') [40-48]. The implications of this approach are far-reaching and open up a new avenue for cancer immunotherapy in human patients. The current state of the art of tumour vaccines prepared by this method is summarized below. The most effective and convenient means for increasing the membrane microviscosity of tumour cells are incorporation of cholesteryl hemisuccinate [39,55] or application of hydrostatic pressure [46]. The duration of the treatment by these
means is relatively short (about 1 h) and for most cells it does not affect viability. A schematic representation of these treatments and their putative effect on the expression of membrane-associated antigens is shown in Fig. 1. It indicates three possible types of vaccine products: cholesteryl hemisuccinate-treated cells (Vx), pressurized cells (VII) and the material shed from pressurized cells (V.I).
IVB1. Cholesteryl hemisuccinate-treated cells (1/1) Incorporation of cholesterol into tumour cell membranes requires relatively long treatment and somewhat unfavourable conditions. Alternatively, one can use charged natural cholesterol esters which can intercalate into the membrane lipid layer relatively fast and induce an increase in lipid microviscosity, similar, though not identical, to that obtained with cholesterol [39]. Among these, cholesteryl hemisuccinate, a natural fatty-acid-like cholesterol ester, is most suitable for manipulation of cell plasma membranes [39-47]. Marked aug-
.'..'-:'..:'..5"..-..
,~
%, ~,~%
?°~°%°~":'
o 2 o :...-...::. ° ° o° o ':#i.
o
o
oo
o
° °o° Vnv
~-TAA
~
-MHC
Fig. 1. Three types of tumour vaccine obtained by treatment of tumour cells with cholesteryl hemisuccinate, pressure or their combination and their proposed effect on antigen expression. Vl, cholesteryl hemisuccinate-treated cells, TAA are more exposed. Vn, pressure-treated cells, TAA are more exposed and aggregated, presumably around MHC. Vm, shed tumour immunogens, including soluble and aggregated TAA and other membrane proteins.
258
mentations in expression of membrane receptors [47-48], antigens [46] or transport channels [39] were accomplished by incorporation of cholesteryl hemisuccinate. In relation to augmentation of tumour immunogenicity, cholesteryl hemisuccinate presumably affects TAA similarly to other membrane antigens. The unequivocal immunogenic potency of cholesteryl hemisuccinate-treated irradiated tumour cells was demonstrated by prophylactic immunization against subsequent challenge with viable untreated tumour cells in a series of mouse and hamster syngeneic tumours [55-59]. Moreover, immunization with vaccine V I after tumour implantation, which is more relevant to human cancer, was also found to be very effective [56,57]. The results of immunotherapy with V~ of mice bearing Lewis lung carcinoma (3LL) are shown in Fig. 2 (From Ref. 57). These results indicate the great potency of V~ in eliminating lung metastasis of this tumour. The immunotherapeutic potential of V~ in human cancer has been tested by delayed-type hypersensitivity (skin reaction) with autologous tumour cells isolated from solid tumours immediately after
r. in|
t
U d R
T L u n
I
g
n c o
w
NN
o b
-
control Immunlzed
P
I
u 0
b
u
a
b
Fig. 2. Suppression of pulmonary metastases in mice infected with 3LL tumour. The primary growth in the footpad was excised at a stage of metastatic dissemination and immunotherapy consisting of 107 cholesteryl hemisuccinate-enriched irradiated 3LL cells was given by intraperitoneal injection at the day of excision and after 6 days. In control animals untreated 3LL cells were used for immunotherapy. Metastasis was scored by lung weight, [125I]UdR incorporation and number of metastatic nodules, all indicating a substantial reduction of metastatic mass (from Ref. 57, by permission).
surgery, and then enriched with cholesteryl hemisuccinate and irradiated [59,60]. In about 70% of the cases the cholesteryl hemisuccinate-treated cells elicited a marked skin reaction, while untreated cells or irrelevant cells (e.g., lymphocytes) treated with cholesteryl hemisuccinate displayed almost no reaction [59,60]. Most of the patients who did not respond to V~ were in an advanced stage of the disease and were practically anergic [59,60]. IVB2. Pressurized cells (VIi) Hydrostatic pressure has a unique potential in manipulating membrane structure (see section II). In practice, pressures of up to about 1500 atmospheres can be well tolerated by most cells provided that the system is air-free. When subjected to a pressure of such a magnitude the membrane lipids are condensed to a level of about twice the microviscosity [46] and the microfilaments and microtubules are practically dissociated [32]. Under such conditions, where the lipid layer is rigidifled and diffusion barriers are released, most membrane proteins are freely displaced vertically or laterally, and some of them are even shed off [46]. The ensuing reshuffling of organization enables proteins with some affinity (e.g., TAA to TAA or TAA to MHC) to associate with each other and to form more-or-less stable complexes. Upon slow release of pressure the lipid microviscosity returns to its initial level and most of the cytoskeletal elements reassemble [32]. However, the initial organization of the membrane proteins is only partially recovered, partly because of the loss of shed material and partly because of the reimposed diffusional restrictions by the membrane cytoskeleton. These may bear important implications for the expression of TAA. Preliminary results from our laboratory [61] indicate that pressurized and then irradiated tumour cells of specific lines become strong tumour vaccines (VH) with a potency which can exceed that of Vl (see Table II). IVB3. Shed tumour immunogens (Vzzz) In line with vertical displacement notion, the increase in lipid microviscosity can exceed a critical level where proteins are shed off to the external medium [46]. Excessive rigidification can be brought about by massive incorporation of cholesteryl hemisuccinate [43] or by high hydro-
259 T A B L E II P O T E N C Y O F T H R E E T U M O R VACCINES (VI, V H A N D VxH) IN I N C R E A S I N G SURVIVAL IN F O U R H A M S T E R A N D MOUSE SYNGENEIC TUMOURS Animals were first treated twice (107 irradiated cells or their equivalent) and then challenged with viable tumour cells. The potency of immunization is qualitatively presented as mean day of 50% survival (D-50) and quantitatively by the relative immunity parameter. RIP = ((D-50 with treatment)/(D-50 without treatment))- 1. RIP > 0 indicates acquired immunity [55]. CHS, cholesteryl hemisuccinate. T u m o r cells
Hamster pancreatic carcinoma (CBP) Hamster pancreatic carcinoma (LSP-1) Mouse mastocytoma (P-815) Mouse T-lymphoma (EL-4)
No treatment
Untreated cells
CHS-treated cells (Vj)a
Pressurized cells (Vrl)
Supernatant of VII (Vil 0
D-50
RIP
D-50
RIP
D-50
D-50
D-50
RIP
45
0
44
= 0
78
n.d.
51
0.1
58
35
0
36
-- 0
38
n.d.
70
1.0
58
14-16
0
14-16
-- 0
30
1.0
n.d.
n.d.
16
0
20
22
0.38
36
0.25
RIP
0.7
--- 0
RIP
1.25
21
Ref.
57
0.31
61
a Other examples can be found in Ref. 55.
static pressure, or better by a combination of moderate cholesteryl hemisuccinate incorporation followed by pressure application [46]. The shed material is a mixture of single proteins which stay in solution through the support of bound phospholipids which are shed off concomitantly, and aggregated membrane proteins or membrane fragments. These can be separated and isolated by conventional centrifugation or gel filtration methods. Shedding of active membrane proteins by lipid 'hyperrigidification' surpasses the main hurdle in isolation of functional membrane proteins the obligatory use of detergents. Shed tumour immunogens (Vln) may be void of the M H C components and therefore could be specific to the kind of tumour from which they were prepared but not restricted for autologous use only. This bears some practical advantage over V~ or V n. In the current state of the art the actual potency of V m is hard to evaluate, as it has been tested in only a few cases (see Table II) and in a few skin tests in humans.
IVC. Variabilities in oaccine potency with tumour type
Inasmuch as the tumour vaccines described above are relatively new in the field of cancer research the comparison between them should await more data. From the few cases where such a comparison could be made (see Table II) it may only be concluded that each cell line may possess a specific type of potent vaccine. Indeed, for practical reasons it may be advisable to use a mixture of the three vaccines which may also evoke some profitable synergistic effects. Another attractive variation which has not yet been tested is vaccine preparation made of allogeneic cells. Here the tissue markers of the vaccine could act as additional adjuvant and may thus augment the potency of the vaccine. If this route proves successful, tumour vaccines could be prepared from banks of human tumour cells grown in tissue culture which offers obvious advantage over the use of autologous cells.
260
IVD. Rectification of immune competence by lipid treatment 6
At the advanced stages of cancer, when most body functions deteriorate, the immune competence declines and the patient approaches a state of anergy. At such a stage the immune reaction against tumour development is negligible and the application of tumour vaccine for skin test or therapy becomes futile. It is not yet clear what is the etiological factor responsible for the decline in responsiveness of the leukocytes in cancer patients. One possibility is that it relates to improper lipid composition of the cell plasma membranes, notably increase in cholesterol level. Such a situation prevails in aged individuals whose leukocytes are of high cholesterol/phospholipid and their immune reactivity is markedly suppressed [62]. The excess cholesterol in leukocytes from aged mice or men can be extraced by phosphatidylcholine [63] or better with a mixture designated 'Active Lipid', consisting of phosphatidylcholine, phosphatidylethanolamine and neutral lipids, all from egg yolk [64,65]. This treatment may be applied in vivo by hyperalimentation and was found to be effective even when given per os [64,65]. Old mice and men who received Active Lipid demonstrated a substantial recovery of their immune responsiveness [64,65]. It remains to be tested whether indeed Active Lipid can enhance leukocyte reactivity to specific, cancer-associated antigens and thus rectify the immune competence of cancer patients. This may add an essential therapeutic modality to cancer immunotherapy of advanced cases. When combined with tumour vaccine or other immunotherapeutic agents the overall beneficial effect of the integrated regimen could be synergistic, and may, one hopes, lead to tumour regression. References 1 Shinitzky, M. and Henkart, P. (1979) Int. Rev. Cytol. 60, 121-147 2 Van Blitterswijk, W.J., Van Hoeven, R.P. and Van der Meer, B.W. (1981) Biochim. Biophys. Acta 644, 323-332 3 Shinitzky, M. and Yuli, I. (1982) Chem. Phys. Lipids 30, 261-282 4 Shinitzky, M. (ed.) (1984) in Physiology of Membrane Fluidity, Vols. I and II, CRC Press, Boca Raton, FL 5 Van Blitterswijk, W.J. (1984) in Physiology Membrane
7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
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261 39 Yuli, I., Wilbrandt, W. and Shinitzky, M. (1981) Biochemistry 20, 4250-4256 40 Borochov, H. and Shinitzky, M. (1976) Proc. Natl. Acad. Sci. USA 73, 4526-4530 41 Gerson, D.F. (1982) Biophys. J. 37, 145-147 42 Maroudas, N.G. (1984) J. Theor. Biol. 107, 243-248 43 Wunderlich, F., Ronai, V., Speth, J., Seelig, J. and Blumen, A. (1975) Biochemistry 18, 251-255 44 Cherry, R.J., Muller, U., Holenstein, C. and Heyn, M.P. (1980) Biochim. Biophys. Acta 596, 145-151 45 Shinitzky, M. and Souroujon, M. (1979) Proc. Natl. Acad. Sci. USA 76, 4438-4440 46 Muller, C.P. and Shinitzky, M. (1981) Exp. Cell Res. 136, 53-62 47 Heron, D.S., Shinitzky, M., Hershkowitz, M. and Samuel, D. (1980) Proc. Natl. Acad. Sci. USA 77, 7463-74 48 Heron, D.S., Israeli, M. Hershkowitz, M., Samuel, D. and Shinitzky, M. (1981) Eur. J. Pharmacol. 72, 361-364 49 Shiku, H., Takahashi, T., Oettgen, H.F. and Old, L.J. (1976) J. Exp. Med. 144, 873-881 50 Lackmann, O.J. and Sikora, K. (1978) Nature 271,463 51 Austin, F. and Boone, C.W. (1979) Adv. Cancer Res. 30, 301-340 52 Goodnight, J.A., Jr. and Morton, D.L. (1978) Annu. Rev. Med. 29, 231-283 53 Baldwin, R.W. and Pimm, M.V. (1978) Adv. Cancer Res. 28. 91-147
54 Zinkernagel, R.M. and Doharty, P.C. (1979) Adv. Immunol. 27, 52-80 55 Shinitzky, M., Skornick, Y. and Haran-Ghera, N. (1979) Proc. Natl. Acad. Sci USA 76, 5313-5316 56 Shinitzky, M. and Skornick, Y. (1982) Dev. Cancer Res. 7, 61-68 57 Skornick, Y., Gorelik, E. and Sindelar, W.F. (1982) Surgical Forum 33, 396-398 58 Skornick, Y., Kurman, C.C. and Sindelar, W.F. (1984) Cancer Res. 44, 946-946 59 Skornick, Y., Danciger, E., Rozin, R.R. and Shinitzky, M. (1981) Cancer Immunol. Immunother. 11, 93-96 60 Skornick, Y., Dresdale, A.R. and Sindelar, W.F. (1983) J. Natl. Cancer Inst. 70, 465-467 61 Richert, L., Or, A., Klausner, J. and Shinitzky, M. submitted for publication 62 Rivnay, B., Bergman, B., Shinitzky, M. and Globerson, A. (1980) Mech. Age. Dev. 12, 119-126 63 Rivnay, B., Orbital-Harel, T., Shinitzky, M. and Globerson, A. (1983) Mech. Age. Dev. 23, 329-336 64 Lyte, M., Rabinovich, H., Klajman, A. and Globerson, A. (1982) lmmunobiology 163, 258 (abstract) 65 Shinitzky, M., Lyte, M., Heron, D.S. and Samuel, D. (1983) in Intervention in the Aging Process (Regelson, W., ed.), Vol. 3, pp. 175-187, Alan Liss Inc., New York