Proc. Nati. Acad. Sci. USAVol. 83, pp. 6829-6833, September 1986Cell Biology

Loss of intercalated membrane particles by treatmentwith phorbols

(intramembranous particles/phorbol esters)

DOROTHEA ZUCKER-FRANKLIN AND ZEENAT F. NABIDepartment of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016

Communicated by Michael Heidelberger, May 20, 1986

ABSTRACT Because brief exposure to phorbol estersrenders normal cells vulnerable to deformation and cytolysis bylymphocytes, it was postulated that these tumor promotersmight cause a hitherto unrecognized physical alteration inmembrane architecture. To investigate this possibility, fourtissue culture cell lines (K-562 erythroleukemia cells, melano-ma cells, N1121 adult fibroblasts, and normal fetal fibroblasts)and three blood cell types (lymphocytes, monocytes, andplatelets) were subjected to freeze-fracture analysis before andafter brief treatment with phorbol myristate acetate. Phorbolmyristate acetate caused a 50% reduction of intramembranousparticles associated with the external leaflet (E face) of theplasma membrane of every cell except platelets. In contrast, nochange in size or number of intramembranous particles asso-ciated with the protoplasmic membrane leaflet (P face) wasevident. Since the platelet membrane is known to be turned"inside out," as regards the partition coefficient of the intra-membranous particles, the disparity between the results ob-tained with platelets and other cells may serve to determine thenature of intramembranous particles affected by phorbols.Also, since phorbols affect primarily glycolipids and/or glyco-proteins anchored in the external membrane leaflet, thesefindings may provide a useful tool for future exploration ofmembrane structure.

Numerous studies have been conducted on the effects oftumor-promoting phorbol esters on mammalian cells. Themost interesting and far-reaching observations have dealtwith the finding that these agents cause a protracted activa-tion of protein kinase C, presumably the basis for manifoldalterations in cell physiology (1-3). Morphologic studies ofcells treated with phorbols have been less numerous, perhapsbecause the observed changes have been largely nonspecific,i.e., degranulation, vacuolization, clumping, blebbing, en-hanced pinocytosis, and lateral redistribution of membraneglycoproteins (4-6). In our laboratory, phorbol myristateacetate (PMA) has been employed primarily to render theminority of tumor cells resistant to lysis by natural killer cellsvulnerable to attack (7, 8). Although the low concentrationsand short exposures used did not appear to alter the ultra-structure or proliferative capacity of the target cells per se,addition of natural killer cells caused massive conjugation,deformation, and emperipolesis. Moreover, PMA-treatedtarget cells became subject to lysis not only by natural killercells, but also by T8 lymphocytes, which as a rule, requireprior sensitization, i.e., antibody and complement, to be-come cytotoxic for tumor cells. This suggested that phorbolesters could have a major effect on the structural integrity ofplasma membranes that may not yet have been recognized.Freeze-fracture analyses were carried out on two tumor celllines, two normal cell lines, and three peripheral blood cell

types before and after incubation with PMA. In every type ofcell with the exception of platelets, PMA caused a remark-able reduction in intramembranous particles (IMP) associat-ed with the external leaflet (E face) of the plasma membranewhile the size and distribution of IMP of the protoplasmicleaflet (P face) were not affected. This observation is partic-ularly intriguing because it has been recognized in severallaboratories that the partition coefficient of the plateletmembrane IMP is the reverse of that in other cells, i.e., theplatelet plasma membrane appears to be turned "inside out"(9, 10). We mention this because the disparity in the PMAresponse of platelet IMP when compared with other cells mayprovide new insights into the biochemical makeup of thesestructures and membrane organization in general.

MATERIALS AND METHODSCells. Neoplastic cells were (i) K-562 erythroleukemia cells

(used routinely as a standard for natural killer cell cytolyticactivity) grown in suspension culture in RPMI 1640, supple-mented with 10% (vol/vol) heat-inactivated fetal calf serum;(ii) a human melanoma cell line (Rob) studied extensively byus and described in detail elsewhere (11). Normal cells were(iii) an adult fibroblast line (N1121), derived from normalhuman skin (and obtained from American Type CultureCollection); (iv) a fetal fibroblast line prepared in our labo-ratory from a 15-week-old human abortus as reported (7, 8).Freshly prepared blood cells consisted of purified (v) lym-phocytes, (vi) monocytes, and (vii) platelets, which wereobtained from heparinized peripheral blood of volunteers andwere isolated by routine procedures.Treatment with PMA and its Analogs. PMA and inactive

analogs of PMA, 4a-phorbol 12,13-didecanoate and 4p-phorbol (Sigma), were dissolved in 100% ethanol (2 mg/ml).Dilutions were made with RPMI to a final concentration of0.01%. Control cells were incubated in ethanol diluentwithout PMA. The cells were washed at least twice beforeincubation with the phorbols (200 ng/ml) in serum-free RPMIfor 1 hr at 37TC. The cells were washed again prior to fixationin 3% (vol/vol) glutaraldehyde in phosphate buffer, afterwhich they were processed for freeze-fracture or thin sectionelectron microscopy. Target cells consisted either of mela-noma cells or K-562 cells and were incubated with lympho-cytes in a ratio of 1:100 for 2 hr or overnight. Cytotoxicityassays are not described here because they were not relevantto the present study and have been reported (7, 8).

Freeze-fracture and Electron Microscopy. Following fixa-tion for a minimum of 2 hr, the cells were thoroughly washedand resuspended in 25% (vol/vol) glycerol for 2 hr at roomtemperature. The glycerinated cells were quick frozen withFreon 22 and further cooled in liquid N2- as described (10).Membrane cleavage was carried out in a Balzer high vacuum

Abbreviations: PMA, phorbol myristate acetate; IMP, intramem-branous particles; E face, external leaflet of the plasma membrane;P face, protoplasmic leaflet of the plasma membrane.

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6830 Cell Biology: Zucker-Franklin and Nabi

FIG. 1. (A) Melanoma cell treated with PMA in suspension, washed, and incubated with lymphocytes that adhered to the target cell,penetrated, and emperipolesed. N, nucleus of melanoma cell. (x2300.) (B) Melanoma cell from a sample cultured in a monolayer, exposed to

PMA for 1 hr, washed three times, after which lymphocytes were added in situ. The specimen was flat-embedded to show the remarkabledeformation of target cell membrane by effector cells. Note that a lymphocyte has invaginated the nucleus (arrow). (x 1200.)

freeze etch unit BAF-300 (Hudson, NH) in a vacuum of 10-6 90°, respectively. After thawing, the replicas were cleared bytorr (1 torr = 1.333 x 102 Pa) at -1000C. The cleaved surfaces soaking with Clorox overnight, followed by multiple washeswere shadowed with platinum and carbon at angles of 430 and in diluted Clorox, acetic acid, and distilled water, as de-

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FIG. 2. (A and B) Freeze-fracture replicas of the P faces of melanoma cell plasma membranes before (A) and after (B) treatment with PMA.(C and D) Replicas of E faces of melanoma cells from the same sample as A and B, before (C) and after (D) exposure to PMA, respectively.In D, large areas of membrane are devoid of particles. When comparing C with D, the impression may be gained that it is the smaller particlesthat have been lost. (x90,000.)

Proc. Natl. Acad. Sci. USA 83 (1986)

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Proc. Natl. Acad. Sci. USA 83 (1986) 6831

scribed (10). A Siemens Elmiskop 1A electron microscopewas used to view the replicas. All electron micrographs wereobtained by an uninformed observer at original magnificationof x 15,000 with an accelerating voltage of 60 kV. Membraneareas, which because of their relationship to the whole cellcould be clearly identified as belonging either to the P face orE face of the plasma membrane, were enlarged photograph-ically to a final magnification of x 150,000 to facilitatecounting of IMP. Each experiment was performed in tripli-cate. Particles of each suitable replica were counted in adouble blind manner by two individuals. The paired Student'st test was used to evaluate statistical significance. An aliquotof each specimen was also dehydrated and embedded inPoly/Bed 812 for thin sectioning. The sections were stainedwith uranyl acetate and lead citrate.

RESULTS

Morphology of Cells. When thin sections of PMA-treatedand untreated specimens were examined blindly, no obviousultrastructural difference was detected in any ofthe cells withthe exception of platelets. Changes in shape displayed byplatelets as a consequence of exposure to PMA have beenpublished (12). Despite the fact that no morphologicalchanges were seen when any of the other cells were treatedwith PMA, incubation of such cells with lymphocytes result-ed in a remarkable deformation of their surface membraneand even emperipolesis into their cytoplasm (Fig. 1 A and B).Lymphocytes did not interact with or change the shape ofuntreated control target cells. The phenomenon occurredbefore any lytic event became detectable morphologically orby isotope release from labeled cells. Fig. 1 is presented tolend significance to the freeze-fracture studies reportedbelow.

Freeze-fracture. Representative replicas of melanoma cellplasma membranes before and after exposure to PMA areshown in Fig. 2 A-D. There was no obvious change in thenumber or size of IMP associated with the P face. Asexpected, the E face of control melanoma cells had fewerIMP than the P face. The remarkable reduction of IMPassociated with the E face that followed exposure to PMAcame as a surprise and was readily noted, even on cursoryinspection. Illustrations of representative replicas of K-562cell membranes before and after exposure to PMA are shownin Fig. 3 A-D. A similar reduction of particles associated withthe E face of PMA-treated cells is apparent. Precise quanti-fication of the particles confirmed that PMA treatmentcaused roughly a 50% decrease in the number of E face-associated IMP of every cell with the exception of platelets(Table 1). (In the interests of space, no replicas of plateletmembranes have been illustrated because control and exper-imental samples were indistinguishable). It is noteworthy thatthe results obtained with the physiologically inactive phorbolanalogs 4a-phorbol 12,13-didecanoate and 4p-phorbol weresimilar to those obtained with PMA. The ethanol diluent hadno effect on IMP in the absence of the phorbols. Because ofthe heterogeneity in the size and irregularity in the circum-ference of the IMP, accurate size measurements of thepopulation of particles affected by PMA treatment have notyet been possible. However, on gross inspection of electronphotomicrographs by three uninformed observers, it wasconcluded that PMA treatment did not affect particle size.

DISCUSSION

Although the scientific literature is replete with reportsdetailing the effects of phorbol esters on mammalian cellmembranes, as far as we could ascertain, freeze-fracture

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temperature (18), low pH (19), and treatment with glycerol ordimethyl sulfoxide without pinor fixation (20). On the otherhand, such an important physiologic event as the "capping"

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6832 Cell Biology: Zucker-Franklin and Nabi

Table 1. Number of IMPs before and after PMA treatment

IMPs, no. per jZm2 of membrane

Cell type Control P face PMA P face Control E face PMA E face

Melanoma 1019.5 ± 20.2 1047.4 ± 22.0 310.1 ± 9.0 162.1 ± 7.8K-562 1229.7 ± 32.0 1177.2 ± 29.7 669.7 ± 29.4 310.7 ± 14.2N-1121 fibroblasts 770.0 ± 34.4 652.0 ± 16.4 454.0 ± 12.0 249.5 ± 13.1Fetal fibroblasts 524.5 ± 21.5 557.0 ± 27.6 368.8 ± 27.1 223.0 ± 29.3Lymphocytes 511.2 ± 37.0 574.1 ± 31.4 232.7 ± 22.5 123.5 ± 11.8Monocytes 982.6 ± 25.1 980.0 ± 30.0 456.5 ± 19.7 271.6 ± 14.0Platelets* 527.5 ± 8.6 553.4 ± 13.2 880.1 ± 13.0 882.1 ± 14.4

*It should be noted that human blood platelets are the only mammalian cells described to date in whichthe partition coefficient of the IMP is reversed, i.e., more IMP are associated with the E face than theP face ofthe plasma membrane. The statistical difference between the number ofIMP on P faces beforeand after treatment with PMA was not significant, whereas the difference ofIMP on the E faces beforeand after treatment with PMA was significant with a P value <0.001 in all instances.

phenomenon, which involves lateral relocation of peripheralmembrane components, or platelet aggregation, which isaccompanied by extreme membrane deformation, is notassociated with any changes in the number and distributionof the IMP (9, 10, 18). Nevertheless, it is generally held thatthe IMP represent structures ofproteinaceous nature that areintercalated into the hydrophobic matrix of the membranebilayer and either anchor or constitute transmembrane pro-teins that respond to cytoplasmic and/or extracellular stim-uli. The asymmetry of the IMP distribution in most mamma-lian cells, i.e., the fact that the P face has roughly twice asmany particles as the E face, is taken to reflect the dispositionof various glycoproteins, phospholipids, and other mem-brane components localized, respectively, in the external orcytoplasmic aspect ofthe membrane by other means (21, 22).The discrepancy of the results obtained with platelets couldbe attributable to a reversed "sidedness" of their plasmamembrane as reflected by the reversed partition coefficient ofthe IMP reported by several investigators (9, 10). In thisregard, it should be mentioned that the aminophospholipidslike phosphatidylserine and phosphatidylethanolamine arenot readily accessible from the outside of intact platelets (23).If short exposure to PMA were to affect primarily phospho-lipids or proteins in the outer lipid bilayer, a differentbehavior of platelet IMP is perhaps to be expected. Thequestion at hand, however, is how the loss of IMP from theE face of the cells following treatment with PMA is to beinterpreted. First, it should be reiterated that the loss appearsto be real. The number of particles on the P face did notchange nor was there an obvious increase in the size of theindividual particles. Thus, a movement of particles from theE to the P face was not likely. Since the phorbol esters arevery lipophilic and may partition among membrane lipids,changes in the physical properties of the lipid bilayer couldaffect the distribution and orientation of proteins therein. Itis, for instance, conceivable that a decreased membraneviscosity would lessen the stability of a molecule whosemajor portion extends to the exterior. Small peptides thatmay not traverse both bilayers would be most vulnerable toan increase in membrane fluidity of this nature. If thisassumption were correct, the nature of the glycoprotein orlipid lost from the membrane might differfrom cell to cell. Forinstance, the "free" portion of band 3 of the erythrocytemembrane that copurifies with lipid moieties (24) would be acandidate for such release from PMA-treated erythrocytes.This is subject to experimental verification. No loss ofglycoprotein bands was seen when membrane extracts ofPMA-treated cells were subjected to NaDodSO4/PAGE.However, a decrease in surface sialic acid has been noted byothers when lymphocytes were treated with PMA (5), and aloss of fibronectin occurs when fibroblasts are exposed tophorbol esters (25). Sialic acid and fibronectin are likely to be

peripheral membrane components. Indeed, in preliminaryexperiments with N-[3H]acetylmannosamine-labeled K-562and melanoma cells (26, 27), we have found that there is a60% reduction in surface sialic acid that neuraminidase canrelease after the cells were treated with PMA (unpublisheddata). Obviously, several approaches will have to be used toidentify the membrane moieties that have been lost. Thelabel-fracture method of Pinto da Silva (28) seems especiallyapplicable to this problem. Since the effect ofPMA is knownto be reversible, it will also be of interest to examine whetherthe physical state of the membrane that permits release ofsome of its components can be exploited for the insertion ofothers.

The authors thank Ms. Susan Dittmar for her unfaltering patiencein the tedious collection of the freeze-fracture data. This researchwas supported by Grants CA 34378 and AM 12274 from the NationalInstitutes of Health.

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