soaking with Clorox overnight, followed by multiple washes were shadowed with platinum and carbon at angles of430 and in diluted Clorox, acetic acid, and ...
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6829-6833, September 1986 Cell Biology
Loss of intercalated membrane particles by treatment with phorbols (intramembranous particles/phorbol esters)
DOROTHEA ZUCKER-FRANKLIN AND ZEENAT F. NABI Department 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 esters renders normal cells vulnerable to deformation and cytolysis by lymphocytes, it was postulated that these tumor promoters might cause a hitherto unrecognized physical alteration in membrane architecture. To investigate this possibility, four tissue culture cell lines (K-562 erythroleukemia cells, melanoma cells, N1121 adult fibroblasts, and normal fetal fibroblasts) and three blood cell types (lymphocytes, monocytes, and platelets) were subjected to freeze-fracture analysis before and after brief treatment with phorbol myristate acetate. Phorbol myristate acetate caused a 50% reduction of intramembranous particles associated with the external leaflet (E face) of the plasma membrane of every cell except platelets. In contrast, no change in size or number of intramembranous particles associated with the protoplasmic membrane leaflet (P face) was evident. Since the platelet membrane is known to be turned "inside out," as regards the partition coefficient of the intramembranous particles, the disparity between the results obtained with platelets and other cells may serve to determine the nature of intramembranous particles affected by phorbols. Also, since phorbols affect primarily glycolipids and/or glycoproteins anchored in the external membrane leaflet, these findings may provide a useful tool for future exploration of membrane structure.
types before and after incubation with PMA. In every type of cell with the exception of platelets, PMA caused a remarkable reduction in intramembranous particles (IMP) associated with the external leaflet (E face) of the plasma membrane while the size and distribution of IMP of the protoplasmic leaflet (P face) were not affected. This observation is particularly intriguing because it has been recognized in several laboratories that the partition coefficient of the platelet membrane IMP is the reverse of that in other cells, i.e., the platelet plasma membrane appears to be turned "inside out" (9, 10). We mention this because the disparity in the PMA response of platelet IMP when compared with other cells may provide new insights into the biochemical makeup of these structures and membrane organization in general.
Numerous studies have been conducted on the effects of tumor-promoting phorbol esters on mammalian cells. The most interesting and far-reaching observations have dealt with the finding that these agents cause a protracted activation of protein kinase C, presumably the basis for manifold alterations in cell physiology (1-3). Morphologic studies of cells treated with phorbols have been less numerous, perhaps because the observed changes have been largely nonspecific, i.e., degranulation, vacuolization, clumping, blebbing, enhanced pinocytosis, and lateral redistribution of membrane glycoproteins (4-6). In our laboratory, phorbol myristate acetate (PMA) has been employed primarily to render the minority of tumor cells resistant to lysis by natural killer cells vulnerable to attack (7, 8). Although the low concentrations and short exposures used did not appear to alter the ultrastructure or proliferative capacity of the target cells per se, addition of natural killer cells caused massive conjugation, deformation, and emperipolesis. Moreover, PMA-treated target cells became subject to lysis not only by natural killer cells, but also by T8 lymphocytes, which as a rule, require prior sensitization, i.e., antibody and complement, to become cytotoxic for tumor cells. This suggested that phorbol esters could have a major effect on the structural integrity of plasma membranes that may not yet have been recognized. Freeze-fracture analyses were carried out on two tumor cell lines, two normal cell lines, and three peripheral blood cell
MATERIALS AND METHODS Cells. Neoplastic cells were (i) K-562 erythroleukemia cells (used routinely as a standard for natural killer cell cytolytic activity) grown in suspension culture in RPMI 1640, supplemented with 10% (vol/vol) heat-inactivated fetal calf serum; (ii) a human melanoma cell line (Rob) studied extensively by us and described in detail elsewhere (11). Normal cells were (iii) an adult fibroblast line (N1121), derived from normal human skin (and obtained from American Type Culture Collection); (iv) a fetal fibroblast line prepared in our laboratory from a 15-week-old human abortus as reported (7, 8). Freshly prepared blood cells consisted of purified (v) lymphocytes, (vi) monocytes, and (vii) platelets, which were obtained from heparinized peripheral blood of volunteers and were isolated by routine procedures. Treatment with PMA and its Analogs. PMA and inactive analogs of PMA, 4a-phorbol 12,13-didecanoate and 4pphorbol (Sigma), were dissolved in 100% ethanol (2 mg/ml). Dilutions were made with RPMI to a final concentration of 0.01%. Control cells were incubated in ethanol diluent without PMA. The cells were washed at least twice before incubation with the phorbols (200 ng/ml) in serum-free RPMI for 1 hr at 37TC. The cells were washed again prior to fixation in 3% (vol/vol) glutaraldehyde in phosphate buffer, after which they were processed for freeze-fracture or thin section electron microscopy. Target cells consisted either of melanoma cells or K-562 cells and were incubated with lymphocytes in a ratio of 1:100 for 2 hr or overnight. Cytotoxicity assays are not described here because they were not relevant to the present study and have been reported (7, 8). Freeze-fracture and Electron Microscopy. Following fixation for a minimum of 2 hr, the cells were thoroughly washed and resuspended in 25% (vol/vol) glycerol for 2 hr at room temperature. The glycerinated cells were quick frozen with Freon 22 and further cooled in liquid N2- as described (10). Membrane cleavage was carried out in a Balzer high vacuum
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Abbreviations: PMA, phorbol myristate acetate; IMP, intramembranous particles; E face, external leaflet of the plasma membrane; P face, protoplasmic leaflet of the plasma membrane. 6829
6830
Proc. Natl. Acad. Sci. USA 83
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(1986)
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 remarkable deformation of target cell membrane by effector cells. Note that a lymphocyte has invaginated the nucleus (arrow). (x 1200.)
90°, respectively. After thawing, the replicas were cleared by soaking with Clorox overnight, followed by multiple washes in diluted Clorox, acetic acid, and distilled water, as de-
freeze etch unit BAF-300 (Hudson, NH) in a vacuum of 10-6 torr (1 torr = 1.333 x 102 Pa) at -1000C. The cleaved surfaces were shadowed with platinum and carbon at angles of 430 and
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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 particles that have been lost. (x90,000.)
Cell
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6831
Proc. Natl. Acad. Sci. USA 83 (1986)
scribed (10). A Siemens Elmiskop 1A electron microscope was used to view the replicas. All electron micrographs were obtained by an uninformed observer at original magnification of x 15,000 with an accelerating voltage of 60 kV. Membrane areas, which because of their relationship to the whole cell could be clearly identified as belonging either to the P face or E face of the plasma membrane, were enlarged photographically to a final magnification of x 150,000 to facilitate counting of IMP. Each experiment was performed in triplicate. Particles of each suitable replica were counted in a double blind manner by two individuals. The paired Student's t test was used to evaluate statistical significance. An aliquot of each specimen was also dehydrated and embedded in Poly/Bed 812 for thin sectioning. The sections were stained with uranyl acetate and lead citrate.
RESULTS Morphology of Cells. When thin sections of PMA-treated and untreated specimens were examined blindly, no obvious ultrastructural difference was detected in any of the cells with the exception of platelets. Changes in shape displayed by platelets as a consequence of exposure to PMA have been published (12). Despite the fact that no morphological changes were seen when any of the other cells were treated with PMA, incubation of such cells with lymphocytes resulted in a remarkable deformation of their surface membrane and even emperipolesis into their cytoplasm (Fig. 1 A and B). Lymphocytes did not interact with or change the shape of untreated control target cells. The phenomenon occurred before any lytic event became detectable morphologically or by isotope release from labeled cells. Fig. 1 is presented to lend significance to the freeze-fracture studies reported below. Freeze-fracture. Representative replicas of melanoma cell plasma membranes before and after exposure to PMA are shown in Fig. 2 A-D. There was no obvious change in the number or size of IMP associated with the P face. As expected, the E face of control melanoma cells had fewer IMP than the P face. The remarkable reduction of IMP associated with the E face that followed exposure to PMA came as a surprise and was readily noted, even on cursory inspection. Illustrations of representative replicas of K-562 cell membranes before and after exposure to PMA are shown in Fig. 3 A-D. A similar reduction of particles associated with the E face of PMA-treated cells is apparent. Precise quantification of the particles confirmed that PMA treatment caused roughly a 50% decrease in the number of E faceassociated IMP of every cell with the exception of platelets (Table 1). (In the interests of space, no replicas of platelet membranes have been illustrated because control and experimental samples were indistinguishable). It is noteworthy that the results obtained with the physiologically inactive phorbol analogs 4a-phorbol 12,13-didecanoate and 4p-phorbol were similar to those obtained with PMA. The ethanol diluent had no effect on IMP in the absence of the phorbols. Because of the heterogeneity in the size and irregularity in the circumference of the IMP, accurate size measurements of the population of particles affected by PMA treatment have not yet been possible. However, on gross inspection of electron photomicrographs by three uninformed observers, it was concluded that PMA treatment did not affect particle size.
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DISCUSSION Although the scientific literature is replete with reports detailing the effects of phorbol esters on mammalian cell membranes, as far as we could ascertain, freeze-fracture
temperature (18), low pH (19), and treatment with glycerol or dimethyl sulfoxide without pinor fixation (20). On the other hand, such an important physiologic event as the "capping"
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Proc. Natl. Acad. Sci. USA 83
(1986)
Table 1. Number of IMPs before and after PMA treatment IMPs, no. per jZm2 of membrane P Control E face PMA E face Cell type Control face PMA P face 1019.5 ± 20.2 310.1 ± 9.0 162.1 ± 7.8 Melanoma 1047.4 ± 22.0 669.7 ± 29.4 310.7 ± 14.2 1229.7 ± 32.0 1177.2 ± 29.7 K-562 770.0 ± 34.4 454.0 ± 12.0 249.5 ± 13.1 652.0 ± 16.4 N-1121 fibroblasts 368.8 ± 27.1 223.0 ± 29.3 524.5 ± 21.5 557.0 ± 27.6 Fetal fibroblasts 511.2 ± 37.0 232.7 ± 22.5 123.5 ± 11.8 574.1 ± 31.4 Lymphocytes 456.5 ± 19.7 271.6 ± 14.0 982.6 ± 25.1 980.0 ± 30.0 Monocytes 880.1 ± 13.0 882.1 ± 14.4 527.5 ± 8.6 553.4 ± 13.2 Platelets* *It should be noted that human blood platelets are the only mammalian cells described to date in which the partition coefficient of the IMP is reversed, i.e., more IMP are associated with the E face than the P face of the plasma membrane. The statistical difference between the number of IMP on P faces before and after treatment with PMA was not significant, whereas the difference of IMP on the E faces before and after treatment with PMA was significant with a P value
