(CANCER RESEARCH 49. 4842-4845. September I. I989|

Relationship between Cell Membrane Potential and Natural Killer Cell Cytolysis inHuman Hepatocellular Carcinoma Cells1

Douglas Stevenson, Richard Binggeli,2 Roy C. Weinstein, James G. Keck, Min Chan Lai, and Myron J. Tong

Liver Center, Huntington Memorial Hospital, Pasadena, California; and the Departments of Anatomy and Cell Biology and Otolaryngology, ííead.and Neck Surgery,University of Southern California School of Medicine, Los Angeles, California

ABSTRACT

One of the body's natural defense mechanisms against tumor cells is

lysis of the invading cell by cytotoxic T-cells and natural killer (NK)cells. Five human hepatocellular carcinoma cell lines were found to havedifferent sensitivities to killing by peripheral blood monocytes in a '( r

release assay. This killing was demonstrated to be due to NK cell lysis.Electrical recording measurements of the membrane potentials of thesefive cell lines showed different values for each line, all below valuesreported for normal hepatocytes. Correlation between mean cell membrane potential, and sensitivity to NK lysis, revealed an inverse relationship. In this study we demonstrate that the lower the mean membranepotential of a human hepatocellular carcinoma cell line, the more sensitiveit is to NK cell cytolysis. Cell surface positive potential did not correlatewith NK cytolysis and only a weak correlation was found between cellmembrane negative potential and cell surface positive potential betweencell lines.

INTRODUCTION

Human PBM' contain a number of effector cells to which

different lytic activities are ascribed. One of these cells thatmediates lysis is the NK cell. NK cytolysis of a target cell is amultistep mechanism involving, initially, interaction betweenrecognition structures on the NK cell, and membrane structureson the NK-sensitive target. Subsequent stages involve the lysisof the target cell by means of potent proteases (1, 2). It hasbeen demonstrated that many cancer cells, and a number ofvirus-infected cells are sensitive to NK cytolysis (3). It has alsobeen shown that some normal cells, including human fetalfibroblasts (4), fetal thymus, and bone marrow cells (5) are alsolysed by NK cells. Why some cells are sensitive to this lysis andothers are not, has been the subject of some speculation.

All living cells have a resting voltage across the plasmamembrane, the inside being negative with respect to the surrounding medium. The membrane potential of several tumorcell lines have been compared with their normal cell counterparts. Generally, proliferating cells, both normal and cancerous,have smaller membrane potentials than normal resting cells (6,7), which may have potentials as large as —¿�90mV (8). Rathepatocellular carcinomas have been reported to have lowermembrane potentials than normal rat hepatocytes (9).

The purpose of this study was to determine if a relationshipexists between the negative membrane potential of a target celland the ability of NK cells to destroy the target. We used severallines of human hepatocellular carcinomas and K562 cells tostudy this relationship.

Received 8/12/87; revised 5/1/89; accepted 6/8/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore he hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This study is supported by a Grant from the Kenneth J. Norris Foundation,and funds donated by Vadam Covington, Henry \V. Dodge and Donald McC'ombs.

2To »horn requests for reprints should be addressed, at Department ofAnatomy and Cell Biology. University of Southern California. School of Medicine, 1330 San Pablo Street, Los Angeles. CA 90033.

3The abbreviations used are: PBM, peripheral blood monocytes; NK. naturalkiller; HCC, human hepatocellular carcinoma; FBS. fetal bovine serum; PBS,phosphate buffered saline; IFNii, 7b2-interferon; BLM. bilayer lipid membrane.

MATERIALS AND METHODS

Cells and Culture Media. The five HCC cell lines used in the studywere the PLC/PRF/5 (10). HepG2, Hep3B (11), HA22T/VGH (12),and MAHLAVU (13). The cells were grown in Earle's minimumessential medium containing 10% heat-inactivated FBS and 10 mivi4-(2-hydroxyethyl)-l-piperazineethansulfonicacid buffer. The media alsocontained penicillin 100 lU/ml and streptomycin 100 iig/ml.

The human lymphoma cell line K562 was grown in RPMI 10% FBSand contained penicillin 100 IU/ml and streptomycin 100 ^g/ml.

Electrical Recording Methods. Cells were grown to confluence inCorning 60-mm tissue culture plates. During the electrical recordingsessions the cells were maintained in their dishes at 37°Cin a constant-

temperature hollowed aluminum heating block and were gassed withflowing, warm, humidified 95% air and 5% CO;. The cells were observed continuously under phase-contrast microscopy and were penetrated under visual monitoring with high-impedance glass micropipets(50-200 megohm impedance with tip potentials less than 5 mV filledwith 3 M KC1. The micropipets were led through silver-silver chlorideKCI-agar electrodes into a high impedance amplifier. A reference silver-silver chloride KCI-agar electrode was placed in the cell medium.Recording was done in a shock-mounted vibration-free Faraday cage.

Two electrical measurements were recorded: the membrane surfacepositive potential obtained when the electrode made contact with thecell membrane, and the cell negative membrane potential obtainedwhen the electrode penetrated the plasma membrane. Cell recordingshad to produce stable potentials for a minimum of 20 s while the cellshad to remain morphologically unchanged in order to be acceptable.

Cytotoxicity Assay. Cytotoxicity was measured using the "Cr release

assay technique of Brunner et al. (14). Briefly, target cells were incubated at a concentration of 1 x IO6 in 500 ¿Jof their appropriategrowth medium with 500 ¿jCiof MCr (International Chemical Nuclear,Irvine, CA 92715). After incubation for l h at 37'C, the cells were

pelleted, washed, and resuspended in growth medium at a concentrationof 1 x IO5 cells/ml. The cells were dispersed in 100-^il aliquots inCorning 96-well round-bottom tissue culture plates. PBM cells wereseparated from heparinized blood on Ficoll-Paque (Pharmacia FineChemicals, Piscataway, NJ). The cells were washed, resuspended ingrowth medium and added in 100 ¿ilvolumes to the MCr-labeled targets

to give PBM/target ratios of 100:1, 50:1, 25:1, and 5:1. Backgroundwas determined by incubating target cells in 200 n\ growth medium,and total incorporation by incubating the target cells in 100 ¿¿1growthmedium and 100 ¿Uof 1% Triton X-100.

After incubation at 37°Cfor 3.5 h, the plates were centrifuged and

100 nl of supernatants from triplicate samples were counted in a gammacounter. Specific lysis was calculated using the formula.

% Specific lysis =Test counts - backgroundTotal counts —¿�background x 100

NK Cell Depletion. NKH-1A antigen is expressed on a subpopulationof peripheral blood large granular lymphocytes, including cells withnatural killer activity. This antigen is not expressed on B-cells, T-cells(T3+ cells), granulocytes, monocytes, and erythrocytes.

PBM were depleted of NK cells using monoclonal antibody NKH-1A (Coulter Immunology, Hialeah, FL) and complement. Before performing the MCr-release assay, 6 x IO6PBM were incubated with 120fi\ NKH-1 A monoclonal antibody and 120 //I of growth medium atroom temperature for 45 min. The cells were pelleted and washed with10 ml PBS, they were then resuspended in 1:2 dilution of rabbitcomplement (Cappel Worthington Biochemicals, Malvern, PA), ingrowth medium and incubated at 37°Cfor 1 h. After washing with 10

ml PBS to remove complement, the cells were resuspended, counted.

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MEMBRANE POTENTIAL AND NK CELL LYSIS

and added to the target cells in the appropriate ratios.Target cells were treated with valinomycin and KC1 as hyperpolar-

izing and depolarizing agents, respectively, to determine their effectson NK cytolysis. Target cells were also treated with interferon which isknown to protect them from NK lysis (16) to determine its effect onmembrane potential.

Valinomycin was purchased from Sigma Chemical Corporation, St.Louis, MO.

Recombinant human IFN« was purchased from Kirby-Warrick,Pharmaceuticals Limited, Mildenhall, Bury St. Edmunds, Suffolk, England.

RESULTS

When the membrane potentials of the five HCC cell lineswere measured, their mean values varied from a low of —¿�9.6mV to a high of —¿�27.7mV. Mean values for surface-positive

potentials showed little variation within these five cell lines, thelowest mean value obtained being 12.7 mV while the highestwas 18.9 mV. Table 1 shows the mean membrane and surfacepositive potentials and SEM values of the five HCC cell linesstudied. Data on K562 cells is given as a comparison.

The membrane potential of individual cells within an HCCcell population varied greatly. In the 95 Hep3B cells measured,membrane potentials varied from as low as —¿�lmV to as highas —¿�24mV. The majority of the cells, however, had membranepotentials between —¿�6and —¿�8mV. By comparison, in the 94PLC/PRF/5 cells, potentials as high as —¿�42mV were observed.

A distribution of membrane potentials within these two celllines is shown in Fig. 1, where the number of cells of a givenmembrane potential are plotted as a percentage of the totalnumber of cells recorded.

When membrane and surface positive potentials were examined on a cell-by-cell basis within cell lines, they were found tobe uncorrelated (e.g., correlation coefficient in Hep3B cells-0.05, correlation coefficient in PLC/PRF/5 cells -0.02).

The five HCC cell lines also showed differences in cytotox-icity when treated with PBM in a 3.5-h MCr-release assay.

Three of the HCC cell lines responded poorly to PBM cellchallenge with varying low levels of cytolysis, while two, theHep3B and HepG2 were more sensitive to cell lysis. Thesedifferences are illustrated in Fig. 2. Data on K562 cells areshown as a comparison.

Table I Mean negative membrane potentials and surface-positive potentials offive HCC cell lines

Cells were grown to confluency in Corning 60-mm tissue culture plates, andmaintained at 37°Cin a humidified 95% air, 5% CO? atmosphere during therecording session. A reference silver-silver chloride KC'1-agarelectrode was placed

in the cell medium. A high-impedance glass micropipet (50-200 megohm impedance with tip potentials less than 5 mV) fillwed with 3 m KCI was used to makecontact with the cell membrane to measure surface potential, then to penetratethe cell membrane to measure transmembrane potential. The micropipets wereled through a silver-silver chloride KCI-agar electrode into a high impedanceamplifier. Recordings were done in a shock-mounted vibration-free Faraday case,while the cells were observed continuously under phase contrast microscopy. Cellpotentials had to produce stable recordings for a minimum of 20 s while the cellsremained morphologically unchanged in order to be acceptable. Mean and standard error of the mean (SEM) values are shown. Values for K562 cells are shownas a comparison.

Membrane potential Surface positive

CelllineHep3BHepG2PLC/PRF/5MAHLAVUHA22T/VGHK562No.

ofindividualcellsrecorded9562941226640Mean-9.6-12.3-17.6-26.0-27.7-9.5SEM0.40.70.81.40.80.87No.

ofindividuacellsrecorded422130302510Mean

SEM12.715.718.913.115.9.1.3.4.0.525.7

2.71

ü2 20

fin H H HH2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 36 40 42

Membrane Potential (-mV)

Fig. 1. Membrane potential distribution among 94 Hep3B cells (D) and 95PLC/PRF/5 cells (Q).

ü 40

enae

K562 Hep3B Hep G2 PLC/PRF/S MAHLAVU HA22T/VGH

Fig. 2. Cytotoxicity of five HCC cell lines and K562 cells as measured byspecific "Cr-release in a 3.5-h assay. 1 x 10* target cells were incubated at 37"Cfor I h in 500 fil Earle's minimum essential medium (MEM) 10% FBS containing500 ><Ci"Cr. The cells were pelleted, washed twice, and resuspended in growthmedia. 100-fil aliquots containing 1 x IO4cells were dispersed in Corning 96-wellround-bottom tissue culture plates. PBM were separated from heparinized bloodby centrifuging over Ficoll-Paque. The PBM were pelleted, »ashed,and added totriplicate wells of target cells in 100-^1 aliquots to give PBM to target ratios of100:1.50:1, 25: Land 5:1. Target cells were incubated in 200^1 of Earlc's MEM

10% FBS to determine background and total incorporation was determined byincubating target cells in 100 ¿ilof Earle's MEM 10% FBS and 100 pi 1% TritonX-100. After incubation, the plates were centrifuged and 100 nl of the supernatantscounted in a gamma counter. The bar graphs for each cell line represent the meanvalue of three or more experiments. PBM/effector 100:1 (D). 50:1 (D). 25:1 (D).

Table 2 Effect ofNKH-IA monoclonal antibody on the abiliti- of PBM to IvseHepJB cells

Specific 5lCr release (%),

effector/target

Effector 100:1 50:1 25:1 12.5:1

PBMNKH-1A treated PBM

35.45.1

29.94.7

21.42.8

12.71.0

PBM were depleted of NK cells by treatment with NKH-1 A,a monoclonal antibody to NK cells (15) followed by complement. This resulted in almost complete elimination of targetcell cytotoxicity, suggesting that the NK cell was responsiblefor target lysis. Table 2 shows the percentage specific MCr-

release from Hep3B cells treated with PBM, or PBM whichhad been preincubated with NKH-1 A antibody and complement. When Hep3B cells were incubated with 100 units of IFN/ml overnight, prior to incubation with PBM, lysis of the cellsis drastically reduced. This result is consistent with the effectorbeing a NK cell, as Welsh et al. (16) showed that tumor target

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MEMBRANE POTENTIAL AND NK CELL LYSIS

cells treated with IFN, are protected against lysis by NK cells.Table 3 shows the specific 5'Cr release from IFNa treated and

untreated Hep3B cells.When the membrane potentials of the five HCC cell lines are

plotted against their cytotoxicity, an inverse relationship between these parameters is apparent (Fig. 3). The closer themean membrane potential approaches that of the theoreticalvalue of a normal resting cell (-36 mV) (17), the less cytotoxiceffect achieved by the NK cell. The HA22T/VGH with a meanmembrane potential of -27.7 mV was the least sensitive to NK

cell lysis, while the Hep3B with a mean membrane potential of—¿�9.6mV was the most sensitive. A regression line of the plotwould intersect the membrane potential ordinate well below the-36 mV theoretical membrane potential for cell division proposed by Binggeli and Weinstein (17). A statistical correlationof membrane potential and NK cytolysis is -0.88 for the five

HCC lines.On examining NK cell activity on a number of cell lines

established from PLC/PRF/5 nude mouse xenografts, one cellline was found to be more sensitive to NK lysis when comparedto the parent cell (19 versus 7%, respectively, at 100:1 effector/target ratio). Measurement of the membrane potential of thiscell line showed that the mean membrane potential was —¿�12.0mV as compared to —¿�17.6mV for the parent cell line.

In the experiment where we pretreated Hep3B cells withIFNa cell lysis decreased at effector/target ratio of 100:1, from21% for the untreated cells to 6.1% for the IFNa-treated cells.The mean membrane potential of the IFNa-treated cells increased to -22.7 mV as compared to -9.8 mV for the controlcells. Treatment of target cells with the hyperpolarizing agent,

Table 3 Effect ofinterferon pretreatmenl of target cells on NK kill andmembrane potential

Table 4 Effect of 10 ¡IMvalinomycin on NK kill and membrane potential ofHep3B cells

Specific 51Cr release (%),

effector/target

100:1 50:1 25:1 12.5:1m.p.(mV)

Hep3BIFN-treated Hep3B

21.06.1

16.82.9

13.82.8

10.12.7

-9.8-22.7

.aü 30

0 5 10 15 20 25 30

Membrane Potential (-mV)

Fig. 3. Relationship between membrane potential and NK cell activity. Thesix points on each line represent values for K562, Hep3B, HepG2, MAHLAVU,PLC/PRF/5 and HA22T/VGH cells, respectively (from left to right). Four PBMto target cell ratios are shown: 100:1 (O), 50:1 (A), 25:1 (D), 5:1 (V).

Specific !'Cr release,

effector/targetHep3B

Valinomycin-treatedHep3B100:152.6

28.650:143.921.325:132.414.55:110.13.6m.p.

(mV)-11.6

-28.6

Table 5 Effect of 50 min KCI on NK kill and membrane potential ofHA22T/VGH cells

Specific "Cr-release,

effector/target5

miviKC150 HIMKCI100:12.42.350:10.91.725:11.4 0.55:10.5 1.0m.p.

(mV)-26.8-15.5

valinomycin resulted in decreased NK cell killing. Table 4 showsthe effect of 10 UM valinomycin on NK kill and membranepotential of Hep3B cells. In each of these instances, a decreasein one of the measured parameters resulted in a correspondingincrease in the other. Treatment of cells with high externalconcentrations of KCI as depolarizing agent, however, had littleor no effect on NK kill, despite the fact that changes in membrane potentials were observed (Table 5).

DISCUSSION

The membrane potential of the five HCC cell lines are aillower than those reported for normal resting hepatocytes (6, 9,18-20). This is in agreement with the hypothesis that tumorcells have lower transmembrane potentials than normal restingcells (8, 17). Mourad et al. (9), showed that during rat hepato-carcinogenesis, membrane potentials of hepatocytes decreasedfrom -28 to -15 mV.

Several nonspecific properties of a tumor target cell havebeen implicated in their sensitivity to lysis by NK cells. Cellsurface hydrophobicity (21), sialic acid composition (22), gly-coprotein composition (22, 23), and cell membrane repairmechanism (24) have all been reported to affect NK cell cytolysis. This study suggests that the membrane potential of atarget cell plays a vital role in the ability of an NK cell to lysethe target. The higher the mean membrane potential of a targetcell line, the less kill achieved by NK cytolysis. From the datacorrelating cell lysis and membrane potential, we propose thatgiven a normal distribution of membrane potential within a cellpopulation, little or no lysis would occur if the mean membranepotential was greater than —¿�31mV. This value is below the—¿�36mV theoretical threshold of membrane potential for cell

division proposed by Binggeli and Weinstein (17) based on anextensive review of the literature on cell membrane potentials.

The high cytotoxicity of K562, Hep3B, and to a lesser extent,HepG2, could well be a reflection of the number of low membrane potential cells present in their cell populations. Possiblyif the membrane potential of a cell falls below a certain criticalvalue, irrespective of whether that cell is a virus infected cell,tumor cell, or embryonic cell, then that cell may become sensitive to NK lysis.

We found a significant reduction in the mean membranepotential of HA22T/VGH cells in the presence of 50 HIMKCI.If the KCI is removed and the cells washed, however, the meanmembrane potentials of the cells revert to their original valuein a very short time." For this reason, the "Cr-release assay was

done in the presence of 50 IHM KC1, and no difference in

* Unpublished data.

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MEMBRANE POTENTIAL AND NK CELL LYSIS

cytotoxicity was observed. This result could be explained if theexternal KC1 is also acting on the NK cell. DeCoursey et al.(25) and Cabalan and coworkers (26), described voltage-gatedpotassium channels in NK cells and proposed that the channelswere necessary for the lytic ability of the NK cell.

In the presence of 10 mivi valinomycin, the mean m.p. ofHep3B cells increased. When valinomycin is removed and thecells washed, it takes almost an hour for the cells to revert totheir original mean membrane potential.4 We were therefore

able to pretreat the target cells with valinomycin, wash, thenperform the NK assay, and at least have target cells with higherm.p. during the first hour of the 3.5-h *'Cr-release assay. Under

these conditions a reduction in NK kill was observed.The ability of an NK cell to lyse a target may be determined

by the flow of ions through voltage-gated channels in responseto the equilibrium current between the NK and target cell. Thiswould explain why NK cells have been shown to bind to targetcells with no resultant lysis (27). Many fetal or embryonic cellswhich are rapidly dividing during the mitotic cycle, depolarizetheir membrane potentials and are attacked by NK cells fromadults. NK cells from fetuses and neonates, however, haveseverely reduced cytolytic activity (28). Treatment of these NKcells with interleukin 2, a maturation factor for NK cells,increases their lytic ability to the adult normal range (29).Changes in membrane, resulting in membrane potentialchanges could explain this if NK cell membrane potential isalso involved in their killing ability. Changes in cell membranepotentials might also explain the findings of Dennert andcoworkers (30) who demonstrated that cell lines sensitive toNK lysis exhibited marked increases in cytolysis in the presenceof concanavalin A, while insensitive target cell lines respondwith only slight increases in lysis. It is possible that cells withlow membrane potential are more sensitive to change in membrane potential than cells with high membrane potential. Alternatively, high and low membrane potential cells may be equallysensitive to changes in potential. A small drop in potential in acell line with an abundance of low membrane potential cellswould drive a large number into the NK cell kill range. Asimilar change in cells with a high mean membrane potential,which have few cells close to this critical range, would result inlittle increase in target cell cytolysis.

Using isolated BLM from K562 cells Vassilev and coworkers(31) showed that NK cells act specifically with BLM inducingan increase in membrane conductance. This effect was morepronounced if the NK cells had been treated with interferon orsodium selenite. Under these conditions they observed breakdown of the BLM within 20 min. They postulated that pore-forming molecules (cytolysin, etc.) were inserted into the BLM.If this is the primary mechanism of NK action, one couldreadily see why differences in membrane potentials between theNK cell and the target cell would be important. The higher thedifferential, the more "flow" of pore-forming molecules from

NK cell to target. Further studies with other membrane potential modulating agents may contribute to our better understanding of the mechanisms of NK cytolysis.

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1989;49:4842-4845. Cancer Res   Douglas Stevenson, Richard Binggeli, Roy C. Weinstein, et al.   Killer Cell Cytolysis in Human Hepatocellular Carcinoma CellsRelationship between Cell Membrane Potential and Natural

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