[CANCER RESEARCH 40, 1830-1835, June 1980]0008-5472/80/0040-0000$02.00

Cellular Potentials of Normal and Cancerous Fibroblasts andHepatocytes

Richard Binggeli1 and Ivan L. Cameron

Department of Anatomy, University of Southern California, Los Angeles, California 90033 (R. B.], and Department of Anatomy, University of Texas Health ScienceCenter at San Antonio, San Antonio, Texas 78284 [I. L. C.]

ABSTRACT

Several lines of investigation point to differences in electricalproperties between normal and cancerous cells. Several tumorlines have low-resting membrane potentials. A few comparisonshave been made between normal and tumor cells within thesame tissue cell type. This study compares the cellular ortransmembrane potential of hepatocytes and fibroblasts in bothnormal and tumor cells.

High-impedance micropipets were used to record intracellularly in vivo in Buffalo rat hepatocytes and Morris 7777hepatoma cells, as well as A/J mouse corneal fibroblasts andpoorly differentiated fibrosarcoma cells.

Rat hepatocytes had a mean membrane potential of —37.1± 4.3(S.D.)mVcomparedto —19.8 ± 7.1 mVinthehepatoma

cells. Mouse corneal fibroblasts measured —42.5±5.4 mV,while cells of mouse fibrosarcoma were —14.3 ±5.4 mV. Themembrane potentials of the tumor cells were lower in bothinstances than in their normal counterpart (statistically significant at p = 0.001 for both tissue cell types).

This supports the notion that lower cellular or membranepotentials may play a significant role in the altered physiologyof the tumor cell.

INTRODUCTION

The notion that cancerous tissues or cells may be in someway electrically different from their normal counterpart extendsat least back to the work of Ambrose et a!. (2), who foundincreased electrophoretic mobility of cancerous kidney andliver cells, and Schaefer and Schanne (5), who described alower membrane potential in human cervical carcinoma thanthat found in any cell at that time.

Cone (9) hypothesizes that a lowered membrane potentialcauses increased proliferation rates of both normal and cancerous cells.

Although the cellular or transmembrane potential has beenrecorded in many normal and abnormal cells by Altman andKatz (1), relatively few studies have attempted to pair normaland abnormal cell types within the same tissue cell. Amongthem are Tokuoka and Morioka (19), who compared cells ofthe gastric mucosa with gastric cancer cells, Balitsky andShuba (3), who recorded from muscle and rhabdomyosarcoma,Limberger (12), who recorded from liver and hepatoma, andJamakosmanovic and Loewenstein (10), who recorded fromthyroid and thyroid cancer. In all instances, lower values werereported for the cancerous cells.

I To whom requests for reprints should be addressed, at the Department of

Anatomy, University of Southern California School of Medicine, 2025 ZonalAvenue, Los Angeles, Calif. 90033.

Received September i 9, 1979; accepted February 26, 1980.

Reports of resting membrane potential in fibroblasts varyfrom a low of —0.6mV (6) to intermediate values of —14.7 mV(4) and —16 mV (13) to higher values of —55mV (14) and— 70 to — 75 mV (1 8) in a variety of mouse, hamster, and

human cells, all recorded in tissue culture.Liver cell recordings in vitro have produced values around

—33mV (7, 8). Most in vivo recordings have produced measurements of around —51 mV in both rat and dog (5, 8, 11, 12,16).

This study investigates the resting cellular (transmembrane)potential of 2 matched cell types in vivo: the hepatocyte andone type of hepatoma; and the fibroblast and a poorly differentiated fibrosarcoma.

MATERIALS AND METHODS

Cell Types

Young adult Buffalo rats were used for studying both normalhepatocytes and Morris hepatoma 7777 cells. A/J mice wereused to study resting fibroblasts and a spontaneous and transplantable type of poorly differentiated fibrosarcoma.

Surgical Procedure

Hepatocytes. Each rat was anesthetized with 35 mg ofsodium pentobarbital per kg. For the normal liver recordings,the right upper peritoneal cavity was opened with both a mediansagittal incision and a transverse incision, and the animal wasplaced in a prone position on a constant-temperature aluminumplatform. The liver was moved to the surface, and the surrounding skin was weighted to stabilize the liver against respiratorymovements. A drip of 0.9% NaCIsolution was started to preventdrying. For the tumor recordings, the same anesthetic dosagewas used. Those animals with 2- to 4-cm-diameter tumors(hepatomas) on the posterior dorsal flank were used. Eachanimal was placed in a prone position. The overlying skin wasincised and retracted, and the tumor was cleansed of denseconnective tissue.

The inferior portion of the animal was stabilized with weightsto minimize movements.

Fibroblasts. For a normal populationof resting fibroblasts,the stromal layers of the mouse cornea were used. In order toapproach these cells, mice were given 60 mg of sodium pentobarbitol per kg, and the heads were immobilized using eitherweighted hemostats or stereotaxic head holders. The poorlydifferentiated fibrosarcomas were in the same position on themice as were the hepatomas, and they were approached in thesame manner. The tumors measured about 15 to 20 mm indiameter.

Recording. Intracellular recordingswere done with pulledglass micropipets filled with 3 M KCI. The pipets had tip

1830 CANCERRESEARCHVOL. 40

Research. on November 23, 2020. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

CeIlularPotentialsMean

cellularp0-tential

inNo. ofNo. ofNo.ofInvivo celltypemVS.D.SE.cellssitesanimalsRat

hepatocytes—37.i4.30.57083Rathepatoma—19.87.10.797102Mouse

corneal fibro —42.55.4i.03i73blastMouse

fibrosarcoma— 1 4.35.40.9331 i2

Membrane Potentials of Normal and Cancer Cells

resistances of from 75 to 150 megohms. These were mountedin a glass tube and bridged to silver-AgCI wires through KCIagar gel. The indifferent electrode was a 2-mm-diameter glasstube filled with KCI-agar gel which protruded slightly out theend; the tube led to a silver-AgCI wire which in turn led toground. This indifferent electrode was placed in the peritonealcavity on the partially opened abdomen or placed s.c. on thedorsum of the animal. The active electrode was led through thematching silver-AgCI-agar gel bridge to a high-impedance firststage of a BME MP-4 electrometer amplifier and from there toboth a Tektronix storage oscilloscope and a Grass D.C. penrecorder.

In the liver, parenchymal cells predominate over stromalelements by more than 9:1 ; in addition, the stromal cells,fibroblasts and endothelial cells, among others, are generallysmaller. Statistically, then, the chances of penetrating othercell types are relatively remote. In the case of the cornealstroma, the only cell type present is the fibroblast. Once thecorneal epithelial cells are passed with the electrode (easilydetected because of the short distance traversed and the rapidoscillations caused from quickly passing from one cell to another), there was a relatively large traverse as, successively,one stromal cell after another was encountered in a slowersequence.

The electrodes were advanced under direct stereomicroscopic observation using an hydraulic microdrive with 1 smresolution. Electrode resistance was frequently checked, andany broken or plugged electrodes were immediately replaced.

Even with restrictions on movement imposed by clamping,weights, or stereotaxic apparatus, some movements precludedlonger recordings. Still, a single cell recording had to be 5 to10 sec long, as a minimum criterion, to be accepted as valid.

In the tumors, which frequently had tough connective tissuecapsules and necrotic centers, care was taken to record fromdepths below the capsule but no deeper than 500 to 1000 @zmbelow the surface.

RESULTS

The results of the measurements of cellular or transmembrane potential in the 4 cell types are listed in Table 1. FromTable I , it can be clearly seen that within both cell types, thefibroblasts and hepatocytes, the membrane potentials of thecancerous cells are significantly lower than those of theirnormal counterparts. When tested statistically with Student's ttest, the difference is significant beyond the p = 0.001 level.

Charts 1 and 2 are frequency histograms of the recordedmembrane potential values that are normalized as percentagesof total cells recorded. In this way, even though there areunequal sample sizes, the distributions can be directly compared.

The distributions of normal and cancerous hepatocytes inChart I show some overlapping values with the hepatoma cellspresenting a broader (platykurtic) and larger range distribution.In the case of the fibroblasts shown in Chart 2, there is nooverlap in values with an exclusive bimodal distribution.

Figs. 1 to 4 are photomicrographs of the 4 types of tissuesused in the recordings. In Figs. 1 and 2, showing the liver andthe hepatoma, respectively, notice the cells in mitosis in thehepatoma. Figs. 3 and 4 show the entirely different organizationof fibroblasts in the normal cornea and in the fibrosarcoma. A

Table i

dividing cell (arrow) is seen in the fibrosarcoma. The cornealfibroblasts are grouped in extended layers arranged betweenthe fascicles of collagen of the corneal stroma. By contrast,the cells of the fibrosarcoma are far less organized, and theamount of collagen present is greatly reduced.

Charts 3 and 4 are polygrams of rat hepatocytes and rathepatoma and of mouse fibroblasts and fibrosarcoma, respectively. Charts 3 and 4 show records of mouse and rat liver cellsand fibroblast membrane potentials recorded in vivo. In Chart3 are samples of both normal rat liver hepatocytes and hepatoma cells. Numerous irregularities and movement artifacts canbe seen, indicating the major difficulty with in vivo recording.However, several cells achieve the criterion of holding a relatively steady potential for approximately 10 sec. In Chart 3A ofa record from a normal liver penetration, it should be notedthat, as the record moves from left to right, the electrode isadvancing over 200 @tminto the liver. With the cells as close asthey are, there is very little time spent in the extra cellularspace (the brief ‘‘spike'‘returns to zero potential). Also noticethe relative uniformity of the potential values as the electrodepasses from cell to cell. Chart 3B, taken from a record from ahepatoma, shows a tendency for the potentials to fade ratherquickly and for the period between successful penetrations tobe greater.

Chart 4A is a sample from a single penetration of a mousecornea showing 3 cell penetrations that were too brief to becounted (arrows), along with 4 that were sustained enough.These followed a period of oscillation during the penetration ofthe epithelium and Bowman's membrane. After recording from5 to 6 fibroblasts, the electrode would penetrate into theanterior chamber of the eye and stay at zero potential.

Chart 4B is a record taken from the superficial layers of afibrosarcoma in a mouse with 5 cell recordings with somemovement artifact and some changes in background noise.

In all of the records, the superimposed dark bar indicatesthe period in which the electrode has penetrated and recordedfrom one cell.

DISCUSSION

While there have been several reports in the past 2 decadesindicating differences in transmembrane potential between normal and cancer cells, they have been scattered and not presented in an interactive and integrative way. Since the development of a model correlating the membrane potential withmitogenic rate by Cone (9), there has not been an ‘‘adequatelycomprehensive, systematic data' ‘collection.

Schaefer and Schanne (15) demonstrated a difference inwhat they believed to be cancerous smooth muscle from thecervix from about —40mVin vivo to —15 mVin vitro. Limberger

JUNE 1980 1831

Research. on November 23, 2020. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

HFl brosarcoma

R. Binggeli and I. L. Cameron

(1 2) compared normal and cancerous liver cells and found

them to be —51mV and about —44mV, respectively. Jamakosmanovic and Loewenstein (10) found normal and cancerousthyroid cells to be —47mV and about —25mV, respectively,while Balitsky and Shuba (3) described normal and cancerousmuscle cells to be —89mV and —16 mV, respectively. Tokuokaand Morioka (19) found normal and cancerous gastric cells todiffer from —34mV to —24mV.

To this group, we add additional studies on 2 distinct celltypes, the fibroblast and the hepatocyte, for both normal andcancerous cells.

Since the first use of the Ling-Gerard electrode, there havebeen several technical innovations which have made recordingmore reliable and which explain some of the variation in theearlier literature. Most notable have been the development ofthe very-high-impedance amplifier and the technique of beveling micropipets. Both of these allow for a smaller, sharper

20

Cl)

@ 15

0

0C— 10

z0

electrode which enables an easier, gentler penetration intosmaller cells. Only the former was used in this study, a highimpedance amplifier; but it enabled the use of 75- to 150-megohm resistance electrodes. These smaller electrodes canbe used to penetrate larger numbers of cells and hold thepotentials longer for more accurate and reliable recordings.Even so, the main restraint in holding cells for a sufficient timewith these electrodes is the presence of movement, either fromthe respiratory or cardiac movements of the animal or secondarily of the recording surface, table top, or even the laboratorybuilding itself. Some of the values in the literature are artificiallylow for 2 probable causes: (a) the use of electrodes that aretoo large (2 to 30 megohms); and (b) mistaking the smallpotential shift which takes place immediately before penetration as the resting membrane potential. Still, some others, inorder to report the positive values seen in some of the literature,must be recording the positive shift that is often seen whiledimpling the membrane during in vitro recordings under directobservation.

Chart 2. Frequency histogram of recorded membrane potential values ofnormal and cancerous fibroblasts.

10 20

20

30 40

Normal Fibroblast

50

Cl) 15

0

I—100

z0

5.

[1Hepatoma[: NormalHepatocyte

M ILLIVOLT5N I LLIVO

Chart 1. Frequency histogram of recorded membrane potential values ofnormal and cancerous hepatocytes plotted as percentages of the total number ofcells in each category.

B

Chart 3. Polygraph records of normal rat hepatocytes, (A), and rat Morris 7777 hepatoma cells, (B). The time marker recorded 10-sec intervals. Bottom bars, totaltime elapsed inside a single cell; arrows, points where cells penetrated too briefly to be accepted.

B

Chart 4. Polygraph records of normal mouse corneal fibroblast, (A), and mouse fibrosarcoma, (B). Bottom bars, total time elapsed inside a single cell; arrows,points where cells penetrated too briefly to be accepted.

mV

— 40

A

mV

— 50

1832 CANCERRESEARCHVOL. 40

Research. on November 23, 2020. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

Membrane Potentials of Normal and Cancer Cells

In this study, animal movement most frequently terminatedthe recording of a single cell within at the most 1 or 2 mm and,more frequently, within 2 to 5 sec, so that several values afterpenetration had to be discarded because they did not hold for5 to 10 sec. Several potentials declined steadily after penetration, indicating a damaged membrane.

The mean value for the fibroblastsin this study was —42.5mV. This agrees well with the unpublishedobservations of—41.4 mV for quail fibroblasts and —33.6 mV for mousefibroblasts recorded in tissue culture cells in the laboratory ofA. L. Binggell, R. C. Weinstein, and E. C. Hughes. The regularityof the comeal recordings from penetration to penetration cor

relates well with the high degree of organization within thelayers of comeal stroma of this resting cell population.

Our result of —37.1mV for normal hepatocytes differs fromresults obtained in the literature, which average —51 mV.Interpretations include possible differences in the strain ofanimals;Limberger(12) noticeddifferences in the mean varying from —46mV to —62mV within the individuals in hissample. Another factor could be the almost universal use ofurethan as the anesthetic in the other studies versus the use ofpentobarbital here. Two less probable explanations include thepossibilities that either cooling or mild ischemia caused by theexternalization and immobilization of the liver could have produced the lower values in our study. Schanne and Coraboeuf(16) and Biedermann (5) both point to the great sensitivity ofthe liver to anoxia or ischemia, demonstrating a significant dropin potential within 10 to 20 mm after circulatory interruption.

The histologicaldifferences in organization of the normalhepatocytes and those from the hepatoma may explain thedifferences in holding time for the 2 cell types. The relativedisorganization of the cells in the hepatoma (Fig. 2) and theirsmaller size may be responsiblefor the longer time intervalsbetween penetrations and the shorter time of holding withinone cell, whereasthe closerappositionof cells and the knownjunctional complexes between normal hepatocytes undoubtedly contributesto the short intervals between penetrationsand the relativeuniformityof potentialsin nearby cells.

The variance in the frequency histograms(Charts 1 and 2)illustratesseveral importantpoints.The first,as Limberger(12)and Schaefer and Schanne (15) point out, is the size of thevariance or the range. This points to the fact that even withinnormal cell populations the variance is large, indicating avariety of states of activity by some measure (perhaps eithermetabolically or proliferatively), within each tissue. It should benoted that, both in variance and in range, the samples taken inthis study are smaller in all cases by about one-half than thosein Limberger or Schaefer's report, with ranges of approximately—50mV to —60mV and standard deviations of 15 mV to 20mV.

Within the normal and cancerous hepatocytes, the rangeshavea slightdegree of overlap,but thisdoes not in eithercaseextend Into the mean values for either cell. The narrowerdistribution of the normal cells may point to a more restrictedrange of activitieswithin the normal liver or, conversely, thatthe liver tumor cells may have internally a greater variety ofactivity and not just a lower mean.

The ranges of the normaland transformedfibroblastshaveexclusivedomains,indicatinga wider separationof properties.The standard deviations of the 2 groups are comparable,indicating about the same range within each group despite the

greater separation of the means.Some of the variance in the data may be due to methodolog

ical problems. Errors of recording almost always result invalues which are lower than the true values. From direct observation in tissue culture studies, damage to the membrane ofthe penetrated cell often produces an instant drop in potential,accompanied by dramatic morphological changes. That this isprobably not significant in this study is indicated by the relativelack of skewness toward the lower side in the distributions. Theslight exceptions are the small subgroups in the normal hepatocytes and the fibrosarcoma cells. Only the latter contributeto the significance of the differences, however.

Several findings from the study of cancer cells may contribute to these differences (1). Since properties of the membraneare generally thought to give rise to the normal resting transmembrane potential, one of several alterations in the membranemay give rise to the reduction, such as changes in cell to cellinteraction and changes in intrinsic membrane proteins eitherin the presence, absence, or mobility of those proteins.

Several studies point to the membrane sodium channel or toaltered sodium transport as a strategic site for alteration. Cone(9) hypothesized higher intracellular sodium in cancer cells,and recently Smith et al. (17) reported higher levels of sodiumin cancerous hepatocytes as revealed by X-ray microanalysis.

Cone (9) has hypothesized that downward shifts in restingmembrane potential give rise to mitogenic activity. Cells thatnormally have proliferative potential have resting voltages ofaround —40mV to —60mV. When called upon, however,these cells may normally drop their potential to —10 mV to—20mV or lower during their active mitotic phase. Both fibroblasts and hepatocytes fall within these categories. Cone'shypothesis further speculates that cancer cells are those withpotentials that have been blocked in the —10- to —20-mVrange by whatever mechanism and that this range is associatedwith their continuous mitogenic activity.

Several questions are raised by the type of correlative datafound in the present study. Are proliferation rates proportionalto membrane potential? Or is there a threshold trigger-level formitotic activity? Are any other aspects of cancer, such asmetastatic activity or high metabolic rate, related to membranepotential? Is it the potential itself, i.e. , the fall in the concentration of anions intracellularly, or is it the concentration of specificanions or cations such as sodium that creates the conditionsfor mitosis? Are these membrane potential changes with increasing proliferation only epiphenomena with some prior common triggering mechanism?

REFERENCES

i . Altman, P. L., and Katz, 0. (Eds.) Electrical properties of nonexcitable cells.In: Biological Handbooks, Cell Biology, Vol. I, pp. 117-1 21 . Bethesda, Md.:Federation of American Societies for Experimental Biology, 1976.

2. Ambrose, E. J., James, A. M., and Lowick, J. H. Differences between theelectrical charge carried by normal and homologous tumour cells. Nature(Lond.), 177: 576—577,1956.

3. Balitsky, K. P., and Shuba, E. P. Resting potential of malignant tumour cells.Acta Unio. Int. Contra Cancrum, 20: 1391-1 393, 1964.

4. Bard, J., and Wright. M. 0. The membrane potential of fibroblasts in differentenvironments. J. Cell. Physiol., 84: 141—145,1975.

5. Biedermann, M. Das Verhalten der Membranpotentiale von Leberzellen derRatte währendund nach Gefàssunterbindungen.Acta Biol. Med. Ger., 21:827-833, 1968.

6. Chowdhury, T. K. Effects of concanavalin A on cellular dynamics andmembrane transport. Adv. Exp. Med. Biol., 55: 187-206, 1975.

7. Claret, B., Claret, M., and Mazet, J. L. Ionic transport and membrane

JUNE 1980 1833

Research. on November 23, 2020. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

potential of rat liver cells in normal and low chloride solutions. J. Physiol.(Lond.), 230: 87-1 01 . 1973.

8. Claret, M., and Coraboeuf, E. Membrane potential of perfused and isolatedrat liver. J. Physiol., 210: 137P—i38P,1970.

9. Cone, C. D. Unified theory on the basic mechanism of normal mitotic controland oncogenesis. J. Theor. Biol., 30: 151—181 , 1971.

10. Jamakosmanovic, A., and Loewenstein, W. Intercellular communication andtissue growth. Ill. Thyroid cancer. J. Cell Biol., 38: 556-561 , 1968.

11. Lambotte, L., and Saey, A. Liver-cell resting membrane potential as a directviability assay of preserved organs. Br. J. Surg., 56: 693, 1969.

12. Limberger, J. Messung von Membranpotentialen normaler Leber-Parenchymzellen und hepatocellulärerLeber-Carcinome der Ratte. Z. Krebsforsch., 65: 590-599, i 963.

13. Nelson, P. G., Peacock, J., and Minna, J. An active electrical response infibroblasts. J. Gen. Physiol., 60: 58—71, 1972.

14. Sachs, H. G., and McDonald, T. F. Membrane potentials of BHK (babyhamster kidney) cell line: ionic and metabolic determinants. J. Cell. Physiol.,80:347—358, i973.

15. Schaefer, H., and Schanne, 0. Membranpotentiale von Einzelzellen in Gewebekulturen. Naturwissenschaften, 43: 445, 1956.

16. Schanne, 0., and Coraboeuf, E. Potential and resistance measurements ofrat liver cells in situ. Nature (Lond.), 210: 1390—1391 , 1966.

17. Smith, N. R., Sparks, R. L., Pool, T. R., and Cameron, I. Differences in theintracellular concentrations of elements in normal and cancerous liver cellsas determined by X-ray microanalysis. Cancer Res., 38: 1952—1959, 1978.

18. Swift, M. R., and Todaro, G. J. Membrane potentials of human fibroblaststrains in culture. J. Cell. Ptsysiol., 71: 61—64,1968.

I 9. Tokuoka, S.. and Morioka, H. The membrane potential of the human cancerand related cells (I). Gann, 48: 353—354,1957.

CANCERRESEARCHVOL. 401834

R. Binggeli and I. L. Cameron

Research. on November 23, 2020. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

‘

C,..,.@

a'.

t I

‘-S@

;‘

.. I ‘W.

@ m

Membrane Potentials of Normal and Cancer Cells

@:†‘:a@

C@@

‘

1@

,@a

,% e.

!V'5e@,‘-a,@

L@ .@Lê,S.-@ ;4@@.C I

•@@‘le'.@'@

.,@. #. ..‘, *

.5@ ‘, ‘1 @‘

.,

@- @-

S

Fig. 1. Normal rat liver. H & E, x 450.Fig. 2. Morris 7777 hepatoma cells near the tumor capsule. Arrows, 2 dividing cells; one is in metaphase, the other in prophase. H & E, X 450.Fig. 3. Cross-section of the mouse cornea. The stratified squamous epithelium is at the top. The avascular stroma with dispersed fibroblasts is shown with the

cells lodged In narrow clefts among the parallel bundles of collagen fibrils. There appear to be about 4 to 6 fibroblasts that one might encounter with an electrodepassing normal to the corneal surface. The corneal endothelium is seen at the bottom. H & E, x 450.

Fig. 4. SectIon of the poorly differentiated fibrosarcoma. Arrow, dividing tumor cell at the telophase stage of mitosis. H & E. x 450.

V

S. ‘

4

0:0 45

I'

I ‘@@ ‘‘‘@@‘?

;1@V

;r―.5

@1ft

‘4

@.; @‘

k'@t

!

JUNE 1980 1835

@:‘•@& t, ‘@p

S@)Ø(;3 S

Research. on November 23, 2020. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

1980;40:1830-1835. Cancer Res   Richard Binggeli and Ivan L. Cameron  HepatocytesCellular Potentials of Normal and Cancerous Fibroblasts and

  Updated version

  http://cancerres.aacrjournals.org/content/40/6/1830

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/40/6/1830To request permission to re-use all or part of this article, use this link

Research. on November 23, 2020. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from