Effect of cell shape on proteinase secretion by epithelial cells

H. L. HONG and D. M. BRUNETTE*

Department of Oral Biology, Faculty of Dentistry, University of British Columbia, 2199 Westbrook Mall, Vancouver, British Columbia,Canada V6T 1Z7

* Author for correspondence

Summary

Cell proliferation has been found to correlatewith increased secretion of proteinases, such asplasminogen activator, in several different cellpopulations. In addition, the shape of the cellmay also play a role in regulating proteinasesecretion. However, the relationship between cellproliferation, cell shape and proteinase secretionhas not been studied in diploid epithelial (E) cellscultured from porcine periodontal ligament (PL).We have modified PLE cell shape by physicalmeans, such as growth on less-adhesive substrataand mechanical stretching, and by exposure tocholera toxin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Neutral proteinase and plasmin-ogen activator secretion were found to correlate

with cell shape, the more round the cells, thegreater the amount of proteinase secreted. PLEcells, stimulated to proliferate by cholera toxin ordibutyryl cyclic AMP, were more spread thancontrol cells, but secreted less neutral proteinaseand plasminogen activator. TPA stimulated cellproliferation slightly but, in contrast to choleratoxin, increased cell rounding and the secretionof neutral proteinase and plasminogen activator.Thus proteinase secretion was related more tocell shape than to cell proliferation.

Key words: epithelial cells, cell shape, proteinases, cellproliferation.

Introduction

For several transformed or malignant cell populations,tumour growth in vivo has been shown to be corre-lated with increased secretion of proteinases such asplasminogen activator (PA) in vitro (Mak et al. 1976;Butler et al. 1979; Mira-y-Lopezeia/. 1983; Pollacksal. 1975). This correlation between growth and pro:

teinase secretion may be important in understandingtumour invasion because there is considerable evi-dence that tumour invasion is accompanied by proteol-ysis (Woolley, 1984; Quigley, 1979). It is not clear,however, whether the increased amount of proteinasessecreted by transformed cell populations is associatedspecifically with the increased rate of proliferation, orwhether other cellular properties, such as cell shape,are involved. Cell shape has been shown to regulateproteinase secretion in normal synovial fibroblasts(Aggeler et al. 1982), and has also been implicated inthe regulation of proliferation of normal cells in vitro(Folkman & Moscona, 1978). This study was designedto investigate the relationship between cell prolifer-ation, cell shape and proteinase secretion. Epithelial

Journal of Cell Science 87, 259-267 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

cells were used in this study since epithelial culturesdiffer in several ways from fibroblast cultures and it isfrom epithelium that the majority of human cancersarise (Weinsteinet al. 1978). Cell shape was altered byphysical means, including growth on less-adhesivesubstrata (poly(HEMA)-coated dishes and groovedsurfaces) and mechanical stretching. Cells were stimu-lated to proliferate by cholera toxin and dibutyrylcyclic AMP, which also caused cells to flatten, and by12-O-tetradecanoylphorbol-13-acetate (TPA), whichincreased cell rounding. Secretion of PA and neutralproteinase (NP) by PLE cells (epithelial cells culturedfrom porcine periodontal ligament) was found to berelated more to cell shape than to rate of proliferation.

Materials and methods

Cell culturePLE cells were obtained from porcine periodontal ligamentas described by Brunette et al. (1976) and separated fromfibroblasts by their tendency to be more resistant thanfibroblasts to removal by trypsin (Owens, 1974) or by acollagenase separation technique (Kanoza et al. 1978). The

259

cells were cultured in a minimal essential medium (a-MEM) plus 15 % foetal calf serum (FCS) (Flow, MD) withpenicillin G (Sigma, St Louis, MO), lOOfjgml"1, genta-mycin (Sigma), 50 fig ml" ' , and amphoteracin B (Gibco,Grand Island, NY), 3/igml"1, at 37°C in a humidifiedatmosphere of air plus 5 % CGv

For assaying proteinase secretion, PLE cells were platedon 60mm Falcon dishes at 4X 10s cells/dish and incubatedovernight in a"-MEM + 15% FCS. The medium was re-moved and replaced with a modified low serum medium withlmM-K+ and01mM-Ca2 +, designated /3-MEM, plus 0-5%dialysed FCS (described by Brunette, 1984a), for 2-4 daysto permit sufficient secretion of enzyme for the activity tobe assayed. /3-MEM was used in these studies because itsupports PLE cell growth at low concentrations of serum inthe presence of growth promoters such as cholera toxin. Itwas desirable to keep the amount of serum low in theseexperiments because serum contains inhibitors to protein-ases. At each time point of medium collection, two cultureswere trypsinized and counted on an electronic cell counter(Coulter Electronics, Inc., Hialeah, Florida) to monitor cellgrowth. In some experiments, as noted in the text, /3-MEMwas supplemented by the addition of 1 ngml"1 cholera toxin(Sigma), or 0 5 mM-dibutyryl cyclic-3'5'-adenosine mono-phosphate (BtjcAMP) (Sigma), or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (Sigma) at either lngml"1 , orlOngml"1. PLE cells plated at 4X105 cells/dish were pro-cessed for scanning electron microscopy (SEM) as describedby Brunette et al. (1983).

Physical methods for altering cell shapeMechanical stretching. Mechanical stretching was ap-

plied using the method of Hasegawa et al. (1985). PLE cellswere plated on Petriperm dishes (Tekmar, Cincinnati, OH).Stretching the flexible plastic membrane of the Petripermdish, to which the cells are firmly attached by hemidesmo-somes, stretches the cells and makes them more flattenedthan cells in unstretched dishes (Brunette, 19846). Con-versely, growing the cells on a stretched membrane and thentaking the Petriperm dish off the template allows the plasticto return to its initial state and the attached cells to becomemore rounded. PLE cells were plated at 8x 105 cells/dish inff-MEM + 15 % FCS and grown for 3 days under theconditions stated below. For the detection of proteinasesecretion the cells were washed twice with /3-MEM + 0-5 %dialysed FCS and incubated in the same medium for 2 days.

To determine the effect of stretching two groups wereused. (1) Unstretched control cultures: the cells weregrown on Petriperm dishes over the experimental period.(2) Stretched cultures: the Petriperm dishes were stretchedduring the 2-day proteinase collection period. To determinethe effect of relaxing cells grown on a stretched membraneanother two groups were used. (1) Controls: the cells wereplated and grown on a stretched membrane and the dishremained stretched during the 2-day proteinase collectionperiod. (2) Relaxed: the cells were plated and grown on astretched membrane for 3 days and then the dishes weretaken off the templates during the 2-day proteinase collec-tion period.

Preparation of grooved substratum. With the exception ofmacrophages (Rich & Harris, 1981), cultured cells adopt a

more spherical shape when cultured on grooved (Rovenskyet al. 1971) or rough substrata. Silicon wafers with V-shapedgrooves were produced as described by Brunette et al.(1983). The V-shaped grooves were 79 jxm wide at the top ofthe V and 60fim deep; the grooves separated by flat areas13 [im in width. Impressions of the groove pattern on thesilicon wafer were made with Exaflex (G-C Dental Indus-trial Corp., Japan). The groove pattern was then reproducedonto Epotek resin (Epoxy Technology Inc., Billerica, MA)in 60 mm Falcon dishes. Control dishes with a flat surfacewere also made with Epotek resin.

Preparation of poly (HEMA) plates. Cell shape was alsoaltered by changing the culture dish surface adhesiveness.Tissue culture dishes were coated with various dilutions of a12% stock solution of poly-2-hydroxyethyl methacrylate(poly(HEMA)) (Polysciences, Inc., Warrington, PA) ac-cording to the method of Folkman & Moscona (1978).Following the convention of Folkman & Moscona (1978) thepoly(HEMA) concentrations stated are the amounts ofdilution of the 12% (v/v) stock solution in ethanol. Forexample, to obtain a concentration of 10~3, 0 ' lml of thestock solution was added to 99'9ml ethanol.

AssaysCyclic AMP. PLE cell cultures were processed as de-

scribed by Oey et al. (1974) and intracellular cyclic AMPwas determined using the Becton Dickinson cAMP radio-immunoassay kit (125I) (Becton Dickinson Immunodiagnos-tics, Orangeburg, NY).

Neutral proteinase assay. The harvested media wereconcentrated 20 times using Millipore CX-10 filters (Mr

cutoff 10000) (Millipore Corp., Bedford, MA) and dialysedagainst 50mM-Tris-HC1 buffer containing 200mM-sodiumchloride and 5 mM-calcium chloride (pH7-5) at 4°C. Theneutral proteinase activity in the media was measured usingA20C0II (Calbiochem, San Diego, CA) as the substrate, asdescribed by Pettigrewe* al. (1978). Activation of the latentenzyme was achieved by the addition of mersalyl acid(Sigma) to the assay at a final concentration of 2mM. Allassays were done in duplicate and a mersalyl activationcontrol was included. The degradation of Azocoll was linearwith respect to enzyme concentration as well as incubationtime up to 56h. As defined by Werb & Reynolds (1974), 1unit of enzyme activity was equivalent to 1 mg Azocollsplubihzed per h at 37°C.

Plasminogen activator assay. The two-stage assay de-scribed by Jackson et al. (1981) was used to determine PAactivity. In the first stage, plasminogen is converted intoplasmin by PA and in the second stage, the plasmin acts onthe substrate S-2251. The reaction is initiated by adding20/il of the sample to 40 jJ.\ of plasminogen (Kabi, Stock-holm, Sweden) ( lun i tmF 1 ) . After 30min, 80^1 of thesubstrate mixture (S-2251 (Kabi) ( 5 m g m r ' ) and 1-77M-NaCl in 32mM-Tris- HC1 buffer mixed in a 2:3 ratio) wasadded. The second stage of the reaction was terminated after30min by the addition of 0-8ml of 40% acetic acid. Allassays were done in duplicate and included controls withbuffer only and without plasminogen. The standard curvewas prepared using increasing concentrations of strepto-kinase (Behring Institute, Marburg, West Germany). Theassay was linear with respect to increasing amounts of the

260 H. L. Hong and D. M. Brunette

sample up to an absorbance of 0'9 at 405 nm. Activities wereexpressed as IU 10~5 cells. The coefficient of variation forthe NP and PA assays was 3 % and 6%, respectively.

Statistical analysisIn experiments where replicate cultures were used, the datawere analysed using the analysis of variance (ANOVA). Inother experiments, the sign test (Zar, 1974) was used to testwhether the differences observed under different conditionswere due to chance alone.

Results

Effect of cell population density

As cell population density has been found to influencemany properties of epithelial cells in culture includingproliferation (Zetterberg & Auer, 1970), prostaglan-din production (Bireket* al. 1980) and dome formation(Young et al. 1978), the effect of cell populationdensity on proteinase secretion was investigated. PLEcells were plated at 2, 4, 8, 12 and 15 (X 10s) cells/dishin /S-MEM + 0-5% dialysed FCS. These conditionskept the cells in a non-proliferative state. The amountof activity present in the medium was assayed at days 7and 10. As the cell population density increased, bothNP (Fig. 1) and PA secretion (Fig. 2), measured inunits 10~scells, decreased. Therefore, experimentswere designed so that the cell densities were normallyin the plateau regions of the curves where the effects ofcell population density on proteinase secretion weresmall.

Effect of cholera toxin and dibutyryl cyclic AMPPLE cells were induced to proliferate by the additionof cholera toxin at Ing ml"1 to cell cultures asdescribed by Brunette (1984a). By day 10, there was asignificant (P<0-01) , increase in cell number in thecholera toxin-treated group (Fig. 3). Cholera toxin-

=5 12

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2 6 10 14Cell density (xlO5 cells/dish)

Fig. 1. Effect of cell population density on NP activity.The cells were plated at 2, 4, 8 and 15 (XlO5) cells/dish.NP activities on days 7 (A) and 10 (•) are from onerepresentative experiment that was repeated. The trendswere the same in both experiments.

treated cells also became more flattened compared tocontrol cultures as described by Brunette (1984a).Thus in this experiment, both an increase in cellproliferation and an altered cell shape were seen.Secretion of proteinases, however, was reduced sig-nificantly (P<0-05). Cholera toxin-treated cells pro-duced, on average, 69% of the amount of NP and27 % of the PA produced by control cultures (Fig. 3).

As expected, cholera toxin increased intracellularcAMP levels in PLE cells; at day 1 of culture, PLEcells in the presence of cholera toxin had 0-52pmolcyclic AMP per 10s cells compared to control cells,which had 0-18 ± 0-03 pmol cyclic AMP per 10s cells(±S.D.). This difference in cyclic AMP level wasstatistically significant (P<0-01).

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Fig. 2. Effect of cell population density on PA activity.The cells were plated as described in Fig. 1. The PAactivities on days 7 (A) and 10 ( • ) are from onerepresentative experiment that was repeated. The trendswere the same in both experiments.

2 0 0 *

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Fig. 3. Effect of cholera toxin. E cells were incubated in /3-MEM + 0-5 % DFCS. Cholera toxin was added at1 ngml"1. Cell counts ( • ) , NP activity (A) and PAactivity (•) of cholera toxin-treated cultures are given as %of the values in control cultures. Cell number in thecholera toxin-treated cultures increased from 4-2X105 cellsper dish on day 3 to 8-2X105 cells per dish on day 10. Thedata represent values from two experiments; error barsindicate S.E.

E-cell shape and proteinase secretion 261

The effect of Bt2cAMP on PLE cell growth wassimilar to that of cholera toxin. Cell numbers increasedtwofold by day 7; with the increase being statisticallysignificant (P<0-001). The morphological responsewas also similar; the cells in the Bt2cAMP-treatedcultures were found to be more flattened than cellscultured in 0-MEM + 0-5 % DFCS (dialysed FCS).

Proteinase secretion was reduced significantly(P<0-05) in the Bt2cAMP-treated cultures. NP se-cretion by Bt2cAMP-treated cells averaged 47 % ofthe NP activity and 27 % of the PA activity of con-trol cultures (Fig. 4). Thus both cholera toxin andBt2cAMP increased cell numbers and cell spreading,but reduced secretion of NP and PA.

Effect of TPA

TPA at both 1 ngml"1 and lOngmP 1 caused a smallbut detectable increase in cell number. Culturestreated with TPA at 1 n g m P 1 showed an increase of29 % in cell numbers while cultures treated with TPAat lOngmP 1 showed an increase of 13 % (compared tountreated controls). As found by Wigler & Weinstein(1976), TPA at lOngml"1 also caused the cells toretract slightly and to appear more round. PLE cellstreated with TPA at 1 ngml"1 produced an increase of103 % in NP activity compared to the control over a4-day culture period. Cultures treated with TPA atlOngmP1 produced increased NP by 127% and PAby 272% relative to the controls.

Because TPA increased cell numbers and alsoincreased the secretion of NP and PA, whereas choleratoxin and Bt^cAMP increased cell number but de-creased the secretion of NP and PA, there appeared tobe no relationship between cell proliferation and thesecretion of these enzymes. However, a flattened cellshape produced by cholera toxin and dibutyryl cyclic

2 0 0 *

2 4 6Time (days)

Fig. 4. Effect of Bt2cAMP. Duplicate cultures wereincubated in /3-MEM + 0-5 % DFCS. Bt2cAMP was addedat 0-5 mM. Cell counts ( • ) , NP activity (A) and PAactivity ( • ) of Bt2cAMP-treated cultures are given as % ofthe values in control cultures. Error bars indicate S.E.

AMP was correlated with reduced secretion of protein-ases (cholera toxin and dibutyryl cyclic AMP exper-iments), while a more rounded cell shape produced byTPA was correlated with increased secretion of pro-teinases.

In order to study the relationship between cell shapeand proteinase secretion independently of cell growth,physical means of changing cell shape were applied toPLE cells grown under non-proliferative conditionsOS-MEM + 0-5 % DFCS). PLE cell shape was modi-fied by three different physical means: (1) mechanicalstretching, (2) growth of cells on grooved substrata,(3) growth of cells on poly(HEMA)-coated dishes.

Effect of mechanical stretching

Electron-microscopic sections of the interface betweenthe cell and plastic show hemidesmosome-like struc-tures indicating tight attachment of the cells to theplastic surface. Stretching the dish also stretches theattached PLE cells and they become more flattened(Brunette, 19846). By removing the dishes from thetemplate, a release in tension could be obtainedenabling stretched cells to relax.

Stretched PLE cells produced 25 % (average of twoexperiments) of the PA secretion produced by un-stretched cells (Table 1). The effect of relaxingstretched cells compared to controls, which remainedstretched, was an increase of 80% (average of threeexperiments) in PA secretion (Table 1). Statisticalanalysis was done using the sign test. The difference inPA values between the treated and the control on allfive occasions that the experiments were carried outwas in the expected direction. The probability of thisoccurrence happening due to chance alone is less than0-05.

The effect of stretching on NP secretion was investi-gated in an experiment in which ten groups of fourdishes each were plated with cells at 8x 10s cells/dish(Table 1). Five groups were stretched and the otherfive groups remained unstretched as controls. To getsufficient cells for this experiment, a ninth subculturewas used, which was older than that normally used inthese experiments. The cells from the ninth subcul-ture did not demonstrate significant amounts of PAactivity but detectable amounts of NP were present.

The stretched cells produced 5-9 ±0-16(S.E.)X1O~3 units of NP per 10s cells compared to7-7 ± 0-16 (s.E.)X 10~3 units per 105 cells produced bycontrol cultures. The difference was statistically sig-nificant when tested by ANOVA (P< 0-005).

In summary, making the cells more flattened bystretching resulted in a decrease in proteinase se-cretion, while making PLE cells more rounded by

262 H. L. Hong and D. M. Brunette

relaxing stretched cells resulted in an increased pro-teinase secretion.

Grooved substrata

In this experiment, PLE cells were made more roundby growing them on a substrate with 60 fim V-shapedgrooves with a repeat spacing of 92|Um (Fig. 5). PLEcells cultured on grooved substratum secreted moreNP and PA than cells grown on a smooth flat surface(Fig. 6). Analysis of the data using the sign testindicates that the increase in NP activity was statisti-cally significant (P<0-05).

Poly(HEMA)-coated dishes

Folkman & Moscona (1978) found that fibroblastsbecome increasingly more spherical when cultured ondishes coated with increasing concentrations of poly-(HEMA). A similar effect was found for PLE cells.PLE cells cultured on dishes coated with 10~3 dilutionof the poly(HEMA) stock, were much less spread andmore spherical than cells grown on tissue cultureplastic, but the change in cell shape was only justdetectable at a 5 X104 dilution. The effects on PLE cellmorphology caused by coating dishes with poly-(HEMA) thus occurred over a narrow range of poly-(HEMA) dilution.

Under the conditions used, there was no increase incell number but the amount of NP activity secretedinto the medium significantly increased with increas-ing poly(HEMA) concentration (Fig. 7) (P< 0-001,using ANOVA). PA secretion also increased withpoly(HEMA) concentration but the effect was some-what smaller (Fig. 8).

Thus, the changes in cell shape with mechanicalstretching and relaxation, grooved substrata and poly-(HEMA)-coated dishes demonstrated the same trend;the more flattened cells secreted less proteinase whilethe more spherical cells secreted increased amounts ofproteinase.

Discussion

In these experiments PLE cell shape was altered byphysical and chemical means. The advantage of alter-ing cell shape by physical means, such as mechanicalstretching or growth on a less-adhesive substratum, isthat the multiple effects known to be produced by theuse of some chemical agents, such as TPA (Weinsteinet al. 1977; Makman et al. 1972) or cholera toxin(Brunette, 1984a), are avoided. A disadvantage ofusing the physical techniques of altering cell shape isthat, in contrast to agents such as cholera toxin wherethe biochemical effects of treatment have been de-scribed in considerable detail (Johnson, 1982), thebiochemical and structural mechanisms associatedwith physical methods used to alter cell shape arelargely unknown and may be complex. For example,diverse cytoskeletal elements such as the intermediatefilament nuclear matrix complex (Ben Ze'ev, 1985) oractin (Werb et al. 1986) have been implicated intransducing changes in cytoskeletal architecture todifferences in gene expression. In the absence of a firmknowledge of the mechanism it is possible that thephysical techniques used to alter cell shape alsoaffected another cellular property or mechanism in-volved in proteinase secretion. This possibility be-comes more unlikely when it is noted that a similarrelationship between cell shape and proteinase se-cretion was observed in cells whose shape had beenaltered by chemical agents with different mechanismsof action. The general rule arising from altering PLEcell shape by either physical or chemical means is thatthe more spherical the cell, the more proteinasesecreted.

The results reported here are similar in part to theobservations of Aggeler et al. (1984), who reportedthat agents or conditions that altered rabbit synovialfibroblast morphology, including TPA and growth onpoly(HEMA), induced the production of procollage-nase and NP. Although growth on poly(HEMA)dishes and TPA treatment also increased PA secretion

Table 1. Effect of mechanical stretching or relaxation on proteinase secretion

PA activity(1CT3 IU per 105 cells)

NP activity(10~3 units per 105 cells)

Stretching experimentsControls

Stretched

Relaxation experimentsControlRelaxed

7-6 (4-8)1-9 (0-1)

3-4 (0-98)5-8(1-35)

7-7 (0-16)5-9 (0-16)

Values represent the amount of proteinase (mean (s.E.)) in media collected for 2 days under each condition. Experiments measuring PAactivity used PLE cells in the fourth to eighth subculture. The stretching experiment was performed twice, the relaxation experiment threetimes; five dishes were used for each condition in each experiment. NP activity was assayed in ninth subculture PLE cells using 20 dishesfor each condition.

E-cell shape and proteinase secretion 263

by rabbit synovial fibroblasts, cytochalasin B stimu-lated collagenase secretion only, and epidermal growthfactor caused an increase in PA activity alone. Aggeleret al. (1984) thus concluded that it was unlikely thatthe production of the two proteinases was under closecoordinated control in rabbit synovial fibroblasts.Similarly, in our study it was found that, althoughsimilar trends in enzyme secretion as a function of cellshape were observed for PA and NP, the size of theeffect on the secretion of the two enzymes often

differed. Moreover, in later subcultures of PLE cells,PA activity was sometimes not detectable whereas NPactivity remained. Thus, it appears that for PLE cellsprecise coordinated control of NP and PA is unlikely.

The decrease in proteinase secretion caused byincreasing PLE cell population density is similar to cellbehaviour observed with other epithelial cell popu-lations and other functions. For example, the pro-portion of cells secreting prostaglandin (Birek et al.1980) or initiating DNA synthesis (Zetterberg &

Fig. 5. Scanning electron micrographs of E cells cultured on Epotek. A. Resin has been moulded into a series of V-shapedgrooves. Note that many of the cells are round, while others are elongated in the direction of the grooves. B. Smooth flatsurface.

264 H. L. Hong and D. M. Brunette

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0-2 1

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30

3 7Time (days)

Fig. 6. Effect of grooved substrata. E cells were plated at8x 105 cells/dish. Proteinase activity from cultures grownon flat substrata (open bars) and grooved substrata(stippled bars) as measured on days 3 and 7. The datashown are from one representative experiment that wasrepeated twice. The trends were the same in allexperiments.

0 1CT3 5x10-"poly(HEMA) concentration

10-

Fig. 7. Effect of poly(HEMA) concentration on NPactivity. Duplicate groups of E cells were plated in dishesprecoated with various concentrations of poly(HEMA)solution. The medium was collected on days 3 (open) and7 (striped) and assayed for NP activity. Error bars indicateS.E.

Auer, 1970) decreases with population density. Simi-larly, a number of metabolic functions in mature rathepatocytes have been shown to be regulated by cellpopulation density (Jurin & McCune, 1985; Naka-mura et al. 1983). The mechanisms underlying these

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0 10~5 5xlO"4 10"'poly(HEMA) concentration

Fig. 8. Effect of poly(HEMA) concentration on activity.Duplicate groups of E cells were plated in dishes precoatedwith various concentrations of poly(HEMA) solution.Medium collected on days 3 (open) and 7 (striped) wereassayed for PA activity. Error bars indicate S.E.

effects are not known, but the effect of cell populationdensity must be considered when designing exper-iments that include treatments that influence cellmultiplication.

The addition of cholera toxin, Bt2cAMP or TPA toa medium containing a low concentration of serumstimulated cell proliferation and permitted direct com-parisons of proliferating and quiescent cells. Becauseserum contains inhibitors to proteinases, the use of alow-serum medium enabled NP activity to be assayedin the proliferating or quiescent cultures. AlthoughTPA increased both PLE cell number and PA and NPsecretion, cholera toxin and Bt2cAMP caused anincrease in PLE cell proliferation but decreased theamount of PA and NP secreted. Our observations,obtained with normal diploid PLE cells (Brunette etal. 1979), thus do not provide support for the hypoth-esis, based originally on observations on tumour cells(Mak et al. 1976; Butler et al. 1979; Mira-y-Lopez etal. 1983), that PA secretion is positively correlatedwith cell multiplication.

However, it should be noted that the production of ahigh level of PA in culture is a distinctive property ofmany transformed cells, and that the PLE cells used inthis study secrete only small amounts of this enzyme.It is possible that PA secretion is regulated differentlyin transformed cells than in the normal cells used inthis study.

In summary, six methods of changing cell shapeincluding chemical (cholera toxin, Bt2cAMP, TPA) orphysical (mechanical stretching, poly(HEMA) dishes,grooved substrata) stimuli, consistently showed that,irrespective of the method used, the more-flattenedcells secreted less proteinase than the more-roundedcells. There was, however, no consistent relationshipbetween the amount of proteinase secreted and the rateof proliferation. Thus for diploid PLE cells proteinase

E-cell shape and proteinase secretion 265

secretion was related more to cell shape than to cellproliferation.

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{Received 31 July 1986 -Accepted 14 October 1986)

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