Cell, Vol. 50, 1131-l 137, September 25, 1987, Copyright 0 1987 by Cell Press

Cell Migration Is Essential for Sustained G rowth of Keratinocyte Colonies: The Roles of Transforming G rowth Factor-a and Epidermal G rowth Factor Yann Barrandon and Howard Green Department of Physiology and Biophysics Harvard Medical School 25 Shattuck Street Boston, Massachusetts 02115

Summary

In common methods of cell cultivation, multiplication takes place in cells distributed uniformly or in small colonies and the number of cells Increases exponen- tlally. In contrast, an Isolated colony of coherent epi- dermal keratinocytes, as it grows larger, departs dras- tically from exponential growth, and instead Increases its radius at a constant rate over time. The rate of Increase of colony radius is &fold greater in the pres- ence of epidermal growth factor (EGF) and lo-fold greater in the presence of transforming growth factor- a (TGF-a): the resulting megacolonles may become 30-50 times greater In area and cell number than colonies grown in the absence of the growth factors. Growth of a colony depends on outward migration of the rapidly proliferating cells located In a thin rim close to the colony perimeter. The effect of EGF and TGF-a in promoting multiplication must depend on their ability to increase the rate of this cell migration.

Introduction

A polypeptide growth factor is defined as an agent promot- ing cell proliferation: its interaction with an external recep- tor is thought to lead to intracellular changes preparing the cell for DNA synthesis and division. Epidermal growth factor (EGF) stimulates the proliferation of epidermal and other epithelial t issues in animals (Cohen, 1962, 1965) as well as a number of cell types in culture (for review see Carpenter and Cohen, 1979). For this reason, it may have a role as an agent promoting wound healing (Brown et al., 1966). The polypeptide transforming growth factor-a (TGF- a) (De Larco and Todaro, 1976; Anzano et al., 1963) pos- sesses sequence homology to EGF (MassaguB, 1963a; Marquardt et al., 1964; Derynck et al., 1964) and binds to the same cell surface receptor (De Larco and Todaro, 1960; Massague, 1963b). In the analysis of the events fol- lowing ligand-receptor interaction and leading to prolifer- ation, the possible importance of cell movements is usu- ally ignored.

Under suitable culture conditions, an isolated single hu- man epidermal keratinocyte gives rise to a colony with high frequency (Barrandon and Green, 1965). Such a colony consists of a stratified epithelium composed of co- herent cells. In the absence of perturbations by other colo- nies, the growth dynamics of the isolated colony can be studied in detail.

We have now examined keratinocyte growth in single colonies as they enlarge and form megacolonies. These

studies reveal the indispensability of cell migration in or- der to sustain multiplication. They also show that the abil- ity of the polypeptides EGF and TGF-a to promote cell growth cannot be explained without considering their ef- fects on cell migration.

Results

Rocking of Keratinocyte Cultures to Promote the Growth of Large Colonies Each of a number of 25 cm* flasks containing lethally ir- radiated 3T3 cells was inoculated with a single keratino- cyte (Barrandon and Green, 1965). After 6 days of station- ary incubation, and for the duration of the experiment, some of the flasks were placed on a commercial platform rocker (Bellco Corp) set to 9 oscillations per min. In the ab- sence of added EGF, this made no difference in the rate of colony growth, but in the presence of EGF or lGF-a, colonies in the rocked cultures grew more rapidly than those remaining stationary (Table 1). By 16 days, the colo- nies in the rocked cultures were 50% larger. As was con- cluded from earlier experiments on the multiplication of 3T3 cells (Stoker and Piggott, 1974; Dunn and Ireland, 1964). the rocking must relieve some diffusion-limited transfer in the medium, possibly of the growth factor itself. Most subsequent experiments on cell growth and migra- tion were carried out on rocked cultures, but similar results were obtained using stationary cultures.

Effect of EGF and TGF-a on Colony Growth: the Formation of Megacolonies Since different colonies may have different growth poten- tial (Barrandon and Green, 1967), experiments on the ef- fects of the growth factors were carried out using founding cells derived from a single clone. This resulted in very uni- form growth of the 6 to 10 colonies in each group. As reported earlier (Rheinwald and Green, 1977), the pres- ence of EGF from day 1 to day 6 after inoculation did not affect the cell growth rate; the mean doubling time of 6 colonies was 16.6 hr in the absence of EGF and 19.0 hr in its presence. By 6-10 days, when growth of the colony be- gan to deviate from exponential, the effects of both EGF and TGF-a became appreciable (Figure 1A). By 13 days, colonies grown in the presence of either agent were 10 times larger than those grown in their absence; by 24 days, colonies in the presence of EGF were 30 times larger and those in the presence of TGF-a were 50 times larger. A colony larger than 1 cm* (1.6 x lo5 cells) was defined as a megacolony: only in the presence of EGF or TGF-a were epidermal cells able to grow into a megacol- ony (Figure 2).

In a group of three experiments, the effect of TGF-a was always more powerful than that of EGF, but the difference between the two polypeptides did not appear until the colonies reached 14 days of age. By 24 days, the colonies grown in the presence of TGF-a were larger by 45% (Fig- ure 2). The greater effect of TGF-a was observed at all con-

Cell 1132

Table 1. Effect of Rocking Cultures on Colony Growth

Day

Area of Colonies (mm?

No EGF

Stationary Rocked

+ EGF (IO nglml)

Stationary Rocked

6 0.150 0.108 0.188 0.191 8 0.497 0.419 1.06 1.18

10 1.97 1.87 3.84 4.66 12 5.30 5.71 18.1 24.8 14 13.9 14.8 44.8 65.0 16 24.1 20.3 92 148 20 35.8 30.4 248 401

All values are the means of 5 or 6 colonies. Colony area may be con- verted to cell number using the factor of 1800 cells per mm2 (Barran- don and Green, 1985).

centrations between 1 and 100 nglml; there is therefore no concentration of EGF that exerts as great an effect as a saturating concentration of TGF-a (30 nglml). The EDs0 for both polypeptides was 3-6 nglml.

Colony Growth by Constant Increase in Radius Within 8 days after inoculation, keratinocyte colonies fail to maintain exponential growth. This is because cells generated by multiplication within the interior of the colony tend to leave the basal layer and terminally differentiate. In order that cells generated by multiplication remain in the basal layer and continue to multiply (Rhein- wald and Green, 1975), they must, even though they re- main coherent, migrate centrifugally at the perimeter of the colony. If there were no cell migration, a colony would expand very slowly, since cell division alone could expand the colony by little more than 1 cell diameter per day. The rate of cell migration must therefore become a limiting fac- tor in the expansion of the colony. After the colony be- comes 6-12 days old, it enters a phase of linear growth, in which its radius increases at a constant rate (Figure 1B). In rocked cultures, this rate is 0.18 mm per day in the ab- sence of growth factors, 1.42 mm per day in the presence

of EGF, and 1.79 mm per day in the presence of TGF-a. In the latter case, the migration distance of cells at the pe- rimeter is equal to about 70 cell diameters per day.

Direct Demonstration of the Effect of EGF and TGF-a on Keratinocyte Migration in Small Colonies In the absence of the growth factors, a growing colony contains closely packed cells that begin to stratify rather early. When EGF or TGF-a is added to a culture containing a 7 day colony grown in the absence of these factors, the cells begin to move centrifugally and the cell density be- comes greater at the periphery of the colony than near its center. This cell migration can be measured as an in- crease in colony area within 15 min of the addition of either polypeptide (Figure 3). Within 2 hr, the effect of TGF-a is clearly the more powerful. In the short time span of this experiment, the contribution of proliferation to increase in colony area is small.

The change in appearance of a single colony during the period following the addition of TGF-a is shown in Figure 4. The colony is obviously larger within 60 min, and the cell density in the central region is reduced as the cells migrate to the perimeter.

Cells Contributing to Expansion of a Large Colony Are Located Close to the Colony Perimeter To locate the cells contributing to colony expansion, differ- ent portions of a megacolony growing in an unrocked petri dish were removed in sectors. The two circles on the megacolony illustrated in Figure 5 indicate the location of the colony perimeters at 18 and 21 days after inoculation of the founding cell. These perimeters are separated by an average distance of 3.0 mm, corresponding to the 3 day radial enlargement in unrocked cultures. At 21 days, one sector of the colony was removed from the center to the 18 day perimeter, leaving a rim of 3 mm; another sector was removed so as to leave a rim of about 1 mm.

The colony was then refed with medium containing TGF-a, allowed to grow for an additional 2 days, and then fixed and stained. It can be seen that regardless of

Figure 1. Effect of EGF and TGF-a on Growth of Megacolonies

Single sister cells were isolated from an 8 day old clone (strain AY, third culture), and each was reinoculated into a separate dish contain- ing supporting 3T3 cells. Addition of the growth factors to the medium was begun 8 days later and repeated with each refeeding at intervals of 3 days. Each point is the mean size of 9 or 10 colonies. The standard error of the means was 4.9%. Open triangles, no growth factor added; open circles, human EGF was added to 30 rig/ml at each feeding; solid circles, human TGF-a was added to the same concentration in EGF equivalents, as determined by receptor binding assay (Derynck et al., 1984). The rela- tion between colony area and cell number over 4 orders of magnitude is approximately 1800 cells per mm*.

8 12 16 20 24

Colony Age (days) Colony Age (days)

~jy3ation and Growth of Keratinocytes

f3

C Figure 2. Appearance of Megacolonies

A colony grown under each condition in the experiment of Figure 1 was fixed at 24 days and stained with rhodamine. (A) Addition of TGFa (30 @ml): the principal colony has an area of 22.2 cm2. (8) Addition of EGF (30 @ml): colony has an area of 15.5 cm*. (C) Neither growth factor: colony has an area of 0.47 cm2. Bar indicates 1 cm. Small sat- ellite colony in (A) is the result of ceil detachment from the principal colony during the experiment (Barrandon and Green, 1985).

whether sectors were removed or not, colony expansion was similar. The mean increase in radius was 1.80 and 1.87 mm for the untouched sectors and 1.80 and 1.84 mm for the 3 and 1 mm rims. It was concluded that ceils lo- cated more than 1 mm internal to the perimeter did not contribute to colony expansion and that the 1 mm rim therefore contained all the cells that migrated outward to expand the perimeter.

Distribution of Multiplying Dells in the Keratlnocyte Megacolony To determine the location of multiplying cells, a megacol- ony was labeled with [Wlthymidine. As thymidine pene-

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Figure 3. The Growth Factors Rapidly Promote Centrifugal Cell Migration

Single colonies were grown for 7 days in the absence of the growth fac- tors. The areas of the colonies were measured, and the cells were refed with medium containing TGF-a (solid circles) or EGF (open cir- cles) at 30 @ml. At close intervals, the areas of the colonies were remeasured, and the radii were calculated. In the controls, to which no growth factor was added (triangles), the increase in colony area in 3 hr was about 12%. corresponding to the expected increase in cell num- ber if the cells were growing exponentially with a doubling time of 17 hr. The increase in colony area in the presence of TGF-a during the same interval was about 60% or 5 times larger. Each point is derived from the mean area of 3 colonies.

trates slowly to the basal cells of a keratinocyte colony (Dover and Potten, 1983), the culture was incubated with [14C]thymidine for 24 hr, then fixed and exposed to photo- graphic emulsion.

In the internal part of the colony, there were clusters of labeled nuclei but the average frequency of labeled nuclei was low. The progeny of the internal labeled cells must be destined for terminal differentiation. In contrast, in the re- gion close to the perimeter virtually 100% of the cell nuclei were labeled (Figure 8). The average width of this rim of uniformly labeled cells measured at 15 points was 0.19 mm. The width of the rim of intense proliferation did not change as the colony grew: when two colonies whose areas differed by la-fold were labeled, it was found that the widths of the rims of intense proliferation were iden- tical.

The cells of the rim of intense proliferation are the most migratory. This was shown by pulse-chase experiments in which the colonies were labeled in the usual way and after 24 hr, the medium was removed, the culture was washed, and growth was allowed to continue for 24 hr in medium containing unlabeled thymidine. At the end of this period, the cells at the perimeter were again the most uniformly

Cell 1134

Figure 4. Change in Appearance of a Single Colony after the Addition of TGF-a

A colony was grown for 7 days without growth factor. At time 0. the colony was photographed and TGF-a was added to 30 nglml. Photo- graphs taken 60, 120, and 160 min later show obvious enlargement of the colony and thin- ning of its interior. The shape of the perimeter changes owing to the cell movement, but the colony is shown in constant orientation with re- spect to fixed external reference points, not visible in the photographs.

labeled. The cells in the rim of intense proliferation there- fore retain their position at the advancing perimeter.

Expansion of the colony depends on continuing genera- tion of cells at a rate sufficient to maintain cell density be-

1

Figure 5. Cells Contributing to Colony Expansion

The perimeter of a megacolony was marked in ink at day 16 and again at day 21. Two sectors of cells were removed from the colony on day 21, leaving rims of 3 mm (lower right) and 1 mm (upper left). On day 23, the colony was fixed and stained. The advance of the colony perim- eter was not significantly different in any of the four sectors. Inward cell migration into the empty sectors was probably prevented by the trenches made in the surface of the dish at the two perimeters in order to remove the cells within the sectors.

hind the advancing perimeter. What proportion of the necessary cells are generated within the rim of intense proliferation? Cells approaching the perimeter more closely than 1 mm cannot be removed with much ac- curacy, so it is not possible to answer the question in this way. At first glance it seems impossible for cells within a rim of 0.2 mm to make an important contribution toward expanding the colony by 1.0 mm within a day, considering that the shortest doubling time of keratinocytes in ex- ponentially growing microscopic colonies is about 17 hr. However, it was found by counting nuclei stained with Hoechst nuclear dye that the cell density within the 0.2 mm rirh of intense proliferation was twice that in the inter- nal region of the colony immediately adjacent to the rim but outside the zone of stratification.

The dynamics of colony expansion are illustrated in Fig- ure 7. At the perimeter of the colony is a rim containing rap- idly proliferating and outwardly migrating cells. In un- rocked cultures growing in the presence of TGF-a, this rim is 0.2 mm wide and in 1 day it moves outward 1 mm. The proliferating cells remain concentrated in the advancing rim. Some of their progeny will be left behind along the path of outward migration and will thereafter sustain a lower rate of multiplication. Since the cells in the rim of in- tense proliferation are present at approximately twice the density in the more internal region, they can, as they ad- vance, leave a substantial number of cells behind. Other cells are probably contributed from a position somewhat deeper in the colony, but not from more than 1 mm behind the perimeter.

It might be expected that in colonies grown in the ab- sence of EGF and TGF-a the rim of intense proliferation would be narrower. This was not the case: instead, the rim was wider, but diffuse and less homogeneously labeled,

$Ftion and Growth of Keratinocytes

Figure 6. The Rim of Intense Cell Proliferation in an Expanding Mega- colony

A single keratinocyte colony, grown for 21 days in a stationary petri dish, was labeled for 24 hr by the addition of 5 uCi of [Wjthymidine. The colony was then fixed, stained with Hoechst nuclear dye, and ex- posed to photographic emulsion for 7 days.

The upper part of the photograph shows the large stained nuclei of the now sparsely distributed supporting 3T3 cells, most of which are unlabeled. The lower part of the photograph shows a region of the colony interior not far from the perimeter and outside the densely strati- fied core of the colony; the keratinocytes are largely within a mono- layer. The nuclei are all stained, but a few are rendered invisible by their overlying silver grains. Between this zone and the perimeter of the colony lies a nearly uniformly black rim containing the most rapidly proliferating keratinocytes. Even though the cell density in this rim is greater than in the immediately adjacent internal part of the colony, none of the stained nuclei can be seen, because of the overlying silver grains. The few visible nuclei are those of 3T3 cells excavated by the advancing edge of the keratinocyte colony. The width of the rim of in- tense proliferation is 0.19 mm.

suggesting that in the absence of EGF and TGF-a, mul- tiplying cells were unable to advance sufficiently to re- main at the perimeter.

Discussion

The experiments described here show that a keratinocyte colony grows by a combination of cell multiplication and cell migration. The phase of exponential growth lasts for only a short time and is replaced by a process of linear growth in which the radius increases daily by a constant amount. This is because colony growth is limited by a con- stant rate of outward migration of the keratinocytes located

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in a thin rim at the perimeter of the colony. When the cul- ture conditions are changed, the radial increase in rocked cultures ranges from 0.18 to 1.79 mm per day; the most im- portant determinant is the presence of EGF or TGF-a.

As implied by earlier experiments (Rheinwald and Green, 1977), the effects of EGF and TGF-a show that these factors cannot be viewed as simple mitogens, as has generally been supposed. Effects of EGF on cell mo- tility have been reported earlier, but not related to cell mul- tiplication. For example, the rapid induction of lamellipo- dia and filipodia by EGF (Chinkers et al., 1979) may be related to enhanced migration. Directed migration of cul- tured intestinal epithelial cells increases up to lOO-fold in the presence of EGF (Blay and Brown, 1985). In small ker- atinocyte colonies, cell growth is exponential and not af- fected by the presence of EGF or X%-a. This supports the idea that these agents affect multiplication only when cell migration becomes a limiting factor in the expansion of the colony. EGF and TGF-a may therefore be looked on as migration factors whose action is essential to sustained proliferation. The evidence for this interpretation is as fol- lows. First, advance of the perimeter of a large colony re- quires cell migration of up to 70 cell diameters per day. Second, addition of TGF-a or EGF to a colony grown in its absence produces detectable colony expansion within 15 min and marked expansion at 3 hr. These effects are much too rapid to be the result of increased proliferation. Third, the rate of increase of colony radius is constant in large colonies both in the presence and absence of the growth factors as would be expected if advance of the pe- rimeter depended on a fixed rate of migration. Finally, pulse-chase experiments show that the rim of intense proliferation close to the perimeter contains the most migratory cells.

Comparative measurements show that, as in some re- sponses by other cells types (Stern et al., 1985; lbbotson et al., 1986) TGF-a is a more powerful agent than EGF. The response of keratinocytes to EGF or TGF-a is very specific since, in contrast to fibroblasts (Hollenberg and Cuatrecasas, 1973), no other growth factors including serum are able to substitute for them (Rheinwald and Green, 1977). An agent of this class is therefore essen- tial to the large scale cultivation of keratinocytes for grafting humans with epidermal defects (Gallico et al., 1984).

FigureZ Cell Migration during Colony Ex- pansion

,*-7 /--- ““r. ,/’ ,A I I

,A , I j -. --._ Diagram shows the advance of the colony pe-

,/’ 1 ,’ 1 ‘\ rimeter from left to right during the period of

-----L?...-, I I ,.--:1-i._.-- / I I I

1 1 day in unrocked cultures. The rim of intense

I I I proliferation migrates outward 1 mm fin the presence of TGF-a and without rocking): Since the cell density in the rim is twice that of the in-

I r im ; I r im I

day x / ; dayxtl 1 terior of the colony and the cells may double in less than 17 hr, enough progeny may be left be-

I (2.9xlO~ j- I.4 x 10’ cells/mm2 I

-1 (2.9x10’ I hind the advancing rim to cover much of the , cells/mm? I j cells/mm’) I I zone traversed by the rim. A few cells in this I k 0.2mm * I

0.8 mm ye 0.2mm >/ zone may originate by migration of cells origi- nally located behind the zone of rapid prolifera- tion but within 1 mm of the perimeter. The zone of stratification and terminal differentiation in the colony lies to the left of the diagram.

Cell 1136

These experiments strongly support the projected use of EGF or TGF-a as an agent promoting epidermal wound healing (Brown et al., 1986; Schultz et al., 1987). The pe- rimeter of a megacolony separates a zone of coherent epi- thelium from a cell-free zone to be covered by cell migra- tion and proliferation. This discontinuity resembles the edge of an epidermal wound (Odland and Ross, 1968) where cell migration is similarly required to extend the epidermal covering. The rate of this process should be much increased if the cells are exposed to EGF or TGF-a. For optimal effect, this exposure must be continuous, as experiments on megacolonies have shown that the effect of TGF-a and EGF is of very short duration: when they are withdrawn from the medium, the rate of colony expansion during the subsequent day is reduced by about 90% to what is typical in the absence of the growth factor. It seems unlikely that constant exposure to optimal concentrations of EGF or TGF-a has been achieved in animal experi- ments. Nevertheless, studies on the rate of epidermal regeneration following partial thickness burns, while only semiquantitative, have shown the effectiveness of EGF and TGF-a as well as the greater potency of TGF-a (Brown et al., 1986; Schultz et al., 1987).

Measurement of cell growth and migration in isolated colonies may be used to measure with precision the bio- logical effects of modification of the EGF or TGF-a mole- cule and to screen for other agents active in epidermal wound healing. The megacolony also seems well suited for investigation of the properties of TGF-a that permit it to exert a stronger response than EGF at the very same receptor.

Experimental Procedures

Cultivation of Epidermai Keratinocytes Cultivation was performed as previously described, using lethally ir- radiated 3T3 ceils to support multiplication (Rheinwaid and Green, 1975). The composition of the medium has been described (Aiien- Hoffmann et al., 1984; Simon and Green, 1985). The medium was changed every 3 days. Keratinocytes were obtained from trypsinized secondary cultures of strain AY, derived from the foreskin of a newborn.

Single Ceil isolation A single ceil was aspirated into a Pasteur pipet under an inverted mi- croscope (Barrandon and Green, 1985). it was then carefully deposited at the center of a culture vessel containing lethally irradiated 3T3 ceils. If f lasks were used, they were allowed to stand for 30-45 min to permit the single ceils to settle before the flasks were returned to the incu- bator.

Measurement of Colony Area A microscopic colony was photographed, and its area was measured with an image-analyzing system (Microplan ii, Nikon) on the photo- graphic print. The value was corrected for microscopic and photo- graphic enlargement. The perimeter of a macroscopic colony was out- lined on the bottom of the culture dish under a binocular microscope. The outline was then transferred to a piece of paper from which the area measurement was made using the digitizing tablet.

Staining Cultures were routinely fixed with 3.7% buffered formaldehyde and stained with 1% rhodamine B. For counting cell nuclei, living or fixed colonies were stained with 10m6 M Hoechst nuclear dye 33342 for 30 min at 3pC (Lalande et al., 1981). The nuclei of small colonies were counted directly under a Zeiss microscope equipped with epifiuores- cence, using a 25x water immersion objective or a 10x objective. Ker-

atinocyte nuclei were identified by their homogeneous fluorescent pat- tern characteristic of human cells, in contrast to the spotted pattern of (mouse) 3T3 cells. if a colony contained too many nuclei for direct counting, it was photographed using 3000 ASA high speed Polaroid film and the nuclei were counted on the print. Where the colony was very large, composite photographs were made.

EGF and TGF-a in most experiments, both agents were added to cultures beginning on the third to sixth day after inoculation of the founding keratinocyte. Hu- man EGF (Urdea et al., 1983) derived from yeast was a gift of Dr. Pablo Vaienzuela of Chiron inc. Human TGF-a, (Derynck et al., 1984) derived from E. coli was kindly provided by Drs. Rik Derynck and Marjorie Winkler of Genentech. Inc.

Removal of Parts of a Colony After the medium was removed from a petri dish containing a single megacoiony, the dish was inverted and the perimeter of the megacol- ony was outlined on the bottom under a binocular microscope. The area of the colony was divided into four sectors. The dish was then rein- verted, and the inner portion of some sectors was defined by cutting the colony with a warm scalpel blade. The epitheiium covering the in- ner portions was then gently removed with a rubber policeman, and any residue was washed away. The remainder of the megacoiony was then fed with medium containing the growth factor and allowed to grow for 2 more days before the colony was fixed and Stained with 1% rhoda- mine B. The increase in radius of each sector was then calculated from the increase in the area.

Autoradiography Each culture was washed 3 times with the Duibecco-Vogt modification of Eagle’s medium (serum-free). It was then fed with this medium sup- plemented with all the additives used in regular cultivation and human TGF-a at 30 nglml. Five uCi of [methyi-14C]thymidine (53.9 mCi/mMol, New England Nuclear) was then added. After 24 hr, each culture was washed with PBS and processed according to Stein and Yanishevsky (1979). It was then covered with a thin layer of NTBP nuclear track emulsion (Kodak) and exposed for 7 days at 4OC. The emulsions were developed with D19 developer (Kodak) and fixed. The ceils were then stained at room temperature with the nuclear dye Hoechst 33342.

To check that the radiographic images were not affected by absorp- tion of l3 particles in the epitheiium, representative colonies were em- bedded in poiybed-araldite, detached from the dish, and exposed to photographic emulsion at the basal surface (Dover and Potten, 1983). The appearance of the radioautographs was identical to those ob- tained in the more usual manner.

These investigations were aided by grants from the National Cancer institute and the National institute of Arthritis, Diabetes, Digestive and Kidney Diseases and by a gift from Johnson and Johnson. We are grateful for crit icisms by Drs. Harvey Greenspan and Rik Derynck and the skillful technical assistance of Karen W. Easley and Jennifer Gill.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received May 1, 1987; revised July 9, 1987.

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