JOURNAL OF CELLULAR PHYSIOLOGY 152302-309 (1992)

Epidermal Growth Factor Dependence and TGFa Autocrine Growth Regulation in Primary Rat Tracheal Epithelial Cells PATRICE C. FERRIOLA,* ALICE T. ROBERTSON, DAVID W. RUSNAK, RICHARD DIAUGUSTINE, AND PAUL NETTESHEIM €pithe/ia/ Carcinogcn~sisGroup, Ldboratories of Pulmonary Pathobiology (P.C.F., A.T. R., D.W.R., P.N.) and Biochemical Risk Analysis (R.D.1, National Institute of Environmerital Health Sciences, Research Triangle Park, North Carolina 27709 We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a T L F a autocrine pathway is operating in these cells. Primary KTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGFiBPE (CSFM). Removal of EGFiBPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology i s significantly altered compared to cells propagated in CSFM. Addition of ECF and/or BPE to cultures propagated in SFM minus ECFIBPE restores maximum cell density. The concentration of TGFa peptide in media conditioned by cells propagaied without EGFIBPE is lower than the concentration in the media of CSFM cultures. TGFa mKNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGFiBPE i s inhibited by neutralizing TGFa antiserum and by a tyrphostin compound that blocks TGFaiEGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize T t i F a as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGFiBPE for sustained proliferation. 0 1992 Wiley-Liss, Inc

The peptide growth factor TGFa, a structural and progress t o a neoplastic phenotype (Ferriola et al., functional homologue of EGF, has been implicated in 1990). We have recently demonstrated autocrine the proliferation of many cancer cells via autocrine growth regulation by TGFa in chemically transformed growth regulation (see Derynck, 1988). The autocrine RTE cells (Ferriola et al., 1991). These neoplastic RTE hypothesis proposes that neoplastic cells develop auton- cells do not require exogenous EGF for proliferation in omy from normal growth constraints by overexpressing vitro, secrete TGFcu, and express functional TGFaiEGF and utilizing their own growth factors (Sporn and Rob- receptors. Neoplastic RTE cell proliferation is deerts, 1985). Most studies of the TGFa pathway have creased by TGFa antiserum and by tyrphostin comfocused on its function in transformed or neoplastic pounds that inhibit tyrosine kinase activity of the cells and i t is presently unclear what role TGFa plays TGFaiEGF receptor. These data indicate that cndogein proliferation of normal cells. Expression of TGFa and nous TGFa can mediate the proliferation of neoplastic its receptor have been detected in normal cells and tis- RTE cells in the absence of exogenous EGF or TGFa. In sues, including the brain (Wilcox and Derynck, 19881, contrast to neoplastic RTE cells, primary RTE cells regastrointestinal tract (Malden et al., 19891, kidney (Go- quire exogenous EGF for maximum proliferation in mella, et al., 1989), keratinocytes (Coffey e t al., 1987; culture even though they express TGFa transcripts Cook et al., 1991), and mammary gland epithelium (Ferriola et al., 1989). Therefore, in the present study (Snedeker et al., 19911, yet it is unclear whether TGFa we have examined EGF dependence and TGFa producis synthesized by these cells as a n autocrine or para- tion and function in primary RTE cells in order to decrine factor, or how this function changes during neo- termine whether endogenous TGFa functions differently in primary versus neoplastic RTE cells. plastic transformation. We are studying the role of EGF and TGFa signalling during multistep carcinogenesis of rat tracheal epithelial cells in vitro. The RTE cell system is well suited for examining growth factor pathways in normal cells and Received November 25,1991; accepted March 3, 1992. identifying alterations in these pathways as cells “To whom reprint requestsicorrespondence should be addressed. Q 1992 WlLEY-LISS,

INC

EFFECTS OF EGF AND ENDOGENOUS TGFa ON PRIMARY RTE CELLS

MATERIALS AND METHODS Cells and cell culture RTE cells were isolated from 10- to 14-week-old Fischer 344iNCR rats. Animals were sacrificed by CO, asphyxiation and their tracheas were removed, filled with 1%Pronase solution (type 5 , Sigma Chemical Co., St. Louis, MO), and incubated for 18-20 hours a t 4°C. RTE cells were collected through sterile nylon mesh, centrifuged, and resuspended in Ham's F-12 media containing 5 ng/ml EGF and 5 pg/ml transferrin (Collaborative Research, Waltham, MA), 5 pgiml insulin, 0.1 pgiml hydrocortisone, 0.1 pgiml cholera toxin, 50 pM ethanolamine and phosphoethanolamine, 0.8 mM CaC12, 1.5 mg/ml bovine serum albumin essentially globulin free (Sigma), 0.1 mg protein/ml of bovine pituitary extract (BPE), and 15 mM Hepes, pH 7.3 (Sigma). This medium is designated CSFM for complete serumfree media. RTE cells were plated onto uncoated 60 mm tissue culture dishes at 5 x lo4 cells per dish and propagated in a humidified 5% C0,/95% air environment a t 37°C. Media was first changed either 1or 2 days after plating and subsequently every two unless indicated otherwise. To determine morphology, attached cells were fixed with 70% ethanol for 5-10 minutes and stained with 10% Giemsa stain for 5 minutes. Cell number was determined by trypsinizing 3-5 replicate dishes and counting using a hemacytometer. Bovine pituitary extract (BPE) was prepared from whole pituitaries (Pel Freeze, Rogers, AK) by homogenization of pituitaries in a Waring blender in phosphate-buffered saline, centrifugation, and filtration of supernatant. Protein content was determined by BioRad protein assay (Bio-Rad Laboratories, Richmond, CAI. Protein concentration was between 8 and 15 mg/ml in the stock solution. When indicated, EGF and BPE were omitted from growth media and medium was designated SFM--E/-B. Both EGF and BPE were removed since BPE preparations may contain EGF or TGFa-like proteins (Kobrin et al., 1987) and since we have previously demonstrated that BPE can supplant EGF requirements by primary RTE cells (Ferriola et al., 1989). Analysis of TGFa RNA expression Total cellular RNA isolated from primary RTE cells was isolated by the method of Chirgwin et al. (19791, and the level of TGFa gene expression was determined by northern analysis of total RNA (Lehrach et al., 1977). Total RNA (10-20 pg) was electrophoresed through 1%agaroseiformaldehyde gels, transferred to Nitroplus 2000 (MSl, Inc., Westboro, MA), and hybridized to "P-y-dCTP labeled rat TGFa cDNA as described previously (Ferriola et al., 1989). The rat TGFa cDNA was a 2.3 kb ECO R1 fragment which included the coding sequence of the TGFa precursor and part of a 3'untranslated region (Lee et al., 1985). Equal loading of RNA samples was assessed by photographing ethidium bromide-stained gels prior to transfer and using a laser densitometer to scan the negatives. Autoradiographs of the Northern blots were also scanned by the densitometer and gene expression was normalized to RNA loading. The use of housekeeping genes such as GAPDH or actin to control for RNA loading was not

303

advisable since we found t h a t the expression of these genes in RTE cells changed with time in culture (not shown). Measurement of TGFa immunoreactivity in conditioned media Cells were propagated in CSFM or SFM--E/-B and media was changed every 48 hours. At various times of culture, media from 2-3 dishes were collected and pooled and cultures were washed with 1volume of PBS, which was added to pooled media. Protease inhibitors were added and media was frozen at -20°C until processing. Media was concentrated 15-fold using Centricon spin tubes (3,000 MW cutoff, Amicon, Danvers, MA). Concentrated media was then assayed for TGFa activity using a commercially available RIA kit according to manufacturer's specifications (Peninsula Labs, Belmont, CAI. Cell proliferation a s s a y s The effect of tyrphostin and TGFa antiserum on 'Hthymidine incorporation by RTE cells was determined using a method described by Freshney (1986). Triplicate cultures were treated for 24 hours with control media or media containing tyrphostin or TGFa antiserum. During the last four hours of treatment, 3H-thymidine (final activity, 5 pCi/ml; Amersham, Arlington Hts., IL) was added to the cultures. At the end of the incubation period, cells were washed with cold 10% TCA and lysed in SDS/NaOH. Acid insoluble radioactivity was determined by scintillation counting. Cell counts were determined for each treatment and incorporation expressed as DPM/105 cells. For studies examining the effect of chronic tyrphostin treatment on RTE cell proliferation, tyrphostin was added to cultures propagated in either CSFM or SFM3 - B on day 2 after plating, and fresh tyrphostin was added with media changes every 2 days a s indicated in results. At the indicated time in culture, cells from triplicate dishes per condition were trypsinized and counted using a hemacytometer. The tyrphostin EGF-receptor kinase inhibitor 3, 4,dihydroxy benzylidine malonitrile (Tyr-8) (Yaish et al., 1988) was synthesized as previously described (Ferriola et al., 1991) and dissolved in ethanol as a n 0.02 M stock solution. Ethanol in culture media did not exceed 0.15% and was added to control cultures. Rabbit polyclonal antiserum to rat TGFa was purchased from Peninsula Labs. According to the supplier, the antiserum had a n IC50 of -30 pgitube in a homologous radioimmunoassay at a dilution ofl:10,000. A synthetic TGFa peptide (amino acids 34-50) was competitive in this radioimmunoassay (unpublished observation). Lyophilized antiserum was reconstituted in distilled water and dilutions from 1:500 to 1:3,000 were used for TGFa neutralization studies. Control cultures contained dilutions of normal rabbit serum (GIBCO Lab, Grand Island, NY).

RESULTS BPE and EGF dependence of p r i m a r y RTE cells Proliferation of p r i m a r y RTE cells cultured in CSFM versus SFM--E/-B. All experiments designed to study EGF dependence of primary RTE cells

FERRIOLA ET AL.

304

I

41 c11

106

1

P ( 1 85%)

?

. ( 1 00%)

LT W

m

5

CSFM

z

1044

0

,

,

,

10

, 20

DAYS IN CULTURE Fig. 1. Time course of RTE cell proliferation in the presence or absence of EGF and BPE in growth media. Cells were plated at 5 x lo4 cellsidish in CSFM ( 0 )or in SFM--E/-B (01. For other conditions, cells were plated in CSFM and then media was changed to SFM--Ei-B on day 1 (O), day 3 (A1 or day 6 (D) after plating. Cells from 3-5 dishes each were counted per point. For all conditions, media was changed every 2 days.

included removal of BPE as well as EGF since we have previously shown that BPE can supplant EGF requirements by primary RTE cells (Ferriola et al., 1989) and other studies have reported that bovine pituitary cells contain TGFa immunoreactivity (Kobrin et al., 1987). In a receptor competition assay using EGV5T neoplastic RTE cells, BPE inhibited I2'1-EGF binding (data not shown). Using TGFa to construct a standard curve, we have calculated that in CSFM containing 8-15 mgiml of BPE protein there is 0.2 to 0.4 ng/ml(O.O3-0.06 nM) of TGFa-like activity. Growth curves for RTE cells plated and propagated in CSFM versus SFM--E/ B are shown in Figure 1. RTE cells plated in SFM--E/-B demonstrate very limited proliferation and do not exceed a cell density of 2 x lo4 cellsi60 mm dish (1,000 cells/cm2).However, when cells are first plated in CSFM and subsequently propagated in SFM- E/-B, significant proliferation occurs, although maximum cell density is reduced compared to cultures propagated continuously in CSFM. There is no significant difference between maximum cell densities when EGF and BPE are removed 1, 3 , or 6 days after plating. Proliferation of cultures propagated in SFMEKB begin to deteriorate after 4 weeks in culture, whereas proliferation of CSFM cultures can be sustained for at least 8 weeks (data not shown). These proliferation characteristics were dependent on a frequent schedule of media changes (every 2 days). To determine whether cultures propagated in SFM-E/-B were still responsive to the proliferative effects of exogenous EGF, we added CSFM or SFM containing either EGF or BPE to cultures propagated in SFM--Ei -B. Addition of exogenous EGF, BPE, or both to primary RTE cells propagated in SFM--E/-B for 3-14 days allows cells to reach plateau cell densities similar

SFM--E/-B

I

Kf

BpE

CSFM

MEDIA ADDITION ON DAY 6 TO SFM-UB CULTURES

I

Fig. 2. Effect of EGP, BPE or CSFM addition on proliferation of primary RTE cells propagated in SFM--E/-B. Cells were plated a t 5 x lo4 cells/dish in CSFM. Media was replaced 1 day after plating with either fresh CSFM or SFM--E/-B. Six days after plating, cultures propagated in SFM--Ei-B received either CSFM or SFM containing EGF 150 nglml) or BPE ( 2 X ). Cell numbers were determined on day 10. Values from a representative experiment in which 3-5 replicate dishes were counted are shown. Numbers in parenthesis above bars represent proliferation as a percent of contro1 (CSFM) proliferation.

to the densities of cells propagated continuously in CSFM. Figure 2 shows a representative experiment in which primary RTE cells plated in CSFM and switched to SFM--E/-B on day 1received either 50 ngiml EGF, 2x the control BPE concentration or CSFM on day 6. Cell numbers were determined on day 10 and compared to cultures propagated in CSFM. Morphology of p r i m a r y RTE cells cultured in CSFM v e r s u s SFM--E/ B Figure 3 shows that both cell (Fig. 3D) and colony (Fig. 3B) morphology of cultures propagated in SFME/-B are significantly different from the morphology of cultures propagated in CSFM (Fig. 3A,C) (EGF and BPE were removed 1day after plating cells). At day 9 in culture, colonies of cells propagated in CSFM are larger than those in cultures in SFM--E/-B and consist of a heterogeneous population of spread cells and more compact, densely staining cells. In contrast, colonies from cultures propagated in SFM--E/- B are more homogeneous and consist of mostly compact, densely staining cells with a larger nucleus to cytoplasmic ratio than cells propagated in CSFM. Removal of EGF alone resulted in similar alterations in morphology (data not shown). Cells propagated in CSFM show very abundant and organized actin stress fibers, whereas cells propagated in SFM--E/-B show disorganized and diffuse actin staining (data not shown, P.C. Ferriola and C. Carter). As with the effects of EGFiBPE on proliferation. addition of EGFiBPE to cultures propagated in SFM--E/-B at any time from midlog through plateau induces a reversal of the SFM--E/-B morphology such that cells and cultures look similar to cultures propagated in CSFM (data not shown).

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EFFECTS OF EGF AND ENDOGENOUS TGFa ON PRIMARY RTE CELLS

A

B

C

D

Fig, 3. Morphology of primary RTE cultures and cells propagated in CSFM or SFXI- E:- B. A and B show primary RTE cultures 9 days after plating. The cells in A were propagated in CSFM with media changes every other day. Cells in B were propagated in SFM--E/-B beginning one day after plating. C and D show lOOx magnifications of the cultures in A and B, respectively.

Inhibition of primary RTE cell proliferation by TGFa antiserum and tyrphostin In order to determine whether the proliferation observed for primary RTE cells in the absence of exogenous EGF/BPE is dependent upon autocrine TGFa, we used TGFa antiserum and a TGFaiEGF receptor tyrosine kinase inhibitor, Tyr-8, to inhibit effects of the endogenous factor. These agents have been used previously to inhibit TGFa signalling in transformed RTE cells (Ferriola et al., 1991). The effects of a n antirat TGFa antiserum (which is not immunoreactive with EGF) on 3H-thymidine incorporation by RTE cells propagated in CSFM or SFM--E/-B are shown in Figure 4. The mean 3H-thymidine incorporation for cultures propagated in CSFM was 5.9 2 2.7 x lo4 DPMI10' cells and 5.8 2.9 x lo4 DPM/105 cells for cultures propagated in SFM--E/-B. In cultures propagated in CSFM, a 1:1,000 dilution (or 1:500, data not shown) of antiserum did not significantly reduce thymidine in-

*

control

I :loo0

control

1 .lo00

]

antisera dilution

----.1

culture condition

1:500

- - - - - C S F M - - - - - -----minus BPElEGF

Fig. 4. Effect of TGFcv antiserum on proliferation of primary RTE cells. Cells were plated at 5 x 10" cellsidish in CSFM. Media was changed 1 day after plating and then every other day. Half of the cultures received SFM--E/-B on day 1. On day 7 cultures were exposed to TGFa antiserum or normal rabbit sera for 24 hours. 3Hthymidine incorporation was measured as described in Materials and Methods. Values represent means i the SD for 2 experiments.

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TABLE 1. Effect of Tyr-8 on proliferation of primary RTE cells' % decrease in thymidine incorporation (Mean +S.D.)

"l'yr-81 Media CSFM

0

0 55 -e 12

10

87

30 0 10 30

SFM--E/-B

5

10

0 18 t 15 71 ? 16

'All cells were plated in CSFM at a r e l l density of 6 X lo4 cellddish. One day aftcr plating, media was changed and half the cultures received SFM--E:-B media. All cultures subsequently received fresh media every other day Five days after plating. cultures received 0, 10, or 30 FM Tyr-8 with frcsh media; 20 hours later, 3H-thymidine was added to cultures and incorporation assayed 4 hours later as described In Materials and Methods. Data represent mean values from 5 separate expcrimcnts.

TABLE 2 Accumulation of TGFa in media conditioned by primary RTE cells] Growth ohase

Cellsidish i x lo5)

ng TGFcti106cells

CSFM log plateau MINUS BPEIEGF 1% plateau

4.3 t 0.1 21.0 * 4.0 1.4

3.4

i 0.8 L 1.4

0.98

* 0.07

0.23

+

0.09"

1.65 i 0.27 0.40 ? O.O6*

[TGFa] (OM) 18.2 t 2.1 17.4 i 2.3 8.1 ? 4.2"* 6.7 ? 3.1**

'Cells were plated a t 5 x 104cells/dishin CSFM. Media was changed 1day after plating and then every other day. Half of the cultures received SFM--E/-B media on day 1. Media was collected for assay by RIA a s described In Materials and Methods. Cultures in log phase of growth were between %lo days postplating and cultures in plateau phase were between 18-22 days postplating. Values represent the mean 2 S.D. of 3 cxprriments. *Significantly different from log phase value. P .: 0.01, **Significantly difkrenl from CSFM values,P < 0.05

5-10 pM for cells in CSFM and between 2-5 pM for cells in SFM- E/-B.

TGFa expression by primary RTE cells TGFa accumulation in conditioned media. TGFa accumulation in media conditioned by primary RTE cells propagated in CSFM or in SFM--E/-B was measured by RIA. The results summarized in Table 2 104 show that the amount of TGFa accumulation per lo6 cells is 4-fold higher during logarithmic growth than during plateau growth regardless of the culture medium used for cell propagation. However, the concen103 tration of TGFa in the media is essentially the same in both log and plateau phase cultures. Cells propagated in SFM--EI-B accumulate slightly higher levels of TGFa protein per cell compared to accumulation in 102 I CSFM cultures, although the TGFa media concentra0 10 20 tion in SFM--E/-B cultures is -50% lower than the concentration of TGFa in CSFM cultures. ITYRPHOSTIN] pM TGFa mRNA expression. Figure 6 (left panel) Fig. 5. Effect of chronic Tyr-8 exposure on proliferation of RTE cells. shows the northern analysis of TGFa RNA from RTE Cells were plated i n CSFM a t 5 x lo4 cellsidish. Media was replaced two days after plating with either fresh CSFM (0)or SFM--Ei-B ( 0 ) cells propagated for 3 weeks in CSFM or SFM--EI-B (EGFIBPE removed 24 hours after plating cells). Levels with or without t h e concentrations of Tyr-8 indicated. Media was changed every other day with or without Tyr-8 a n d cell numbeddish of TGFa message are similar in cells propagated in the was determined for all conditions on day 11after plating. two different media. TGFa transcript levels in cells plated in SFM--E/-B are also similar to levels in CSFM cultures (data not shown). corporation, presumably because exogenous EGF was Since the amount of TGFa accumulation per cell deavailable to mediate proliferation. In contrast, antise- creases as a function of time in culture (Table 2) we rum dilutions of 1:1,000 and 1:500 decreased thymidine examined whether TGFa RNA levels in primary RTE incorporation by -50% in cultures propagated in SFM- cells change over a 5-week time course in culture. A representative northern blot is shown in Figure 6 (right -E/- B. The effects of the tyrphostin, Tyr-8, on proliferation panel). Levels of TGFa RNA decrease 70% following of primary RTE cells propagated in CSFM and SFM- the first week in culture and remain a t this decreased -E/-B are shown in Table 1 and Figure 5. The mean level for the remainder of the time course. In the course 3H-thymidine incorporation for the experiments in- of these studies we found that the total RNA yield (pg cluded in Table 1was 1.4 * 0.4 x lo5 DPM/105 cells for RNA/107 cells) for primary RTE cells decreases significultures propagated with CSFM and 6.1 5.3 x lo4 cantly as a function of time in culture. Thus the amount DPM/105 cells for cultures propagated with SFM--EI of total RNA1107 cells recovered on day 6 is 8-fold -B. Addition of 10-30 pM Tyr-8 to cultures signifi- higher (433 p.g/107 cells) than on day 35 (53 pg/107 cantly inhibited 3H-thymidine incorporation by cells cells). propagated in CSFM and in SFM--EI-B. Likewise, the DISCUSSION proliferation of RTE cells was inhibited by chronic (8 Two objectives of this study were to examine the EGF days) treatment with Tyr-8 as shown in the dose response curve in Figure 5. Chronic Tyr-8 treatment sig- dependence of primary RTE cells and to determine nificantly inhibited proliferation of RTE cells propa- whether TGFa is an autocrine growth factor for these gated in CSFM and SFM--E/-B with IC50's between cells. We have previously demonstrated that proliferalo5

I

+

EFFECTS OF EGF AND ENDOGENOUS TGFa ON PRIMARY RTE CELLS

307

- TGFa 4.5 kb

Growth CSFM minus Condition: EGFBPE

Daysin CuIture:

6

14

21

28

35

Fig. 6. Northern analysis of TGFa expression in primary RTE cells. Cells were propagated in CSFM or in SFM--E/-B and total RNA extracted and electrophoresed as described in Materials and Methods. Ethidium bromide staining of total RNA is shown below blots.

tion of primary RTE cells is dependent on exogenous rum and by a tyrphostin compound. Our results, thereEGFiBPE even though these cells produce TGFa (Fer- fore, indicate that primary RTE cells, like neoplastic riola et al., 1989). In this previous study we also exam- RTE cells, express and utilize a functional TGFa autoined EGFiBPE dependence of immortalized, neoplastic crine pathway. In contrast, however, neoplastic RTE RTE cell lines and reported t h a t in contrast to the pri- cells do not require EGF and BPE in the media at the mary RTE cells, most neoplastic RTE cells did not re- time of plating (Ferriola et al., 1991) and the levels of quire exogenous EGFiBPE for proliferation. We there- TGFa transcripts and TGFa accumulation in condifore hypothesized that neoplastic RTE cells utilized tioned media are greater in transformed RTE cells than endogenous TGFa, which supplanted the requirements in primary RTE cells (Ferriola et al., 1989). Therefore, for exogenous EGF/BPE, and in support of this hypoth- EGF/BPE dependence of primary RTE cells could be esis, we demonstrated that proliferation of immortal- explained in part by limitations on TGFa availability. ized RTE cells was dependent on a n autocrine TGFa This idea is supported by our data showing that addipathway (Ferriola et al., 1991). In the present study we tion of EGF to primary RTE cultures propagated in have further studied EGF dependence and TGFa pro- SFM--E/-B stimulates proliferation and higher cell duction in primary RTE cells in order to understand the densities. We also examined whether the level of TGFa producdifferences in the TGFaIEGF pathway between normal tion by primary RTE cells was regulated by different and neoplastic RTE cells. Primary RTE cells plated in media lacking EGF or growth conditions. EGF and TGFa induce TGFa exBPE do not proliferate significantly. Removal of EGF/ pression in keratinocytes (Coffey et al., 1987), hepatoBPE shortly after cell RTE plating in CSFM, however, cytes (Raymond et al., 19891, and pituitary (Mueller results in significant proliferation. Similar results et al., 1989) and in normal human mammary epithelial have been reported by Cook e t al. (1991) for human cells, removal of EGF from low density cultures results keratinocytes in which exogenous EGF is required for in decreased TGFa expression (Bates et al., 1990). initiating clonal growth, but not after colonies are es- Therefore, we examined whether TGFa production by tablished, presumably due to production of autocrine RTE cells propagated in SFM--EI-B is reduced comTGFa. We hypothesize that in RTE cultures estab- pared to cells propagated in CSFM. We found that both lished in the absence of exogenous EGF/BPE, there is mRNA expression and peptide accumulation on a per not enough endogenous TGFa available to support pro- cell basis are similar in cells propagated with and withliferation. Once cultures are proliferating, however, out EGFiBPE, suggesting t h a t EGFiBPE is not reEGFiBPE can be removed due to the availability of quired to induce or maintain TGFu production in priautocrine TGFa. In support of this hypothesis, we show mary RTE cells. However, since cell densities are lower that proliferation of primary RTE cells propagated in for cultures propagated in SFM-- Ei B, the TGFa conSFM--E/-B is inhibited by a neutralizing TGFa antise- centration in the media is -50% lower than in the

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media of CSFM. This lower TGFa concentration may limit proliferative capacity of the cells if secreted TGFa induces a proliferative effect on the entire culture rather than by a direct effect on the individual cell synthesizing the factor. Finzi et al. (1987) studied this indirect (“feeding”) versus direct effect of secreted TGFa in NIH3T3 cells transfected with human TGFa and concluded that growth stimulation by TGFa was mediated by the culture effect, possibly due to the rapid secretion and diffusion of TGFa away from the cells producing the growth factor. Therefore, lower concentrations of TGFa in the media of cells propagated in SFM--EI-B compared t o CSFM may contribute to lower cell densities. Regardless of whether RTE cells are propagated in SFM--E/-B or in CSFM, TGFa secretion per cell is highest during the rapid log growth phase and decreases as proliferation attenuates in plateau phase. TGFa RNA levels also decrease as a function of time in culture. Mechanisms for this decrease in TGFa production as a function of time in culture are unknown, and we are currently investigating how this regulation occurs. Cook et al. (1991) have indicated that TGFa production decreases in confluent normal human keratinocytes in culture, suggesting that this phenomenon may occur in other normal epithelia. However, Bates et al. (1990) found no decrease in TGFa expression in normal mammary epithelia in culture as a function of growth state. Interestingly, transformed RTE cells do not down regulate TGFa production in the same way as primary RTE cells, indicating that there are differences in regulation of TGFa between primary and transformed RTE cells (Robertson, Ferriola, and Nettesheim, unpublished observations). We are also investigating the level of membrane bound versus secreted TGFa in primary and transformed RTE cells in order t o examine whether cell associated TGFa can function in an autocrine capacity. Although production of TGFa by primary RTE cells decreases with time, the concentration of TGFa in the culture medium remains the same in both log and plateau cultures, since the cell density is higher in plateau phase cultures (in contrast, TGFa concentrations are higher in transformed RTE cell plateau cultures; Robertson et al., in preparation). If there are concomitant changes in the TGFaiEGF receptor affinity or number with time in culture, TGFa availability to its receptor might change. Studies by Rizzino et al. (1990) and Holley et al. (1977) show that TGFaiEGF receptors are down-regulated as cell cultures reach plateau cell densities. We have found no difference in the level of receptor mRNA over growth in culture (unpublished results) but are currently examining whether receptor kinetics change in primary RTE cells as a function of time in culture in order to determine whether down regulation of TGFa production in primary RTE cells is relevant t o overall cell proliferation and survival. In addition t o affects on proliferation, EGFiBPE removal significantly affects the morphology of primary RTE cells and colonies, suggesting that the exogenous factors play a role in extracellular matrix production andior cytoskeleton integrity of RTE cells. EGF has been shown to affect the spreading and migration of epithelial cells from liver (Bade and Feindler, 1988),

intestine (Blay and Brown, 19851,and in keratinocytes (Barrandon and Green, 1987) by modulating synthesis of extracellular matrix components. Although some studies suggest that EGF effects on cell spreading and proliferation may occur independently (Yardin et al., 1982), it is well known that cell shape is a critical regulator of cell proliferation (Folkman et al., 1978; Gospadarowitz et al., 1978). Presently we are examining the effect of EGF and TGFa on several extracellular matrix and cytoskeletal proteins to determine the mechanisms involved in EGF-induced morphological changes in primary RTE cells. We find significant disorganization of extracellular matrix and cytoskeletal components in cultures propagated without EGF (P.C. Ferriola and C.A. Carter, unpublished observations), indicating that whereas the amount of TGFa produced by primary RTE cells can support cell proliferation, it is not sufficient to maintain normal cell shape. In conclusion, primary RTE cells are able to utilize an autocrine TGFa pathway, which appears to be regulated as a function of cell growth. In contrast to neoplastic RTE cells, the level of TGFa produced by primary RTE cells is not sufficient to support maximum proliferation, since exogenous EGFiBPE induces higher cell densities and prolonged culture survival. In addition, the morphology of RTE cultures propagated without exogenous EGF is significantly different from cultures propagated with the growth factor. These data indicate significant differences between the TGFlv autocrine pathway in primary RTE cells and transformed RTE cells, indicating that there are alterations in regulation of components of the TGFa autocrine pathway as RTE cells progress to the neoplastic phenotype.

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