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[CANCER RESEARCH 46, 917-921, February 1986] Inhibition of Carcinogen-altered Rat Trachea! Epithelial Cells by Normal Epithelial Cell-conditioned Medium1 M. Terzaghi-Howe2 and C. McKeown Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 ABSTRACT There is mounting evidence that normal cells can either inhibit the growth of carcinogen-altered cells and/or affect progression to a neoplastic phenotype. This effect(s) has been observed both in vivo in intact rat trachea! tissues and in rat trachéalepithelial cell cultures. The inhibition of carcinogen-altered cells in culture appears to be associated with the production of an acid and heat stable, dithiothreitol sensitive, nondialyzable protein pro duced by normal trachea! epithelial cells or esophageal epithelial cells in primary culture. It was found to be optimally produced by 3-4-week-old cultures of normal epithelial cells. In the pres ence of a 1:4 dilution of normal cell conditioned medium, the colony forming efficiency of a sensitive cell line is decreased 5fold. Biochemical properties of the inhibitor are similar to those associated with type ßtransforming growth factor. inhibitors as mediators of the observed cell interaction. In experiments carried out previously in our laboratory we observed that the frequency of dimethylbenz(a)anthracene-exposed tracheas which contained potentially neoplastic cells, de tected in culture, far exceeded the frequency of tracheas which ultimately developed tumors over the life span of the animals (16,17). These data suggest that in the intact tissue only a small fraction of the "initiated" potentially neoplastic cells ever ex presses the neoplastic phenotype. It is likely that this observation reflects important controlling effects exerted by the surrounding normal cells as well as hormonal and immunological effects on potentially transformed populations. In this report we evaluate the influence of primary trachea! and esophageal epithelial cell cultures on the survival and growth of cultured carcinogen-altered trachéalepithelial cells. Normal cells inhibited carcinogen-altered cells in culture indirectly by means of a dithiothreitol sensitive, heat and acid stable, non-dialyzable inhibitor present in normal-cell conditioned medium. INTRODUCTION There has long been evidence suggesting that the behavior of "initiated," preneoplastic as well as neoplastic cells is determined not only by the genetic makeup of the cell but also by the environment. Of particular interest is the possibility that sur rounding normal cells might be capable of limiting proliferation and/or expression of the neoplastic phenotype of carcinogenaltered cell populations (1, 2). Both in vivo (1-5) and in culture (6-8) there is evidence that preneoplastic as well as neoplastic cells can be affected by surrounding normal cell populations. It is not entirely clear whether the influence of normal cells on altered-cell behavior is mediated via a direct effect requiring close cell contact and/or an indirect effect involving stable diffusible factors. Similarly it is not clear that interactions observed in vivo and in culture involve a common mechanism(s). One possible mode of interaction involves formation of gap junctions between 2 cell populations (e.g., 9, 10). There have also been reports of diffusible growth factors associated with normal cell populations such as liver (11), mammary gland (12), kidney epithelial cells (13,14), and placenta (15) which could be involved. In cell culture, interactions between normal and carcinogen-altered cells typically require close prox imity of the interacting populations (e.g., 6, 7). This would tend to implicate the importance of factors other than stable, diffusible Received 7/22/85; revised 10/22/85; accepted 10/24/85. 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 U.S.C. Section 1734 solely to indicate this fact. 1Research was sponsored by the USPHS under Interagency Agreement 401268-82, Grant 5R01 CA34695 awarded by the National Cancer Institute, Depart ment of Health and Human Services, and the Office of Health and Environmental Research, U. S. Department of Energy, under contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc. 2 To whom requests for reprints should be addressed, at Biology Division, Oak Ridge National Laboratory. P. 0. Box Y, Oak Ridge. TN 37831. MATERIALS AND METHODS Carcinogen-altered TEC3 populations were cocultured with estab lished primary cultures of normal TEC or fed with diluted CM harvested from established primary cultures of normal TEC or EEC. The effect of normal cells on carcinogen altered cells was determined by changes in colony forming efficiency and growth rate (colony size). Cells. Normal trachéalepithelial cells and cell lines used were initially obtained from 6-10-week-old F344 rats bred and housed in specific pathogen-free rooms in the Biology Division. Normal primary cultures of TEC and EEC were established as de scribed previously for TEC (18,19). Briefly, lumina were filled to extension with 0.1% Pronase (type VI, Sigma Chemical Co.) submerged in Ca2*, Mg2+ free Hanks' balanced salt solution and incubated at 37°C for 30 min. Each trachea was then flushed with 5 ml Ca2+ Mg2* free Hanks' balanced salt solution plus 0.5% bovine serum albumin. Cells were washed twice by centrifugation and seeded at a density of 103 to 104 viable cells/60-mm tissue culture dish containing 3T3 feeder cells. Feeder layers were prepared 24 hr in advance by seeding with 2.5 x 104 irradiated (5000 rad X-ray) 3T3 cells (18, 20). Preneoplastic and neoplastic TEC are carried as established lines. The lines designated NGC2 cl.1 (2), XR600 cl.6 (2). and N120 cl.5 (2)A were initiated by exposure to rV-methyl-W-nitro-A/-nitrosoguanidine, 600 rads X-ray, and 120 rads neutron exposure of F344 TEC, respectively. These lines are considered preneoplastic in that they, at the indicated passage level, do not grow in soft agarose or yield tumors in immunosuppressed syngeneic rats. The cell lines designated 8-1-2 (21), IC-12, IC-17, and 6-2 ag.cl.4 were initiated by exposure of F-344 rat trachéalepithelium to A/-methyl-A/'-nitro-A/-nitrosoguanidine (21) or dimethylbenz(a)anthracene (17). These lines are considered neoplastic in that, at the designated passages, they grow in soft agarose and yield tumors in immunosup pressed syngeneic rats. 3The abbreviations used are: TEC, trachéalepithelial cells; EEC, esophageal epithelial cells; CM, conditioned medium; RCFE, relative colony-forming efficiency; CFE, colony-forming efficiency; NTE, normal trachéalepithelial; TGF, transforming growth factor. CANCER RESEARCH VOL. 46 FEBRUARY 1986 917 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1986 American Association for Cancer Research. GROWTH INHIBITION BY NORMAL All Å“il lines and normal primary TEC and EEC are maintained in Ham's hydrocortisone (0.1 Mg/ml). epidermal growth factor (5 ng/ml), transferrin (50 Mg/ml), 3 x 10~5 mivi selenium, pyruvate (100 Mg/ml). putrescine, lipoic acid, linoleic acid, and amino acids (22), and gentamycin (50 ßg/ ml). Unless otherwise specified, chemicals were purchased from Sigma Chemical Co., St. Louis, MO. Medium was changed every 3-4 days. Coculturing of Normal and Carcinogen-altered TEC. Normal primary cultures established as described above were used to investigate pos sible direct effects of normal cells on cocultured carcinogen-altered cell lines. At 1-4 weeks after seeding 103 to 2.5 x 103 normal TEC, 50 Age of primary cultures (weeks)81-2 2-3 3-4No. of nor mal TEC plated*103 no. of normal TEC colon ies012 2.5 x 103 103 2.5 x 103 103 2.5 x 103Average 31 6 20 3 4Relative colony efficiency0IC-1 forming 2e0.6 0.25 1.1 0.80.4 0.05 0.6 0.25 0.2 0.35XR600'0.30.15 Duration of cocultures in terms of age of primary cultures of normal TEC. All cultures were seeded with IC-12 or XR600 at the beginning of each interval and scored 1 week later, i.e., at the end of each interval. b Number of normal trachéalepithelial cells plated at time 0. c Number of normal TEC colonies with some proliferating cells remaining. All colonies remaining by 3-4 weeks primarily consist of terminally differentiated cells which are insoluble in SDS and /3-mercaptoethanol. The average diameter of the persisting colonies ranged from 2-3 mm in 1-2-week cultures to 6-8 mm in 3-4week primary TEC cultures. Average of triplicate dishes for each point. dRCFE = CFE (number of colonies -t- number of viable cells seeded) in clonogenic units of the test cell line were seeded into the normal TEC primary culture dishes. Controls were seeded into empty tissue culture dishes or dishes containing irradiated feeder cells only. At 1 week dishes were fixed and stained. The effect of normal cells on the growth and survival of the cell line was evaluated in terms of relative colony size and the relative colony forming efficiency (RCFE = CFE when cocultured with normal TEC •*CFE in control cultures). Colonies containing greater than 20 cells were scored. Normal Cell-conditioned Medium Effects on Survival and Growth of Carcinogen-altered TEC. Conditioned medium was harvested at weekly intervals from 1-5-week normal primary cultures initially seeded with 103-104 normal TEC. Medium harvested during the indicated time cocultures divided by the CRE of the cell line alone. A 10% variation in duplicate CFE dishes was observed. Thus, a compounded variation in relative CFE of 20% cannot be considered significant. For example, RCFEs of 1.1 and 0.8 are not considered to be significantly different from 1.0. 8 RCFE in neoplastic cell line IC-12. ' RCFE in preneoplastic cell line XR600. interval was stored separately and diluted 1:1 to 1:10 with complete Ham's F-12. Cultures of carcinogen-altered cell lines containing approx imately 50 clonogenic units were fed 24 h after seeding with complete Ham's F-12 (controls) or with CM diluted in complete Ham's F-12. Effects is observed when IC-12 and XR600 are cocultured with 2-3week-old normal TEC cultures. This pattern of maximum inhibi of CM on survival and growth were evaluated as described above. For each series of experiments pooled lots of CM were used and the potency was assayed in XR600 cell cultures. Only lots which gave an RCFE of 0.2 on XR600 at a 1:2 dilution were used. Specificity of Inhibitor Production. Conditioned medium was har vested from exponentially growing and confluent normal primary cultures of rat trachéalfibroblasts, XR600, IC-12, and EEC. Conditioned medium was diluted 1:4 in complete Ham's F-12 and assayed for inhibitor on the tion (lower RCFE) in early and late cultures and minimum inhibi tion (higher RCFE) in 2-3-week-old cultures was observed in 4 separate experiments involving different densities of normal cells. The larger the number of normal cells seeded, the greater the degree of inhibition of both IC-12 and XR600 (Table 1). With age, sensitive cell line XR600 and the resistant line IC-12. Characterization of the Factor(s) Present in Normal TEC Condi tioned Medium. Preliminary experiments designed to characterize the factor(s) present in normal cell CM were carried out. As above, a pooled lot of inhibitory CM was used in these experiments. Prior to assaying for inhibitor, CM was treated as follows. Maximally inhibitory conditioned medium harvested from 4-week-old primary TEC was treated as follows: (a) heated to 75-80°C for 10 min; (b) dialyzed through spectrapor membrane tubing (3787-D12; A. H. Thomas Co., Philadelphia, PA) with a pore size of 12,000 dallons against 60 volumes Ham's F-12 changed daily; (c) sequentially frozen and thawed; (d) filtered through a 0.20-Mm membrane filter; (e) acidified to pH 3.6 with 1 N HCI for 1 h at 4°C, followed by neutralization with 1 N NaOH (23); and (f) incubated at room temperature for 2 h with 0.065 M dithiothreitol followed by dialysis for 3 days in Hank's balanced salt solution to remove the dithiothreitol (23). Treated CM was then diluted, and the inhibitor level was assayed in an appropriate cell line as described above. Controls used were maintained in complete Ham's F-12 treated and diluted in the same manner. RESULTS Inhibition of Carcinogen-altered Cells Cocultured with Pri mary Cultures of Normal TEC. Primary cultures of normal TEC established for 1-4 weeks were seeded with 50 clonogenic units of a neoplastic cell line (IC-12) or a preneoplastic cell line (XR600). The results of two such experiments involving 2 densities of normal TEC are summarized in Table 1. The RCFE of both IC12 and XR600 are maximally depressed in early (1-2-week-old) and late (3-4-week-old) primary cultures. Minimal effect on CFE RESEARCH CELLS Table 1 Effect of primary cultures of normal trachéalepithelial cells on the plating efficiency of cocultured carcinogen-altered-tracheal-epithelial cells F-12 plus 5% fetal bovine serum (M. A. Whitaker, Bioproducts, Walkersville, MD), insulin (0.5 nQ/m\) (Calbiochem-Behring Corp., La Jolla, CA), CANCER EPITHELIAL a number of the colonies of normal TEC terminally differentiate and either remain attached or are shed into the medium. In all experiments the neoplastic cell line IC-12 was somewhat less sensitive to inhibition by the same density of normal TEC than was the preneoplastic cell line XR600. For example, in 3-4week-old cultures when an average of 4 TEC colonies persist, XR600 had an RCFE of 0.15, while IC-12 had an RCFE of 0.35 (Table 1). Effect of Normal Cell Conditioned Medium on the Survival and Growth of Carcinogen-altered TEC. In order to determine whether the inhibitory effect of normal cells on cocultured carcin ogen-altered cells was a direct effect (involving cell contact) or an indirect effect (mediated via diffusible stable factors), normal cell conditioned medium was tested for growth inhibition on carcinogen altered cells. The first experiment involved evaluating the influence of pri mary culture age on the production of inhibitory conditioned medium (Table 2). As in experiments involving cocultures of normal and altered cells, the cell line XR600 was more sensitive to inhibition than IC-12 (Table 2). The relative CFE of XR600 in the presence of a 1:2 dilution of 4-5-week-old CM was 0.2. In contrast IC-12 was essentially uninhibited (RCFE = 0.95). With increased age of the normal primary culture from 1-5 weeks the conditioned medium became increasingly inhibitory to XR600, with the RCFE decreasing from 1.1 to 0.2 with increasing age of NTE cultures. The RPE of line IC-12 was apparently unaffected by aging of the NTE cultures (Table 2). Likewise, addition of CM at time 0 when IC-12 were seeded did not influence the insensitivity to CM. The extent of inhibition of XR600 is also reflected VOL. 46 FEBRUARY 1986 918 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1986 American Association for Cancer Research. GROWTH INHIBITION BY NORMAL Table 2 CELLS Table3 Effect of conditioned medium harvested from 4-week normal TECcultures on a series of preneoplastic and neoplastic trachéal epithelial cell lines A pooled lot of conditioned medium from 4-5-week primary cultures of normal TEC was mixed at a 1:4 ratio with complete Ham's F-12. Cells were fed with complete Ham's F-12 (controls)or diluted CM, 24 h after seeding. Effect of conditioned medium harvested from 1-5-week-old normal cultures on the plating efficiency of trachéalepithelial cell lines Age of primary culture (weeks)81-2 EPITHELIAL colony forming efficiency6IC-12"1.1 2-3 0.6 1.1 3-4 1.0 0.26 4-5Relative 0.2 0.95XR600*1.1 " Interval for collection of conditioned medium from normal TEC in terms of age of primary culture of normal TEC. RCFE = CFE (number of colonies + number of viable cells seeded)in cultures plus conditioned medium divided by the CFE of the cell line in complete medium. Less than 20% deviation from 1.0 is not considered significant (see Table 1). c RCFE in neoplastic cell line IC-12. " RCFE in preneoplastic cell line XR600. colony forming efficiency00.2 Preneoplastic XR600cl.6(2)p.10 NGC2, d.1(2) p.21 N120d.5(2)Ap.8Neoplastic 3060 0.13 0.80.95 IC-12p.30 IC-17p.18 45 0.8 8-1-2p.65 28 1.00.8 6-2 ag.d.4 p.5CF«2625 39Relative * CFE of cell populations in complete medium without conditioned medium. Relative colony forming efficiency = no. of colonies In dishes with CM •+• no. of colonies in controls of the same ceil line. Each number is the average of 2-4 dishes. Deviations of «20%from a RCFE = 1.0 are not considered significantly different from 1.0 (seeTable 1). Table 4 Effecf of conditioned medium harvested from diverse cell populations on the relative colony forming efficiency of TEC lines Source of conditioned medium'4-5-week-old normal TEC Rat trachéal fibroblasts 1.0 1.2 Primary normal rat esoph0.951.0 0.31.0 ageal cells IC-12 XR600IC-1200.9 1.1RCFE"XR600"0.2 1.0 " Mediumconditioned for 3-5 days by the designatedcell population and diluted 1:4 in complete medium. 6 RCFE = colony forming efficiency(numberof colonies + number of viablecells Fig. 1. Culture dishes of the preneoplastic cell line XR600 (fop row) and the neoplastic cell line IC-12 (bottom row) given complete Ham's F-12 (frames A and D), Ham's F-12 plus a 1:4 dilution of normal trachéal fibroblast conditioned medium (frames B and E), or Ham's F-12 plus normal TEC conditioned medium (frames C and F). Dishes were seeded with 400 (XR600) or 200 (IC-12) cells and were fed the appropriate medium 24 h after seeding. One week later dishes were fixed in methanol and stained with Giemsa in the colony size (Fig. 1, frames A, C, D, and F). XR600 colonies are smaller when grown in diluted CM (Fig. 1, frame A versus frame C). IC-12 is essentially unaffected (Fig. 1, frame D versus frame F). In order to investigate the specificity of cell sensitivity to inhibitor a variety of preneoplastic and neoplastic cell populations were cultured in the presence of a 1:2 dilution of 4-5-week-old NTE-CM (Table 3). In general neoplastic cell populations appear to be less sensitive than preneoplastic cell populations to NTEassociated inhibitors, although one preneoplastic cell line tested [N120 cl.5 (2)] was also uninhibited. In all cases where the RCFE is decreased, colony size (i.e., growth rate) was also decreased. Specificity of Inhibitor Production. It was of interest to de termine the tissue/cell specificity of inhibitor production. Media conditioned by diverse confluent and exponentially growing cell populations were tested for production of inhibitor on the inhibi tor-sensitive line XR600 and the inhibitor-resistant line IC-12. The data indicate that inhibitor is not detected in CM of carcin ogen-altered TEC or of trachea! fibroblasts (Table 4). The results were not affected by the growth state of the cultures. In contrast, primary normal EEC appear to produce inhibitor to which IC-12 CANCER RESEARCH seeded)incultures plusconditionedmediumdividedby the colonyformingefficiency of the cell line in complete medium. c RCFE in neoplastic cell line IC-12. " RCFEin preneoplasticcell line XR600. is resistant and XR600 is sensitive (Table 4). In the presence of fibroblast conditioned medium, cell growth is enhanced in the line XR600, but no enhancement is observed in the neoplastic line IC-12 (Fig. 1, frames B and E). Preliminary Characterization of Inhibitor Present in Normal TEC Conditioned Medium. In order to characterize the inhibitor present in normal TEC medium, a uniform lot of 4-5-week-old TEC culture medium was treated with dithiothreitol, heated, acidified, frozen, filtered, diluted, or dialyzed and then tested for inhibitor activity on the inhibitor-sensitive line XR600 (Table 5). The data indicate that the inhibitor survives heating, acidification, freezing, and filtration of CM. The inhibitory CM appears to be quite potent in that dilution as much as 1:10 is associated with a small increase in RCFE from 0.2 at a 1:1 dilution, to an RCFE of 0.4 at a 1:10 dilution. Treatment of CM with dithiothreitol was found to destroy the inhibitor in that following treatment the RCFE approaches 1.0 (Table 5). DISCUSSION We have found that normal TEC and EEC populations in culture release a substance into the medium which is inhibitory to the growth of preneoplastic TEC in culture. Inhibition is observed both in terms of a decreased CFE (Tables 1 to 3) as VOL. 46 FEBRUARY 1986 919 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1986 American Association for Cancer Research. GROWTH INHIBITION BY NORMAL EPITHELIAL CELLS present in conditioned medium, are involved in the inhibition Tables Effect of various treatments on inhibitor present in normal TECconditioned observed in cell cocultures or simply that cocultures are more medium sensitive for the assay of inhibitors. Preliminary data obtained in Treatment8Control0Dialysis"Heat8AckfDithiothreitof5Freezing'1 our laboratory involving coculture of neoplastic and normal TEC in vivo in reconstituted trachéalgrafts (19, 26) suggest that both neoplastic and preneoplastic cells are inhibited by direct or close contact with normal TEC. It is interesting to speculate on the possible role, if any, of an inhibitor such as the one observed in normal epithelial cell conditioned medium, in the intact trachea or esophagus. While it is not clear what population in vivo might be analogous to those cells predominating in 3-5-week-old normal TEC cultures, Filtration'Dilution*1:11:21:41:10RCFE"0.20.30.30.31.00.2 0.20.20.30.30.4 "A pooled lot of conditioned medium collected for 1 week in 4-5-week-old primary cultures of normal TEC was treated prior to testing for inhibitor on the sensitive line XR600. 6 Relativecolony forming efficiency = CFE of XR600 in the presence of treated CM divided by the CFE in similarlytreated complete medium. c Untreatedconditioned medium tested on XR600 at a 1:1 dilution with complete medium. " Dialysisfor 3 days (daily changes of 60 volumes Ham's F-12). 8 80°for 10 min. ' Samples of CM were acidified to pH 3.5 with 1 N HCI, incubated for 1 h at 5°C,and then returned to pH 7.5 with 1 N NaOH. 9 CM was incubated at room temperature for 2 h with 0.065 M dithiothreitol followed by 3 days dialysis against Hanks' balanced salts to remove the dithio threitol. h Twice frozen and thawed. ' 0.2-^m membranefilter. ' Dilution factor = parts CM to parts of complete medium. well as decreased growth rate (Fig. 1, frames A and C) in sensitive TEC lines. It is of interest to note that in experiments involving cocultured normal TEC-target cell populations (Table 1) as well as in con ditioned medium experiments (Tables 2 and 3) maximum inhibitor production is observed in aged primary TEC cultures at a time when cells appear to be terminally differentiating. A marked increase in the number of comified envelopes (cell structures which are insoluble in detergent and 0-mercaptoethanol; Refs. 24 and 25) is observed at this time. In contrast, maximally proliferating TEC populations such as those observed in 2-3week-old cultures do not appear to produce inhibitor. It is not clear why there is a difference in inhibition detected in 1-2-weekold cell cocultures (Table 1) versus that detected in conditioned medium experiments at the same time (Table 2). An inhibitory effect, in particular on the preneoplastic line XR600, is observed in cocultures at 1-2 weeks. This was not observed in conditioned medium experiments. It is conceivable that during the first week of culture there may be terminal differentiation of a small cell subpopulation which is associated with inhibitor production and cell cocultures may be more sensitive for detection of low levels of inhibitor. It is also possible that another mode of cell inhibition is involved in cell cocultures which is not mediated by stable, diffusible factors released into the culture medium. In experiments involving conditioned medium it is clear that neoplastic cells tend to be less sensitive to inhibitor than are the preneoplastic TEC tested (Table 3). This was observed whether CM was added at time 0 or 24 h after seeding the test cell population. In contrast, in cell-coculture experiments both neo plastic and preneoplastic cell growth and survival were de creased in the presence of normal TEC. The preneoplastic cell line XR600, however, was somewhat more sensitive to inhibition. This could imply that other factors, in addition to diffusible factors CANCER RESEARCH a logical guess might be the differentiated cells (i.e., cilliated and/ or goblet cells) present in the intact trachea or keratinizing epithelium in the esophagus. It was observed (Fig. ÃŒB) that rat trachéalfibroblast conditioned medium stimulates the growth of the preneoplastic cell line tested but not the neoplastic line IC12 (Fig. 1£).One might postulate that in the intact organ the proliferating basal epithelial cell layer, adjacent to the fibroblasts, is maximally stimulated. As cells leave the basal cell layer and become differentiated they perhaps are more under the influence of inhibitors associated with terminally differentiated layers of the intact tissue. Such a model would not be unique to the esopha gus or trachea (2). One might further speculate that as cells progress from a normal to a preneoplastic or neoplastic state they become less sensitive to those factors which mediate orderly cell proliferation and differentiation in the intact tissue. Transforming growth factors, operationally defined as peptides which can reversibly induce anchorage independent growth in nonneoplastic cells, have been isolated from normal (15) and transformed (27) cells. A growth inhibitor described by Holley ef al. (13, 14) was found to be closely related to platelet-derived type ßtransforming growth factor by Tucker et al. (28). More recently Roberts ef al. (29) have found that TGF-/3 can be a bifunctional regulator of cell growth in that it can either function as an inhibitor or an enhancer of cell growth depending on the target cells involved and the particular conditions of the assay. Within this context it is of interest to speculate on the possibility that the inhibitor herein described is related to /î-TGF4 and the growth inhibitor described by Holley ef al. (13). A number of biochemical characteristics of the TEC growth inhibitor are similar to those of TGF-0. For example, heat and acid stability as well as instability in dithiothreitol are characteristic of TGF-ß(e.g., 27, 28). The predominant biological effect of the TEC associated inhibitor observed, namely inhibition of cell growth in culture by the native form (i.e., unacidified) of the inhibitor (23), while often not typical of TGF-/3 may reflect the diversity of effects associated with TGF-0 (29). We are currently attempting to identify and further characterize the inhibitor produced by aged primary cul tures of TEC as well as determine what, if any, role this inhibitor might play in vivo in the intact tissue. ACKNOWLEDGMENTS The authors acknowledge the skillful secretarial assistanceof Charlotte Rains. 4Serum-free inhibitory conditioned medium from esophageal and trachéal epi thelial cell cultures was kindly assayed for TGF-tf by Earl Branum in the laboratory of Dr. Harold Moses, Mayo Clinic, Rochester, MN. Both samples contained signif icant levels of TGF-/3.Control, non-inhibitory samples of CM did not contain levels ofTGF-d VOL. 46 FEBRUARY 1986 920 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1986 American Association for Cancer Research. GROWTH INHIBITION BY NORMAL REFERENCES 1. Pierce, G. B., and Fenneil, R. H., Jr. Latent carcinoma and carcinoma in situ. Nati. Cancer. Inst. Monogr., 44: 99-101, 1974. 2. Potter, V. R. Cancer as a problem in intercellular communication: Regulation by growth-inhibiting factors (Chalones). Prog. Nucleic Acid Res. Mol. Biol., 29: 161-173,1983. 3. DeOme, K. B., Miyamoto, M. J., Osbom, R. C., Guzman, R. C., and Lynn, K. 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