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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.
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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
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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
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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
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1986 American Association for Cancer Research.
Inhibition of Carcinogen-altered Rat Tracheal Epithelial Cells by
Normal Epithelial Cell-conditioned Medium
M. Terzaghi-Howe and C. McKeown
Cancer Res 1986;46:917-921.
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