[CANCER RESEARCH 52. 5641-5646. October 15. 1992]
Particular Types of Tumor Cells Have the Capacity to Convert Transforming
Growth Factor ßfrom a Latent to an Active Form1
Hidekazu Takiuchi, Tsuyoshi Tada, Xiao-Fei Li, Masato Ogata, Tatehiko Ikeda, Shigeyoshi Fujimoto,
Hiromi Fujiwara,2 and Toshiyuki Hamaoka
Biomedicai Research Center, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565 ¡H. T., T. T., X-F. L., M. O., H. F., T. H.]; Research
Laboratories, King Brewing Co., Lid., Kakogawa 675-01 [T. I./; and Department of Immunology, Kochi Medical College, Nangoku 783 [S. F.¡,Japan
ABSTRACT
We investigated the capacities of various tumor types to generate an
active versus latent form of transforming growth factor ß
(TGF-/8) in its
culture supernatants (SNs). Tumor cell lines were divided into three
types depending on the form and magnitude of TGF-0 detected in their
culture SNs: some (2 of 7 lines) generated mostly an active form (Type
A); others (4 of 7) generated exclusively a latent form (Type B); and the
remaining line (1 of 7) produced only marginal levels of active/latent
TGF-0 (Type C). When Type A tumor cells were cultured at lower
numbers, cultures failed to generate active TGF-/3. However, the addi
tion of Type B tumor cell culture SNs containing only a latent form of
TGF-/3 resulted in the generation of the potent activity of active TGF-/3.
This capacity was observed for another Type A tumor but not for other
types (Type B and Type C). An active form of TGF-/8 was detected in
culture SNs of Type A tumor cells as early as 3-6 h after the addition
of Type B tumor culture SNs. The emergence of an active form of TGF-/3
was also observed in cultures of Type A tumor cells, the protein syn
thesis of which was almost completely inhibited by pretreatment with
cycloheximide. Moreover, the Type B tumor SN used for the induction
of active I (tl -,i activity was found to contain latent I (.1 -/; with an
apparent molecular weight of about 200,000. Type A tumor cells were
also capable of generating active TGF-/3 by the addition of recombinant
TGF-0 of latent form with a small molecular weight (about 60,000),
although the generation of active TGF-/3 was much weaker after the
addition of small latent TGF-/3 than after the addition of large latent
TGF-/3. Taken collectively, these results indicate that particular types of
tumor cells have the capacity to generate an active form of TGF-/3 and
that such capacity can be attributed to their potential to convert TGF-0
from a latent (mainly large type) to an active form.
INTRODUCTION
TGF-/33 was initially characterized by its ability to support
the anchorage-independent
growth of nontransformed fibroblasts (1, 2). More recent studies have demonstrated that this
unique growth factor can induce a variety of responses in target
cells. These include (a) a growth-modulatory effect on various
cell types depending on whether they are mesenchymal, epithe
lial, or endothelial (3-7); (b) the capacity to regulate the differ
entiation of several cell types (8-10); and (c) various immunoregulatory properties of the immune system (11-15). Whereas
many cell types have the potential to produce TGF-0, they have
been reported to secrete TGF-/3 in an inactive (latent) form
(3, 16-19). Therefore, the physiological relevance of TGF-/J as
a growth-regulatory/immunomodulatory
factor appears to rest
on the regulation of its activation. Although latent TGF-0 can
be activated by physicochemical (20, 21) or enzymatic treat-
ments (22), the physiological activation in vivo of latent TGF-/3
is totally unclear. Concerned with the generation of active
TGF-/3, there are only a few reports which observed that an
activated (active) form of TGF-0 is generated in vitro in culture
SNs from some types of transformed (23) and nontransformed
cells (24, 25). It is unclear, however, whether TGF-/3 is actually
produced in an active form or whether latent TGF-0 produced
by these cells is converted to an active form in their cultures. An
investigation of these matters could contribute to a better un
derstanding of the biological significance of TGF-ßfunction.
The present study investigated the differential capacities of
various tumor cell types to produce active versus latent forms of
TGF-/3 and some of the mechanisms underlying the generation
of active TGF-/3 in particular types of tumor cell cultures. The
results demonstrate that seven tumor cell lines were divided
into three types based on the generation of active versus latent
TGF-/3 in their culture SNs, i.e., active (Type A), latent (Type
B), or low-producer (Type C). Under conditions in which shortterm (3-6 h) cultures of lower numbers of Type A tumor cells
failed to produce an active form of TGF-/3 in their SNs, the
addition of SNs from Type B tumor cell cultures resulted in the
generation of a potent activity of active TGF-/3. The addition of
portions of the same SNs into Type B or C tumor cell cultures
did not lead to the generation of active TGF-/3. Moreover, it was
found that the generation of active TGF-/3 in Type A tumor
cultures was inducible even after pretreatment of tumor cells
with cycloheximide and by the addition of latent TGF-/3-containing fractions from Type B tumor culture SNs or a latent
form of recombinant TGF-0. These results indicate that partic
ular types of tumor cells have the capacity to generate an active
form of TGF-0 in their cultures by activating the latent TGF-/3
they produce.
MATERIALS
AND METHODS
Tumor Cell Lines. The following tumor cell lines were used: Rous
sarcoma virus-induced fibrosarcomas CSA1M (BALB/c origin) (26)
and S826 (B10.A origin) (27); a subline of MH134 hepatoma (C3H/He
origin) (28), MH134-E3; methylcholanthrene-induced fibrosarcomas
(C3H/He origin) MCH-4-C2 and MCH-11-A1 (29); and Meth A fib
rosarcoma and LSTRA T-cell leukemia.
Cells and Reagents. MvlLu cells for TGF-/3 assays were obtained
from American Type Culture Collection (Rockville, MD). rTGF-/3 and
anti-TGF-/3 antibody were purchased from King Brewing Co., Ltd.
(Kakogawa, Japan). Normal rabbit IgG was obtained from Cappel Lab
oratories (West Chester, PA). rTGF-£in a latent form isolated from
TGF-/J gene-transfected CHO cell culture SN by high-performance
liquid chromatography was provided by the research laboratories of
Received 4/.V92: accepted 8/4/92.
King Brewing Co., Ltd.
The costs of publication of this article were defrayed in part by the payment of
Preparation of Tumor Cell Culture SNs and Treatment with Acid.
page charges. This article must therefore be hereby marked advertisement in accord
Tumor cell culture SNs were obtained 1 day after culturing tumor cells
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by Special Project Research-Cancer Bioscicnce from the Ministry of
in a 1-ml volume of FBS-free RPMI 1640 medium in 24-well culture
Education, Science and Culture. Japan.
plates
(Corning no. 25820; Corning Glass Works, Corning, NY). Treat
2 To whom requests for reprints should be addressed.
ment of SNs was performed by dialyzing against two changes of l M
3 The abbreviations used are: TCF-rf. transforming growth factor li; rTGF-0.
acetic acid and then dialyzing against phosphate-buffered saline (pH
recombinant TGF-tf; SN, supernatant: FBS. fetal bovine serum; CHO, Chinese
hamster ovary: |'H]Thd (TdR). pH|lhymidine.
7.4). This procedure was described as acid treatment.
5641
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ACTIVE CONVERSION
Detection of TGF-0 Activity (MvlLu Cell Growth Inhibition Assay).
The growth inhibition assay was performed with slight modifications
according to the original method by Cheifetz et al. (30) as previously
described (31). Briefly, Mvl Lu cells (1 x IO4)were cultured with diluted
samples or rTGF-0 in a 0.2-ml volume of RPMI 1640 medium con
taining 5% FBS in 96-well microplates (Corning no. 25860) for 24 h in
a CO2 incubator. Cells were pulse-labeled with 20 kBq pHJThdfTdR)
for the final 4 h, and the incorporated radioactivity was measured.
Results are shown as the mean cpm ±SE of triplicate cultures. In some
experiments, ['HJThd uptake was expressed by the percentage prolif
OF TGF-pf
3H-TdR Incorporation
or MvlLu
c«Us(cpm.xtcr3)
eration of Mvl Lu cells. Percentage proliferation was calculated as fol
lows:
% of proliferation of Mvl Lu cells in an experimental group
'H-TdR uptake in experimental group
= 100 x
uptake in control group
Gel Filtration of Culture SN with Sephacryl S-300 Column Chroma
tograph). Gel filtration of culture SN was carried out by using a
2.0 x 100 cm Sephacryl S-300 column equilibrated in phosphate-buff
ered saline (pH 7.2). One liter of culture SN obtained by culturing
M H134-E3 tumor cells for 24 h in the absence of FBS was concentrated
by ultrafiltration using YM10 membrance (Amicon Corp., Lexington,
MA). Three ml of the concentrated sample were applied to the column
and eluted with phosphate-buffered saline (pH 7.2) at a flow rate of 10
ml/h. The fractions of each 6-ml volume were collected and tested for
active and total TGF-/3 activities before and after acid treatment of each
fraction, respectively.
Treatment of CSA1M Tumor Cells with Cycloheximide. CSA1M
cells were treated with 5 Mg/ml of cycloheximide for 6 h at 37°Cin a
CO2 incubator. Cells were washed with leucine-free RPMI 1640 twice
and used for experiments after suspension in leucine-free RPMI 1640
supplemented with 10% FBS. The inhibition of protein synthesis by
cycloheximide-treated cells was confirmed by the following separate
experiments. Cycloheximide-treated CSA1M cells (1 x 104/well) were
cultured for 6 h in 96-well microplates in leucine-free RPMI 1640
supplemented with 10% FBS and 40 kBq/well of |3H]leucine. Cells
were harvested, and the incorporated radioactivity was measured. The
mean cpm (SE) over background in untreated and cycloheximidetreated CSA1M cells were 11781 (1.08) and 219 (1.08), respectively.
RESULTS
Production of an Active versus Latent Form of TGF-/8 Activity
by Various Tumor Cell Lines. We have examined the capacity
of various tumor cell lines to generate an active and/or latent
form of TGF-/3 in its culture SNs. SNs were obtained 1 day after
cultures of seven tumor cell lines (5 x 10s cells/well) in FBSfree RPMI 1640 in 24-well culture plates, and portions of each
SN were treated with acid. TGF-/3 activity in these untreated
(crude) and acid-treated SNs was assessed by the growth inhi
bition assay using Mvl Lu mink epithelial cells. Fig. 1 demon
strates that potent TGF-/3 activity is detected in crude SNs from
two of seven tumor cell cultures. Such activity is comparable to
total TGF-0 activity detected in acid-treated SNs, indicating
that these culture SNs contain TGF-/3 mostly in an active form.
Crude SNs from four tumor cell cultures contained only mar
ginal levels of active TGF-0, but they exhibited strong activities
after acid treatment. This represents a latent form of TGF-ß.
The results also show marginal TGF-/3 activity in MCH-11-A1
cultures even in the acid-treated SN. Thus, the above seven
tumor cell lines are divided into three types based on the pro
duction of active versus latent TGF-0: Type A, active TGF-ß
producers (CSA1M and S826); Type B, latent TGF-/3 produc-
Fig. 1. Production of an active and/or latent forms of TGF-0 by various tumor
cell lines. Various tumor cell lines (5 x 10-Vwell)were cultured for 1 day in a 1-ml
of FBS-free RPMI 1640 medium in 24-well culture plates. TGF-/3 activity in an
active form or in total (active and latent forms) was detected in crude (untreated)
or acid-treated culture SNs by the MvlLu cell growth inhibition assay. MvlLu
cells (1 x IO4) were cultured for 24 h in the presence of tumor cell culture SNs at
indicated concentrations. Cells were pulse-labeled with 20 kBq ['HVThd, and
incorporated ¡'H]Thd was determined. The vertical dashed lines represent the
background of [-"HJThd uptake in the presence of medium instead of tumor
culture SNs (negative control). The value of ['H]Thd uptake in the presence of
1 ng/ml rTGF-fi (positive control) was 564.
No. ot CSA1M cells cultured
Fig. 2. Generation of active TGF-fi in SNs from cultures of CSA1M cells with
SNs of various tumor cultures. Graded numbers of CSA1M cells were cultured in
the absence (O) or presence of different concentrations (A, 12.5%; O, 25%; V,
50%) of crude MH134-E3 SN (A), Meth A SN (B), or MCH-1 I-AI SN (O in a
total volume of 1 ml for 24 h. Culture SNs harvested were submitted directly to
the MvlLu growth inhibition assay without acid treatment. [3H]Thd uptake of
MvlLu cultures receiving various groups of CSA1M culture SNs (25%) was
expressed by percentage proliferation of Mvl Lu cells. Percentage proliferation of
MvlLu cells = 100 x pH]Thd uptake in MvlLu culture added with CSA1M
SN/['H]Thd uptake in control culture (MvlLu culture added with medium). •¿,
percentage proliferation of MvlLu cells in the presence of 1 ng/ml rTGF-/3
(positive control).
ers (MH134-E3, MCH-4-C2, Meth A, and LSTRA); and Type
C, low-producer (MCH-11-A1).
Enhanced Generation of Active TGF-/9 by Cultures of Type A
Tumor Cells with SNs from Type B but Not from Type C Tumor
Lines. We examined dilution effects of cultured Type A tumor
cells. Fig. 2A illustrates that decreased numbers of Type A
(CSA1M) tumor cells reduced their ability to generate active
TGF-0 in SNs. The results also demonstrate that the addition
of SNs from Type B (MH134-E3) tumor lines to CSA1M cul
tures at low cell concentrations (5 x IO4 or 105/well) resulted in
the generation of high levels of active TGF-/3 in culture SNs
under conditions in which the same number of CSA1M cells by
themselves generated only marginal or weak levels of active
TGF-/ÃŽ.
We further investigated (a) whether cultures of lower num
bers of CSA1M cells can generate active TGF-ßwhen supple
mented with other types of tumor cell culture SNs and (b)
whether lower numbers of other types of tumor cell lines can
also induce the generation of active TGF-0. Fig. 2 (B and C)
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ACTIVE CONVERSION
shows that a similar pattern of active TGF-0 generation was
observed when CSA1M cultures were supplemented with other
Type B tumor (Meth A) culture SNs (Fig. 2B). In contrast, the
addition to CSA1M cells of Type C tumor (MCH-11-A1) cul
ture SNs containing only marginal amounts of active/latent
TGF-/3 failed to induce the enhanced generation of active
TGF-/3 (Fig. 2Q. In the experiments of Fig. 3, culture SNs of a
MH134-E3 tumor (Type B) were included in cultures of Type A
(S826) and Type C (MCH-11-A1) tumor cell lines. The gener
ation of active TGF-0 was induced only in cultures of the Type
A tumor cell line S826. Thus, these results indicate that the
enhanced generation of active TGF-0 is inducible by Type A but
not by other types of tumor cell lines only when supplemented
with SNs containing large amounts of latent TGF-0 (SNs from
Type B tumor lines).
Generation of Active TGF-/3 after Short-Term Cultures of
CSA1M Cells with SNs Containing Latent TGF-/3. We next
investigated the time course of the generation of active TGF-/3
by CSA1M cultures supplemented with latent TGF-/3-containing SNs. At various times after the initiation of CSA1M cul
tures with the MH134-E3 SN, culture SNs were recovered and
tested for an active form of TGF-0 activity. When CSA1M
cultures were not supplemented with MH134-E3 SN, these
cells again generated a potent activity of active TGF-/3 24 h after
culturing (data not shown), but they generated only marginal
levels of active TGF-/3 3 or 6 h after culturing (Table 1). Under
these conditions, the supplementation with the latent TGF-ßcontaining MH134-E3 SN resulted in the generation of high
magnitudes of active TGF-/3 as early as 3 or 6 h after culturing.
The proportion of conversion in these cultures was approxi
mately 85% of the total amount of latent TGF-|8 that was in
cluded in the input MH134-E3 SN.
Because TGF-/3 activity in crude and acid-treated culture
SNs as well as SNs of CSA1M cultures supplemented with
MH134-E3 SN was assessed by the growth inhibition assays
using MvlLu cells, we confirmed the presence of TGF-0 mol
ecules in these culture SNs by examining the blocking of such
TGF-/3 activity by anti-TGF-0 antibody. The results in Table 2
demonstrate that TGF-/S activity as detected in culture SNs by
the growth inhibition assays can be neutralized considerably or
almost completely by anti-TGF-/3 antibody, indicating the pres
ence of TGF-0 in culture SNs tested.
A
o
1
5x10«
10s
5x105
5x10«
105
5x 105
No. of tumor cells cultured
Fig. 3. Differential capacities of various tumor lines to generate active TGF-/3
after cultures with latent TGF-0-containing SNs. Graded numbers of S826 (A)
and MCH-11-A1 cells (B) were cultured with 50% (V), 25% (D). 12.5% (A), or 0%
(O) MH134-E3 SN for 24 h. •¿,
percentage proliferation of MvlLu cells in the
presence of l ng/ml rTGF-ß(positive control).
OF TGF-fl
Table 1 Detection of active TGF-ß
in SNs 3 or 6 h after culturing CSA1M
cells with latent TGF-il-containing SN
[3H]Thd uptake of MvlLu cells (cpm)
Period of CSA1M cultures*
Group1
to
cultures"Medium
CSA1M
h14,299
±2,044
±423
MH134-E3SN 12.5%
9,465 ±2.962
8,026 ±44
2
MH134-E3
SN
25.0%
3.638
±
873
2,255
±202
34Addition
MH134-E3SN50.0%3
2,683 ±1856h14,617 2,490 ±293
"Various concentrations of MH134-E3 SN were added to CSA1M cultures
(5 x lOVwell).
* CSA l M cultures were conducted for 3 or 6 h, and SNs from these cultures were
submitted to the MvlLu growth inhibition assay at 25%. The values of |'H]Thd
uptake in the presence of medium instead of SNs from CSA1M cultures (negative
control) and in the presence of 1 ng/ml or rTGF-rf (positive control) were 13116
and 458, respectively. The values of ['H]Thd uptake upon addition of acid-treated
MH134-E3 SN (total amount of TGF-/J) were 889 (50%) and 2668 (25%).
Table 2 Blocking of TGF-0 activity in various SNs by anti-TGF-ßantibody
|'H]Thd uptake of MvlLu cells"
(cpm)
Sample"Medium
IgG6078
controlrTGF-0Crude
antibody1855
90340
±
ng/ml
0.125
ng/ml25.0%
±70
±31686
698
2032324
±
192392
±
±281
4149
1372496
±
±626
4408
4765899
±
CSA1M
SNAcid-treated
21.5%25.0%
SNAcid-treated
MH134-E3
12.5%25.0%
±33
±71374
834
±165
5545916
5968
±
Meth A
SNSN*fromCSAlM
12.5%25.0%
±76
891249
718 ±
±364
±4824645
61
32
+ 318
±250
Cultures receiving
MH134-E3 SNConcentration0.25
12.5%Normal 2562 ±210Anti-TGF-tf
5630 ±557
" Mvl Lu cells were cultured with various samples at indicated concentrations in
the presence of normal (control) rabbit IgG or anti-TGF-rf antibody (25 >ig/ml).
*SN was obtained 6 h after culturing CSA1M cells (5 x lOVwell) with 25%
MH134-E3SN.
Generation of TGF-0 Activity by the Addition of MH134-E3
SN to CSA1M Cells Pretreated with Cycloheximide. The fact
that TGF-/3 activity emerged in CSA1M cultures as early as 3-6
h after supplementation with latent TGF-/3-containing SN sug
gests that the generation of active TGF-/3 may not be ascribed to
de now synthesis of this cytokine by CSA1M cells themselves.
We directly investigated this by examining whether the gener
ation of active TGF-/3 is influenced by inhibiting the protein
synthesis of responding CSA1M cells through pretreatment
with cycloheximide. CSA1M cells were treated with 5 Mg/ml
cycloheximide for 6 h. The efficacy of this treatment was con
firmed as described in "Materials and Methods." More than
98% of protein synthesis during an additional 6 h was found to
be inhibited in these treated cells. Untreated and cycloheximide-treated CSA1M cells (5 X 105/well) were cultured with
various concentrations of MH134-E3 SN for 6 h. The emer
gence of active TGF-0 activity in each culture is summarized in
Fig. 4. The results demonstrate that active TGF-0 was gener
ated in CSA1M cultures supplemented with MH134-E3 SN
irrespective of whether responding CSA1M cells had been
pretreated with cycloheximide, indicating that the emergence
of active TGF-/3 is not ascribed to de novo synthesis of this
cytokine.
Determination of the Types of Latent TGF-0 that Are Con
verted to an Active Form by CSA1M Cells. We finally inves
tigated whether the emergence of active TGF-0 in CSA1M
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ACTIVE CONVERSION OF TGF-fì
100
5x104
105
5x105
5X104
105
5x105
No. of CSA1M cells cultured
Fig. 4. Generation of active TGF-rf is not affected by pretreatment of CSA IM
responding cells with cycloheximide. CSA l M cells were untreated or treated with
5 wg/ml of cycloheximide for 6 h. These untreated (A) or treated (B) CSA1M cells
(5 x lOVwell) were cultured with various concentrations of MH134-E3 SN (O,
0%; A, 12.5%; D, 25%; V, 50%) for 6 h. The efficacy of cycloheximide treatment
was confirmed beforehand (detailed in "Materials and Methods"). •¿,
percentage
proliferation of MvlLu cells in the presence of 1 ng/ml rTGF-/3 (positive control).
CSA 1M cultures (Fig. 6). The addition of latent rTGF-/3 at a
smaller amount (2 ng/ml) did not lead to the enhanced gener
ation of active TGF-/3. Considerable activity of active TGF-/3
was generated when a larger amount (10 ng/ml) of latent rTGF-ßwas added. However, such an activity was comparable to
or slightly weaker than that obtained by the addition of 25%
MH134-E3 SN (Fig. 2A). In order to compare the efficacy in
the active conversion between large and small types of latent
TGF-0, we determined the amount of TGF-/3 contained in
MH134-E3 SN. The results of Fig. 7 show that MH134-E3 SN
contains 1-2 ng/ml TGF-/3, mostly in a latent form. Thus,
25-50% of the MH134-E3 SN containing 0.5-1.0 ng/ml of
large latent TGF-0 can be efficiently converted into an active
form to generate the potent activity of TGF-/3, as observed in
Figs. 2 and 4, whereas only marginal amounts of TGF-/3 are
converted from comparable amounts (1-2 ng/ml) of small la
tent TGF-/3, as in Fig. 6. Thus, CSA1M cells have the potential
to convert TGF-0 from a latent to an active form. Although
they are capable of converting both large and small types of
440»D 232kD
I
I
100
50
1
2
3
4
5
6
7
8
9
10 11 12
13 14 15 16 17
18
Fraction No.
Fig. 5. Gel filtration profile of latent TGF-/J activity in MH134-E3 SNs on
Sephacryl S-300. The concentrated sample (3 ml) of FBS-free MH134-E3 SN
( 1000 ml) was applied to a Sephacryl S-300 column. The column was eluted with
phosphate-buffered saline (pH 7.4), and 6-ml fractions were collected. Untreated
(crude) samples (•)or samples after acid treatment from each fraction (O) were
tested for TGF-ÕÕ
activity. Molecular markers used were as follows: bovine serum
albumin, M, 67,000; aldolase, M, 158,000; catalase. M, 232,000; ferritin. M,
440,000.
cultures with MH134-E3 SN depends on the addition of a
latent form of TGF-/3 in the SN, and if this is the case, which
types of latent TGF-/3 are converted to an active form. The
concentrated sample of FBS-free MH134-E3 SN was applied to
a Sephacryl S-300 column, and a portion of each fraction from
the chromatography was treated with acid. Fig. 5 shows the
profile of TGF-/3 activities that were detected in each untreated
or acid-treated fraction. The results show that each of the un
treated fractions contained no TGF-0 activity, whereas some
fractions exhibited potent TGF-0 activity after acid treatment,
indicating the inclusion of latent TGF-/3 in these fractions. The
addition to CSA1M cultures of a sample from these latent
TGF-ß-containing fractions instead of the whole MH134-E3
SN resulted in the generation of appreciable levels of active
TGF-0 activity (data not shown). The results of Fig. 5 demon
strate that such a latent form of TGF-/3 has an apparent mo
lecular weight of approximately 200,000.
Additional experiments were performed to investigate
whether the emergence of active TGF-/3 is induced in CSA1M
cultures by the addition of another type (small size) of TGF-/3.
The small size of TGF-0 was obtained as a partially purified
sample from the culture SN of TGF-/3 gene-transfected CHO
cells. Various amounts of rTGF-/3 (latent form) were added to
5x10"
105
5x105
No of CSA1M cells cultured
Fig. 6. Generation of active TGF-/3 in CSA1M cultures supplemented with a
latent form of rTGF-0. CSA1M cells were cultured for 6 h in the absence (O) or
presence of 2 (A) or 10 (G) ng/ml latent rTGF-0 (column-purified sample of
TGF-/J gene-transfected CHO SN). SNs collected were tested for an active form
of TGF-/3 activity. •¿.
percentage proliferation of MvlLu cells in the presence of
I ng/ml rTGF-0 (positive control).
3H-TdR uptake ol MvlLu cells (cpm. x103)
medium
TGF-p 1 0 ng/ml
TGF-p 0 5 ng/ml
TGF-p 025 ng/ml
TGF-p 0 12 ng/ml
TGF-p 006 ng/ml
ng/mlMH134-E3SN(A/N)50%
TGF-p 003
MH134-E3SN(A/N)25%
MH134-E3SN(A/N)
125%
MH134-E3 SN (A/N| 6 2%
MH134-E3 SN (A/N) 31%
1H3
MH134-E3 SN (A/N) 16%MIHyHHHyZHHHh-
5
10
Fig. 7. Concentration of total TGF-Öcontained in the MH134-E3 culture SN.
MvlLu cells were cultured in the presence of various concentrations of acidtreated MH134-E3 SN or active rTGF-fi as control.
5644
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ACTIVE CONVERSION
OF TGF-tf
TGF-/3 gene-transfected CHO SN). Moreover, the generation
of active TGF-0 under the above conditions was induced even
when treatment of CSA l M tumor cells with cycloheximide had
DISCUSSION
been performed prior to culturing to inhibit their de novo pro
tein synthesis. These observations indicate that particular types
TGF-/3 is produced by most cell types and affects the growth
of cells can convert TGF-/3 from a latent (secreted) to an active
and differentiation of many cell types. This cytokine is secreted
in a latent form that is unable to bind to its receptors. Six form.
The form of latent TGF-ßused for the supplementation to
different types of TGF-/3 receptor have been identified. It is also
CSA1M
cultures should also be considered. Two different
known that two of these mediate the signal of TGF-ßand that
forms of latent TGF-ßhave been identified (37). Platelet-de
either or both of these two are deleted on some types of tumor
rived latent TGF-/3, a large latent TGF-/S complex, is composed
cells, leading to the failure of TGF-/3-mediated signal transduction (32). Therefore, central issues in investigating the TGF-/3- of three different subunits: mature TGF-/3, the NH2-terminal
remnant of the TGF-/3 precursor, and M, 125,000-160,000
mediated regulation of various cellular events have been (a) to
analyze the mechanisms by which latent TGF-/3 secreted is TGF-0-binding protein (38, 39). In contrast, latent TGF-ßob
converted into an active form and (b) to determine how the tained as recombinant materials from TGF-/3 gene-transfected
distribution/expression of different types of TGF-/3 receptors is cells such as CHO cells is a small latent complex, composed of
controlled. The data obtained in this study demonstrate the only mature TGF-ßand the NH2-terminal remnant of the
following: (a) a particular type of tumor cells is capable of TGF-0 precursor (37, 40). Thus, the NH2-terminal remnant of
the TGF-/3 precursor is primarily responsible for TGF-/3 la
generating active TGF-/3 in its culture SNs; (b) whereas these
tumor cells alone fail to do this either in cultures at lower cell tency. Although the binding protein is further equipped for the
numbers or in short-term cultures, they succeed under such former type of latent TGF-ß,it remains to be elucidated which
type(s) of function is exerted by this protein. It has not been well
conditions when the cultures are supplemented with latent
investigated which form of latent TGF-ßis produced by various
TGF-/3-containing SNs from another type of tumor cell culture;
(c) the emergence of active TGF-0 in the above cultures is not types of transformed and nontransformed cells. In this context,
affected by pretreatment of responding tumor cells with cyclo- our results demonstrate that MH134-E3 SN, used mainly in
this study as a source of tumor cell-derived latent TGF-/3, con
heximide; and (d) in these cultures the generation of active
tains a larger form of TGF-/3. The results also illustrate that
TGF-0 is much more efficient upon supplementation with tu
mor cell-derived samples containing a large (A/r 200,000) type such a larger latent form can be much more efficiently con
verted into an active form than a small latent form of TGF-ß.
of latent TGF-/3 than with a small type of latent TGF-0 ob
Although it has not been directly determined which form of
tained as a recombinant sample. Thus, the present results indi
cate that a particular type of tumor cell has the capacity to latent TGF-ßis produced by CSA1M and S826 cells with the
convert TGF-/3 from a latent to an active form. In general, an active conversion capacity, this could be speculated on as fol
lows. The total amounts of TGF-/3 contained in these tumor cell
active form of TGF-/3 has been known to be converted from
secreted latent TGF-/3 by extremes of pH or by treatment with SNs as detected by acid treatment were approximately 1-2
ng/ml or less than these concentrations. CSA1M cells failed to
selected chaotrophic agents including sodium dodecyl sulfate
convert such concentrations of small latent TGF-/3. However,
and urea (20, 21, 33), suggesting that access to acidic microenmost of the TGF-ßproduced in culture SNs of these Type A
vironments may activate TGF-/3 in vivo. Alternatively, the find
tumors was already converted into active form when harvested.
ing that exposure to proteolytic enzymes, especially to plasmin,
activates latent TGF-/3 (22) provides another possible mecha
Thus, CSA1M cells might be visualized as producing mainly a
nism for in vivo regulation. It also appears that removal in vitro large form latent TGF-/3 and as converting it into an active
of the carbohydrate structures in the remnant of the TGF-ß form. This does not, however, exclude the possibility that
precursor by glycosidase treatment produces biologically active CSA1M and MH134-E3 tumor cells are unable to produce a
TGF-/3 from the latent complex (34).
small form of latent TGF-ßas well. Recent studies of Miyazono
Apart from the physicochemical mechanisms of TGF-0 acti
et al. (41) demonstrated that latent TGF-/3 with binding protein
vation, it has been reported that some types of cells generate an (large form) can be more efficiently secreted through plasma
active form of TGF-ßin culture SNs. These include keratimembranes by host cells. Therefore, it is still possible that the
nocytes (35), monocytes/granulocytes
(36), endothelial cells above two types of tumor cells as well as Type C tumors have
cocultured with pericytes (24), and breast cancer cells (23). We the potential to produce a small type of TGF-ß,although this
type of TGF-/3 is less efficiently secreted into culture SNs.
have also shown that two tumor cell lines used here (CSA1M
and S826) generated active TGF-ßin 24-h cultures at a higher
There are five different forms of TGF-ß(32). The types of
cell concentration (5 x 105/well). It should be noted that this TGF-/3 isoforms produced by various tumor types used here
was observed in cultures containing no special stimulant. This were not determined in the present study. Further studies will
contrasted with the fact that active TGF-/3 was detected in be required to directly determine this point and to investigate
cultures of the above-mentioned cell types when stimulants
whether there is a correlation between TGF-ßisoforms and the
such as retinoic acid or hormone were present. Importantly, it efficacy of its active conversion.
was found that the CSA1M tumor cells were capable of gener
Our results illustrate that particular types of tumor cells have
ating active TGF-ßby using exogenously administered latent
the ability to generate an active form of TGF-ßin culture SN.
TGF-0 . This was demonstrated by defining conditions under
This is considered to be based on its capacity to convert into an
which the CSA1M cells with potential to generate an active active form from latent TGF-ß.In general, molecular mecha
form of TGF-/3 in their own cultures generate only marginal
nisms for the active conversion are currently unclear. It has long
levels of active TGF-/3 and by detecting the potent activity of been speculated that the cell-associated active conversion de
active TGF-/3 depending on supplementation with latent TGF-/3 pends on the activity of proteolytic enzymes such as plasmin.
(latent TGF-/3-containing fractions of tumor culture SNs or However, the addition of various protease inhibitors to CSA1M
5645
latent TGF-/ÃŽ,the former type is much more efficiently con
verted by these cells than the latter type of latent TGF-/3.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1992 American Association for Cancer Research.
ACTIVE CONVERSION OF TGF-rf
cultures did not prevent the active conversion.4 Although a
recent paper (42) suggested its potential mechanism (the in
volvement of mannose 6-phosphate receptor in the activation
process), such mechanism was also found to be uninvolved
in our present model.4 Thus, the mechanism in this model
remains to be determined. Nevertheless, our results add to a
growing list of the cell-associated generation of active TGF-0
and provide a potential explanation for the cell-associated ac
tivation of latent TGF-/3. Thus, the present approach could
provide an intriguing model for investigating the regulation of
TGF-0 functions through the analyses of the molecular mech
anisms underlying the active conversion of TGF-/3.
ACKNOWLEDGMENTS
The authors are grateful to K. Katayatna for her expert secretarial
assistance.
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5646
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Particular Types of Tumor Cells Have the Capacity to Convert
Transforming Growth Factor β from a Latent to an Active Form
Hidekazu Takiuchi, Tsuyoshi Tada, Xiao-Fei Li, et al.
Cancer Res 1992;52:5641-5646.
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