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Proc. NatL Acad. Sci. USA78(1981) 1045 Table3. EffectsofTPA,saccharin, andcyclamateonEGFbinding Conditions Cells, no. 125I-EGF, 1'I-EGF '25I-EGFbound,%of 1'2I-EGFadded

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Saccharin epidermal - PNAS

Proc. NatL Acad. Sci. USA78(1981) 1045 Table3. EffectsofTPA,saccharin, andcyclamateonEGFbinding Conditions Cells, no. 125I-EGF, 1'I-EGF '25I-EGFbound,%of 1'2I-EGFadded

Proc. NatL Acad. Sci. USA
Vol. 78, No. 2, pp. 1042-1046, February 1981

Cell Biology

Saccharin and cyclamate inhibit binding of epidermal
growth factor

(tumor promotion/phorbol esters/hormone response control unit/carcinogenesis)

L. S. LEE

General Electric Company, Corporate Research and Development, P.O. Box 8, Schenectady, New York 12301

Communicated by Charles P. Bean, November 3, 1980

ABSTRACT The binding of '5I-labeled mouse epidermal parently is not due to a direct competition between TPA and
growth factor (EGF) to 18 cell lines, including HeLa (human car- EGF for the active site of either the EGF receptor or the TPA
cinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat receptor (7). Recent studies also revealed that EGF binding is
liver), HF (human foreskin), GM17 (human skin fibroblasts), XP accompanied by a host of dynamic changes in the cell mem-
(human xeroderma pigmentosum fibroblasts), and 3T3-LI (mouse brane including patching, internalization, and down-regulation
fibroblasts), was inhibited by saccharin and cyclamate. The human (19-23). Although the specific physiological roles of EGF and
cells were more sensitive to inhibition by these sweeteners than EGF receptors are not well understood, it seems plausible that
mouse or rat cells. EGF at doses far above the physiological levels the regulation of EGF levels in the body and the regulation of
reversed the inhibition in rodent cells but not in HeLa cells. In EGF receptors in the cells could be important events in tumor
HeLa cells, the doses of saccharin and cyclamate needed for 50% promotion.
inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deox-
yglucose, sucrose, and xylitol did not inhibit EGF binding. Pre- The carcinogenicity of saccharin and cyclamate has received
vious studies have shown that phorbol esters, strongly potent tu- considerable attention because of their widespread use as ar-
mor promoters, also inhibit EGF binding to tissue culture cells. tificial sweeteners (24-29, *). Much work has been done on
To explain the EGF binding inhibition by such greatly dissimilar their mutagenicity in bacteria (30-31), their tumorigenicity in
molecules as phorbol esters, saccharin, and cyclamate, it is sug- animals (24-28, t), and their epidemiological correlations with
gested that they operate through the activation of a hormone re- human cancers (32-38). The reported effects of these artificial
sponse control unit. sweeteners are confusing and contradictory. It seems more
likely that saccharin and cyclamate function as weakly potent
Cancer-inducing agents have been classified as initiators or pro- tumor promoters rather than initiators when administered to
moters according to their effects on the carcinogenic process animals at high doses (29, 39, 40), although this remains to be
(1-5). A tumor promoter is a substance which, when repeatedly confirmed. The molecular action of these two compounds is
applied to animals previously exposed to a tumor initiator, unknown. Identification of the molecular target in the cell may
greatly enhances the production of tumors (1-5). Extensive provide new insight in the elucidation of their tumor-promoting
studies have revealed that initiators interact with cellular DNA, activity.
but the initial biological events in the action of tumor promoters
are less-well understood (2, 3). Current knowledge of tumor Accordingly, it seemed worthwhile to determine whether
promotion is largely derived from studies of the biological ef- some of the effects exerted by TPA, such as the inhibition of
fects exerted by the potent tumor promoter 12-0-tetradeca- EGF binding, might also be exerted by them. I report here that
noylphorbol 13-acetate (TPA) (1-5). The effects exhibited by such an effect occurs within 1 hr of exposure of human, mouse,
such promoters at hormone-like levels in cellular systems have hamster, rat, and dog cells in culture to saccharin or cyclamate
been used to search for the receptor systems involved in tumor and that it is not due to the toxicity of these compounds at the
promotion (3, 6). It recently was shown that phorbol esters in- levels used.
hibit the binding of epidermal growth factor (EGF) to cellular
receptors and that their potencies in the inhibition of EGF MATERIALS AND METHODS
binding correlate well with their effects as tumor promoters on
mouse skin (6, 7). Materials. Murine EGF was purchased from Collaborative
Research, Waltham, MA. Other radioactive compounds were
Mouse EGF is a peptide of molecular weight 6000 (8, 9). It from New England Nuclear. TPA was from Consolidated
exhibits all the biological activities ascribed to human EGF and Chemical, Biewster, NY. Cell culture materials were from
competes specifically for binding to human EGF receptors (10, GIBCO. Saccharin and cyclamate were obtained from Sigma,
11). It can stimulate proliferation of both ectodermal and me- except for saccharin preparation lot 1022 and the purified sac-
sodermal cells in culture, increase deoxyglucose transport, in- charin that were originally used and distributed by Toxicology
duce the enzyme ornithine decarboxylase, and stimulate pros- Research Division of the Health Protection Branch, Health and
taglandin synthesis (12-15). Rose et aL (16) have shown that Welfare, Canada (25). All other chemical reagents were from
EGF (2-5 ,ug/g of body weight) can enhance skin tumor in- Sigma. Saccharin and cyclamate stock solutions were prepared
duction in mice. Many of the effects induced by EGF are also
exerted by TPA (6, 7). The reason for such parallelism in bio- Abbreviations: TPA, 12-0-tetradecanoylphorbol 13-acetate; EGF, ep-
logical activity is not clear at this time. However, it has been idermal growth factor; ME medium, Eagle's mimimal essential
shown that TPA can synergistically enhance the mitogenic effect medium.
of EGF (17, 18) and that TPA inhibition of EGF binding ap- * Stavric, B. (1977) in Proceedings, Toxicology Forum on Saccharin
(Center for Continuing Education, Univ. of Nebraska, Medical Cen-
The publication costs of this article were defrayed in part by page charge ter, Omaha, NE, May 9), pp. 73-80.
payment. This article must therefore be hereby marked "advertise- t Wisconsin Alumni.Research Foundation (1973) Long Term Saccharin
ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Feeding in Rats, Final Report (WARF, Madison, WI).

1042

Cell Biology: Lee Proc. Natl. Acad. Sci. USA 78 (1981) 1043

in Dulbecco's phosphate-buffered saline. TPA stock solutions obtained from Collaborative Research and had a specific activity
were prepared in dimethyl sulfoxide. of 55,000 cpm/ng.

Cell Culture. The media used in culturing different cell lines RESULTS
were as follows: A, Eagle's minimal essential medium (ME
medium) plus 5% fetal calf serum; B, ME medium plus 10% The dose-response relationships for the inhibition of EGF
heat-inactivated fetal calf serum; C, ME medium plus 5% heat- binding by addition of saccharin and cyclamate 10 min before
inactivated fetal calf serum; D, Dulbecco's modified ME me- 125I-EGF are shown in Fig 1. The doses causing 50% inhibition
dium plus 10% calf serum; E, F-12 medium plus 10% heat-in- were about 3.5 mg/ml for saccharin and 9.3 mg/ml for cycla-
activated fetal calf serum; F, Dulbecco's ME medium plus 10%
fetal calf serum; G, Dulbecco's ME medium plus 10% heat-in- mate, when 2 ng of 125I-EGF was used in binding to 3.65 x 10'
activated fetal calf serum; H, F-12medium plus 10% calf serum;
and I, F-12 medium plus 10% fetal calf serum. All cultures were HeLa cells. Saccharin inhibition of EGF binding was not due
to impurities in the preparations because lot 1022 and purified
grown at 370C in humidified 5% C02/95% air. The cells were saccharin [used in the Canadian studies (25)] gave the same re-
sults in our assays. Inhibition was negligible when the dose of
counted with a Coulter Counter (model Zb). saccharin was less than 1 ,ug/ml or that of cyclamate was less
Human carcinoma HeLa cells were grown in medium D.
than 10 pug/ml (Fig. 1 Insets). Slight variation of this 50% in-
HTG was a thioguanine-resistant mutant of HeLa. The K22
epithelial cell line was isolated from liver of normal young adult hibition dose may occur with different doses of EGF or cell
Lew/Mai rats (41) and was grown in medium E. The HTC cell numbers. Similar potencies of saccharin and cyclamate in EGF-
line was originally isolated from Morris hepatoma 7288C in- binding inhibition were also observed in other human cell types
duced in a BUF rat by an aromatic amine carcinogen and was tested. For instance, in GM17 the 50% inhibition doses were
4.6 and 10.3 mg/ml for saccharin and cyclamate, respectively;
grown in medium H. in XPII they were 7.6and 11.6 mg/ml, respectively. When the
The IAR27 and IAR20-PCI epithelial cell lines were derived binding assay was done in the presence of unlabeled EGF at
levels (200 ng/ml) far above the physiological level, together
from normal 10-week-old BD VI rats (42) and provided by R. with either saccharin at 3 mg/ml or cyclamate at 10 mg/ml, this
Montesano. At 24 weeks in culture, they did not form tumors inhibition was still observed in HeLa cells (Fig. 2). On the other
but at 60 weeks in culture they did. They were maintained in hand, there may be species differences (see below).
medium I. Abbreviations used: IARI, IAR20PCI-24 week, pas-
sage 14; IAR2, IAR20PCI-40 week, passage 21; IAR3, IAR27- 1001 9
24 week, passage 5; IAR4, IAR27-60 week, passage 5. 100 .- 0 -0

MDCK (dog kidney cells) were grown in medium G. HF 90
(human foreskin fibroblasts) were grown in medium F; passage e
11 was used. 3T3 (mouse fibroblasts) were grown in medium
C; 3T3-BP was a line transformed from 3T3 by treatment with 80 80
benzo[a]pyrene and was grown in medium A. GM17 was a hu-
man skin fibroblast line derived from a man with a translocation 0.1 1 10 100 1000
between chromosomes 15 and 18 and was grown in medium B.
CHO (Chinese hamster ovary) cells were grown in medium F. 0 JAg1MI
XP cells were skin fibroblasts of human xeroderma pigmento-
sum; they were obtained from the American Type Culture Col- 60
lection and were maintained in medium B [XPI, ATCC-CRL
1162 (WoMec), passage 30, sex M, age 27; XPII, ATCC-CRL 40 [
1233 (Jay Tim), passage 15, sex F, age 7].
20 -
Binding Assay. The binding assay with l"I-labeled EGF
(125I-EGF) was performed as described (7). The binding buffer 0
consisted of ME medium plus (per liter): 1 g of bovine serum 1-.
albumin, 0.1 g of KCl, 0.1 g of KH2PO4, 4.0 g of NaCl, and
1.06 g of Na2HPO4-7H2O. The binding was carried out with 0
a 55-min incubation at 37°C. Nonspecific binding was deter-
mined by measurement in the presence of unlabeled EGF (2 e0- 2 4 6 8 10 12 14
,ug/ml) and this value was subtracted from the value obtained
with the labeled ligand alone. All assays were performed twice u Cyclamate, mg/ml
and the values presented are means. Duplicates agreed within
C4.
10%. 0
Iodination of EGF. This was performed by using the chlo-
-o
ramine-T method according to Carpenter and Cohen (10) with
slight modifications. Briefly, 3 mCi (1 Ci = 3.7 x 1010 becque- ~Z4

rels) of carrier-free Na'25I solution (17 Ci/mg) was adjusted to 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Saccharin, mg/ml
pH 7.5 and mixed with 0.1 ml of EGF in Dulbecco's phosphate-
FIG. 1. Effectof cyclamate (Upper) or saccharin (Lower) on binding
buffered saline 20 ,ug/ml. Then, 12.5 ,ug of chloramine-T in of EGF to HeLa cells. HeLa cells (3.65 x 106) were assayed for EGF
binding with 2 ng of lmI-EGF and various amounts of sodium cycla-
water was added; after 45 sec at room temperature, the reaction mate or saccharin included in the binding buffer (1.5 ml).
was stopped with 25 ,ug of sodium metabisulfite, 0.1 ml of KI
(100 mg/ml) was added, and the reaction vial was washed three
times with 0.1 ml of KI solution. The labeled EGF was then
isolated with a Bio-Gel P-2 column. The elution buffer con-
tained 0.1% bovine serum albumin in 3ulbecco's phosphate-
buffered saline. Lot A had a specific activity of 13,000 cpm/
ng; lot B had a specific activity of 61,739 cpm/ng; lot C was

1044 Cell Biology: Lee Proc. Natl. Acad. Sci. USA 78 (1981)

100 Table 1. Effects of various reagents on EGF binding to HeLa cells
80
40 Reagents 25I-EGF
60 -
bound, cpm
x
Xylitol 6237
0 100 300' 500 5768
2-Deoxy-D-glucose 5557
0 6283
L-Glucose 6541
Unlabeled EGF, ng 1253
f-D(+)-Glucose 2333
20 902
a-D(+)-Glucose 5973
0.5 6179
Sodium saccharin 6097
Free ldI-EGF, ng Sodium cyclamate
FIG. 2. Dependence of EGF-binding to HeLa cells on the concen- TPA
trations of EGF. (Upper) 125I-EGF (23.8 ng) and various amounts of Sucrose
unlabeled EGF were used in the binding to 3.1 x 106 HelLa cells in the Phosphate-buffered saline
presence of saccharin at 3 mg/ml ( x), cyclamate at 10 mg/ml (o), or Dimethyl sulfoxide

solvent alone (e). (Lower) 12,51-EGF (1-5 ng) was used in the binding The assay was performed with 1.6 x 10 HeLa cells and 64,680 cpm.
to 3.1 x 10' HeLa cells in the presence Lof saccharin at 3 mg/ml (x),
of 125I-EGF (13,000 cpm/ng). Amounts of the reagents added simul-
cyclamate at 10 mg/ml (o), or solvent alone (e).
taneously with 1"I-EGF to the binding buffer: TPA, 100 ng/ml; di-
When other sweeteners such as xylitol, sucrose, or glucose
analogs were tested at a dose of 13.3 mg/ml, no effects on the methyl sulfoxide, 0.01%; all other reagents, 13.3 mg/ml.
EGF binding were observed (Table 1).
assay. TPA inhibited the binding of physiological levels of EGF
Table 2 shows the effects of saccharin, cyclamate, and TPA to various cells in various degrees, confirming previous findings
on the growth and detachment of human cells HeLa and XPII. (6, 7).
None of these compounds showed an effect on the adhesion of
human cells when cultures were exposed to these agents for 1 The inhibition was seen with physiological levels of EGF and
hr under the assay conditions. In agreement with previous ob-
servations, TPA showed slight inhibition of the growth of HeLa milligram per milliliter levels of saccharin and cyclamate in
cells (7). On the other hand, growth inhibition was observed normal cells (XPI, XPII, 3T3-L1, IAR3, IAR1) and tumor cells
after 2 days of incubation when either saccharin or cyclamate
at 1.7 mg/ml was included in the culture medium. I have not (HeLa, IAR2, IAR4), in fibroblasts (GM17) and epithelial cells
determined whether growth inhibition resulted from inhibition (MDCK and K22), and in cells derived from liver (K22) and
of EGF binding, from impurities in the preparation, or from kidney (MDCK) as well as from human skin fibroblasts of both
the metabolites of saccharin or cyclamate. sexes (XPI and XPII). The binding of EGF at 92.86 ng/ml to
IAR3 cells was not inhibited by saccharin at 13.3 mg/ml, al-
The effects of TPA, saccharin, and cyclamate on EGF-bind- though the binding of EGF at 3.89 ng/ml to these cells was
ing to cells derived from various species and cell types are listed inhibited by this saccharin dose. In addition, dose-response
in Table 3. These results have been confirmed by dose-response studies showed that saccharin or cyclamate at less than 6.0 mg/
studies. Because the number -of EGF receptors or the affinity ml had no effect on the binding of EGF at 3.89 ng/ml to IAR3
cells. Similarly, binding with EGF at 19.6 ng/ml was not sig-
of mouse EGF for the receptors on different cells may vary nificantly inhibited by saccharin at 5 mg/ml or cyclamate at 6.6
mg/ml when mouse 3T3 cells were used in the binding assays.
greatly, various amounts of 12 1-EGF had to be used in the assay On the other hand, appreciable inhibition by these compounds
for assessing EGF binding and its inhibition by tumor pro- was observed in mouse 3T3-L1 cells (Table 3). It thus seems that
moters in different cells. Whenever ~possible, however, nearly
physiological levels of EGF (about 1 ng/ml) were used in the certain mouse fibroblast and rat liver cells are less sensitive to

the inhibition of the EGF binding by saccharin and cyclamate
than are human cells. Also, in human cell lines such as HeLa

Table 2. Effects of TPA, saccharin, and cyclamate on adhesion and
growth of human cells

Adhesion test,* Growth

no. cells x 10-' test,t no.
cells x 10-'

Reagents HeLa XPII HeLa

Dimethyl sulfoxide 2.07 ± 0.11 0.35 ± 0.04 3.80 ± 0.39
(0.01%) 1.99 ± 0.03 0.33 ± 0.04 3.74 ± 0.06
2.01 ± 0.21 0.34 ± 0.02 3.08 ± 0.65
None 1.91 ± 0.12 0.32 ± 0.01 2.62 ± 0.40
Saccharin 2.17 ± 0.09 0.33 ± 0.04 2.79 ± 0.23
Cyclamate
TPA

Results are shown as mean ± SD.
* The adhesion test was performed by determining the cell numbers
in dishes after treating the cells with the indicated reagents in the

binding buffer (saccharin, 13.3 mg/ml; cyclamate, 13.3 mg/ml; TPA,

100 ng/ml) at 370C for 60 min in 5% C02/95% humidified air and

then washing them three times with 5 ml of cold binding buffer.
t For the growth test, 1.4 x 106 HeLa cells were plated 24 hr before
the medium was replaced with the same growth medium plus the

indicated reagents (saccharin, 3 mg/ml; cyclamate, 3 mg/ml; TPA,
100 ng/ml) and the cell numbers were determined 3 days later.

Cell Biology: Lee Proc. NatL Acad. Sci. USA 78 (1981) 1045

Table 3. Effects of TPA, saccharin, and cyclamate on EGF binding

Conditions

Cells, no. 125I-EGF, 1'I-EGF '25I-EGF bound, % of 1'2I-EGF added

x 10-6 ng lot Cells Control TPA Saccharin Cyclamate

3.6 2.0 A HeLa 17.7 1.5 4.1 7.0
1.1 12.37
0.8 3.0 A HF 1.7 0.64 0.50 0.42
0.68 4.04
0.9 2.5 B GM17 3.7 0.58 0.81 1.22
3.7 18.68
3.3 2.03 B XPI 2.86 0.85 1.79 1.37
2.1 186.76
1.14 4.64 B XPII 3.89 0.54 0.91 1.58
1.2 0.85
1.3 3.89 A HTG 8.0 2.1 2.1 1.22
1.24 12.4
2.0 0.58 B K22 2.11 0.83 1.39 1.12
1.76 1.6
1.8 1.6 A HTC 0.047 0.019 0.033 0.026
1.2 2.5
2.7 249 B IAR1 2.18 0.57 1.73 0.97
0.91 3.89
C IAR2 1.11 0.21 0.35 0.18

B IAR3 0.99 0.11 0.65 0.36

B IAR4 0.67 0.13 0.24 0.13

C MDCK 4.58 0.18 1.10 0.99

B 3T3 1.88 0.81 1.32 1.06

B 3T3-BP 0.19 0.10 0.12 0.07

B 1OT1/2 3.06 0.20 3.00 1.61

A CHO 0.037 0.000 0.019 0.007

B 3T3-L1 0.61 0.05 0.17 0.14

The binding assay was performed 3 days after the cells had been plated in 5-cm petri dishes. The concentrations of the re-
agents included in the binding buffer (1.5 ml) were: TPA, 100 ng/ml; saccharin, 13.3 mg/ml; cyclamate, 13.3 mg/ml.

and XPII, saccharin at 13.3 mg/ml was more potent than cy- shown that TPA tumor-promoting doses (100 to 1 nM in tissue
clamate at 13.3 mg/ml whereas in other species, such as IAR
cells (rat) and 10T1/2 (mouse), the converse was true. culture or 0.1-10 /ig on mouse skin) are also in the same range
as the doses required to inhibit EGF binding. It seems that a
DISCUSSION class of promoting compounds which operate through inter-
action with the EGF receptors may be detected with the EGF
These results have shown that the EGF receptor system may binding inhibition assay, although a dose-related condition may
be an important molecular target with which saccharin and cy- be required (see below).
clamate interact. The observation that saccharin and cyclamate
inhibit EGF binding in vitro raises several important questions. In this sense, whether saccharin or cyclamate can be accepted
(a) Is saccharin or cyclamate a tumor promoter? (b) Is EGF bind- as tumor promoters based on EGF-binding inhibition or EGF-
ing inhibition a useful assay system for the activity of tumor binding assay can be established as a testing system based on
promoters? (c) If the answers to questions a and b are "Yes," their tumor promoting activity is better approached from re-
what biological properties can account for the observed corre- phrasing questions c and d as follows: (e) What is the biological
lation between tumor promotion and the EGF binding inhi- property that can account for the 125I-EGF binding inhibition
bition among various structurally related and unrelated sub- by the structurally related phorbol esters (tricyclic diterpene
stances such as phorbol esters, saccharin, and EGF? (d) If the esters) and structurally unrelated saccharin (1,2-benzisothia-
answers to questions a and b are "No," what is the reason for zolin-3-one 1,1-dioxide), cyclamate (cyclohexanesulfamic acid),
the observed "apparent" correlation between EGF-binding in- and EGF (epidermal growth factor), without referring to their
hibition by the tumor promoting phorbol esters and by the sus- carcinogenic properties? Also, () if such a biological property
pected promoters saccharin and cyclamate? can be identified, under what conditions can this biological
property be related to tumor promotion and under what con-
At present, there is no definite answer to question a. Al- ditions will it not be related to tumor promotion?
though it is more likely that saccharin and cyclamate are weak
tumor promoters with very weak initiating activity (29, 39, 40) In order to answer question e, it should be pointed out that
which may not conform to the strict criterion established with phorbol esters bind to a receptor that is distinct from the EGF
potent carcinogens, this has not been generally accepted receptor (7, 43). Cyclamate and saccharin do not bind to the
(24-42). On the other hand, at present there are not enough TPA receptor (unpublished results), nor do they share any
data to permit the unequivocal claim that the EGF binding assay structural similarity with EGF. It seems reasonable to expect
can be used without conditions for the testing of the activity of that all cells contain some sort of a hormone response control
all tumor promoters. To establish such an assay system would unit that regulates the abilities of a certain group of receptors
require testing of saccharin or cyclamate analogs whose carcin- to participate in their hormone-binding processes and modu-
ogenicities are established unequivocally, as well as the testing lates the expression of the receptor response signals. Such a
of all the known tumor initiators and promoters. Nevertheless, control unit would normally be interacting with the specific
EGF binding is inhibited by tumor-promoting phorbol esters. binding products of the hormones or growth factors with their
Furthermore, EGF per se is tumor promoting at high doses. respective receptors, such as the EGF-receptor complex or the
It is interesting to point out that the doses of saccharin or cy- binding product(s) of TPA and the receptor(s) sensitive to-it.
clamate required to demonstrate their tumorigenic activity are Thus, the formation of one receptor complex would exert,
very high (0. 1 to 0.001 M in tissue culture or 5% in diet in animal through this control unit, an effect on the availability of another
tests) (26, 39), as are the doses required to demonstrate signif- receptor. The observed inhibition of EGF binding by saccharin
icant EGF binding inhibition. Previous work (1-5, 39) has and cyclamate suggests that this hormone response control unit
can also be activated by simple organic molecules at sufficiently
high concentrations.

1046 Cell Biology: Lee Proc. Natd Acad. Sci. USA 78 (1981)

With the above hypothesis to answer question e, we can de- 18. Dicker, P. & Rozengurt, E. (1978) Nature (London) 276, 723-726.
rive a reasonable theory to answer question f: namely, in cells 19. Das, M. & Fox, C. F. (1978) Proc. Natl Acad. Sci. USA 75,
that have been exposed to a tumor initiator, prolonged and ex-
cess activation of the hormone response control unit can also 2644-2648.
affect the permanent changes in gene expressions that are in- 20. Schlessinger, J., Shechter, Y., Cuatrecasas, P., Willingham, M.
volved in tumor promotion. The classical observation that low
doses of tumor initiators produce negligible numbers of tumors C. & Pastan, I. (1978) Proc. Natl Acad. Sci. USA 75, 5353-5357.
in the absence of exogenous tumor promoters (1-3) indicates 21. Haigler, H., Ash, J. F., Singer, S. J. & Cohen, S. (1978) Proc. Natl
that endogenous growth factors, such as EGF, do not have tu-
mor-promoting activities at normal physiological levels even Acad. Sci. USA 75, 3317-3321.
though tumor promotion has been reported at very high levels 22. McKanna, J. A., Haigler, H. T. & Cohen, S. (1979) Proc. Natl
(16). Thus, the degree of stimulation of the hormone response
control unit required for tumor promotion may require that Acad. Sci. USA 76, 5689-5693.
some threshold be exceeded. 23. Maxfield, F. R., Davies, P. J. A., Klempner, L., Willingham, M.

Further studies are required to deduce the way the hormone C. & Pastan, I. (1979) Proc. Natl Acad. Sci. USA 76, 5731-5735.
response control unit responds to stimulation by various com- 24. Hicks, R. M., Wakefield, J. St. J. & Chowaniec, J. (1975) Chem-
pounds in different systems under various conditions, which
may shed more light on the tumor-promoting activity of sac- Biol Interact. 11, 225-233.
charin or cyclamate. 25. Arnold, D. L., Moodie, C. A., Grice, N. C., Collins, S. M.,

Thanks are due to C. Kania and M. Lynch for technical help. McGuire, P. F. & Munro, I. C. (1977) Long Term Toxicity of
Orthotoluene-sulfonamide and Sodium Saccharin in the Rat: An
1. Berenblum, I. (1975) in Cancer: A Comprehensive Treatise, ed. Interim Report (Toxicology Research Division, Health Protection
Becker, F. F. (Plenum, New York), Vol. 1, pp. 323-344. Branch, National Health and Welfare Ministry, Ottawa, Canada).
26. Division of Pathology, Food and Drug Administration (1973) Sub-
2. Boutwell, R. K. (1974) CRC Crit. Rev. Toxicol. 2, 419-443. acute and Chronic Toxicity and Carcinogenicity of Various Dose
3. Slaga, T. J., Sivak, A. & Boutwell, R. K., eds. (1978) Mechanisms Levels of Sodium Saccharin, Final Report (Government Printing
Office, Washington, DC), pp. 169-170.
of Tumor Promotion and Cocarcinogenesis, Carcinogenesis: A 27. Munro, I. C., Moodie, C. A., Krewski, K. & Grice, H. C. (1975)
Comprehensive Survey (Raven, New York), Vol. 2. Toxicol Appl Pharmacol 32, 513-526.
4. Weinstein, I. B., Yamasaki, H., Wigler, M., Lee, L. S., Fisher, 28. Friedman, L., Richardson, H. L., Richardson, M. E., Lethco, E.
P. B., Jeffrey, A. & Grunberger, D. (1979) in Carcinogens: Iden- J., Wallace, W. C. & Sauro, F. M. (1972)J. Natl Cancer Inst. 49,
tification and Mechanisms of Action, eds. Griffin, A. C. & Shaw, 751-764.
C. R. (Raven, New York), pp. 399-418. 29. Hicks, R. M., Chowaniec, J. & Wakefield, J. St. J. (1978) in Mech-
5. Hiatt, H., Watson, J. D. & Winston, J. A. (1977) Origins of Hu- anisms of Tumor Promotion and Cocarcinogenesis, Carcinogen-
man Cancer (Cold Spring Harbor Laboratory, Cold Spring Har- esis: A Comprehensive Survey, eds. Slaga, T. J., Sivak, A. & Bou-
bor, NY), Vol. 4. twell, R. K. (Raven, New York), Vol. 2, pp. 475-489.
6. Lee, L. S. & Weinstein, I. B. (1978) Science 202, 313-315. 30. Batzinger, R. P., Ou, S. L. & Bueding, E. (1977) Science 198,
7. Lee, L. S. & Weinstein, I. B. (1979) Proc. Natl Acad. Sci. USA
76, 5168-5172. 944-946.
8. Cohen, S. (1965) Dev. Biol 12, 394-407.
9. Savage, C. R., Jr. & Cohen, S. (1972) J. Biol Chem. 247, 31. Arnold, D. L., Moodie, C. A., Stavic, B., Stoltz, D. R., Grice,
7609-7611. H. C. & Munro, I. C. (1977) Science 197, 320.
10. Carpenter, G. & Cohen, S. (1979) Annu. Rev. Biochem. 48,
32. Howe, G. R., Burch, J. D., Miller, A. B., Morrison, B., Gordon,
193-216. P., Weldon, L., Chambers, L. W., Fodor, G. & Winsor, G. M.
(1977) Lancet ii, 578-581.
11. Carpenter, G. & Cohen, S. (1976)J. Cell Biol 71, 159-171.
33. Miller, A. B. & Howe, G. R. (1977) Lancet ii, 1221-1222.
12. Stastny, M. & Cohen, S. (1970) Biochim. Biophys. Acta. 204, 34. Simon, D., Yen, S. & Cole, P. (1975) J. Natl Cancer Inst. 54,
578-589.
587-591.
13. Hooker, J. K. & Cohen, S. (1967) Biochim. Biophys. Acta 138, 35. Clark, J. P. (1978) J. Am. Med. Assoc. 240, 349-355.
347-356.
36. Morgan, R. W. & Jain, M. G. (1974) Cancer Med. Assoc. J. 111,
14. Barnes, D. & Colowick, S. P. (1976)J. Cell Physiol 89, 633-640.
1967.
15. Levine, L. & Hassid, D. F. (1977) Biochim. Biophys. Res. Com- 37. Wynder, E. L. & Stellman, S. D. (1980) Science 207, 1214-1216.
mun. 76, 1181-1187.
38. Kessler, I. I. (1976)J. Urol 115, 143-146.
16. Rose, S., Stahn, R., Passovoy, D. S. & Herschman, H. (1976)
Experientia 32, 913-915. 39. Mondal, S., Brankow, D. W. & Heidelberger, C. (1978) Science
201, 1141-1142.
17. Frantz, C. N., Stiles, C. D. & Scher, C. D. (1979) Proc. Am. Assoc.
Cancer Res. 20, 207 (abstr.). 40. Cohen, S. M., Arai, M., Jacobs, J. B. & Friedel, G. M. (1979)
Cancer Res. 39, 1207-1217.

41. Weinstein, I. B., Orenstein, J. M., Gebert, R., Kaighn, M. E.
& Stadler, U. C. (1975) Cancer Res. 35, 253-263.

42. Montesano, R., Drevon, C., Kuroki, T., Saint Vincent, L., Hen-
dleman, S., Sanford, K. K., DeFeo, D. & Weinstein, I. B. (1977)
J. Natl. Cancer Inst. 59, 1651-1658.

43. Delclos, K. B., Nagle, D. S. & Blumberg, P. M. (1980) Cell 19,
1025-1032.


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