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[CANCER RESEARCH 51, 4539-4543, September 1, 1991]
Effect of the Bowman-Birk Protease Inhibitor on the Expression
of Oncogenes inthe Irradiated Rat Colon1
William H. St. Clair2 and Darei K. St. Clair
Experimental Radiation Oncology, Department of Radiology, Bowman
Gray School of Medicine of Wake Forest University, Winston-Salem,
North Carolina 27157
ABSTRACT
In this study, we tested the influence of i.p. Bowman-Birk
proteaseinhibitor (BBI) administration on oncogene expression in
unirradiatedand irradiated rat colonie mucosa. Total cellular RNA
was collected fromthe colonie mucosa, and the levels of c-myc,
c-fos, c-Ha-ras, c-EGFR,and c-actin mRNA were examined by standard
dot and Northern blotanalyses. The data demonstrate that BBI is
capable of preventing radiation-induced overexpression of c-myc and
c-fos without interfering withthe constitutive expression of these
2 genes. It was also determined thatBBI did not interfere with
either radiation-induced overexpression of c-Ha-rai and c-EGFR or
the constitutive expression of c-Ha-ros, c-EGFR,or c-actin. The
data demonstrate that the anticarcinogenic BBI selectivelyinhibits
the overexpression of c-myc and c-fos while not affecting cryptcell
proliferation. These results suggest that a protease is involved in
thepathway for enhanced c-myc and c-fos expression and that
proteaseinhibitors such as BBI can interrupt this pathway.
INTRODUCTION
Studies have indicated that both naturally occurring
andsynthetic protease inhibitors have the capacity to inhibit
carci-nogenesis in vivo and in vitro (reviewed in Refs. 1 and 2).
Forexample, the soybean-derived BBI,3 an inhibitor of both
trypsin
and chymotrypsin (3), has been shown to suppress experimentally
induced cancers in animals. BBI in the diets of animalshas been
shown to suppress dimethylhydrazine-induced colonand liver
carcinogenesis (4, 5), dimethylbenz[a]anthracene-in-duced cheek
pouch carcinogenesis in hamsters when appliedtopically (6), and
3-methylcholanthrene-induced lung tumorswhen administered by gavage
or i.p. injection (7). In vitro studieshave demonstrated that
protease inhibitors including BBI arealso capable of preventing
transformation of cultured cellsinduced by either physical or
chemical carcinogens (8-13).
These same anticarcinogenic protease inhibitors also suppress a
number of other phenomena that have been associatedwith animal
carcinogenesis and malignant transformation ofcultured cells, such
as: c-myc expression (14-16), ras-inducedcellular transformation
(17), chromosomal aberrations occurring in cells from Bloom's
syndrome patients (18), and H2U2
formation in phorbol ester activated neutrophils (19).
Previousstudies have shown that the level of c-myc mRNA is reduced
inproliferating C3H/10T'/2 cells that were grown in a
mediumcontaining anticarcinogenic protease inhibitors, such as
anti-pain, BBI, and leupeptin (14, 15, 20). Protease inhibitors
thatdo not have the ability to reduce radiation-induced
transformation, such as soybean trypsin inhibitor, elastatinal, and
a-l-antitrypsin, had little or no effect on the expression of
c-myc
Received 3/4/91; accepted 6/18/91.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 inaccordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
' This research was supported in part by NIH Grant RR-05404 and
funds
from the Department of Radiology, Bowman Gray School of Medicine
of WakeForest University.
2To whom requests for reprints should be addressed, at
Department of
Radiology, Bowman Gray School of Medicine, Wake Forest
University, MedicalCenter Boulevard, Winston-Salem, NC 27157.
3The abbreviation used is: BBI, Bowman-Birk protease
inhibitor.
RNA in proliferating C3H/10T'/2 cells (20).
The colonie epithelium of rodents has been utilized as amodel
for the investigation of cellular proliferation,
migration,differentiation, and carcinogenesis (reviewed in Refs. 21
and22). It is well recognized that ionizing radiation disrupts
thenormal homeostasis of the gut mucosa. Radiation-induced
epithelial cell killing is followed by a compensatory increase
incrypt cell proliferation (21-24). Radiation has also been usedto
induce colonie carcinomas in rodents (25, 26). We havepreviously
shown that abdominal irradiation led to a compensatory hyperplasia
of the colonie mucosa of mice and an over-expression of c-myc
during the time of increased crypt cellproliferation (16). BBI
administered by gavage in conjunctionwith abdominal irradiation
inhibited the overexpression of c-myc RNA but had no effect on
radiation-induced crypt cellproliferation (16). Although both in
vivo and in vitro studiesdemonstrate that chemopreventive protease
inhibitors preventc-myc expression, little is known about the
effect of protease
inhibitors on the expression of other oncogenes that are
thoughtto be relevant for cellular proliferation and
carcinogenesis.
The present study was designed to further evaluate the effectof
i.p. BBI administration on oncogene expression in the co-Ionic
mucosa of rats following abdominal irradiation. Resultsof this
study demonstrate that BBI administration did not
affectcompensatory crypt cell proliferation but prevented the
over-expression of c-myc and c-fos in the regenerating colonie
mucosa following abdominal irradiation. The expression of c-Ha-ras
and c-EGFR was unaffected by the protease inhibitortreatment.
MATERIALS AND METHODS
General Procedures. Five- to 6-week-old Fischer 344 rats
(HarÃ-anSprague Dawley, Indianapolis, IN) were used in this
investigation.Upon arrival at the laboratory, the rats were housed
in an environmentally controlled room and had free access to food
and water. Followinga 1-week acclimation period, 5 rats were
randomly assigned to each of4 treatment groups. Rats assigned to
groups A and B received i.p.injections of sterile saline every
other day for 20 days or until they werekilled. Rats assigned to
groups C and D received i.p. injections of asoybean extract
containing BBI (hereafter referred to as BBI, 25 mg/kg, filter
sterilized; Central Soya Company, Fort Wayne, IN) everyother day
for 20 days or until the rats were killed. One day after thefirst
saline or BBI injection, all rats were anesthetized with an
i.p.injection of sodium pentobarbital (40 mg/kg), and the rats of
groups Aand C were sham irradiated. The rats of groups B and D were
whole-abdomen irradiated with 11 Gy in a specially constructed jig
thatshielded the remainder of the body. The radiation was delivered
by amodified Mark IV Cs-137 7-irradiator (J. L. Shepard and
Associates,San Fernando, CA) at a dose rate of 9.63 Gy/min.
dpm/Crypt. One h prior to being killed, the rats received 0.5
^Ci/gbody weight of tritiated thymidine (200 /iCi/ml solution; 6.0
Ci/mmol)via i.v. injection. After the labeling period, the rats
were killed by anoverdose of pentobarbital and the entire colon
removed and rinsed withchilled saline. Approximately 0.5 cm of
transverse colon was removed,fixed in Clarke's solution, and at a
subsequent time hydrated, hydro-
lyzed in l N HC1, then stained with Schiffs reagent. Fifty colon
cryptswere isolated in triplicate from each segment by a
microdissection
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EFFECT OF BOWMAN-BIRK ON ONCOGENE EXPRESSION
technique originally described by Hagemann et al. (27) and
modifiedby Hanson et al. (28). The isolated crypts were placed into
mini-scintillation vials containing 150 p\ 0.5 N NaOH. After the
crypts weresolubilized, 3 ml scintillation cocktail (Ecolume; ICN
Biomedicai, Inc.,Irvine, CA) were added, each vial counted, and the
results expressed asdpm/crypt.
RNA Isolation. The remainder of the colons were split open
longitudinally and the colonie mucosa removed from the underlying
sub-mucosa and muscularis with a microscope slide. The colonie
mucosafrom the animals of each treatment group were pooled and
total cellularRNA was collected by the method of Chirgwin et al.
(29) and thenstored at -80°Cuntil used.
Northern and Dot Blot Hybridization. Fifty ^g of total cellular
RNAwere blotted onto a nitrocellulose filter using a dot blot
manifold(Schleicher and Schuell, Keene, NH). Filters were baked at
80°Cin avacuum oven for 2 h, then hybridized with 32P-labeled cDNA
usingstandard methods (30) and subsequently exposed to X-ray film.
Dotblots were used to select time points to be compared by Northern
blotanalysis. The probes used in this investigation c-myc , v-fos,
v-Ha-ras,\-erbB (EGFR), and actin were obtained from Oncor, Inc.
(Gaithers-burg, MD).
For Northern analysis, 3-5 ¿¡8P°'y(A+) RNA were isolated
fromtotal RNA using an oligo dT column and size fractionated on a
1%formaldehyde agarose gel (31) and transferred to a nitrocellulose
filter.The filters were hybridized to a -"P-labeled c-myc, or v-fos
probe as
described above.
RESULTS
General Observations. Fig. 1 shows the effect of the
varioustreatment protocols on rat body weight. Rats receiving
i.p.administration of saline or BBI gained weight steadily
throughout the investigation. Rats receiving 11 Gy of abdominal
irradiation in addition to i.p. administration of saline or
BBIexhibited a decline in body weight that reached its minimumvalue
6-10 days after irradiation. Body weights of the irradiatedrats
then gradually increased toward that of the unirradiatedrats.
Because protease inhibitors may lead to pancreatic hypertrophy,
the pancreas from each animal was weighed at the time ofautopsy.
Table 1 lists the mean pancreas weights from rats ineach treatment
group. Rats treated with 11 Gy abdominalirradiation exhibited a
reduction in pancreas weight thatreached a nadir at 7 days after
irradiation. The irradiatedpancreases slowly gained weight
throughout the remainder ofthe investigation. BBI administration
had no effect on thepancreas weight of irradiated or unirradiated
rats.
dpm/Crypt. To examine the influence of the various treatments on
crypt cell proliferation, the uptake of [3H]thymidine
by the colonie crypts was monitored at the time of
autopsy.Abdominal irradiation led to a time-dependent change in
theproliferative activity of the colonie epithelium, as indicated
bythe uptake of [3H]thymidine in the colonie crypts (Table 2).
Rats subjected to 11 Gy of abdominal irradiation exhibited
amarked but transient increase in crypt cell proliferation
(dpm/crypt), which peaked at 7 days postirradiation. The
compensatory increase in crypt cell proliferation then rapidly
returned tocontrol levels. Administration of BBI i.p. had no
influence oncither the control level of crypt cell proliferation or
the radiation-induced compensatory increase in crypt cell
proliferationas measured by the uptake of tritium (Table 2).
RNA Expression. Hybridization analysis of dot and Northernblots
revealed that 11 Gy of abdominal irradiation led to anincrease in
the expression of c-myc, c-fos, c-Ha-ras, and c-
EGFR mRNA. Dot blots of total cellular RNA were used todetermine
when oncogene expression was most enhanced and
OiJU-1
30°"oE-0
250-i0
200-1
*în1OU0
UNTREATED•BBI .
D 11.0 Gy••11.0 Gy + BBI 0Ao
IB••00000°mSa
Biy" -i.Bi
i i i i i ii05 10 15 20 25 30 35 4
DAYSFig. 1. Effect of BBI and radiation on body weight. BBI
treatment had no
detrimental effect on the general health of the unirradiated
rats as demonstratedby the gain in body weight compared to
unirradiated rats that did not receiveBBI.
Table 1 Rat pancreas weight in g (mean ±1 SE) as a function of
radiation andBBI treatment
The effect of BBI and/or abdominal irradiation on pancreatic
weight wasdetermined 3, 7, 10, 14, 21, and 35 days after
irradiation. At the time of autopsy,the pancreas of each rat was
removed, freed from fat and lymph nodes, andweighed. Administration
of BBI did not affect pancreatic weight in unirradiatedor
abdominally irradiated rats.
Time after7-irradiation 0 Gy 0 Gy + BBI 11 Gy 11 Gy + BBI
Day 3Day 7Day 10Day 14Day 21Day 350.975
±0.026(1.1
77 ±0.039
1.213 + 0.062).979±
0.025 0.937.105 + 0.066 0.568.108 ±0.050 0.777.019 ±0.022
0.758.043 ±0.036 0.903.137 + 0.076 0.8390.078
0.870 ±0.0250.016 0.586 + 0.0900.066 0.768 ±0.0210.070 0.823
±0.0820.104 0.727 + 0.0930.167 0.821 ±0.024
Table 2 Proliferative activity per crypt (dpm/crypt ±I SE) as a
function ofradiation and BBI treatment
The total proliferative activity per colonie crypt as monitored
by the incorporation of tritium (dpm) 60 min after an i.v.
injection of tritiated thymidine.Tritium content per crypt was
measured at multiple times after abdominalirradiation. BBI did not
influence the proliferative activity per crypt in either
theunirradiated or irradiated colon.
Time afterT-irradiationDay
3Day 7Day 10Day 14Day 21Day 35OGy2.8
±0.32.4
±0.2
2.6 ±0.40
Gy +BBI2.1
±0.23.4±0.7
1.8 ±0.21.9 ±0.22.5 ±0.32.1 +0.311
Gy5.6
±0.913.3 ±4.14.9 ±1.32.1 ±0.23.1 ±0.52.5 + 0.51
1 Gy +BBI5.7
+ 0.912.9 + 3.02.7 ±0.32.7 ±0.42.4 ±0.22.5 ±0.3
whether BBI would affect the expression of the
oncogenes.Expression of c-myc was greatest 7 days postirradiation.
Northern analysis confirmed that c-myc mRNA was elevated 7
daysafter irradiation; a represenative blot is shown in Fig. 2.
Den-sitometry indicated that c-myc expression in the colonie
mucosaexhibited a 2.2-fold increase at 7 days after irradiation
whencompared to untreated rats (Table 3). Expression of c-myc
fromthe colonie mucosa of rats treated with BBI alone or
radiationplus BBI resulted in no change relative to the untreated
animals(Table 3). Thus, following abdominal irradiation, the peak
inc-myc mRNA expression occurred simultaneously with increased
crypt cell proliferation. Northern blots of mRNA collected 3 days
after irradiation revealed that radiation-inducedoverexpression of
c-myc was preceded by a substantial increasein c-fos expression
(Fig. 2). Densitometry of Northern blotsfrom 3 days after treatment
revealed that 11 Gy led to a 6.3-fold increase in c-fos expression,
whereas BBI alone and 11 Gyplus BBI produced no change in c-fos
expression relative to the
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EFFECT OF BOWMAN-BIRK ON ONCOGENE EXPRESSION
DISCUSSION
B
A considerable body of evidence indicates that certain protease
inhibitors are able to suppress carcinogenesis and
cellulartransformation (4-13). However, little is currently known
regarding the mechanism by which protease inhibitors functionto
suppress carcinogenesis. Because protease inhibitors suppress both
physically and chemically induced carcinogenesis ina variety of
experimental systems, it suggests that protease
B
Fig. 2. Northern analysis of c-myc expression in the colonie
mucosa at 7 daysfrom (A) untreated rats, (B) rats treated with 11
Gy abdominal irradiation, (C)rats treated with BBI, or (/') rats
treated with BBI and 11 Gy abdominalirradiation.
*Table 3 Relative oncogene expression in the colonie mucosa from
the various
treatment groupsLevels of messeage RNA were determined by
scanning densitometry. The
values represent the mean RNA levels ±SE from 3 dot or Northern
blots relativeto the RNA levels from the untreated colons.
UntreatedBBI11 Gy11 Gy + BBIc-myc°1.0
1.13 ±0.09'
2.18 ±0.091.01 ±0.05CC-fos'1.0
0.91 ±0.11'
6.34 ±0.160.99 ±0.22'c-Ha-ras*1.00.99
±0.03'
1.83 ±0.161.82 + 0.08c-EGFR41.01.30
±0.24'
2.96 ±0.423.35 ±0.53
" Message RNA levels of c-myc as evaluated at 7 days
irradiation.* Message RNA levels of c-fos, c-Ha-ras, and c-EGFR as
evaluated at 3 days
after irradiation.'Significantly different from the 11-Gy
treatment group as determined by
Student's nest (P < 0.01).
untreated group (Table 3). BBI administration appeared to
havelittle effect on the constitutive level of c-myc or c-fos
mRNA
(Figs. 2 and 3). However, i.p. administration of BBI
completelyprevented the radiation-induced overexpression of both
c-mycand c-fos (Figs. 2 and 3) without interfering with the
compen
satory increase of crypt cell proliferation (Table 2).Abdominal
irradiation also led to an increased expression of
c-Ha-ras and c-EGFR in the colonie mucosa. A represenativedot
blot shows that the expression of c-Ha-ras peaked at 3 daysafter
irradiation (Fig. 4); similar results were found for c-EGFR(data
not shown). Densitometry of c-Ha-ras dot blots at 3
daysposttreatment demonstrated a 1.8-, 1.0-, and 1.8-fold changein
c-Ha-ras expression in the groups treated with 11 Gy alone,BBI
alone, and 11 Gy plus BBI, respectively, when comparedto the
untreated group (Table 3). Analysis of RNA dot blotsfrom 3 days
after treatment revealed a 3.0-, 1.3-, and 3.4-foldchange in c-EGFR
expression relative to the untreated group,
in the groups treated with 11 Gy alone, BBI alone, and 11 Gyplus
BBI, respectively (Table 3). BBI administration had noeffect on the
constitutive or radiation-induced expression of c-Ha-ras or c-EGFR
(Fig. 4). Expression of ß-actin,a normal
structural gene, was found not to be affected by radiation
and/or BBI administration and demonstrated approximately
equalloading of RNA (Fig. 5).
Fig. 3. Northern analysis of c-fos expression in the colonie
mucosa at 3 daysfrom (A) untreated rats, (B) rats treated with 11
Gy abdominal irradiation, (C)rats treated with UBI, or ill] rats
treated with BBI and 11 Gy abdominalirradiation.
A B C D
10» • I
* * I
••4• • • 35
14
21
Fig. 4. Dot blot analysis comparing the levels of c-Ha-ros RNA
from coloniemucosa at 3, 7, 10, 14, 21, and 35 days in (A)
untreated rats, (B) rats treated with11 Gy abdominal irradiation,
(C) rats treated with BBI, or (D) rats treated withBBI and 11 Gy
abdominal irradiation.
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EFFECT OF BOWMAN-BIRK ON ONCOGENE EXPRESSION
A B
Fig. 5. Northern analysis of c-actin expression in the colonie
mucosa at day 7from (A) untreated rats, (B) rats treated with 11 Gy
abdominal irradiation, (C)rats treated with Ulti, or (/M rats
treated with BBI and 11 Gy abdominalirradiation.
inhibitors antagonize a critical step in the process of
carcino-genesis. Central to the eventual understanding of the
mechanism of anticarcinogenesis by protease inhibitors will be
theidentification of cellular targets that are affected or
regulatedby the anticarcinogenic protease inhibitors.
Equally important to the prevention of cancer is the
assurancethat the chemopreventive agent itself does not pose a
healthrisk. In the investigation reported here, rat body and
pancreasweights were monitored, because protease inhibitors have
previously been associated with impaired growth of animals
andpancreatic hypertrophy (32, 33). The results of this study
demonstrate that BBI when administered by i.p. injection has
nodeleterious effect on body weight and does not lead to
pancreatichypertrophy (measured by pancreatic weight). These
results areconsistent with earlier findings that demonstrate that
BBI administered by gavage or as part of the rodents' diet did
not
affect body weight or pancreas size (4, 5, 16). The fact that
BBIdid not affect body weight indicates that the BBI treatmentused
in this study did not interfere with normal pancreaticfunction or
normal proteolytic digestion.
Numerous oncogenes have been associated with carcinogen-
esis. It has been observed that 2 distinct
complementationgroups, often represented by the myc and ras
families of oncogenes, are together capable of transforming primary
cells invitro (34-36), indicating that the biological contributions
ofmembers from each of the different complementation groupsof
oncogenes are important for the development of a transformed
phenotype. Recently it was reported that transformationof NIH3T3
cells after transfection with Ha-ras was inhibitedby treatment with
a variety of protease inhibitors includingantipain (17). Although
antipain suppressed malignant transformation, it had no effect on
the transfection frequency of theactivated ras oncogene (17).
Furthermore, antipain was effectivefor inhibiting ras-induced
transformation only during the pro-liferative phase of the
experiment following transfection. Thisstudy of Garte et al. (17)
suggested that transformation ofNIH3T3 cells by a mutated ras
oncogene involves multiplestages, and at least one stage involved
events, which occurduring cellular proliferation, that are
susceptible to inhibition
by protease inhibitors. The results of the present
investigationindicate that BBI prevented the overexpression of
c-myc and c-fas in the colonie mucosa by a mechanism that did not
influencecrypt cell proliferation.
An interesting feature of the investigation reported here isthe
spectrum of gene expression affected by BBI administration.BBI
prevented radiation-induced overexpression of c-myc andc-fos
without interfering with the constitutive expression ofthese 2
genes. This indicates that enhanced expression of c-mycand c-fos is
dependent upon a protease that is inhibited by BBI,whereas the
constitutive expression of these 2 genes is independent of this
proteolytic regulation. The mechanism by whichBBI prevents the
overexpression of c-myc and c-fos has notbeen defined. However,
there are a number of points in theregulatory cascade of c-myc and
c-fos that potentially may beaffected by protease inhibitors.
Signal transduction from thecytoplasm to the nucleus primarily
involves a translocation ofproteins between the 2 compartments
(reviewed in Ref. 37).For example, steroid hormones are shuttled
from a receptor atthe membrane through the cytoplasm into the
nucleus. Peptidegrowth factors, such as PDGF, bind to a cellular
receptor andinitiate the transduction of a signal that ultimately
results in achange in RNA transcription (37). Growth factors have
beenshown to rapidly increase the transcription of c-myc and
c-fosthrough a signal transduction pathway (38-40). Thus, BBI
mayact on a proteolytic dependent step along the signal
transduction pathway to prevent the overexpression of c-myc and
c-fos.In addition, negative transcription factors have been
postulatedto exist for both c-myc and c-fos (reviewed in Refs. 41
and 42).Perhaps BBI suppresses the overexpression of these 2
oncogenes by preventing the proteolytic degradation of
represserproteins. Thus, there are a number of candidate sites for
BBIto act in preventing the overexpression of c-myc and c-fos.
In contrast, both the constitutive expression and the
over-expression of the 2 membrane genes evaluated in this study,
c-Ha-ras and c-EGFR, were unaffected by BBI administration.The
results of this study are consistent with the possibility thatBBI
administration may suppress the overexpression of one"type" of
oncogene, thereby blocking the pathway of carcino-
genesis. Elucidation of the biochemical mechanism by
whichprotease inhibitors interact with oncogenes should provide
valuable information regarding the nature of the
carcinogenesisprocess and methods for its arrest.
ACKNOWLEDGMENTS
The authors wish to thank Darlene Cantrell for secretarial
assistancein the preparation of this manuscript, Dr. Donna Garrison
and NancyRagland for editing the manuscript, and Ellen Hensen for
the photography. We also thank Dr. Kenneth Wheeler for his helpful
comments.The Bowman-Birk protease inhibitor was generously provided
by theCentral Soya Company of Fort Wayne, IN.
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1991;51:4539-4543. Cancer Res William H. St. Clair and Daret K.
St. Clair of Oncogenes in the Irradiated Rat ColonEffect of the
Bowman-Birk Protease Inhibitor on the Expression
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