[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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EFFECT OF BOWMAN-BIRK ON ONCOGENE EXPRESSION

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