Carclnogenesis vol.16 no. 10 pp , 1995

Carclnogenesis vol.16 no. 10 pp.2487-2492, 1995

K-ras mutations in lung tumors from A/J and A/jXTSG-p53 Fj

mice treated with 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone

and phenethyl isothiocyanate

Steven A.Matzinger, Keith A.Crist, Gary D.Stoner

,

Marshall W.Anderson

, Michael A.Pereira

,

Vernon E.Steele

, Gary J.Kelloff, Ronald A.Lubet

and

Ming You

Medical College of Ohio, Toledo, OH 43699, 'Ohio State University, Columbus, OH 43210, 2St Mary's Hospital and Medical Center, Grand Junction, CO 81501, 'Environmental Health Research and Testing Inc., Lexington, KY 40503 and 4National Cancer Institute, Rockville, MD 20892,

USA

^To whom reprint requests should be sent at: Department of Pathology, Medical College of Ohio, 3000 Arlington Avenue, Toledo, OH 43699, USA

The purpose of this study was to evaluate the effects of

the loss of a p53 allele and phenethyl isothiocyanate

(PEITC) pre-treatment on the tumorigenicity of

4-(methyl-nitrosamino)-l-(3-pyridyl)-l-butanone (NNK) and K-ras

mutation frequency in a hybrid mouse model. Male

TSG-p53 'knock-out' mice were bred with A/J female mice

to produce (A/jXTSG-p53) Fj mice either homozygous

(p53+/+) or heterozygous (p53+/-) for p53 aJleles. These

mice, together with female A/J mice, were treated at 6-8

weeks of age with NNK or dosed with PEITC prior to

administration of NNK. The A/J mice treated with NNK

had a 100% incidence of lung tumors, with 9.7 ± 3.4

tumors/mouse. A/J mice pre-treated with PEITC prior to

NNK administration had 3.5 ± 2.1 lung tumors/animal,

although the incidence remained at 100%. In

(A/JXTSG-p53) F, mice with either thep53(+/-) or p53(+/+) genotype

PEITC pre-treatment significantly decreased tumor

inci-dence (100 to 40 and 36%, respectively) and multiplicity

(2.0 ± 0.5 to 0.5 ± 0.4 and 2.1 ± 0.5 to OS ± 0.4,

respectively), indicating that PEITC is an effective

chemo-preventive agent in both A/J mice and (A/jXTSG-p53) F!

mice. Analysis of lung tumor DNA from A/J mice treated

with NNK or NNK/PEITC indicated that 15 of 17 (88%)

and 20 of 23 (87%) of the tumors, respectively, contained

G—»A transitions at the second base of codon 12 in the

K-ras gene. Similarly, in lung tumors from (A/jXTSG-p53)

F, mice treated with NNK or NNK/PEITC 29 of 30 (96%)

and 9 of 10 (90%), respectively contained G->A transitions

at the second base of codon 12 of the K-ras gene. No

mutations of the p53 gene were found in any of the tumors

analyzed, suggesting minimal involvement of this gene in

the development of lung adenomas. These data indicate

that absence of ap53 allele in (A/jXTSG-p53) Fj mice does

not alter the incidence or multiplicity of NNK-induced lung

tumors and that PEITC inhibition of NNK

tumori-genesis does not affect the frequency or spectrum of K-ras

gene mutations found consistently with NNK

carcino-genesis.

'Abbreviations: NNK, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone;

PEITC, phenethyl isothiocyanate; NNAL, A'-nitroso alcohol; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism.

Introduction

Important environmental factors associated with the

develop-ment of human cancers include tobacco smoking and diet

(1). Tobacco smoking is a well-established risk factor for

lung cancer (2) as tobacco smoke contains >4000

com-pounds, of which 43 have been shown to induce tumors

in laboratory animals (3,4). The most potent A/-nitrosamine

contained in cigarette smoke is 4-(methylnitrosamino)-l-(3

pyridyl)-1-butanone (NNK*). NNK induces a spectrum of

tumors in laboratory animals (5) and has been suggested to be

an important etiological factor in tobacco-related human cancer

(6). NNK requires metabolic activation, which occurs by

a-hydroxylation of its methylene and methyl groups, resulting

in the formation of pyridyloxobutylated or methylated DNA

adducts, respectively (5,7). Mouse lung adenomas have not

previously been shown to contain mutations of the p53 gene

(8-11), but predominantly contain K-ras mutations (GC—»AT

transition) at the second base of codon 12, although mutations

have also been found at codon 61 with high doses of NNK

(12,13). The specific GC-)AT transition at the second base of

codon 12 appears to result from C^-methylguanine adduct

formation (14). In addition to inducing tumors of the lung,

NNK induces tumors of the forestomach of A/J mice when

administered by gavage at a dose of 8 mg/mouse (15).

Phenethyl isothiocyanate (PEITC) has been shown to inhibit

NNK-induced lung tumorigenesis in A/J mice and in F344

rats (16). PEITC exerts its inhibitory effects by competitively

binding to and chemically inactivating P450 isozymes that

metabolically activate NNK (17). No studies have been

pub-lished regarding the effect of PEITC on the profile of K-ras

mutations induced by NNK in mouse lung tumors. The

metabolism of NNK has been extensively studied and

essen-tially follows three major pathways (18). Briefly, NNK can

undergo carbonyl reduction to produce A/-nitroso alcohol

(NNAL). A/-Oxidation of the pyridine ring of NNK and NNAL

yields NNK A/-oxide and NNAL /V-oxide. Finally, keto alcohol

is a product of the methyl hydroxylation of NNK, and

ketoaldehyde is formed by hydroxylation of the a-methylene

group. In general, PEITC pre-treatment does not affect the

carbonyl reduction of NNK to produce NNAL (19). PEITC

inhibits jV-oxidation of NNK, as well as the a-hydroxylation

pathways for metabolism of NNK (19). This inhibition leads

to an abundance of unmetabolized NNK and NNAL (20).

NNAL or NNK may cause injury to DNA via an unknown

minor metabolic pathway when these normal metabolic

path-ways are blocked by PEITC prior to NNK administration.

Furthermore, the methyl hydroxylation pathway is normally

four times as active as the methylene hydroxylation pathway

(21) and Hamilton et al. provided evidence that following

PEITC pre-treatment NNK was still metabolized to the keto

alcohol metabolite by methyl carbon hydroxylation (20).

Reac-tion of the diazohydroxide intermediate produced by a-methyl

hydroxylation with DNA, possibly through a cyclic oxonium

intermediate (22), gives an adduct of unknown structure that

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S.A.Matzinger el al

could be considered as an alternative mechanism for DNA

damage when levels of C^-methylguanine and 0

-methylthymi-dine formation are negated by PEITC administration.

Proto-oncogene activation and tumor suppressor gene

in-activation are important steps in tumor development (23). Of

the existing tumor suppressor genes p53 and the retinoblastoma

{Rb-1) gene are the two most studied (24,25). In a variety of

human rumors, including lung, and in tumor-derived cell lines

the p53 and Rb-1 genes are frequently inactivated (25-27).

Furthermore, mutations in these genes are associated with

tumor progression (23,24,28-32). Caamano et al. recently

summarized p53 alterations in human and laboratory animal

tumors and tumor-derived cell lines (33). Transgenic mice that

foster a mutant p53 gene exhibit increased incidences of lung,

bone and lymphoid tumors (34), whereas mice that lack a

functional p53 gene as a result of replacement by homologous

recombination (p53 knock-out mice) develop normally, but are

highly susceptible to tumor formation (35).

The objectives of the present study were to: (i) investigate

the effects of PEITC on the NNK-induced K-ras mutation

spectrum in lung tumors from both A/J and (AJiXTSG-p53)

F, mice; (ii) to investigate the role of the p53 gene in lung

tumor induction by examining for mutations in this gene in

adenomas from A/J and (A/JXTSG-p55) F, mice treated with

NNK. We also determined whether starting the carcinogenic

process with an inactive p53 allele predisposes the animal to

an increased chance of losing the remaining allele in mouse

lung tumors.

Materials and methods

Chemicals

NNK was purchased from Chemsyn Science Laboratories (Lenexa, Kansas). PEITC was purchased from Aldrich Chemical Co (Milwaukee, Wisconsin). PEITC and NNK were assayed by reverse-phase HPLC to assure a punty >98.5% (36).

Typing of the A/JXTSG-p53 F, Mice

Female A/J mice 6-7 weeks of age were obtained from NCI Harlan Sprague Dawley (Frederick Cancer Research Facility, Frederick, MD) and were crossed with male TSG-p53 mice from Genepharm International (Mountain View, CA). TSG-p53 mice were created by gene targeting to replace a portion of exon 5 of the p53 gene with a Pol II-neo expression cassette construct (64). TSG-p53 mice, derived by homologous recombination in an embryonic stem cell line of 129/Sv genetic background mice and subsequently back-crossed five times to C57BL/6 mice, are heterozygous for a disrupted p53 allele (64). At 5 weeks of age the (A/JXTSG-p5J) F, mice were identified by tail tattoo and partial tailectomy was performed to allow for genotypic characterization. DNA was isolated as described below and was subjected to polymerase chain reaction (PCR) amplification utilizing a set of three primers: primer I (5'-TGGGACAGCCAAGTCTGTTATGTGCACG-3') is a sense primer that anneals to the 3:-end of exon 4 of the murine p53 gene; primer 2 (5'-GTCTCACGACCTCCGTCATGTGCTGTGA-3') is an antisense primer that anneals in the middle of exon 5 of the murine p53 gene; primer 3 (5'-CGATGCCTGCTTGCCGAATATCATGGTG-3') is a sense primer that anneals within the neo gene construct, which disrupts exon 5 of the TSG-p53 mouse. A fragment of 976 bp was amplified in the wild-type sequence \p53(+l+)\ using primers 1 and 2, however, when the neo gene construct is present a diagnostic 300 bp fragment is generated using primers 1 and 3 (Figure 1). Mice heterozygous [p53(+/-)] for an intact p53 gene will demonstrate both fragments on PCR amplification using all three primers and the following parameters: 35 cycles of 94°C, 1 min; 68°C, 2 min; 72°C, 2 min (Figure 1). Lung adenoma assay

Female A/J mice and (A/JXTSG-/?53) F, mice were quarantined for 2 weeks prior to use. All mice were housed in a Bioclean laminar flow room in groups of four in solid bottom microisolated cages on hardwood bedding. They were kept in an environmentally controlled room (24 ± 1°C, 12/12 h light/dark cycle) for the duration of the study and fed AIN-76A diet (Teklad, Madison, WI) ad libitum. The biossay followed conditions previously described (37). Six- to 8-week-old mice were administered PEITC (5 nmol/day) or com oil

1

- 976 bp

- 300 bp

Fig. 1. Genotyping of the (AJJXTSG-p53) F, mice by PCR. Lane 1, 976

and 300 bp PCR-amplified DNA fragments from a (A/JXTSG-p5J) F| mouse heterozygous for an intact p53 gene \p53( +/-)]. Lane 2, 300 bp PCR-amplified DNA fragment from a TSG-p53 F| mouse homozygous for inactive p53 gene alleles [p53(-/-)\. Lane 3, 976 bp PCR-amplified DNA fragment from a (A/JXTSG-p5J) F, mouse homozygous for an intact p53 gene \p53(+/+)]

by gavage for 4 days consecutively. Two hours after the final dose of PEITC or corn oil on day 4 NNK (10 |imol/mouse) or saline vehicle was administered by intraperitoneal injection. Seventeen weeks after NNK dosing mice were sacrificed and pulmonary adenomas were counted. Tumors were dissected out with a portion of the tumor fixed for histopathology; the remaining tumor was snap frozen in liquid nitrogen.

DNA isolation

DNA was isolated from tumors by incubation overnight with proteinase K at 37°C in a buffer consisting of 10 mM Tris, 400 mM NaCl, 2 mM EDTA and 0 46% (w/v) sodium dodecylsulfate. The crude nucleic acids were then precipitated by addition of saturated NaCl and ethanol (38). The molecular weight of the DNA recovered was estimated by electrophoresis on a 1.0% agarose gel with appropriate markers.

PCR

Oligomers that flank codons 12 and 13, 61 or 117 and 119 of the K-ras gene, as well as exons 5-8 of the murine p53 gene, were synthesized using the solid phase phosphoramidite method (DNA Model 39IB synthesizer; Applied Biosystems). The sequences of PCR primers have been described previously (13,39). As described by Saiki et al. (40), the 100 uj PCR reaction mixture contained -100 ng genomic DNA, 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 2.5 mM MgCI2, 0 001% gelatin, 100 u_M each deoxyribonucleoside

triphosphates (dATP, dCTP, dTTP, dGTP), 2.5 U Taq DNA polymerase (Promega, Madison, WI) and 40 pmol each primer. The reaction mixture was overlaid with 75 nl sterile mineral oil and subjected to 40 cycles of PCR amplification using a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT). Each cycle consisted of 1 min denaturation at 95°C, 2 min annealing at 55°C and 2 min extension at 72°C. The amplified DNA was purified by agarose gel electrophoresis (1.2%) and subsequently concentrated by the Qiaex Method (Qiagen, Chatsworth, CA).

Single-strand conformational polymorphism (SSCP)

Mutations in the K-ras gene and the p53 gene were determined by SSCP analysis as previously described (41-43), with minor modifications. One microliter (-5 ng) of the purified DNA was end-labeled with [y-32P)ATP by

T4 polynucleotide kinase (Pharmacia, Piscataway, NJ) in a 10 |il mixture containing 10 mM Tris-acetate and 50 mM potassium acetate. Ten microliters of stop buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF) were added and the mixture healed to 95°C for 5 min, chilled on ice for 1 min and 3 uj promptly applied to a 16% non-denaturing polyacrylamide gel. Electrophoresis was performed at 30 W for

18-24 h at 4°C. The gels were placed on X-ray film at -80°C for 3-24 h. Direct sequencing analysis

The direct sequencing of PCR products was carried out as described by Tindall and Stankowski (44). Sequencing primers were end-labeled with [•y-32P]ATP by T4 polynucleotide kinase, then annealed to 20 ng amplified

DNA by heat denaturation at 95°C for 5 min and subsequent chilling on ice for 5 min. Sequenase (a modified form of T7 DNA polymerase; USB, Cleveland, OH) (1.5 U) was added to the reaction mixture and divided into four tubes, each containing 3 |il 80 nM deoxyribonucleoside triphosphates and 8 |iM dideoxyribonucleoside triphosphates. The reaction time was 5 min at 37°C, followed by addition of formamide dye mix to terminate the reaction.

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4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone tumorigenidty

Table I. Effects of PEITC on NNK-induced pulmonary adenomas in A/J and (A/JxTSG-p53) F, mice

Treatment group Mouse Mice with lung tumors (%) Lung tumors/mouse ( ± SD)

NNK NNK/PEITC NNK (j>53+/+) NNK (p53+/-) NNK/PEITC (p53+/+) NNK/PEITC (p53+/-) Ml A/J (A/JXTSG-p5i)F, (AJiXTSG-p53) F, (A/JXTSG-p5J) F, (A/JXTSG-p55) F, 10 15 10 10 10 10 100 100 100 100 36C 40c 9.7 ± 3.4 3.5 ± 2.1' 2.1 ± 0.5b 2.0 ± 0.5b 0.5 ± 0.4d 0.5 ± 0.4d

"Significantly (P <0.0001) fewer tumors than treatment with NNK alone by Student's West.

bSignificantly (P < 0 0001) fewer tumors than NNK treatment in A/J mice by Student's r-test.

Significantly (P < 0.0001) less than that of NNK (p53+/+) as determined by Fisher's exact test.

dSignificantly (/» < 0.0001) fewer tumors than treatment with NNK alone by Student's Mest.

'Significantly (P < 0.0001) less than that of NNK (p53+/-) as determined by Fisher's exact test. The samples were heated to 94°C for 5 min and separated on an 8% denaturing

polyacrylamide gel. The gel was dried and placed on X-ray film for 16-24 h. Statistical analysis

One way ANOVA of ranks followed by the Student Newman-Keuls multiple range test were used to determine the difference in the number of pulmonary adenomas per mouse between control and treated groups. The significance of these differences was determined by Student's Mest with adjustment of error for the number of comparisons. Fisher's exact test was used to determine the significance of the differences in the incidence of tumors between treated and control groups. All tests were two sided.

Results

Lung adenoma assays

The effects of PEITC on NNK-induced lung tumors in A/J

and (A/iXTSG-p53) F, mice are shown in Table I. A/J mice

treated with NNK alone demonstrated 100% tumor incidence

and developed an average of 9.7 lung adenomas/mouse.

Pre-treatment with PEITC at 5 (imol/day for 4 days did not reduce

the incidence of lung tumors in A/J mice, however, tumor

multiplicity decreased from 9.7 to 3.5 tumors/mouse. None of

the A/J mice treated with PEITC/saline developed tumors,

while A/J mice treated with corn oil/saline developed 3 tumors

in a total of 29 mice at the week 17 sacrifice. Tumor incidence

of 100% was observed in (AJJXTSG-p53) F, mice dosed with

10 umol NNK, similar to results in A/J mice. However, the

number of adenomas/mouse was significantly decreased when

compared with A/J mice, being 2.0 and 2.1 in mice with

the genotypes p53(+/-) and p53(+/+), respectively. The

proportion of mice with tumors, as well as the number of

tumors/mouse, decreased -70% (100 to 38% and 2.0 to 0.5,

respectively) with PEITC pre-treatment in (AJ]XTSG-p53) F,

mice regardless of the p53 genotype. No tumors were found

in (A/JXTSG-p53) F, mice treated with PEITC/saline or with

corn oil/saline.

K-ras mutations in mouse lung adenomas

To investigate the frequency and mutational spectrum of the

K-ras gene in A/J and (A/JXTSG-p55) F, mouse lung tumors

induced by NNK, DNA from the tumors was first screened

for mobility shifts using the PCR-SSCP method. SSCP analysis

identifies mutations in denatured

P-labeled DNA fragments

on high resolution, non-denaturing polyacrylamide gels. Fifteen

of 17 (88%) and 29 of 30 (97%) mouse lung tumors from

NNK-treated A/J and (A/JXTSG-p55) F, mice were found to

have a single mobility shift within the first exon of the K-ras

gene. This characteristic mobility shift has been shown to

represent a GC—»AT transition at the second base of codon 12

in the K-ras gene and a representative autoradiogram is

depicted in Figure 2. Results are shown in Table II. PEITC

Mutant

Shift-Single Stranded

DNA

Fig. 2. Detection of K-ras mutations by PCR-SSCP analysis. Lant 1,

positive control (known G—>A transition at the second base of codon 12 in the K-ras gene). Lane 2, negative control taken from a normal A/J mouse lung. Lane 3, exon 1 of K-ras from an A/J mouse lung tumor induced with NNK alone showing a positive mobility shift. Lane 4, exon 1 of K-ras from an A/J mouse lung tumor induced with NNK alone showing no mobility shift. Lane 5, exon 1 of K-ras from an (A/JXTSG-p55) F| mouse lung tumor induced with NNK alone showing a positive mobility shift.

Table II. K-ras mutations in mouse lung tumors treated with NNK and

NNK/PEITC

Treatment Mouse No. of tumors Activated K-ras Codon 12 (GGT->GAT) NNK NNK/PEITC NNK NNK/PEITC A/J A/J (A/JXTSG-/>53) (A/JXTSG-/>5J) F, F, 17 23 30 10 15 20 29 9 (88%) (87%) (97%) (90%) 15 20 29 9

treatment did not affect the frequency of this codon 12

mutation, since 20 of 23 (87%) and 9 of 10 (90%) tumors

from A/J and (AJixTSG-p53) F, mice, respectively, were

found to have the same mobility shift as those tumors taken

from mice treated with NNK alone. No mobility shifts were

found in the second or third exons of the K-ras gene nor were

any mutations detected in normal A/J or (AJ}XTSG-p53) F]

mouse lung tissues.

Direct sequence analysis was performed to confirm the

presence or absence of the G-»A transition at the second base

of codon 12 of the K-ras gene, the characteristic activating

mutation associated with NNK tumorigenesis. All of the A/J

and (£J]XTSG-p53) F

mouse lung tumors positive for a

mobility shift induced by NNK alone or NNK + PEITC were

found to have a GC—»AT transition at the second base of

codon 12 in the K-ras gene (data not shown). All of the

shift-negative samples from the NNK or NNK + PEITC groups

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S.A.Matzlnger et at

were sequenced. No mutations in the first, second or third

exons of the K-ras gene were detected.

p53i mutations in mouse lung adenomas

Lung tumor DNA from (A/JXTSG-p5J) F, mice was also

examined for mutations in exons 5-8 of the p53 gene by SSCP

analysis. No mobility shifts were detected. Direct sequencing

of the PCR products from these tumors yielded no mutations

in either (A/JXTSG-p53) F

p53(+/-) or (A/JXTSG-p53)

F,-p53(+/+) mice (data not shown).

Discussion

The inhibition of tumorigenesis in A/J mouse lung by

adminis-tration of PEITC prior to NNK dosing has been demonstrated

in several prior studies (17,45-50). This study demonstrates

that mice with an inactive p53 allele did not predispose the

animal to an increased chance of losing the remaining allele

via mutation induced by NNK. p53 alterations did not play a

significant role in the early stages of mouse and hamster lung

tumorigenesis (8-11,39,63). Chen et al. analyzed 59

NNK-induced A/J mouse lung adenomas and found no mutations in

exons 5-8 of the p53 gene (8). Goodrow et al. investigated

35 primary CD-I mouse lung adenomas induced with

N-nitrosodiethylamine or 7,12-dimethylbenz[a]anthracene and

found no p53 mutations (9). Oreffo et al. found only one lung

tumor with a p53 mutation out of 30 adenomas analyzed from

hamsters treated with NNK for 26 and 40 weeks (10,11).

These results imply that alterations in the p53 gene are

infrequent at the adenoma stage. In contrast, 25% of lung

carcinomas from (C3HXA/J) F, mice treated with NNK were

found to have a loss of heterozygosity at the p53 locus (63).

Hegi et al. (39) detected p53 alterations in seven of 54

methylene chloride-induced lung carcinomas in B6C3 F| mice.

Thus, p53 alterations may be involved in the progression of

mouse lung tumorigenesis.

In the design of this study we crossed TSG-p53 (C57BL/

6J) mice heterozygous for an inactive p53 allele with A/J mice

highly susceptible to chemical and spontaneous lung tumors.

Our intent was to investigate whether starting the carcinogenic

process with an inactive p53 allele predisposes (he animal to

an increased chance of losing the remaining p53 allele via

mutation concurrent with treatment with NNK. Loss of the

remaining p53 allele would then predispose these animals to

an increase in tumor incidence and multiplicity and possibly

a faster conversion to the malignant phenotype. However, a

similar lung tumor incidence and multiplicity was induced by

NNK in (A/JXTSG-/75J) F

p53(+/+) and (A/JXTSG-p53)

F

p53(+/-) mice. Furthermore, no p53 mutations were found

in any of the lung adenomas analyzed. These data indicate

that p53 does not play a significant role in the early stages of

lung tumor induction of mice treated with the tobacco-specific

nitrosamine NNK. Kemp et al. (64) investigated the role

of p53 in tumor induction in vivo utilizing a mouse skin

carcinogenesis model. This group concluded that loss of a

single p53 allele has no effect on benign tumor formation,

but increased the frequency of progression from benign to

malignant tumors (64). Complete loss of p53 appeared to

decrease the incidence of benign tumors, but greatly enhanced

the rate of malignant progression (64). In our laboratory we

found that methylnitrosourea-induced benign tumor incidence

and multiplicity were the same in (A/JXTSG-p55) F, mice

with the genotype p53(+/-) or p53(+/+) (unpublished data).

In the present study no mutations in the p53 gene were

discovered in lung adenomas harvested at 17 weeks post-NNK

treatment in A/J or (A/JXTSG-/?5J) F, mice. In addition, no

lung adenocarcinomas were found at this time point. This lack

of more highly progressed tumors is probably secondary to

the fact that NNK treatment does not target the p53 gene, but

instead targets the murine K-ras gene in the early stages of

mouse lung tumorigenesis. Thus the remaining wild type p53

allele in the heterozygous mice is not inactivated. Studies

allowing for longer follow up periods post-carcinogen

adminis-tration in the (AJJXTSG-p53) F, mouse may allow us to

further investigate the role of p53 in the progression of lung

adenomas to the malignant phenotype, since p53 alterations

were detected in mouse lung carcinomas (39) and p53

'knock-out' (either p53-l- or p53+l—) was found to significantly

increase the malignant conversion of mouse skin papillomas

(64).

In this study we also found that treatment of mice with

NNK and PEITC did not affect the spectrum and frequency

of G—>A transitions at the second base of codon 12 in the

K-ras gene. Several research groups have provided evidence

supporting the hypothesis that non-genotoxic agents can alter

the mutational profile of a tumor cell population. In a previous

study conducted in this laboratory we discovered that A/J mice

treated with NNK and butylated hydroxytoluene, a lung tumor

promoter, had a significant decrease in the number of tumors

carrying an activated K-ras gene (58). Two other studies

examined the effects of chemopreventive compounds on the

mutational profile of a tumor cell population. Llor et al. found

that supplemental dietary calcium decreased the incidence

of K-ras mutations in 1,2-dimethylhydrazine-induced colon

tumors from 36% to zero (59,60). This anti-mutagenic effect

was subsequently abolished by vitamin D deficiency (59). In

this study treatment of mice with NNK and PEITC, whether

A/J or (AJiXTSG-p53) F, hybrids, did not affect the yield

of G—»A transitions associated with NNK carcinogenesis

(8,12,58). The lack of an effect of PEITC upon the yield of

activated K-ras genes is consistent with the mechanism of

action of PEITC as an inhibitor of mouse lung cytochrome

P-450 isozymes responsible for the metabolic activation of NNK

to methylating and pyridyloxobutylating species (17,61,62).

NNK lung tumorigenesis has been demonstrated in several

animal species, including rat, hamster and mouse (5,51).

Various strains of mice have been shown to have different

susceptibilities to lung tumors induced by chemical carcinogens

(52). The A/J mouse is the most sensitive strain with regard

to both spontaneous and chemically induced lung tumors (53—

56). In contrast, C57BL/6J mice are very resistant to both

spontaneous and chemically induced lung tumor formation

(54-56). When treated with 10 umol NNK we detected 80%

(9.7 versus 2.0) fewer tumors in (A/JXTSG-/?5J) hybrids as

compared with strain A/J mice treated with the same dose of

NNK. The reasons for this difference in tumor multiplicity

might include the following, (i) The genetic make up of the

(AJ5XTSG-p53) F, hybrids used in this study. Based on

Malkinson's three pulmonary adenoma susceptibility gene

model for lung tumor susceptibility, there is a strong locus

whose effect on tumor multiplicity is greater than that of the

other two loci (56). (ii) Different rates of metabolism of NNK

to its active form by the P450 system may exist in the hybrid

versus the A/J mouse (57). However, it is not clear at present

what is involved in lung tumor susceptibility to NNK.

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4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone tumorigenlcity

Acknowledgements

The authors acknowledge Michelle Truesdale, Marshonna Forgues, Laura A.Kresty and Mats Femstrom for their excellent technical assistance and Joel C.McClurg, Robert Blumenthal and Randall Ruch for critical reading of this manuscript. This work was supported by NCI Master Agreements N01-CN-25495-01 and N01-CN-25495-O2, as well as NIH grant CA-58554.

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Received on April 5. 1995; revised on June 22, 1995; accepted on June 22, 1995

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