H-ras oncogene mutations during development of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland cancer
Minshu Yu and
Elizabeth G. Snyderwine1
Chemical Carcinogenesis Section, Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA
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Abstract
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Laser capture microdissection, polymerase chain reaction-restriction fragment length polymorphism analysis, and DNA sequencing was used to detect H-ras codon 12 and 13 mutations during the stages of mammary gland cancer development in rats exposed to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in cooked meat. Ten oral doses of PhIP (75 mg/kg, p.o., once per day) were administered to adolescent female SpragueDawley rats and mammary glands examined histologically for intraductal proliferations (IDPs), carcinoma in situ and carcinomas 714 weeks later. Mammary gland epithelial cells from normal tissue and distinct lesions were collected from glass slides and analyzed for mutations. H-ras codon 12/13 mutations were detected in 73%, 75%, 100%, and 100% of normal mammary glands, IDPs, carcinoma in situ, and carcinoma, respectively, after PhIP treatment. The spectrum of activating mutations included G35 to A or C base substitution mutations in codon 12, and G37 to T or A base substitution mutations in codon 13. The spectrum of H-ras mutations was similar among normal mammary gland from PhIP treated rats, preneoplastic lesions, and carcinomas. Furthermore, the spectrum of mutations was consistent with the involvement of PhIP-guanine adduct formation. The results support the notion that mutations in H-ras codons 12 and 13 are largely PhIPDNA adduct-induced and involved in the initiation and development of mammary gland cancer in rats exposed to PhIP.
Abbreviations: dG-C8-PhIP, N-deoxyguanosin-8-yl-PhIP; DMBA, 7,12-dimethylbenz[a]anthracene; IDPs, intraductal proliferations; LCM, laser capture microdissection; NMU, nitrosomethylurea; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine .
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Introduction
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Chemically-induced rat mammary gland carcinogenesis is a valuable experimental model system for understanding the biology of breast cancer (1). Using the classical chemical carcinogens 7,12-dimethylbenz[a]anthracene (DMBA) and nitrosomethylurea (NMU), the pathogenesis and stages of breast cancer development have been described in rats (25). Rat mammary gland carcinomas arise from ductal elements that progressively develop intraductal hyperplasia, atypical ductal hyperplasia, carcinoma in situ and carcinoma (15). The ability to examine the temporal sequence of breast cancer development, and the similarities between rat and human breast cancer in pathogenesis make the rat an excellent model for studying the human disease.
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) belongs to the class of heterocyclic amine mutagens found in the human diet specifically in cooked meat (68). In rodent carcinogenicity bioassays, PhIP has been shown to be a mammary gland carcinogen after chronic dietary administration and by short-term gavage (911). Multiple oral doses of PhIP administered to adolescent female SpragueDawley rats induce about a 50% incidence of mammary gland tumors within 25 weeks (11). PhIP is a procarcinogen that is metabolically activated to reactive ester derivatives that form DNA adducts, the major adduct being N-deoxyguanosin-8-yl-PhIP (dG-C8-PhIP) (reviewed in ref. 12). PhIPDNA adducts induce characteristic mutations in in vitro assays and in vivo in the mammary gland of Big Blue rats, transgenic rats carrying a mutational reporter gene (1215). However, the critical genes harboring PhIPDNA adduct-induced mutations and hence potentially involved in the initiation of mammary gland cancer by PhIP are not yet known.
One candidate gene potentially associated with the initiation of chemically-induced rat mammary gland cancer is H-ras. H-ras is a GTP-binding protein which functions as a molecular switch, active in the GTP-bound form and inactive in the GDP-bound form, that transduces extracellular signals from the plasma membrane to the nucleus (16). Oncogenic mutations in H-ras decrease the ability of Ras to interact with the GTPase activator protein (GAP) effectively leaving Ras in the `on' position and continuously activating downstream signals including those for growth and proliferation (17,18). Studies by Barbacid and colleagues first reported that NMU-induced rat mammary gland carcinomas carry a specific G35 to A transition mutation in codon 12 of H-ras and proposed that this mutation contributed to the initiation of carcinogenesis (19,20). Carcinomas induced by NMU have a high frequency of oncogenic H-ras mutations with up to 90% showing the G35 to A transition, a mutation characteristic for the alkyl DNA adduct of NMU (20,21). Prior to the development of neoplasia, the G35 to A35 transition mutation has also been found in the normal mammary gland of NMU-treated rats and in intraductal proliferations (IDPs), further supporting the notion that the H-ras G35 to A transition mutation participates in the early stages of NMU-induced carcinogenesis (2226).
PhIP-induced rat mammary gland carcinomas have been shown to harbor H-ras mutations including the G35 to A transition mutation and other guanine mutations in codons 12 and 13 (2729). However, it is not yet known whether H-ras is mutated during the early stages of PhIP-induced mammary gland carcinogenesis. H-ras codons 12 and 13 contain multiple G:C base pairs, and G:C rich regions are susceptible to PhIPDNA adduct-induced mutations (15). H-ras is therefore a good candidate for a gene involved in the initiation of mammary gland cancer by PhIP. In the current study, laser-capture microdissection (LCM) was used to collect epithelial cells from the normal mammary gland of PhIP-treated and control rats, as well as IDPs, carcinoma in situ, and mammary gland carcinomas arising after PhIP treatment. The frequency and spectrum of H-ras codon 12 and 13 mutations was examined after polymerase chain reaction (PCR) amplification, restriction fragment length polymorphism (RFLP) and DNA sequencing. To our knowledge, no previous study has used LCM to examine H-ras mutations during early stages of PhIP-induced rat mammary gland carcinogenesis. This study was undertaken to provide a view to the critical genomic alterations associated with the initiation and development of PhIP-induced rat mammary gland cancer.
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Materials and methods
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Animals
PhIPHCl was purchased from Toronto Research Chemicals (North York, ON, Canada). Female SpragueDawley rats were obtained from the Animal Production Area, Frederick Cancer Research and Development Center (Frederick, MD, USA). Throughout the study, rats were given food and water ad libitum and maintained on a 12 h dark12 h light cycle. At 43 days of age, 30 rats were administered PhIP (75 mg/kg, p.o., dissolved in water, 5 ml/kg) once per day (days 15 and 812) over a 14 day period according to our previously described protocol (10,11). Ten control rats were given vehicle only. Rats were provided NIH laboratory chow before and during dosing. Two days after the final dose of PhIP (or vehicle) rats were then placed on a defined high-fat diet consisting of 23.5% corn oil identical to that described previously (10).
Collection of mammary glands and histopathology
Rats were killed at 7, 9, 10, 12 and 14 weeks after the last dose of PhIP or vehicle. The six pairs of mammary glands were excised taking care to collect each gland region separately. Tissue was fixed in 70% ethanol, embedded in paraffin, and cut into 5 micron sections onto glass slides. Slides were stained with hematoxylin and eosin and examined under light microscopy. Preneoplastic lesions and carcinomas were classified according to criteria described previously (5,30).
Laser capture microdissection
The LCM of mammary glands was performed according to NIEHS Laser Capture Microdissection Guidelines (http://dir.niehs.nih.gov/dirlep/lcm/guidelines.html#lcmpet) using the facilities available at the NIH Clinical Center LCM Core Laboratory (http://dir.nichd.nih.gov/lcm/lcm.htm). Each microscopic field was dissected by successive laser shots at a pulse power of 40 mW and at 30 microns in diameter. An estimated 300600 total cells were transferred to the undersurface of a film attached to an Eppendorf tube cap. Normal mammary gland samples included both ductal and lobular epithelial cells. Epithelial cells from several IDPs found in one field were routinely pooled, and epithelial cells from carcinomas in situ were collected separately. In carcinomas, care was taken to insure that only epithelial cells and not surrounding stroma was collected. Within each sample category (i.e. normal, IDP, carcinoma in situ, and carcinoma) every microdissected sample was from a distinct mammary gland. After LCM, the cap with adherent epithelial cells was inserted into an Eppendorf tube containing digestion buffer (50 µl buffer containing 0.04% Proteinase K, 10 mM TrisHCl (pH 8.0), 1 mM EDTA, and 1% Tween-20), and the tube placed upside down to facilitate digestion during a 1012 h incubation at 37°C. After brief centrifugation, the reaction was heated to 95°C for 3 min to inactivate the proteinase K, and the mixture used directly for PCR.
Analysis of codon 12 and 13 H-Ras mutations
Mutation analysis was carried out as described by Roberts-Thomson and Snyderwine (28) involving PCR, RFLP, and DNA sequencing. Briefly, codon 12 and 13 of the H-ras gene was initially amplified by PCR using primers identical to Takahashi et al. (31). A single DNA fragment was purified by agarose gel electrophoresis and gel extraction, and digested with MnlI (New England Biolabs, Beverly, MA). Since a MnlI site is found in wild-type codon 12 and 13, mutations in this region destroy the MnlI site and generate a mutant fragment of 125 bp. This fragment was further purified through a 2% agarose gel and re-amplified with the previously described primers (28). Mutant fragments were cloned into a plasmid pCR 2.1 vector (Invitrogen, Carlsbad, CA). If no mutant fragment band was visible on agarose gel, sham bands were isolated and cloned into the plasmid vector. Three to five clones were selected at random and sequenced by fluorescent labeled dye-terminator chemistry and analyzed on an Applied BioSystems 377 automatic sequencer (Perkin-Elmer Cetus). Sequencing was carried out at the NCI's DNA Sequencing MiniCore Facility (http://neoplasia.nci.nih.gov/dna/index.html).
Ras mutation frequency and spectrum
The H-ras mutation frequency and spectrum of H-ras mutations per microdissected sample (Figures 2 and 3
, respectively) were derived from the raw sequence data shown in Table I
. Plasmid vector clones from the same microdissected mammary gland sample occasionally harbored an identical H-ras mutation and mutation spectrum (Figure 3
) was determined by counting a repeated codon 12/13 H-ras mutation only once for each microdissected sample. A microdissected sample was considered as harboring an H-ras mutation when at least one plasmid vector clone had a mutant H-ras.

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Figure 2. Frequency of H-ras mutations in control mammary gland and in histologically normal ducts, IDPs, carcinoma in situ, and carcinomas from PhIP-treated rats. Mutations in H-ras codons 12 and 13, and mutations overall (codons 12 and 13 combined) were determined by RFLP and DNA sequencing. Data are the percentage of codon 12, codon 13, and combined total mutations found in each histological structure. The number of mammary structures examined is represented in the figure, and 12 structures were collected per rat. Carc, carcinoma; in situ, carcinoma in situ; control, normal mammary gland from untreated rats.
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Figure 3. Spectrum of H-ras (codons 12 and 13) mutations in histologically normal ducts, IDPs, carcinoma in situ, and carcinomas from PhIP-treated rats. Mutations in H-ras codons 12 and 13, and mutations overall (codons 12 and 13 combined) were determined from DNA sequencing. For each sample, plasmid vector clones containing identical mutations were counted only once. Data are the percentage of specific mutations observed in normal mammary gland, IDPs, carcinoma in situ, and carcinomas from PhIP-treated rats.
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Table I. H-ras mutations detected in laser microdissected mammary gland samples from control and PhIP-treated rats
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Statistical analysis
Statistical calculation was carried out by
2-square analysis and by the z-test using SigmaStat, version 2, (Jandel Scientific Software, San Rafael, CA).
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Results
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Representative mammary gland samples collected for analysis of H-ras mutations are shown in Figure 1
. The normal mammary gland of virgin female rats consisted primarily of ducts lined with a single layer of epithelial cells (Figure 1A, B
). Occasionally, small lobules comprised of ~710 clusters of tubules were also seen. IDP, considered to be the earliest histopathological change observed in the rat mammary gland parenchyma during carcinogenesis (3,5), was detected in mammary glands from PhIP-treated rats but not in control rats (Figure 1C, D
). IDPs were primarily found in PhIP-treated rats at 7 weeks, the earliest time point examined after PhIP administration. Histologically, IDPs showed multiple layers of epithelial cells surrounding the duct and protruding into the lumen. Carcinomas in situ were larger than IDPs and were detected histologically at several time points including 7, 9, 10 and 14 weeks after PhIP dosing. The lumen of carcinomas in situ were expanded with epithelial cells growing in patterns similar in appearance to palpable carcinomas (Figure 1E, F
). Tubulopapillary carcinomas were the major tumor type observed in PhIP-treated rats (Figure 1G, H
). In these carcinomas, epithelial growth occurred inward forming papillae and/or tubular structures surrounding small lumens. Carcinomas were found mainly at 9, 10, and 14 weeks after dosing.

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Fig. 1. Hematoxylin and eosin stained sections of representative lesions and normal rat mammary gland. (A, B) Normal ductal structures of the mammary gland (100x and 200x, respectively). (C) (D) Mammary ducts with hyperplasia found in PhIP-treated rats (200x and 400x, respectively). (E, F) Carcinoma in situ found in PhIP-treated rats (100x, 200x, respectively). (G, H) Typical tubulopapillary carcinomas detected after PhIP-treatment (100x and 200x, respectively).
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Since different types of H-ras codon 12 and 13 mutations may be present in PhIP-induced rat mammary gland carcinomas (28), DNA from each microdissected sample was cloned into a plasmid pCR2.1 vector prior to sequencing as described in Materials and methods. Table I
shows the raw data from the sequencing analysis of all plasmid vector clones. From this data, mutation frequency in control mammary gland and in normal, preneoplastic, and neoplastic mammary gland from PhIP-treated rats was derived (Figure 2
). The overall frequency of H-ras mutation (codons 12 and 13 combined) was 20% in control mammary gland from untreated rats, and this frequency rose to 73%, 75%, 100%, and 100% in normal mammary gland, IDPs, carcinoma in situ, and carcinoma, respectively, from PhIP treated rats. The overall mutation frequency in carcinomas was significantly higher than in control (z-test, P < 0.05). Codon 12 mutations were found in mammary gland samples from both control and PhIP-treated rats, however, the frequency of codon 12 mutations increased after PhIP exposure and with the progression of neoplasia. The frequency of codon 12 mutations was significantly higher in carcinoma than in normal control mammary gland (z-test, P < 0.05). Codon 13 mutations were found after PhIP treatment in all lesions as well as in histologically normal tissue, but these mutations were not detected in normal mammary gland from control rats. After PhIP treatment, there was a statistically significant increase in the frequency of codon 13 mutations and the overall frequency of mutations in histologically normal mammary gland in comparison to control mammary gland (z test, P < 0.05). Codon 13 mutations were also detected in IDPs, carcinoma in situ and carcinomas at frequencies of 75%, 60% and 60%, respectively, values that were slightly higher than that seen in normal tissue from PhIP-treated rats (45%).
In addition to the differences in mutation frequency, the spectrum of H-ras mutations were different between mammary gland from control and PhIP-treated rats. In the control mammary gland, the only mutation detected was a G35 to C transversion in codon 12 (Table I
). In contrast, after PhIP treatment, both G35 to C and G35 to A mutations were detected in histologically normal gland. The codon 13 mutations, which were only found after PhIP treatment, included transitions and transversions especially at G37 and to a lesser extent at G38 in histologically normal tissue. The spectrum of mutations found in normal mammary gland, preneoplastic and neoplastic lesions after PhIP treatment was largely similar (Figure 3
). After exposure to PhIP, the predominant mutations found in normal, preneoplastic and neoplastic epithelium were guanine base substitutions. G:C to C:G transversions were the major mutation accounting for 4050% of all mutations. G:C to A:T transition and then G:C to T:A transversion mutations were the next most prevalent comprising 2230% and 1822% of total mutations, respectively. Interestingly, adenine mutations were found at a low frequency in IDPs, carcinoma in situ and carcinomas but were not detected in normal epithelial cells from PhIP-treated rats. For example, in carcinomas, 13 clones carried silent mutations at A36 with 5 showing A:T to T:A transversions and 8 showing A:T to G:C transitions (Table I). It is notable that silent mutations were always found in DNA samples that also carried activating H-ras mutations. Adenine mutations may be associated with instability in PhIP-induced carcinomas (32).
The G:C to C:G transversion mutation at G35 detected in control mammary gland appeared to be a novel naturally harbored activating mutation in mammary gland from SpragueDawley rats. Three of 75 total clones that were sequenced from microdissected normal mammary gland samples harbored this mutation, suggesting that this mutation was harbored in a low percentage of mammary epithelial cells (Table I
). This H-ras mutation was not detected in DNA isolated from whole liver of PhIP-treated rats (eight rats, 17 clones, 13 clones per rat), and it was not detected in microdissected skeletal muscle tissue that surrounded the mammary gland parenchyma (28 clones sequenced, Table I
). Since this mutation was only found in the mammary gland epithelium, it did not appear to be a methodological artifact involving PCR amplification or DNA sequencing. Furthermore, the mutation was found in DNA isolated from whole mammary gland carcinomas indicating that the mutation was probably not introduced by LCM. Previous studies have supported the notion that the codon 12 5'-GCA-3' alanine substitution in H-ras is transforming (18). In addition, this mutation has been found previously in urethane-induced skin carcinoma in mice (33).
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Discussion
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Using LCM, PCRRFLP, cloning and sequencing, H-ras mutations were detected in 10 of 10 PhIP-induced rat mammary gland carcinomas. Previous studies have reported various frequencies of H-ras mutations in PhIP-induced mammary gland carcinomas (2729). Our laboratory previously reported a 26% frequency of H-ras mutations in PhIP-induced carcinomas using DNA isolated from whole tumors (28). However, with LCM we found an incidence of 100%. Rat mammary carcinomas, although largely epithelial, contain stromal elements that may have diluted tumor DNA with wild-type H-ras. LCM appears to have facilitated detection of H-ras mutations in PhIP-induced carcinomas by enriching for epithelial cells harboring these mutations. By LCM methods, we were furthermore able to examine the frequency of H-ras mutations during the stages of mammary gland cancer development for the purpose of providing insight into the role of H-ras in the progression of the mammary gland epithelium to malignancy. H-ras mutations were detected in normal tissue, IDPs, and carcinoma in situ in PhIP-treated rats. Although a low frequency of H-ras mutations was also found in normal mammary gland from untreated rats (20%), after PhIP exposure, the frequency of H-ras mutations increased in normal mammary epithelium to 73% (Figure 2
). Seventy-five percent of IDPs carried an H-ras mutation, and the frequency rose to 100% in carcinomas in situ and carcinomas. Therefore, PhIP exposure and PhIP-induced mammary gland carcinogenesis was associated with an increase in H-ras mutations such that 100% of carcinomas in situ and carcinomas carried a mutant H-ras. These findings are consistent with a major contribution of H-ras in the initiation and progression of PhIP-induced rat mammary gland carcinogenesis.
The induction of mammary gland carcinogenesis by PhIP requires metabolic activation and formation of PhIPDNA adducts (12). PhIPDNA adducts including the C8-guanine adduct, the major adduct, have been detected at relatively high levels in the mammary gland of rats that develop cancer (34). As demonstrated by previous studies in Big Blue rats, PhIPDNA adducts induce characteristic mutations in vivo in the mammary gland (15). In the paradigm of the multistep model of chemical carcinogenesis, adducts induce mutations in critical genes that are responsible for the initiation of carcinogenesis. However, the genes associated with initiation of mammary gland carcinogenesis by PhIP have remained unknown. The data from the current study are consistent with the notion that H-ras is a critical target gene for the initiation of mammary gland carcinogenesis by PhIPDNA adduct-induced mutations. Under an identical treatment regimen for PhIP and within a similar time frame for mutation analysis, there are distinct similarities between the mutation spectrum observed in the lacI mutational reporter gene in mammary glands of Big Blue rats (15) and seen in the current study in the H-ras gene. In Big Blue rats, we found that PhIP-induced mutations occur preferentially at guanine bases with 43% of all guanine mutations occurring at guanine nucleotides adjacent to another guanine. In H-ras codons 12 and 13, the nucleotides harboring mutations, G35, G37, and G38, meet these general specifications. In addition, 12 out of 109 sites showing mutations in the lacI gene of Big Blue rats carried a guanine mutation in 5'-GGA-3', a mutation in a sequence context coinciding with the major site of mutation at G35 in H-ras. Mutations at four of these lacI sites specifically showed G:C to A:T transitions, and one site harbored a G:C to C:G transversion; these are the same mutations found at G35 of H-ras. One site in the lacI gene showed a recurrent G:C to T:A transversion mutation at 5'-AGG-3', a mutation that matched the major mutation found in G37 of H-ras codon 13. Furthermore, 9 out of 109 sites in the lacI gene carried a mutation (predominately G:C to T:A transversions) at 5'-GGC-3', a sequence context identical to the G38 mutation site in H-ras codon 13. Taken together, these data provide evidence to support the role of PhIPDNA adducts in H-ras-induced mutations in the mammary gland and to link PhIPDNA adducts to the initiation of mammary gland carcinogenesis via activating mutations in the H-ras gene.
Cha et al. (35) detected a G35 to A transition mutation in H-ras codon 12 in the mammary gland of Fischer 344 rats that were not exposed to carcinogen. They proposed that subsequent exposure to NMU facilitates the expansion of cells carrying this pre-existing mutation rather than directly inducing this mutation through DNA adduct formation. Although in the current study this mutation was not detected in normal control SpragueDawley rat mammary gland, we did detect a G:C to C:G transversion mutation at G35 of codon 12, which appeared to be a novel naturally harbored mutation. The frequency of this mutation also appeared to increase after PhIP exposure and with the development of preneoplastic and neoplastic lesions. For example, three of 75 vector clones (4%) that were sequenced carried this mutation in control mammary gland, while after PhIP treatment, 9 of 49 clones (18%) showed mutation in normal mammary gland, and 30 of 116 clones (26%) carried this mutation in carcinomas (Table I
). Therefore akin to the situation with NMU, it appears possible that PhIP exposure may be associated with an expansion of epithelial cells harboring this specific G35 pre-existing mutation. However, other codon 12 mutations and mutations in codon 13 which were not found in control rat mammary gland, appear from mutation spectra data to be PhIPDNA adduct induced. Indeed, the G:C to T:A transversion mutation that occurs predominantly in codon 13 is the major mutation induced by the C8-guanine adduct of PhIP (14).
The results from this study are compatible with the notion that mammary epithelial cells with H-ras mutation have a growth advantage that may facilitate mammary gland cancer development. This conclusion is supported by the progressive increase in the frequency of H-ras mutations in the mammary gland concurrent with the development of neoplasia following PhIP exposure (Figure 2
). Furthermore, the similarity in the mutation spectrum in normal epithelium after PhIP treatment with the spectra found in IDPs, carcinomas in situ and carcinomas suggests that initiated cell populations carrying H-ras mutations were expanded with carcinoma development (Figure 3
). Previous studies indicate that resistance to chemically-induced mammary gland carcinogenesis in the Copenhagen rat strain is associated with an inability of cells with H-ras mutations to undergo clonal expansion (22,24). The findings from this study bolster the concept that expansion of cells carrying activated H-ras is important for chemically-induced rat mammary gland cancer development. Likewise, the presence of H-ras mutations in rat mammary gland cancers induced by a variety of structurally distinct chemical carcinogens including NMU, DMBA (36), and PhIP, and the detection of H-ras mutations in the early stages of PhIP carcinogenesis, provide strong support for the significance of the H-ras gene in chemically-induced rat mammary gland carcinogenesis.
Studies in transgenic mice expressing activated H-ras and in rats infected with v-H-ras via the mammary gland central duct show that activated H-ras is associated with a modest number of mammary carcinomas after a long latency period (37,38). Consistent with these findings, we have found that PhIP induces a relatively low tumor multiplicity with on average 2.1 tumors arising per rat (11) despite the high percentage of mammary glands carrying an H-ras mutation. These data are compatible with the conclusion that additional genetic alterations are required to complement activated H-ras during mammary gland carcinogenesis (37). Further study is required to determine the genetic alterations induced by PhIP that may cooperate with mutated H-ras.
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Notes
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1 To whom correspondence should be addressed at: Chemical Carcinogenesis Section, Laboratory of Experimental Carcinogenesis, Building 37, Room 4146, 37 Convent Dr. MSC 4262, National Cancer Institute, NIH, Bethesda, MD 20892, USA Email: elizabeth_snyderwine{at}nih.gov 
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Acknowledgments
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The authors thank Dr Mark Miller, head of NCI's DNA Minicore Facility, for DNA sequencing analysis.
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Received May 8, 2002;
revised July 3, 2002;
accepted September 5, 2002.