ras gene mutations are absent in NMU-induced mammary carcinomas from aging rats

Todd A. Thompson1, Jill D. Haag and Michael N. Gould1,2

Department of Oncology, McArdle Laboratory for Cancer Research and
1 Environmental Toxicology Center, University of Wisconsin–Madison, Madison, WI 53706, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carcinoma induction in the rat mammary carcinogenesis model is age dependent. In this study, mammary cancer susceptibility and ras gene activation were investigated in rats exposed to N-methyl-N-nitrosourea (NMU) at 2, 6, 8 and 15 months. Animals were resistant to NMU-induced mammary tumor development when exposed at 6 and 8 months of age, whereas a significant number of mammary carcinomas developed in animals exposed to NMU at 2 and 15 months of age. G35->A35 activating mutations in the Harvey ras gene were found only in mammary carcinomas from rats exposed to NMU at 2 months of age, but not in tumors that developed in animals exposed to NMU at 15 months of age. No G35->A35 activating mutations were present in the Kirsten ras gene of any of the mammary tumors. Additional analysis of exons 1 and 2 of the Harvey ras gene from mammary carcinomas that developed in animals exposed to NMU at 15 months of age did not reveal any other activating mutations in this gene. In mammary carcinomas from animals exposed to NMU at 2 months of age, the frequency of mammary carcinomas with mutations in the Harvey ras gene was independent of the time from which the tumor first appeared. Therefore, age at the time of carcinogen exposure plays a critical role in both breast cancer susceptibility and the molecular events that contribute to mammary carcinoma development.

Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; NMU, N-methyl-N-nitrosourea; RFLP, restriction fragment length polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer, for the most part, is a disease of aging. While most cancers develop in older women, the time at which these cancers are initiated is less clear. Both human (1) and rodent (2) data suggest that the susceptibility to the initiation of breast cancer by environmental carcinogens is a function of age. This function is not of a continuous nature, but involves windows of certain age ranges in which induction is most probable. For example, the age of highest susceptibility of humans to breast cancer induction by ionizing radiation is that of the immature girl. This susceptibility drops off continuously with age with only a slight trend to transient increased risk preceding menopause (1). In rats, the best documented age of susceptibility to model chemical carcinogens including both polycyclic hydrocarbons, such as 7,12-dimethylbenz[a]anthracene (DMBA), and alkylating agents, such as N-methyl-N-nitrosourea (NMU), surrounds the period of sexual maturation (3).

In the NMU-induced rat mammary carcinogenesis model, up to 90% of mammary carcinomas have been reported with G35->A35 transition mutations in codon 12 of the Harvey ras gene (4). The high frequency in which activating Harvey ras gene mutations are found in these cancers suggests that they occur early in the development of NMU-induced rat mammary carcinomas (4,5). We have previously quantified the frequency of Harvey ras gene mutations in NMU-exposed female rats and found that approximately one in 43 000 mammary stem-like cells have activating Harvey ras gene mutations following NMU exposure (6). Therefore, activating mutations in the Harvey ras gene are believed to be important molecular lesions in the etiology of rat mammary carcinomas.

In this study, the susceptibility of inbred Wistar–Furth rats to NMU-induced mammary carcinogenesis over a broad age range was investigated. NMU-specific activating mutations in the Harvey ras gene from all resultant mammary tumors were determined.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal maintenance and carcinogen administration
Virgin female Wistar–Furth rats (Harlan Sprague–Dawley, Indianapolis, IN) received 30 mg/kg NMU in acidified saline by tail-vein injection at 2, 6, 8 or 15 months of age. The 30 mg/kg NMU dose was used since it maximizes the number of mammary carcinomas produced with those harboring Harvey ras gene mutations (7). Untreated females were used as controls to monitor background mammary tumor development. Rats were maintained at the University of Wisconsin Animal Care Facility and received lab chow (Teklad, Madison, WI) and acidified water ad libitum. Animals were palpated every 2–4 weeks for 34 weeks after NMU exposure. All animals remaining in the study up to 70 weeks after NMU administration were killed, except untreated control animals, which were maintained until 19 months of age to determine background mammary tumor incidence in the Wistar–Furth rat strain.

Mammary tumor preparation and assessment
During the course of the study, mammary tumors ranging in size from 10 to 20 mm in diameter were surgically resected and divided with sections either fixed in formalin for histopathological analysis or stored at –80°C for molecular analysis. After tumor resection, animals were returned to the study. At necropsy, all mammary tumors were removed, prepared and analyzed as indicated above.

Harvey ras gene and Kirsten ras gene mutation analysis
An allele-specific oligonucleotide hybridization (ASOH) assay, a designed diagnostic restriction fragment length polymorphism (RFLP) assay and DNA sequencing were performed to screen for mutations in the Harvey and Kirsten ras genes. For ASOH, the region surrounding codon 12 of the ras genes was PCR amplified and transferred to Gene Screen Plus nylon membranes (NEN/DuPont), as described previously (6). Membranes were probed using a 32P-end-labeled probe (~105 c.p.m./ml) that specifically anneals to PCR product with a G35->A35 mutation (5'-gcgctgaaggcgtggaaa-3') in the Harvey ras gene under stringent conditions. After probing, membranes were imaged on a PhosphorImager (Molecular Dynamics) and analyzed using ImageQuant software (Molecular Dynamics). Kirsten ras gene activation was assessed using the designed diagnostic RFLP method, as described previously (8). For complete mutational analysis of mammary carcinomas from rats exposed to NMU at 15 months of age, a 5' primer (5'-tggctagggcctggctaagt-3') and 3' primer (5'-tgccgggtcttggctgatgt-3') that generate an 870 bp fragment spanning exons 1 and 2 of the rat Harvey ras gene were PCR amplified. The PCR product was purified using a Chroma Spin-100 column (Clontech, Palo Alto, CA), sequenced using an ABI Prism BigDye cycle sequencing kit (Applied Biosystems, Foster City, CA) with the same 5' primer used for PCR amplification and analyzed using an ABI 373 Automated DNA sequencer (Applied Biosystems, Foster City, CA). DNA from an NMU-induced rat mammary tumor with a G35->A35 mutation in the Harvey ras gene or DNA from an NMU-induced rat colon tumor with a G35->A35 mutation of the Kirsten ras gene were used as positive mutation controls and rat-tail DNA was used for negative controls.

Statistical analysis
Kaplan–Meier survival curve estimates were determined for each group. The time without tumor was determined for each age group and used to compare the number of expected and observed events using a log-rank test. Pair-wise comparisons were also made using a log-rank test. If global comparisons at 25.5 or at 34 weeks were significant for tumor multiplicity, then a one-way analysis of variance for global comparison of each group was performed, followed by least significant difference pair-wise comparison. A Fisher exact test was used to determine the significance of Harvey ras gene activation among tumor groups.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mammary tumorigenesis in Wistar–Furth rats exposed to NMU at 2, 6, 8 or 15 months of age
The multiplicity of mammary tumors of all histopathological classes developing in the rats was determined up to 34 weeks following NMU exposure (Figure 1aGo). In rats exposed to NMU at 2, 6, 8 and 15 months of age, an average of 0.91, 0.25, 0.06 and 1.22 mammary tumors per rat were present at 34 weeks, respectively. No mammary tumors developed in untreated control rats over the course of this study. The number of tumors that developed by 34 weeks in rats exposed to NMU at 2 and 15 months of age were significantly different from untreated control rats (P < 0.05). The mean time to first tumor for rats exposed to NMU at 2, 6, 8 and 15 months of age was found to be 26, 30, 32 and 22 weeks, respectively. The latency of mammary tumor development was significantly greater for rats exposed to NMU at 8 months of age compared with those exposed at 2 (P = 0.005) and 15 months (P = 0.004) of age, whereas no significant difference in the latency of tumor development was found in rats exposed to NMU at 2 and 15 months of age.



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Fig. 1. Multiplicity analysis of mammary tumors of all histopathological types (a) and mammary carcinomas only (b) that developed in Wistar–Furth rats exposed to NMU at 2, 6, 8 and 15 months of age. Control Wistar–Furth rats did not develop mammary tumors over the course of this study.

 
Histopathological analysis of all mammary tumors was performed to identify carcinomas from other mammary tumors. These data were used to plot the multiplicity of mammary carcinomas up to 34 weeks following NMU exposure (Figure 1bGo). At 34 weeks of NMU administration, all mammary tumors from rats exposed to NMU at 2 months of age were identified as carcinomas, giving 0.91 carcinomas per rat. One mammary carcinoma developed in rats exposed to NMU at 6 months. Sixty-four percent of the mammary tumors that developed in rats exposed to NMU at 15 months of age were carcinomas, resulting in an average multiplicity of 0.78 carcinomas per rat at 34 weeks. No mammary carcinomas were observed by 34 weeks in rats exposed to NMU at 8 months of age or in untreated controls. The mean time to first carcinoma for rats exposed to NMU at 2, 6 and 15 months of age was found to be 26, 32 and 24 weeks, respectively.

ras gene activation in NMU-induced mammary tumors from rats exposed to NMU at 2, 6, 8 or 15 months of age
The majority of mutations found in rat mammary carcinomas following NMU exposure are found in the Harvey ras gene and are uniformly observed as G35->A35 transition mutations (4), although a small percentage of activating mutations in the Kirsten ras gene have previously been reported in mammary carcinomas that developed in rats exposed to NMU as neonates (8). Therefore, all mammary tumors produced in this study were screened for the presence of G35->A35 mutations in both the Harvey ras and Kirsten ras genes. Table IGo summarizes data on the frequency of ras gene mutations identified from the various mammary tumor types produced throughout this study from all age groups. Ten of 23 mammary tumors (44%) from rats exposed to NMU at 2 months of age were found with G35->A35 mutations in the Harvey ras gene (Table IGo; Figure 2a and cGo). No G35->A35 Harvey ras gene mutations were observed in mammary tumors from rats exposed to NMU at 6, 8 or 15 months of age (Table IGo; Figure 2bGo). A second study was performed to further investigate mammary tumor development in rats exposed to 30 mg/kg NMU at 15 months of age. Again, no G35->A35 mutations in the Harvey ras gene were observed in the mammary tumors derived from this study (Figure 2cGo). Sequencing analysis of exons 1 and 2 of the Harvey ras gene, the regions where activating mutations of the Harvey ras gene have previously been identified (9), revealed no additional activating mutations in mammary carcinomas taken from rats exposed to NMU at 15 months of age (unpublished data). No G35->A35 mutations were observed in the Kirsten ras gene in any of the resultant mammary tumors (Table IGo). The frequency of mutations in the Harvey ras gene found in mammary carcinomas that developed throughout the experiment in rats exposed to NMU at 2 months of age was independent of the time the carcinoma was first palpated (Table IIGo).


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Table I. Mammary tumor histopathological typing from each NMU-exposed group and the number of tumors with Harvey ras or Kirsten ras G35->A35 mutations
 


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Fig. 2. ASOH (a and b) and designed diagnostic RFLP (c) analysis of G35->A35 mutations in the Harvey ras gene from NMU-induced mammary tumors that developed in Wistar–Furth rats exposed to NMU at 2 (a and c) or 15 months (b and c) of age. (A and B) H-ras, negative control DNA; H-ras+, G35->A35 Harvey ras positive control; PCR, PCR negative control; T1–T12, mammary tumor DNA. Arrow indicates samples identified with G35->A35 mutations in the Harvey ras gene. (c) Lane 1, G35->A35 Harvey ras positive control; lane 2, negative control DNA; lanes 3–13, mammary tumors from rats exposed to NMU at 2 months of age; lanes 14–23, mammary tumors from rats exposed to NMU at 15 months of age. Arrow indicates position of restriction fragment indicating the presence of a G35->A35 mutation in the Harvey ras gene. Samples in lanes 4, 9, 12 and 13 are identified as having a G35->A35 mutation in the Harvey ras gene.

 

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Table II. Evaluation of Harvey ras gene activation in mammary carcinomas from rats exposed to NMU at 2 months age compared with the time the carcinoma was first observed
 
Comparative histopathological analysis of NMU-induced mammary tumors
Mammary tumors that developed in rats administered NMU at 2 months of age were predominantly papillary carcinomas (Figure 3aGo). However, the mammary tumors that developed in the group exposed to NMU at 15 months of age varied widely and included papillary carcinomas (Figure 3bGo), carcinosarcomas (Figure 3cGo), cribriform carcinomas (Figure 3dGo) and comedocarcinomas (Figure 3e and fGo).



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Fig. 3. Histopathological analysis of mammary tumors from Wistar–Furth rats exposed to NMU at 2 (a) and 15 months of age (bf). (a) Mammary papillary carcinoma from a female Wistar–Furth rat exposed to NMU at 2 months of age. Neoplasms that developed in female Wistar–Furth rats exposed to NMU at 15 months of age identified as a papillary carcinoma (b), carcinosarcoma (c), cribriform carcinoma (d) and comedocarcinomas (e and f). Hematoxylin and eosin stained slides; magnification x230.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Variations between rat mammary carcinogenesis studies include the type and dose of carcinogen administered, the age at the time of exposure, and the strains of rats investigated. Due to variations among studies, it is difficult to make direct comparisons between them. Yet, in general, it has been observed that sexually maturing rats are more susceptible to mammary carcinogenesis than adult rats. In this study, female Wistar–Furth rats showed an age-dependent difference in NMU-induced mammary tumor development, which is consistent with previous studies (3). For example, animals administered NMU at 2 months of age developed an average of 0.9 mammary carcinomas per rat within 34 weeks of exposure, which was in agreement with previous reports of carcinoma development in the Wistar–Furth rat strain using a 30 mg/kg NMU dose (7). In contrast, only one mammary carcinoma occurred in animals exposed at 6 months of age and no carcinomas developed in rats exposed to NMU at 8 months of age. Therefore, adult female rats exposed to chemical carcinogens at 6 and 8 months of age are less susceptible to mammary carcinoma development than animals exposed to carcinogen during sexual development.

In contrast to the resistance of mammary carcinoma induction observed in middle-aged adult rats, older rats that received NMU at 15 months developed an average of 1.2 mammary tumors per rat at 34 weeks following NMU exposure. Sixty-four percent of these mammary tumors were carcinomas, resulting in 0.78 carcinomas per rat at 34 weeks, which was not significantly different than the number of carcinomas that developed in rats exposed to carcinogen at 2 months of age (i.e. during sexual development). Previously, Sinha et al. (2) reported that 440-day-old (i.e. ~14-month-old) female Sprague–Dawley rats exposed to DMBA developed a 37.5% incidence of mammary carcinomas. These investigators also reported that the age-dependent development of DMBA-induced mammary carcinomas was directly proportional to the labeling index of the mammary gland at the time of carcinogen exposure. Therefore, it was suggested that the proliferative status of the mammary gland at the time of carcinogen exposure was important for mammary carcinogenesis. Older female rats experience senescent anovulation (i.e. constant estrus) that coincides with increased serum prolactin levels (10,11). Thus, mammary gland restructuring and hormonal fluctuations may play a role in the susceptibility of older rats to mammary carcinogenesis compared with the relative resistance of mammary cancer development found in middle-aged adult female rats.

NMU-induced activating mutations in the Harvey ras gene of mammary carcinomas are well established and are uniformly observed as G35->A35 transition mutations in the second base pair of codon 12 (9). Zarbl et al. (4) found that 60–92% of mammary carcinomas that developed following NMU exposure had mutations in the Harvey ras gene, dependent on the rat strain used. This high frequency of Harvey ras gene activation was further demonstrated in Sprague–Dawley rats exposed to NMU at 21 days of age, where 98% of resultant mammary carcinomas were found with G35->A35 mutations in the Harvey ras gene (12). We have previously reported that the frequency of Harvey ras gene mutations present in NMU-induced mammary carcinomas is inversely proportional to the NMU dose administered (7). Thus, the frequency of activating Harvey ras gene mutations present in mammary carcinomas that develop following NMU exposure may vary depending on the strain and physiologic status of rat studied, the age of NMU exposure and the dose of NMU administered.

This hypothesis is supported in the current study where 53% of the mammary carcinomas that developed in female Wistar–Furth rats exposed to NMU at 2 months of age were found with G35->A35 mutations in the Harvey ras gene. No activating mutations were observed in the Harvey ras gene of mammary carcinomas from the group exposed to NMU at 15 months of age. Yet, no difference in the percentage of mammary carcinomas with activating mutations in the Harvey ras gene in rats exposed to NMU at 2 months of age was found relative to the time the tumor was first observed. That is, even carcinomas that arose beyond 59 weeks (i.e. 15 months after NMU exposure) in rats exposed to NMU at 2 months of age had mutations in the Harvey ras gene. Thus, the time of carcinogen exposure is critical to the presence of Harvey ras gene mutations in rat mammary carcinogenesis.

We have shown previously that in rats exposed to NMU at 7–8 weeks of age, the percentage of mammary carcinomas with Harvey ras gene mutations decreases when carcinogenesis is promoted with prolactin (7). The level of serum prolactin in rats has been reported to increase with age (10,11). Thus, the lack of ras gene mutations observed in mammary carcinomas from rats exposed to carcinogen when aged may stem from the promotion of carcinoma development by pathways that are independent or downstream of pathways that would otherwise involve ras gene activation. Therefore, physiological differences between young and older rats may account for differences in mammary carcinogenesis and the molecular events that contribute to mammary cancer development.

The histopathology of mammary tumors that developed in rats exposed to carcinogen when older was more variable than the carcinomas that developed in sexually maturing rats. For example, the majority of mammary carcinomas that developed in rats exposed to carcinogen during sexual development were papillary in morphology whereas those arising following NMU exposure at 15 months were more varied and included comedocarcinomas, carcinosarcomas and cribriform carcinomas. In addition, adenomas and fibroadenomas were observed in older, but not younger, animals given NMU. This parallels human breast cancer where breast cancer in younger women is mostly ductal carcinoma whereas cancers in older women are more varied (13). Since activating mutations in the Harvey ras gene were only observed in mammary carcinomas from animals exposed to NMU during sexual maturation, activated Harvey ras may contribute predominantly to papillary carcinoma development instead of other histopathological types of mammary carcinomas.

The standard rat mammary carcinogenesis model (i.e. carcinogen exposure during sexual maturation) has been criticized because the predominant molecular signature observed in this model is Harvey ras activation, whereas ras activation by mutation is almost never observed in human breast cancers. Our current results suggest that ras activation in rat mammary carcinogenesis is not simply the result of NMU exposure, but also involves the age-related cellular physiology and development of the mammary gland. It is thus possible that the lack of ras activation in most human mammary cancers results from the lack of significant carcinogen exposure during the developmental window that is equivalent to that present in the mammary gland of the sexually maturing rat.

In summary, an age dependence in NMU-induced mammary carcinogenesis was observed. Rats aged 6 or 8 months when exposed to NMU were resistant to mammary tumor development, whereas carcinogen exposure in sexually developing and aged animals resulted in the efficient production of mammary tumors. Interestingly, while NMU-induced carcinomas were found in sexually developing and older rats, activation of the Harvey ras gene was only observed in carcinomas from rats exposed to NMU during sexual development. Therefore, the incidence of carcinogen-induced mammary cancer development and the molecular events that contribute to mammary carcinogenesis may vary with age.


    Notes
 
2 To whom correspondence should be addressedEmail: gould{at}oncology.wisc.edu Back


    Acknowledgments
 
The authors would like to thank Dr Debra MacKenzie for review of this manuscript, Wendy Kennan, Patricia McGuire and Jon Houtman for technical assistance, Dr Kelly Clifton for histopathological analysis, and Dr Mary Lindstrom for statistical analysis. This work was supported by NIH Grant CA77527 and Fellowship DAMD17-94-J-4104 awarded to T.A.T. by the US Army Medical Research and Material Command.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Land,C.E. (1997) Radiation and breast cancer risk. In Aldaz,C.M., Gould,M.N., McLachlan,J. and Slaga,T.J. (eds) Etiology of Breast and Gynecological Cancers. Wiley Liss, New York, pp. 1–16.
  2. Sinha,D.K., Pazik,J.E. and Dao,T.L. (1983) Progression of rat mammary development with age and its relationship to carcinogenesis by a chemical carcinogen. Int. J. Cancer, 31, 321–327.[ISI][Medline]
  3. Russo,J., Russo,I.H., Rogers,A.E., Van Zwieten,M.J. and Gusterson,B. (1990) Tumours of the mammary gland. In Turusov,V.S. and Mohr,U. (eds) Pathology of Tumours in Laboratory Animals. IARC Scientific Publications no. 99, IARC, Lyon, pp. 47–78.
  4. Zarbl,H., Sukumar,S., Arthur,A.V., Martin-Zanca,D. and Barbacid,M. (1985) Direct mutagenesis of Ha-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats. Nature, 315, 382–385.[ISI][Medline]
  5. Sukumar,S., Notario,V., Martin-Zanco,D. and Barbacid,M. (1983) Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature, 306, 658–661.[ISI][Medline]
  6. Zhang,R., Haag,J.D. and Gould,M.N. (1991) Quantitating the frequency of initiation and cH-ras mutation in in situ N-methyl-N-nitrosourea-exposed rat mammary gland. Cell Growth Differ., 2, 1–6.[Abstract]
  7. Zhang,R., Haag,J.D. and Gould,M.N. (1990) Reduction in the frequency of activated ras oncogenes in rat mammary carcinomas with increasing N-methyl-N-nitrosourea doses or increasing prolactin levels. Cancer Res., 50, 4286–4290.[Abstract]
  8. Kumar,R., Sukumar,S. and Barbacid,M. (1990) Activation of ras oncogenes preceding the onset of neoplasia. Science, 248, 1101–1104.[ISI][Medline]
  9. Barbacid,M. (1987) ras genes. Annu. Rev. Biochem., 56, 779–827.[ISI][Medline]
  10. Wilkes,M.M., Lu,K.H., Fulton,S.L. and Yen,S.S.C. (1978) Hypothalamic–pituitary–ovarian interactions during reproductive senescence in the rat. In Finch,C.E., Potter,D.E. and Kenny,A.D. (eds) Parkinson's Disease—II. Aging and Neuroendocrine Relationships. Plenum Press, New York, pp. 126–147.
  11. Demarest,K.T., Moore,K.E. and Riegle,G.D. (1982) Dopaminergic neuronal function, anterior pituitary dopamine content and serum concentrations of prolactin, luteinizing hormone and progesterone in the aged female rat. Brain Res., 247, 347–354.[ISI][Medline]
  12. Lu,J., Jiang,C., Mitrenga,T., Cutter,G. and Thompson,H.J. (1998) Pathogenic characterization of 1-methyl-nitrosourea-induced mammary carcinomas in the rat. Carcinogenesis, 19, 223–227.[Abstract]
  13. Walker,R.A., Lees,E., Webb,M.B. and Dearing,S.J. (1996) Breast carcinomas occurring in young women (<35 years) are different. Br. J. Cancer, 74, 1796–1800.[ISI][Medline]
Received May 10, 2000; revised June 14, 2000; accepted June 14, 2000.