Resistance to mammary tumorigenesis in Copenhagen rats is associated with the loss of preneoplastic lesions
James E. Korkola1 and
Michael C. Archer1,2,3
1 Departments of Medical Biophysics and
2 Nutritional Sciences, Faculty of Medicine, University of Toronto, 150 College Street, Toronto M5S 3E2, Canada
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Abstract
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The resistance of Copenhagen (Cop) rats to mammary tumor development has recently been linked to three loci, but the genes have yet to be cloned and the mechanism of resistance is still largely unknown. In order to determine the cellular events associated with resistance, we prepared mammary whole mounts from Cop and susceptible Wistar Furth (WF) rats 0, 15, 30, 45 and 60 days after treatment with 50 mg/kg N-methyl-N-nitrosourea (MNU). At 15 days, treated rats of both strains had significantly more undifferentiated structures [terminal end buds (TEBs)] and significantly fewer differentiated structures [alveolar buds (ABs)] than untreated rats. Treated Cop rats, however, had significantly more TEBs and fewer ABs than age-matched, treated WF rats. Histological analysis of preneoplastic lesions tentatively identified from the whole mounts showed that like WF rats, Cop rats developed early preneoplastic lesions [intraductal proliferations (IDPs)] by 15 days post-MNU treatment. Unlike IDPs from WF rats, however, the IDPs in Cop rats then decreased in number until they were absent 60 days post-MNU treatment. Furthermore, they failed to progress into more advanced lesions such as ductal carcinomas in situ (DCIS). Finally, we found G
A activating mutations in codon 12 of the Ha-ras gene in 60% of IDPs from Cop rats and 75% of IDPs from WF rats. Our results show that resistance in Cop rats is not due to a target cell population for the carcinogen that is smaller than in susceptible rats or to the failure of the carcinogen to inhibit mammary gland differentiation. Furthermore, we have shown that Cop rats develop preneoplastic IDPs that harbor Ha-ras mutations but, unlike IDPs in susceptible strains, they fail to progress and ultimately disappear.
Abbreviations: ABs, alveolar buds; Cop, Copenhagen; DAH, ductal alveolar hyperplasia; DCIS, ductal carcinoma in situ; DH, ductal hyperplasia; DMBA, 7,12-dimethylbenz[a]anthracene; H & E, hematoxylin and eosin; HANs, hyperplastic alveolar nodules; IDPs, intraductal proliferations; mcs, mammary carcinoma suppressor; MNU, N-methyl-N-nitrosourea; PBS, phosphatebuffered saline; PCR, polymerase chain reaction; SD, SpragueDawley; TEBs, terminal end buds; WF, Wistar Furth.
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Introduction
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Studies of families with hereditary breast cancer have resulted in the identification of abnormalities in several genes such as Brca-1 (1), Brca-2 (2) and p53 (3) that confer susceptibility to the disease. Resistance to breast cancer, on the other hand, is difficult to study in humans and, as a result, little is known about the contributing genetic factors. Chemical induction of mammary tumors in rats, however, provides an excellent model to study resistance (4). Carcinogens such as N-methyl-N-nitrosourea (MNU) (5) or 7,12-dimethylbenz[a]anthracene (DMBA) (6) are able to induce multiple mammary tumors in susceptible strains. Several strains of rat, however, are resistant to mammary tumor induction by a number of carcinogens including MNU and DMBA. The Copenhagen (Cop) rat is the most widely studied of these resistant strains (7). Resistance to mammary tumorigenesis in the Cop rat has recently been localized to three loci by linkage analysis (8), but the putative mammary carcinoma suppressor (mcs) or related genes have not yet been cloned and the mechanism of resistance is still largely unknown.
The pathogenesis of rat mammary tumors has been well characterized for susceptible strains treated with a single dose of DMBA (9,10) or MNU (1113). Following carcinogen exposure at 50 days of age when the mammary gland is not yet fully differentiated, there is an enlargement in the size of some terminal end buds (TEBs) that are thought to contain the target cells for the carcinogen (9). The resulting structures, known as intraductal proliferations (IDPs), are approximately twice the size of TEBs and are first observed 23 weeks after carcinogen exposure (9). Histological analysis shows that IDPs have 610 cell layers surrounding the central lumen, compared with the 34 cell layers typically present in a TEB (9,11). IDPs also have several other distinguishing characteristics such as infiltration of inflammatory cells, desmoplastic reaction in the stroma and luminal debris (14). During carcinogenesis, a number of IDPs progress to form ductal carcinomas in situ (DCIS) that have a more neoplastic phenotype. DCIS are distinguishable from IDPs by their increased size and the presence of multiple luminal spaces (14). A subpopulation of the DCIS develops into adenocarcinomas, the most common tumor in this model. Other structures, such as hyperplastic alveolar nodules (HANs), may arise following carcinogen treatment (13,15,16). HANs, however, appear not to be causally related to mammary cancer development (15), but instead, may develop into non-malignant structures such as adenomas (13).
The Ha-ras oncogene, activated by a G
A transition at the second nucleotide of codon 12, is found in the majority of MNU-induced rat mammary carcinomas (17). Indeed, this mutation may be the initiating event that occurs as a direct result of MNU exposure (17). In a series of experiments designed to investigate the molecular basis of resistance of the Cop rat, we have demonstrated that the extent of methylation by MNU of the Ha-ras gene and expression levels of this gene are equivalent in susceptible and resistant strains (18). Furthermore, the rates of formation and repair of the pro-mutagenic DNA adduct O6-methylguanine are not different in the two strains (18). These results suggest that the Ha-ras gene is not protected from methylation by MNU in the Cop rat and, therefore, should be susceptible to mutational activation. Indeed, we found that activated Ha-ras is present in the mammary glands of resistant rats following MNU treatment (19).
Our results indicate that the resistance of the Cop rat to mammary tumorigenesis is not due to a defect in initiation but rather appears to be due to suppression of the growth of preneoplastic cells. It is not known, however, whether the mutated Ha-ras allele in Cop rats occurs only in sporadic cells that fail to grow, or whether the initiated cells are able to form IDPs or even DCIS. Furthermore, if these preneoplastic lesions are capable of forming, it is not clear at what stage their growth is inhibited or whether they are eventually lost. Thus, the goal of this study was to examine the cellular changes that take place in the mammary glands of Cop rats following MNU treatment in order to determine at what stage during carcinogenesis resistance occurs.
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Materials and methods
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Animals and carcinogen treatment
Cop and Wistar Furth (WF) rats aged 67 weeks (Harlan SpragueDawley, Frederick, MD or San Diego, CA) were maintained on a 12 h light/dark cycle and were fed Harlan Teklad rodent diet (6% fat) and water ad libitum. After 1 week of acclimatization (i.e. at 78 weeks of age), they were randomized into three groups per strain. The first group of five rats was killed prior to carcinogen treatment for whole mount analysis (day 0 controls). The second group of 16 rats was treated i.p. with 50 mg/kg MNU (Ash-Stevens, Detroit, MI). The MNU was dissolved in 0.05% acetic acid in normal saline at a concentration of 10 mg/ml and used immediately. The third group of 16 rats received vehicle only. At 15, 30, 45 and 60 days post-MNU treatment, four rats randomly selected from each of these last two groups were killed for whole mount analysis.
Whole mount analysis
The whole mount procedure we used has been described previously by Russo et al. (9) and Thompson et al. (12). Briefly, rats were anesthetized using 75 mg/kg ketamine hydrochloride (Rogar/STB, London, Ontario, Canada) and 6 mg/kg xylazine (MTC Pharmaceuticals, Cambridge, Ontario, Canada) in normal saline administered i.m., and then killed by cervical dislocation. The pelts with the mammary glands still attached were fixed in 10% buffered formalin for 24 h. The glands were dissected from the pelts, defatted in acetone for 24 days, then hydrated through an ethanol series and left in distilled water overnight on a rocking platform. The following day, the glands were stained in 0.025% toluidine blue (Sigma, St Louis, MO) for 2 h, rinsed in water and washed in methanol then 70% ethanol for 30 min each. The stained glands were fixed in 4% ammonium molybdate for 30 min, washed in distilled water, then stored in distilled water overnight. The following day, the glands were dehydrated through an ethanol series and stored in xylenes overnight. They were then sealed in plastic bags containing methyl salicylate, coded and scored for TEBs, alveolar buds (ABs), hyperplastic alveolar nodules (HANs), IDPs, DCIS and tumors. Numbers of TEBs and ABs from untreated rats (day 0), untreated rats (day 15) and treated rats (day 15) were compared using a two-way ANOVA followed by Tukey's post hoc test.
Histological analysis
Any structures tentatively identified in the whole mounts as IDPs or DCIS were dissected from the glands. They were cleared in two changes of toluene, followed by two changes in molten paraffin wax before being embedded in paraffin wax. Sections (4 µm) were mounted on slides coated with poly-L-lysine (Sigma) and stained with hematoxylin and eosin (H & E) for histopathological analysis. Lesions were identified using the criteria of Russo et al. (14) and Sakai and Ogawa (11). Briefly, IDPs were identified by their increased cross-sectional diameter, number of cells and number of cell layers compared with TEBs, as well as by infiltration of inflammatory cells. DCIS were identified by the above criteria as well as the presence of multiple luminal spaces and a further size increase. For statistical analysis of the number of lesions per mammary gland chain, we performed a two-way ANOVA using strain and time as the variables followed by post hoc comparisons using Tukey's test.
DNA isolation and PCR analysis of mutant Ha-ras
DNA for PCR amplification was prepared from paraffin-embedded tissues by a modification of the method of Greer et al. (20). Briefly, slides of serial sections from paraffin blocks known to contain IDPs from histological analysis were deparaffinized through two changes of xylenes, rehydrated in 100% ethanol followed by 75% ethanol, then placed in phosphate-buffered saline (PBS). Lesions on the slides were located under a dissecting microscope, scraped off using a sterile pipette tip and placed in 200 µl of digestion buffer (200 µg/ml proteinase K, 50 mM TrisHCl, pH 7.5, 1% Triton X-100, 10 mM EDTA), and incubated at 37°C for 2.5 h. After inactivating the proteinase K by boiling for 10 min, the samples were concentrated by precipitation with ethanol using tRNA as a carrier, resuspended in 20 µl ddH2O, and stored at 76°C prior to use.
For analysis of MNU-induced G
A mutations at the second nucleotide of codon 12 in the Ha-ras gene, we used the PCR/liquid hybridization and gel retardation assay we have previously described (19,21), with three modifications. First, we did not pre-digest the DNA with the restriction endonuclease Mnl I, which cleaves the normal but not mutant alleles. Second, the reaction was carried out in a total volume of 50 µl instead of 100 µl, using 20 µl of the DNA solution described above. Third, we used Pfu polymerase (Stratagene, La Jolla, CA) instead of Taq or Vent polymerases. Pfu eliminated the background we observed with Taq polymerase (21), presumably due to its higher fidelity, and led to higher levels of amplification than Vent. Overnight digestion of 5 µl of the PCR mixture with 1 U of the restriction endonuclease Xmn I (New England Biolabs, Beverley, MA) followed by labeling with a 20mer probe and separation on a 10% polyacrylamide gel (19:1) resulted in 53 and 18 bp bands for samples containing mutant Ha-ras and a 71 bp band for wild-type Ha-ras.
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Results
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We prepared whole mounts from the cervicalthoracic and abdominalinguinal mammary chains of both resistant Cop and susceptible WF rats at 0, 15, 30, 45 and 60 days post-MNU treatment. In preliminary counts prior to histological confirmation of coded samples from both mammary chains, we were able to detect more putative lesions in the abdominalinguinal region. This was somewhat surprising, since it has been reported that the cervicalthoracic chain is less differentiated than the abdominalinguinal chain (22) and is known to be more susceptible to tumor formation (5). The cervicalthoracic chain, however, has an associated muscle layer that stains very darkly with toluidine blue making the ductal tree difficult to visualize and analyze. Therefore, we decided to restrict our analysis to the abdominalinguinal mammary chain. Thompson et al. (12) analyzed whole mounts only from the abdominalinguinal region of SpragueDawley (SD) rat mammary glands for similar reasons.
In susceptible rats, it has been observed that the mammary glands of treated animals are less differentiated than those of age-matched, untreated controls, the treated animals having a larger number of TEBs and fewer ABs (23). We found that there was no difference in the number of TEBs or ABs between the WF and Cop controls prior to carcinogen treatment (day 0). At 15 days post-MNU treatment, however, mammary glands from both Cop and WF rats had significantly more TEBs/mm2 and significantly fewer ABs/mm2 than the age-matched untreated rats (Figure 1A and B
). Although there were no differences in the number of TEBs/mm2 and ABs/mm2 in untreated Cop rats compared with age-matched, untreated WF rats at either day 0 or 15, there were significant differences in both these parameters between the treated groups, with Cop rats being less differentiated than the WFs (Figure 1A and B
).

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Fig. 1. Number of (A) TEBs/mm2 and (B) ABs/mm2 in Cop and WF rats from untreated (day 0), untreated (day 15) and MNU-treated (day 15) animals. All bars not sharing the same letter differ significantly from each other (P < 0.05). t, treated; u, untreated.
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An example of two putative IDPs in a whole mount from a Cop rat 15 days post-MNU treatment is shown in Figure 2A
. Compared with normal TEBs, these structures are clearly larger and stain more darkly and, indeed, both of these structures were confirmed to be IDPs histologically. A typical histological section of an IDP from a Cop rat is shown in Figure 2B
. The lesion is ~180 µm in diameter, has some areas 710 cell layers thick, and contains luminal debris, all of which are characteristics of IDPs (11,14). Two DCIS in a whole mount of a WF rat 45 days post-MNU are shown in Figure 2C
. These structures still retain the club-like shape of TEBs and IDPs, but are significantly larger (~250 µm in diameter) than the IDPs. A histological section of a DCIS from a treated WF animal is shown in Figure 2D
. In addition to its larger size, the main characteristic that distinguishes this lesion from an IDP is the presence of distinct multiple luminal spaces. As well, there is an inflammatory reaction in the stroma, which is also characteristic of these lesions (14).
We next quantified the IDPs and DCIS present in the abdominalinguinal mammary chain of all of the rats killed on days 15, 30, 45 and 60 days following MNU administration. We microdissected from the whole mounts, embedded in paraffin and sectioned suspected lesions from the 16 Cop and 16 WF rats. This analysis showed the presence of 36 IDPs, one DCIS and two cysts in the Cop rats and 46 IDPs, 17 DCIS, one fibroma, one adenocarcinoma and three cysts in the WF rats. The average diameter of the IDPs was 150 ± 6.1 µm for the Cop rats and 160 ± 12 µm for the WF rats with average cell numbers of 200 ± 12 and 210 ± 12, respectively (all values are means ± SEM). Anderson et al. (13) classified terminal structures >130 µm as abnormal in SD rats, which is in good agreement with our findings for IDPs. The average diameter of all the DCIS in WF rats was 370 ± 52 µm, whereas the one DCIS we observed in a Cop rat was 600 µm in diameter. We did not observe any IDPs in the untreated controls at any of the time points. Furthermore, we did not observe HANs in the treated rats at any time points, although we did observe cysts that arise from the same alveolar structures as HANs (13). In agreement with our observations, in rats treated with MNU, neither Sakai and Ogawa (11) nor Thompson et al. (12) reported finding HANs, whereas Anderson et al. (13) found fewer than one HAN per rat.
A time-course for the appearance of preneoplastic lesions (IDPs and DCIS) confirmed by histology is shown in Figure 3
for both strains. At 15 and 30 days post-MNU treatment the lesions were exclusively IDPs. There was an average of two DCIS in each of the WF rats at 45 and 60 days post-MNU. In contrast, we observed only one DCIS in all of the Cop rats and that appeared at day 60. It is clear that IDPs are present in the mammary glands of Cop rats 15 days post-MNU treatment. Moreover, the number of IDPs in Cop rats was not different from WFs at this time. Unlike the WF rats, however, the number of lesions in the Cop rats decreased at each subsequent time point until there were significantly more preneoplastic lesions in the WF rats at 60 days post-MNU than in the Cop rats (P < 0.01). Furthermore, the number of lesions at this time in the treated Cop rats was not significantly different from the untreated controls, which had no lesions. It should be noted that Russo and Russo (10) have reported that at 610 weeks post-DMBA treatment, as many as 30 IDPs are present per gland pair in SD rats, whereas the greatest number we observed was two per gland pair. It is likely that this difference is caused by the different carcinogens used, since others (11,13) have reported that preneoplastic lesions are less prevalent in the glands of MNU-treated rats than in those treated with doses of DMBA that produce similar numbers of tumors.

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Fig. 3. Number of preneoplastic lesions (IDPs and DCIS) in Cop and WF animals at various times following MNU treatment.
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We analyzed both Cop (Figure 4A
) and WF (Figure 4B
) DNA isolated from sections of IDPs for G
A mutations at the second nucleotide in codon 12 of the Ha-ras gene. Normal liver DNA was used as a negative control (Figure 4A and B
, lane 1) and DNA from an MNU-induced mammary tumor that contained a mutated Ha-ras allele was used as a positive control (Figure 4A and B
, lane 2). Further controls included a sample with no added DNA to show that there was no contamination of any components of the reaction mixture (Figure 4A and B
, lane 3) and DNA from two TEBs isolated from the whole-mount of a 50-day-old untreated WF rat (Figure 4B
, lanes 4 and 5). Neither of these TEBs contained a mutated Ha-ras allele. We found that Ha-ras was mutated in six of 10 IDPs from Cop rats (Figure 4A
, lanes 413) and in six of eight IDPs from WF rats (Figure 4B
, lanes 613).

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Fig. 4. (A) Ha-ras analysis of IDPs from Cop rats. Lanes 13, negative control (normal liver DNA from a WF rat), positive control (mammary tumor DNA from an MNU-treated SD rat) and blank control (no DNA), respectively; lanes 413, DNA samples amplified from sections confirmed to be IDPs. (B) Ha-ras analysis of IDPs from WF rats. Lanes 13; negative control (normal liver DNA from a WF rat), positive control (mammary tumor DNA from an MNU-treated SD rat) and blank control (no DNA), respectively; lanes 4 and 5, DNA samples amplified from sections containing normal TEBs; lanes 613, DNA samples amplified from sections confirmed to be IDPs.
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Discussion
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In order to further our understanding of the mechanisms underlying genetic resistance to breast cancer, we compared the cellular structure of the mammary glands of susceptible WF rats and resistant Cop rats before and after carcinogen treatment. TEBs contain the most rapidly proliferating cells in the mammary gland and these cells are thought to be the targets for the carcinogen (9). Thus, resistance could be caused by Cop glands having fewer TEBs. We showed, however, that untreated Cop rats had the same number of TEBs as untreated, age-matched WF rats prior to carcinogen treatment. Mammary gland differentiation has been shown to be inhibited in susceptible rats following carcinogen treatment (23) but the state of differentiation following treatment of resistant animals is unknown. Different rates of mammary gland differentiation could affect the growth and progression of initiated cells. Our results show, however, that differentiation is inhibited in both strains following carcinogen treatment. At 15 days post-MNU treatment, the number of TEBs and ABs per mm2 in both strains of treated rats was significantly different from untreated controls, the treated rats, as expected, being less differentiated. Unexpectedly, differentiation in the resistant Cop rats was inhibited to a greater extent by the carcinogen than in the susceptible WF rats. We conclude that resistance in Cop rats is not caused by either a smaller target cell population for the carcinogen compared with susceptible rats or to the failure of the carcinogen to inhibit mammary gland differentiation.
We next measured the appearance of IDPs, the first clearly observable preneoplastic lesions that develop in the rat mammary gland (9,10). In susceptible strains, a subpopulation of IDPs are thought to progress to form DCIS and ultimately adenocarcinomas (9,10). Our results clearly show the presence of IDPs in the abdominalinguinal mammary glands of both WF and Cop rats 15 days post-MNU treatment. Indeed, at this time point, there were approximately the same number of IDPs in the Cop rats as in the WFs. The number of IDPs in the Cop glands, however, decreased with time until none could be detected at day 60. In contrast, the number of IDPs in WF mammary glands increased to day 30, then remained roughly constant to day 60. At this time the number of lesions was significantly higher than in Cop rats. Moreover, we observed an average of two DCIS in the mammary glands of WF rats on days 45 and 60, whereas we saw only one DCIS in 16 Cop rats, and that was detected at day 60. We conclude that the putative mcs gene(s) does not prevent formation of IDPs in Cop rats, but rather causes the loss of IDPs and/or inhibits their development.
We have previously shown that DNA from whole mammary glands of Cop rats contains Ha-ras mutations 30 days post-MNU treatment (19). Since ~65% of IDPs from MNU-treated SD rats contain Ha-ras mutations (11), it seemed likely that the presence of similar mutations in IDPs from Cop rats would account for our previous observations. Although we could not achieve amplification of DNA from all of our IDP sections, we were able to show that six of 10 IDPs from Cop rats and six of eight IDPs from WF rats contained a mutated Ha-ras gene. These numbers are in good agreement with the results of Sakai and Ogawa (11) and indicate that the mutated Ha-ras genes that we previously observed were probably present in IDPs. This notion is strengthened by our observations of similar kinetics for the appearance and disappearance of IDPs in this study and of mutant Ha-ras alleles in the previous study (19).
One of several mechanisms may account for the disappearance of IDPs in Cop rat mammary glands. First, it is possible that initiated cells within the preneoplastic lesions are removed by an immunosurveillance mechanism. We believe this is unlikely, however, since we have recently provided evidence that T cell immunity is not involved in resistance to mammary tumorigenesis in Cop rats (24). We also have presented some evidence that NK cells are not involved (24). Second, it is possible that there is a difference in the kinetics of cell proliferation and loss in the IDPs between susceptible and resistant strains. In order for growth of a lesion to occur, the proliferative rate must be greater than the rate of cell loss and, indeed, the proliferative rate in IDPs has been shown to be higher than in TEBs (10). If the increased proliferation rate in IDPs of Cop rats is not maintained such that cell death (apoptosis) then occurs at a higher rate, the lesion will shrink in size. Similarly, if the apoptotic rate in Cop lesions increases so that it becomes greater than the proliferative rate, then the IDP will be eliminated. This type of mechanism accounts for the inhibition of growth of micro-metastases during the suppression of angiogenesis when the apoptotic rate increases 7-fold compared with non-inhibited tumors (25). We are currently measuring the proliferation and apoptotic rates to determine if these factors play a role in Cop resistance. A third potential mechanism to account for IDP loss is remodeling, a process that may account for the loss of preneoplastic foci in the liver (26). According to this mechanism, preneoplastic cells are not lost, but rather redifferentiate to yield a more normal phenotype. This reversion is thought to occur when the promoting stimulus is withdrawn (26). Redifferentiated cells are capable of being promoted to form preneoplastic hepatic foci again if the promoting stimulus is restored (27). In the mammary gland, the major stimulus for tumor development is provided by estrogenic hormones (28). The levels of these hormones in Cop rats, however, have been shown to be similar to those in other strains (29). Thus, if remodeling occurs, it is unlikely to be caused by a lack of promotional stimulus. Finally, it has been postulated that different subsets of IDPs may exist (10). The first set, known as IDP (i), are initiated but not promoted and do not progress to form tumors in susceptible animals. Instead, tumors ultimately form from a second set of IDPs that are initiated plus promoted [IDP (i + p)]. One characteristic that distinguishes these two types of lesion is the inflammatory response, with IDP (i + p) having a more marked mast cell and lymphocyte infiltration in the surrounding stroma than IDP (i). Indeed, there appears to be a 3-fold increase in mast cells surrounding IDP (i + p) compared with IDP (i) (10). Furthermore, Russo has postulated that mast cells may play a role in promoting the growth of IDP (i + p), since these cells can release factors that stimulate cell division and angiogenesis (10). If lesions in Cop rats do not elicit a mast cell response, the IDPs may not be exposed to necessary factors that stimulate tumor formation. We are currently investigating the possibility that there is a differential mast cell response in the IDPs of Cop rats compared with those of WFs using specific staining techniques.
The formation of mammary tumors in the rat has been reported to follow a well-defined developmental pattern. IDPs arise from TEBs and progress to DCIS and eventually adenocarcinomas (10). Alternative mechanisms of tumor formation in proximal regions of the gland via ductal hyperplasia (DH) and ductal alveolar hyperplasia (DAH) have been suggested (13). Whereas most of the lesions we observed were at the periphery of the gland where TEBs are located, some IDPs and DCIS were found in regions in which alveolar buds were more prevalent than TEBs. We still classified these as IDPs and DCIS based on their histopathology, but it is possible that these are the same structures that Anderson et al. (13) describe as DH and DAH. In support of this notion, Anderson et al. (13) reported that many DAH and DH have large bulbous endings, much like IDPs, possibly leading to confusion in their classification. Neither Thompson et al. (12) nor Sakai and Ogawa (11) observed DH or DAH in the glands of animals treated with MNU.
In summary, we have shown that resistance to mammary carcinogenesis in Cop rats is not due to either a target cell population that is smaller than in susceptible rats or to a failure of the carcinogen to inhibit mammary gland differentiation. Furthermore, our results show that Cop rats develop preneoplastic IDPs that harbor mutated Ha-ras alleles, but these fail to progress to form more advanced lesions such as DCIS and ultimately disappear.
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Acknowledgments
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The authors wish to thank Dr J.Russo for his help and valuable discussions, Diana Booth for her help with the preparation and sectioning of samples, and Geoffrey Wood and Amit Ghoshal for their assistance with the animal work and whole mount staining. This work was supported by a grant from the Canadian Breast Cancer Research Initiative. J.E.K. is supported in part by an Ontario Graduate Scholarship. MCA is the recipient of a Natural Sciences and Engineering Research Council of Canada Industrial Research Chair and acknowledges support from the member companies of the Program in Food Safety (University of Toronto).
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Notes
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3 To whom correspondence should be addressed Email: m.archer{at}utoronto.ca 
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Received August 6, 1998;
revised October 15, 1998;
accepted October 30, 1998.