Tissue-specific resistance to cancer development in the rat: phenotypes of tumor-modifier genes

Geoffrey A. Wood1, James E. Korkola1,3 and Michael C. Archer1,2,4

1 Department of Medical Biophysics and
2 Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, 150 College Street, Toronto M5S 3E2, Canada


    Abstract
 Top
 Abstract
 Introduction
 Mammary gland carcinogenesis
 Hepatocarcinogenesis
 Peripheral nervous system...
 Summary and conclusions
 References
 
Resistance to carcinogenesis in the rat is both strain- and tissue-specific. The phenotypic characteristics of resistance in the mammary gland, liver and peripheral nervous system (PNS) are strikingly similar. In all three tissues, initiation is intact with subsequent formation of preneoplastic cells and lesions. In the mammary gland and PNS, activation of the Ha-ras and neu proto-oncogenes, respectively, takes place. A number of different modifier genes are involved in resistance, many of which appear to be tissue-specific in their action with no overlap between strains. A single resistance phenotype, however, involving the formation, growth and subsequent loss of preneoplastic lesions is common to all three tissues of resistant strains. In the PNS, there is evidence that preneoplastic cells are eliminated by apoptosis or immunosurveillance. In the mammary gland and liver, the immune system is not involved in the loss of preneoplastic lesions and there are no clear differences between susceptible and resistant strains in the kinetics of proliferation and apoptosis of preneoplastic cells. The evidence to date favors a mechanism in which preneoplastic cells from these tissues undergo a process of remodeling/redifferentiation to yield cells with a normal phenotype. Identification of human homologues of rodent tumormodifier genes will result in a better understanding of cancer development and potentially provide new strategies for prevention and therapy.

Abbreviations: 2-AAF, 2-acetylaminofluorene; BD, Berlin–Druckrey; BN, Brown Norway; Buff, Buffalo; Cop, Copenhagen; CTL, cytotoxic T-cells; DEN, diethylnitrosamine; DMBA, dimethylbenz[a]anthracene; ENU, ethylnitrosourea; F344, Fischer F344; GST, glutathione S-transferase; HGF, hepatocyte growth factor; IDP, intraductal proliferation; LE, Long–Evans; 3'-Me-DAB, 3'-dimethylaminoazobenzene; MHC, major histocompatability complex; MMP, matrix metalloproteinase; MNU, methylnitrosourea; NK, natural killer; PH, partial hepatectomy; PNS, peripheral nervous system; RH, resistant hepatocyte; T3, triiodothyronine; SD, Sprague–Dawley; SHR, spontaneously hypertensive rat; WF, Wistar–Furth; WK, Wistar–Kyoto.


    Introduction
 Top
 Abstract
 Introduction
 Mammary gland carcinogenesis
 Hepatocarcinogenesis
 Peripheral nervous system...
 Summary and conclusions
 References
 
The etiology of most, if not all, human cancers is multifactorial and ill defined. Genetic predisposition as well as physiological and environmental risk factors affect cancer development, such that the mechanistic basis of susceptibility is highly complex. A small fraction of human tumors are caused by the germline transmission of defective alleles of tumor suppressor genes such as BRCA1, BRCA2 and p53 (1). The overwhelming majority of human cancers, however, are sporadic, arising as a consequence of spontaneous or induced mutations in proto-oncogenes and/or tumor suppressor genes. These cancers show no obvious familial inheritance patterns and it has been suggested that multiple, low penetrance genes segregate to confer differences in predisposition between individuals (2). The polygenic characteristics of inheritance as well as variations in penetrance will make identification of these tumor-modifier genes in humans by linkage analysis difficult, if not impossible. Genes that confer resistance or susceptibility to cancer development, however, are amenable to study in rodents. Characterization of resistance/susceptibility phenotypes in such models will lead to a better understanding of the etiology of human cancer. Identification of resistance/susceptibility genes in rodents will enable the identification of human homologues of such genes that will have potentially important applications in cancer prevention and therapy. Here we review three rat models of tissue-specific resistance to cancer development (mammary gland, liver and peripheral nervous system) that share a common resistance phenotype involving the formation and subsequent loss of preneoplastic lesions.


    Mammary gland carcinogenesis
 Top
 Abstract
 Introduction
 Mammary gland carcinogenesis
 Hepatocarcinogenesis
 Peripheral nervous system...
 Summary and conclusions
 References
 
General characteristics
Chemically induced mammary carcinomas in the rat have been particularly useful in furthering our understanding of human breast cancer development (3,4). Both the histopathological progression and hormone responsiveness of rat carcinomas are similar to breast malignancies in women (5). Tumors can be readily induced in 50-day-old virgin female rats of susceptible strains by a single injection of a chemical such as methylnitrosourea (MNU) or dimethylbenz[a]anthracene (DMBA) (6). For example, following treatment of Sprague–Dawley (SD) rats with 50 mg/kg MNU at 50 days of age, when the mammary glands are developing during puberty, mammary adenocarcinomas develop within 4–6 months in almost 100% of the animals. More than 85% of these tumors harbor the Ha-ras oncogene (7). Mammary carcinomas in the rat are generally dependent on ovarian and pituitary hormones for both induction and growth (3,4,6,8).

In this model, susceptibility varies considerably among different strains of rat (Table IGo and references therein). Many strains such as SD, Wistar–Furth (WF) and Buffalo (Buff) are highly susceptible, developing multiple tumors following treatment as described above. Some strains such as Fischer F344 and Wistar develop less than one carcinoma per rat on average with a longer latency period and are classified as intermediate in their susceptibility. Other strains such as Copenhagen (Cop), Wistar–Kyoto (WK) and DRH rats are highly resistant, rarely, if ever, developing tumors after treatment.


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Table I. Examples of susceptibilities of different strains of rat to chemical carcinogenesis in the mammary gland, liver and peripheral nervous system
 
Cop rats are the most widely studied of the resistant strains. These rats are resistant to the induction of mammary adenocarcinomas by a variety of means. Injections of MNU, DMBA (9,10), estrogen (11,12), diethylstilbestrol (11,13), direct exposure of the gland to DMBA (9,14) or radiation (10) all fail to induce mammary tumors. Furthermore, Cop rats do not develop spontaneous mammary tumors (10). As discussed below, Cop rats are also resistant to chemically-induced liver cancer, but they do develop tumors at other sites such as the bladder following implantation of diethylstilbestrol in the scapular region (13) or intravesical administration of MNU (15), and leukemia following DMBA treatment (9). They also develop spontaneous thymic tumors (16,17). It is clear, therefore, that resistance to tumorigenesis is organ specific.

The resistance phenotype
The first mechanistic studies on the resistance of the Cop rat to mammary carcinogenesis clearly demonstrated that there is no difference between susceptible and resistant rats in a series of factors including animal growth rate, mammary gland growth rate and estrogen, progesterone and prolactin profiles (18). It is also clear that resistance in Cop rats is not due to a defect in the enzymes of carcinogen metabolism. The profile of DMBA activation and DNA adduct formation in Cop rat mammary epithelium is similar to that from susceptible rats (19) and MNU, a direct acting mammary carcinogen, fails to induce tumors (9,18). Furthermore, the difference between resistant and susceptible rats is not due to a difference in the formation or repair of carcinogen–DNA adducts. We showed that the kinetics of formation and repair of O6-methylguanine in DNA from mammary epithelial cells are essentially identical in highly susceptible Buff and Cop rats (20). In addition, the extent of methylation by MNU of a restriction fragment containing exons 1–4 of the Ha-ras gene in mammary gland DNA, as well as the level of expression of Ha-ras expression in mammary tissue, are similar in the two strains (20). These various studies suggested that the Ha-ras gene in Cop mammary glands is not protected from carcinogen modification and should be susceptible to mutational activation.

In order to test this hypothesis, we developed a sensitive PCR-based method to detect the presence of low levels of Ha-ras mutations and to estimate the fraction of cells containing a mutant allele in individual glands. Using this method, we were able to detect G->A transitions at the second nucleotide of codon 12 of Ha-ras in the mammary glands of both highly susceptible Buff and Cop rats 30 days after MNU treatment (21). The mutations were uniformly distributed amongst individual mammary glands and were present in purified mammary epithelial cells. These results demonstrated that the complete resistance of the Cop rats is not due to the inability of the Ha-ras gene to undergo mutational activation. In Buff rats, however, the fraction of cells containing a mutated Ha-ras allele increased by a factor of 10–1000 between 30 and 60 days after MNU treatment, whereas in Cop rats, there was no significant increase in the mutant cell fraction during this time period (21). We concluded that the resistance of the Cop rat is due to the inability of mammary epithelial cells containing a mutated Ha-ras allele to undergo sustained clonal expansion.

This notion has been strengthened by our recent work showing that Cop rats develop approximately the same number of putative preneoplastic intraductal proliferations (IDPs) as highly susceptible WF rats up to 30 days post MNU (22,23). Unlike IDPs from WF rats, however, the IDPs from Cop rats thereafter begin to decrease in number such that by 37 days, there are significantly fewer lesions in Cop glands. This decrease in number continues until, by 60 days post treatment, there are none present. Furthermore, G->A activating mutations in codon 12 of the Ha-ras gene are present in 60% of IDPs from Cop rats and 75% of IDPs from WF rats, indicating that the Ha-ras mutations we observed previously in whole mammary glands and epithelial cell preparations were most likely present in IDPs. Presence of mutated Ha-ras in IDPs explains our observation of similar kinetics for the appearance of IDPs and of mutant Ha-ras alleles (21,22).

These various results clearly demonstrate that carcinogen treatment leads to initiation of carcinogenesis in the mammary gland with the formation of preneoplastic lesions in both susceptible and resistant rats. In susceptible rats, however, these lesions rapidly grow and progress to form ductal carcinomas in situ and finally adenocarcinomas, whereas similar preneoplastic lesions in resistant rats fail to progress and ultimately disappear.

Consistent with this model developed in Cop rats, though with somewhat different kinetics, is the behavior of preneoplastic lesions in the spontaneously hypertensive rat (SHR). SHR rats are derived from the WK strain and are also highly resistant to mammary carcinogenesis. In SHR rats, small (1–3 mm) palpable nodules begin to develop about 4 weeks post-DMBA treatment (24). By 7 weeks, 60–70% of the rats have such nodules, but thereafter they regress and by 12 weeks, they have virtually disappeared.

Mechanism of loss of preneoplastic lesions
A mechanism that could account for the observation that IDPs are lost from the glands of Cop rats is recognition and elimination of preneoplastic cells by the immune system. To determine if the immune system is involved in resistance, a number of years ago two groups performed similar transplantation experiments (18,25,26). In one set of experiments for example, Cop mammary epithelial cells, mammary epithelial cells from highly susceptible inbred SD rats, or a mixture of the two preparations were transplanted into the cleared mammary fat pads of CopxSD F1 animals. The transplanted cells were then directly treated with carcinogen. No tumors developed if Cop cells were transplanted, but tumors did develop if SD or a mixture of Cop and SD cells were transplanted. It was concluded that the genes determining resistance act within the Cop mammary parenchyma and that neither the immune system nor paracrine effects are likely to account for resistance (18,25,26).

The results of these transplantation experiments, however, do not rule out an involvement of the immune system in resistance. Immune recognition and removal of abnormal cells depends not only on the immune system of the host, but also the transplanted cells themselves (27). In order to test definitively whether or not Cop resistance is dependent on T-cells, we crossed Cop rats with an athymic nude rat to produce F1s, that were interbred to produce F2 animals, some of which were athymic with partial Cop background (27). Following treatment with MNU, we observed no difference in the incidence of carcinomas between the euthymic and athymic F2s. These results show unequivocally that T-cells are not involved in Cop resistance. Interestingly, the results showed that nude rats are likely to carry resistance genes.

We have also shown that no mammary tumors develop in MNU-treated Cop rats that are administered carrageenan chronically (27). Carrageenan has been shown to produce a substantial decline in natural killer (NK) cell activity in treated animals (28). While this evidence is not definitive, it seems unlikely that NK cells play a role in resistance. Antibodies produced by B-cells would most likely have eliminated both Cop and WF cells in the F1 animals receiving transplants described previously (18,25), suggesting that B-cells are not responsible for resistance. Thus, these various studies indicate that it is unlikely that the immune system is involved in the elimination of preneoplastic mammary epithelial cells from Cop rats.

Another mechanism to account for the loss of preneoplastic cells in resistant rats would be a change in cell kinetics within IDPs. We have recently shown, however, that a change in the balance of cell proliferation and apoptosis does not appear to account for the disappearance of Cop IDPs (23), although it is possible that small perturbations in the rates of cell loss and/or cell growth may occur that would be undetectable in short-term assays. Such changes could have profound effects over the long period of carcinogenesis. Consistent with the notion that IDPs are not lost by apoptosis, however, are our findings that by 60 days post MNU treatment, virtually all IDPs have been lost (22), but at this time the fraction of cells containing a mutated Ha-ras allele is similar to that at 30 days when numerous IDPs are present (21). These results suggest that rather than being lost by apoptosis, the preneoplastic cells in Cop IDPs may undergo a process of redifferentiation or remodeling to yield cells with a more normal phenotype. Stronger evidence for this mechanism is described below on the mechanism of resistance to hepatocarcinogenesis.

The resistance genotype
Breeding experiments in which Cop rats were crossed with highly susceptible inbred strains initially indicated that a single, autosomal dominant gene, confers resistance (9,10,26). Subsequent linkage analysis localized the gene to chromosome 2, but also indicated that resistance is polygenic (29). Recently, an expanded linkage analysis has shown that there are four loci linked to resistance, designated Mcs1–4 (for mammary carcinoma susceptibility loci 1–4) (Table IIGo) (30). Mcs1 is located on chromosome 2 and is thought to modulate tumor number in a semi-dominant manner, since there is an effect of gene dosage (animals homozygous for the Mcs1 gene are more resistant than heterozygous animals that, in turn, are more resistant than rats that lack the Mcs1 gene completely). This raises the possibility that the Mcs1 gene may be either a defective sensitivity gene or a semi-dominant tumor suppressor gene. Mcs2 and Mcs3 are located on chromosomes 7 and 1, respectively, and act in a dominant manner to suppress carcinoma formation. These two loci, however, are located in large segments of DNA (36 and 30 cM, respectively) so there may be more than one resistance locus within each of these linkage groups. Mcs1, -2 and -3 appear to act in an additive manner to produce resistance to mammary carcinogenesis. The fourth locus, Mcs4, is located on chromosome 8 and seems to act to increase the number of tumors in animals carrying this allele. None of the loci correspond to any known genes associated with human breast cancer, although a polymorphism in the human genomic region homologous to the rat Mcs4 region is associated with increased breast cancer risk in African–American women (30).


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Table II. Mammary, liver and peripheral nervous system susceptibility loci
 
Lan et al. (31) have recently shown that the Mcs1, -3 and -4 loci identified in the Cop rat do not contribute significantly to resistance in the WK rat. Three new resistance loci, Mcs5, -7 and -8, on chromosomes 5, 10 and 14, respectively, identified in the WK rat, are not involved in the resistance of the Cop rat (Table IIGo). Interestingly, the Cop Mcs2 and WK Mcs6 resistance loci have large overlapping regions on chromosome 7, although it is not yet clear whether Mcs2 and -6 are identical. The authors conclude that their data suggest that susceptibility to mammary carcinogenesis is influenced by a wide variety of genes and gene interactions.

In a `candidate gene' approach, we have recently examined the expression of cyclin D1 within IDPs and other lesions from Cop and WF rats (23). Cyclin D1 has been shown to be important in the transition from the G1 to the S phase of the cell cycle and perturbations in this control point can lead to neoplastic transformation (32). Indeed, cyclin D1 is frequently over-expressed in both human (33,34) and rat (35) mammary cancers and is thought to be an important factor in their development. This notion was strengthened by studies showing that mice engineered to over-express cyclin D1 in their mammary glands develop hyperplastic lesions and eventually mammary carcinomas (36). Furthermore, it has been shown that over-expression of cyclin D1 may be an important event in determining whether preneoplastic lesions go on to develop into malignant or benign lesions in humans (37). Immunohistochemical staining of preneoplastic lesions showed that cyclin D1 is frequently over-expressed in WF lesions 37 days post MNU treatment, but not in Cop lesions from the same time (23). There was no correlation between the level of cyclin D1 expression and cell proliferation in either strain, a result that agrees with other studies showing no correlation between these parameters in rat mammary carcinomas (35) or in human cancers (37). The over-expression of cyclin D1 in WF but not Cop lesions suggests that this gene may play a role in resistance, although this concept clearly requires further study.

In other candidate gene studies, Hsu and Gould (38) showed that the putative suppressor gene Krev-1 (rap1A) and its related gene rap1B are unlikely to be involved in the tumor resistant phenotype of the Cop rat. Over-expression of casein genes has been associated with the resistance phenotype (39,40), although the biological significance of this association is not yet clear.


    Hepatocarcinogenesis
 Top
 Abstract
 Introduction
 Mammary gland carcinogenesis
 Hepatocarcinogenesis
 Peripheral nervous system...
 Summary and conclusions
 References
 
General characteristics
Susceptibility to hepatocarcinogenesis varies considerably among different strains of rat (Table IGo). Strains such as Fischer F344, that are commonly used in hepatocarcinogenesis studies, are highly susceptible to a variety of carcinogens and consistently develop multiple liver nodules and carcinomas, while other strains such as SD and Wistar are intermediate in their susceptibility (41). Several strains show a relatively high degree of resistance to hepatocarcinogenesis. A study done several decades ago showed that Cop rats are resistant to liver tumor formation by long-term feeding of 2-acetylaminofluorene (2-AAF) (42). In that study, almost all F344, August and Marshall rats developed liver tumors. Although 2 of 10 Cop rats eventually developed liver tumors, they outlived all treated rats from the other strains and even many untreated control rats. The Cop tumors were not characterized with respect to their type or level of malignancy, but since the tumors did not affect mortality, it is unlikely that they were malignant. The DRH rat strain has also been shown to be relatively resistant to the induction of liver tumors, with only 1 of 8 rats developing a tumor after dietary 2-AAF (43). Furthermore, this strain is resistant to the induction of liver tumors by the carcinogen 3 2-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) (44,45). Indeed, DRH rats were developed by treating susceptible Donryu rats with 3'-Me-DAB, selecting resistant rats and inbreeding them for 10 years (46). A third strain, the Brown Norway (BN), has recently been found to be highly resistant to preneoplastic hepatic nodule formation by the resistant hepatocyte protocol described below (47).

The resistance phenotype
Protocols that separate the initiation and promotion phases of hepatocarcinogenesis have greatly facilitated studies of the cellular mechanisms involved in these processes. The resistant hepatocyte (RH) protocol that is used in many of the studies discussed in this review has been particularly useful (48,49). In this protocol, rats are first treated with a necrogenic initiating agent such as diethylnitrosamine (DEN). After a brief recovery period, they are administered the mitoinhibitory agent 2-AAF followed by surgical or chemical (CCl4) partial hepatectomy (PH). The proliferative stimulus of the PH results in the growth of putative initiated cells that, unlike normal hepatocytes, are resistant to the mitoinhibitory effects of 2-AAF. Some of the foci of these resistant cells in susceptible rats grow to form nodules and eventually carcinomas. The foci, nodules and tumors commonly have enhanced expression of enzymes such as glutathione S-transferase (GST) 7-7 and {gamma}-glutamyltransferase. Formation and growth of the preneoplastic lesions can hence be readily quantified by immunohistochemistry.

Using the RH protocol, resistant Cop and BN rats have been shown to develop foci of putative preneoplastic hepatocytes, indicating that the initiation of hepatocarcinogenesis is intact. These lesions, however, fail to develop into nodules (47,50). The kinetics of growth of GST 7-7-positive lesions has been most extensively studied in Cop rats (50). Lesion growth during the first 7 days following PH is not different in Cop and susceptible F344 rats. At this time, ~5% of the liver volume is occupied by lesions in both strains. By day 14 following PH, all F344 livers have many pale yellowish-white nodules clearly visible on the surface and in sections. In contrast, Cop livers are essentially free of gross lesions. This difference is also clear by immunohistochemical analysis. Cop rats show no significant increase in lesion volume on day 14 compared with day 7, while F344 rats have ~8-fold more liver occupied by lesions on day 14 than day 7. By day 21, F344 lesions occupy over half the liver volume, whereas Cop lesions only occupy ~6%. Furthermore, >85% of the Cop lesions at this time are undergoing remodeling, a process whereby the lesions revert to normal appearing liver (discussed in more detail in the next section).

Initiation in BN and DRH rats is also not different from susceptible controls and, as in Cop rats, foci of initiated cells fail to progress. BN rats have ~12-fold less liver volume occupied by GST 7-7-positive lesions than F344 rats 4 weeks after PH in the RH protocol (47). Similarly, DRH rats treated with DEN followed by promotion with 3'-Me-DAB and PH have ~10-fold less liver section area occupied by GST 7-7 lesions than susceptible Donryu rats 5 weeks following PH (51).

Resistance to hepatocarcinogenesis, at least in Cop rats, is not due to a defect in the enzymes of drug metabolism. DEN and CCl4 are at least as hepatotoxic in Cop as in F344 rats and 2-AAF is equally mitoinhibitory to Cop and F344 hepatocytes (52). While metabolism of DEN has not been evaluated in BN rats, it is unlikely to be a factor in their resistance since they are susceptible to the formation of preneoplastic lesions (47). Although there are some differences in P450 enzyme expression following 3'-Me-DAB treatment in DRH and Donryu rats, the levels of DNA adducts (43) as well as the number of lesions per unit area of liver sections (51) are not different between the two strains, indicating that the resistance of DRH rats is not likely to be caused by a defect in drug metabolism.

These various studies in Cop, BN and DRH rats all show that carcinogen treatment leads to the formation of preneoplastic foci as in susceptible rats. In susceptible rats, however, these lesions grow rapidly and progress to form liver nodules and finally cancer, whereas in resistant rats, the lesions fail to progress beyond the microscopic level.

Mechanism of loss of preneoplastic lesions
As in the case of the mammary gland, the immune system has been evaluated as a mechanism by which preneoplastic liver lesions are eliminated from resistant rats. In a recent study, the resistant DRH rat was shown to have a higher T-cell response than the closely related, but susceptible, Donryu rat (53). This study, however, showed that the T-cell response of DRH rats is similar to SD and Wistar rats that are of intermediate susceptibility (41), indicating that T-cell function does not correlate with the level of resistance. In a similar study to that described above for the mammary gland in which we crossed Cop rats with an athymic nude rat, we have recently shown that T-cells are not involved in Cop resistance in the liver (54).

It is clear that the overall rate of lesion growth in the liver is influenced by the rates of cell division and cell loss (55,56). It might be expected, therefore, that there would be significant differences in the labeling index and/or apoptotic index between the resistant and susceptible strains. We have shown, however, that there are no differences in these parameters (50). The labeling indices of hepatocytes both within GST 7-7-positive lesions and in the surrounding parenchyma of Cop and F344 rats were not different between the strains up to 21 days post PH. The hepatocytes within lesions had the same high labeling index in both strains on day 2 post PH, and maintained this growth advantage over surrounding hepatocytes to the same extent in both strains at least until day 7. By day 14 post PH, there was no growth advantage for GST 7-7-positive hepatocytes in either strain. Furthermore, we showed that the apoptotic indices within lesions are not significantly different in the two strains up to 21 days following PH. These various results suggest that resistance is not caused by significantly lower rates of proliferation or higher apoptotic rates within Cop lesions compared with F344 lesions.

Following promotion with the RH protocol, most lesions of susceptible rats eventually begin to lose expression of GST 7-7 and other marker enzymes, their borders become indistinct, and they revert to normal appearing liver in a process termed remodeling or redifferentiation (57,58). A few persistent lesions never remodel and these persistent lesions are thought to be sites from within which cancer eventually develops. In contrast to F344 lesions that typically remodel in weeks to months following promotion by the RH protocol, we have shown that Cop lesions remodel very early (50). By 14 days after 2-AAF/PH treatment, ~70% of Cop lesions show evidence of remodeling compared with only ~14% of F344 lesions and by day 21, >85% of lesions are remodeling in Cop rats compared with ~20% in F344 rats. Lesions undergoing remodeling are effectively losing preneoplastic cells, but not by cell death. Cells within lesions that remodel lose expression of GST 7-7 and integrate into the parenchyma. They are then undetectable by immunohistochemistry and appear as normal hepatocytes. Thus, precocious remodeling could account for the regression of Cop lesions despite the lesions having proliferation and apoptotic rates similar to F344 lesions.

Strong evidence that an increase in the rate of remodeling can inhibit rat hepatocarcinogenesis comes from a recent study using the thyroid hormone triiodothyronine (T3) (59). Following the RH protocol, 1 week of dietary T3 administration was able to induce a 4-fold reduction in the percentage of liver section area occupied by GST 7-7-positive lesions despite inducing an increase in proliferation in the lesions. There was no difference in the apoptotic index of lesions between the treated and control rats and the mechanism of T3 inhibition appeared to be completely due to an increase in remodeling. When dietary T3 was given for 1 week/month for 7 months following the RH protocol and rats were killed 7 months after the completion of treatment, the incidence of hepatocellular carcinoma was reduced from 100 to 50% and lung metastases were completely inhibited. From this study it is clear that remodeling alone can have dramatic effects on both preneoplastic lesion growth and the formation of liver tumors.

Brown Norway rats also show precocious lesion remodeling compared with F344 rats both at 4 and 28 weeks after RH promotion (47). The authors of this study suggest that this difference could partly account for the differences in susceptibility of the F344 and BN rats. Although lesion remodeling has not been examined directly in DRH rats, the amount of GST 7-7 mRNA in the livers of DRH rats has been shown to be lower than in Donryu rats following promotion (51). A decrease in GST 7-7 mRNA has been proposed as a marker of lesion remodeling (60). Overall, these data suggests that a mechanism of resistance involving precocious remodeling of preneoplastic liver lesions could be common to Cop, BN and DRH rats.

A further factor that may contribute to the resistance of Cop rats to the growth of preneoplastic lesions is oval cell migration. Oval cells are thought to be bipotential cells that can differentiate into either bile duct cells or hepatocytes (61–63). We showed that compared with F344 livers in which there is extensive migration of oval cells from the portal tract into the liver parenchyma following PH, oval cells in Cop livers fail to migrate and remain localized in the periportal area (50). Since local production of growth factors by oval cells could contribute to the growth of preneoplastic lesions (64–69), the weak oval cell response we observed in Cop rats could play a role in their resistance, although much more evidence is needed to substantiate this notion.

The resistance genotype
Breeding studies in which Cop, BN or DRH rats were crossed to susceptible strains have shown that the F1 generations are resistant to liver lesion growth, but not to the same extent as the resistant parental strains, indicating an incompletely dominant mode of inheritance (47,51,70). Using CopxF344 F1's and backcrosses, we have shown that Cop resistance is polygenic and likely involves a minimum of four genes, one of which may exhibit recessive epistasis (70). Preliminary linkage analyses of BN and DRH rats have revealed that several genes are involved in resistance of these strains. BN resistance loci (Hcr1, Hcr2 and Hcr3) reside on chromosomes 10, 4 and 8, respectively (Table IIGo), with possible additional loci on chromosomes 5 and 8 (71). Two susceptibility loci, Hcs1 and Hcs2, reside on chromosomes 7 and 1, respectively. DRH resistance in the liver shows linkage to several loci on chromosomes 1 (Drh1a, b and c) and 4 (Drh2a and b) (45).

The results of the above studies suggest that resistance in all of these strains appears to be inherited in a similar polygenic, incompletely dominant manner. Although some resistance loci from different strains are on the same chromosome, most are too far removed from each other to be the same gene. Hcr2, Drh2a and Drh2b, however, all confer resistance to hepatocarcinogenesis and are located between 46.45 and 54.33 cM from the top framework of chromosome 4. Thus, it is possible that Hrc2 is the same gene as either Drh2a or Drh2b.


    Peripheral nervous system carcinogenesis
 Top
 Abstract
 Introduction
 Mammary gland carcinogenesis
 Hepatocarcinogenesis
 Peripheral nervous system...
 Summary and conclusions
 References
 
General characteristics
In sensitive strains of rats such as SD, tumors of the peripheral nervous system (PNS) can be induced by a single transplacental (72) or perinatal (73) injection of 80 mg/kg N-ethyl-N-nitrosourea (ENU). Essentially 100% of the rats develop malignant schwannomas of the trigeminal nerve by 250 days of age. Sensitivity to the induction of these tumors, however, varies markedly between rat strains (Table IGo). The most studied are Berlin–Druckrey (BD) rats, a group of 10 inbred strains (BDI to BDX). Of these strains, BDIX and BDIV are the most susceptible and resistant, respectively (74,75). Virtually all BDIX rats given ENU shortly after birth develop schwannomas while similarly treated BDIV rats develop no tumors (74,75). PNS carcinogenesis has also been studied in WF and Long–Evans (LE) rats, though less extensively than in the BD strains. Six months following a perinatal subcutaneous injection of 40 mg/kg ENU, the incidence of schwannomas of the trigeminal nerve is ~20% in WF rats, whereas LE rats have an incidence of ~90% (76). Although WF rats were considered to be resistant in this study, in light of the high resistance of the BDIV strain, WF rats should now be considered intermediate in their susceptibility.

The resistance phenotype
ENU-induced trigeminal nerve schwannomas have a characteristic T:A->A:T transversion at nucleotide 2012 of the neu/erbB-2 oncogene that is thought to be the initiating event in tumorigenesis (73,77). This mutation can be found at all stages of tumor development, from single, putatively initiated cells, through to malignant tumors. The target cells for ENU in the trigeminal nerve are thought to be immature Schwann cells near the nerve–brain junction that, when harboring a mutant neu gene, exhibit unrestrained proliferation.

To estimate the proportion of cells carrying a neu mutation at various times after initiation, the fraction of mutant alleles in microdissected tissue has been determined (78). BDIV rats that are resistant to tumorigenesis are clearly not resistant to initiation and actually have a higher proportion of mutant neu alleles than susceptible BDIX rats up to roughly 100 days post ENU. From day 100 onwards, however, BDIV rats begin to lose cells carrying mutant alleles until these cells are almost undetectable by 250 days. In contrast, BDIX rats have progressively higher proportions of mutant alleles and eventually form malignant tumors that necessitate killing of the rats by 250 days.

Histologically, the formation and disappearance of preneoplastic cells in BDIV rats and the progression to tumors in BDIX rats parallel results at the molecular level described above. The cells carrying mutant neu alleles appear in groups termed early atypical proliferates that consist of irregularly distributed cells with an increased nuclear/cytoplasmic ratio (73,77). BDIV rats are clearly not resistant to initiation, forming atypical proliferates, but lose these preneoplastic cells at later times, while in BDIX rats, lesions continue to grow into larger nests of atypical cells and eventually tumors (78).

Mechanism of loss of preneoplastic cells
In contrast to resistance in the liver and possibly the mammary gland, the loss of lesions in BDIV rats appears to be exclusively caused by elimination of preneoplastic cells (78). If remodeling caused the disappearance of lesions, the cells carrying mutant neu alleles would remain in the neural tissue. This is not the case, however, since the fraction of mutant alleles decreases until it is practically undetectable. The removal of preneoplastic cells by cytotoxic T-cells or natural killer cells has been suggested as a potential mechanism of resistance in BDIV rats (78). In support of this mechanism, ENU-induced rat schwannomas have been shown to induce a specific T-cell response, although they also secrete factors that inhibit T-cell proliferation (79). Apoptosis of preneoplastic cells has also been proposed as a mechanism by which atypical proliferates may be lost in BDIV rats (78). No measurements of apoptosis have been conducted, however, to confirm this notion.

The resistance genotype
BDIVxBDIX F1 rats are relatively resistant to ENU-induced PNS carcinogenesis, but inheritance is incompletely dominant with ~20% of the F1s developing tumors (75). Loss of heterozygosity and linkage analysis has revealed two schwannoma resistance loci, Dis1 and Mss1, located ~30 cM apart on chromosome 10 (Table IIGo) (75). The linkage analysis was targeted only to chromosome 10 and the authors of this study concluded that several other unlinked genes of differing functionality in DBIV and DBIX rats might modulate resistance. Crosses of WF (intermediate in susceptibility) and susceptible LE rats had tumor incidences in between the two parental strains and a three-loci model of inheritance of resistance best fits the tumor incidence data (76).


    Summary and conclusions
 Top
 Abstract
 Introduction
 Mammary gland carcinogenesis
 Hepatocarcinogenesis
 Peripheral nervous system...
 Summary and conclusions
 References
 
The phenotypic characteristics of resistance to carcinogenesis in the mammary gland, liver and PNS are strikingly similar in the rat strains that have been studied. In all three tissues, initiation is intact with subsequent formation of preneoplastic cells and lesions. In the mammary gland and PNS, activation of the Ha-ras and neu proto-oncogenes, respectively, takes place. Resistance occurs in all three tissues because preneoplastic lesions grow for a while, but then fail to progress and begin to disappear (Figure 1). If the same resistance phenotype occurs in humans, we would predict that the presence of preneoplastic lesions would not necessarily indicate that cancer would develop.

In the rat mammary gland and liver, the immune system has been demonstrated not to be involved in the loss of preneoplastic lesions and there are no clear differences between susceptible and resistant strains in the kinetics of proliferation and apoptosis of preneoplastic cells. The evidence to date favors a mechanism in which preneoplastic cells from these tissues undergo a process of remodeling/redifferentiation to yield cells with a normal phenotype. In the PNS, however, preneoplastic cells do not remodel and are instead eliminated, perhaps by apoptosis or immunosurveillance (78).

It is clear that resistance to carcinogenesis is both strain- and tissue-specific. Of the strains reviewed here, the most striking example is the WK rat that is resistant to mammary gland cancer, but is highly susceptible to the development of liver cancer (Table IGo). A number of strains are highly susceptible in one tissue, but of intermediate susceptibility in another. Perhaps the best examples are the SD and F344 rats that are among the most commonly used laboratory strains. SD rats are highly susceptible to mammary gland carcinogenesis, but are only of intermediate susceptibility to hepatocarcinogenesis, whereas F344s are the opposite. Cop and DRH rats are resistant to both mammary gland and liver tumorigenesis.

From these observations as well as results from breeding experiments and linkage analysis, it is clear that susceptibility and resistance involve a number of genes. Of the genes thought to be involved (Table IIGo), only Mcs2 and Mcs6 on chromosome 7 and Hcr2 and Drh2a,b on chromosome 4 have overlapping regions and may be the same genes. Mcs3 and Drh1 both confer resistance and are localized on the same area of chromosome 1, but are sufficiently far apart that they are unlikely to be the same gene. Hcr1 is at the opposite end of chromosome 10 from Dis1 and Mss1. The other resistance and susceptibility genes all seem to be tissue-specific in their action, with no overlap between strains. It is of interest that despite the large number of different modifier genes involved, a single resistance phenotype involving the formation and subsequent loss of preneoplastic lesions appears to be common to all resistant strains in all three tissues (Figure 1).

The feral rat has been shown to be resistant to mammary carcinogenesis (26). This suggests that many inbred strains may have inadvertently been selected for susceptibility, whereas resistant strains have retained the wild-type characteristic. Thus, susceptible strains may carry defective wild-type resistance genes that have been bred to homozygosity, some of which are recessive and some of which are dominant. The dominant genes are detected by linkage analysis as susceptibility loci, whereas the recessive genes appear phenotypically as a lack of wild-type resistance and the locus appears to be a resistance locus. Alternatively, resistant strains may have inadvertently been selected for dominant resistance loci, though this seems less likely.

Now that the cancer resistance phenotype has been characterized in the rat, efforts are being directed to cloning the genes involved and understanding their interactions. This should be greatly facilitated by the completion of the rat genome-sequencing project. It is anticipated that knowledge of the mechanisms of susceptibility–resistance in the rat will lead to the identification of human tumor-modifier genes that will, in turn, result in a better understanding of human cancer development.


    Notes
 
3 Present address: Cancer Center, University of California, San Francisco, CA 94143-0808, USA Back

4 To whom correspondence should be addressed Email: m.archer{at}utoronto.ca Back


    Acknowledgments
 
This work was supported by a grant from the Canadian Breast Cancer Research Initiative.


    References
 Top
 Abstract
 Introduction
 Mammary gland carcinogenesis
 Hepatocarcinogenesis
 Peripheral nervous system...
 Summary and conclusions
 References
 

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Received May 29, 2001; revised August 20, 2001; accepted September 14, 2001.