The Mcs7 quantitative trait locus is associated with an increased susceptibility to mammary cancer in congenic rats and an allele-specific imbalance.

Identification of high-penetrance breast cancer genes such as Brca1 has been accomplished by analysing familial cases. However, these genes occur at low frequency and do not account for the majority of genetic risk. Identification of low-penetrance alleles that occur commonly in populations may benefit from unbiased genome-wide screening. One such approach uses linkage studies in rodent models to identify homologous human candidates. The Wistar Kyoto (WKy) rat is resistant to mammary carcinomas induced with 7,12-dimethybenz[a]anthracene (DMBA), whereas the Wistar Furth (WF) strain is susceptible. Previous genome-wide linkage studies in crosses of these strains identified three WKy resistance quantitative trait loci, Mcs5, Mcs6 and Mcs8, and one predicted to increase susceptibility, Mcs7. The Mcs7 region on rat chromosome 10 (RNO10) is orthologous to human 17q, a common site of genetic aberrations in breast cancer. Here, we establish the independent phenotype conferred by Mcs7 using congenic rats carrying the WKy Mcs7 locus on a WF background. Tumor multiplicity was significantly higher ( approximately 50%) in DMBA-treated congenics homozygous and heterozygous for the WKy allele at the Mcs7 locus, compared to controls. We also investigated allelic imbalance (AI) in mammary carcinomas from (WKy x WF)F1 rats and Mcs7 heterozygous congenics. Of the four known WKy Mcs loci tested, only Mcs7 displayed AI. The pattern of AI in carcinomas from both F1 and Mcs7 congenic rats was similar, suggesting a WF allelic loss. Together, these data suggest that one or more breast cancer-related genes are located within the dominantly acting WKy allele at the Mcs7 locus.

Genetic loci controlling breast cancer susceptibility in the Wistar-Kyoto rat.

In this study, the Wistar-Kyoto (WKy) rat was genetically characterized for loci that modify susceptibility to mammary carcinogenesis. We used a genetic backcross between resistant WKy and susceptible Wistar-Furth (WF) rats as a panel for linkage mapping to genetically identify mammary carcinoma susceptibility (Mcs) loci underlying the resistance of the WKy rat. Rats were phenotyped for DMBA-induced mammary carcinomas and genotyped using microsatellite markers. To detect quantitative trait loci (QTL), we analyzed the genome scan data under both parametric and nonparametric distributional assumptions and used permutation tests to calculate significance thresholds. A generalized linear model analysis was also performed to test for interactions between significant QTL. This methodology was extended to identify interactions between the significant QTL and other genome locations. Chromosomes 5, 7, 10, and 14 were found to contain significant QTL, termed Mcs5, Mcs6, Mcs7, and Mcs8, respectively. The WKy alleles of Mcs5, -6, and -8 are associated with mammary carcinoma resistance; the WKy allele of Mcs7 is associated with an increased incidence of mammary cancer. In addition, we identified an interaction between Mcs8 and a region on chromosome 6 termed Mcsm1 (modifier of Mcs), which had no significant main effect on mammary cancer susceptibility in this genetic analysis.

The genetic components of susceptibility to breast cancer in the rat.

The rat is an extremely valuable model for studies of inherited susceptibility to breast cancer because the characteristics of rat mammary cancer and human breast cancer are so similar. There are now several rat models for studying sensitivity versus resistance, or cell autonomy versus non-cell-autonomy, for spontaneous and induced mammary cancers. It is known that the tumor-resistant Cop [20, 21] and WKy [8] strains carry dominant resistance genes that inhibit both spontaneous and induced mammary tumors. The WF and SD strains are known to carry dominant sensitivity genes that appear to increase susceptibility to induced but not spontaneous mammary tumors. The presence of both resistance and sensitivity genes in the Cop strain is intriguing, and provides a unique model for studying the interactions of both types of genes. It appears that the resistance genes together are at least partially dominant over the sensitivity gene in this model since the F1 rats develop only a few tumors. Yet another strain, the F344, has an intermediate sensitivity and has been shown to carry neither sensitivity or resistance genes. Thus, all these models and data indicate that sensitivity genes are not necessary for the development of mammary tumors, and neither are they sufficient. However, loss of resistance gene function is necessary but is not sufficient for mammary tumor development. Studies have shown that the sensitivity and resistance genes act directly within the mammary epithelial cells rather than globally in the rat. The products of these genes also do not appear to act at early steps in the carcinogenic process because there have been no observed effects of these genes on carcinogen metabolism or DNA adduct formation. It would appear that these genes act at later stages of mammary carcinogenesis. Identification and isolation of these genes should aid our understanding of the inherited components of human breast cancer. With the increasing availability of genetic markers and large-insert libraries for the rat genome, genetic and physical mapping studies are now a reality for the genes involved in mammary carcinogenesis of the rat. Such studies have already revealed the multigenic nature of this cancer, supporting the idea that the limited penetrance of BRCA1 and BRCA2 in human breast cancer is due to loci that modify the effects of the sensitivity genes. Assuming that human homologues of the Mcs genes exist, cloning the genes and defining the human homologues may provide a way to identify the risk for breast cancer development in women. Analysis of the function of such genes may also lead to the development of new drugs for chemoprevention and/or therapy of this lethal disease.

Linkage mapping of rat chromosome 5 markers generated from chromosome-specific libraries.

Seventy-six novel microsatellite markers with various simple sequence repeat (SSR) motifs are reported in this paper. They were generated on the basis of non-radioactive library screening procedures from flow-sorted rat Chromosome (Chr) 5-specific DNA, and were mapped in three rat backcross populations. Fifty-four of these markers mapped to Chr 5, while the other 22 mapped to other chromosomes of the rat genome. The marker D3Uwm8 is a new microsatellite marker for the rat syndecan 4 (ryudocan) gene. A genotyping protocol based on agarose gel electrophoresis is also provided in this paper.

A comparative analysis of allelic imbalance events in chemically induced rat mammary, colon, and bladder tumors.

In this paper, patterns of allelic imbalances (Als) in chemically induced rat mammary, colon, and bladder tumors from (Wistar Furth x Fischer 344)F1 rats are described and compared. Male F1 rats were administered azoxymethane (AOM), and colon tumors were collected at 58 wk after treatment. Female F1 rats were given either N-nitroso-N-methylurea (NMU) or N-butyl-(hydroxybutyl)-nitrosoamine (BBN), and mammary and bladder tumors were collected at 15 and 52 wk after treatment, respectively. DNA was extracted from a subset of 18 of the largest tumors from each group, and a genome scan was performed by using polymerase chain reaction and 90 polymorphic microsatellite markers. Als, such as loss of heterozygosity, gene duplication, and microsatellite instability, were observed at low frequencies in all of the tumor models. Thirty random Als were observed in the AOM-induced colon tumors but only four in the NMU-induced mammary tumors. In both these models, all the tumors were classified as adenocarcinomas, and most of the Als observed were confined to single tumors with atypical histopathology. In contrast, 27 random Als were identified in the BBN-induced bladder tumors. Als were observed in both transitional-cell carcinomas and papillomas, although most were in the carcinomas. Statistical analysis of the Al data revealed no significant nonrandom Als within or among the tumor models, although several of the infrequently observed Al events identified in the rat tumors may also be observed in the corresponding human tumor type.

Genetic mapping of the rat Lcn2 gene to chromosome 3.

The expression of rat 24p3, encoded by the Lcn2 gene, has been associated with rat mammary carcinomas initiated by the neu oncogene (Stoesz and Gould, 1995). In this study, we assign the Lcn2 gene to rat chromosome band 3q12 by genetic linkage analysis.

Mapping of 55 new rat microsatellite markers from chromosome-specific libraries.

Fifty-five novel rat microsatellite markers were isolated from libraries specific for rat chromosomes (Chrs) 1, 2, and 7. The markers were mapped in three backcross rat populations. Thirty of these markers mapped to Chrs 1, 2, or 7, while the other 25 mapped to other chromosomes. New markers for two genes, liver-specific transporter gene (Livtr) and insulin-responsive glucose transporter (Glut4), were also mapped to rat Chrs 9 and 10, respectively. Three provisionally assigned markers from previous studies were also confirmed. Detailed methodologies for the generation and enrichment of clones containing repeat sequences and for the isolation of chromosome-specific markers are presented, since they represent unique combinations and modifications of previous protocols. Such methods and the newly presented markers should be useful for both specific and general mapping studies in the rat.

Genetic identification of multiple loci that control breast cancer susceptibility in the rat.

We have used a rat model of induced mammary carcinomas in an effort to identify breast cancer susceptibility genes. Using genetic crosses between the carcinoma-resistant Copenhagen (COP) and carcinoma-sensitive Wistar-Furth rats, we have confirmed the identification of the Mcs1 locus that modulates tumor number. We have now also identified two additional loci, Mcs2 and Mcs3. These three loci map to chromosomes 2, 7, and 1, respectively, and interact additively to suppress mammary carcinoma development in the COP strain. They are responsible for a major portion of the tumor-resistant phenotype of the COP rat. No loss of heterozygosity was observed surrounding the three loci. A fourth COP locus, Mcs4, has also been identified on chromosome 8 and acts in contrast to increase the number of carcinomas. These results show that mammary carcinoma susceptibility in the COP rat is a polygenic trait. Interestingly, a polymorphism in the human genomic region homologous to the rat Mcs4 region is associated with an increased breast cancer risk in African-American women. The isolation of the Mcs genes may help elucidate novel mechanisms of carcinogenesis, provide information important for human breast cancer risk estimation, and also provide unique drug discovery targets for breast cancer prevention.

Genetic linkage mapping of 11 novel DNA markers and the ceruloplasmin (Cp) gene on rat chromosome 2.

We have mapped 11 novel, anonymous genetic markers to rat chromosome 2. The rat ceruloplasmin gene (Cp) had been previously mapped to chromosomes 2 and 7q11-->q13 by two different methods. To resolve the assignment and to localize the Cp gene on the rat genetic linkage map, we used linkage analysis to confirm that rat Cp lies on chromosome 2.

Cloning, genetic mapping and expression studies of the rat Brca1 gene.

The breast cancer gene BRCA1 has previously been cloned from both human and mouse. We cloned a fragment of the rat Brca1 homologue in order to map it and explore its biological function. Partial cDNA fragments of the rat Brca1 homologue were isolated by RT-PCR. Sequence analysis revealed that the RING-finger domain is well conserved among rat, mouse and human. Rat Brca1 mRNA was expressed in most tissues studied with the highest level in testis, consistent with studies in human and mouse. Next, intron 6-containing DNA fragments were amplified by PCR from WKY and WF rat strains. The splicing sites between exon 6 and exon 7 are conserved between rat and human. Partial sequencing of the rat Brca1 intron 6 revealed a polymorphism of a pentanucleotide TTTTG repeat between the WKY and WF strains. With this intragenic microsatellite marker, we were able to map precisely the rat Brca1 gene to chromosome 10 using a genetic linkage study of (WKY x WF)F1 x WF backcross rats. Brca1 cosegregates with marker BAND3A, and is flanked by R5123 and R5842. Using this polymorphic marker, we also investigated the loss of heterozygosity (LOH) of the Brca1 microsatellite marker in carcinogen- or radiation-induced mammary carcinomas in (WF x F344)F1 female rats. No LOH or somatic microsatellite instability was detected in 18 DMBA-induced tumors studied. Only one LOH of the F344 allele was observed in 26 radiation-induced tumors tested. Ribonuclease protection assays demonstrated that Brca1 mRNA levels are similar in normal rat mammary glands and mammary carcinomas of various etiologies, including those induced by DMBA, NMU, activated-neu and activated-ras oncogenes.

Genetic identification of Mcs-1, a rat mammary carcinoma suppressor gene.

Women have inherited differences in their susceptibility to breast cancer, but the genes underlying this variation are difficult to identify. We have approached the problem of identifying breast cancer susceptibility genes by using a rat model. Inbred rat strains display differential susceptibilities to mammary carcinogenesis; the Copenhagen (COP) rat is resistant, while the Wistar-Furth (WF) rat is susceptible to induction of mammary tumors by 7,12-dimethylbenz[a]anthracene. Genetic breeding studies have shown that tumor resistance in the COP rat is a dominant phenotype, termed the rat mammary carcinoma suppressor trait. As a step toward defining the basis of this resistance, we undertook genetic mapping of this phenotype in a (WF x COP)F1 x WF backcross by studying a large collection of microsatellite and minisatellite polymorphisms. A total of 114 genetic markers, covering approximately 75% of the rat genome, were genotyped in the backcross progeny. A marker on rat chromosome 2 was found to show linkage to the resistance phenotype. Genetic linkage was demonstrated both in a qualitative analysis (in which rats were defined as resistant if they developed 0 tumors and sensitive if they developed two or more tumors; LOD score, 4.0) and in a quantitative trait locus analysis (in which tumor number was used as the quantitative phenotype; LOD score, 3.8). We infer the existence of a gene, Mcs-1, on rat chromosome 2 that suppresses mammary carcinogenesis.

Bivariate flow karyotyping, sorting, and peak assignment of all rat chromosomes.

A bivariate flow cytometric rat karyotype was established from second- and third-passage Copenhagen (Cop) rat embryo cell cultures. Chromosome suspensions from such cells (2n = 42 chromosomes) yielded bivariate flow karyotypes composed of 14-18 peaks, 10 of which were sortable into pools of single chromosome types. Conditions affecting resolution of peaks (including the length of colcemid treatment of cells and various combinations of fluorescent and nonfluorescent dyes) were optimized. Using chromosome suspensions from second-passage cultures of adult Cop male and female ear fibroblasts, peaks representing the X and Y chromosomes were identified. Assignment of chromosomes was accomplished by polymerase chain reactions of flow-sorted chromosomes using primers from mapped genes. Availability of this characterized rat flow karyotype should prove useful for assignment of genes to chromosomes as well as generation of chromosome-specific libraries in cloning assigned genes.

Transplacental transmission of a leukemogenic murine leukemia virus.

Recombinant inbred BXH-2 mice spontaneously produce a B-tropic murine leukemia virus (MuLV) beginning early in life and have a high incidence of spontaneous myeloid leukemia. These traits are not characteristic of the progenitor strains (C57BL/6J and C3H/HeJ) or of 11 other recombinant inbred BXH strains. Genetic analysis has shown that the virus is not transmitted through the germ line, suggesting that the virus is passed from one generation to the next by horizontal transmission. An additional ecotropic proviral locus was detected in some mice of this strain after several generations of inbreeding. We show that BXH ecotropic virus was transmitted to other strains when fostered on viremic BXH-2 mice and that these mice go on to develop tumors of hematopoietic origin. Our earlier finding that virus is expressed early in gestation suggested that the ecotropic MuLV is also transmitted in utero. In order to determine the stage at which the ecotropic MuLV is transmitted in utero, preimplantation stage embryos were transferred to the uteri of recipient ecotropic virus-negative mice. These mice were found to be negative for the presence of the ecotropic MuLV, suggesting that transplacental transmission of the ecotropic virus readily occurs in BXH-2 mice. Although other viruses, including human lentiviruses, are transmitted across the placental barrier, transplacental transmission of MuLV is a rare event. Thus, the BXH-2 mouse strain may contribute to our understanding of the mechanism of transplacental transmission and pathogenesis and offers a potential new model for use in drug therapy of exogenously transmitted viruses related to lentiviruses.

Relationship of polymorphisms near the rat prolactin, N-ras, and retinoblastoma genes with susceptibility to estrogen-induced pituitary tumors.

Chronic treatment of rats with the synthetic estrogen diethylstilbestrol is known to induce the formation of pituitary tumors, and such tumor induction is highly dependent on the strain of rat used. We examined three previously discovered restriction fragment length polymorphisms in rats to determine whether these correlated with susceptibility to tumor formation. The results indicate that the presence of particular alleles of the polymorphic N-ras and retinoblastoma (Rb) genes does not correlate with tumor susceptibility. A polymorphism upstream of the rat prolactin (Prl) gene is due to the presence or absence of an Alu-like sequence. Results of this study indicate that animals bearing the allele lacking this Alu-like insertion are more likely to develop larger pituitary tumors in response to diethylstilbestrol than are animals in which the Prl allele contains the insertion. In addition, we show that the N-ras, Rb, and Prl genes are dispersed in the rat genome and that the polymorphic alleles of the Prl genes are segregating as classical Mendelian alleles. These results suggest that the difference in the Prl gene itself or in some closely linked gene is related to tumor resistance or susceptibility.

Restriction fragment length polymorphisms in the rat N-ras and retinoblastoma genes: their identification and characterization.

We have observed restriction fragment length polymorphisms for the N-ras and retinoblastoma (Rb) genes between rat strains that are susceptible or resistant to induction of pituitary tumors by diethylstilbestrol (DES). Thirteen other proto-oncogenes tested displayed the same restriction patterns between all samples. The N-ras polymorphism is observed with more than one restriction nuclease and the N-ras and Rb polymorphisms are not a result of DES treatment. The N-ras polymorphism is inherited in a Mendelian fashion and there appear to be two separate loci in the rat genome that are detected by the N-ras probe. For both the N-ras and Rb genes there is an 'extra' fragment in the tumor-resistant animals that is not seen in the susceptible rats. These polymorphisms may indicate regions of the genome that play a role in determining susceptibility to pituitary tumors or any of a number of other chemically induced tumors.

Steroid hormone receptors and oncogenes.

Steroid hormones exert diverse effects on normal growth and development through the action of specific intracellular receptor molecules. These receptors are thought to function as trans-acting regulatory proteins by interacting with chromatin and modulating the transcription of specific genes in target cells. Another class of molecules, the viral oncogenes (v-oncs) encode proteins which are associated with the transformation of normal cells to cancer cells. The cellular oncogenes (proto-oncogenes), from which the viral oncogenes were derived, may have important roles in the growth and differentiation of normal cells. Steroid hormones, directly or in conjunction with altered oncogene products, also appear to be important in the growth and differentiation of cancer cells. Recently it has been determined that the amino acid sequences of the steroid receptors are similar to the erb-A oncogene product, which subsequently has been found to be a thyroid hormone receptor. These results have led to the hypothesis that the steroid receptors and the erb-A product are members of a superfamily of transcription factors which regulate normal and malignant growth and differentiation. Similarities and functional interactions between steroid receptors and oncogene products will be the focus of this review. The results to date imply that these two classes of molecules have interesting and important inter-relationships.