Jill D. Haag

Production of knockout rats using ENU mutagenesis and a yeast-basedscreening assay

The rat is a widely used model in biomedical research and is often the preferred rodent model in many areas of physiological and pathobiological research. Although many genetic tools are available for the rat, methods to produce gene-disrupted knockout rats are greatly needed. In this study, we developed protocols for creating N-ethyl-N-nitrosourea (ENU)-induced germline mutations in several rat strains. F1 preweanling pups from mutagenized Sprague Dawley (SD) male rats were then screened for functional mutations in Brca1 and Brca2 using a yeast gap-repair, ADE2-reporter truncation assay. We produced knockout rats for each of these two breast cancer suppressor genes.

Mammary carcinoma regression induced by perillyl alcohol, a hydroxylated analog of limonene

The monoterpene perillyl alcohol has been shown to induce the regression of 81% of small mammary carcinomas and up to 75% of advanced mammary carcinomas initiated by 7,12-dimethylbenz(a)anthracene (DMBA) in the Wistar-Furth rat. Dietary perillyl alcohol was greater than 5 times more potent than the monoterpene limonene at inducing tumor regression. Perilly alcohol is rapidly metabolized in the rat, as is limonene. Rats chronically fed perillyl alcohol had the same circulating plasma metabolites as rats fed limonene; however, the levels of these metabolites found in the plasma were higher for perillyl alcohol-fed rats. For example, rats given a 2% perillyl alcohol diet for 10 weeks had plasma levels of terpene metabolites of 0.82 mM whereas those fed a 10% limonene diet for the same period had blood levels of 0.27 mM. It thus appears that the increased potency of perillyl alcohol over limonene in causing tumor regression may be dee at least in part to differences in the pharmacokinetics of these two monoterpenes. We feel that perillyl alcohol is a good candidate for clinical testing of anticancer efficacy in humans.

Heterogeneous expression of the lipocalin NGAL in primary breast cancers

We have previously shown that neu oncogene‐initiated rat mammary carcinomas uniquely over‐express neu‐related lipocalin (NRL), a member of the calycin protein superfamily. Here, we characterize the putative human homolog of NRL, neutrophil gelatinase‐associated lipocalin (NGAL). ngal gene expression was found at moderate levels in only 2 of 17 human tissues examined, breast and lung. When breast cancers were examined for NGAL mRNA and protein levels, they were found to exhibit heterogeneous expression. NGAL levels varied in these tumors from undetectable to exceeding those in normal breast parenchyma. Immuno‐histochemical analysis confirmed the presence of NGAL within breast carcinoma cells but detected only low levels of this protein in normal ductal epithelium. In contrast, large amounts of the protein were localized to the lumen of normal breast ducts in the vicinity of NGAL‐expressing tumors. Interestingly, unlike NRL in rat mammary carcinomas, no significant association between NGAL expression and HER‐2/neu activation was found in human breast tumors. In contrast, a significant correlation between NGAL expression in breast cancer was found with several other markers of poor prognosis, including estrogen and progesterone receptor‐negative status and high proliferation (S‐phase fraction). NGAL levels were stratified as high or low in breast cancers from a cohort of node‐positive patients with known outcome. No significant association between NGAL expression and disease‐free or overall survival was observed. Int. J. Cancer (Pred. Oncol.) 79:565–572, 1998.

A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer

Progress toward the understanding and management of human colon cancer can be significantly advanced if appropriate experimental platforms become available. We have investigated whether a rat model carrying a knockout allele in the gatekeeper gene Adenomatous polyposis coli (Apc) recapitulates familial colon cancer of the human more closely than existing murine models. We have established a mutagen-induced nonsense allele of the rat Apc gene on an inbred F344/NTac (F344) genetic background. Carriers of this mutant allele develop multiple neoplasms with a distribution between the colon and small intestine that closely simulates that found in human familial adenomatous polyposis patients. To distinguish this phenotype from the predominantly small intestinal phenotype found in most Apc-mutant mouse strains, this strain has been designated the polyposis in the rat colon (Pirc) kindred. The Pirc rat kindred provides several unique and favorable features for the study of colon cancer. Tumor-bearing Pirc rats can live at least 17 months, carrying a significant colonic tumor burden. These tumors can be imaged both by micro computed tomography scanning and by classical endoscopy, enabling longitudinal studies of tumor genotype and phenotype as a function of response to chemopreventive and therapeutic regimes. The metacentric character of the rat karyotype, like that of the human and unlike the acrocentric mouse, has enabled us to demonstrate that the loss of the wild-type Apc allele in tumors does not involve chromosome loss. We believe that the Pirc rat kindred can address many of the current gaps in the modeling of human colon cancer.

Human metabolism of the experimental cancer therapeutic agentd-limonene

Abstractd-Limonene has efficacy in preclinical models of breast cancer, causing >80% of carcinomas to regress with little host toxicity. We performed a pilot study on healthy human volunteers to identify plasma metabolites of limonene and to assess the toxicity of supradietary quantities ofd-limonene. Seven subjects ingested 100 mg/kg limonene in a custard. Blood was drawn at 0 and 24 h for chemistry-panel analysis and at 0, 4, and 24 h for limonenemetabolite analysis. On-line capillary gas chromatography/mass spectrometry (GC/MS) analysis indicated that at least five compounds were present at 4 h that were not present at time zero. Two major peaks were identified as the rat limonene metabolites dihydroperillic acid and perillic acid, and two minor peaks were found to be the respective methyl esters of these acids. A third major peak was identified as limonene-1,2-diol. Limonene was a minor component. At a dose of 100 mg/kg, limonene caused no gradable toxicity. Limonene is metabolized by humans and rats in a similar manner. These observations and the high therapeutic ratio of limonene in the chemotherapy of rodent cancers suggest that limonene may be an efficacious chemotherapeutic agent for human malignancies.

A comparison of tocopherol and tocotrienol for the chemoprevention of chemically induced rat mammary tumors

Two forms of vitamin E, tocopherol and tocotrienol, were tested for chemopreventive activity in two chemically induced rat mammary-tumor models. When mammary tumors were induced by 7,12-dimethylbenz(a)anthracene (DMBA, 50 mg/kg), only the tocotrienol group had a statistically significant increase in tumor latency. There was no effect of either compound on tumor multiplicity. When tumors were induced by N-nitrosomethylurea (NMU, 30 mg/kg), neither analogue of vitamin E modified latency, whereas tocotrienol increased tumor multiplicity. In summary, neither vitamin analog had a major impact on mammary-tumor development after tumor induction with either DMBA or NMU.

Rat Mcs5a is a compound quantitative trait locus with orthologous human loci that associate with breast cancer risk

Breast cancer risk is a polygenic trait. To identify breast cancer modifier alleles that have a high population frequency and low penetrance we used a comparative genomics approach. Quantitative trait loci (QTL) were initially identified by linkage analysis in a rat mammary carcinogenesis model followed by verification in congenic rats carrying the specific QTL allele under study. The Mcs5a locus was identified by fine-mapping Mcs5 in a congenic model. Here we characterize the Mcs5a locus, which when homozygous for the Wky allele, reduces mammary cancer risk by 50%. The Mcs5a locus is a compound QTL with at least two noncoding interacting elements: Mcs5a1 and Mcs5a2. The resistance phenotype is only observed in rats carrying at least one copy of the Wky allele of each element on the same chromosome. Mcs5a1 is located within the ubiquitin ligase Fbxo10, whereas Mcs5a2 includes the 5′ portion of Frmpd1. Resistant congenic rats show a down-regulation of Fbxo10 in the thymus and an up-regulation of Frmpd1 in the spleen. The association of the Mcs5a1 and Mcs5a2 human orthologs with breast cancer was tested in two population-based breast cancer case-control studies (≈12,000 women). The minor alleles of rs6476643 (MCS5A1) and rs2182317 (MCS5A2) were independently associated with breast cancer risk. The minor allele of rs6476643 increases risk, whereas the rs2182317 minor allele decreases risk. Both alleles have a high population frequency and a low penetrance toward breast cancer risk.

On the statistical analysis of allelic-loss data

This paper concerns the statistical analysis of certain binary data arising in molecular studies of cancer. In allelic-loss experiments, tumour cell genomes are analysed at informative molecular marker loci to identify deleted chromosomal regions. The resulting binary data are used to infer properties of putative suppressor genes, genes involved in normal cell cycling. Various factors can complicate this inference, including background loss of heterozygosity, spatial (that is, within chromosome) dependence of the binary responses, non-informativeness of markers, covariates such as protein levels or tumour histology, heterogeneity of cells within tumours, and measurement error. We focus on the first three factors, discussing methods for statistical inference that separate background loss from significant loss. We outline the extension to other inferences, such as comparison questions and the relationship to covariates. Using characteristic features of tumourigenesis, we present a framework for the stochastic modelling of allelic-loss data, and build models within this framework; in particular, we propose a simple model that has chromosome breaks at locations of a Poisson process, and preferential selection cells with inactivated suppressor genes. We illustrate these methods on allelic-loss data from induced rat mammary tumours and human bladder cancers.

Mammary glands of sexually immature rats are more susceptible than those of mature rats to the carcinogenic, lethal, and mutagenic effects of N-nitroso-N-methylurea.

Knowing that the prepubertal period is a time of enhanced susceptibility for radiation‐induced human breast cancer, we used the Fischer 344 rat model to explore the age‐differential susceptibility of the mammary gland to the carcinogenic, lethal, and mutagenic effects of two structurally diverse chemical carcinogens, N‐nitroso‐N‐methylurea (NMU), and 7,12‐dimethylbenz(a)anthracene (DMBA). Mammary carcinoma incidences and multiplicities were significantly greater in immature than mature NMU‐treated rats while mammary carcinoma incidences and multiplicities were significantly lower in immature than mature DMBA‐treated rats. The survival of mammary clonogens of mature NMU‐treated rats in limiting dilution transplantation assays was greater than that of the survival of mammary clonogens of immature NMU‐treated rats. No differences were found in the survival of mammary cells from immature and mature rats exposed to DMBA. Although there were no mutation spectra differences, mammary epithelial cells of immature NMU‐treated rats had greater mutation frequencies than those of mature NMU‐treated rats. Together these results support the hypothesis that the mammary gland of immature rats is more susceptible to the carcinogenic, lethal, and mutagenic effects of alkylating agents represented by NMU in a carcinogen‐class‐specific manner. Further, the results suggest the importance of mechanistic and epidemiological studies of the susceptibility of the prepubertal breast to specific carcinogens such as alkylating agents.

Allelic imbalance in mammary carcinomas induced by either 7,12‐dimethylbenz[a]anthracene or ionizing radiation in rats carrying genes conferring differential susceptibilities to mammary carcinogenesis

To identify and compare the genetic lesions associated with tumorigenesis in rats carrying the mammary carcinoma suppressor (MCS) 1 gene, we induced mammary carcinomas in (Wistar Furth (WF) × Copenhagen (Cop))F1 rats by using either 7,12‐dimethylbenz[a]anthracene (DMBA) or radiation. The tumors were screened for allelic imbalances by using polymerase chain reaction and 65 polymorphic microsatellite markers spanning the genome. No allelic imbalance was detected at the mapped location of MCS‐1 on chromosome 2; however, a scan of the genome revealed random allelic imbalances in the radiation‐induced tumors. In addition, non‐random loss of heterozygosity (LOH) on chromosome 1 in the DMBA‐induced tumors was documented. We then screened three other subsets of DMBA‐ and radiation‐induced mammary carcinomas from (WF × Fischer (F344))F1, (Wistar Kyoto×F344)F1, and (F344×Cop)F1 rats for imbalance on chromosomes 1 and 2. No allelic imbalance was detected in the MCS‐1 region of chromosome 2 in any of the tumors screened. Nonrandom imbalance on chromosome 1 was detected but only in the DMBA‐induced tumors from the (F344×Cop)F1 rats. Thus, only Cop‐derived F1 rats have mammary tumors with the chromosome 1 imbalance; however, the imbalance does not favor the Cop parental allele. We also analyzed the DMBA‐induced tumors with LOH at chromosome 1 for Ha‐ras codon 61 mutation and found no association. These results suggest that loss of the MCS‐1 Cop allele is not required for tumor formation, that the genetic background of the F1 rat appears to influence the type of genetic lesion identified in the mammary tumors, and that there is no association between Ha‐ras codon 61 mutation and chromosome 1 imbalance in our model system.