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The Wistar-Kyoto (WKY) rat strain expresses high levels of beta-casein in its virgin mammary glands. We found that the onset of beta-casein overexpression (BCO) occurs at 6 weeks of age, with morphological differentiation of the mammary gland detectable at 7 weeks of age. BCO was previously shown to be cell autonomous; however, we found that adrenal and ovarian hormones were permissive and necessary for the expression of the BCO phenotype, indicating that the genetic variation that initiates BCO from within the mammary epithelium can only manifest BCO in the presence of virgin hormone levels. Sequencing of the WKY and Wistar-Furth (WF) rat beta-casein promoters showed them to be identical. Culture of primary rat mammary epithelial cells (RMEC) under lactogenic conditions revealed that expression of beta-casein was independent of epidermal growth factor (EGF) in RMEC from virgin WKYv, but was dependent in WFv, RMEC. RMEC from a pregnant WFp responded similarly to WKYv RMEC, suggesting that EGF-independent beta-casein expression occurs naturally in differentiated rat mammary epithelium. However, induction of beta-casein expression in RMEC from immature WKY rats was also independent of EGF, indicating that the induction as well as maintenance of BCO do not require EGF. We suggest that an EGF-independent signaling pathway, arising from a trans-acting inherited effector(s), underlies BCO.

The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21(Cip1/WAF1), bax, bad, and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a potential role for transforming growth factor beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-related genes were induced and temporally regulated during POH treatment: (a) c-jun and c-fos were transiently induced within 12 h of chemotherapy; (b) TGF-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta type I and II receptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland.

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.

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.

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.

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) x 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 x Fischer (F344))F1, (Wistar Kyoto x F344)F1, and (F344 x 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 x 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.

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.

Monoterpenes, including limonene and its in vivo rat plasma metabolites, have been shown to be inhibitors of protein isoprenylation of small G proteins, including p21 ras. In addition, dietary limonene has been shown to be capable of preventing the development and causing the regression of chemically induced mammary carcinomas, many of which contain activated ras oncogenes. On the basis of these observations, it was hypothesized that a possible mechanism by which limonene exerts its effects on the chemoprevention and regression of mammary tumors involves the inhibition of protein isoprenylation of the small G protein p21. In the first study, we asked whether dietary limonene was able to prevent the development of mammary carcinomas which were induced using direct retroviral gene transfer of v-Ha-ras into the mammary parenchyma in situ. Limonene modified neither the rate of gene transfer nor the stability of gene expression. However, limonene did greatly inhibit the formation of mammary carcinomas induced by the insertion of activated ras. In a follow-up study, we asked whether chemoprevention by limonene was preferentially effective against a subset of chemically induced mammary carcinomas with activated ras. Rats were fed limonene to prevent the development of N-nitroso-N-methylurea-induced mammary tumors, a majority of which contain the activated Ha-ras oncogene. As expected, limonene administration increased the latency period and lowered the frequency of mammary carcinoma development as compared to controls. However, tumor characterization revealed that limonene treatment did not alter the percentage of carcinomas with activated ras. These studies are consistent with the above studies in that limonene is effective in preventing mammary carcinomas with activated ras. Interestingly, carcinomas without activated ras were prevented to the same extent as those with the activated oncogene.

The monoterpenes represent a potentially new class of breast cancer therapeutic agents. We have shown that d-limonene induces the regression of advanced rat mammary adenocarcinomas. These regressing tumors have an increased cellular concentration of both the mannose-6-phosphate/insulin-like growth factor II receptors and transforming growth factor beta 1. The terpene-induced regression of mammary tumors may result in part from the mitoinhibitory and differentiation properties of active transforming growth factor beta 1. Furthermore, the activation of transforming growth factor beta 1 in these tumors is likely to be facilitated by the increased concentration of the mannose-6-phosphate/insulin-like growth factor II receptors in the mammary tumor cells. Tumors not responding to terpene therapy lacked a rise in the mannose-6-phosphate/insulin-like growth factor II receptor level which may relate to the fact that this gene is hemizygous due to maternal imprinting.

Dietary administration of the monocyclic monoterpenoid d-limonene causes complete regression of both dimethylbenz[alpha]anthracene- and N-nitroso-N-methylurea-induced rat mammary carcinomas. Carcinomas regress when limonene is added to the diet either when the tumor is small and still capable of spontaneously regressing or when it is large and progressed beyond the stage when it is susceptible to spontaneous regression. The limonene dose-tumor regression response relationship is steep. Significant regressions are not observed at 5% dietary levels, while a majority of tumors completely regress above a 7.5% dietary level. Limonene appears to act in a cytostatic fashion. Its removal from the diet results in a significant number of tumor recurrences. Regressing tumors have a unique histopathological appearance that is not associated with gross cytotoxicity, immune cell involvement, or apoptosis. Preliminary analysis suggests a remodeling/redifferentiation event underlying regression. The underlying mechanism of action of limonene in causing tumor regression is unknown. However, it should be noted that limonene can selectively inhibit the isoprenylation of small G proteins. Monoterpenoids such as limonene represent a novel class of anticancer drugs with the potential to cause tumor regressions with limited toxicity.

Mammary carcinogenesis is a multistep process consisting minimally of initiation and promotion/progression stages. The rate-limiting stage in the carcinogenesis process is undetermined but can in part be addressed by estimating the frequency of initiation, a heritable early event. Here, we use an in vivo limiting dilution transplantation assay to estimate initiation frequency in a rat mammary epithelial stem-like cell population that was exposed in situ to 50 mg/kg N-methyl-N-nitrosourea (NMU) administered i.v. We estimate that this dose resulted in the killing of 65% of exposed mammary cells. Known numbers of cells surviving NMU exposure were grafted into fat-pads of recipient rats in which the cells grew and differentiated into structurally and functionally normal mammary glands. Recipient rats were hormonally manipulated to provide maximal promotion of initiated cells. Mammary carcinomas developing at graft sites were quantitated over a 2-year period. Based on these results, we estimate that at least 1 surviving NMU-exposed mammary cell in 7,200 was initiated. Seventeen % of these graft site carcinomas had an activated H-ras oncogene with a G to A mutation in codon 12. This suggests that at least 1 mammary cell in 43,000 was mutated in this fashion by in situ exposure to NMU. These data suggest that cH-ras represents approximately 1 of 5 of the initiation events produced by NMU exposure of rat mammary glands.

The role of c-Ha-ras-1 oncogene activation in the multistage biological process of N-methyl-N-nitrosourea (NMU)-induced mammary carcinogenesis was investigated. The average yield of NMU-induced mammary tumors in Wistar-Furth rats was altered by modification of either the initiation or promotion/progression stage of carcinogenesis. Initiation was varied by the use of different doses of NMU from 20 to 50 mg/kg. Tumor yield was increased with increasing NMU doses. However, the frequency of mammary tumors with activated c-Ha-ras-1 decreased in a linear fashion with increasing NMU doses. Promotion/progression was varied by increasing prolactin levels starting approximately 2 weeks after NMU administration. This hormonal manipulation increased tumor yield, while reducing the frequency of tumors with activated ras. It is postulated that ras activation represents one of several possible mechanisms by which NMU initiates mammary carcinogenesis. Furthermore, initiated cells without activated ras are more dependent on epigenetic promotional events provided by either prolactin or NMU than are ras-initiated cells.