Estrogen receptor-positive mammary tumorigenesis in TGFalpha transgenic mice progresses with progesterone receptor loss.

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFalpha) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFalpha mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/-) p53 mice did not acquire mammary lesions, p53+/- mice carrying the NRL-TGFalpha transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFalpha expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR-, and when combined with a heterozygous p53 genotype, ER-/PR-.

Characterizing a rat Brca2 knockout model.

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2-/- rats are 100% viable and the vast majority live to over 1 year of age. Brca2-/- rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2-/- rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

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.

On the utility of pooling biological samples in microarray experiments.

Over 15% of the data sets catalogued in the Gene Expression Omnibus Database involve RNA samples that have been pooled before hybridization. Pooling affects data quality and inference, but the exact effects are not yet known because pooling has not been systematically studied in the context of microarray experiments. Here we report on the results of an experiment designed to evaluate the utility of pooling and the impact on identifying differentially expressed genes. We find that inference for most genes is not adversely affected by pooling, and we recommend that pooling be done when fewer than three arrays are used in each condition. For larger designs, pooling does not significantly improve inferences if few subjects are pooled. The realized benefits in this case do not outweigh the price paid for loss of individual specific information. Pooling is beneficial when many subjects are pooled, provided that independent samples contribute to multiple pools.

On parametric empirical Bayes methods for comparing multiple groups using replicated gene expression profiles.

DNA microarrays provide for unprecedented large-scale views of gene expression and, as a result, have emerged as a fundamental measurement tool in the study of diverse biological systems. Statistical questions abound, but many traditional data analytic approaches do not apply, in large part because thousands of individual genes are measured with relatively little replication. Empirical Bayes methods provide a natural approach to microarray data analysis because they can significantly reduce the dimensionality of an inference problem while compensating for relatively few replicates by using information across the array. We propose a general empirical Bayes modelling approach which allows for replicate expression profiles in multiple conditions. The hierarchical mixture model accounts for differences among genes in their average expression levels, differential expression for a given gene among cell types, and measurement fluctuations. Two distinct parameterizations are considered: a model based on Gamma distributed measurements and one based on log-normally distributed measurements. False discovery rate and related operating characteristics of the methodology are assessed in a simulation study. We also show how the posterior odds of differential expression in one version of the model is related to the ratio of the arithmetic mean to the geometric mean of the two sample means. The methodology is used in a study of mammary cancer in the rat, where four distinct patterns of expression are possible.

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.

ras gene mutations are absent in NMU-induced mammary carcinomas from aging rats.

Carcinoma induction in the rat mammary carcinogenesis model is age dependent. In this study, mammary cancer susceptibility and ras gene activation were investigated in rats exposed to N:-methyl-N:-nitrosourea (NMU) at 2, 6, 8 and 15 months. Animals were resistant to NMU-induced mammary tumor development when exposed at 6 and 8 months of age, whereas a significant number of mammary carcinomas developed in animals exposed to NMU at 2 and 15 months of age. G35-->A35 activating mutations in the Harvey ras gene were found only in mammary carcinomas from rats exposed to NMU at 2 months of age, but not in tumors that developed in animals exposed to NMU at 15 months of age. No G35-->A35 activating mutations were present in the Kirsten ras gene of any of the mammary tumors. Additional analysis of exons 1 and 2 of the Harvey ras gene from mammary carcinomas that developed in animals exposed to NMU at 15 months of age did not reveal any other activating mutations in this gene. In mammary carcinomas from animals exposed to NMU at 2 months of age, the frequency of mammary carcinomas with mutations in the Harvey ras gene was independent of the time from which the tumor first appeared. Therefore, age at the time of carcinogen exposure plays a critical role in both breast cancer susceptibility and the molecular events that contribute to mammary carcinoma development.

Phase I clinical and pharmacokinetic study of perillyl alcohol administered four times a day.

We conducted a phase I dose-escalation trial of perillyl alcohol (POH; NSC 641066) given p.o. on a continuous four times a day basis to characterize the maximum tolerated dose, toxicities, pharmacokinetic profile, and antitumor activity. Sixteen evaluable patients with advanced refractory malignancies were treated at the following doses: level 1 (L1), 800 mg/m2/dose; L2, 1200 mg/m2/dose; L3, 1600 mg/m2/dose. POH was formulated in soft gelatin capsules containing 250 mg of POH and 250 mg of soybean oil. The predominant toxicities seen were gastrointestinal (nausea, vomiting, satiety, and eructation), which were dose limiting. There appeared to be a dose-dependent increase in levels of the two main metabolites, perillic acid and dihydroperillic acid. No significant differences were seen whether the drug was taken with or without food. There was a trend toward decreasing metabolite levels on day 29 compared with days 1 and 2. Peak metabolite levels were seen 1-3 h post ingestion. Metabolite half-lives were approximately 2 h. Approximately 9% of the total dose was recovered in the urine in the first 24 h, the majority as perillic acid. Evidence of antitumor activity was seen in a patient with metastatic colorectal cancer who has an ongoing near-complete response of > 2 years duration. Several other patients were on study for > or = 6 months with stable disease. The maximum tolerated dose of POH given continuously four times a day was 1200 mg/m2/dose. Gastrointestinal toxicity was dose limiting, although significant interpatient variability in drug tolerance was seen.

Induction of AP-1 activity by perillyl alcohol in breast cancer cells.

Monoterpenes display chemopreventive and therapeutic activity in rat mammary tumor models. Monoterpenes can also inhibit cell growth and induce apoptosis of cultured cells. In this study, the monoterpene perillyl alcohol (POH) was found to induce transient expression of the c-jun and c-fos genes transcriptionally. POH also transiently induced phosphorylation of c-Jun protein. These events were associated with transcriptional activation of an AP-1-dependent reporter gene. These results suggest that POH might affect c-Jun activity via the Jun N-terminal kinase/stress-activated protein kinase pathway and modulate expression of AP-1 target genes.

Neutrophil gelatinase-associated lipocalin in normal and neoplastic human tissues. Cell type-specific pattern of expression.

Neutrophil gelatinase-associated lipocalin (NGAL) has recently been identified in myeloperoxidase-negative neutrophil granules. Members of the lipocalin family are thought to bind and transport small lipophilic molecules such as retinoids and roles in cell regulation have been proposed. Recently, NGAL has also been demonstrated in the colonic mucosa in certain pathologic conditions. The aim of this study was to examine the distribution of NGAL in normal and neoplastic tissues by immunohistochemistry. Interestingly, NGAL was found in a variety of normal and pathological human tissues. A cell type-specific pattern of expression was seen in bronchus, stomach, small intestine, pancreas, kidney, prostate gland, and thymus. The comparative analysis of the putative rat homologue neu-related lipocalin showed a very similar pattern of expression with the exception of pancreas and kidney. Neoplastic human tissues showed a very heterogeneous expression of NGAL protein. High NGAL levels were found in adenocarcinomas of lung, colon and pancreas. In contrast, renal cell carcinomas of various subtypes and prostate cancers contained low NGAL levels. Lymphomas and thymic tumours were negative for NGAL immuno-labeling. Knowledge about the location of NGAL in normal cells and in disease states provides the first clues towards understanding its biological function.

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.

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.

Precocious differentiation of the virgin Wistar-Kyoto rat mammary gland.

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.

Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol.

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.

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.

Radiobiological studies using synchrotron-produced ultrasoft X-rays.

Ultrasoft X-rays have been extensively used to explore radiobiological mechanisms surrounding cell killing. These studies for the most part have been linked to a small number of X-ray energies. Recently, this field of study has been broadened by the availability of synchrotron-produced ultrasoft X-rays which can be produced at any desired energy. We have taken advantage of the University of Wisconsin Synchrotron to reexamine two fundamental radiobiological questions: Dose RBE vary with different ultrasoft X-ray energies? Dose the fraction of the nuclear volume exposed to equal total X-ray energy modify cell cytotoxicity? The first study focuses on the survival of Chinese hamster V79 and mouse C3H10T1/2 cells irradiated with synchrotron-produced 273 eV and 860 eV ultrasoft X-rays. These two energies, which are available by multilayer monochromatization of the synchrotron output spectrum, exhibit equal attenuation within living cells. Such an isoattenuating energy pair allows the direct examination of how biological effectiveness varies with the energy of the ultrasoft X-rays. In comparing survival results, we find similar biological effectiveness of these two energies for both the C3H10T1/2 and the V79 cells. These results are no consistent with previous findings of increasing RBE with decreasing ultrasoft X-ray energies. In addition, after correcting for mean nuclear based on measurements of cell thickness obtained with confocal microscopy, we find no significant differences in survival between the two ultrasoft X-ray energies and 250 kVp X-rays. These results suggest that RBE does not increase with decreasing energy of ultrasoft X-ray between 860 eV and 273 eV. In a second study we introduced an method which allows partial-volume irradiation of live cells using synchrotron-produced ultrasoft X-rays and micro-fabricated irradiation masks. The masks were made by X-ray lithography at the University of Wisconsin Synchrotron Radiation Center, and they consist of 1.85-micron-wide stripes of gold 1.35 microns apart plated onto thin silicon nitrate membranes. When placed adjacent to mylar on which live cells are plated, these masks allow cells to be irradiated in a striped pattern with dimensions much smaller than the cell nuclei. Using 1340 eV synchrotron-produced X-rays, we compare the survival of cells subjected to uniform irradiation and cells subjected to partial-volume irradiation. Our results show that, at equal mean dose to the nucleus (i.e. equal total energies deposited), survival is not statistically different for the two treatments over a wide range of doses. Thus, imparting equal energies to smaller intranuclear volumes does not appear to modulate cell killing.

Kinetics of mammary clonogenic cells and rat mammary cancer induction by X-rays or fission neutrons.

Following the hormonal treatment of rats with high prolactin levels and glucocorticoid deficiency (Prl+/Glc-) for 48 days (Day +48), total recoverable mammary DNA was increased by more than sevenfold, tritiated thymidine uptake by nearly fourfold, and total mammary clonogens by about fivefold. Irradiation with 4, 40, and 80 cGy X-rays on Day +48 increased total mammary carcinomas per rat-day-at-risk linearly with dose, and 40 and 80 cGy significantly decreased first carcinoma latency. A dose of 40 cGy X-rays before hormone treatment (Day -1) yielded tumor latencies and frequencies insignificantly different from unirradiated controls but significantly different from those when the dose was given on Day +48. Total carcinomas per rat-day-at-risk were fitted better by a function of dose to the power 0.4 than by a linear function after exposure to 1, 10. and 20 cGy fission neutrons, and 10 and 20 cGy significantly shortened the time to appearance of the first cancer. In contrast to results with X-rays, 10 cGy neutrons on Day -1 yielded tumor frequencies and latencies insignificantly different from 10 cGy neutrons on Day +48. The carcinogenic action of X-rays, but not of neutrons, was thus influenced by total clonogen numbers and/or proliferation rates.

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.

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.

Harvey ras results in a higher frequency of mammary carcinomas than Kirsten ras after direct retroviral transfer into the rat mammary gland.

Exclusive activation of either the Harvey-, Kirsten-, or N-ras gene is often found in human and rodent cancers, although the mechanisms responsible for tissue-specific ras gene activation are poorly understood. In this study, the contribution of ras gene expression and Ras protein activity to the tissue-specificity of ras gene activation was investigated using the rat mammary carcinogenesis model where ras activation, when it occurs, is exclusively in the Harvey ras gene. Differential ras gene expression was examined in mammary tissue from virgin, pregnant, and lactating rats. Harvey ras expression was 1.5-2-fold higher than Kirsten ras or N-ras at each adult stage of development, with the highest ras levels expressed during pregnancy. The modest difference in total mRNA expression found between the independent members of the ras gene family is unlikely to fully account for the exclusive tissue-specificity of Harvey ras activation observed in rat mammary carcinogenesis. Thus, the role of Ras protein specificity was studied by infecting the mammary gland of virgin rats in situ with replication-defective retroviral vectors expressing either the activated or wild-type forms of Harvey- or Kirsten-ras. A 7-14-fold higher number of mammary carcinomas was observed after infection with vectors expressing the G35 to A activated Harvey ras gene product compared with those expressing G35 to A activated Kirsten ras. Mammary carcinomas also developed from infusion of vectors expressing wild-type Harvey ras, but not wild-type Kirsten ras. These data suggest the importance of the Ras protein itself in determining the specificity of the highly homologous Ras family members in organ-specific carcinogenesis.