Rat and human mammary tissue can synthesize choline moiety via the methylation of phosphatidylethanolamine.

The normal mammal requires large amounts of choline for maintenance and growth of tissue mass. Since milk, the only food for neonates, has many-fold higher free choline concentration than does maternal plasma, it is possible that mammary gland can synthesize choline molecules. The only known mammalian pathway for the synthesis de novo of choline molecules is catalysed by phosphatidylethanolamine N-methyltransferase (PeMT), which synthesizes phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor. We identified PeMT activity in rat mammary tissue, and differences in affinities for substrate, as well as in activities as a function of pH, suggest that at least two distinct enzyme activities are involved [i.e. one catalysing the methylation of PtdEtn to form phosphatidyl-N-methylethanolamine (PtdMeEtn) and the other catalysing the methylation of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) to form PtdMe2Etn and PtdCho, respectively]. The relationships between AdoMet concentrations and PtdCho formation from endogenous PtdEtn in rat mammary homogenate were complex: a sigmoidal component (with a Hill coefficient of 2.2), requiring 55 microM-AdoMet for half saturation (Vmax. = 9 pmol/h per mg of protein), and a high affinity component (Kapparent = 8.7 microM and Vmax. = 3.8 pmol/h per mg of protein) were identified. When exogenous PtdMe2Etn was added as substrate, PtdCho formation exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (Kapparent = 9 microM, Vmax. = 85 pmol/h per mg of protein). In the presence of endogenous substrates, the rates of PeMT-catalysed PtdCho formation within homogenates of rat mammary tissue were similar in tissue from lactating and non-lactating animals. When exogenous PtdMe2Etn was added to homogenates of rat mammary tissue, tissue from lactating rats made twice as much PtdCho as did tissue from non-lactating rats. Isolated mammary epithelial cells also exhibited PeMT activity; the rate of formation of PtdCho was much greater in intact versus broken cells. We also identified PeMT activity in homogenates of mammary tissue from non-lactating humans. The rate of PtdCho formation was of similar magnitude to that seen in rat tissue. This evidence supports the hypothesis that some of the choline found in milk could have been synthesized de novo in the mammary gland.     

A study of iodothyronine 5'-monodeiodinase activities in normal and pathological tissues in man and their comparison with activities in rat tissues

The present study was undertaken to investigate the peripheral iodothyronine 5'-monodeiodination in different human and rat tissues. We studied iodothyronine 5'-monodeiodinase type I (5'-DI) activity in liver, kidney, intestine, right cardiac atrium and skeletal muscle and we compared the results with those in rat tissues. Lodothyronine 5'- monodeiodinase type II (5'-DII) activity was studied in normal and ischemic human heart and in rat normal myocardium and brain. The 5'-DI activity (fmol/min x mg protein) in liver and kidney was significantly higher (p < 0.001, ANOVA) in normal rat tissue than in human. However, no significant differences were observed in 5'-DI activity between normal and tumoral human intestine or between intestinal tissue of man and rat. 5'-DI activity in normal human skeletal muscle was significantly higher than that in rat skeletal muscle (p < 0.05). The 5'-DI activity was lower in human ischemic myocardium when compared to normal myocardium either in humans (p < 0.05) or rat (p < 0.001). The Km of 5'-DI was significantly lower in rat than in human kidney and liver (p < 0.05). We conclude that 1) 5'-DI is distributed widely among extrathyroidal human and rat tissues and 5'-DII activity is detectable both in human and rat heart; 2) 5'-DI activity in liver and kidney is lower in man than in rat; 3) 5'-DI activity in the skeletal muscle is higher in man than in the rat; 4) 5'-DI activity is decreased in tumoral tissues of human liver and kidney and in ischemic myocardium, while no significant difference was found between human and rat cardiac 5'-DII activity.     

Expression of nuclear hormone receptors, their coregulators and type I iodothyronine 5'-deiodinase gene in mammary tissue of nonlactating and postlactating rats

The aim of the study was to test the hypothesis that expression of retinoid receptors (RARalpha, RARbeta, RARgamma), rexinoid receptors (RXRalpha, RXRbeta), thyroid hormone receptors (TRalpha, TRbeta), estrogen receptors (ERalpha, ERbeta), nuclear receptor coregulators (N-CoR, SRC-1, SMRT), and in addition type I iodothyronine 5'-deiodinase (5'-DI), EGFR and erb-B2/neu would be different in mammary postlactating tissue in comparison with that of nonlactating mammary gland. Using RT-PCR, we have shown that expression of RARalpha, RXRalpha,TRalpha, ERalpha,ERbeta,N-CoR, SRC-1, SMRT and EGFR in rat was significantly increased in postlactating mammary gland when compared to that of nonlactating mammary tissue. Postlactating mammary glands were found to express all RAR and RXR subtypes studied when compared to nonlactating mammary tissues that express exclusively RARalpha and RXRalpha subtypes. Enhanced expression of a number of nuclear hormone receptors, their coregulators in mammary tissue of postlactating rats in comparison with nonlactating animals identify a potential role for retinoid, thyroid and estrogen signalling pathways also after lactation period.     

Hypercholesterolemia and tissue-specific differential mRNA expression of type-1 5'-iodothyronine deiodinase under different selenium status in rats

Type-1 5'-iodothyronine deiodinase (5'-DI) is responsible for conversion of T4 to T3. Selenium (Se) is an integral part of this enzyme. Keeping in view the strong association between atherosclerosis and hypothyroidism, the present study examined the behavior of 5'-DI in liver, aorta and thyroid during hypercholesterolemia following different Se status, i.e., Se deficiency (0.02 ppm), adequate (0.2 ppm) and excess dose (1 ppm) in SD male rats. Animals were fed a control or high-cholesterol diet (2%) for 1 and 2 months. 5'-DI activity and mRNA expression was measured by RIA and RT-PCR respectively. In liver and aorta, 5'-DI expression significantly decreased with the Se-deficient and the high-cholesterol diet. The trend was opposite in thyroid, i.e., mRNA expression increased significantly during selenium deficiency and with a high-cholesterol feeding. But with 1 ppm Se supplementation, the 5'-DI expression increased in all the three tissues. The present study indicates that hypercholesterolemia along with selenium deficiency is co-responsible for differential regulation of 5'-DI enzyme in thyroidal vs. extrathyroidal tissues. Distinct regulation of 5'-DI in the thyroid reflects the clinical importance of this selenoprotein during hypercholesterolemia as this enzyme is essential for T3 production, which further has a vital role in the maintenance of lipid metabolism.     

Comparison of alkaline phosphatase activity in normal, lactating and neoplastic rat mammary tissue

1. Alkaline phosphatase activity in NMU-induced rat mammary tumours was compared with activity in normal and lactating mammary gland. 2. Both tumour and normal mammary alkaline phosphatase were sensitive to heat inactivation and inhibition by phenylalanine. 3. Specific activity of enzyme in tumours was comparable to normal mammary tissue. 4. Mammary gland alkaline phosphatase increased markedly in late pregnancy and early lactation. 5. Bromocryptine treatment had no effect on enzyme activity in lactating mammary gland.     

The expression of beta-1,3 galactosyltransferase and beta-1,4 galactosyltransferase enzymatic activities in the mammary gland of the tammar wallaby (Macropus eugenii) during early lactation

The regulation of beta-1,3 galactosyltransferase (3betaGalT) and beta-1,4 galactosyltransferase enzymatic (4betaGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two beta-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4betaGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3betaGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3betaGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17beta-oestradiol (E). The activity of 4betaGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3betaGalT and 4betaGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.     

Enzymic changes in rabbit and rat mammary gland during the lactation cycle

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.     

Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland

A recently published method for purification of a new inhibitor of growth of mammary cells in vitro from bovine mammary gland has been modified to yield several hundred μg of inhibitor per kg of glandular tissue. The inhibitory effect exerted by this preparation to Ehrlich ascites mammary carcinoma cells fulfilled all biological criteria of specificity established earlier for preparations obtained by other means, the most important being that the inhibitory effect is abolished by the epidermal growth factor and insulin. The preparation is shown to consist mainly of a protein of 13 kDa which appears to be not glycosylated. An antiserum raised in mice against the inhibitor is demonstrated to be specific for the 13 kDa bovine mammary gland protein. Neutralization of the inhibitory activity by the specific antiserum strongly supports the view that the 13 kDa protein is indeed the carrier of inhibitory activity. First data on tissue distribution obtained with an enzyme-linked immunosorbent assay revealed a high concentration of the anti-inhibitor-antiserum-reactive antigen in bovine lactating but not in non-lactating mammary gland tissue and in milk fat globule membranes. Some reactivity was also found in bovine lung. These data are interpreted with respect to a possible physiological significance of the growth inhibitor.     

Competing addition and hydrolysis of the cytidylylcytidylyladenosine terminal residues of transfer ribonucleic acid isolated from the non-lactating bovine mammary gland

1. The enzyme fraction obtained from the pH5 enzyme of non-lactating bovine mammary gland between 40 and 100% ammonium sulphate saturation markedly inhibited the AMP-incorporating activity of rat liver nucleotide-incorporating enzyme. This inhibitory effect has been attributed to high nuclease activity which can be partially removed by adsorption of the enzyme fraction on to calcium phosphate gel. 2. The degradation action of the calcium phosphate-purified enzyme is confined mainly to the terminal trinucleotide sequence -pCpCpA of tRNA, its effect being analogous to that of venom phosphodiesterase. This enzyme is heat labile and very readily loses its degradative activity. 3. Treatment of the enzyme fraction with Macaloid results in complete removal of the phosphodiesterase, leaving an enzyme capable of incorporating AMP into tRNA. 4. Transfer RNA extracted from non-lactating bovine mammary gland in the presence of polyvinyl sulphate and Macaloid is able to accept amino acids with an efficiency 30% of that shown by lactating bovine mammary-gland tRNA isolated under identical conditions.     

The expression of β-1,3 galactosyltransferase and β-1,4 galactosyltransferase enzymatic activities in the mammary gland of the tammar wallaby (Macropus eugenii) during early lactation

The regulation of β-1,3 galactosyltransferase (3βGalT) and β-1,4 galactosyltransferase enzymatic (4βGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two β-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4βGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3βGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3βGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17β-oestradiol (E). The activity of 4βGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3βGalT and 4βGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.     

Inhibition of T3 production in levothyroxine-treated female mice by the root extract of Convolvulus pluricaulis.

An investigation was made to evaluate the role of Convolvulus pluricaulis root extract in the regulation of hyperthyroidism in female mice. Its possible site of action was also studied. L-Thyroxine treatment for 30 days increased serum concentrations of thyroxine (T4) and triodothyronine (T3). The activity of hepatic 5'-monodeiodinase (5'-DI) and glucose-6-phosphatase (G-6-Pase) was also enhanced. On the other hand, administration of the plant extract either alone or with L-T4, decreased serum T3 concentration and the activity of hepatic 5'-DI and G-6-phase, without marked alteration in hepatic lipid peroxidation, indicating the possible regulation of hyperthyroidism by the plant extract. It appears that the action of the plant extract on thyroid function is primarily mediated through the inhibition of 5'-DI enzyme activity.     

Angiotensin converting enzyme from sheep mammary, lingual and other tissues

Occurrence of angiotensin converting enzyme (ACE) in mammary gland and tongue taste epithelium was demonstrated for the first time. Six times higher ACE activity in lactating mammary gland, than non-lactating mammary gland, suggested pregnancy and lactation hormonal dependent expression of ACE in female mammals. ACE activity was highest in choroid plexus, less in spinal cord and moderate in cerebrum, medulla, cerebellum and pons. Distribution of ACE in different regions of skin, kidney and among other tissues was different. Presence of ACE in adrenal glands, pancreas, bone marrow and thyroid gland indicated functions other than blood pressure homeostasis for this enzyme.     

Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of (14)C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of (14)C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105 degrees for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.     

Augmented efficacy of tamoxifen in rat breast tumorigenesis when gavaged along with riboflavin, niacin, and CoQ10: effects on lipid peroxidation and antioxidants in mitochondria

Reactive oxygen species (ROS) play a major role in causing mitochondrial changes linked to cancer and metastasis. Uptake of antioxidants by tissue to reduce the ROS production could be instrumental in controlling cancer. Tamoxifen (TAM), a nonsteroidal anti-estrogen drug most used in the chemotherapy and chemoprevention of breast cancer. Riboflavin, niacin and coenzyme Q10 (CoQ10) are proved to be potent antioxidants and protective agents against many diseases including cancer. The objective of this research is to determine the therapeutic efficacy of combinatorial therapy on mammary carcinoma bearing rats in terms of the mitochondrial lipid peroxidation and antioxidant status especially MnSOD. Female albino rats of Sprague-Dawley strain were selected for the investigation. Mammary carcinoma was induced with 7,12-dimethyl benz(a)anthracene (DMBA: 25 mg), and the treatment was started by the oral administration of TAM (10 mg/kg body weight/day) along with riboflavin (45 mg/kg body weight/day), niacin (100 mg/kg body weight/day) and CoQ10 (40 mg/kg body weight/day) for 28 days. The levels of lipid peroxides, activities of enzymic and non-enzymic antioxidants were measured in the mitochondria isolated from the mammary gland and liver of control and experimental rats. Rats treated with DMBA showed an increase in mitochondrial lipid peroxidation (mammary gland 52.3%; liver 25.1%) accompanied by high malondialdehyde levels along with lowered activities of mitochondrial enzymic antioxidants [superoxide dismutase (mammary gland 19.9%; liver 24.8%), catalase (mammary gland 50%; liver 19.7%), glutathione peroxidase (mammary gland 47.8%; liver 31.1%)] and non-enzymic antioxidants [reduced glutathione (mammary gland 14.3%; liver 13.3%), Vitamin C (mammary gland 6.49%; liver 21.4%) and E (mammary gland 20.3%; liver 22.2%)]. Administration of combinatorial therapy restored lipid peroxide level and the activities of enzymic and non-enzymic antioxidants to near normalcy. In addition, antitumour activity was also found to be enhanced which is evident from the increased expression of tumour suppressor gene MnSOD thereby preventing cancer cell proliferation. These results suggested that TAM treatment is the most effective during co-administration of riboflavin, niacin and CoQ10 in terms of mitochondrial antioxidant and antitumour activity.     

Oxytocin binding by myoepithelial cell membranes from involuted mammary tissue

Oxytocin binding activity of myoepithelial cell membranes from mammary tissue was measured under a variety of different experimental conditions. Mammary tissue from non-lactating rats bound oxytocin with a Kd of 9.2 +/- 1.6 nM (+/- S.E.) and indicates that receptors are retained by the myoepithelial cells in a non-lactating state. Ovariectomy of non-lactating rats did not depress the binding activity of the membranes. Administration of the estrogenic compounds estradiol-17 beta and diethylstibestrol at doses which affect uterine weight and are known to increase uterine oxytocin binding did not influence the binding activity of the myoepithelial cells. This indicates that the oxytocin receptors of the mammary gland are not under the same endocrine control as the uterine receptors.     

Functional analysis of mouse keratin 8 in polyoma middle T-induced mammary gland tumours

Keratin 8 and 18 are commonly used as tumorigenic markers for various types of carcinomas. They are known to be involved in cell migration, cell invasiveness, plasminogen activity and drug and radiation resistance. To ascertain a potential function for simple epithelium keratins in mammary adenocarcinoma in vivo, keratin-8-deficient mice (mK8) were mated with transgenic mice carrying the middle T oncogene driven by the MMTV promoter. The resulting mK8 knockout and control progeny carrying the middle T transgene developed mammary gland tumours with the same incidence. However, the onset of palpable mammary gland tumours occurred earlier in mK8 mutant than in control mice. This effect was prominent in males where the onset in control animals is delayed overall, because of the lower hormonal inducibility of the MMTV promoter. Metastatic foci were observed in the lungs of all females and of a few males, idependently of the genotype. Histological analysis revealed no morphological differences of the tumorigenic cells in primary tumours nor in metastatic foci. As expected, keratin 8 was absent in the mK8 tumours. Keratin 7 (mK7), keratin 18 (mK18) and keratin 19 (mK19) protein were observed in both primary and metastatic foci. These results constitute the first in vivo analysis of the role of simple epithelium keratins in mammary carcinogenesis. It demonstrates that the latency, but not the incidence nor the morphological features, of PyV middle T-induced mammary gland tumours is affected by keratin 8 deficiency     

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation

The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.     

Iodinated bovine prolactin. Characterization and binding to mammary gland cells.

Iodinated bovine prolactin (2.6 iodine atoms/molecule; labelled with a trace of 125I to give a specific activity of 0.041 muCi/mg) was prepared by the chloramine T method. It was active in two bioassays (pigeon crop sac and dispersed mouse mammary cell), though somewhat less active than the unmodified hormone. In an immunoassay, iodinated prolactin was more effective than the unmodified hormone at displacing 125I-prolactin from antibody. High specific activity 125I-prolactin (1 iodine atom/molecule; 70 muCi/microgram) was used for autoradiographic studies on the binding of prolactin to mouse mammary cells. In vivo the labelled hormone found in the mammary gland was associated with membranes of mammary epithelial cells and with alveolar lumen contents. In vitro 125I-prolactin was shown to bind to dispersed mouse mammary epithelial cells.     

Lactation-induced WAP-SV40 Tag Transgene Expression in C57BL/6J Mice Leads to Mammary Carcinoma

Two transgenic lineages were generated by directing the expression of SV40 T antigen to the mammary gland of inbred C57BL/6J mice using the whey acidic protein (WAP) promoter. In one lineage, WAPTag 1, multiparous female mice developed mammary adenocarcinoma with an average latency period of 13 months. The histopathological phenotype was heterogeneous, tumours occurred in a stochastic fashion, normal tissue was located next to neoplastic tissue, the mammary tumours usually developed and were remarkably similar to that observed in human cases. In addition, male and virgin females developed a poorly differentiated SV40 T antigen-positive soft tissue sarcoma, also at 13 months of age. In the other lineage, WAPTag 3, some parous females developed mammary tumours, but most mice succumbed to osteosarcomas arising from the os petrosum at 5.5 to 6 months of age and on necropsy, renal adenocarcinomas were also found. Appearance of these unexpected tumour types demonstrates the non-specific e...