Mammary gland carcinoma-related increase of type I iodothyronine 5'-deiodinase activity in Sprague-Dawley rats.

Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.     

Coexistence of different tissue tumourigenesis in an N-methyl-N-nitrosourea-induced mammary carcinoma model: a histopathological report in Sprague-Dawley rats

N-methyl-N-nitrosourea (MNU), a highly potent carginogen, is widely used to generate mammary tumours in murine species. In a model of MNU-induced mammary carcinogenesis using immature female Sprague-Dawley rats, large mammary tumours (largest dimension > or =0.5 cm) were obtained within a very short period of time. In addition, in the rats bearing MNU-induced mammary carcinomas, there were a number of tumours whose origins were not from mammary tissue but from several different tissues and from mammary non-epithelial tissue. The tumours were of mesenchymal or epithelial origin and they were located in the inguinal region. These tumours were diagnosed as fibroadenoma, combined tubular adenoma and fibroadenoma, hyperkeratotic papilloma, keratinous cyst and malignant peripheral nerve sheath tumour (MPNST) with smooth muscle differentiation. The occurrence of these other tumours in addition to the development of the mammary carcinomas may be attributed to a direct local effect of the intraperitoneal administration of MNU during the sexual development of the immature rats. In the MNU-induced mammary tumour model, coexistence of tumourigenesis in various non-mammary tissues should be considered an important factor that may interfere with experimental procedures and results and also the quality of life of the tumour-bearing animals.     

Histological evaluation of rat mammary tumours after treatment with retinoic acid analogues — phytol, TTNPB and vitamin D3 analogue seocalcitol

The effects of retinoic acid analogue — phytol (precursor of retinoic X receptors ligand), TTNPB (retinoic acid receptor agonist) and seocalcitol (EB1089, analogue of vitamin D₃) in mammary tumours of Sprague-Dawley rats induced with 1-methyl-1-nitrosourea (MNU) were investigated. Treatment with phytol, TTNPB and seocalcitol may have some protective and therapeutical effects on malignant processes. Treatment with these components in combination of TTNPB and phytol or seocalcitol and phytol inhibited progression of MNU-induced tumours of the rat mammary gland, and also induced decrease of tumour burden and volume in comparison with treated control group. Treatment of rats with the above compounds had no effect on malignity and invasiveness of carcinomas.     

Effects of astaxanthin supplementation on chemically induced tumorigenesis in Wistar rats

ABSTRACT: BACKGROUND: Astaxanthin (ASTA) is a fat-soluble xanthophyll with powerful antioxidant functions. It is extracted from e.g. salmon, an important food source for certain human populations known to have a reduced risk of tumor development. It is possible that ASTA plays a role in cancer chemoprevention in such populations. The purpose of this study was to investigate the effects of dietary ASTA on chemically induced mammary tumorigenesis using N-methyl-N-nitroso-urea (MNU) in immature Wistar rats. METHODS: Thirty-six 37 days old juvenile female Wistar rats were at random allocated to 4 groups of which Groups 1 and 2 received a single dose of 55 mg MNU/kg body weight. The effects of ASTA was evaluated by giving rats of Groups 2 and 4 a dose of 50 mg ASTA/kg/day for the entire duration of the study. Group 3 rats received feed added alimentary oil.Necropsy and histopathological examinations were carried out on each rat 14 months after the administration of MNU. Haematological values and antioxidative status were determined. Oxidative stress was evaluated by monitoring superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in hepatic tissue. Lipid peroxidation and carbonylation of proteins was determined in protein extracts from the liver. RESULTS: Tumor development occurred only in rats of Groups 1 and 2, i.e. MNU exposed animals. Frequency of tumor development in general and average number of tumors per animal were insignificant between these two groups. Mammary gland tumors developed in equal frequencies in Group 1 and 2 rats, respectively. Although only rather few tumors were found in the mammary glands, a substantial number of other tumors were found in Group 1 and 2 rats, but at equal rates.Biochemical analyses showed significant higher levels of GPx, malondialdehyde and dinitrophenylhydrazine in Group 1 rats that for rats in all other groups thus indicating protective effects of ASTA on MNU induced hepatic oxidative stress. CONCLUSIONS: Supplementation with ASTA did not reduce tumorigenesis induced by MNU in Wistar rats. However, supplementation with ASTA seemed to have anti-inflammatory effects.     

Reduction of 1-methyl-1-nitrosourea-induced tumor burden with DNA vaccines encoding mutated ras epitopes and the costimulatory molecule B7.1

1-methyl-1-nitrosourea (MNU), a well characterized carcinogen, was used to induce adenocarcinomas in rat mammary gland. 150 days after the first injection of MNU, the animals were treated with DNA minigene vaccines encoding ras T cell epitopes together with the co-stimulatory molecule B7.1 (CD 80). Five injections with a biolistic device (gene gun) in monthly intervals significantly reduced the tumor burden. A therapeutic effect could be measured with both, DNA vaccines encoding ras epitopes and B7.1, as well as with a DNA vaccine expressing solely the B7.1 molecule thus indicating the potential of genetic vaccination for turnor treatment.     

Quantification of epithelial cell differentiation in mammary glands and carcinomas from DMBA- and MNU-exposed rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.     

A comparative study on the protective effects of 17beta-estradiol, biochanin A and bisphenol A on mammary gland differentiation and tumorigenesis in rats

Mammary tissue differentiation and tumorigenesis were studied in female rats following subcutaneous injection at 2, 4 and 6 days after birth with low or high doses of 17beta-estradiol (0.1 or 10 microg; E2), biochanin A (0.1 or 10 mg; BCA) or bisphenol A (0.1 or 10.0 mg; BPA). Half of the rats were killed on day 35 to analyze the terminal end bud (TEB), terminal duct (TD) and alveolar bud (AB) of the mammary tissue. The remaining rats were injected, ip, with a dose of 50 mg/kg of N-nitroso-N-methylurea (MNU) at 7 weeks of age and sacrificed 26 weeks later. The incidence and multiplicity of mammary tumors (MT) decreased among all three different treated groups, dose-dependently. However, the pattern of mammary gland differentiation varied. No significant difference was observed after E2 administration. TEB decreased dose-dependently in BCA treated groups and the number of TD and AB were suppressed significantly in BPA high dose group.     

Intratracheal synthetic CpG oligodeoxynucleotide causes acute lung injury with systemic inflammatory response

Bacterial genome is characterized by frequent unmethylated cytosine-phosphate-guanine (CpG) motifs. Deleterious effects can occur when synthetic oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides (CpG-ODN) are administered in a systemic fashion. We aimed to evaluate the effect of intratracheal CpG-ODN on lung inflammation and systemic inflammatory response. C57BL/6J mice received intratracheal administration of CpG-ODN (0.01, 0.1, 1.0, 10, or 100 μM) or control ODN without CpG motif. Bronchoalveolar lavage (BAL) fluid was obtained 3 or 6 h or 1, 2, 7, or 14 days after the instillation and subjected to a differential cell count and cytokine measurement. Lung permeability was evaluated as the BAL fluid-to-plasma ratio of the concentration of human serum albumin that was injected 1 h before euthanasia. Nuclear factor (NF)-κB DNA binding activity was also evaluated in lung homogenates. Intratracheal administration of 10 μM or higher concentration of CpG-ODN induced significant inflammatory cell accumulation into the airspace. The peak accumulation of neutrophils and lymphocytes occurred 1 and 2 days after the CpG-ODN administration, respectively. Lung permeability was increased 1 day after the 10 μM CpG-ODN challenge. CpG-ODN also induced nuclear translocation of NF-κB and upregulation of various inflammatory cytokines in BAL fluid and plasma. Histopathology of the lungs and liver revealed acute lung injury and liver damage with necrosis, respectively. Control ODN without CpG motif did not induce any inflammatory change. Since intratracheal CpG-ODN induced acute lung injury as well as systemic inflammatory response, therapeutic strategies to neutralize bacterial DNA that is released after administration of bactericidal agents should be considered.     

Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs

Bacterial DNA with CpG motifs can efficiently stimulate the vertebrate immune system. Thus, synthetic oligodeoxynucleotides that contain such CpG motifs (CpG-ODN) are currently used in preclinical and clinical studies to develop new allergy or cancer therapies and vaccine adjuvants. Recent animal studies indicate that CpG-ODN therapies can also be used for successful treatment of infections caused by bacteria, parasites or viruses. In these experiments, innate and adaptive immune responses against pathogens were augmented by CpG-ODN and subsequently induced resistance against infectious diseases. The stimulation of dendritic cells played a central role for the therapeutic effect of CpG-ODN. However, CpG-ODN can also have negative side effects, which accelerate disease progression in some viral infections. Clinical studies with CpG-ODN will determine their potential for the therapy of infectious diseases in humans.     

The critical DNA flanking sequences of a CpG oligodeoxynucleotide, but not the 6 base CpG motif, can be replaced with RNA without quantitative or qualitative changes in Toll-like receptor 9-mediated activity

Double- and single-stranded oligodeoxynucleotides containing unmethylated cytosine-guanosine (CpG) dinucleotides (CpG-ODN) activate immune cells via TLR9. In this report we synthesized hybrid DNA-RNA molecules (HDR) in order to further explore the structure-immune function relationship of CpG-ODN in TLR9 signaling and the potential immunomodulatory properties of RNA. We demonstrate that replacement of the deoxyadenosine flanking sequences, critical for the immune activating properties of CpG-ODN, with a similar number of adenosines, although not guanosines, cytosines, or uracils, maintains complete immunostimulatory activity of the hybrid oligonucleotide in vitro, whereas a similar RNA replacement of even 1 base of the required unmethylated 6 base DNA motif (purine-purine-CpG-pyrimidine-pyrimidine) results in a complete loss of activity. Regardless of whether the critical flanking sequence was RNA or DNA there was no significant change in the quantitative or qualitative immune-stimulating activity, or TLR-specificity of the resulting sequences, thus underscoring the relatively permissive functional role of the flanking sequence, and the more specific role of the motif in mediating TLR9 signaling. These data further support a potential role for RNA in immunomodulation.     

Oligodeoxynucleotides containing CpG motifs induce low levels of TNF-alpha in human B lymphocytes: possible adjuvants for Th1 responses

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) represent potential adjuvants for specific immunotherapy of type I allergies because they foster Th1-like immune responses. However, previous work has shown that CpG-ODN induce systemically active levels of TNF-alpha in murine macrophages. The goal of the present study was to evaluate the release of TNF-alpha in human cells by a CpG-ODN proven to induce Th1 immune responses in cells from atopic individuals and in mice. CpG-ODN induced TNF-alpha in cells from atopic and healthy individuals. However, the amounts were low, as determined by comparison with commonly used Ags. Intracellular cytokine staining of PBMC revealed that CpG-ODN-induced TNF-alpha derived exclusively from B lymphocytes. TNF-alpha contributed to the CpG-ODN-augmented proliferation and Ig synthesis in PBMC, but was not involved in IFN-gamma synthesis. In conclusion, our findings indicate that certain CpG-ODN induce low amounts of TNF-alpha in human B lymphocytes and may therefore be used to modulate Th2-biased immune responses in allergic patients.     

Role of steroid hormones and prolactin in canine mammary cancer

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.     

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     

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.     

Ca2+-stimulated ribonuclease. A new marker enzyme of differentiated rat mammary tissues.

A unique ribonuclease, active only in the presence of Ca2+, was present in lactating mammary gland and milk of the rat. This enzyme was absent from virgin-rat mammary gland and non-mammary tissues of lactating rats. The presence of moderate activity in differentiated mammary tumours, together with an increase in activity in normal tissue parelleling development of mammary function, identify this enzyme as a marker of mammary differentiation in the rat.