Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea

Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL-60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias.     

Transforming growth factor-alpha in carcinogen-induced F344 rat hepatic foci

Transforming growth factor-alpha (TGF alpha) is a positive growth regulator in epithelial cells, including hepatocytes. Overexpression of TGF alpha has been associated with increased growth and malignancy of end-stage cancers in humans and rodents. The overall aim of this study was to characterize TGF alpha staining in diethylnitrosamine-induced hepatic foci from male F344 rats with the hematoxylin and eosin (H and E) histological phenotype. The association between the individual focal DNA synthesis labeling index and the presence of TGF alpha was also examined. Hepatic foci were identified as eosinophilic, basophilic, clear cell, or mixed cell. Of these foci, 37.5% labeled positive for TGF alpha. There were distinct differences in the pattern of TGF alpha labeling between the different H and E histological phenotypes. Intense, uniform TGF alpha labeling was observed in eosinophilic foci. Basophilic foci labeled for TGF alpha diffusely uniform throughout the cytoplasm. In clear-cell foci, TGF alpha labeling occurred primarily along the periphery of the cell membrane. In mixed-cell foci, labeling occurred both along the periphery and diffusely throughout the cytoplasm. On those slides stained, glutathione-S-transferase (placental; GSTP) was detected in almost all eosinophilic and mixed-cell foci, whereas approximately half of the basophilic and clear-cell foci stained for GSTP. The presence of GSTP in a focus was not always associated with the presence of increased TGF alpha protein. All rat hepatic adenomas and the one carcinoma labeled positive for TGF alpha. Increased levels of TGF alpha protein were associated with increased DNA synthesis labeling index. The number of TGF alpha-positive foci with the highest DNA synthesis labeling indices were statistically higher than those with lower levels of DNA synthesis labeling. Although characteristic staining patterns for TGF alpha were associated with specific histological subtype, the role that TGF alpha plays in the progression of focal lesions to neoplasia requires further definition. High levels of TGF alpha protein appear to be acquired sometime during the hepatocarcinogenic process. It may be that early lesions that acquire high levels of TGF alpha are the ones to develop into hepatocellular carcinoma (e.g., hepatocellular carcinoma is determined very early in the carcinogenic process). It is apparent that further work is needed to delineate the role of TGF alpha in both rodent and human hepatocarcinogenesis.     

Potent growth inhibitory activity of zidovudine on cultured human breast cancer cells and rat mammary tumors

Originally designed as an antitumor agent, zidovudine (AZT) has exhibited only marginal tumor growth inhibitory activity. Recently, three abstracts have described positive clinical outcomes for a small number of patients with advanced breast cancer treated with weekly infusions of either methotrexate or cisplatin and AZT. Consequently, we conducted a preclinical study of the anti-breast cancer and anti-mammary tumor activity of AZT. Here we have demonstrated that AZT, alone, has a preferential in vitro and in vivo effect on breast and mammary cancer cells. It is 1000 times as potent as an inhibitor of the in vitro growth of the human breast cancer cell line MCF-7 (IC50 = 10 +/- 5 nM) than of the growth of the T-cell leukemia cell line CEM (IC50 = 14 +/- 2 microM). A novel mechanism for this preferential effect on growth is indicated by the 3-4-fold increase in production of phosphorylated AZT (mono-, di-, and triphosphate) in MCF-7 relative to CEM. We extended these in vitro observations to in vivo studies in rats and found that AZT is a potent in vivo inhibitor of the growth of methylnitrosourea-induced rat mammary tumors without any apparent toxic effects on internal organs. These preclinical results demonstrate, for the first time, that AZT has significant anti-breast cancer activity and strongly suggest that the clinical usefulness of this drug is worthy of investigation.     

Alterations in the Ha-ras-1 and the p53 pathway genes in the progression of N-methyl-N-nitrosourea-induced rat mammary tumors

Glutamate is the most prominent excitatory neurotransmitter in the retina and brain. It has become clear that the physiology of many glial cells, including retinal Müller cells, is modified by a host of neurotransmitters, including glutamate. The experiments presented here demonstrate that Müller cells isolated from the tiger salamander retina have metabotropic glutamate receptors that, when activated, lead to the release of calcium ions (Ca2+) from intracellular stores. The Ca2+-sensitive fluorescent dye, Fura-2, and video imaging microscopy were used to monitor changes in cytosolic calcium ion concentration ([Ca2+]i) evoked by glutamate (30-50 microM), (1S,3R)-ACPD (50-200 microM), quisqualate (10-50 microM), and L-AP4 (5-100 microM). Bath application of each of these metabotropic receptor agonists in the absence of extracellular Ca2+ resulted in an increase in [Ca2+]i that often began in the distal end of the cell and occurred later in the endfoot. This wavelike increase in [Ca2+]i is reminiscent of the Ca2+ waves evoked in these cells by other Ca2+ releasing agents such as ryanodine and caffeine. Extracellular application ofATP also evoked increases in [Ca2+] in Müller cells. The presence on Müller cells of receptors for retinal neurotransmitters, such as glutamate and ATP, demonstrates that these glial cells can respond to changes in the retinal extracellular environment and hence neuronal activity. Since Müller cells span almost all layers of the retina, they are likely to be exposed to most retinal neurotransmitters. The Ca2+ waves evoked in Müller cells by neurotransmitters could represent a form of signaling from the outer retinal layers to the inner ones.     

Membrane glycoprotein changes in primary mammary tumors associated with autonomous growth

Primary and transplanted mammary tumors of the GR mouse were explanted in tissue culture and grown in the presence of radioactive fucose. Labelled membrane glycopeptides were isolated and compared by cochromatography with differentially labelled glycopeptides from normal mammary gland tissue. Differences with controls in the glycopeptide elution profiles were observed in autonomous, hormone-independent tumors but were absent in histologically similar tumors which required a continuous hormonal stimulus for growth. The results suggest that alterations in membrane glycopeptides are associated with the capacity of autonomous, hormone-independent growth of murine adenocarcinoma cells.     

Importance of progesterone in DNA synthesis of pregnancy-dependent mammary tumors in mice

Various purines were investigated with the absorption model, according to Stricker, their kD-values calculated and compared with one another. The diffusion speed constants showed to be dependent upon the lipoid solubility and the polarity of the drugs. The experimental methods recommended by Stricker were analyzed.     

Insulin does not promote rat mammary carcinogenesis

Indirect evidence from both epidemiological studies and animal experiments suggests that insulin may promote breast cancer development. In this study, we directly tested for a promoting effect of insulin on mammary carcinogenesis in Sprague-Dawley rats. Fifty day-old female rats received an i.p. injection of 37.5 mg/kg methylnitrosourea (MNU). Five days later, the animals were randomized into two groups. One group received insulin injections five times/week until the time of death, while the other control group received similar injections of normal saline. Over the course of 26 weeks following MNU treatment, the mammary tumour incidence in the insulin-treated group did not differ significantly from the saline-treated controls. Furthermore, the number of tumours per tumour-bearing rat did not differ between groups. Our results demonstrate that insulin is not a promoter of mammary carcinogenesis in this model.     

Computer-assisted image analysis of intratumoral vessel density in mammary tumors from dogs

OBJECTIVE: To determine whether intratumoral microvessel density can be used to distinguish benign from malignant mammary tumors in dogs and to predict the outcome of surgical treatment for small volume (< 3-cm diameter) tumors. SAMPLE POPULATION: Tissue sections from 58 mammary tumors (42 malignant and 16 benign) from dogs. PROCEDURE: Mammary tumors were stained by immunohistochemistry for factor VIII-related antigen. Computer-assisted image analysis was used to determine intratumoral vessel density in immunostained areas. Total vascular density (TVD), calculated from 3 non-overlapping fields, was analyzed for correlation with patient or tumor histomorphologic characteristics, and results obtained by surgical treatment of small volume tumors. RESULTS: Mean TVD of malignant tumors was significantly greater than that of benign tumors. Total vascular density was not correlated with patient age, sex, reproductive status, clinical tumor stage, or histologic type. For small volume (< 3-cm diameter) malignant tumors, mean TVD was higher in tumors that recurred after surgery than in tumors that did not recur; however, TVD was not predictive of time to recurrence. CONCLUSION AND CLINICAL IMPLICATIONS: Immunohistochemistry and computer-assisted image analysis allowed objective quantitation of intratumoral microvessel density in formalin-fixed, paraffin-embedded tissue sections. Tumors with high TVD were more likely to recur after surgical treatment than tumors with low TVD suggesting that TVD measurements can be used by the clinician, in addition to histologic type and clinical stage, to predict prognosis after surgical treatment. These data also provide rationale for use of antiangiogenesis strategies for treatment of malignant mammary tumors in dogs.     

Combined effect of cyclocytidine and lentinan of spontaneous mammary tumors in mice

Cytotoxic cytidine analog, cyclocytidine, and antitumor immunopotentiator, lentinan, offered an opportunity of testing the effect of combined modality of cytocidal agent and immunopotentiator on spontaneous mammary tumors of mice. Lentinan did not improve the therapeutic effect of cyclocytidine; it only prevented the early toxic death of mice due to cyclocytidine in one strain of mice but not in the other strain.     

Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-ras mammary carcinoma model in female (FVB/N x Mus musculus castaneus)F1 mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH ( approximately 38%) for markers on chromosome 4. A marked allelic bias was observed, with M. musculus castaneus alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-ras expression, and for which a significant functional difference may exist between the M. musculus castaneus and FVB/N alleles. Provisional subchromosomal localization of this gene, designated Loh-3, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.     

Telomerase activity in the normal and neoplastic rat mammary gland

The 1-methyl-1-nitrosourea-induced rat mammary tumor model system is well studied, reproducible, and widely used. We have investigated whether these tumors possess higher telomerase activity than normal mammary tissue. Using the telomeric repeat amplification protocol assay, we found significantly higher telomerase activity in 36 mammary carcinomas than in 72 mammary glands of virgin rats. The level of telomerase activity in virgin rats was unaffected by strain, age, stage of the estrous cycle, or ovariectomy. However, mammary glands obtained from pregnant rats exhibited telomerase activity comparable to that found in the tumors, possibly reflecting the high epithelial content of these tissues. Indeed, isolated epithelial cells from virgin and pregnant mammary glands and from carcinomas had similar telomerase activities. Thus, telomerase activity is constitutive in the rat mammary epithelium and is not a unique characteristic of malignant transformation in this tissue. These results underscore the importance of attributing biochemical properties to specific cell types in a tissue, a situation not paralleled in the interpretation of data from in vitro models.     

Inhibition of rat mammary tumorigenesis by dietary cholesterol

Mitochondrial trifunctional protein deficiency, a recently identified disorder of fatty-acid oxidation, may show characteristic features such as peripheral neuropathy, pigmentary retinopathy, and acute fatty liver degeneration in pregnant women with an affected fetus. We describe a patient with trifunctional protein deficiency whose clinical picture consisted of severe calcium and phosphate abnormalities caused by hypoparathyroidism.     

Neonatal modification of endocrine functions and mammary carcinogenesis in the rat

Effects of neonatal androgenization or neonatal ovariectomy in female rats on endocrine functions and mammary tumourigenesis are examined. Pituitary gonadotrophin contents (both LH and FSH) are significantly lower in neonatally androgenized rats (TT) and significantly increased in neonatally ovariectomized rats (NO) when compared with controls of the same age. Plasma and pituitary prolactin levels are higher in TT rats than in the control rats of the same age, but the difference is not significant. Mammary tumours developing in TT rats after DMBA treatment are predominantly fibroadenomata, and lactogenesis in TT rats occurs almost entirely in those receiving DMBA treatment. Neonatal ovariectomy in female rats protects against subsequent induction of mammary cnacer by DMBA. The relationship between neonatal modification of endocrine functions and mammary tumourigenesis is discussed.     

Interleukin-10 gene transfer inhibits murine mammary tumors and elevates nitric oxide

Transfection of cDNA for IL-10 into line 66.1 murine mammary tumor cells results in marked suppression of tumor growth and metastasis. Others have reported that nitric oxide has potent antitumor activity and IL-10 is known to regulate the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We identified nitric oxide production in mammary tumors as indicated by electron paramagnetic resonance detection of nitric oxide-hemoglobin (NO-Hb). IL-10 expression resulted in elevated levels of NO-Hb in mammary tumors. Immunohistochemical examination of mammary tumors for iNOS protein revealed few positively staining cells in parental or control neo-transfected tumors but strong iNOS staining in all IL-10 transfected tumors, consistent with the NO-Hb data. To determine if mammary epithelial tumor cells themselves, express nitric oxide synthase activity, cultured tumor cells were treated with pro-inflammatory cytokines and nitrite accumulation was assessed in the conditioned medium. All IL-10 producing cell lines accumulated uM concentrations of nitrite in response to short term (24 hr) cytokine stimulation. Cells not expressing IL-10 (parental and neo-transfectants) accumulated no nitrite under similar culture conditions. After longer stimulation (48 hr), parental and 66-neo cells accumulated lower amounts of nitrite. IL-10 gene transfer is associated with increased iNOS protein expression and enzymatic activity detected both in vitro and in vivo. Our findings suggest that the antimetastatic and antitumor activity of IL-10 is related to enhanced production of nitric oxide.