Susceptibility of the mammary gland to carcinogenesis. III. The cell of origin of rat mammary carcinoma.

The rat mammary gland epithelium is composed of three cell types: dark cells (DCs), intermediate cells (ICs), and a layer of myoepithelial cells (MCs), which are evenly distributed along the mammary gland tree in rather constant proportions. The present study was carried out for a determination of the effect of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) on the distribution and proliferative activity of these cell populations. The proportion and the DNA labeling index (DNA-LI) of each cell type were determined in terminal end buds (TEBs), terminal ducts (TDs), alveolar buds (ABs), and alveoli of the normal Sprague-Dawley rat mammary gland and in intraductal proliferations (IDPs) and carcinomas removed at selected intervals after DMBA administration. DMBA-induced changes in cell distribution were limited to TEBs and TDs, whereas ABs and alveoli were unaffected. The alterations consisted in an increment in ICs from 11% in TEBs and TDs to 90% in tumors and a decrease in DCs from 77% in TEBs and TDs to 7% in tumors. MCs were relatively unaffected. The DNA-LI of DCs, which in the normal gland TEB was 14%, was depressed by DMBA to 6%, whereas the DNA-LI of ICs remained unchanged from the basal level of 40% during the process of carcinogenesis. The progressive increment in number of ICs with a steady DNA-LI suggested that the IC is the target cell of the carcinogen and the cell of origin of mammary carcinomas.     

Stem cells in mammary development and carcinogenesis

Recently, substantial progress has been made in the identification and characterization of stem and progenitor cells in the mouse and human mammary gland. Furthermore, there is increasing evidence that a variety of neoplasms, including breast cancer, may result from transformation of normal stem and progenitor cells. Consistent with this model of carcinogenesis, a breast cancer stem cell population, with the phenotype CD24-CD44+ lineage, was recently identified utilizing flow-cytometry based cell sorting and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografts. As few as 200 cells of this cancer stem cell population were capable of generating tumors in animals, whereas the bulk of the tumor population was tumorigenic only when implanted in high numbers. Like their normal counterparts, the cancer stem cells have the ability to self-renew, driving tumorigenicity and possibly recurrence and metastasis, and have the ability to differentiate, generating the heterogeneity of the tumors. This stem cell model of carcinogenesis has important implications for understanding the basic biology of breast cancer, as well as other cancers. Furthermore, the concept of cancer as a disease of stem and progenitor cells has profound implications for the development of new strategies for cancer prevention and therapy.     

Characterization of N-ethyl-N-nitrosourea--induced mammary tumors in the rat.

A single intraperitoneal inoculation of 180 mg/kg N-ethyl-N-nitrosourea (ENU) into 30-day-old outbred Sprague-Dawley (CD) rats resulted in an 90% incidence of mammary tumors (MTs) after an average latent period of 93 days, with an 85% rate of malignancy. The incidence, induction period, number of tumors per rats, and the rate of malignancy were reduced in rats ovariectomized prior to or shortly after (5 days) ENU exposure. Over 70% of the ENU-induced MTs regressed following ovarioectomy. The malignant MTs developed from nodules located in the terminal end buds (TEB), which are believed to contain the most susceptible cell population for chemical carcinogens. The serum calcium was elevated above control values in 92% of intact rats evaluated with MTs in the absence of bone metastases. No correlation was demonstrated between the volume of MTs or total number of MTs and the serum calcium level. The ENU-induced MT is a reproducible animal model that can be used in the investigation of the early changes in mammary cells (especially TEB) and the interrelationships between the carcinogen and ovarian hormones associated with neoplastic transformation.     

A proposed stem cell research policy

The aspirations of scientists and patients for human embryonic stem cell (hESC) research in the U.S. motivate attention to the nitty-gritty of law and regulation and its confluence with such moral consensus as lies within our reach. Federal law and regulation form a tangle. Analysis yields several conclusions not widely appreciated. A legislative enactment is the rate-limiting step of federally funded research, the restriction of research imposed by the previous administration's policy as reprised in current proposals fails to achieve its objective of avoiding complicity in embryo sacrifice, the current administration's policy is another failed noncomplicity scheme under which research cannot be expanded without demolishing its putative justification, and the Food and Drug Administration has already effectively interdicted procreative cloning. While it is not plausible to deny complicity in embryo sacrifice when performing or funding hESC research, one can justify sacrifice of some embryos by an argument whose premises are consistent with a wide range of moral and religious views. This paper proposes a rule of public policy providing for the use of donated embryos barred from the womb. This rule would optimize research while manifesting its moral justification. The rule is suitable for implementation by any government that funds hESC research. The rule's justification provides a cogent argument for such incremental steps toward its implementation as become politically feasible from time to time.     

Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most prevalent carcinogenic heterocyclic amines in the environment, targeting the colon, prostate, pancreas, and mammary gland in rodents. Chemopreventive effects of synthetic and naturally occurring compounds on PhIP-induced rat mammary carcinogenesis were investigated in a series of experiments. In a PhIP feeding model, groups of 20-21 female F344 rats each, were treated with 0.02% PhIP alone or PhIP plus 0.5% 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), 1% green tea catechins, 1% alpha-tocopherol, 0.1% ellagic acid, or 1% chlorophyllin, each in the diet, or 0.1% caffeine in drinking water for 52 weeks. To assess the mechanism of HTHQ and caffeine inhibition of PhIP-induced carcinogenesis, effects of these compound on the in vitro metabolic activation of PhIP were examined in the presence of S9 mix. In the next series of experiments, the PhIP intragastric dose model was applied to allow separate investigation of the effects of chemicals during the initiation and postinitiation periods. In these experiments, female Sprague-Dawley rats were given eight intragastric doses of 100 mg/kg body weight during the first 4-8 weeks for initiation. Either during initiation or after initiation, or only after initiation, animals were treated with either corn or perilla oil at doses of 5 and 20%, conjugated fatty acid derived from safflower oil (CFA-S) or perilla oil (CFA-P) at a dose of 1%, arctiin at doses of 0.02 and 0.2% in the diet, or sodium nitrite (NaNO(2)) at a dose of 0.2% in drinking water. In the PhIP feeding model, administration of PhIP alone for 52 weeks induced adenocarcinomas in 40% of rats, but the incidence was remarkably reduced to 5% by the simultaneous treatment with 0.5% HTHQ, a strong lipophilic phenolic antioxidant, or to 10% by 0.1% caffeine. Administration of 1% chlorophyllin exerted similar, albeit weaker, effects. alpha-Tocopherol at a dose of 0.5% only reduced the multiplicity of carcinomas, and 1% green tea catechins only the mean size of mammary tumors. In a metabolic activation study of PhIP, HTHQ and caffeine clearly inhibited the formation of metabolites. In the PhIP gastric dose model, among the naturally occurring compounds examined, a plant lignan arctiin, perilla oil, which contains a large amount of n-6 alpha-linolenic acid, and CFA-S or CFA-P inhibited mammary tumor development, particularly in the postinitiation period, although a clear dose response was not observed. Treatment with 0.2% NaNO(2) in the initiation period was found to lower the volume of mammary tumors. The present results indicate that a number of compounds may be candidate chemopreventive agents against PhIP-induced mammary carcinogenesis, acting through different mechanisms and depending on the stage of carcinogenesis.     

Endocrinologic milieu and susceptibility of the rat mammary gland to carcinogenesis.

The incidence of DMBA-induced mammary carcinomas in Sprague-Dawley rats depends upon their previous reproductive histories. Young virgin rats (YV) are highly susceptible to the carcinogen, while old virgin rats (OV) are less susceptible, and parous rats (P) are resistant. The authors performed endocrinologic studies in these three groups of rats in order to determine whether the different susceptibility to carcinogenesis, according to the reproductive history, is or is not related to the hormonal milieu. The pituitary, the ovaries, the adrenals, and the mammary glands were processed for light microscopy. Pituitary prolactin (PRL), follicle-stimulating hormone, and luteinizing hormone cells were immunostained by peroxidase-antiperoxidase and quantitated with an image analyzer. Radioimmunoassays of serum and pituitary PRL and serum estradiol were also done. The results showed no differences in the hormonal milieu of YV, OV, and P rats at the time of carcinogen treatment. Several changes were observed after DMBA administration, the most conspicuous being 1) hyperplasia of pituitary PRL cells, 2) high serum PRL levels, 3) nodular hyperplasia of the adrenal cortex, 4) high serum estradiol levels, and 5) lack of adrenal necrosis in P rats and some OV rats. These modifications did not correlate with the degree of susceptibility of YV, OV, and P rats to carcinogenesis, supporting the concept of the importance of the mammary gland differentiation at the moment of carcinogen administration. (Am J Pathol 1982, 109:47-56).     

A recurring pattern of chromosomal aberrations in mammary gland tumors of MMTV-cmyc transgenic mice.

Mice carrying the MMTV-cmyc transgene develop mammary tumors at 9 to 12 months of age. Little is known about karyotypic changes in this model of human breast cancer. We have developed and applied molecular cytogenetic techniques to study chromosomal aberrations that occur in these tumors, namely, comparative genomic hybridization and spectral karyotyping. Cell lines from eight tumors were established and analyzed, four of which carried a heterozygous p53 mutation. All of the tumor cell lines revealed increases in ploidy and/or multiple numerical and structural chromosomal aberrations. No consistent differences were observed between cmyc/p53+/+ and cmyc/p53+/- tumors, suggesting that cmyc induces karyotype instability independent of p53 status. Loss of whole chromosome (Chr) 4 was detected in five of the eight tumors. Parts of Chr 4 are syntenic to human 1p31-p36, a region that is also deleted in human breast carcinomas. Four tumors carried translocations involving the distal portion of Chr 11 (syntenic to human chromosome arm 17q), including two translocations T(X;11), with cytogenetically identical breakpoints. We compare the pattern of chromosomal aberrations with human breast cancers, find similarities in several syntenic regions, and discuss the potential of an interspecies cytogenetic map of chromosomal gains and losses.     

Solid and mucinous varieties of so-called mammary carcinoid tumors.

Three neoplasms that had histologic features reminiscent of carcinoid tumors of other sites were encountered in a review of 3,300 examples of invasive mammary cancer in women (.09%). One of these showed cytoplasmic argyrophilia. This, as well as the two putative carcinoids, lacked argentaffinity. Attention is directed to the occurence of variable numbers of argyrophilic cells in eight of 19 so-called mucinous cancers of the breast studied. Further, neurosecretory-type granules were observed in cells of all four mucinous cancers suitably prepared for electron microscopic examination. The possible reasons for the lack of universal argyrophilic reactions in these lesions is discussed. It is concluded that there may be two types of mammary carcinoid tumors, the solid and mucinous varieties. No patient who had the latter type had experienced treatment failure after five years of observation. Various numbers of ductal epithelial cells in four of 45 examples of banal fibrocystic disease showed cytoplasmic argyrophilia, and neurosecretory-type granules were found in two of eight examples suitably prepared for electron microscopic examination. Whether this demonstration establishes the existence of precursor elements for the development of the carcinoid tumors is at present uncertain.     

Tumour angiogenesis and p53 protein expression in mammary phyllodes tumors.

We examined 186 phyllodes tumors (106 benign, 51 borderline, 29 malignant) for angiogenesis by assessing stromal microvessel density by the hot spot method and assessing p53 protein expression; we correlated these factors with stromal cellularity, margin status, nuclear pleomorphism, mitosis, and stromal overgrowth. Increased degree of malignancy in phyllodes tumors is associated with increased patient age and tumor size. Microvessel density and p53 protein expression also showed a similar increase with malignancy. Using a logistic regression model, microvessel density was shown to be useful in predicting malignancy in phyllodes tumors, independent of key criteria of stromal overgrowth, nuclear pleomorphism, and mitosis. Microvessel density showed correlation with stromal cellularity and margin status, suggesting an interrelationship between these parameters. P53 protein expression showed a positive correlation with microvessel density, suggesting possible overlap in the underlying mechanism of these two factors in the pathogenesis of phyllodes tumors. The numbers of recurrences and metastases are small in our series, and no significant difference was demonstrated in microvessel density and p53 protein expression compared with the primary. We conclude that microvessel density and p53 are useful as independent criteria in evaluating malignancy in phyllodes tumors.     

Molecular analysis of rat mammary carcinogenesis: an approach from carcinogenesis research to cancer prevention

A rat strain carrying the human c-Ha-ras proto-oncogene is highly susceptible to chemically induced mammary carcinogenesis. All the transgenic rats develop preneoplastic mammary lesions within 20 days of an injection of N-methyl-N-nitrosourea, and mammary carcinomas appear within 8 weeks of treatment with a variety of chemical carcinogens. In this review, we summarize molecular aspects of mammary carcinogenesis in transgenic rats and the potential application of this model for studies of breast cancer prevention.     

Some factors affecting selective adherence of microorganisms in the bovine mammary gland.

Some parameters affecting the adherence of microbes to the ductular epithelium of the bovine mammary gland were studied. Adherence increased from teat sinus to lactiferous sinus to the large ducts, and cells from the lactiferous sinus to the large ducts, and cells from the lactiferous sinus were used for all other experiments. There was no difference in adherence to cells from different quarters of the same cow, but there were significant differences between cows. Scanning electron microscopy suggested that the cells of the ductular epithelium undergo dynamic changes that probably result in secretion and/or desquamation. Adherence to cells could be demonstrated only at a late stage of these changes. The adherence of organisms associated with mastitis was studied using an in vitro test. Adherence generally paralleled prevalence as cause of disease, with Staphylococcus aureus and Streptococcus agalactiae adhering best. Strain variation suggested that virulence was related to adherence with S. agalactiae and S. dysgalactiae but not with S. aureus. It is proposed that specific adherence is an important aspect of pathogenesis of mastitis due to S. aureus and S. agalactiae.     

Role of vitamin a in chemical carcinogenesis of the mammary gland

The effect of feeding rats with large doses of vitamin A on the concentration of the polycyclic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) and its metabolites in various organs and in the blood and also on the rate of metabolism in the liver of rats after intravenous injection of the carcinogen were studied. In hypervitaminosis A the quantity of DMBA and its metabolites was found to be considerably reduced in all the organs tested and in the blood. The rate of DMBA metabolism in the liver of the animals increased with an increase in the dose of vitamin A.Deceased.Department of Biophysics, Biological Faculty, Moscow University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 273–275, March, 1979.     

Viral carcinogenesis in a pronephric cell line. An ultrastructural study.

Herpesvirus recovered from cell fractions of the spontaneous Lucké renal tumor of adult Rana pipiens were used to infect a cell line derived from pronephroi of the same species. Viruses and virus-associated structures previously found in the primary renal tumor were observed, including nuclear inclusions of capsids with single or double membranes and capsids with nucleoids often within nuclear sacs. Embedded within the clumped and marginated chromatin were 55-nm tubular elements and associated unit membrane structures. Virus-associated, 35-nm tubular elements were also seen. The cytoplasm contained single, enveloped nucleoid virus and clusters of virus within cytoplasmic vesicles. Other cytoplasmic inclusions were dense, virus-associated, 25-nm filaments, virus particles within myeloid bodies, and possible viral budding from tubular organelles.     

Vascular tumors of the mammary gland

Summary A total of five haemangiosarcomata and two benign haemangiomas arising in the mammary gland have been studied electron microscopically and by histochemical techniques. Malignant tumors were mainly composed of endothelial cells reactive to alkaline phosphatase and adenosine triphosphatase, and of pericytes and undifferentiated mesenchymal elements. A juvenile haemangioma showed a more structured wall with an increase of endoplasmic reticulum and filaments, and a diminution of membrane modulations and rod-like tubular bodies. A cavernous haemangioma showed an ultrastructure very similar to normal vessels.The ultrastructural and histochemical data suggest a blood vessel origin of mammary angiosarcomas and show that vascular neoplasms of the breast, benign or malignant, are composed of a combined proliferation of the different cell types present in the vessel wall, as described in other organs.Juvenile haemangioma and cavernous haemangioma show a more complex structure in their walls with stratification of the different types of cells and a continuous wall. The features of endothelium in juvenile haemangioma suggest a low functional activity rather than morphological de-differentiation.