SUMMARY: Mammary glands are enzymatically dissociated and the resulting tissue digest enriched for epithelial cells by isopycnic banding on a density gradient of Percoll. The cells are embedded within a rat tail collagen gel matrix and fed with the appropriate medium. Growth and differentiation are superior in such a system when compared to culture on plastic, using identical media.

The examination of rectoanal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens for the diagnosis of transmissible spongiform encephalopathies has been described in sheep, elk, and small numbers of mule and white-tailed deer. Previous sample numbers have been too small to validate examination of this type of tissue as a viable antemortem diagnostic test. In this study, we examined RAMALT collected postmortem from 76 white-tailed deer removed from a farm in Wisconsin known to be affected by chronic wasting disease (CWD) and from 210 free-ranging white-tailed deer harvested from an area in Wisconsin where the overall prevalence of CWD among the deer was approximately 4 to 6%. The results of immunohistochemical (IHC) staining of the RAMALT sections were compared to the results of IHC staining of sections from the brain stem at the convergence of the dorsal motor nucleus of the vagus nerve, sections of the medial retropharyngeal lymph nodes (RLNs), and sections of tonsil (sections of tonsil only from captive animals were tested). The sensitivities of the IHC staining test with RAMALT sections were 81% for the captive animals and 91% for the free-ranging animals. False-negative results were usually associated with early infection, indicated by a low intensity of immunostaining in the obex and/or a polymorphism at PRNP codon 96. While the RLN remains the tissue of choice for use for the diagnosis of CWD in white-tailed deer, the results of the present study further support the use of RAMALTs collected antemortem as an adjunct to testing of tonsil biopsy specimens and surveillance by necropsy for the screening of farmed deer which have been put at risk through environmental exposure or exposure to deer with CWD.Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that has been reported in free-ranging and captive Rocky Mountain elk (Cervus elaphus nelsoni), mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and moose (Alces alces shirasi) in North America (1,23,27,28). Clinical signs of CWD do not appear until very late in the infection process (11), and as the successful management of CWD appears to be dependent on the early detection and elimination of infected individuals (8), a reliable diagnostic test for the identification of infected animals during the early preclinical stage is of paramount importance. Currently, diagnosis is generally dependent on the finding of PrP CWD , the abnormal isoform of the cellular prion protein (PrP C ), in obex and/or lymphoid tissues of animals collected postmortem. Antemortem evaluation of tonsil biopsy specimens has been shown to detect preclinical CWD in deer (17,26,29), but the collection of tonsil tissue is not a universally practical procedure for use in the field, in that it requires general anesthesia and highly trained personnel to collect adequate samples. On the basis of the findings of recent studies that have shown that scrapie-associated PrP (PrP Sc ) is deposited in the rectoanal mucosa-associated lymphoid tis...

Mouse mammary epithelial cells can be transformed in primary cultures to preneoplastic and neoplastic states when treated with N-methyl-N-nitrosourea (MNU). Mammary carcinomas arising from MNU-induced hyperplastic alveolar nodules (a type of mouse mammary preneoplastic lesion) contained transforming c-Ki-ras genes when examined by the NIH 3T3 focus assay. Hybridization of allele-specific oligonucleotides to c-Ki-ras sequences amplified by the polymerase chain reaction demonstrated the presence of a specific G-35--->A-35 point mutation in codon 12 in each of the NIH 3T3 foci as well as the mammary carcinomas. This mutation resulted in the substitution of the normal glycine with an aspartic acid. Furthermore, this mutation in the c-Ki-ras proto-oncogenes was also detected in 9 of 10 hyperplastic alveolar nodules. These results demonstrate that the specific c-Ki-ras mutation is a preneoplastic event in MNU-induced mouse mammary carcinogenesis.Cancer is recognized as a disease arising from a perturbation of gene function. Over 30 different cellular genes, called proto-oncogenes, have been investigated intensely because of their involvement in tumorigenesis. Of the proto-oncogenes identified in human and animal tumors, members of the ras gene family, c-Ha-ras, c-Ki-ras, and N-ras, are by far the most frequent (1, 2, 19). The ras genes are commonly activated by single point mutations in codons 12, 13, and 61 (1, 2). In rodent tumors induced by chemical or physical carcinogens, the nature of the mutational activation of ras proto-oncogenes has shown a remarkable correlation with the known mutagenic properties of the causative agents (1). For example, mammary tumors in rats induced by Nmethyl-N-nitrosourea (MNU) contain c-Ha-ras proto-oncogenes activated by G--A transitions (26,29). Mouse hepatic neoplasms induced by a variety of carcinogens contain c-Ha-ras proto-oncogenes activated by an alteration corresponding to the mutational specificity of each carcinogen (27). Therefore, the identification of the critical gene mutations directly involved in cancer is becoming feasible by use of animal model systems.We have recently developed a transformation system in which mouse mammary epithelial cells in serum-free primary collagen gel cultures are transformed by treatment with chemical carcinogens (11,18). Cells treated with carcinogens form mammary lesions when transplanted into a cleared mammary fat pad, an anatomically natural transplantation site from which mammary epithelial cells have been removed surgically (6). This in vitro-in vivo system facilitates a stepwise study of the transformation process at the molecular level as the transformants progress through morphologically well-defined preneoplastic stages (11,17,18). Furthermore, the molecular effects upon transformation by various factors that control growth and differentiation of mammary cells can be directly studied under well-defined conditions. In the present studies, in vitro MNU-induced mouse mammary preneoplasias and neoplasias were analyzed for the * Corres...

High-efficiency neoplastic transformation of mouse mammary epithelial cells in primary collagen gel culture was induced by N-methyl-N-nitrosourea (MNU). Mammary epithelial cells, isolated from virgin BALB/c mice, were embedded within collagen gels and grown in a serum-free medium containing prolactin, progesterone, and linoleic acid.The cells were then treated with MNU on day 3 of culture and subsequently at weekly intervals for up to 4 weeks. Eleven to 14 days after the final carcinogen treatment, the cells were removed from the collagen gels and i jected into the cleared mammary fat pads of syngeneic hosts to assay for transformed cell populations. A single exposure or multiple exposures of these cells to MNU was effective in inducing tumorigenic cells that produced palpable tumors as early as 6 weeks after transplantation. Two treatments with MNU (100 ,ug/ml) were optimal for neoplastic transformation and produced tumors in 79% of the injected fat pads. All the tumors originated at the site of injection and had extensive central necroses. Histological examination indicated that the tumors were mammary carcinomas. Secondary transplantation of tumor pieces into intact mammary glands produced palpable carcinomas of the same histology within 1-S weeks. Control cells cultured for the same periods of time as MNU-treated cells produced only ductal outgrowths that were morphologically similar to those found in the mammary glands of adult virgin hosts. This system provides a distinct means to study the mechanism of mammary neoplastic transformation at cellular and molecular levels.Mammary neoplasias induced by chemical carcinogens have been studied extensively in animal model systems. These in vivo studies have provided invaluable information about effective carcinogens, susceptible strains, the sensitive age groups, hormonal and dietary influences, and the presence of preneoplastic stages in mammary tumor development (1-3). It has been difficult, however, to directly study the mechanisms underlying the neoplastic transformation of mammary cells because of inherent complexities present in vivo.In vitro transformation systems provide a distinct opportunity to directly study the mechanisms of transformation under well-controlled conditions because (i) mammary epithelial cells with or without their accompanying stroma can be treated with carcinogens, (ii) the direct effects of different hormones and growth factors on transformation can be studied, (iii) the molecular effects of carcinogens can be examined, and (iv) the cellular origin of various mammary tumors observed in vivo can be elucidated. Many attempts have been made to develop a suitable system to transform mammary cells in cell and organ cultures (4-10). In previous studies transformed mammary cells were identified by enhanced growth potential (4-6) or anchorage independence (7) in cell culture, induction of alveolar lesions (8, 9) in organ culture, and induction of preneoplastic lesions or carcinomas (9, 10) in animal hosts. Unfortunately, these systems...

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