Neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to N-methyl-N-nitrosourea.

Abstract

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...

The stroma as a crucial target in rat mammary gland carcinogenesis

“…Miyamoto et al reported tumor formation after mammary epithelial cells were exposed to NMU in vitro and injected into cleared fat pads (Miyamoto et al, 1988). These authors used a cell inoculum one order of magnitude higher than the one we used and a different cell purification method.…”

“…Non-epithelial cells were successfully removed by treating the plates with a 0.025% trypsin and 0.01% EDTA solution. Five days before being transplanted into recipient animals, the mammary epithelial cells were treated with SFM containing either vehicle or 50 µg/ml NMU for 1 hour at 37°C (Miyamoto et al, 1988). The cells were then rinsed twice with SFM and fresh SFM was added.…”

“…A different batch of mammary epithelial cells prepared following this protocol was used for each of the four experiments. The dose of NMU used in the in vitro experiments was selected following Miyamoto et al (Miyamoto et al, 1988).…”

Catechol estrogens as biomarkers for mammary gland cancer

“…Another widely used experimental system to study mammary tumorigenesis is the model in which tumors were induced in the Fischer 344 rats by a single dose of N-nitrosomethylurea (NMU) (35,36). On the other hand, Miyamoto et al (37) observed neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to NMU. Furthermore, the highest number of tumors per animal induced by DMBA was observed when the carcinogen was given to animals when they were 40 to 46 days of age, a period when terminal end buds were most actively differentiating into alveolar buds (38).…”

Cell proliferation and tumor formation induced by eserine, an acetylcholinesterase inhibitor, in rat mammary gland

“…In vitro model systems have been extensively used to study the processes of initiation, progression and metastasis. Several authors have developed models to study these processes (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Most of them give a unique opportunity for studying mammary gland changes that can occur by the effect of different exogenous and endogenous substances.…”