Charles M. McGrath

Mammary Neoplasia in Mice

Publisher Summary This chapter summarizes briefly the past literature on the studies of spontaneous mouse mammary cancer and discusses in detail the discoveries that have been made only recently. Mammary tumor studies on mice began with the first description of such tumors in a wild mouse in the year 1854. This is an area of tumor biology that was studied first by pathologists, was nurtured by geneticists and endocrinologists, and is only lately being pursued vigorously by investigators from other disciplines such as virology, immunology, biochemistry, molecular biology. Mouse mammary tumors offer many advantages for tumor biological studies. The chapter also talks about mammary tumor virus (MTV), their hormonal influences on mouse mammary neoplasia, and the two categories of inbred strains of mice employed in mammary cancer research—susceptible strains and resistant strains. The mouse mammary tumor system still remains an area of interest to researchers from different disciplines. One of the great advantages of this system remains the amenability of the host animals to laboratory experimentation.

Estrogen responsive proliferation of clonal human breast carcinoma cells in athymic mice

A clone of MCF-7 (MCF-7ED), a cell in continuous in vitro cultivation derived from an estrogen-responsive human breast carcinoma, requires estrogen supplementation for progressive exponential (double time: 50--85 h) tumor growth in the mammary fat of athymic mice. The plasma concentration of estradiol which stimulated exponential growth of MCF-7ED corresponded to physiologic premenopausal levels in women. The tumors were carcinomas with murine and MCF-7ED cells intermixed. MCF-7ED cells could be repurified in subcultures of mixed tumors. Comparative studies of breast and non-breast cell lines showed concordance between the presence of estradiol receptor, sensitivity to the anti-estrogen tamoxifen for growth in vitro, and estradiol dependence for tumorigenic growth in athymic mice. Progesterone alone did not stimulate MCF-7ED growth, but acted synergistically with estrogen. Progesterones action was to decrease tumor latent period, not to increase final tumor incidence or growth rate. Under estrogen-deficient conditions, condsitions approximating postmenopausal status in women, (10(-10) M in plasma), a dormant state was established between MCF-7ED cells and murine mammary stroma which could be maintained several months. The dormant state could be broken by introduction of estradiol, but not progesterone. This system should be useful for defining host and cancer cell determinants in estrogen-responsive breast cancer growth.

A simplified method for passage and long-term growth of human mammary epithelial cells.

SummaryA method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue are plated in medium containing 1.05 mM Ca++ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca++ to overcome “renewal inhibition” and to stimulate growth. In low Ca++ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca++, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca++, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of (linear) growth. Cells released from monolayer were greater than 90% viable and yielded 105 cells/cm2 of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin for dissociation. Long-term free-floating cells were typical mammary epithelium: (a) they formed domes and exhibited renewal inhibition, (b) they produced ductlike formations in collagen gels, (c) they contained epithelium-specific keratin filaments, and (d) they were diploid.

Calcium regulation of normal human mammary epithelial cell growth in culture

SummaryThe concentration of Ca++ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca++ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca++ to less than 0.08 mM.The reduction of Ca++ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca++ on growth and differentiation were specific (Mg++ and Mn++ variations were without effect) and reversible and in many respects resembled Ca++ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca++-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca++ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu.

The etiology of breast cancer.

A dominant focus of the research programs at our own and other laboratories remains the identification and characterization of the populations at high risk to breast cancer. The goal: the early detection of the disease and, ultimately, its biological control.

A common mouse mammary tumor virus integration site in chemically induced precancerous mammary hyperplasias

Mammary carcinomas can be induced by chemical and hormonal as well as viral carcinogens. Irrespective of the class of inducer, these tumors develop in discrete stages, of which alveolar hyperplasia is one of the earliest identifiable. Since carcinogenesis by the mammary tumor virus is now thought to involve proviral activation of adjacent cell genes at specific loci, we sought to determine if a similar mechanism also played a role in chemical and hormonal carcinogenesis and if its role was stage specific. Three high-tumor-incidence BALB/c hyperplastic alveolar nodule outgrowths of two different etiologies were found to have exogenous mouse mammary tumor virus proviruses integrated at the same site in the genome. This common site of integration is not within the bounds of the int-1 and int-2 loci into which proviruses detected at these loci are clustered in MMTV-induced mammary tumors. All three HANs are commonly impaired in end-point differentiation. We propose that mouse mammary tumor virus integration at this site is responsible for a specific abnormality in differentiation associated with the preneoplastic phenotype.

Estrogen induced growth of human breast cancer cells (MCF-7) in athymic nude mice is enhanced by secretions from a transplantable pituitary tumor

MCF-7, a human breast carcinoma cell line, was maintained s.c. in female athymic nude mice for a period of 5-6 weeks. Administration of estrogen (s.c. pellet of 17 beta-estradiol and estrone in drinking water, 0.5 mg/l) to these mice resulted in sustained (P less than 0.001) growth of MCF-7 tumors. Grafting of a prolactin and growth hormone secreting rat pituitary tumor to the estrogen-treated mice resulted in an increased (P less than 0.05) rate of MCF-7 tumor growth. MCF-7 did not grow in athymic nude mice grafted with rat pituitary tumor alone or in mice without hormone treatment (controls). Thus, secretions of pituitary hormones alone are not capable of promoting in vivo growth of MCF-7 although such secretions significantly enhance estrogen-induced growth of this cell line. A synergism between pituitary hormones and estrogen for in vivo growth of a human breast carcinoma has been demonstrated in this study.

Mouse mammary tumor virus DNA sequences in tumorigenic and nontumorigenic cells from a mammary adenocarcinoma.

Abstract If BALB/c mice are foster nursed on C311 mice, which contain milk-borne mouse mammary tumor virus (MMTV) associated with a high mammary tumor incidence, their tumor incidence rises from

Changes in casein sequence abundance in DNA of mouse mammary tumors

Abstract The casein gene content of mouse mammary tumor virus-induced mammary tumors was found to be elevated, apparently in proportion to the increase in MTV proviral copy number. Conversely, mammotropic hormone-induced mammary tumor DNA was deficient in casein genes in 2 of 3 tumors. Globin gene abundance, however, in both normal and malignant mammary tissues was unchanged, regardless of changes in casein gene content. These results suggest that perturbations have occurred in a common region of DNA from mammary tumors with 2 different etiologies.