In conjunction with our studies on mammary tumorigenesis in feral and inbred mice, our efforts have focused upon understanding the cellular basis of malignant progression in the mammary gland. Taking the point of view that mammary carcinomas arise as clonal populations of transformed tissue-specific stem cells and their differentiating progeny, we have initiated a long-term project aimed at elucidating the cellular, molecular, and genetic events underlying mammary epithelial cell growth, regeneration, and functional development. The reproductive capacity of the mammary epithelial stem cell is reduced coincident with the number of symmetric divisions it must perform. In a study of FVB/N mice with the transgene, WAP-TGFb1, we discovered that mammary epithelial stem cells were prematurely aged due to ectopic expression of TGF-b1. To test whether premature aging of mammary epithelial stem cells would have an impact on susceptibility or resistance to mammary cancer, female littermates from FVB/N X WAP-TGF-b1 mating were injected with mouse mammary tumor virus (MMTV) at 8-10 weeks of age. A total of 44 females were inoculated, maintained as breeders and observed for tumor development for up to 18 months. Only one mammary tumor appeared in 17 TGF-b1 females while 15 were collected from 29 wild type sisters. Premalignant mammary epithelial cells in infected glands were identified by transplantation of single cell (1x105) suspensions into nulliparous hosts and testing for hyperplastic outgrowth. Although the number of positive takes was significantly reduced with TGF-b1 cells, both MMTV-infected TGF-b1 and wild type cells produced hyperplastic outgrowths suggesting that premalignant transformation was achieved in each group. The results suggest a positive correlation between the procreative life-span of mammary epithelial stem cells and mammary cancer risk. An in vivo transplantation system was used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically, mouse mammary epithelial cells with distinctly limited developmental potentials were identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. Mammary gland biologists have long assumed that differentiated secretory epithelial cells undergo programmed cell death at the end of lactation and that the alveolar compartment is reconstituted from undifferentiated precursor cells in subsequent pregnancies. It is generally agreed that the remodeled gland in a parous animal resembles that of a mature virgin on the morphological level. However, a number of physiological differences have been noted in comparing the responses of mammary epithelia from nulliparous versus parous females to hormonal stimulation and carcinogenic agents. Here, we present genetic evidence that an involuted mammary gland is fundamentally different from a virgin gland despite its close resemblance in the morphology. This difference results from the formation of a new mammary epithelial cell population that originates from differentiating cells during pregnancy. In contrast to the majority of fully committed alveolar cells, this epithelial population does not undergo cell death during involution and remodeling following a lactation period. We show that these cells can function as alveolar progenitors in subsequent pregnancies, and they can play an important role in functional adaptation in genetically engineered mice, which exhibit a reversion of a lactation-deficient phenotype in multiparous animals. In transplantation studies, this parity-induced epithelial population shows the capacity for self-renewal and contributes significantly to the reconstitution of the resulting mammary outgrowth (i.e. ductal morphogenesis and lobulogenesis). We propose that this parity-induced population contributes importantly to the biological differences between the mammary glands of parous and nulliparous females.
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