Cellular proliferation and differentiation are well coordinated biological processes, that are mutually exclusive. It is, however, unclear how the decline in one is causally related to augmentation of the other. The need to gain critical insights regarding these mechanisms is borne out by the fact that an uncoupling of this otherwise interdependent process is an obligatory step in the generation of the transformed phenotype i.e., tumors arise because of the failure of normal cells to achieve differentiation. The goal of this proposal is to define the mechanisms responsible for decreased cell proliferation following the onset of mammary differentiation by studying the regulation and role of progesterone receptor (PgR) during mammary development. We chose PgR as a model gene because (a) it is developmentally regulated such that once the mammary epithelial cell achieves full functional differentiation during lactation there is no detectable levels PgR, and (b) as a receptor for progesterone, it has the potential to mediate progesterone action, which is to promote, mammary epithelial growth and differentiation.
The specific aims are: I) To examine the molecular mechanisms responsible for estrogen (E2) dependent PgR gene expression in undifferentiated mammary glands and those responsible for impeding its expression during lactation and (2) to examine the role of PgR in mediating mammary growth and differentiation. To achieve specific aim 1, we plan to (a) identify the DNA elements on the murine PgR gene responsible for its positive and negative regulation by testing the various DNA sequences in transfection assays (b) test the functional role of these elements in developmental regulation by nuclear micro-injection into mammary epithelial cells and subsequently cultured in the presence or absence of lactogenic hormones and (c) study the interaction of estrogen receptors and other cellular factors from E2- sensitive (non-lactating) and insensitive (lactating) mammary glands with the various DNA regulatory elements using in vivo foot printing and gel retardation assays. Normal mammary epithelial growth and differentiation, in addition to hormones, involves a complex interplay between the epithelium and its surrounding connective and adipose tissue (referred to as the fat pad). Also, mammary PgR exists in two molecular forms, the 'A' and 'B' forms which are known to differentially regulate progesterone responsive promoters. As such to understand the role of PgR in mammry growth and differentiation, (specific aim #2) we have to examine the effects of the two receptor forms on the two tissue types alone and in combination which is best served by a transgenic system.. Therefore, we propose to (a) construct transgenic mice expressing either 'A' or 'B' forms of PgR, and examine mammary glands of these mice by various morphological and functional criteria and (b) transplant mammary epithelial cells of these transgenic mice into heterologous fat pads and subsequently examine the chimeric tissues for their potential for proliferation and differentiation. We anticipate that these studies will provide critical insights into the mechanisms responsible for the interdependent processes of mammary proliferation and differentiation and hence its derangement in cancer.
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