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The susceptibility of the mammary gland to carcinogenesis is influenced by its normal development, particularly during developmental stages such as puberty and pregnancy that are characterized by marked changes in proliferation and differentiation. Protein kinases are important regulators of proliferation and differentiation, as well as of neoplastic transformation, in a wide array of tissues, including the breast. Using a RT-PCR-based cloning strategy, we have identified 41 protein kinases that are expressed in breast cancer cell lines and in the murine mammary gland during development. The expression of each of these kinases was analyzed throughout postnatal mammary gland development as well as in a panel of mammary epithelial cell lines derived from distinct transgenic models of breast cancer. Although the majority of protein kinases isolated in this screen have no currently recognized role in mammary development, most kinases examined were found to exhibit developmental regulation. After kinases were clustered on the basis of similarities in their temporal expression profiles during mammary development, multiple distinct patterns of expression were observed. Analysis of these patterns revealed an ordered set of expression profiles in which successive waves of kinase expression occur during development. Interestingly, several protein kinases whose expression has previously been reported to be restricted to tissues other than the mammary gland were isolated in this screen and found to be expressed in the mammary gland. In aggregate, these findings suggest that the array of kinases participating in the regulation of normal mammary development is considerably broader than currently appreciated., (Copyright 2000 Academic Press.)

Calcium is an important second messenger in eukaryotic cells. Many of the effects of calcium are mediated via its interaction with calmodulin and the subsequent activation of Ca(2+)/calmodulin-dependent (CaM) kinases. CaM kinases are involved in a wide variety of cellular processes including muscle contraction, neurotransmitter release, cell cycle control, and transcriptional regulation. While CaMKII has been implicated in learning and memory, the biological role of the other multifunctional CaM kinases, CaMKI and CaMKIV, is largely unknown. In the course of a degenerate RT-PCR protein kinase screen, we identified a novel serine/threonine kinase, Pnck. In this report, we describe the cloning, chromosomal localization, and expression of Pnck, which encodes a 38-kDa protein kinase whose catalytic domain shares 45-70% identity with members of the CaM kinase family. The gene for Pnck localizes to mouse chromosome X, in a region of conserved synteny with human chromosome Xq28 that is associated with multiple distinct mental retardation syndromes. Pnck is upregulated during intermediate and late stages of murine fetal development with highest levels of expression in developing brain, bone, and gut. Pnck is also expressed in a tissue-specific manner in adult mice with highest levels of expression detected in brain, uterus, ovary, and testis. Interestingly, Pnck expression in these tissues is restricted to particular compartments and appears to be further restricted to subsets of cells within those compartments. The chromosomal localization of Pnck, along with its tissue-specific and restricted pattern of spatial expression during development, suggests that Pnck may be involved in a variety of developmental processes including development of the central nervous system., (Copyright 2000 Academic Press.)

The observation that normal pathways of differentiation and development are invariably altered during the process of carcinogenesis implies an intrinsic relationship between these processes. This relationship is particularly evident in the breast, as exemplified by the existence of endocrine risk factors for breast cancer that are related to the timing of normal developmental events. Understanding the mechanisms by which normal developmental events alter breast cancer risk is a central focus of our laboratory. Herein, we describe three approaches being taken in our laboratory toward defining the molecular basis of this relationship. These include: determining the roles played by the tumor suppressor genes, BRCA1 and BRCA2, in the normal differentiation and development of the breast; studying the function of three novel protein kinases identified in our laboratory in mammary epithelial development; and defining the molecular and cellular changes that occur in the breast as a result of reproductive events known to influence breast cancer risk.

Germline mutations in the putative tumor suppressor gene, BRCA1, predispose women to dramatically elevated risks of breast cancer, while germline mutations in the structurally unrelated gene, BRCA2, predispose both men and women to breast cancer. Recent studies have suggested an important developmental role for the murine homologue of BRCA1 in the regulation of proliferation and differentiation. At the present time, however, little is known about the developmental role of BRCA2 or the regulation of its expression in vivo. We have determined the spatial and temporal pattern of expression of the murine homologue of BRCA2 during fetal development, in adult tissues, and in the mammary gland during postnatal development. Our results indicate that Brca2 mRNA expression is highest in proliferating cellular compartments, particularly those undergoing differentiation. In the breast, Brca2 expression is developmentally regulated and is induced during puberty and pregnancy and as a result of parity. Surprisingly, in multiple fetal and adult tissues the spatial and temporal pattern of Brca2 mRNA expression is virtually indistinguishable from that of Brca1, despite the fact that these genes display no homology. These observations suggest that Brca2 is involved in the processes of proliferation and differentiation in the mammary gland and other tissues, and that Brca1 and Brca2 mRNA expression may be regulated by similar pathways and stimuli in multiple cell types. Interestingly, however, our analysis reveals that Brca1 and Brca2 expression are differentially regulated during the development of specific endocrine target tissues, such as the testis during spermatogenesis and the breast during pregnancy. In addition, the ratio of mRNA expression in the mammary glands of adult females relative to adult males is significantly greater for Brca1 than for Brca2. These observations imply that Brca1 and Brca2 mRNA expression are differentially regulated by sex hormones. In order to test this hypothesis, we have analyzed the expression of these two breast cancer susceptibility genes in ovariectomized mice treated with 17beta-estradiol and progesterone. Our results demonstrate that the up-regulation of mRNA expression in the breast by ovarian hormones is significantly greater for Brca1 than for Brca2. These observations suggest that the gender-specific differences in phenotype associated with germline mutations in BRCA2 versus BRCA1 may be related to the differential regulation of these genes by sex hormones.

We have analyzed the expression of the breast cancer susceptibility gene, Brca2, in mammary epithelial cells as a function of proliferation and differentiation. Our results demonstrate that Brca2 mRNA expression is tightly regulated during mammary epithelial proliferation and differentiation, and that this regulation occurs coordinately with Brca1. Specifically, Brca2 mRNA expression is up-regulated in rapidly proliferating cells; is down-regulated in response to serum deprivation; is expressed in a cell cycle-dependent manner, peaking at the G1/S boundary; and is up-regulated in differentiating mammary epithelial cells in response to glucocorticoids. In each case, an identical pattern of expression was observed for Brca1. These results indicate that proliferative stimuli modulate the mRNA expression of these two breast cancer susceptibility genes. In addition, the coordinate regulation of Brca1 and Brca2 revealed by these experiments suggests that these genes are induced by, and may function in, overlapping regulatory pathways involved in the control of cell proliferation and differentiation.

We have examined the developmental expression of the murine breast and ovarian cancer susceptibility gene, Brca1, to investigate its role in the control of cell growth and differentiation. Specifically, we have analysed Brca1 expression during embryonic development, in adult tissues, and during postnatal mammary gland development, particularly in response to ovarian hormones. Our results suggest that Brca1 is expressed in rapidly proliferating cell types undergoing differentiation. In the mammary gland, Brca1 expression is induced during puberty, pregnancy, and following treatment of ovariectomized animals with 17 beta-estradiol and progesterone. These observations imply that Brca1 is involved in the processes of proliferation and differentiation in multiple tissues, notably in the mammary gland in response to ovarian hormones.