Overexpression of the A Form of Pr Pra Causes Transformation of Mammary Epithelial Cells

It is well established that PR exists in two molecular forms commonly referred to as the 'A' and 'B' forms whose ratio varies among target tissues (9). In adult rodents, the mammary gland ratio of the 'A':'B' forms is 3:1 (10). In-vitro studies have shown that the 'A' and 'B' forms can have different functions in the same cell. In addition, the PR activity of the individual form can vary among different types of cells (11). Furthermore, depending on the cell, the 'A' form can either inhibit or enhance the activity of the PR'B' form (11). Based on these observations, it is believed that appropriate cellular responsiveness to progesterone is dependent on regulated expression and/or activity of the two PR forms (12). Therefore, an aberration in normal mammary development may result from inappropriate progesterone signaling due to an imbalance in the expression and/or activities of the two PR forms. The imbalance, in turn, may have implications for mammary oncogenesis. To test this hypothesis, our laboratory created transgenic mice in which the native PR 'A':'B' ratio was altered by introducing additional PR 'A' or 'B' forms as transgene. A binary system was used for the expression of the transgene as shown in Figure 1. Detailed descriptions of the constructs have been published (13, 14).

Figure 1. Schematic representation of plasmid construction for the binary system. (A) Insertion of the GAL-4 gene into the CMV promoter expression plasmid containing simian virus (SV) 40 splice and polyadenylylation sequences. (B) mPR cDNA (A form with only ATG 2) containing inttron 1 and SV40 splice and polyadenylation sequences fused to UAS-TATA fragment containing four GAL-4 binding sites. E, EcoRl. (C) mPR cDNA (B form with only ATG1) containing inttron 1 and SV40 splice and polyadenylation sequences fused to UAS-TATA fragment containing four GAL-4 binding sites.

Mammary development is abnormal in transgenic mice carrying an imbalance in the native ratio of 'A':'B' forms by overexpressing either the 'A' or 'B' form (referred to as PR-A and PR-B transgenics, respectively) (13, 14). In particular, PR-A transgenic mammary glands exhibit excessive ductal growth, and morphological and histological features associated with transformed cells, i.e., loss in basement membrane integrity and cell-cell adhesion. The aberrant patterns of ductal growth were not restricted to the offspring of any particular founder mouse, establishing that the phenotype was due to the transgene expression, and not due to their integration into any unique site on the genome (15).

In a series of comprehensive studies, Medina, et al. identified certain molecular markers unique to mammary epithelial cells at various stages of transformation. In particular, they showed that in mouse mammary epithelial cells, a decrease in p21 expression, without an increase in cyclin D1, is indicative of immortalization, and precedes the onset of hyperplasia/preneoplasia (16). Among the characteristics that distinguish the presumptive immortalized epithelial cells from those in hyperplasia/preneoplasia are increases in cyclin D1 expression and cell proliferation, and a decrease in ERa expression (17).

Figure 2. Morphological and histological characteristics of wild type and PR-A in adult transgenic mice. Whole-mounts (A-C) and histology (D-G) of mammary glands from adult (10-14 weeks old) control PR-A transgene-negative (A and D) and PR-A transgenic mice (B, C, and E-G) are shown. In B and C, open arrows point to thick ducts, while solid arrows point to clustered buds at the tip of ducts. Note that, in contrast to the monolayer associated with the duct from wild type mice (D), the ducts in PR-A transgenics are composed of multilayered cells (E, F, and G). In E, the arrow points to an indistinct epithelial-stromal boundary, and in F, the arrow points to disorganized masses of cells at the tip of a duct.

Figure 2. Morphological and histological characteristics of wild type and PR-A in adult transgenic mice. Whole-mounts (A-C) and histology (D-G) of mammary glands from adult (10-14 weeks old) control PR-A transgene-negative (A and D) and PR-A transgenic mice (B, C, and E-G) are shown. In B and C, open arrows point to thick ducts, while solid arrows point to clustered buds at the tip of ducts. Note that, in contrast to the monolayer associated with the duct from wild type mice (D), the ducts in PR-A transgenics are composed of multilayered cells (E, F, and G). In E, the arrow points to an indistinct epithelial-stromal boundary, and in F, the arrow points to disorganized masses of cells at the tip of a duct.

In mammary glands of PR-A transgenics, ducts with normal histology, displayed a decrease in p21 expression (Figure 3A) without an elevation in cyclin D1 (Figure 3B), or cell proliferation (Figure 3C). Similar to ducts with normal histology, in mammary dysplasias of PR-A transgenic, there was a decrease in p21 expression (Figure 3A), but was accompanied by an increase in cyclin D1 expression (Figure 3B) and cell proliferation (Figure 3C), and a decrease in expression (Figure 3D). Therefore, these studies establish that mammary glands of PR-A transgenics contain at least two distinct populations of transformed epithelial cells: one, corresponding to immortalized cells, in early stages of transformation, present in the ducts with normal histology; and two, cells in later stages of transformation associated with dysplasias.

An imbalance in the native ratio of PR A:B isoforms also exists in PR-B transgenic mice, but the mammary phenotype of these mice is distinct from that of the PR-A transgenics (14). In fact, PR-B mammary glands have impaired ductal elongation. Analyses of these mammary glands did not reveal any detectable changes in either the number of BrdU-positive cells or the expression of cyclin D1, p21, or ERa (18).

Figure 3. Cell proliferation, cyclin-Dl, p21, and ERa expression in mammary glands of wild-type and PR-A transgenic mice. Panel A shows the expression profiles of p21 in a mammary duct of wild type mice (I), a mammary duct of PR-A transgenic with normal histology (II), an abnormal mammary duct of PR-A transgenic (III), and (IV) the absence of immunoreactivity with irrelevant mouse IgG (top), and deletion of primary antibody (bottom). Scale bar represents 20 pm. Panel B shows the number of cyclin D1-positive cells in normal (ND) and abnormal ducts (AD). Panel C shows the rate of cell proliferation, using BrdU-positive cells as index, in ND and AD. Panel D shows the number of ERa-positive epithelial cells in ND and AD.

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