Even though the breast is influenced by a myriad of hormones and growth factors [111-118], estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium [111,112, 115]. Estradiol acts locally in the mammary gland, stimulating DNA synthesis and promoting bud formation, probably through an ER-mediated mechanism . It is also known that the prevailing metabolic condition of an individual animal or human may significantly influence mammary gland responses to hormones. In addition, the mammary gland responds selectively to given hormonal stimuli for either cell proliferation or differentiation, depending upon specific topographic differences in gland development. In either case, the response of the mammary gland to these complex hormonal and metabolic interactions results in developmental changes that permanently modify both the architecture and the biological characteristics of the gland [111,113]. The fact that the normal epithelium contains receptors for both estrogen and progesterone lends support to the receptor-mediated mechanism as a major player in the hormonal regulation of breast development. The role of these hormones on the proliferative activity of the breast, which is indispensable for its normal growth and development, has been for a long time, and still is, the subject of heated controversies [117-125]. There is little doubt, however, that the proliferative activity of the mammary epithelium in both rodents and humans varies with the degree of differentiation of the mammary parenchyma [111-114, 126-128]. In humans, the highest level of cell proliferation is observed in the undifferentiated lobules type (Lob 1) present in the breast of young nulliparous females [111-114]. The progressive differentiation of Lob 1 into lobules types 2 (Lob 2) and 3 (Lob 3), occurring under the hormonal influences of the menstrual cycle, and the full differentiation into lobules type 4 (Lob 4), as a result of pregnancy, leads to a concomitant reduction in the proliferative activity of the mammary epithelium [111-114, 126-128]. The content of ERa and progesterone receptor (PgR) in the lobular structures of the breast is directly proportional to the rate of cell proliferation, being also maximal in the undifferentiated Lob 1, and decreasing progressively in Lob 2,Lob 3, and Lob 4 (Fig. 5) . Cell proliferation, as determined as the percentage of cycling cells that are positively stained with Ki67 antibody as a brown nuclear reaction characteristic of DAB
Fig. 6. Lob 1 ductules of the human breast. The single-layered epithelium lining the ductule contains Ki67 positive cells (brown nuclei), and ER positive cells (red-purple nuclei) (x40)
stain, is most frequently found in Lob 1 (Fig. 5) . The percentage of positive cells is reduced by three fold in Lob 2, and by more than ten fold in Lob 3 (Fig. 5) . In all cases, the proliferating cells are almost exclusively found in the epithelium lining ducts and lobules. Only occasionally are positive cells found in the myoepithelium, or in the intralobular and interlobular stroma. The same pattern of reactivity is also observed in tissue sections incubated with the ERa and PgR antibodies. Positive cells are found exclusively in the epithelium. The number of cells positive for ERa or PgR is highest in the Lob 1, and decreases progressively in Lob 2 and Lob 3 (Fig. 5) .
It should be noted, however, that it remains unclear from the above studies whether the cells that are positive for steroid receptors are those that are proliferating. The use of the double staining procedure for Ki67 and ERa or PgR has allowed us to quantitatively determine in the same tissue sections the spatial relationship between those cells that are proliferating and those that react with the ERa or PgR antibody. The double stained cells appear purple-red in color due to the alkaline phosphatase-vector red staining (Fig. 6).
The number of cells that express ERa and/or PgR is similar to that of cells positive for Ki67, and the highest percentage of positive cells is also observed in Lob 1 for both steroid hormones. The percentages of ERa- and PgR-positive cells in Lob 1 are 7.5% and 5.7%, respectively, which do not differ significantly (Fig. 5). The percentages of ERa- and PgR-positive cells in Lob 2 are reduced to 3.8% and 0.7%, respectively, and become negligible in Lob 3 (Fig. 5).
Of interest is the observation that even though there are similarities in the relative percentages of Ki67-, ERa- and PgR-positive cells, and in the progressive reduction in the percentage of positive cells as the lobular differentiation progresses, those cells positive for Ki67 are not the same as those positive for ERa or PgR. Very few cells, less than 0.5% in Lob 1, and even fewer in Lob 2 and Lob 3, appear positive for both Ki67 and ERa (Ki67+ER) (Fig. 5). This double reactivity is identified by the darker staining of the nuclei, which appear dark purple-brown. Whereas the percentage of cells double labeled with Ki67 and
ERa (Ki67+ER) decreases gradually from Lob 1 to Lob 3, the percentage of cells exhibiting double labeling with Ki67 and PgR antibodies (Ki67+PgR) is high in Lob 2 but low in Lob 1 and Lob 3 (Fig. 5). As a result, Ki67+PgR-positive cells is lower than the percentage of Ki67+ER-positive cells in Lob 1, but the percentages of Ki67+PgR- and Ki67+ER-positive cells become quite similar in Lob 2 and Lob 3 (Fig. 5).
Our data indicate that the contents of ERa and PgR in the normal breast tissue, as detected immunocytochemically, vary with the degree of lobular development, but are linearly related to the rate of cell proliferation of the same structures. The utilization of a double labeling immunocytochemical technique to stain the same tissue section for steroid hormone receptors and Ki67 proliferating antigen has allowed us to conclude that the expression of the receptors occurs in cells other than the proliferating cells, confirming results reported by others . The findings that proliferating cells are different from those that are ERa- and PgR-positive support data that indicate that estrogen controls cell proliferation by an indirect mechanism. This phenomenon has been demonstrated using supernatant of estrogen-treated ERa-positive cells that stimulates the growth of ERa-negative cell lines in culture. The same phenomenon has been shown in vivo in nude mice bearing ER-negative breast tumor xenografts [130, 131]. ERa-positive cells treated with antiestrogens secrete transforming growth factor-^ that inhibits the proliferation of ERa-negative cells . The findings that proliferating cells in the human breast are different from those that contain steroid hormone receptors explain many of the in vitro data [133-135]. Of interest are the observations that while the ERa-positive MCF-7 cells respond to estrogen treatment with increased cell proliferation, and that the enhanced expression of the ERa by transfection also increases the prolifer-ative response to estrogen [133-136], ERa-negative cells, such as MDA-MB 468 and others, when transfected with ERa, exhibit inhibition of cell growth under the same type of treatment [134-137]. Although the negative effect of estrogen on those ERa-negative cells transfected with the ERa has been interpreted as an interference of the transcription factor used to maintain estrogen independent growth , there is no definitive explanation for their lack of survival. However, it can be explained by the finding that proliferating and ERa-positive cells are two separate populations. Further support is the finding that when Lob 1 of normal breast tissue are placed in culture, they lose the ERa-positive cells, indicating that only proliferating cells that are also ERa-negative can survive and constitute the stem cells [137,138].
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