乳腺上皮细胞微量元素转运机制研究进展

新生儿生长发育所需的微量元素主要从母乳中获得,微量元素参与了机体的许多生命活动,如酶的活性、细胞增殖及分化等。乳腺上皮细胞含有多种微量元素转运体系,如锌离子转运体系（Zip／ZnT）、铁离子转运体系（DMTl／FPN）和铜离子转运体系（Ctrl／ATP7）。在分泌乳汁的同时,这些转运蛋白对锌、铁、铜等微量元素的吸收、转运和分泌起着重要的作用。同时这些微量元素的转运及代谢受到多种因素的调控,使母乳中微量元素含量达到动态稳定,以满足新生儿生长发育各阶段对微量元素的需求。对近年来锌、铁、铜三种微量元素在乳腺上皮细胞内转运机制的研究进展进行综述。     

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction.

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk-promoter or the MMTV-promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone-responsive element in mammary cells.     

Quantitative analysis of the glucocorticoid receptor-DNA interaction at the mouse mammary tumor virus glucocorticoid response element

Purified glucocorticoid receptor (GR) from rat liver was used for a quantitative analysis of the protein-DNA interaction at specific GR-binding segments within the 5'-long terminal repeat of the mouse mammary tumor virus. A truncated receptor was generated and used to demonstrate formation of heterodimeric GR, which furthermore was shown to be in rapid equilibrium with receptor-monomer. The relative affinity for GR binding to specific GR sites versus random calf thymus DNA was approximately 2 x 10(3). At equilibrium a free GR concentration of 3 x 10(-10) M was required for half-maximal saturation of the two functionally important DNA sites within the mouse mammary tumor virus 5'-long terminal repeat. Although these two DNA segments act synergistically in mediating hormonal response, we did not detect cooperative GR binding to these regions in vitro. However, GR bound cooperatively within the downstream binding region. Similarly, GR was unable to facilitate factor binding to a neighboring nuclear factor 1 site, another essential element in the promoter. In contrast, nuclear factor 1 binding was inhibited slightly by GR.     

Differential gene activation by glucocorticoids and progestins through the hormone regulatory element of mouse mammary tumor virus

The hormone regulatory element (HRE) of mouse mammary tumor virus can mediate activation of an adjacent promoter by glucocorticoids and progestins. A detailed comparison of the DNA binding of receptors for both hormones using DNAase I footprinting and methylation protection detects clear differences in their interactions with the HRE region between positions -130 and -100. Binding studies and gene transfer experiments with a variety of mutants covering the entire HRE demonstrate differences in the relevance of the individual sequence motifs for induction by each hormone. The influence of changes in the angular orientation of receptor binding sites is also different for glucocorticoid and progesterone induction. In transfection experiments with mutated HREs, we find a functional cooperation between the receptor binding sites that does not correlate with variations in the in vitro affinity of the receptors for the corresponding DNA fragment.     

A novel cis-acting element in Her2 promoter regulated by Stat3 in mammary cancer cells

Stat3 plays important roles in the development of breast malignancies and oncogenesis. In the present study, a palindromic cis-acting element displaying repression activity in breast cancer cells expressing low level of Her2 was found in Her2 promoter. Deletion analysis showed that the novel element was located within Pal2 region spanning nucleotides -529 to -505. The sequence analysis of Pal2 region revealed a DNA sequence (TTAAGATAA) homologous to the binding site of Stat3, starting from position -529 to -521bp. By reporter assay, Pal2 was found to be regulated by constitutive activated Stat3C. A stimulatory effect both on Her2 mRNA and protein expressions was observed in MCF-7 cells stably expressing Stat3C, suggesting that Stat3 regulated Her2 expression. Using ChIP assays the binding of Stat3 to Her2 promoter was confirmed. The data obtained in this study indicate constitutive activated Stat3 regulates Her2 expression. Further investigation of differential effects of Stat3 exerting on breast cancer cells expressing Her2 at different levels will provide more insights into the roles of Stat3 in Her2 expression as well as the regulation of diverse biological activities.     

A composite enhancer element directing tissue-specific expression of mouse mammary tumor virus requires both ubiquitous and tissue-restricted factors

Mouse mammary tumor virus (MMTV) expression is restricted primarily to mammary epithelial cells. Sequences responsible for both the mammary-specific expression of MMTV and the activation of cellular oncogenes are located within two enhancer elements at the 5'-end of the long terminal repeat. Whereas the Ban2 enhancer (-1075 to -978) has been well characterized, the mammary-specific enhancer of MMTV from -956 to -862 has only recently been recognized as a key determinant of mammary-specific oncogene activation by MMTV. The present study identifies and characterizes three binding sites located within this element. Transient transfection of deletion and mutation constructs shows that all three sites contribute to the basal expression of MMTV in mammary cells. One of the binding activities (footprint I) is restricted to mammary cells, whereas the other two sites bind factors found in both mammary and nonmammary cells. The multimerized mammary-specific enhancer of MMTV on its own can enhance a minimal promoter in a mammary-specific fashion. However, the FpI binding site alone cannot mediate this effect. Thus, it is the binding of multiple factors in a combinatorial fashion that mediates the mammary-restricted expression of MMTV.     

Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas

ABSTRACT: BACKGROUND: Mammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffinembedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas. RESULTS: The highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p =0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells. CONCLUSIONS: Malignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding.     

A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis

The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin⁻CD24^medCD29⁺ cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin⁻CD24⁺CD29^hi), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin⁻CD24^medCD29⁺ cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin⁻CD24^medCD29⁺ cell population possessed low proportion of stem cells. In summary, heterogeneous Lin⁻CD24^medCD29⁺ cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment.     

Analysis of mammary tumors for cytochemical evidence of endogenous mammary peroxidase.

The purpose of this study was to determine whether or not endogenous mammary peroxidase can serve as a cytochemical marker to distinguish ovarian hormone-dependent from ovarian hormone independent mammary tumors. Spontaneous mammary tumors arising in virgin C3H and GR mice (hormone independent tumors) and hormone-dependent mammary tumors arising during pregnancy in GR mice were examined. None of these tumors contained mammary peroxidase. Mammary tumors induced in Sprague-Dawley rats with methylnitrousourea (MNU) and dimethylbenzanthracene (DMBA) were also examined. These tumors included hormone-dependent and hormone independent ones. Several of the DMBA-induced hormone-dependent tumors contained a few peroxidase-positive cells, but the hormone independent tumors were negative. All of the MNU-induced tumors examined were negative for mammary peroxidase. Twenty human breast tumors (malignant and non-malignant) removed from women at surgery, were also negative for mammary peroxidase. Our results indicate that endogenous mammary peroxidase cannot be used to distinguish hormone-dependent from hormone independent mammary tumors.