The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ERtargeted genes encoding E2F1 and cyclin D1, which promote progression through the G 1 /S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells. N uclear receptors, which include steroid and nonsteroid hormone receptors, comprise a superfamily of DNA-bound transcriptional regulators that are activated in response to specific small lipophilic ligands and that play major physiological roles in cell growth, differentiation, and homeostasis (reviewed in references 10 and 25). Estrogen receptor ␣ (ER␣) is the key activator that leads to growth of the mammary glands during adolescence, as well as during pregnancy, in response to elevated plasma estrogen levels. Among the hormone-responsive genes transcribed under the control of ER␣ is another steroid hormone receptor, progesterone receptor (PR), which in concert with ER␣, plays an important role in mammary gland development (5).The metazoan Mediator/TRAP coactivator complex is a master transcriptional coregulator composed of about 30 subunits and is structurally subdivided into head, body, and tail modules. It constitutes a subcomplex of the RNA polymerase II holoenzyme and integrates a wide variety of intracellular signals through specific interactions of activators with specific Mediator subunits that reside predominantly at its tail module (reviewed in references 4, 6, 15, 20, and 24). We have proposed a multistep model for nuclear receptor-induced transcriptional activation (15). In this model, histone-modifying coactivators that possess either histone acetyltransferase or histone methyltransferase activities first interact with ligand-bound nuclear receptors, and chromatin structure is subsequently relaxed. Then an exchange of coactivators takes place and the Mediator is bound to nuclear receptors through two canonical LxxLL nuc...

BACKGROUND: Antigen retrieval, a crucial technique for immunostaining, is often carried out on formalin-fixed, paraffinembedded (FFPE) tissue sections. The role of antigen retrieval in immunostaining of ethanol-fixed smears remains unclear.The authors evaluated the effects of 2 common antigen retrieval procedures, heat-induced antigen retrieval and proteaseinduced antigen retrieval, for immunostaining using a broad panel of antibodies. METHODS: Papanicolaou-stained ethanol-fixed smears from 36 surgical specimens were immunostained with 43 antibodies. Three widely used heat-induced antigen retrieval solutions, namely, citrate buffer (pH 6.0 and pH 7.0) and ethylenediaminetetraacetic acid solution (pH 8.0) for heat-induced antigen retrieval, and pronase were used. The staining results were compared between the ethanolfixed smears and the corresponding FFPE tissue sections. RESULTS: Heat-induced antigen retrieval was essential for all the 9 antibodies examined against nuclear antigens, and for 7 of 26 antibodies against cytoplasmic and cell membrane antigens. Superior results were obtained using lower-pH heat-induced antigen retrieval solutions for ethanol-fixed smears than was the case for FFPE tissue sections; use of citrate buffer (pH 6.0) was optimal for most antibodies. For 17 antibodies against cytoplasmic/cell membrane antigens, satisfactory results were obtained even without antigen retrieval on the ethanol-fixed smears, whereas antigen retrieval was necessary for detection on the FFPE tissue sections. Proteaseinduced antigen retrieval frequently exerted deleterious effects on ethanol-fixed smears. Despite antigen retrieval, detection of 2 lymphocytic markers failed on ethanol-fixed smears. This limitation was overcome by heat-induced antigen retrieval on formalin vapor-fixed smears. CONCLUSIONS: In ethanol-fixed smears, most of the antibodies can be immunostained successfully without antigen retrieval treatment or mild heat-induced antigen retrieval using citrate buffer (pH 6.0). The optimal antigen retrieval condition for each antibody must be individually determined. Cancer (Cancer Cytopathol) 2012;120:167À76. V C 2012 American Cancer Society.KEY WORDS: ethanol-fixed smear, formalin vapor-fixed smear, immunocytochemistry, heat-induced antigen retrieval, protease-induced antigen retrieval.

Objective In The Paris System for Reporting Urinary Cytology (TPS), the important cytomorphological features for diagnosing high‐grade urothelial carcinoma (HGUC) are a nuclear‐to‐cytoplasmic (N:C) ratio exceeding 0.7, hyperchromasia, coarse chromatin, and irregular nuclear borders. However, quantitative cytomorphological assessments of HGUC cells using SurePath slides are rare. Therefore, we evaluated HGUC cells on SurePath slides quantitatively using a digital image analysis system and compared these data with ThinPrep data. Methods The same urine samples were divided into two aliquots and used to prepare SurePath and ThinPrep slides. We used ImageJ to measure the N:C ratio, hyperchromasia, and irregular nuclear borders for HGUC cells on SurePath and ThinPrep slides. Results The total number of analysed HGUC cells on SurePath slides was 981, versus 889 on ThinPrep slides. Hyperchromasia and irregular nuclear borders were significantly more severe on SurePath than on ThinPrep slides. Conversely, the N:C ratio did not differ between the methods. Additionally, HGUC cells with N:C ratios exceeding 0.7 were present on almost all slides for both methods. Conclusions Our data indicated the reasonableness of using the N:C ratio as the major criterion for TPS on both SurePath and ThinPrep slides, and an N:C ratio cut‐off of 0.7 as suitable for identifying HGUC cells. However, the severity of hyperchromasia and irregular nuclear borders differed between the processing methods.

Our findings implicate a potential role for IDO activity and a maternal immunological reaction against an allogeneic fetus in the etiology of pre-eclampsia.

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