2.6 Flow cytometric steroid receptor analysis

Schütte, B.; Scheres, H M; Goeij, A.F.P.M. De; Rousch, Mat; Blijham, Geert H.; Bosman, Fred T.; Ramaekers, Frans C.S.

doi:10.1016/s0079-6336(11)80080-x

Cited by 8 publications

(10 citation statements)

References 16 publications

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“…However, in the present study, we did not observe any major cell-cyclerelated changes in ER expression of cells stained simultaneously for both DNA content and ER expression. This observation is in agreement with the results of Schutte et al (13).…”

Section: Discussionsupporting

confidence: 94%

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Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues

et al. 1999

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Section: Discussionsupporting

confidence: 94%

“…These receptor-negative cells effectively lower the MFC value for any given sample (14). Various modifications of the fixation procedures and/or permeabilization have been employed to increase immunostaining, with limited success (11,13,14).…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues

et al. 1999

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“…The flow-cytometric method has been shown to compare favourably with the radioligand-binding assay (P = 1 x 10-5) usually used to detect ER in breast tumours. The monoclonal antibody DAKO-ER 1D5, which reacts with the N-terminal domain (A/B region) of the receptor (Kumar et al, 1987), has been shown to give a good correlation immunohistochemically with results obtained by biochemical quantification (Schutte et al, 1992).…”

mentioning

confidence: 95%

The effect of 3-week tamoxifen treatment on oestrogen receptor levels in primary breast tumours: a flow cytometric study

Brotherick

Browell²,

Shenton³

et al. 1998

Br J Cancer

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Summary The effect of 3-week, preoperative tamoxifen treatment on oestrogen receptor (ER) levels, expressed by primary breast tumours, was examined. Patients (age-matched) with breast cancer, confirmed by fine-needle aspiration, were either treated with 20 mg ml-' oral tamoxifen per day or received no medication in the 3-week interval between assessment and surgery. Quantification of ER using flow cytometry was performed on the surgically removed tumour samples from tamoxifen-treated (n = 40) and control (n = 38, untreated) patient groups. The tumours were mechanically disaggregated, and saponin treatment rendered these cells permeable to antibodies. Using dualparameter labelling with a FITC-conjugated antibody (NCL-5D3) directed against cytokeratin 8/18/19 and a biotinylated antibody (DAKO-ER 1 D5) directed against the oestrogen receptor, ER quantification was determined on a number of receptors per cell basis. Using QC quantum bead standards, ER levels in the epithelial cell population, the non-epithelial cell population and the whole-cell population (ER +) were calculated. ER levels were significantly lower in the total cell population than tamoxifen-treated patients (P = 0.002) when compared with the control (untreated) group. By using a gating procedure using 5D3 antibody positivity, a significantly lower level was detected on examining the cytokeratin-positive population alone (P = 0.006). Using a complementary gating technique, ER levels were quantified in the cytokeratinnegative cell population. Examination of this group of cells showed no significant difference between the levels of oestrogen receptor found in the tamoxifen-treated and untreated groups (P = 0.4). We have demonstrated that ER levels can be monitored by flow cytometry. ER levels in patients treated with tamoxifen 3 weeks before operation are significantly lower than in a comparative group of patients who received no drug. Furthermore, the most significant difference in receptor levels is seen by quantification of total ER levels expressed by all the tissue.

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“…The presence of ER␣ receptors was determined in MEC suspensions by microwave permeabilization (22) and flow cytometry (23). MEC were harvested with trypsin (0.025%), washed and 10 6 cells fixed in 2% paraformaldehyde for 30 min at 4 C, washed and resuspended in 0.01 mol/ liter citrate buffer, pH 6.0, and microwaved for 30 sec on High (500W), washed and resuspended in PBS/1% FCS.…”

Section: Er␣ Expression By Flow Cytometrymentioning

confidence: 99%

17β-Estradiol Up-Regulates Vascular Endothelial Growth Factor Receptor-2 Expression in Human Myometrial Microvascular Endothelial Cells: Role of Estrogen Receptor-α and -β

Gargett

Zaitseva

Bucak

et al. 2002

The Journal of Clinical Endocrinology & Metabolism

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Estrogen has a cardiovascular protective role in women due in part to its effect on the vasculature. The roles of the two estrogen receptors (ERs), ERalpha and ERbeta, in the vascular actions of estrogen are unclear, as are effects of estrogen on microvascular endothelial cells (MEC) derived from sex steroid-responsive tissues. The present study demonstrates that 17beta-estradiol, but not progesterone, increases vascular endothelial growth factor (VEGF) receptor (VEGFR) expression on human myometrial MEC measured using biotin-recombinant human (rh) VEGF(165) and flow cytometry. This response occurred in a time- and dose-dependent manner, with significantly increased rhVEGF(165) binding at 3 h and maximal responses between 0.1 and 10 nmol/liter 17beta-estradiol, which was blocked by the antiestrogen ICI 182,780. Approximately 60% of samples demonstrated this response to 17beta-estradiol. All samples of myometrial MEC expressed both ERbeta mRNA and protein demonstrated by semiquantitative RT-PCR and Western blotting. However, ERalpha mRNA and protein were expressed in only 13 of 21 MEC samples. There was a significant association between ERalpha expression in myometrial MEC and their ability to respond to 17beta-estradiol by increasing rhVEGF(165) binding. 17beta-estradiol increased VEGFR-2 expression in ERalpha-expressing MEC isolates, which also demonstrated increased rhVEGF(165) binding, but failed to have these effects on ERalpha negative samples. Similarly, 17beta-estradiol augmented VEGF-induced MEC proliferation in ERalpha-expressing MEC samples, which was blocked by ICI 182,780. These observations suggest that 17beta-estradiol increases VEGFR-2 expression on human myometrial MEC promoting endothelial cell proliferation, an effect that varies between subjects and appears to be mediated primarily by ERalpha.

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2.6 Flow cytometric steroid receptor analysis

Cited by 8 publications

References 16 publications

Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues

Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues

The effect of 3-week tamoxifen treatment on oestrogen receptor levels in primary breast tumours: a flow cytometric study

17β-Estradiol Up-Regulates Vascular Endothelial Growth Factor Receptor-2 Expression in Human Myometrial Microvascular Endothelial Cells: Role of Estrogen Receptor-α and -β

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