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This report describes the separation and characterization of estrogen receptors (ER) according to their degree of hydrophobicity and surface charges. Molybdate-stabilized [3H]ER from rabbit uterine cytosol was sequentially purified by passage through a size-exclusion pre-column, an anion-exchange column, and a hydrophobic interaction column. With fresh cytosol, a major radioactive peak was eluted from the DEAE columns; a major peak and a minor, less hydrophobic, peak were eluted from the hydrophobic column. In contrast, ER from frozen cytosol showed one peak in the DEAE-column and exhibited four radioactive peaks in the hydrophobic column. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, [3H] tamoxifen aziridine(TA)-labelled ER showed radioactive bands at 62 and 48 kd. The subunits which were characterized by these radioactive bands were successfully separated by the hydrophobic column; the more hydrophobic subunit corresponded to the 62 kd band. The HPLC-purified [3H]TA-labelled ER subunits sediment at a 7.4-8.5S region in a low-salt sucrose gradient. These results show that differential negative surface charge and hydrophobic areas exist in the holo-receptor and its subunits, and the hydrophobic interaction HPLC column separates the two major 8S steroid binding subunits of ER.

A micro-fluorometric ELISA which required only 50 microliter of reagent in each step is reported here for rapid detection of anti-estrogen receptor (ER) antibody production by hybridoma cultures. In this study, several factors which influence the sensitivity of ELISA, e.g., incubation time, substrates, microtiter plates, concentration of coating antigens, choice and concentration of specific antibody and enzyme were evaluated. We observed that micro-fluorometric ELISA using 4-methylumbelliferyl phosphate-phosphatase system and MicroFluor W plates was the most sensitive; it detected readily 1 ng coating antigens or 1 ng antibody during a 15 min incubation. This system was used successfully for the detection of anti-ER antibodies in mouse immune sera, and in spent media from in vitro immunized spleen cells or hybridoma cells. This method is simple, sensitive, rapid and reliable for ER or anti-ER antibody determination.

To determine the frequency of occurrence of "blue bodies" (BBs), 2,010 pulmonary cytology specimens (1,403 sputum and 607 bronchial specimens) from 1985 were reviewed. The smears were examined microscopically by transmitted and polarized light. BBs were extracellular structures, occurring most commonly in clusters but sometimes conglomerated. With Papanicolaou stain, they had a characteristic birefringent laminated brown core and a nonbirefringent blue rim. Chemical microanalysis and energy-dispersive x-ray analysis revealed that their chemical composition was calcium carbonate. A total of 233 specimens contained BBs, with a frequency of 10.5% in bronchial specimens and 12% in sputum. Only 8.6% of the BBs had co-existent pulmonary malignancy. We concluded that BBs were common structures in pulmonary cytology and were not associated with pulmonary malignancy or pulmonary fibrosis in our series. They must be distinguished from contaminants, staining artifacts, and parasitic ova.

The presence of ER in DMBA-tumors was demonstrated by the use of dextran-charcoal assay, sephadex chromatography, sucrose gradient sedimentation, and organ culture techniques. It was found that tumors have binding sites ranging from 10-13 to 10-15 moles/mg protein, and a dissociation constant of ER 10-9 to 10-10 M. In experiments with tumor explants, prolactin-insulin significantly stimulated ER binding capacity, as compared with control without prolactin. This stimulation was tissue-specific and inhibited by progesterone. Insulin had a synergistic effect on prolactin stimulation of ER. Our results presents a plausible explanation for tumor responses to these hormones in vivo. This interaction of prolactin, estrogen, and progesterone may be a common phenomenon for all estrogen-responsive tissues.

Eighty-six cases of primary endometrial carcinoma were assayed for the presence or absence of cytoplasmic estrogen and progesterone receptors by the saturation point dextran-coated charcoal assay. The levels of cytoplasmic progesterone receptors and estrogen receptors were analyzed according to clinical stage, histologic type and grade of the tumor, presence or absence of lymph node metastases, myometrial invasion, and survival. The cases were divided into positive and negative receptor groups with levels chosen of greater than 10 fmol/mg of cytosol protein for progesterone receptor and 5 fmol/mg of cytosol protein for estrogen receptor as discrimination points. Statistically significant survival differences were found between estrogen receptor positive versus estrogen receptor negative patients, progesterone receptor positive versus progesterone receptor negative patients, and estrogen positive-progesterone receptor positive versus estrogen negative-progesterone receptor negative patients. Mean cytoplasmic estrogen and progesterone receptor levels were inversely proportional to grade. This report suggests that treatment protocols should be devised to reflect the prognostic significance of receptor status.

Interaction of estradiol receptor with chromatin was probed by nuclease digestion of receptor-chromatin complex. The complex was formed by incubating partially purified receptor with chromatin. Micrococcal nuclease digestion of the complex released a 7S form of receptor which could be converted to 2.8S form by DNAase I. Digestion of the complex with DNAase I yielded different forms of receptors ranging from 7S to 2.8S depending on the digestion time. Receptor distribution was also examined by isolating nuclei form tissue pre-incubated with radioactive estradiol. Micrococcal nuclease digestion of receptor-filled nuclei released 7S, 5.5S and 3.5S forms of receptors. Collectively, these results indicate that the 7S nuclear receptor may have an associated chromatin fragment which is sensitive to DNAase I activity. The desirable features of receptor-chromatin method over conventional methods for studies relating to receptor interaction at the gene site are discussed.

Twenty-five patients with measurable metastatic breast cancer and assays for estrogen receptor (ER) were studied. Of the 16 ER positive patients on anti-estrogen therapy, one had complete disappearance of all tumor for seven months and seven patients had more than 50% reduction in their measurable tumor for an average duration of 8.8 months. Seven other ER positive patients had stabilization of their tumors for an average interval of 8.4 months. Only one of the 16 ER positive patients progressed promptly. Conversely there was only one partial response in the nine ER negative patients and only two ER negative patients had stabilization of disease. Six out of nine ER negative patients progressed promptly. Correlation existed between the duration of response and absolute estrogen receptor level of the tumor. There may be a positive correlation between the response to antiestrogen therapy and response to endocrine ablation but prospective studies must be done to further define the role of antiestrogens in this regard.

Phospholipids and their acyl group composition in mammary adenocarcinomas and mammary tissue of the same tumor-bearing animals were investigated. Breast adenocarcinoma induced by dimethybenz(a)anthracene exhibited a phospholipid pattern which was different from that of the mammary tissue. Tumor phospholipids had higher proportions of diacyl-GPI, diacyl-GPE, and alkenylacyl-GPE and a lower proportion of diacyl-GPC than the controls. The acyl groups of most phospholipids in tumors showed a marked increase in the proportion of 18:1 and a decrease in the proportion of 18:2. The fatty acid composition of plasmalogen and triglyceride, however, remained unchanged. In spite of the decrease in the proportion of 18:2, there was no apparent difference in the proportion of 20:4 in most of the phosphoglycerides; however, a significant decrease in this fatty acid was noted in diacyl-GPI. Results of this study demonstrated that the membrane phospholipids of mammary adenocarcinoma were altered in respect to acyl group composition. Changes in physical properties of the cell membrane, in turn, could lead to abnormal manifestation of membrane regulated events in tumor cells.

The distribution of phospholipids as well as their fatty acid compositions of rat mammary tissues were examined during pregnant, lactating, and post-weaning periods. There was no apparent change in phospholipids and their acyl groups during the early and late pregnant periods. However, tissue phospholipid composition was different during pregnant, early, and late lactating periods. After parturition, there was a marked increase in the proportion of diacyl-glycerophosphorylcholine in the phospholipids of mammary tissue, but this proportion decreased gradually during lactation. The decrease in diacyl-glycerophosphorylcholine during lactation was marked by a corresponding increase in diacyl-glycerophosphorylethanolamie. Although the shorter chain fatty acids of triglycerides were increased during lactation, only a small proportion of these fatty acids was found in the phosphoglycerides. Marked changes in acyl group composition of individual phospholipids are found during these different physiological stages. In general, there was a rapid decrease in 20:4 and an increase in 18:2 in the major phosphoglycerides during parturition. The proportion of 20:4 in the phosphoglycerides remained low throughout the entire lactating period, while that of 18:2 continued to increase 2-3 fold. Most of the changes in acyl group of the phosphoglycerides during lactation returned to normal ca. 10 days after weaning. A possible relationship of the variation of phospholipid and acyl group compositions in mammary tissue to changes in hormonal pattern during different physiological stages is discussed.

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.