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The structure of the holo enzyme shows that the four chemically identical sub-units are arranged with almost perfect 222 symmetry. There are indications, however, that the active centre regions might only be related in pairs.

Indirect immunofluorescence and immunoperoxidase assays were developed to detect estradiol and progesterone in breast cancer cells. Appropriate controls were used to confirm immunologic specificity. Studies of estradiol binding by human breast cancer cells identified three groups: no detectable binding (25%); all tumor cells exhibiting binding although to different degrees (4%); and tumors with varying numbers of positive and negative cells (71%). Similar observations were made with respect to progesterone binding. The percentage of cells with estradiol binding was correlated with the amount of estrogen receptors (ER) present in the tumor specimens. Post-hormone binding events e.g., nuclear binding of estradiol, were also evaluated. Some tumor cells showing cytoplasmic binding of estradiol did not show nuclear binding of estradiol; such tumors lacked detectable diethylstilbestrol under routine assay conditions, and relatively high concentrations of estradiol were needed to observe estradiol-specific staining. The results suggest that the immunocytochemical assays detect hormone-specific binding, but that the binding is probably due to multiple classes of steroid-binding sites.

An indirect immunofluorescence assay was used to detect estradiol in MCF-7 breast cancer cells to determine if the estradiol-specific fluorescence observed represented estrogen receptor-bound estradiol. Appropriate controls were used to demonstrate the immunological specificity of our assay procedures. Initial studies of estradiol binding in MCF-7 cells were performed at 20 degrees for 1 hr with different concentrations of estradiol. Cytoplasmic and nuclear staining were observed following treatment with 10 nM estradiol, but not with lower concentrations which were nevertheless still sufficient to saturate estrogen receptor. The staining intensity increased with higher estradiol concentration, which is consistent with estradiol binding to lower-affinity binding sites. In order to further determine if estradiol binding by estrogen receptor was being detected, we pretreated MCF-7 cells with 5 nM diethylstilbestrol at 37 degrees for 1 hr to translocate all estrogen receptor to the nucleus and then administered estradiol at varying concentrations for 4 hr at 4 degrees. The estradiol was still primarily detected in the cytoplasm, although virtually all of the estrogen receptor was found to be present in the nucleus by standard [3H]estradiol binding assays. Additional immunochemical studies using sucrose gradient analysis to detect antibody-estradiol-receptor complexes clearly established that these complexes could not be detected. The present results suggest that, although immunocytochemical assays can specifically detect estradiol in MCF-7 cells, the estradiol is bound to lower-affinity binding sites rather than to estrogen receptor. Saturation analyses of intact viable MCF-7 cells performed at 37 degrees for 30 min using [3H]estradiol at concentrations ranging from 0.1 to 93 nM revealed an additional lower-affinity estradiol-binding site besides the receptor, perhaps analogous to the Type II sites reported in the rat uterus and human breast cancers.

We studied natural killer (NK) activity and lymphocyte subsets in 11 active parenteral heroin abusers, 11 long-term methadone-maintained former heroin abusers and 11 apparently healthy individuals. All subjects were males aged 23 to 49 and none had active infectious or inflammatory diseases. All current or former heroin abusers were seronegative for antibody to human immunodeficiency virus. The methadone maintenance patients were socially rehabilitated and had not abused drugs parenterally for at least 10 years. NK activity was determined by a standard Cr-release cytotoxicity assay using K562 cells as targets, and lymphocyte subsets were determined by direct immunofluorescence using flow cytometry. At all three effector-target ratios (100:1, 50:1 and 25:1), NK activity was reduced significantly (P less than .01) in parenteral heroin abusers compared with methadone maintenance patients and apparently healthy individuals. The latter two groups did not differ from each other. Parenteral heroin abusers also had higher absolute numbers of CD2, CD3, CD4 and CD8-positive cells. These data support our hypothesis that significant abnormalities of cellular immunity in parenteral heroin abusers can be normalized by successful long-term methadone treatment.