Presence of estrogen binding sites and growth-stimulating effect of estradiol in the human myelogenous cell line HL60.

In the present study, we investigated the effects of estrogens on the growth of the HL60 line in vitro and the presence of estrogen binding sites in the same cells. Cell proliferation was estimated by cell counts, [3H]thymidine incorporation, and determination of the percentage of cells in the S phase by flow cytometry. Cells maintained in a medium containing physiological concentrations of estradiol (10(-9) M, 10(-8) M, 10(-7) M) exhibited a growth stimulation, shown by an increase in the percentage of cells in the S phase, whereas a pharmacological concentration (10(-6) M) produced a growth inhibition. Furthermore, the addition of the specific antihormone, tamoxifen, inhibited the stimulating effect of the estrogens. Receptor analysis showed the presence of specific estrogen-binding sites with an apparent dissociation constant of about 5.3 X 10(-10) M. The effect of estrogen was therefore associated with the presence of estrogen receptors in the human leukemic cell line HL60.

Distribution of androgen and estrogen receptors among lymphoid and haemopoietic cell lines.

Estrogen receptors (ER) and androgen receptors (AR) were determined in a series of 23 leukemia or lymphoma cell lines including 8 T-cell lines, 12 B-cell lines, and 3 non lymphoid cell lines. The phenotypic characterization of these cells by currently available immunological markers provides an estimate of their stage of differentiation. The result indicate that none of the investigated cell lines bear simultaneously ER and AR. Four were found to bear ER: U266, RPMI 8226, HL60, IARC/310/LT2, and four to express AR: RAJI, IARC/301/LTI, U937, REH6. The presence of cytosolic receptors was always associated with that of nuclear receptors. The expression of either ER or AR is restricted to discrete maturation stages of different haemopoietic cell lineages. Thus AR were found among the most immature lines of the T, B or monocytic lineage but they could be detected neither in the promyelocytic line HL60, nor in the pluripotential K562. The present results are in keeping with the demonstration of AR in some leukaemic blasts or in non Hodgkin's lymphomas. In contrast with AR, ER are present in two myeloma cell lines, in the promyelocytic cell line, HL60 and in a T-cell line bearing the phenotype of mature suppressor/cytotoxic T cells.

Specific estrogen binding sites in human lymphoid cells and thymic cells.

The binding of estrogen in preparations of human peripheral blood mononuclear cells, as well as by splenic and thymic cells is demonstrated by three different approaches (Dextran-coated charcoal method, whole cell assay, and gel filtration on a sepharose 4B column). Scatchard's analysis of [3H]-moxestrol (R2858) and [3H]-estradiol binding proves the existence of a single class of receptor sites having a dissociation constant of 0.18-2.4 X 10(-9) M. Physicochemical properties of the binder, including binding capacity and steroid specificity, are quite similar to those reported for the thymus of small mammalian species or human thymoma.

Sex steroid receptors in peripheral T cells: absence of androgen receptors and restriction of estrogen receptors to OKT8-positive cells.

The immune response has been reported to be modulated by sex hormones in several models, and estrogen receptors have been demonstrated in the human thymus. We therefore investigated the presence of estrogen and androgen receptors among human peripheral T cells; thoracic duct lymph provided large amounts of circulating lymphocytes. Pure T cells were obtained by negative selection by using complement-dependent cytotoxicity with a monoclonal antibody against a monomorphic determinant of class II histocompatibility antigen (HLA-DR). Furthermore, subsets of OKT8-positive and OKT8-negative lymphocytes were selected by using an OKT8-like monoclonal antibody. Sex steroid binding was determined on purified nuclei; no androgen receptors could be demonstrated on peripheral T cells. The cytoplasmic [3H] 17-beta-estradiol-receptor complex was always translocated to the nucleus in vitro within 1 hr at 37 degrees C; no estrogen receptors were demonstrable on purified OKT4-positive subsets. Assuming that estrogen receptors were evenly distributed among OKT8-positive cells, their level could be estimated to be about 40 fmol/mg DNA. The restriction of estrogen receptors to T cells bearing the "suppressor-cytotoxic" phenotype suggests a possible pathway for the modulation of T cell suppressive activities by estrogens.

Characterization of a specific androgen receptor in non-Hodgkin's lymphoma cells.

The use of a competitive binding assay has permitted us to detect a cytoplasmic androgen receptor in cells of node biopsies of several patients suffering from non-Hodgkin's malignant lymphomas (NHML). These same cells appear to contain very low or undetectable numbers of estrogen receptor. The androgen receptors are saturated at approximately 2 X 10(-10) M [3H] 5 alpha DHT and Scatchard analyses of the binding data indicated a high affinity constant (Kd = 0.29 +/- 0.19 10(-9) M). The range of receptor sites is 19-327 fmol/mg protein (median = 42 fmol/mg protein). These cytoplasmic receptors are inactivated at 37 degrees C and pronase. The [3H] 5 alpha DHT receptor complex migrates in linear 5-20% sucrose gradients with a sedimentation coefficient of 7S and this peak is shifted to the 4S region under high salt conditions. Physiochemical properties of the binding including binding capacity and steroid specificity, are quite similar to those reported in target organs.

Androgen, estrogen and progestin binding sites in human leukemic cells.

Sex steroid binding capacity was investigated in malignant cells from 32 patients with acute non-lymphoblastic leukemia (25 patients with acute myeloid leukemia, 4 with subacute leukemia, 3 with chronic myeloid leukemia in blast crisis) and 30 patients with acute lymphoblastic leukemia. Specific binding of labelled steroids was characterized either by competition assay in cytosol fraction or by whole-cell incorporation. In some cases further characterization of the receptor complex was attempted by sucrose gradient centrifugation and gel filtration column. The results show the presence of specific binding sites for dexamethasone (22/32 in non ALL and 30/30 in ALL), for estrogens (11/15 in non-ALL and 5/12 in ALL), for progestins (8/25 in non-ALL and 5/13 in ALL) for androgens when R1881 was used as ligand (8/21 in non-ALL and 5/10 in ALL patients) but only 1/13 non-ALL patients and no ALL patients when labelled 5 alpha DHT was used. These results indicate that the blast cells from patients with acute leukemia contain specific proteins binding steroids with a high affinity. Our results for dexamethasone receptors are similar to those described in the literature in ALL and non-ALL.

Estrogen and progesterone receptors in some human myeloma cell lines and murine hybridomas.

The control of immune responses by sex hormones is well documented but the effect of sex hormones on lymphoid cell subsets is poorly understood. We have investigated the expression of receptors for androgens (AR), estradiol (ER) and progesterone (PR) by human cell lines of the B lymphocyte lineage and by murine myeloma or hybridomas. AR, ER and PR were determined by cytosol and nuclear binding assays. Eleven human lymphoblastoid cell lines obtained by in vitro infection of blood or tonsil B cells with Epstein-Barr Virus (EBV) B95, did not express AR or ER. Similarly, 10 Burkitt's lymphoma cell lines were AR, ER and PR negative with the exception of the pre-B RAJI cells which bear AR. Among 13 cell lines derived from patients with multiple myeloma none expressed AR but five were found to bear ER (20-164 fmol/mg DNA or 5-10 fmol/mg protein). Four of the latter group also bear PR (86-450 fmol/mg DNA). Two mouse hybridomas out of seven tested were ER and PR positive. The MOPC 315 myeloma expressed ER but not PR. The possible functional role of these sex hormone binding sites in cell proliferation and immunoglobulin secretion deserves further investigation.

Effect of physiological concentrations of sex hormones on the formation of "early" sheep red blood cell rosettes by human lymphocytes. Possible relations with the presence of sex-hormone-cytosol-receptors in lymphocytes.

The formation of "early" sheep red blood cell rosettes by normal human peripheral blood lymphocytes was increased by incubation with physiological concentration of 17 beta-Estradiol. This effect was shown to be hormone specific. Whole cell incorporation of radioactive hormones and analysis by gel filtration of cytosols incubated with labeled hormones have shown a saturable fixation of estrogens and androgens. These results raise the possibility that normal human peripheral blood lymphocytes are target cells for sex steroid hormones.