Detection of membrane and soluble interleukin-6 receptor in lymphoid malignancies.

We studied the membrane expression of the gp80 chain of IL-6 receptor (IL-6R) by quantitative flow cytometry in chronic lymphocytic leukaemia (CLL) and leukaemic centrocytic lymphoma using a panel of seven monoclonal antibodies. IL-6R was detected in 18/26 CLL cases and 4/7 lymphoma cases, with a mean antigen density < 3000 molecules/cell. Multiple labelling experiments confirmed the IL-6R expression by neoplastic cells. Specific mRNA was found by RT-PCR in neoplastic cells. A specific ELISA test was designed using two anti-IL-6 receptor MAbs to measure the serum soluble IL-6R (sIL-6R) in CLL (n = 48). B-cell non-Hodgkin's lymphoma (NHL; n = 40), and monoclonal gammopathy (MG; n = 32). SIL-6R was higher in CLL (170 +/- 12.6 ng/ml) in NHL (160 +/- 12 ng/ml) and MG patients (183 +/- 23 ng/ml) than in age-matched controls (100 +/- 5.6 ng/ml; P < 0.001) and higher in high-grade than low-grade NHL. No correlation was noted with a previous treatment. Among CLL cases the patients classified as stage B according to the Binet's staging of the disease had the highest sIL-6R values, thus suggesting a link with tumour cell mass.

Angiotensin receptors from rat liver, brain and pituitary gland. Expression of two subtypes in Xenopus oocytes.

Xenopus laevis oocytes were used to express angiotensin receptors encoded by mRNAs extracted from rat liver, adenohypophysis and brain. Groups of ten mRNA-injected oocytes were loaded with 45Ca2+ and the responsiveness to angiotensin II (A II) and related molecules tested by monitoring 45Ca2+ outflux. A II and angiotensin III (A III) induced a marked and transient increase in 45Ca2+ outflux from mRNA, but not from control, water-injected, oocytes. The increase over basal value of 45Ca2+ outflux during a 5 min application period of A II or A III was used as a response index. Observed responses were of high magnitude, reproducible and dose-dependent. For these reasons, mRNA-injected oocytes constitute a valuable system for investigating the pharmacological properties of angiotensin receptors from tissues of different origin under experimental conditions which eliminate tissue-specific interference which might be encountered in classical binding studies on acellular preparations. We demonstrate a fairly good parallelism between the relative potencies of A I, A II and A III in eliciting an increase in 45Ca2+ outflux from liver and adenohypophyseal mRNA-injected oocytes and the relative affinities of these peptides for binding to liver or adenohypophyseal membranes (A II greater than A III much greater than A I). The predominant receptor subtype expressed by brain mRNA discriminated very poorly between A II and A III, whereas angiotensin receptors expressed by liver or adenohypophyseal mRNA discriminated between AII and AIII very efficiently.

Oxytocin receptors from LLC-PK1 cells: expression in Xenopus oocytes.

Two selective radioligands for oxytocin receptors, [3H]-[4-threonine,7-glycine]oxytocin [( 3H]-[Thr4,Gly7]OT) and 125I-[1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine, 4-threonine, 8-ornithine, 9-tyrosine amide]-oxytocin (125I-OTA), were used to characterize oxytocin receptors from two pig kidney-derived cell lines, LLC-PK1 and LLC-PK1L. [3H]-[Thr4,Gly7]OT and 125I-OTA bind with high affinity (mean Kd values of 14 and 0.06 nM, respectively) to the same population of sites on LLC-PK1 cell membranes [maximum binding (Bmax) of 100 fmol/mg membrane protein]. These sites had the expected ligand selectivity of oxytocin receptors. [3H]-[Thr4,Gly7]OT and 125I-OTA binding sites could be distinguished from V2 vasopressin receptors present on LLC-PK1 and LLC-PK1L cells on the basis of clearly different maximal capacities and ligand selectivities, different sensitivities to insulin and serum, and absence of heterologous downregulation. Oxytocin receptors from LLC-PK1 cells have no functional relationship with adenylate cyclase. [Thr4,Gly7]OT affected neither the basal adenosine 3',5'-cyclic monophosphate (cAMP) content nor the vasopressin-induced cAMP accumulation by LLC-PK1 cells. Xenopus laevis oocytes injected with LLC-PK1 cell mRNA responded to [Thr4,Gly7]OT by an increase in 45Ca2+ outflux; this effect is antagonized by a highly selective oxytocin antagonist.

Estradiol increases the production of alpha 1-antichymotrypsin in MCF7 and T47D human breast cancer cell lines.

alpha 1-Antichymotrypsin (Achy) is an antiprotease of the acute inflammation phase, which is also released by MCF7 human breast cancer cells in culture. Using a fluorimetric assay with the synthetic substrate L-Seryl-L-Tyrosyl-2-N-naphthylamide, we have shown that a medium conditioned by MCF7 cells treated by estradiol inhibits the activity of alpha-chymotrypsin. This inhibition increased when physiological concentrations of estradiol were added to the cells for 2 days. It was due to an increased production of Achy and not to a direct effect of estradiol on alpha-chymotrypsin activity as shown by double immunoprecipitation with an antiserum against human alpha 1-antichymotrypsin. An increased accumulation by estradiol of an antigen located in the cytoplasm of MCF7 cells, which was revealed by immunoperoxidase staining with antibodies to Achy, also indicated that estradiol increased the production of Achy in these cells. Similar immunostaining was observed in a breast cancer tissue. Most of the estrogen regulated 60-68 kDa protein secreted by T47D cells (Chalbos et al. (1982) J. Clin. Endocrinol. Metab. 55, 276-283) was also specifically immunoprecipitated by the antibodies to Achy. Thus, alpha 1-antichymotrypsin is the first protein to be identified which is induced by estradiol and secreted by breast cancer cells.

A cytosol protease from the estrogen-resistant C3H mammary carcinoma increases the affinity of the estrogen receptor for DNA in vitro.

We have previously shown, in the estrogen-unresponsive C3H mouse mammary tumor that the affinity of the estrogen receptor (ER) for calf thymus DNA in vitro is four-times higher than that of uterine ER [Baskevitch, P. P., Vignon, F., Bousquet, C. and Rochefort, H. (1983) Cancer Res. 43, 2290]. By mixing cytosols from this tumor and uterus, we describe a tumor factor capable of increasing ER affinity for DNA, as assayed by DNA-cellulose chromatography and saturation studies. The activity of this factor was inhibited by alpha-chymotrypsin-inhibitors such as N-tosylphenylalanylchloromethane and chymostatin. Using the fluorogenic substrate glutarylglycylglycylphenylalanyl-N-naphthylamide, we assayed such a protease in the C3H mammary tumor cytosol. This protease and the factor altering ER-DNA binding were eluted together from chromatography on DEAE-cellulose, AcA 44, and carboline-agarose and were sensitive to the same inhibitors. The partially purified factor decreases the molecular mass of the estrogen receptor as seen by denaturing electrophoresis after covalent labelling of the ER with [3H]tamoxifen aziridine. We suggest that the increase of ER affinity for DNA and the decrease of ER molecular size are due to the same protease with an alpha-chymotrypsin-like specificity.

Increased DNA binding of the estrogen receptor in an estrogen-resistant mammary cancer.

In the C3H mouse mammary adenocarcinoma, estradiol cannot induce the progesterone receptor, and the tumor growth rate is not decreased by ovariectomy. To find an explanation for this estrogen resistance, we have compared the estrogen receptor (ER) from this tumor to the ER of uterus and of the mammary tumors induced in rats by dimethylbenz(a)anthracene. Since the ER concentration of the C3H tumor is low (congruent to 20 fmol/mg protein), we have used iodoestradiol of high specific activity to label the receptor. Several criteria of ER activation were studied. The dissociation rates of estradiol with or without sodium molybdate were similar in all tissues. In metrizamide isopycnic gradients, ER from rat uterus and C3H tumor had a similar density, both in the presence or absence of DNA. The binding of ER to DNA-cellulose was analyzed by incubating to equilibrium a constant amount of ER with a variable amount of DNA, the cellulose concentration being kept constant. The saturation data were plotted according to the method of Scatchard. The apparent affinity for DNA of the cytosol ER was similar for the rat dimethylbenz(a)anthracene tumors and the uterus (Kd congruent to 10 microM) but was significantly higher for the C3H tumor ER (Kd congruent to 2.3 microM). Neither the substitution of estradiol by iodoestradiol, nor the difference in cytosol protein and ER concentrations, nor the nonspecific steroid binding to DNA-cellulose could explain this result. This difference was confirmed when using DNA-agarose or soluble DNA in sucrose gradients. Finally, the salt concentrations necessary to elute ER from DNA-cellulose columns were 0.20 and 0.28 M for uterine and C3H tumor ER, respectively. To conclude, the C3H tumor has a low content of ER which appears to have a higher affinity for DNA than the ER of estrogen-responsive tissue. We suggest that the reason for the inefficiency of ER in the C3H tumor may be related to its increased affinity for nonspecific DNA sites.

Estrogen-receptor--DNA interaction. Difference between activation by estrogen and antiestrogen.

We have compared the interaction of the cytosol estrogen receptor with calf thymus DNA after its binding to [3H]estradiol or 4-hydroxy[3H]tamoxifen, a high-affinity antiestrogen, in an attempt to find a difference in the nature of these two complexes. The activation of the receptor and its binding to DNA were simultaneously obtained during an 18-h incubation at 0 degree C. The binding of a constant concentration of estrogen receptor to increasing concentrations of adsorbed DNA was examined. Scatchard representation of the data showed that the antiestrogen--receptor complex has a twofold lower affinity for DNA than the estradiol--receptor complex, while the proportion of receptor able to bind DNA was the same whether bound to estradiol or antiestrogen. Similar results were obtained by using soluble DNA and separation of the unbound and DNA-bound receptor on sucrose gradients. These results indicate that the estrogen receptor binds in vitro to non-specific double-stranded DNA with a lower affinity when it has been activated by an antiestrogen than when activated by estradiol.

Isopycnic banding in metrizamide of the uterine cytosol and nuclear estradiol receptor.

We have characterized the cytosol and the nuclear estrogen receptors (RE) of immature lamb uterus and their complexes formed with DNA and chromatin by using metrizamide isopycnic gradients. In low salt, the cytosol RE had a density of 1.238 +/- 0.002 g/cm3. This density was increased by Ca2+-activated proteolysis (1.275 g/cm3) and heat transformation of the receptor (1.257 g/cm3) and lowered by trypsin treatment (1.20 g/cm3), DNA binding (1.20 g/cm3) or molybdate treatment. In high salt (0.5 M KCl) both the native and the heat-"transformed" cytosol RE banded at the same density of 1.26 g/cm3. The 8S RE had the same density when bound to E2 or to 4-hydroxy-tamoxifen. Endometrial nuclei purified after nuclear translocation of RE were digested by micrococcal nuclease to solubilize the nuclear RE under low salt conditions. The majority of the extracted nuclear RE had a density similar to that of the 8S cytosol RE (1.239 +/- 0.002) and thus was different from the proteolyzed and heat "transformed" forms. Conversely, after a slight digestion of the nuclei, RE banded with chromatin at 1.208 +/- 0.001 g/cm3. In low salt, both forms of the nuclear RE, but not the trypsin proteolyzed nuclear RE, were displaced by naked DNA added in vitro. We conclude that the cytosol RE and the free nuclear RE have, in low salt, the same density and DNA-binding ability and that metrizamide isopycnic analysis is a good method for quantifying the interactions of the receptor with DNA and chromatin.