Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components.

We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.

PIP: The hypothesis that variations in the conformation of the progesterone receptor induced by the antiprogesterone RU38486 compared to the progestin ORG2058 may cause differences in the association of the receptor with some of the chromatin components was investigated. The approach to analyzing the functional organization of the receptor in the chromatin was to study the receptor-bound fragments released by nuclear digestion. The physical properties of these receptor-bound chromatin fragments were characterized by sucrose gradient sedimentation and gel filtration. Micrococcal nuclease digestion solubilized 2 receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only 1 receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking resulted in 6.7 S forms for the antiprogestin receptors and 4.4 S forms for the progestin ligated receptors. Use of the sedimentation coefficient and the Stokes radius yielded molecular weights of 202,000 and 116,000 for the antiprogestin and progestin ligated receptors, respectively. Overall, these kinetic studies were unable to explain the variation in responses of the target cell to agonist or antagonist ligands. It is postulated that the antagonist exerts a subtle, but important, conformational change in the receptor, which alters the receptor's ability to associate with some chromatin components and thus affects the normal events in gene expression.

Activation of the insulin-like growth factor 1 signaling pathway by the antiapoptotic agents aurintricarboxylic acid and evans blue.

Aurintricarboxylic acid (ATA), an endonuclease inhibitor, prevents the death of a variety of cell types in culture. Previously we have shown that ATA, similar to insulin-like growth factor I (IGF-I), protected MCF-7 cells against apoptotic death induced by the protein synthesis inhibitor cycloheximide. Here we show that ATA and a polysulfonated aromatic compound, Evans blue (EB), similar to IGF-I, promote survival and increase proliferation of MCF-7 cells in serum-free culture medium. This may suggest a common signaling pathway shared by the aromatic polyanions and IGF-I. Therefore, the ability of these aromatic compounds to activate the signal transduction pathway of IGF-I was examined. We found that ATA and EB mimicked the IGF-I effect on tyrosine phosphorylation of the IGF-I receptor (IGF-IR) and its major substrates, insulin receptor substrate-1 (IRS-1) and IRS-2; induced the association of these substrates with phosphatidylinositol 3-kinase and Grb2; and activated Akt kinase and p42/p44 mitogen-activated protein kinases. ATA and EB competed for IGF-I binding to the IGF-IR. ATA was found to be selective for the IGF-IR, whereas EB also activated the insulin receptor. Upon fractionation of commercial ATA by size exclusion chromatography, we found that fractions that enhanced the intensity of tyrosyl-phosphorylated IRS-1/IRS-2 also increased the survival of MCF-7 cells in the presence of cycloheximide, whereas fractions devoid of IRS phosphorylation activity had no survival ability. Taken together, these results suggest that the survival/proliferation-promoting effects of ATA and EB in MCF-7 cells are transduced via the IGF-IR signaling pathway.

Unoccupied nuclear receptors for estrogen in human endometrial tissue.

PIP: The nuclear content of estrogen-binding sites in normal and pathological endometrial tissue was studied. Extracts of cell nuclei from normal, nonmalignant, and cancerous human endometrial tissues were used, and at 0 degrees centigrade were found to contain material capable of binding estradiol in vitro. Only binding sites unoccupied by endogenous estrogen were determined. Estrogenic receptor (ER) character of this binding was demonstrated by 3 findings: 1) high affinity of binding to estradiol; 2) specificity of binding, competition by diethylstilbestrol and estriol for estradiol binding, and absence of competition by cortisol, progesterone, and 5 alpha-dihydrotestosterone; and 3) sedimentation constant at about 4S in sucrose density gradient. Next, occupied receptors were determined at 30 degrees centigrade. Both occupied and unoccupied receptors were measured by a single saturating dose of 7.5 nM tritiated estradiol with or without a 100-fold excess of diethylstilbestrol to estimate the amount of nonspecific binding. All specimens assayed had unoccupied nuclear receptors. This unoccupied receptor comprised from 9-37% of the total estradiol receptors (cytoplasmic plus nuclear). Such a large percentage of unoccupied nuclear receptors in both normal and pathological endometrial material may indicate that the unoccupied receptor is a necessary product in the normal mechanism of estradiol action.^ieng

Progesterone receptor levels and distribution in different phases of the menstrual cycle and in estrogen-primed postmenopausal endometrium.

Progesterone receptor levels were measured in the cytosol and in the 0.4 M KCl nuclear extract of normal cyclic endometria in 49 women, classified into four categories: proliferative, midcycle, secretory, and estrogen-primed (four to 11 days); [3H]R5020 was used as a ligand. The women in each category were further subdivided into test and control groups; the women in the test group received a progesterone injection one to three hours before tissue collection. Both the nuclear progesterone receptor levels and the portion of the progesterone receptor in the nuclear extract were significantly higher in the test than in the control groups; among the test groups in the four categories, however, these changes were significantly smaller in women in the secretory category than in the other three categories. These results indicate that the progesterone receptor activation-translocation dynamics in human endometrium are dependent upon the hormonal status of the woman.

Tamoxifen increases plasma estrogen-binding equivalents and has an estradiol agonistic effect on histologically normal premenopausal and postmenopausal endometrium.

OBJECTIVE: To determine the effects of single dose tamoxifen on plasma estrogen (E)-binding equivalents and endometrial estradiol (E2) and progesterone (P) receptors. DESIGN: Controlled clinical study. SETTING: Normal human volunteers were studied in an academic research environment. PATIENTS: Premenopausal and postmenopausal women with histologically normal endometrium undergoing curettage or hysterectomy were selected. INTERVENTIONS: Tamoxifen was administered orally; blood and endometrial samples were collected 4 to 96 hours after tamoxifen administration. MAIN OUTCOME MEASURES: Plasma E-binding equivalents, endometrial cytosolic and nuclear E2 and P receptors. RESULTS: (1) Plasma E-binding equivalents increased eightfold at 4 to 24 hours of tamoxifen administration and declined exponentially thereafter, reaching control levels at 73 to 96 hours. Plasma E-binding equivalents were not affected by endogenous E2 levels. (2) Endometrial total E2 and P receptor levels increased in all women 2.9 to 19.2-fold after tamoxifen. (3) Tamoxifen resulted in an increase in the fraction of the E2 receptor measured in the nuclear extract 2.1 to 7.5-fold in midcycle, secretory, and menopausal endometria but not in proliferative endometrium. CONCLUSIONS: (1) Tamoxifen has an E2 agonistic effect on histologically normal human endometrium. (2) Irrespective of the total level of the endometrial E2 receptor, the nuclear capacity of that receptor in vivo is limited (approximately 75% to 80% of the total level).

Progesterone (P) receptor dynamics in estrogen primed normal human cervix following P injection.

Progesterone (P) receptor levels were measured in the cytosol and in the 0.4 M KCl nuclear extract in human cervical tissues with [3H]-R5020 as a ligand and the results compared with those obtained in the myometrium and endometrium of the same uteri. Tissue samples were obtained from 28 women, grouped as follows: group A, 12 premenopausal controls; group B, 7 perimenopausal women who received estrogen 7 to 11 days before operation; and group C, 9 perimenopausal and postmenopausal women who received estrogen as in group B plus P injection 1 to 3 hours before operation. Estrogen administration resulted in a significant rise in total P receptor levels in the cervix, compared with the endometrium and myometrium. P injection after estrogen priming resulted in down-regulation of the P receptor in the cervix to undetectable levels, whereas in the endometrium and myometrium it resulted only in redistribution of the P receptor, with higher nuclear levels and lower cytosol levels. These results indicate the possibility of a different mechanism of regulation of the P receptor in the human cervix, compared with that in the endometrium and myometrium.

Characterization and assay of the nuclear and cytosolic progesterone (P) receptors in Premarin-primed human endometrium and myometrium after single-dose P administration.

Progesterone receptors (RP) were characterized and measured in the 0.4 M KCl nuclear extract and in the cytosol of 7 to 11 days Premarin (Premarin, Ayerst Laboratories, New York, NY) primed human endometrium and myometrium with [3H]R5020 as the ligand. The test group comprised 12 women who received progesterone injection 1 to 3 hours before tissue collection; the control group numbered 10 women. Receptor characteristics in both the endometrium and the myometrium, in the nuclear extract and cytosol in the test group and in the cytosol in the control group, were similar and were demonstrated by (1) high specificity, (2) sedimentation constant of 4.7 to 6.5S in the cytosols and 2.8 to 3.9S in the nuclear extract, (3) saturable affinity with Kd of 2.2 to 3.5 nM and 1.9 to 3.3 nM, respectively. Total RP levels (nuclear extract and cytosol) ranged from 1030 to 13,270 and 1280 to 17,300 fmol/mg DNA, respectively, in the endometria of women in the test and control groups. In 13 of 14 women myometrial total RP levels were higher than in the endometrium. There was a significant difference in the distribution of the RP between the test and the control groups: 50% and 48% of the receptor were measured in the nuclear extract in the endometrium and myometrium, respectively, in the test group, compared with 25% and 21% in the control group (P less than 0.001).

Comparison of the physical properties of the nuclear progesterone receptor, bound to antiprogestin RU 486 or progestin ORG 2058, following limited proteolysis.

The susceptibility of the progesterone receptor, liganded either by the antiprogestin RU 486 or by the progestin ORG 2058, to chymotrypsin and trypsin degradation was investigated. The nuclear fraction was isolated from T47D cells previously exposed either to 0.1 microM [3H]RU 486 or to 0.1 microM [3H]ORG 2058. The proteolytic digestion was performed on the micrococcal nuclease hydrolysate. The molecular weights of the receptor fragments were calculated, in high salt buffer, from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration on an Agarose A-0.5 m column. Micrococcal nuclease solubilized receptor forms with molecular weights of 80,000 and 75,000 for the antiprogestin- or progestin-liganded receptor, respectively. Chymotrypsin degraded these receptor forms to fragments with molecular weights of 23,000 either for the antiprogestin- or progestin-liganded receptor. Similar molecular weights of 23,000 were calculated for the progesterone receptor liganded either by the antiprogestin RU 436 or the progestin ORG 2058 following trypsin cleavage. We conclude that the degradation pattern of the progesterone receptor liganded either by the antiprogestin RU 486 or the progestin ORG 2058 following chymotrypsin or trypsin digestion seems to be similar.

Characterization of the nuclear progesterone receptor in normal Premarin-primed post-menopausal endometrium.

Nuclear progesterone receptors (RPN) were characterised in a 0.4 M KCl nuclear extract of Premarin-primed normal post-menopausal human endometrium after progesterone injection using [3H]R5020 as a ligand. The receptor character of the binding was demonstrated by the following findings: the high specificity for progesterone binding; the high saturable affinity (dissociation constant approx. 2.5 nM) for R5020; the sedimentation coefficient at high salt concentration (approx. 3 S); and the change in distribution of the RP following progesterone administration. These results indicate that normal post-menopausal human endometrium retains the property of translocation (2-step model) or nuclear activation and retention (nuclear receptor model) of the RP if it is exposed to the appropriate oestrogenic and progestational hormonal milieu.

Effect of progesterone injection on progesterone receptor levels and distribution in untreated and short-term Premarin-primed pre-menopausal and post-menopausal endometrium.

Cytosolic and nuclear progesterone receptors (RPC, RPN) were measured in post-menopausal endometria, using [3H]R5020 as the radioligand, and the findings compared with those in pre-menopausal endometria. Total RP levels (RPC + RPN) in post-menopausal endometria were low, i.e. less than 2000 fmol/mg DNA. A 7-11 day course of Premarin (conjugated equine oestrogen) treatment in post-menopausal subjects resulted in RP levels in 11818 +/- 3008 fmol/mg DNA, which were higher than those in proliferative, mid-cycle and Premarin-primed pre-menopausal endometria. Progesterone injection 1-3 h before tissue collection resulted in a change in the distribution of the RP in both premenopausal and post-menopausal Premarin-primed endometria and pre-menopausal proliferative and mid-cycle endometria. Following the progesterone injection RPN levels increased to 57 +/- 9% of the total as compared with 23 +/- 8% in endometrial samples from women who received no progesterone.

Phosphorylation of A 27-kDa protein correlates with survival of protein-synthesis-inhibited MCF-7 cells.

Previously, we have shown that IGF-1, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and aurintricarboxylic acid (ATA) protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). We proposed that phosphorylation of a putative cellular proteins(s) may be involved in this survival mechanism. In the present study we investigated the ability of several agents to induce phosphorylation of cellular proteins and correlated this ability to their survival effect. We found that TPA, ATA, and IGF-1 increased the degree of phosphorylation of a 27-kDa protein in a dose- and time-dependent manner in CHX-treated MCF-7 cells. The ED50 values observed were 25 ng/ml, 40 micrograms/ml and 15 ng/ml for TPA, ATA, and IGF-1, respectively. The effect was measured upon 10 min of cell treatment with each agent; it reached maximum at 60 min and thereafter decreased continuously to control levels. The 27-kDa protein was found in the cytosolic fraction as a phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphoprotein was resolved into two isoforms with pI 5.7 and 5.9. Such characteristics were observed for the small molecular weight heat shock protein HSP27. Indeed, a single band of 27 kDa was detected immunologically with rabbit polyclonal anti-human HSP27. The inactive phorbol ester alpha TPA, epidermal growth factor (EGF), and 8-bromoadenosine 3'5'-cyclic monophosphate (Br-cAMP) did not increase phosphorylation of the 27-kDa protein. Cell survival was measured by exposure of the CHX-pretreated cells to increasing concentrations of the various agents for 60 min, followed by a further incubation for 48 h in the presence of CHX only. TPA, ATA, and IGF-1 were found to enhance cell survival, whereas alpha-TPA, EGF and Br-cAMP did not. Our results indicate a correlation between phosphorylation of a 27-kDa protein, probably HSP27, and enhanced cell survival, suggesting a role for this phosphoprotein in the survival mechanism.

Epidermal growth factor and insulin-like growth factor-1 protect MDA-231 cells from death induced by actinomycin D: the involvement of growth factors in drug resistance.

In the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2 microgram/ml), or by a short-time exposure to a high dose of ACT-D (2 micrograms/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2 micrograms/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor, phorbol esters, and aurintricarboxylic acid are survival factors for MDA-231 cells exposed to adriamycin.

The ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), insulin, 12-O-tetradecanoylphorbol-13-acetate (TPA), and aurintricarboxylic acid (ATA) to protect the human breast cancer cell line MDA-231 from death induced by the anticancer drug adriamycin was investigated. Cell death was induced in the MDA-231 cells either by a short-time exposure to a high dose of adriamycin (2 micrograms.ml-1.1h-1) and further culturing in the absence of the drug, or by continuous exposure to a low dose of adriamycin (0.3 micrograms/ml). Cell death was evaluated after 48 h of incubation by several techniques (trypan blue dye exclusion, lactic dehydrogenase activity, cellular ATP content, transmission electron microscopy, and DNA fragmentation). EGF, TPA, and ATA, each at an optimal concentration of 20 ng/ml, 5 ng/ml, and 100 micrograms/ml respectively, substantially enhanced survival of cells exposed either to a high or low dose of adriamycin. Neither IGF-1 nor insulin, each at concentrations of 20 ng/ml, had an effect on cell survival. The three survival factors enhanced protein synthesis in the untreated cells and attenuated the continuous decrease in protein synthesis in the adriamycin-treated cells. Moreover, the three survival factors protected the MDA-231 cells from death in the absence of protein synthesis (cycloheximide 30 micrograms/ml). These results suggest that EGF, TPA, and ATA promote survival of adriamycin pretreated cells by at least two mechanisms: enhancement of protein synthesis and by a protein synthesis independent process, probably a posttranslational modification effect.

Characterization of the nuclear progesterone receptor in human uterine leiomyoma.

Progesterone binding components were characterized and measured in the 0.4 M KCl nuclear extract of Premarin-primed human leiomyoma (LM) following progesterone injection (n = 5) using [3H]R5020 as the ligand. The receptor character of the binding was demonstrated by: high specificity for progesterone and R5020 binding, high saturable affinity (Kd approximately 2.5 nM) for R5020, sedimentation coefficient in high salt concentration (approximately 3 S), change of distribution of the receptor after progesterone administration, i.e. an increase in the nuclear and decrease in the cytosolic receptor levels. Similar results were demonstrated in the corresponding myometria and endometria. The characteristics of the nuclear progesterone receptor in all three tissues of the test group were similar to those of the cytosolic progesterone receptor in the test group and in the control group (no treatment before operation, n = 5). These results indicate that the nuclear progesterone receptor in human uterine LM shares similar characteristics to those of the myometrium and endometrium.

Analysis of the nuclear estrogen receptor from MCF-7 cells by limited proteolysis.

The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000. Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000. Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000. The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography. About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer. We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively. Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain.

Analysis of the 4-hydroxytamoxifen (4-OHTAM) bound nuclear estrogen receptor from MCF-7 cells by limited proteolysis.

The assumption that a different conformational form was induced in the nuclear estrogen receptor following binding by antiestrogens compared to estrogens was studied by analysing the proteolytic fragments of the receptor following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from MCF-7 cells previously exposed to [3H] 4-OHTAM. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients (S) determined on a sucrose gradient and from the Stokes radii (Rs) estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 155,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 63,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. A similar receptor molecule was released by chymotrypsin from intact nuclei. Digestion of the micrococcal nuclease hydrolysate with trypsin degraded the receptor to a form of a Mr = 67,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. Digestion of intact nuclei with trypsin followed by micrococcal nuclease, solubilized a receptor form of Mr = 80,000 which could be further dissociated with 0.4 M KCl and 3 M urea to a receptor form of Mr = 67,000. This trypsin degraded receptor form seems to be similar in Mr to the chymotrypsin degraded form. On the other hand different receptor fragments of Mr = 33,000 and Mr = 60,000 were excised by chymotrypsin and trypsin respectively from the estradiol ligated estrogen receptor. (Geier et al., J. steroid Biochem. 26 [1987] 35-40.) These results support the assumption of a different conformational form for the antiestrogen ligated receptor, compared to the estrogen ligated receptor since they were differentially susceptible to proteolytic degradation by chymotrypsin.

Physical-chemical properties of the estrogen receptor solubilized by micrococcal nuclease.

The physical-chemical properties of the nuclear estrogen receptor from MCF-7 cells were determined. The receptor was solubilized by micrococcal nuclease. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The amount of receptor released was parallel to the extent of chromatin solubilized, which suggested that the receptor is homogeneously distributed on the chromatin. Following mild nuclease digestion the excised receptor sedimented as an abundant 6-7 S form and as a less abundant approximately 12 S species. The 6-7 S form represented the receptor excised in association with linker DNA, while the approximately 12 S may represent receptor bound to linker DNA which remained associated with the nucleosome. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.6 S. Additional digestion with DNase I did not affect the sedimentation coefficient of the receptor. Sedimentation of the micrococcal nuclease hydrolysate in a 0.4 M KCl sucrose gradient resulted in a 4.2 S receptor form. The same receptor form was extracted from undigested nuclei with 0.4 M KCl. Using Sephadex G-200 column chromatography we have determined the Stokes radii (Rs), molecular weight (Mr) and frictional ratio (f/fo) for the 5.6 S and 4.2 S receptor forms. For the 5.6 S form: Rs = 7.04 nm, Mr = 163,000 and (f/fo) = 1.80. For the 4.2 S receptor, Rs = 4.45 nm, Mr = 77,000 and (f/fo) = 1.46. The ability of the nuclease solubilized 5.6 S receptor to bind DNA was tested using DNA-cellulose column and highly polymerized DNA. About 40% of the applied receptor bound to the column and could be eluted by high salt concentrated buffer. The 5.6 S receptor form was sedimented on sucrose gradient by the highly polymerized DNA. These results suggested that the receptor is bound in chromatin as a dimer or as a monomer in association with other protein(s) which complexed it with DNA.

The effect of castration on the synthesis and secretion of proteins in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

UNLABELLED: The effect of castration on the incorporation of [35S]methionine into secreted proteins in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, was investigated. Biopsy specimens were obtained from 19 tumors, 0, 24, 48, 72, and 96 h after castration. In 14 tumors, castration induced an increase in the incorporation (mean of 5-fold), reaching the maximal level after 24 h (3 tumors), 48 h (7 tumors), 72 h (3 tumors), and after 96 h (1 tumor). In three tumors castration did not alter the incorporation rate, while in two tumors incorporation declined immediately after castration. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the labeled secreted protein showed that castration did not decrease or increase significantly the incorporation of [35S]methionine into any of the major labeled proteins. CONCLUSION: tumor regression following hormonal deprivation is apparently preceded by an increased synthesis of secreted proteins. However, no qualitative differences in any major labeled proteins could be observed.

Characterization and assay of progesterone-binding components in DMBA-induced rat mammary carcinoma tissue after progesterone administration.

Nuclear and cytoplasmic progesterone-binding components were characterized and measured in DMBA-induced rat mammary carcinoma tissue, before and after progesterone administration. Rats, bearing growing tumors, were ovariectomized and then primed for two days with estradiol. Biopsy specimens were taken prior to or following administration of progesterone. Nuclear binding was assayed in the 0.4 M KCl extract of the nuclear fraction using [3H]R5020 as ligand. The receptor character of the binding was demonstrated by: (1) high affinity (Kd approximately 2 nM); (2) specificity: competition by R5020 and progesterone, minimal competition by 17 alpha-hydroxyprogesterone, corticosterone, testosterone and estradiol; (3) sedimentation constant at about 3S in a sucrose density gradient. Similar characteristics displayed the cytoplasmic receptor before and after progesterone administration. Progesterone receptor distribution in the nuclear extract and cytosol were determined in 36 tumors. The levels of total receptors (cytoplasmic plus nuclear) before and after progesterone administration varied widely, however the average values found after progesterone administration were significantly lower, 1.59 +/- 0.20 pmol/mg DNA compared to 2.58 +/- 0.32 pmol/mg DNA. Before progesterone administration only cytoplasmic receptors were found. One hour after progesterone administration a variable amount of the receptor (0-40%) was found in the nucleus of the tumorous tissue. In uteri of the same rats a uniform distribution of receptors (about 40% in the nucleus) was found after progesterone administration. A defect in the translocation process might be considered in the tumors with low receptors level, which suggests that DMBA-tumors may not respond uniformly to progesterone administration.