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.

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.

Induction of apoptosis in MDA-231 cells by protein synthesis inhibitors is suppressed by multiple agents.

In the present study we investigated the ability of several diverse agents to inhibit MDA-231 cell death induced by two different protein synthesis inhibitors, cycloheximide (CHX) and ricin. Cell death was evaluated by several techniques: trypan blue staining, determination of the released lactic dehydrogenase, transmission electron microscopy, and DNA fragmentation. Results from DNA gel electrophoresis and electron microscopy suggest a mechanism of death by apoptosis which terminates in necrosis. Approximately 60% of cell death was induced either by a continuous exposure to 30 micrograms/ml CHX for 48 hr or by a 1-hr exposure to 250 pg/ml ricin followed by a subsequent incubation of 48 hr in the absence of the drug. Cell survival, in the protein synthesis-inhibited cells, was enhanced by the following diverse agents: the growth factors EGF (20 ng/ml) and IGF-1 (20 ng/ml), the protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (5 ng/ml), the protein kinase A activator 8-bromoadenosine 3':5'-cyclic monophosphate (650 micrograms/ml), the nuclease inhibitor aurintricarboxylic acid (100 micrograms/ml), and fetal bovine serum (5%). The survival agents that stimulated protein synthesis in the control untreated cells had no effect on the CHX-inhibited protein synthesis, which indicates that new protein synthesis is not required for cell survival. The same survival agents attenuated the continuous decrease in protein synthesis in the ricin-exposed cells; therefore, the involvement of new protein synthesis in the survival mechanism could not be excluded. The protein kinase C inhibitor staurosporine blocked, in a dose-dependent manner, the survival effect of 12-0-tetradecanoyl-phorbol-13-acetate and EGF, but not that of aurintricarboxyclic acid or fetal bovine serum, in the protein synthesis-inhibited cells. These results provide evidence for several distinctive pathways, the activation of which inhibits MDA-231 cell death induced by protein synthesis inhibitors. Some of these pathways involved activation of protein kinases, probably protein kinase C.

Multiple pathways are involved in protection of MCF-7 cells against death due to protein synthesis inhibition.

Previously we have shown that IGF-I protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). In the present study we investigated the ability of protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the protein kinase A activator 8-bromoadenosine 3'5'-cyclic monophosphate (Br-cAMP), and the enzyme inhibitor aurintricarboxylic acid (ATA) to protect MCF-7 cells against death, due to a continuous presence of CHX. Cell death was evaluated after 48 h of incubation by several techniques (trypan blue staining, release of lactic dehydrogenase, cellular ATP content, transmission electron microscopy, and DNA fragmentation). Apoptosis which terminates in necrosis, characterized this mode of cell death. TPA and ATA at optimal concentrations of 40 ng/ml and 100 micrograms/ml, respectively, reduced cell death to the control level (without CHX), while Br-cAMP at an optimal concentration of 650 micrograms/ml reduced cell death only partially. IGF-1, TPA, and ATA, which stimulated protein synthesis in the control MCF-7 cells, had no effect on protein synthesis in the CHX-treated cells, indicating that the survival effect is not due to new protein synthesis. The protein kinase C inhibitor staurosporine blocked the survival effect of TPA and IGF-1 in a dose-dependent manner, however did not affect the survival effect of ATA. The tyrosine kinase inhibitor genistein blocked the survival effect of IGF-1, but not that of TPA and ATA. Our results provide evidence for several distinctive pathways, the activation of which protects MCF-7 cells against death, due to protein synthesis inhibition.

Insulin-like growth factor-1 inhibits cell death induced by anticancer drugs in the MCF-7 cells: involvement of growth factors in drug resistance.

The involvement of growth factors in cell survival in the presence of anticancer drugs was investigated. Cell death was induced in the human breast cancer cell line MCF-7, by the structurally and mechanistically unrelated chemotherapeutic drugs puromycin, actinomycin D, 5-fluorouracil, cisplatin, and adriamycin. The effect of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and insulin on cell death was evaluated by two different methods: (1) trypan blue dye exclusion test and (2) lactic dehydrogenase release into the culture medium. IGF-1 inhibited cell death induced by each of the diverse drugs in a concentration-dependent manner reaching a maximal effect at 40 ng/ml. Insulin mimicked the effect of IGF-1 only at supraphysiological concentration with an optimal effect at 10,000 ng/ml. EGF had no effect on cell death up to 100 ng/ml. Our finding that IGF-1 specifically enhanced MCF-7 cell survival in the presence of different anticancer drugs suggests the involvement of growth factors in the mechanism of drug resistance.

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.

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)

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.

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.

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.

Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I.

The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.

Characterization of the 4-hydroxytamoxifen (4-OHTAM) bound estrogen receptor of MCF-7 cells solubilized by micrococcal nuclease.

In order to get an insight into the molecular mechanism of antiestrogen action at the chromatin level, we characterized the physical-chemical properties of the chromatin fragments released by micrococcal nuclease digestion of nuclei isolated from MCF-7 cells previously exposed to [3H]4-OHTAM. The [3H]4-OHTAM bound solubilized fragments were characterized in a low ionic strength buffer and in a high ionic strength buffer without and with urea. The following parameters were determined: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Following mild nuclease digestion, the solubilized [3H]4-OHTAM bound receptor sedimented as an abundant 6-7 S form and a less abundant approximately 12 S species. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.2 S, Rs = 7.25 nm and Mr = 155,000. About 45% of the applied receptor bound to a DNA-cellulose column could be eluted by a high salt concentrated buffer. Dissociation of the micrococcal nuclease solubilized receptor in 0.4 M KCl resulted in a smaller receptor form with a 4.9 S, Rs = 5.87 nm and Mr = 119,000. Further dissociation in the presence of 3 M urea resulted in a receptor with a 3.5 S, Rs = 5.78 nm and Mr = 83,000. These results suggested that the antiestrogen bound estrogen receptor in chromatin, is associated with a tightly bound protein component and with an additional less tightly bound protein, complexes with DNA.

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.

Evidence for the origin of the unoccupied oestrogen receptor in nuclei of a human breast-cancer cell line (MCF-7).

The origin of the unoccupied nuclear oestrogen receptor (R(n)) was studied. Three working hypotheses were investigated. (a) R(n) is a dissociation product of the oestrogen occupied nuclear receptor (ER(n)). (b) ER(n) is only partially occupied, so that additional binding may occur at 0 degrees C (the temperature at which oestradiol saturates unoccupied sites). (c) R(n) is derived from the penetration of unoccupied cytoplasmic receptor (R(c)) into the nucleus. The MCF-7 cell line was used as a model in the present investigation. The amount of unoccupied receptors was measured by saturation with 7.5nm-[(3)H]oestradiol at 0 degrees C, whereas the occupied receptors were measured by exchange at 30 degrees C. The cells at preconfluency were exposed to a medium fortified with 10nm-[(3)H]oestradiol for 1h, washed and cultured up to 5 days in fresh growth medium. The distribution of oestradiol receptors was determined before exposure and during the following 5 days. After 1h exposure only ER(n) was found in the nuclear fraction. Thereafter ER(n) declined continuously so that on day 5 it approached 15% of its value measured 1h after exposure. Although after 3 days about 80% of ER(n) disappeared no R(n) appeared, which contradicts hypotheses (a) and (b). On day 4 R(n) and R(c) appeared simultaneously. The appearance of R(n) and R(c) was not prevented by culturing the cells in an oestrogen-free medium, supporting hypothesis (c). Exposure of cells to increasing concentration of [(3)H]oestradiol (0.1-10nm) for 1h resulted in a parallel increase in ER(n) without increasing the amount of unoccupied binding sites, which contradicts hypothesis (b). The present study supports the hypothesis (c), i.e., R(c) may also penetrate the nucleus without binding to oestradiol.

Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7).

1. A method to measure both occupied and unoccupied oestrogen receptors directly in the crude nuclear fraction of the MCF-7 cells was developed. The receptors had high affinity for oestradiol (Kd approx. 0.7 nM) and binding specificity characteristics of oestrogen receptors. 2. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. 3. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. (a) Multiple extractions with Tris buffer released about 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. (b) Nuclei purified by centrifugation through 1.8M-sucrose and treatment with 0.7% Triton X-100, or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. (c) In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred, whereas the amount of unoccupied nuclear receptors did not change significantly and conversely in cells exposed to increasing concentrations of oestradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.

Estradiol binding to nuclear receptors in human breast cancer tissue (MCF-7 cell line) and in dimethylbenz(a)anthracene-induced mammary carcinoma.

PIP: The presence or absence of estrogen receptors in the nuclei of human breast tumor may be a useful tool in determining whether the tumor will or will not respond to endocrine therapy. This paper describes an assay which measures both unoccupied and occupied nuclear receptors in human breast cancer tumors. The assay was predicated on the fact that at low salt concentration, the nuclear receptor is bound to chromatin particles and can be separated from the soluble components containing proteolytic acitivity. Nuclear estradiol receptors were measured in human breast cancer tissue (MCF-7 cell line) and in DMBA (dimethylbenz(a)anthracene-induced rat mammary carcinomas) tumors. Complete translocation of the cytoplasmic receptor in the MCF-7 cells was observed compared to only 35-50% of the cytoplasmic receptors seen in the nucleus of the DMBA tumor after estradiol injection. The study also showed 6 pmol/mg DNA for total unoccupied nuclei and cytoplasmic estrogen receptors, and 25% of it in the nucleus; this finding differed from Zava et al's finding of 2 pmol/mg DNA and 75% in the nucleus, probably because of differing methodology or use of a later passage of cell line. 29 out of the 34 tumors with cytoplasmic receptors were found to contain unoccupied nuclear receptors, indicating that free nuclear receptors are not exceptions. The assay used in this study is currently being used to determine the translocative ability of the cytoplasmic receptors in human breast carcinomas.^ieng

Early changes in the membrane of HeLa cells adsorbing Sendai virus under conditions of fusion.

Adsorption of Sendai virus at high multiplicity (500-1,000 HAU/10(6) cells) to HeLa cells grown in monolayers causes immediate changes in the ion barrier of the cell membrane, as well as changes in the morphology of the virus-treated cells. Within minutes of adsorption the cells begin to lose potassium and an extensive influx of ions into the cells occurs. Concomitantly with these changes, the cell membrane becomes depolarized, and the resting potential across its membrane decreases. Twenty to sixty minutes post adsorption the damage to the cell membrane is repaired, and both the potassium uptake and the resting potential return to their pre-exposure values. Scanning electron-micrographs of Sendai infected cells incubated at 37 degrees C show formation of bridging microvilli in a zipper-like fashion within two to five minutes post-adsorption; 30 to 60 minutes thereafter the majority of cells in the monolayer are fused. Biochemical changes induced by virus adsorption and the role of Ca++ ions in the observed effects are discussed.