Estramustine binds a MAP-1-like protein to inhibit microtubule assembly in vitro and disrupt microtubule organization in DU 145 cells.

The twofold purpose of the study was (a) to determine if a MAP-1-like protein was expressed in human prostatic DU 145 cells and (b) to demonstrate whether a novel antimicrotubule drug, estramustine, binds the MAP-1-like protein to disrupt microtubules. SDS-PAGE and Western blots showed that a 330-kD protein was associated with microtubules isolated in an assembly buffer containing 10 microM taxol and 10 mM adenylylimidodiphosphate. After purification to homogeneity on an A5m agarose column, the 330-kD protein was found to promote 6 S tubulin assembly. Turbidimetric (A350), SDS-PAGE, and electron microscopic studies revealed that micromolar estramustine inhibited assembly promoted by the 330-kD protein. Similarly, estramustine inhibited binding of the 330-kD protein to 6-S microtubules independently stimulated to assemble with taxol. Immunofluorescent studies with beta-tubulin antibody (27B) and MAP-1 antibody (MI-AI) revealed that 60 microM estramustine (a) caused disassembly of MAP-1 microtubules in DU 145 cells and (b) removed MAP-1 from the surfaces of microtubules stabilized with 0.1 microM taxol. Taken together the data suggested that estramustine binds to a 330-kD MAP-1-like protein to disrupt microtubules in tumor cells.

Influence of sex hormones on prostatic secretion protein, a major protein in rat prostate.

Prostatic secretion protein (PSP) or estramustine-binding protein is a major protein in rat ventral prostate. The amount of PSP was measured per mg of cytosolic protein at different ages and after castration or administration of sex hormones. The amount of PSP is relatively low before puberty (25 microgram/mg of protein) but increases at about 28 days of age to about 670 microgram/mg of protein and then decreases to a constant level of about 300 to 400 microgram/mg of protein, which is stable until at least 9 months of age. Following castration, the amount of PSP decreased relatively slowly, but 6 days after castration less than 20% of the original amount of PSP was detected. Treatment with testosterone propionate (1 mg/day) for 2 weeks (starting 2 weeks after castration) restored precastration levels of PSP. It is concluded that PSP is an androgen-sensitive protein, and it is suggested that PSP should be considered as a probe for estimation of androgenic action on the prostate. PSP is similar to the so-called prostatic binding protein as well as to prostatein, and it is quite possible that the three proteins represent one and the same entity.

Expression of an estramustine-binding associated protein in human lung cancer cell lines.

Estramustine-binding protein has previously been demonstrated in normal rat prostatic tissue, in normal human prostate epithelium, and in prostatic carcinomas. It binds specifically estramustine and estromustine, the cytotoxic metabolites of estramustine phosphate (Estracyt), a drug which is used in the treatment of prostatic carcinoma. In this study we have examined the presence of an estramustine-binding associated protein in a panel of human cell lines, representing the major histopathological types of lung cancer. A mouse (murine) monoclonal antiserum developed against rat estramustine-binding protein was used for immunohistochemical detection. Fast protein liquid chromatography was used for biochemical characterization. As judged from the immunohistochemical investigation, estramustine-binding protein was present in large amounts in five of six non-small cell carcinoma cell lines, while seven of eight small cell carcinoma cell lines were essentially negative. Fast protein liquid chromatography analyses of lysated cells from the lung cancer cell lines, incubated with [3H]estromustine, concurred with the results from the immunohistochemical stainings. These data strongly indicate a convincing connection between the immunoreactivity and ligand-binding properties of estramustine-binding protein in the cell lines examined. The presence of an estramustine-binding associated protein in human lung cancer cell lines has implications for further investigations into the biological relevance and the potential for eventual therapeutic applications.

Partial characterization and "quantitation" of a human prostatic estramustine-binding protein.

The [3H]estramustine-binding macromolecule in human prostate was partially characterized using a number of chromatographic procedures. Although human estramustine-binding protein (HEMBP) had a marked tendency to aggregate in several systems, a molecular weight of about 54,000 was determined by gel filtration on Sephacryl S-200 Superfine and high-performance liquid chromatography. A sedimentation-coefficient of about 3.6S was obtained for HEMBP when analyzed by sucrose density gradient centrifugation. Isoelectric focusing in polyacrylamide gels indicated an isoelectric point of 4.7 to 4.8, which was in agreement with the elution position of HEMBP following chromatofocusing on Polybuffer Exchanger 94. Furthermore, HEMBP was eluted from diethylaminoethyl-Sepharose with 0.23 M KCl, was retained by concanavalin A-Sepharose (indicating that HEMBP is a glycoprotein), but did not interact with Affi-Gel Blue. Special efforts were concentrated on establishing that HEMBP was a species distinct from human serum albumin. Separation between the [3H]estramustine-labeled HEMBP and the [3H]estramustine-human serum albumin complex was obtained both on sucrose density gradients by chromatography on Affi-Gel Blue, by chromatofocusing, by gel filtration, by isoelectric focusing, and on concanavalin A-Sepharose by affinity chromatography. Twenty-two of 27 human benign hyperplastic prostate cytosol samples were found to contain protein immunochemically similar to estramustine-binding protein (EMBP) purified from rat ventral prostate as determined by the EMBP radioimmunoassay method. Concentrations from 0.2 to 139.6 ng EMBP per mg of total cytosolic protein (mean, 19.3) were determined. Furthermore, four of seven prostatic cancer specimens as well as two of two normal prostatic specimens were also found to contain rat EMBP-immunoreactive material. The unequivocal demonstration of the presence of a HEMBP is of great potential interest in consideration of estramustine phosphate (Estracyt) therapy against prostatic carcinoma. It is not inconceivable that the concentration of HEMBP in the carcinomatous tissue will be of significance in determining the drug uptake in the malignant tissue.

Binding characteristics of a major protein in rat ventral prostate cytosol that interacts with estramustine, a nitrogen mustard derivative of 17 beta-estradiol.

The tissue distribution of [3H]estramustine, the dephosphorylated metabolite of estramustine phosphate (Estracyt), in the male rat was compared to that of [3H]estradiol 30 min and 2 hr following i.p. administration. In contrast to estradiol, estramustine was found to be efficiently concentrated in the ventral prostate gland by a soluble protein. The binding characteristics of this protein were studied in vitro using cytosol preparations of the gland. With a dextran-coated charcoal technique, the protein was found to bind estramustine with a broad pH optimum between pH 7 and pH 8.5, with an apparent Kd of 10 to 30 nM, and with a binding capacity of about 5 nmol/mg cytosol protein. The estramustine/protein complex was not retained by DNA-cellulose. None of the natural steroids tested inhibited the binding of 10 nM [3H]estramustine by more than 35% (progesterone), even when added in 4500-fold excess. The presence of a nitrogen mustard moiety at position 3 of the steroid was necessary for high-affinity binding to the protein. The protein was calculated to constitute about 20% of the total cytosol protein content.

Estramustine-binding protein and specific binding of the anti-mitotic compound estramustine in astrocytoma.

Estramustine-binding protein (EMBP) is a M(r) 46,000 heterodimeric protein originally isolated from prostatic tissue. It has a demonstrated high affinity for, and selective binding of, estramustine, which is a derivative of 17 beta-estradiol and nornitrogen mustard with antimitotic activity. In this study, we have analysed the expression of an EMBP-like protein in astrocytoma specimens. Immunohistochemistry revealed a pronounced reactivity for EMBP in astrocytoma grades III-IV as well as in metastatic prostatic adenocarcinoma used as positive control. In astrocytoma grades I-II, the expression was weak. The EMBP-like protein was quantified by radioimmunoassay in astrocytoma tumor tissue with higher concentrations in malignant astrocytoma, grades III-IV, compared to grades I-II tumors. Western immunoblotting of immunoaffinity purified EMBP-like protein under nonreducing conditions revealed an immunoreactivity corresponding to M(r) 138,000 and 200,000, indicating a different structure of EMBP in astrocytoma compared to prostatic tissue. Specific binding and the presence of saturable binding sites for 3H-labeled estramustine were demonstrated in astrocytoma tissues expressing EMBP-like protein. Scatchard plot analysis showed a Kd at approximately 30 nM, which suggests a binding affinity for estramustine in the same range as previously reported for EMBP in the prostate. Moreover, the number of estramustine binding sites/g tumor as calculated from the Scatchard plots was well correlated with the EMBP levels determined in the radioimmunoassay. In conclusion, an EMBP-like protein is expressed in astrocytoma. This protein may be responsible for the specific binding of estramustine in the tumor tissue. Whether this specific binding of estramustine is of importance for the cytotoxic effect in glioma cells remains to be evaluated.

Fractionation of the rat ventral prostate with respect to isolation and exocytosis of the prostatic secretion protein.

The ventral prostate was fractionated into one mitochondrial and three microsomal fractions. The different fractions were characterized morphologically and chemically. An interesting finding was that upon homogenization the endoplasmic reticulum membranes often turned 'inside-out' giving rise to microsomes with ribosomes attached to the inside of the vesicles. The secretion of the prostatic secretion protein was studied by means of isotopic pulse labeling using radioactive leucine. Peak radioactivity in the microsomal fraction was obtained at 2 h after injection with a relatively rapid fall. The radioactivity in the secretory fluid displayed a continuous increase up to 8 h followed by a plateau. When prostatic secretion protein was purified from secretory fluid and microsomes using a Con A-Sepharose column it showed a typical precursor-product relationship with an early peak at 60 min in microsomal prostatic secretion protein followed by a peak in secretory fluid at 4 h. Vinblastine blocked the release of labeled secretion protein into the secretory fluid, a phenomenon characteristic for secretory proteins which are exocytosed by means of fusion between secretory granules and the plasma membrane. Following intravenous injection of [3H]estramustine, accumulation was seen in the secretory fluid. Some estramustine probably binds to newly synthesized prostatic secretion protein and follows the same route of intracellular transport and extracellular discharge as does prostatic secretion protein.

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.

Differential uptake of estramustine phosphate metabolites and its correlation with the levels of estramustine binding protein in prostate tumor tissue.

Estracyt (EMP) has been used for the treatment of hormone refractory prostate cancer for many years. Recently, new data from combination studies have given rise to new interest in this old drug. Explanations for the synergy found in the clinic are many, but one major factor may be the previous indication that the drug accumulates in the prostate tumor. We have, therefore, examined the level of the four metabolites, estromustine (EoM), estramustine (EaM), estrone, and estradiol in the tumor and serum of 14 patients with T2 and T3 prostate cancer receiving a single i.v. dose of 600 mg of EMP, about 12 h before radical prostatectomy. Because it has been suggested that the uptake into the prostate tumor is due to binding to the estramustine binding protein (EMBP), we have in addition measured the level of EMBP in the prostate tumor tissue. The main serum and tissue metabolite in all patients was EoM followed by EaM, estrone, and estradiol. The levels for EoM ranged from 63.8-162.8 ng/ml in the serum and from 64.8-1209 ng/ml in the prostate tumor, resulting in a mean ratio for serum to tumor of 1:5. The levels for EaM ranged from 8.3-51.4 ng/ml in the serum and 73.9-563.4 ng/ml in the tumor, giving a mean ratio for serum to tumor of 1:13. The levels of EMBP were higher in T3 tumors than in T2 tumors, 54.1 and 40.7 ng/g tissue, respectively. A significant correlation was found between the levels of EaM (r = 0.60) and the levels of EMBP in the tumor. These data demonstrate that 12 h after a single i.v. dose of 600 mg of EMP the levels of the cytotoxic metabolites EoM and EaM are substantially higher in the tumor than in the serum of the same patient and that a correlation exists between the levels of EaM in the tumor and the levels of EMBP. Thus, this supports the hypothesis that the EMBP is responsible for the retention of EoM and EaM in the prostate tumor.

Studies on phenolic steroids in human subjects. XIV. Fate of a nitrogen mustard of estradiol-17beta.

Estracyt, a conjugate of estradiol-17beta (E2) and a nitrogen mustard, is a compound which recently has been found to be of value in the treatment of cancer of the prostate. The present study concerned itself with the fate of labeled Estracyt administered 1) either as a mixture of 3H-Estracyt, labeled in the estrogen moiety, and 14C-E2 or 2) as doubly labeled Estracyt with 3H in the E2 and 14C in the carbamate part. These studies were performed in the human and baboon, with biliary excretion studies being performed in the latter. In the human studies with singly labeled Estracyt, it was shown that about 10-15% of the compound is hydrolyzed in such a fashion as to yield E2, which is then metabolized and conjugated similarly to the 14C-labeled E2; and that the excretory pattern in the urine of the released E2, including the CCD pattern, did not differ materially from that of the 14C-E2, though it was excreted at about 1/3 the rate of the 14C-E2. The remainder of the injected Estracyt was not accounted for. No intact Estracyt was found in the urine. When doubly labeled Estracyt was administered to human subjects, it was shown that the excretion of the 14C (carbamate moiety) was much lower than that of the 3H (E2 moiety), indicating that hydrolysis of the molecule did, in fact, take place, but that the excretion of the 14C, as well as the rest of Estracyt, must follow a route other than the urine. The most likely route is the biliary excretion of either intact Estracyt and/or its metabolites and conjugates without any significant enterohepatic circulation, with the bulk ultimately being excreted in the stools. The studies in the baboon mirrored those in the human. We were able, however, to demonstrate substantial excretion of singly and doubly labeled Estracyt in the bile, indicating that this may be the major route by which the compound is excreted in the baboon and, by analogy, in the human.

Interaction of estramustine phosphate with microtubule-associated proteins.

We have reported [(1984) Cancer Res., in press] that estramustine phosphate inhibits microtubule assembly and disassembled preformed microtubules. We now present evidence that estramustine phosphate inhibits microtubule assembly by binding to the microtubule-associated proteins. We have found that: additional microtubule-associated proteins relieved the inhibition of assembly by estramustine phosphate; 3H-labelled estramustine phosphate bound predominantly to the microtubule-associated proteins; and the content of the microtubule-associated proteins was reduced in taxol reversed estramustine phosphate-inhibited microtubules.

Binding of vinblastine and estramustine to isolated plasma membrane fractions from human prostate and prostatic tumors.

The binding of vinblastine (VLB) and estramustine (EM) to plasma membranes isolated from human prostate, prostatic tumors as well as from Dunning rat prostatic AT-1 tumors was studied. In addition, the uptake of these drugs in AT-1 tumor cells in culture was examined. Binding of VLB was six-fold lower than that of EM in membrane preparations from all three sources. The uptake of VLB in the intact AT-1 cells was nearly five-fold lower than that of EM. At concentrations comparable to those achieved clinically the binding of EM was 100-fold higher than that of VLB. The data suggest that, owing to a very high membrane concentration of EM relative to that of VLB, the active efflux VLB in drug resistant cells would be impeded. This in turn would lead to a higher accumulation of VLB in cells that actively transport cytotoxic drugs.

Distribution and metabolism of estramustine in HeLa cells and the human prostatic tumour cell line 1013L.

The metabolism of estramustine [estradiol-3-N-bis(2-chloroethyl) carbamate] was investigated in the human prostatic tumour cell line 1013L and the human cervix tumour cell line HeLa S3. Uptake studies revealed that estramustine (EM), and its 17-ketoanalogue estromustine (EoM), differed in their nuclear binding pattern in 1013L cells but not in HeLa cells. Most of the nuclear radioactivity from both EM and EoM was found in the fraction containing the majority of the phospholipids. HPLC studies on EM-treated 1013L cells showed the presence of the oxidized metabolite EoM, in the medium, an enrichment of estradiol and estrone in whole cells and EM and EoM bound to the nuclear protein matrix. Similar studies on the HeLa cell line showed a completely different pattern, no metabolites other than EoM were found in the cell medium and whole cells but several very lipophilic metabolites were found bound to the nuclear protein matrix. On investigation of other tumour cell lines these metabolites were found to be unique to HeLa cells. The results extend our knowledge concerning EM and demonstrate that the cell line 1013L is a relevant model system for studying drugs active against human prostatic tumours.

The effect of estramustine derivatives on microtubule assembly in vitro depends on the charge of the substituent.

Estramustine, and derivatives of estramustine with a charged substituent at position 17 on the estrogen moiety, have been investigated for their effects on bovine brain microtubules in vitro. The negatively charged estramustine phosphate has been found previously to be a microtubule-associated protein (MAP)-dependent microtubule inhibitor [Wallin M, Deinum J and Fridén B, FEBS Lett 179: 289-293, 1985]. In the present study the binding of estramustine phosphate to MAP2 and tau was investigated. Both these MAPs were found to have two to three binding sites for estramustine phosphate which is compatible with the reported number of basic amino acid repeats of these MAPs, considered to be the ultimate tubulin binding domains. The Kd for the binding of estramustine phosphate to MAP2 was estimated to be 20 microM at 4 degrees, and for the binding of tau, 200 microM. The rate of dissociation was very low (T1/2 greater than 2 hr), which indicates that the binding of estramustine phosphate may stabilize the protein-drug complex by changing the protein conformation. Two new negatively charged estramustine derivatives, estramustine sulphate and estramustine glucuronide, were found to be similar MAP-dependent microtubule inhibitors. The concentration for 50% inhibition of assembly was 100 microM for the sulphate derivative, the same as found previously for estramustine phosphate, and 250 microM for the more bulky estramustine glucuronide. A positively charged derivative, estramustine sarcosinate, did not inhibit microtubule assembly or alter the composition of the coassembled MAPs. The morphology of the microtubules was, however, affected. The uncharged estramustine bound to both tubulin and MAPs, but no effects were seen on microtubule assembly, the composition of coassembled MAPs or the microtubule morphology. Our results suggest that only negatively charged estramustine derivatives have a MAP-dependent microtubule inhibitory effect. The two new negatively charged derivatives could therefore be valuable tools in the study of tubulin-MAP interactions. The results also confirm that these interactions between tubulin and MAPs are mainly electrostatic.

Inhibition by estramustine phosphate on estradiol and androgen binding in benign and malignant prostate in humans.

Specific cytoplasmic androgen and estrogen binding has been measured in human benign prostatic hypertrophy and carcinomatous tissue. In vitro support for the binding of estramustine phosphate (Estracyt) to both estradiol- and dihydrotestosterone-binding sites is presented, which in part could explain the clinical effect of estramustine phosphate when pure estrogenic compunds are not effective.

Binding sites for the cytotoxic metabolites of estramustine phosphate (Estracyt) in rat and human pancreas that are distinct from pancreatic estrogen-binding protein.

The interaction of estramustine and estromustine, cytotoxic metabolites of estramustine phosphate (Estracyt), with protein-binding sites in rat pancreatic tissue was examined. These compounds were bound with relatively high affinity (Kd 10 nmol/L) to binding sites constituting 0.02-0.03% of total protein. Further characterization of these binding sites revealed a native molecular weight of 24-30,000 a sedimentation coefficient of 3.4 S, and a heterogenous surface-charge distribution by ion-exchange chromatography. Removal of endogenously bound ligand(s) by acetone precipitation or charcoal treatment increased binding significantly. Similar binding sites were present in two of two human pancreatic tumors, but was low or absent in the only histopathologically normal pancreas examined as well as in serum and pancreatic juice. These binding sites were distinct from the "estrogen-binding protein" reported in normal pancreas from various species, but were similar to the "estramustine-binding protein" (EMBP) in rat ventral prostate with respect to ligand specificity and the positive effect of endogenous ligand removal on binding. Furthermore, specimens demonstrating presence of these binding sites also indicated cross-reactivity with antibodies raised against the latter protein, suggesting an immunochemical relation between estra-/estromustine-binding sites in the pancreas and rat prostate EMBP. The presence of high-affinity sites for estramustine and estromustine in human pancreatic carcinomas make this type of tumor a possible target tissue for compounds that exert antiproliferative as well as antimitotic activity in vitro.

Estramustine binding protein in human brain-tumor tissue.

Estramustine, an estradiol-17 beta and nornitrogen mustard complex, is used in the treatment of advanced prostatic carcinoma. A specific estramustine binding protein (EMBP) is important for its cytotoxic action, and the presence of EMBP has previously been demonstrated in rat and human prostatic cancer tissue. Significant levels of EMBP were detected by radioimmunoassay in human brain-tumor tissue. The EMBP concentrations (expressed as ng/mg protein) in 16 astrocytomas (mean 2.6 ng/mg, range 0.5 to 6.2 ng/mg) and seven meningiomas (mean 5.1 ng/mg, range 0.3 to 9.3 ng/mg) were significantly higher than that found in four samples of epileptic brain (mean 0.7 ng/mg, range 0.5 to 1 ng/mg) and 18 samples of normal brain (mean 0.5 ng/mg, range 0.2 to 1.0 ng/mg). The uptake, metabolism, and antiproliferative effects of the prostatic anticancer agent estramustine have been previously demonstrated in cultured glioma cells. The presence of EMBP may suggest a selective binding and effectiveness in human brain-tumor tissue.

Estramustine binding in rat, baboon and human prostate measured by high pressure liquid chromatography.

High pressure liquid chromatography (HPLC) was used to determine 3H-estramustine (estradiol-17 beta 3N-bis-[2-chlorethyl] carbamate), 3H-17 beta-hydroxy-5 alpha-androstan-3-one (3H-dihydrotestosterone or 3H-DHT), 3H-estradiol-17 beta (3H-E2) and 3H-3 beta-hydroxy-5-pregnen-20-one (3H-pregnenolone) binding in 50(2) microliter of cytosol utilizing a column which separates proteins in the molecular weight range of 2,000 to 70,000 daltons. The rat prostate contains a protein in considerable concentration and with the highest affinity for estramustine (375,000 dpm 3H-estramustine per mg. cytosol protein) among the substances tested. Operationally, we have named this protein "estramustine binding protein" (EBP), though it is very likely similar to other previously described prostatic proteins (e.g., alpha-protein, prostatein, prostatic binding protein). The sensitivity of the HPLC method disclosed EBP-like proteins, but in much lesser concentrations, in some of the other tissues tested. The concentration of these proteins in the human and baboon prostates was much lower (average for the baboon cranial lobe 4800 dpm/mg cytosol protein, with a somewhat higher value for the caudal lobe) than that in the rat gland. The amount of the EBP-like protein was higher in prostatic cancer than in that of benign prostatic hypertrophy (BPH) (range 9350--25,900 vs. 2200--18,900 dpm/mg cytosol protein). In the human, the highest value was found in one normal prostate tested (106,000 dpm/mg cytosol protein).

Expression and partial characterization of estramustine-binding protein (EMBP) in human breast cancer and malignant melanoma.

The expression of the estramustine/estromustine-binding protein (EMBP) in human mammary cancer and malignant melanoma was examined by immunochemical methods and compared with that in endometrial and ovarian cancers. By RIA measurements, EMBP was detected in 6/17 mammary cancers (range 11.3-2,660 ng/g tissue) and 2/3 malignant melanomas (618 and 1,240 ng/g), whereas endometrial (n = 6) and ovarian (n = 3) cancers exhibited non-detectable levels. In breast cancer, EMBP-expressing tumours were all estrogen receptor-negative, suggesting an inverse correlation between EMBP and hormone responsiveness of the tumour. Biochemical characterization revealed properties of EMBP in mammary tumours and melanomas almost identical to those for EMBP purified from rat ventral prostate: i.e. surface-charge distribution by Mono Q/FPLC ion-exchange chromatography, a molecular weight of 50,000 by gel filtration, and a subunit composition by Western blot analysis under denaturing conditions. Finally, the EMBP immunoreactivity was confined to the cytoplasm of malignant cells in breast cancer and melanoma sections by immunohistochemical examination. This is the first study that demonstrates EMBP in mammary cancer and malignant melanoma. Our findings suggest that a mechanism for selective uptake of cytotoxic estramustine and estromustine is prevailing in these malignancies and that monitoring of EMBP in biopsy samples will be of value in defining patients who may benefit from Estracyt treatment.

Estrogen receptor and binding site for estramustine in metastatic malignant melanoma.

Enzyme immuno assay (EIA) of estrogen receptor (ER) has confirmed the results of earlier investigations using steroid binding techniques, namely that ER is present at very low concentrations in samples from metastatic melanoma. Thirty-four of 61 samples (56%) were ER positive with EIA. The corresponding figures using isoelectric focusing (IF) for the steroid binding assay were 16 of these 61 samples (26%). The difference between the methods may be due to difficulties in the interpretation of analytical results for IF at low ER concentration levels or to interference from other 3H-estradiol binding components. Estramustine binding site (EMBS) has been found in samples from 15 of 77 patients (20%) with IF in polyacrylamide gels. Estramustine is, together with estramustine, the cytotoxic metabolite of estramustine phosphate (Estracyt). In analogy to the previously suggested therapeutic significance of estramustine binding protein in the treatment of prostatic cancer, the clinical importance of estramustine phosphate should also be studied in metastatic malignant melanoma in correlation with EMBS status.