Per Björk

The Expression of Estramustine-Binding Protein in the Human Prostatic Cancer Cell Line Du 145 Is Not Androgen Dependent

Estramustine-binding protein (EMBP) constitutes one of the major proteins in the prostatic gland, it binds estramustine and estromustine, the active metabolites of estramustine phosphate (Estracyt). Previous studies in rats have indicated that the expression of EMBP is androgen dependent, with diminishing quantities following castration and estrogen treatment as well as restored pre-castration production upon administration of androgens. In this study, we have used the human prostatic cancer cell line DU 145 transplanted in female and male nude mice. This cell line, which is sex hormone independent, gave rise to subcutaneous tumors in the rats with no difference in growth characteristics between the males and females. The expression of EMBP was analysed by radioimmunoassay, immunohistochemical and Western blot techniques. No difference was seen between the two sexes with respect to EMBP content, demonstrating that the expression of EMBP, in contrast to that reported for the normal prostate, is neither androgen- nor estrogen-dependent in tumor tissues.

Mouse monoclonal antibodies against rat estramustine binding protein

Hybridomas producing antibodies against rat prostatic estramustine binding protein (rEMBP) have been obtained by fusion of spleen lymphocytes from Balb/c mice, immunized with rEMBP, and SP2/0 Ag 14 mouse myeloma cells. Anti-rEMBP IgG producing cultures were identified in a solid phase ELISA using alkaline phosphatase conjugated sheep antimouse IgG. Cultures producing specific antibodies were subcloned and expanded. The produced immunoglobulins were characterized according to interaction with subunits of rEMBP. No interaction was found with [3H]estramustine-human estramustine binding protein. Following development of a radioimmunoassay, tissue distribution of rEMBP in the rat was studied. Despite high specificity, shown by selective interaction with rEMBP (F and S subunits), macromolecules interacting with anti-rEMBP were found in high concentrations in the prostrate and also in low concentrations, in other tissues of the male genital tract, and further in the adrenals, pancreas and submaxillary gland. The high specificity also makes it possible to study the F and S subunit selectively. These results show that macromolecules very similar to rEMBP are present in hormone-sensitive tissues in the rat.

Estramustine-binding protein in meningioma

Abstract The presence of estramustine-binding protein has been suggested to positively correlate with the effect of the cytotoxic drug estramustine, a combination of estradiol and nornitrogen mustard used in the treatment of prostatic carcinoma. This study demonstrates expression of estramustine-binding protein in a series of meningioma using different ligand-based and immunological techniques. Scatchard plot analysis showed specific binding sites for [3H]estramustine in meningioma tissue with a dissociation constant of 22–26 nM. Immunohistochemistry revealed an immunoreactivity in meningioma comparable to that demonstrated in prostatic carcinoma. The mean concentration (n = 6) of estramustine-binding protein in meningioma, as determined by radioimmunoassay was 159 ng/g tissue (range 18–274 ng/g). Moreover, partial characterization using size exclusion chromatography of [3H]estramustine-labeled tumor extracts and Western blot analysis of immunoprecipitated samples indicated that the structure of the estramustine-binding protein in meningioma is similar to that in rat prostate, with three polypeptide components of 10, 14, and 16 kDa, as compared to 8, 10–11, and 12 kDa in rat prostate. In conclusion, the novel observation of estramustine-binding protein and specific binding of estramustine in meningioma justify further evaluation regarding the role of estramustine-binding protein in the growth behavior of meningioma and the potential for estramustine and similar hormone-related drugs in the treatment of relapsing meningioma.

Estramustine-binding Protein in Malignant Glioma in Rat

Estramustine is a chemotherapeutic drug, used in the treatment of prostatic carcinoma. In the prostate, it binds specifically to a 46 kDa glycoprotein called estramustine-binding protein (EMBP), which consists of three polypeptide components; C1, C2, and C3, each coded for by a specific gene. Expression of EMBP and binding of estramustine has also been detected in malignant glioma in both rats and humans. Elevated levels of this protein in astrocytoma have proved to correlate with poor prognosis. In the present work, expression of all three polypeptide components of EMBP was confirmed in an orthotopic rat glioma model with nested reverse transcriptase PCR and Western blot (molecular weights of 8, 10, and 12 kDa). Specific binding of estramustine with a Kd of 40 for male and 50 for female rats, and a total number of binding sites of 0.7 and 0.4 pmol/mg proteins for male and female rats respectively, was demonstrated with Scatchard plot analysis. These binding characteristics are similar to those of prostatic EMBP. Further studies to elucidate how EMBP expression affects the effect of estramustine treatment, and its putative prognostic value is of special clinical interest. The confirmation of BMBP expression in BT4C rat glioma demonstrates its suitability as a model system for such studies.

Hormonal regulation of an estramustine-binding protein in the submaxillary gland of the rat

An estramustine binding protein, in many aspects similar to the prostatic secretion protein (PSP), has partly been characterized in the submaxillary gland of the male rat. The [3H]estramustine-macromolecule complex is found in the void volume of a Sephadex G 200 column, indicating a Stokes radius larger than 52 A. The estramustine binding protein is bound to Concanavalin-A, indicating a glycoprotein structure. Like PSP, the macromolecule complex that is bound to Concanavalin-A inhibits the binding of the androgen-receptor complex to DNA-cellulose. The concentration of the protein is decreased following testectomy or estrogen treatment but can be restored to normal values following testosterone administration. These results strongly indicate that the estramustine binding macromolecule in the submaxillary gland belongs to the same group of proteins as PSP. We have earlier proposed a role for PSP as an intracellular regulator of androgen activity. Based on these new results it is tempting to speculate that androgen sensitive glycoproteins may act in the same way in all androgen sensitive tissues.