Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the reactions between 17-hydroxy and 17-keto steroids. In the present study, the enzyme activities and tissue distribution of 17HSD type 1, type 2 and type 4 were characterized. Furthermore, the role of 17HSD type 1 in estrogen-dependent growth was studied in MCF-7 breast cancer cells which were stably transfected with type 1 cDNA. Endogenous oxidative 17HSD activity found in COS-m6 monkey kidney cells was first compared with that of human placental 17HSD. Cultured COS-m6 cells exclusively possessed oxidative 17HSD activity, converting estradiol (E2) to less active estrone (E1). When placental 17HSD was transfected into these cells, highly reductive activity appeared. The 17HSD enzyme in COS-m6 cells also catalyzed the conversion of testosterone to androstenedione, whereas the placental enzyme was estrogen-specific. These results further proved the existence of different 17HSD isoenzymes. The enzymatic properties and cell- and tissue-specific expression of 17HSD type 1, type 2 and oxidative type 4 were further characterized. The data confirmed that in cultured cells the direction of 17HSD activity is determined by the expression of different isoenzymes and not by the intracellular environment. In addition, the 17HSD type 1 gene expresses two mRNA signals, 1.3 kb and 2.3 kb in size. The expression of 1.3 kb mRNA, but not 2.3 kb mRNA was related to enzyme concentration in all the cell types studied. The type 1 enzyme was expressed in the placenta, ovary and in some breast cancer specimens and in the cell lines originated from these tissues. 17HSD type 2 was more widely expressed in both steroidogenic and in target tissues of steroid action. 17HSD type 4 was expressed in almost all cell lines and in all tissues studied, but no correlation with 17HSD activity was detected. These results suggest that 17HSD type 1 is involved in E2 production in females and 17HSD type 2 is responsible for inactivation of sex steroids. However, the oxidation of 17β-hydroxysteroids seems not to be the primary activity of 17HSD type 4. The mRNAs for 17HSD type 1, type 2 and type 4 were found to be expressed in human mammary epithelial cells. In breast tissue samples both 17HSD type 1 and type 2 were detected by in situ hybridization. Despite the presence of 17HSD type 1 mRNA in human mammary epithelial cells, only oxidative 17HSD activity was detected. The reason for the lack of reductive activity is not yet known. Finally, MCF-7 breast cancer cells were stably transfected with 17HSD type 1 cDNA in order to study the effect of 17HSD type 1 on estrogen-dependent growth. In wild type MCF-7 cells, very low 17HSD activity was detected and E1 did not have any effect on cell growth. In the cells expressing 17HSD type 1, E1 was rapidly converted to E2. Hence in these cells E1 had a similar growth-promoting effect as E2 as a result of the action of 17HSD type 1. The presence of 17HSD type 1 in breast cancer cells may thus be an important factor regulating estrogen exposure and the estrogen-responsive growth of breast cancer tissue.

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Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and ß-thromboglobulin ( ßTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified l¹²⁵ I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, (ßTG, with [¹⁴C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric ßTG with a dissociation con stant of about 4 x 10-8ThesisThesis⁻⁸ M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by -PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and ßTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and ßTG may be mediated in part by serotonin-protein associations. / KMBT_363 / Adobe Acrobat 9.53 Paper Capture Plug-in

In this study the metabolism of the anti-arthritic drug, phenylbutazone, was investigated in female Wistar rats, and the results compared with those of other workers in this field. Two interrelated projects were undertaken. The first covered the pattern of excretion, isolation and characterisation of the metabolites and decomposition products of phenylbutazone in rats dosed post-orally with the drug. It was found that the major route of excretion was via the urine and over 50% of the administered dose was excreted in the first 24 hours by this route. A small percentage of the dose was excreted in the faeces. The following compounds were identified using chromatographic and autoradiographic techniques: p-Hydroxy derivative of phenylbutazone γ-Hydroxy derivative of phenylbutazone in both its molecular forms (ring lactone and straight chain hydroxyl) 4-Hydroxy derivative of phenylbutazone p-γ-Dihydroxy derivative of phenylbutazone p-4-Dihydroxy derivative of phenylbutazone Hydrolysable conjugates (possibly glucuronides) Water soluble non-hydrolysable conjugates. The second project dealt with the quantitation of the water insoluble compounds isolated in the initial work. Using a unique technique, combining inverse isotope dilution assay and spectrophotometric analysis, it was found that the major metabolite was the γ-hydroxy derivative of phenylbutazone, present in both its molecular forms. Oxyphenbutazone was a minor metabolite and the p-γ-dihydroxy derivative of phenylbutazone was present only in very low concentration. These results did not conform with those of previous workers in this field who reported the γ-hydroxy derivative of phenylbutazone, in one molecular form only, as the major metabolite and the dihydroxy derivative as the second metabolite with a higher concentration in the urine than oxyphenbutazone. This disparity could be due to the fact that these workers took no account of the presence of the two molecular forms of the γ-hydroxy derivative of phenylbutazone with their different polarities and different Rf values. The present study showed that the straight chain hydroxyl isomer was probably mistakenly identified as the p-γ-dihydroxy derivative of phenylbutazone. This theory is supported by the fact that the percentage dose recovered by the previous workers of the γ-hydroxy and p-γ-dihydroxy derivatives together equalled the percentage dose recovered in this study of the two molecular forms of the γ-hydroxy derivative. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in