John R. D. Stalvey

Inhibition of 3β-hydroxysteroid dehydrogenase-isomerase in mouse adrenal cells : a direct effect of testosterone

Gonadal steroids modulate adrenal gland size and function in a variety of species, and our previous studies demonstrate that circulating androgens suppress 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) activity in the adrenal cortex of male mice. The present study tests the hypothesis that androgens have a direct, receptor-mediated inhibitory effect on adrenal 3betaHSD. Treatment of cultured adrenal cells from C57BL/6J and C3H/HeJ mice with 0.02-2.0 microM testosterone for 7 days significantly reduces 3betaHSD activity in cells from both strains. However, treatment for 3 days reduces 3betaHSD activity in the adrenal cells from C3H/HeJ, but not C57BL/6J mice. The decreases in 3betaHSD activity in response to testosterone treatment is reflected in decreases in the amount of 3betaHSD immunoreactive protein, such that extended treatment decreases 3betaHSD immunoreactive protein in adrenal cells from both strains, but short-term treatment only decreases 3betaHSD immunoreactive protein in adrenal cells from C3H/HeJ mice. Thus, there appears to be a temporal difference between strains in the effect of the testosterone on 3betaHSD activity and immunoreactive protein. Treatment of the adrenal cells with androgen agonists and an antagonist indicate that the effect of testosterone is androgen receptor mediated. The effect of testosterone appears to be specific for 3betaHSD, since none of the treatments alter P450(scc) in cells from either strain. Testosterone treatment also causes a decrease in the amount of 3betaHSD mRNA. However, in contrast to the effect on activity and immunoreactive protein, there is no strain-related temporal difference because testosterone decreases 3betaHSD mRNA within 24h in adrenal cells from both strains. These results indicate that testosterone can act directly on the adrenal gland to decrease 3betaHSD activity, immunoreactive protein, and mRNA content in mouse adrenal glands, and thus contribute to the sex difference in adrenal function observed in many species.

Motility of select ovarian cancer cell lines: Effect of extra­cellular matrix proteins and the involvement of PAK2

The interaction between tumor cells and extracellular matrix (ECM) proteins influences cell migration and the invasive behavior of cancer cells. In this study, we provide experimental evidence that collagen I and fibronectin affect ovarian cancer cell migration. In vitro wound healing assays and transwell migration assays were used to measure both total wound healing and directionality of individually migrating OV2008 and C13 ovarian cancer cells on glass, collagen I and fibronectin. Involvement of p21-activated kinase 2 (Pak2) in the motility of these cell lines was investigated using a chemical inhibitor as well as siRNA transfection. Culturing ovarian cancer cells on collagen type I (COLL) increased the migratory ability of OV2008 and C13 cells by increasing the directional migration of cells. In contrast, fibronectin (FN) decreased the migratory ability of OV2008 cells by reducing their ability to migrate directionally. When both cell lines are cultured on COLL and treated with increasing concentrations of a PAK inhibitor (IPA-3), there is a dose-dependent response such that there is a decrease in migration with an increase in inhibitor concentration. Further experiments utilizing PAK2 knockdown via siRNA transfection demonstrated significantly reduced migration of OV2008 cells on COLL as compared to those receiving control siRNA. In conclusion, our results indicate that FN and COLL affect the motility of the selected ovarian cancer cells lines and the effect of COLL is likely mediated, at least in part, by PAK2. A better understanding of the molecular events that contribute to tumor invasion and metastasis is crucial for developing novel treatment strategies to improve the long-term survival of women with ovarian cancer. As PAK2 is involved in motility, it should be further explored as a pro-metastatic gene in ovarian cancer.

Subcellular distribution of 3β-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity

Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

Altering culture conditions reveals strain-related differences in activity and immunoreactive isoforms of 3β-hydroxysteroid dehydrogenase-isomerase in mouse Leydig cells

We previously reported that 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity is higher in Leydig cells from C57BL/6J mice than those from C3H/HeJ mice. This study examines whether the differences in 3 beta HSD activity in Leydig cells from the two strains of mice are due to the expression of different 3 beta HSD isoforms and if a specific isoform corresponds with the amount of 3 beta HSD activity under various culture conditions. Leydig cells were plated in Waymouths +15% horse serum (HS) medium. Some cultures were terminated 24 h later after plating (day 1) and assayed for 3 beta HSD activity or immunoreactivity. The remaining cultures were maintained in HS or changed to serum-free medium. We demonstrate for the first time that two 3 beta HSD immunoreactive isoforms are expressed in freshly isolated Leydig cells and those cultured for 1 day. The same two 3 beta HSD isoforms are detectable in both strains. Thus, the previously reported strain-related differences in 3 beta HSD activity are not due to the expression of different isoforms. When cultured for 8 days, the higher molecular weight 3 beta HSD immunoreactive band is no longer detectable in Leydig cells from either strain. When maintained in HS, 3 beta HSD activity in C57BL/6J Leydig cells decreases significantly by day 8, while 3 beta HSD activity in C3H/HeJ Leydig cells does not change through day 8. When Leydig cells were cultured in serum-free medium, 3 beta HSD activity is maintained in cultured Leydig cells from C57BL/6J and significantly increases in C3H/HeJ 3 beta HSD by day 8. These data suggest that HS has a strain-specific inhibitory effect on 3 beta HSD activity, causing a significant decrease in C57BL/6J 3 beta HSD activity and preventing an increase in C3H/HeJ. Densitometric analysis reveals a correspondence between changes in 3 beta HSD activity and the lower molecular weight isoform but not the higher molecular weight isoform. Treatment with cAMP induces the immunoreactive mass of the lower molecular isoform but not the higher molecular isoform of 3 beta HSD. Currently, it is unclear what the function of the higher molecular weight 3 beta HSD isoform is in mouse Leydig cells.