Magnetic resonance imaging of stapes prostheses.

OBJECTIVE: To assess the magnetic resonance imaging (MRI) compatibility of stapes prostheses. DATA SOURCES: A comprehensive review of the English literature evaluating MRI compatibility of stapes prostheses. Multiple series of stapes prostheses from different manufactures placed in a 1.5-tesla MRI field to determine ferromagnetic properties. RESULTS: When MRI was first introduced, reports demonstrated the MRI compatibility of stapes prostheses. The testing was performed on single copies of various prostheses from several manufacturers. Although implant manufacturers have indicated MRI compatibility, several reports of variable ferromagnetic properties of aneurysm clips have been reported. This variability has led to rotation of the clips and hemorrhage in patients with supposed MRI-compatible clips. These findings suggest that testing single stapes prostheses from a manufacturer might not completely assess the safety of MRI on patients with stapes prostheses. We performed MRI compatibility testing on several series of stapes prostheses from Xomed Surgical Products and Smith & Nephew Richards. Two series of Xomed stapes prostheses were found to have ferromagnetic properties. CONCLUSION: Manufacturing variability could lead to stapes prostheses being MRI incompatible. Each prosthesis should be tested before implantation for ferromagnetic properties.

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras.

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.

Glucocorticoid effects on growth, and androgen receptor concentrations in DDT1MF-2 cell lines.

The DDT1MF-2 smooth muscle tumor cell line contains receptors for and is differentially sensitive to androgens and glucocorticoids. Androgens stimulate and glucocorticoids inhibit growth. We now confirm that the latter involves the induction of a block in the G1 phase of the cell cycle. We have developed and characterized in vitro and in vivo a glucocorticoid resistant variant of this cell line, the DDT1MF-2-GR. Glucocorticoids specifically inhibit androgen induced androgen receptor augmentation in DDT1MF-2 cells, but not in the GR variant suggesting that growth inhibition is related to inhibition of androgen receptor augmentation. However, under optimal conditions for cell proliferation, when glucocorticoid inhibited growth is relieved by the exogenous addition of platelet derived growth factor, androgen receptor augmentation is still suppressed. Thus, androgen induced elevation in androgen receptor concentrations is not a prerequisite for cell proliferation. These results imply that in androgen responsive cells, although androgen stimulation of growth can be blocked by antagonism of androgen receptor mediated events, the antagonism can be bypassed by supplying the cells with exogenous growth factors. These results provoke speculation on how cells, which are dependent upon androgens for growth, become autonomous.

Glucocorticoid induction of beta-adrenergic receptors in the DDT1 MF-2 smooth muscle cell line involves synthesis of new receptor.

We have shown that glucocorticoids induce the appearance of beta 2-adrenergic receptors in membranes of the ductus deferens smooth muscle cell line (DDT1 MF-2). A concomitant increase in isoproterenol stimulated adenylate cyclase activity in the absence of exogenously applied GTP was observed as was a significantly increased (p less than 0.05) sensitivity of the adenylate cyclase system to exogenously applied GTP. However, no significant difference in the maximal velocity of adenylate cyclase between control and steroid treatment was measurable in the presence of sodium fluoride. Induction of beta 2-adrenergic receptors in DDT1 MF-2 cells is correlated with the presence of steroid receptors (androgen and glucocorticoid) in the cells since estrogens and progesterones had no effect on receptor levels. Finally, utilizing dense amino acid labeling of cells to measure old versus newly synthesized receptor sites by a density shift method, we have documented that glucocorticoid induction of beta 2-adrenergic receptors involves synthesis of new receptor protein.

Steroid regulation of receptor concentration and oncogene expression.

The DDT1MF-2 cell line was derived from an estrogen/androgen-induced tumor of the hamster ductus deferens. This cell line contains receptors for both androgens and glucocorticoids and its proliferation is differentially sensitive to these classes of steroids. Androgens stimulate cell growth dramatically and augment intracellular androgen receptors, whereas glucocorticoids inhibit growth and prevent androgen receptor augmentation. Androgen receptor augmentation occurs by an androgen-dependent increase in receptor half-life and an increase in the rate of synthesis. Glucocorticoids, in the presence of androgens, reduce both the half-life and the rate of synthesis of androgen receptors. For comparison purposes, a glucocorticoid-resistant mutant (DDT1MF-2-GR) of this cell line has been developed. Unlike the wild type, in this variant glucocorticoids neither arrest cell growth nor inhibit androgen receptor augmentation. Glucocorticoids block growth of the wild type in the G1-phase of the cell cycle. This event can be overcome either by addition of exogenous platelet-derived growth factor (PDGF) or by the addition of concentrated conditioned medium from nonglucocorticoid-treated cells, however, androgen receptor augmentation remains inhibited. The reduced production of PDGF-like growth factors in the presence of glucocorticoids appears to be the result of a decrease in production of mature mRNA with homology to v-sis (the viral oncogene coding for PDGF-like proteins). The level of regulation appears to be posttranscriptional and does not occur in the DDT1MF-2-GR cells. Thus glucocorticoids regulate the autocrine growth of the DDT1MF-2 cells by a mechanism that can be uncoupled from the regulation of androgen receptor augmentation.

Mechanism of the glucocorticoid regulation of growth of the androgen-sensitive prostate-derived R3327H-G8-A1 tumor cell line.

The R3327H-G8-A1 cell line derived from the Dunning rat prostate adenocarcinoma contains both androgen and glucocorticoid receptors. Following steroid deprivation, androgens specifically increase the concentration of their receptors in these cells by approximately 2-fold within 6 h and 3-4-fold in 24 h. In the presence of potent glucocorticoids, androgen receptor augmentation is reduced by 40-50% in the first 6 h and completely inhibited during the subsequent 24 h. This event, which is specific for glucocorticoids, appears to be due to an inhibition of androgen receptor synthesis. Furthermore, glucocorticoids inhibit proliferation of these cells by inhibiting the release of growth factors and arresting them in the G0 or A state of the cell cycle. This inhibition can be overcome by addition of low concentrations of either epidermal growth factor or platelet-derived growth factor; however, the inhibitory effect of the glucocorticoid on androgen receptor augmentation is not released. These results suggest that glucocorticoids arrest cellular proliferation by altering the autoregulation of growth and that this event is not dependent upon inhibition of androgen receptor augmentation.

Glucocorticoids induce a 29 000 Mr protein in DDT1 MF-2 smooth muscle cells but not in the DDT1 MF-2 GR glucocorticoid resistant variant.

We have demonstrated that glucocorticoids induce in DDT1 MF-2 cells by a glucocorticoid mediated mechanism the synthesis of a methionine-cysteine rich protein of 29 000 Mr (p29). Induction of p29 is not observed in DDT1 MF-2 GR glucocorticoid resistant variants which have only 7% of glucocorticoid receptor site per cell compared to wild type cells. Increased synthesis of p29 is specific to glucocorticoids since neither androgens, estrogens, progesterone nor the glucocorticoid antagonist dexamethasone mesylate are effective inducers. Stimulation of p29 synthesis in wild type cells is observed at 10(-10) M triamcinolone acetonide, reaching a maximum at a concentration of 1 X 10(-8) M. The induction of p29 is not a function of glucocorticoid arrest of DDT1 MF-2 cells since DDT1 MF-2 cells promoted to re-enter the cell cycle by 50 ng/ml platelet derived growth factor (PDGF) continue synthesis of p29. Finally, increased levels of p29 translation products are observed in cell free translation assays carried out utilizing poly A+ RNA transcripts isolated from glucocorticoid treated cells. These data suggest that the glucocorticoid stimulation of p29 synthesis is a transcriptional and/or RNA processing event controlled by glucocorticoid receptor complexes.

Effect of aging and cold temperature storage of hamster ova as assessed in the sperm penetration assay.

The penetration of zona pellucida-free hamster ova by human spermatozoa has been used to quantitate sperm penetration potential. However, since mammalian eggs in vitro have limited viability, the effect of in vitro aging on the ability of hamster ova to be penetrated by human spermatozoa was examined. Zona-free ova maintained at room temperature (25 degrees C) lost their ability to be subsequently penetrated with a half-life of 50.1 +/- 8.8 minutes. This was partly the result of removing the zona pellucida by trypsin digestion, since zona-free oocytes in the presence of trypsin inhibitor or zona pellucida-intact oocytes had half-lives of 99.1 +/- 15.2 and 120.5 +/- 17.4 minutes, respectively. Reduction in penetration rates associated with ovum aging did not appear to be due to loss of viability and could be completely prevented by maintaining the ova on ice (4 degrees C). In the presence of TEST-yolk buffer at 4 degrees C, ova retained (100%) their ability to be penetrated for up to 24 hours and were morphologically indistinguishable from fresh ova. These observations show that ovum aging in vitro at 25 degrees C is much greater than previously anticipated. This may result in artifactually low and variable scores in the penetration bioassay.

The effect of prolactin on human BPH epithelial cell proliferation.

Epithelial cell monolayers derived from specimens of human benign prostatic hyperplasia (BPH) tissue by an explant culture technique were cultured with prolactin in the presence and absence of androgens. Proliferation of the cells was measured by both autoradiographic assessment of [3H]-thymidine uptake and stathmokinetic procedures. Prolactin significantly stimulated the growth of these cells in the concentration range 0.5 mIU/ml to 10 mIU/ml but was inhibitory at a concentration of 100 mIU/ml. In the presence of testosterone (1 X 10(-7) M), prolactin at low concentrations (greater than 1 mIU/ml) but not at 10 mIU/ml, the concentration at which all other experiments were performed, produced a further stimulation in the proliferation. The increase in growth seen with cells cultured with 5 alpha-dihydrotestosterone (1 X 10(-7) M) was reduced with addition of prolactin at high concentrations (10-100 mIU). When the fetal calf serum used in the cultures was stripped of endogenous steroids, prolactin still increased cell proliferation, although to a reduced extent. This indicated that the effects of prolactin were not dependent on the presence of androgens.

Mechanism of androgen-receptor augmentation. Analysis of receptor synthesis and degradation by the density-shift technique.

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) contains receptors for, and is stimulated by, androgens. Cells cultured in the absence of androgens maintain a basal level of androgen receptors. Following incubation with various concentrations of the synthetic androgen methyltrienolone (R1881) for 1-6 h, the concentration of these receptors increased from 6.0 to 12.2 fmol/micrograms of DNA, while the equilibrium dissociation constant (Kd) of 0.5 nM for this steroid remained unchanged. The steroid-induced increase in androgen receptor levels was specific for androgens and dependent upon protein synthesis. The mechanism of receptor augmentation was examined by utilization of isotopically dense amino acids to determine rates of receptor appearance and degradation in the presence or absence of [3H]R1881. In the absence of androgens, the half-life of the androgen receptor was 3.1 h, with a rate constant (kD) of 0.22/h. In the presence of 1 nM [3H]R1881, however, the half-life was 6.6 h, with kD = 0.11/h. The rate constant for receptor synthesis (ks) in the absence or presence of [3H]R1881 was calculated to be 1.35 and 2.23 fmol/micrograms of DNA/h, respectively. Thus, androgen-induced androgen-receptor augmentation is explained by an increase both in receptor half-life and in rates of receptor synthesis.

Studies on human spermatozoa with round head syndrome.

The ability of spermatozoa with round head syndrome to penetrate zona pellucida-free hamster ova and to undergo nuclear decondensation was studied in three infertile patients. Whereas spermatozoa from pregnancy-proven donors bound to hamster ova and achieved a penetration rate of 100%, those with round heads did not bind to or penetrate any ova. While spermatozoa nuclear decondensation occurs after ovum penetration and indicates a positive result, it has now been demonstrated that this phenomenon can also be induced by incubating spermatozoa with crushed hamster ova. In addition, nuclei from normal and round-headed spermatozoa decondense in the presence of these ova to a similar degree. These observations suggest that infertility related to round head sperm morphology is associated with an inability to interact with and penetrate the oolemma, rathern than dysfunctional spermatozoan changes following penetration.

Autocrine regulation of growth: I. Glucocorticoid inhibition is overcome by exogenous platelet derived growth factor.

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) expresses c-sis protooncogene mRNA transcripts which encode at least one subunit of the potent mitogenic agent, platelet derived growth factor (PDGF). These cells also synthesize and secrete a protein which is immunologically identical to this growth factor. Therefore, PDGF is implicated in the autocrine regulation of DDT1MF-2 cell proliferation. While androgens also stimulate proliferation and induce an augmentation in androgen receptor levels in DDT1MF-2 cells, glucocorticoids inhibit both events and arrest cells in the G1 phase of the cell cycle. Addition of PDGF overcomes the glucocorticoid cell cycle arrest, but does not diminish the suppressive action on androgen receptor concentration. These findings are consistent with a mechanism by which glucocorticoids regulate DDT1MF-2 cell proliferation through modulation of PDGF expression which is independent of the glucocorticoid effects on androgen receptor concentrations.

Autocrine regulation of growth: II. Glucocorticoids inhibit transcription of c-sis oncogene-specific RNA transcripts.

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) expresses c-sis proto-oncogene poly A+ RNA transcripts which are thought to encode at least one subunit of the potent mitogen platelet derived growth factor (PDGF). We have previously demonstrated that glucocorticoids block DDT1MF-2 cells in G0/G1 stage of the cell cycle, and that exogenously applied PDGF reinitiates cell cycle progression. In this paper we document that glucocorticoids act to inhibit cell cycle progression by inhibiting the expression of c-sis poly A+ transcripts, which we suggest are encoding a PDGF-like molecule for DDT1MF-2 cells.

Conditions influencing human sperm capacitation and penetration of zona-free hamster ova.

The ability of human spermatozoa to penetrate zona-free hamster ova was examined following low-temperature capacitation (4 degrees C) in TES-Tris (TEST)-yolk buffer for periods of up to 66 hours. Results obtained from 66 individuals demonstrated that the number of penetrations per ova were increased by an average of 2.5-fold when spermatozoa were capacitated for 42 hours, as compared with 18 hours. Furthermore sperm with extremely poor penetration rates after 18-hour capacitation was often improved by longer capacitation periods. Patients from our infertility clinic with less than 20 X 10(6) spermatozoa/ml of ejaculate had significantly lower penetration rates when compared with patients and donors with greater than 20 X 10(6) spermatozoa/ml (P less than or equal to 0.001). The observed effects of TEST-yolk buffer appear to be related to its ability to preserve sperm motility over prolonged periods, during which increased capacitation of the total sperm population is achieved.

Differential effects of androgens and glucocorticoids on regulation of androgen receptor concentrations and cell growth.

This report describes androgen induced up-regulation of intracellular androgen receptor concentrations in the DDT1MF-2 cell line derived from the hamster ductus deferens and the R3327H-G8-A1 line derived from the Dunning prostate adenocarcinoma. Within 6 h of exposure to androgens the receptor concentration is increased approximately 2-fold. Incorporation of dense amino-acids and subsequent analysis by sucrose density gradient centrifugation indicates that the observed increase in receptors is possibly due to de novo androgen receptor synthesis. In both cell lines this increase is inhibited by 30-50% within 6 h and completely during a subsequent 18 h period by the potent glucocorticoid triamcinolone acetonide (TA). TA also inhibits the growth of both cell lines and antagonizes the stimulatory effect of androgens. Flow cell cytometry studies indicate that TA blocks the cells in the G1 phase of the cell cycle. This event may be associated with regulation of androgen receptor concentrations.

Proliferation of a highly androgen-sensitive ductus deferens cell line (DDT1MF-2) is regulated by glucocorticoids and modulated by growth on collagen.

Proliferation of the hamster ductus deferens cloned tumor cell line (DDT1MF-2) in monolayer culture is markedly stimulated by androgens in a dose dependent fashion. Furthermore, growth on collagen confers upon these cells a greater dependence on this class of hormones, such that testosterone (10 nM) induces a 15-fold elevation in cell number compared to controls. Addition of either dexamethasone (10 nM) or triamcinolone acetonide (TA; 10 nM) dramatically blocks this stimulation by reversibly arresting the cells in the G1 phase of the cell cycle as assessed by flow cell cytometry. Associated with the decreased growth rate is a change from a rounded to a more flattened morphology that may also implicate cell shape in the regulation of proliferation. These steroid effects presumably are mediated through specific receptor proteins for which dihydrotestosterone (DHT) and TA bind with equilibrium dissociation constants (Kd) of 0.3 and 1.0 nM, respectively. Moreover, not only do androgens increase growth rate but treatment with 1 nM [3H]DHT also results in an elevation in androgen receptor concentration from 1.6 to 3.6 f mol/micrograms DNA in 7 h. Simultaneous treatment with 10 nM TA, however, reduces this increase by 53%. Inasmuch as neither progesterone nor estradiol-17 beta display similar inhibitory activity, this effect also seems to be glucocorticoid specific. These observations may be important in elucidating the mechanism of androgen action and should provide some insight into the role of glucocorticoids in regulating the growth of androgen dependent tissues.

Androgen stimulated elevation in androgen receptor levels is inhibited by the synthetic glucocorticoid triamcinolone acetonide.

Two cloned tumor cell lines derived from the rat prostate and hamster ductus deferens contain receptors for androgens and glucocorticoids. Androgens increase both the rate of proliferation in these cells and induce a doubling in the number of androgen receptors within 6 hours. This elevation in androgen receptors is dependent on protein synthesis. The glucocorticoid triamcinolone acetonide specifically inhibits both these androgen mediated events without altering the equilibrium dissociation constant (Kd) of the androgen receptor for either [3H]-methyltrienolone (Kd = 0.46 nM) or [3H]-dihydrotestosterone (Kd = 0.2 nM). These observations infer that androgen receptor up-regulation is an important facet of androgen action which may be modulated by glucocorticoids.

Proliferation of human prostatic epithelial cells in culture: aspects of identification.

A reproducible explant culture technique has been developed to study in monolayer, the growth of human prostatic epithelium from specimens of tissue removed from patients with benign prostatic hyperplasia. The procedure was used to study hormone responsiveness of prostatic tissue. The cells appear to originate from the progenitors of glandular epithelium and possess epithelial characteristics, as assessed by histologic, electron microscopic and immunocytochemical procedures. Close cell-to-cell contacts, desmosomes and microvilli were visualized in electron micrographs. In addition, we observed immunocytochemical localization of epithelial antigens in the cells which constituted the monolayer by using antisera raised against purified prostatic epithelium. Prostatic acid phosphatase was not detected histochemically in the monolayers and may reflect the non-secretory state of the cells. Growth parameters under various hormonal conditions were assessed by counting metaphase arrested cells, total cell number and measurement of the area covered by the monolayers. Proliferation was significantly increased by the addition of physiological concentrations of either testosterone or 5 alpha-dihydrotestosterone.