James S. Norris

Atrial natriuretic prohormone peptides 1–30, 31–67, and 79–98 vasodilate the aorta

Human prohormone atrial natriuretic peptides 1-30, 31-67, and 79-98 caused vasodilation of porcine aortas which began in 30 seconds and was maximal at 10 minutes. These three peptides were found to be equally potent to atrial natriuretic factor in their vasodilatory activity which was found with or without endothelium present. This vasodilation was associated with a 4 to 5 fold increase in cyclic GMP in the aorta secondary to activation of particulate guanylate cyclase [E.C. 4.6.12]. These data demonstrate that three N-terminal peptide segments of the atrial natriuretic factor prohormone cause vasodilation.

CA 125 antigen in human amniotic fluid and fetal membranes

Abstract The cancer antigen CA 125 is manifest by serous cystadenocarcinoma of the ovary and to a lesser extent by other gynecologic and nongynecologic tumors. Its presence was screened for in normal human fetal tissues and fluids. Appreciable quantities of CA 125 were discovered in amniotic fluid by both a dot blot assay and the commercially available immunoradiometric assay kit. The most likely source of this antigen was found not to be the developing fetus, since antigen was absent from cord blood and fetal urine, but rather the chorionic membrane, which contained significant quantities of the antigen. CA 125 was found in extracts of maternal decidua, but none was found in extracts of placenta or amnion. The CA 125 antigen was determined by gel filtration experiments to be in excess of 700,000 daltons and probably in the range of 2 to 3 × 10 6 daltons. Size heterogeneity based on gel filtration and anion heterogeneity based on anion exchange chromatography have both been demonstrated for the CA 125 molecule. The amniotic fluid antigen is composed of two subunits of approximately 240,000 and 180,000 daltons as detected by iodine 125-labeled OC 125 monoclonal antibody. The antigen may contain additional subunits not detected by the monoclonal antibody. Size and change heterogeneity as well as the poor definition of the subunit bands on polyacrylamide gels also suggest this molecule contains an appreciable carbohydrate component.

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.

In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer

Previous investigations have revealed that bladder cancer cells are generally resistant to Fas-mediated apoptosis by conventional Fas agonists. However, the ability of these cell lines to undergo Fas-mediated apoptosis may have been underappreciated. As a result, we investigated the in vitro efficacy of Fas ligand gene therapy for bladder cancer. Three human bladder cancer lines (T24, J82, and 5637) were treated with the conventional Fas agonist CH-11, a monoclonal antibody to the Fas receptor. Cells were also treated with a replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), AdGFPFasL. A virus containing the GFP gene alone was used to control for viral toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were also evaluated by Western blotting to evaluate poly (ADP-ribose) polymerase, caspase 8, and caspase 9 cleavage and by flow cytometry to determine the presence of coxsackie/adenovirus receptor (CAR). These studies confirmed bladder cancer resistance to cell death by the anti-Fas monoclonal antibody CH-11. This resistance was overcome with AdGFPFasL at a multiplicity of infection (MOI) of 1000 achieving over 80% cell death in all cell lines. Furthermore, greater than 80% cell death was evident in 5637 cells treated with low-dose AdGFPFasL (MOI=10). 5637 cells expressed significantly higher levels of surface CAR than J82 or T24 cells (P<.05). AdGFPFasL is cytotoxic to bladder cancer cells that would otherwise be considered Fas resistant, supporting its in vivo potential. Enhanced sensitivity to AdGFPFasL may be in part due to increased cell surface CAR levels.

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.

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

SummaryProliferation 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/μg DNA in 7 h. Simultaneous treatment with 10 nM TA, however, reduces this increase by 53%. Inasmuch as neither progesterone nor estradiol-17β 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.

Enhanced glycosylation of a 50 kD protein during development of thermotolerance in CHO cells.

During the development of thermotolerance, Chinese hamster ovary cells not only synthesized classical heat shock proteins, but also incorporated [3H]D-glucose or mannose into a glycoprotein with a Mr of approximately 50 kD. The glycosylation of the 50 kD protein correlated with the expression of thermotolerance under conditions when tolerance was induced either by acute or chronic heat conditioning. A phosphoprotein with the same molecular weight as the 50 kD glycoprotein was dephosphorylated immediately after heat conditioning. Both phosphate and glucose label in the ion front were enhanced immediately after heating, and may represent elevated levels of sugar phosphates. However, the composition of the ion front material remains to be determined. The data are consistent with a hypothesis that attributes increased heat resistance of thermotolerant cells to the glycosylation of specific heat-sensitive cellular sites.

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.

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

AbstractWe have shown that binding of 3H-dihydroalprenolol ([3H] DHA) to DDT1 MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [3H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [3H]-DHA binding site of DDT1 cell membranes was isoproterenol (Ki = 0.20 ± 0.07 μM) > epinephrine (Ki = 0.4 ± 0.2 μM) > norepinephrine (Ki = 66.5 ± 5.15 μM) consistent with a β2-selective receptor interaction. Zinterol, a β2-selective antagonist, (Ki = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β1-selective antagonist (Ki = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT1 cells possess a pure p...