A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase

Prostate cancer progresses from a hormone-sensitive, androgen-dependent stage to a hormone-refractory, androgen-independent tumor. The androgen receptor pathway functions in these androgen-independent tumors despite anti-androgen therapy. In our LAPC-4 prostate cancer model, androgen-independent sublines expressed higher levels of the HER-2/neu receptor tyrosine kinase than their androgen-dependent counterparts. Forced overexpression of HER-2/neu in androgen-dependent prostate cancer cells allowed ligand-independent growth. HER-2/neu activated the androgen receptor pathway in the absence of ligand and synergized with low levels of androgen to 'superactivate' the pathway. By modulating the response to low doses of androgen, a tyrosine kinase receptor can restore androgen receptor function to prostate cancer cells, a finding directly related to the clinical progression of prostate cancer.     

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes.

There is no consensus whether hepatic lipid regulatory enzymes play primary or secondary roles in cholesterol cholelithiasis. We have used inbred mice with Lith genes that determine cholesterol gallstone susceptibility to evaluate the question. We studied activities of regulatory enzymes in cholesterol biosynthesis (HMG-CoA reductase), cholesterol esterification (acyl-CoA:cholesterol acyltransferase) and the "neutral" (cholesterol 7alpha-hydroxylase) and "acidic" (sterol 27-hydroxylase) pathways of bile salt synthesis in strains C57L/J and SWR/J as well as recombinant inbred (AKXL-29) mice, all of which have susceptible Lith alleles, and compared them to AKR/J mice with resistant Lith alleles. We determined hepatic enzyme activities of male mice before and at frequent intervals during feeding a lithogenic diet (15% dairy fat, 1% cholesterol, 0.5% cholic acid) for 12 weeks. Basal activities on chow show significant genetic variations for HMG-CoA reductase, sterol 27-hydroxylase, and acyl-CoA: cholesterol acyltranferase, but not for cholesterol 7alpha-hydroxylase. In response to the lithogenic diet, activities of the regulatory enzymes in the two bile salt synthetic pathways are coordinately down-regulated and correlate inversely with prevalence rates of cholesterol crystals and gallstones. Compared with gallstone-resistant mice, significantly higher HMG-CoA reductase activities together with lower activities of both bile salt synthetic enzymes are hallmarks of the enzymatic phenotype in mice with susceptible Lith alleles. The most parsimonious explanation for the multiple enzymatic alterations is that the primary Lith phenotype induces secondary events to increase availability of cholesterol to supply the sterol to the hepatocyte canalicular membrane for hypersecretion into bile.     

Mutually antagonistic effects of androgen and activin in the regulation of prostate cancer cell growth

Activin, a member of the TGFbeta superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.     

Sperm from beta1,4-galactosyltransferase I-null mice exhibit precocious capacitation

Mammalian sperm must undergo a physiological maturation, termed capacitation, before they are able to fertilize eggs. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. In this paper, we describe the capacitation phenotype of sperm lacking the long isoform of beta1,4-galactosyltransferase I (GalT I), a sperm surface protein that functions as a receptor for the zona pellucida glycoprotein, ZP3, and as an inducer of the acrosome reaction following ZP3-dependent aggregation. As expected, wild-type sperm must undergo capacitation in order to bind the zona pellucida and undergo a Ca(2+) ionophore-induced acrosome reaction. By contrast, GalT I-null sperm behave as though they are precociously capacitated, in that they demonstrate maximal binding to the zona pellucida and greatly increased sensitivity to ionophore-induced acrosome reactions without undergoing capacitation in vitro. The loss of GalT I from sperm results in an inability to bind epididymal glycoconjugates that normally maintain sperm in an 'uncapacitated' state; removing these decapacitating factors from wild-type sperm phenocopies the capacitation behavior of GalT I-null sperm. Interestingly, capacitation of GalT I-null sperm is independent of the presence of albumin, Ca(2+) and HCO(3)(-); three co-factors normally required by wild-type sperm to achieve capacitation. This implies that intracellular targets of albumin, Ca(2+) and/or HCO(3)(-) may be constitutively active in GalT I-null sperm. Consistent with this, GalT I-null sperm have increased levels of cAMP that correlate closely with both the accelerated kinetics and co-factor-independence of GalT I-null sperm capacitation. By contrast, the kinetics of protein tyrosine phosphorylation and sperm motility are unaltered in mutant sperm relative to wild-type. These data suggest that GalT I may function as a negative regulator of capacitation in the sperm head by suppressing intracellular signaling pathways that promote this process.     

Measurement of intestinal cholesterol absorption by plasma and fecal dual-isotope ratio,mass balance,and lymph fistula methods in the mouse: an analysis of direct versus indirect methodologies

The rate of intestinal cholesterol (Ch) absorption is an important criterion for quantitation of Ch homeostasis. However, studies in the literature suggest that percent Ch absorption, measured usually by a fecal dual-isotope ratio method, spans a wide range, from 20% to 90%, in healthy inbred mice on a chow diet. In the present study, we adapted four standard methods, one direct (lymph collection) and three indirect (plasma and fecal dual-isotope ratio, and sterol balance) measurements of Ch absorption and applied them to mice. Our data establish that all methodologies can be valid in mice, with all methods supporting the concept that gallstone-susceptible C57L mice absorb significantly more Ch (37 +/- 5%) than gallstone-resistant AKR mice (24 +/- 4%). We ascertained that sources of error in the literature leading to marked differences in Ch absorption efficiencies between laboratories relate to a number of technical factors, most notably expertise in mouse surgery, complete solubilization and delivery of radioisotopes, appropriate collection periods for plasma and fecal samples, and total extraction of radioisotopes from feces. We find that all methods provide excellent interexperimental agreement, and the ranges obtained challenge previously held beliefs regarding the spread of intestinal Ch absorption efficiencies in mice. The approaches documented herein provide quantifiable methodologies for exploring genetic mechanisms of Ch absorption, and for investigating the assembly and secretion of chylomicrons, as well as intestinal lipoprotein metabolism in mice.     

ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.     

Interleukin-6 gene expression is increased in insulin-resistant rat skeletal muscle following insulin stimulation

IL-6 expression in skeletal muscle is stimulated by contractions. We sought to examine whether hyperinsulinaemia increases IL-6 mRNA in skeletal muscle and whether any increase is modified in insulin resistant muscle. We hypothesized that intramuscular IL-6 mRNA would be increased in response to insulin, but such an affect would be unaffected by insulin resistance because the primary insulin sensitive signalling protein responsible for activating IL-6 functions normally in insulin resistant muscle. Transgenic rats over-expressing the gluconeogenic regulatory enzyme phosphoenolpyruvate carboxykinase (PEPCK) were studied. White gastrocnemius muscle samples were obtained under hyperinsulinaemic, euglycaemic clamp (4 mU kg(-1)min(-1) insulin, plasma glucose concentration 4-6 mmol L(-1)) and basal conditions in both PEPCK (basal n=4; insulin n=5) and wild-type (CON) (basal n=5; insulin n=4) rats, which were previously injected with a bolus of 2-[1-14C]deoxyglucose (2-DG) into the carotid artery. Muscle samples were assayed for 2-DG uptake and IL-6 mRNA. No differences in 2-DG uptake or IL-6 mRNA were observed when comparing groups under basal conditions. Under clamp conditions, 2-DG uptake was lower (P<0.05) in PEPCK compared with CON. Insulin stimulation in CON did not change IL-6 mRNA compared with basal levels. In contrast, there was an approximately 8-fold increase (P<0.05) in IL-6 mRNA in insulin-stimulated PEPCK compared with CON basal levels. Insulin stimulation increases IL-6 gene expression in insulin resistant, but not healthy, skeletal muscle, suggesting that IL-6 expression in skeletal muscle is sensitive to changes in insulin in circumstances of insulin resistance. It is likely that the differences observed when comparing healthy with insulin resistant muscle are due to the differential activation of insulin sensitive signalling proteins responsible for activating IL-6.