Sandra W. McLeskey

Expression and function of angiopoietin-1 in breast cancer

Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. We have investigated the role of Ang1 in tumour neovascularization under clinical conditions and in animal models. The expression of Ang1 in clinical breast cancer specimens was analysed by using laser-capture microdissection and reverse transcriptase-linked polymerase chain reaction (RT-PCR) on RNA isolated from the samples. Despite the expression of Ang1 in many human breast cancer cell lines, the gene was expressed in only three of 21 breast cancer clinical specimens, even though its receptor, Tie2, is abundant in the vasculature of all of these tumours. Ang1 was then overexpressed in a human breast cancer cell line (MCF-7) on its own and in conjunction with FGF1, an angiogenic factor shown to be able to increase the tumorigenicity of MCF-7 cells. High concentrations of Ang1 were produced in the conditioned media of the transfected cells (range 156–820u2009ng ml–1). However, in contrast to its physiological role as promoter of angiogenesis, overexpression of Ang1 did not enhance tumour growth, but instead caused up to a 3-fold retardation of tumour growth (P = 0.003).

Electroconvulsive shock but not antidepressant drugs increases α1-adrenoceptor binding sites in rat brain

Treatment of rats with electroconvulsive shock once daily for 10-12 days increased the density of alpha 1-adrenoceptors labeled by [3H]prazosin in homogenates of frontal cerebral cortex. A single treatment did not affect [3H]prazosin binding. Repeated treatment with electroconvulsive shock did not appear to affect alpha 1-adrenoceptor binding in the hippocampus or hypothalamus. Treatment of rats with reserpine also increased [3H]prazosin binding in the frontal cortex. In contrast to electroconvulsive shock, three weeks administration of tricyclic antidepressant drugs, monoamine oxidase inhibitors, or atypical antidepressant drugs did not significantly affect [3H]prazosin binding sites in the frontal cortex. The affinities of antidepressant drugs for alpha 1-adrenoceptors ranged from about 50 nM for tricyclic and atypical antidepressants to about 100 microM for monoamine oxidase inhibitors. The high affinities of the tricyclic and atypical antidepressant drugs for alpha 1-adrenoceptors could contribute to clinical differences between these classes of drugs and monoamine oxidase inhibitors. Furthermore, the electroconvulsive shock-induced increase in alpha 1-adrenoceptors could contribute to differences in clinical effects between this treatment and antidepressant drugs.

Effects of AGM-1470 and pentosan polysulphate on tumorigenicity and metastasis of FGF-transfected MCF-7 cells

Previously, we described FGF-1- or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorigenic and metastatic in untreated or tamoxifen-treated ovariectomised nude mice. In this study, we have assessed the effects of AGM-1470, an antiangiogenic agent, and pentosan polysulphate (PPS), an agent that abrogates the effects of FGFs, on tumour growth and metastasis produced by these FGF-transfected MCF-7 cells. Untreated or tamoxifen-treated ovariectomised mice were injected with FGF-transfected cells, treated with AGM-1470 or PPS, and tumour growth and metastasis analysed. The sensitivity of FGF-transfected and parental MCF-7 cells to AGM-1470 or PPS was also determined in vitro. Both AGM-1470 and PPS inhibited tumour growth in otherwise untreated or tamoxifen-treated mice injected with either FGF- or FGF-4-transfected MCF-7 cells. This effect was more reliably seen in tamoxifen-treated animals. AGM-1470 was about 10(5) times less potent in inhibiting the anchorage-dependent growth of parental MCF-7 or FGF-transfected MCF-7 cells than in inhibiting the growth of human umbilical vein endothelial cells. PPS did not affect the in vitro growth of the transfectants or parental cells. Thus, the growth-inhibitory effect on tumours was in excess of the effect of either drug on the same cells in tissue culture, implying that stromal elements are important determinants of the effects of these drugs. There was a positive correlation between tumour size and the extent of proximal lymph node metastasis. However, neither drug had a significant effect on the extent of metastasis to proximal or distal lymph nodes or lungs. AGM-1470 or PPS may be helpful in cases of breast carcinoma in which angiogenesis is due to expression of FGFs by the tumour cells and may be more effective when combined with tamoxifen.

Identification of muscarinic receptor subtypes present in cerebellar granule cells: Prevention of [3H]propylbenzilyl choline mustard binding with specific antagonists

Subtypes of muscarinic receptors (possible m1 to m5) can be identified by their molecular size, specific effector systems and antagonist specificity. In membranes prepared from primary cultures of cerebellar granule cells, [3H]propylbenzilylcholine mustard [( 3H]PBCM) irreversibly binds to muscarinic receptive proteins, having two major molecular sizes, 92 and 66 kDa. With relatively short periods of incubation (approx. 30 min, 30 degrees C) of [3H]PBCM with atropine, a nonspecific competitive receptor antagonist, the irreversible labeling of these muscarinic proteins by [3H]PBCM could be prevented. Methoctramine, a specific competitive antagonist at muscarinic receptors coupled to inhibition of adenylate cyclase, protected most of the muscarinic receptors having a molecular size of 66 kDa from binding of [3H]PBCM. These 66 kDa receptive proteins are suggested to be muscarinic m2 and m4 subtypes. (-)Quinuclidinyl xanthene-9-carboxylate [(-)QNX], a somewhat specific competitive antagonist at muscarinic receptors coupled to hydrolysis of phosphatidylinositol, prevented the binding of [3H]PBCM to 92 kDa muscarinic receptive proteins and some 66 kDa muscarinic receptive proteins. The 92 kDa receptive proteins are suggested to be the muscarinic m3 subtype and the 66 kDa proteins could be either the m2 or m4 receptor subtype. Lastly, pirenzepine, a nonspecific antagonist at muscarinic receptors mediating inhibition of adenylate cyclase and hydrolysis of PI in these cultures, resembled (-)QNX in preventing binding of [3H]PBCM to the 92 kDa receptive proteins and some 66 kDa receptive proteins. The suggested subtypes of muscarinic receptors, specifically bound by pirenzepine should be the m3 (92 kDa) and the m4 (66 kDa) subtypes, since pirenzepine reportedly exhibits a low affinity for the muscarinic m2 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of methoctramine in blocking muscarinic receptors which inhibit adenylate cyclase in cerebellar granule cells

In primary cultures of cerebellar granule cells, activation of muscarinic receptors stimulates both hydrolysis of phosphatidylinositol (PI) and inhibition of adenylate cyclase. The specificity of three muscarinic receptor antagonists, pirenzepine, methoctramine and (-)quinuclidinyl xanthene-9-carboxylate [(-)QNX], in blocking carbachol-stimulated hydrolysis of PI and inhibition of adenylate cyclase were determined. Pirenzepine was found to be nonspecific in blocking the carbachol-stimulated hydrolysis of PI and inhibition of adenylate cyclase, while methoctramine specifically antagonized carbachol-stimulated inhibition of adenylate cyclase with 600 times greater potency than carbachol-stimulated hydrolysis of PI. (-)Quinuclidinyl xanthene-9-carboxylate was approximately 20 times more potent in blocking the carbachol-stimulated hydrolysis of PI than inhibition of adenylate cyclase. Studies of the ability of these three antagonists to block the binding of [3H]quinuclidinyl benzilate [( 3H]QNB) to muscarinic sites on membranes from cerebellar granule cells, revealed that all three antagonists displayed binding characteristics, characteristic of two binding sites, possibly representing the two types of muscarinic receptors. However, the ratio of the affinities for each of the two binding sites was about ten for pirenzepine, 100 for methoctramine and 650 for (-)QNX. Thus, the specificity of these antagonists, in blocking the inhibition of adenylate cyclase and hydrolysis of PI did not correlate with their specificities obtained with the binding studies with [3H]QNB.(ABSTRACT TRUNCATED AT 250 WORDS)