Profiling human gene expression with cDNA microarrays.

As a result of genomics initiatives worldwide, it has become increasingly easy to obtain cDNA clones representing the 3 ends of many human genes. This unit describes methods that allow these clones to be used as hybridization detectors in a highly parallel assay of gene expression. Protocols are provided for preparing cDNA microarrays, extracting RNA from cells of interest and preparing fluorescently labeled cDNA representations of the message pools, and hybridizing the labeled cDNAs to the microarrays.

Profiling human gene expression with cDNA microarrays.

As a result of genomics initiatives worldwide, it has become increasingly easy to obtain cDNA clones representing the 3' ends of many human genes. This unit describes methods that allow these clones to be used as hybridization detectors in a highly parallel assay of gene expression. Protocols are provided for preparing cDNA microarrays, extracting RNA from cells of interest and preparing fluorescently labeled cDNA representations of the message pools, and hybridizing the labeled cDNAs to the microarrays.

Molecular classification of cutaneous malignant melanoma by gene expression profiling.

The most common human cancers are malignant neoplasms of the skin. Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease. Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm. Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities. Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas. Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.

Maitotoxin-elicited calcium influx in cultured cells. Effect of calcium-channel blockers.

Maitotoxin elicited a marked influx of 45Ca2+ into NIH 3T3 fibroblast cells. The influx was blocked by imidazoles (econazole, miconazole, SKF 96365, clotrimazole, calmidazolium) with IC50 values from 0.56 to 3 microM. Phenylalkylamines (verapamil, methoxyverapamil) and nitrendipine were less potent, and diltiazem was very weak. Among other calcium blockers, the diphenylbutylpiperidines fluspirilene and penfluridol, the diphenylpropylpiperidine loperamide, and the local anesthetic proadifen were quite active with IC50 values of 2-4 microM. The pattern of inhibition of maitotoxin-elicited calcium influx did not correspond to the ability of the agents to block elevation of calcium that ensues through calcium-release activated calcium (CRAC) channels after activation of phosphoinositide breakdown by ATP in HL-60 cells. The imidazoles did block CRAC channels, but fluspirilene, penfluridol, loperamide and proadifen were ineffective. Loperamide actually appeared to enhance influx of calcium via the activated CRAC channels. The imidazoles, in particular calmidazolium, caused an apparent influx of calcium and caused a stimulation of phosphoinositide breakdown in HL-60 cells.

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding, G proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx.

1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A1-adenosine receptors. 2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTP gamma S to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins. 3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin. N-Ethylmaleimide inhibits the response to both peptides. 4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTP gamma S-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects. 5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered by N-ethylmaleimide. 6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.

Muscarinic receptor-mediated tyrosine phosphorylation of phospholipase C-gamma. An alternative mechanism for cholinergic-induced phosphoinositide breakdown.

In Chinese hamster ovary cells transfected with m5 muscarinic receptors, carbachol stimulates both calcium influx and calcium release from intracellular stores. The marine toxin maitotoxin (MTX) elicits a similar response on calcium influx. Carbachol- and MTX-induced calcium influx can be inhibited by the proposed blockers of receptor-operated calcium channels (ROCC), CAI and SK&F 96365. Both carbachol and MTX induce a significant increase in total protein tyrosine phosphorylation, which is dependent on extracellular calcium and can be inhibited by CAI and SK&F 96365. Phospholipase C-gamma was identified as one of the substrates subject to calcium-dependent tyrosine phosphorylation following carbachol or MTX stimulation. Carbachol-induced [3H]inositol trisphosphate formation was partially inhibited by an inhibitor of tyrosine kinases, by removal of extracellular calcium, and by the inhibitor of receptor-operated calcium channels CAI suggesting that phosphorylation of phospholipase C-gamma plays a role in the muscarinic activation of phosphoinositide breakdown. Such an effect of carbachol is reminiscent of effects observed with peptide growth factors and represents a novel alternative signaling pathway for a muscarinic G protein-coupled receptor.

Monocyte-derived interleukin 1: effects on norepinephrine-stimulated aortic contraction and phosphoinositide turnover.

Medium conditioned by silica-stimulated human peripheral blood monocytes expresses vascular suppressive activity. Rat aortic rings, after incubation in conditioned medium, exhibited comprised contraction to stimulation by norepinephrine (NE). Maximal contraction (300 +/- 51 mg tension/mg tissue) and sensitivity (-5.91 +/- 0.23 M [log EC50]) were both reduced in comparison to contraction (762 +/- 66) and sensitivity (-7.42 +/- 0.11) displayed by rings after incubation in control medium. A polyvalent antibody (Ab) against human interleukin 1 (Il-1) neutralized the suppressive activity in conditioned medium. Rings incubated in conditioned medium containing Ab exhibited normal maximal contraction (722 +/- 46) and a partial restoration of sensitivity to NE (-6.91 +/- 0.13). In contrast, incubation of rings in control medium supplemented with recombinant human Il-1 resulted in a dose-dependent suppression of aortic contraction to NE that was analogous to the defects induced by monocyte-conditioned medium. No significant differences in NE-stimulated phosphoinositide hydrolysis were present between rings incubated in Ab-treated or untreated conditioned or control media. The data suggest that monocyte-derived Il-1 may have a significant influence on vascular contractile function and that the mechanism by which Il-1 induces vascular dysfunction cannot be demonstrated to involve inhibition of NE-stimulated phosphoinositide metabolism.