Predominance of a proinflammatory phenotype in monocyte-derived macrophages from subjects with low plasma HDL-cholesterol.

OBJECTIVE: Reduced plasma concentrations of high-density lipoprotein-cholesterol (HDL-C) are a significant risk factor for cardiovascular disease. Mechanisms that regulate HDL-C concentrations represent an important area of investigation. METHODS AND RESULTS: Comparative transcriptome analyses of monocyte-derived macrophages (MDM) from a large population of low HDL-C subjects and age- and sex-matched controls revealed a cluster of inflammatory genes highly expressed in low HDL-C subjects. The expression levels of peroxisome proliferator activated receptor (PPAR) gamma and several antioxidant metallothionein genes were decreased in MDM from all low HDL-C groups compared with controls, as was the expression of other genes regulated by PPARgamma, including CD36, adipocyte fatty acid binding protein (FABP4), and adipophilin (ADFP). In contrast, PPARdelta expression was increased in MDM from low HDL-C groups. Quantitative RT-PCR corroborated all major findings from the microarray analysis in two separate patient cohorts. Expression of several inflammatory cytokine genes including interleukin 1beta, interleukin 8, and tumor necrosis factor alpha were highly increased in low HDL-C subjects. CONCLUSIONS: The activated proinflammatory state of monocytes and MDM in low HDL-C subjects constitutes a novel parameter of risk associated with HDL deficiency, related to altered expression of metallothionein genes and the reciprocal regulation of PPARgamma and PPARdelta.

Delineation of molecular mechanisms of sensitivity to lapatinib in breast cancer cell lines using global gene expression profiles.

Lapatinib (GW572016) is a small-molecule dual inhibitor of epidermal growth factor receptor (ErbB1) and ErbB2 receptor kinase activities currently in phase III clinical trials. We used phosphoprotein and microarray analyses to carry out targeted pathway studies of phosphorylation and gene expression changes in human breast cancer cell lines in the presence or absence of lapatinib. Studies were done in four breast cancer cell lines, two of which were responsive and two of which were nonresponsive to lapatinib. Responsive cell lines, BT474 and SKBr3, constitutively overexpress ErbB2 and show an IC(50) of 25 or 32 nmol/L for lapatinib, respectively. In contrast, nonresponsive MDA-MB-468 and T47D cells expressed a low basal level of ErbB2 and showed IC(50) values in the micromolar range. Cells responsive to lapatinib exhibited strong differential effects on multiple genes in the AKT pathway. After 12 h of exposure to 1.0 micromol/L of lapatinib, AKT1, MAPK9, HSPCA, IRAK1, and CCND1 transcripts were down-regulated 7- to 25-fold in responsive BT474 and SKBr3 cells. In contrast, lapatinib weakly down-regulated the AKT pathway in nonresponsive breast cancer cell lines (<5-fold down-regulation of most genes in the pathway). Furthermore, the proapoptotic gene FOXO3A, which is negatively regulated by AKT, was up-regulated 7- and 25-fold in lapatinib-responsive SKBr3 and BT474 cells, respectively. Phosphorylated Akt and Akt-mediated phosphorylation of FOXO3A also decreased in responsive breast cancer cell lines exposed to lapatinib. Gene expression profiling also revealed that lapatinib stimulated the expression of estrogen and progesterone receptors and modulated the expression of genes involved in cell cycle control, glycolysis, and fatty acid metabolism. In BT474 and T47D cells, which expressed moderate basal levels of the estrogen and progesterone receptors, 1.0 micromol/L of lapatinib induced expression by 7- to 11-fold. These data provide insight into the mechanism of action of lapatinib in breast cancer cells.

Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples.

Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharide-stimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.