Transcriptomic changes in the nasal epithelium associated with diesel engine exhaust exposure.

BACKGROUND: Diesel engine exhaust (DEE) exposure causes lung cancer, but the molecular mechanisms by which this occurs are not well understood. OBJECTIVES: To assess transcriptomic alterations in nasal epithelium of DEE-exposed factory workers to better understand the cellular and molecular effects of DEE. METHODS: Nasal epithelial brushings were obtained from 41 diesel engine factory workers exposed to relatively high levels of DEE (17.2-105.4 µg/m3), and 38 unexposed workers from factories without DEE exposure. mRNA was profiled for gene expression using Affymetrix microarrays. Linear modeling was used to identify differentially expressed genes associated with DEE exposure and interaction effects with current smoking status. Pathway enrichment among differentially expressed genes was assessed using EnrichR. Gene Set Enrichment Analysis (GSEA) was used to compare gene expression patterns between datasets. RESULTS: 225 genes had expression associated with DEE exposure after adjusting for smoking status (FDR q < 0.25) and were enriched for genes in pathways related to oxidative stress response, cell cycle pathways such as MAPK/ERK, protein modification, and transmembrane transport. Genes up-regulated in DEE-exposed individuals were enriched among the genes most up-regulated by cigarette smoking in a previously reported bronchial airway smoking dataset. We also found that the DEE signature was enriched among the genes most altered in two previous studies of the effects of acute DEE on PBMC gene expression. An exposure-response relationship was demonstrated between air levels of elemental carbon and the first principal component of the DEE signature. CONCLUSIONS: A gene expression signature was identified for workers occupationally exposed to DEE that was altered in an exposure-dependent manner and had some overlap with the effects of smoking and the effects of acute DEE exposure. This is the first study of gene expression in nasal epithelial cells of workers heavily exposed to DEE and provides new insights into the molecular alterations that occur with DEE exposure.

Impact of acute exposure to cigarette smoke on airway gene expression.

BACKGROUND: Understanding effects of acute smoke exposure (ASE) on airway epithelial gene expression and their relationship with the effects of chronic smoke exposure may provide biological insights into the development of smoking-related respiratory diseases. METHODS: Bronchial airway epithelial cell brushings were collected from 63 individuals without recent cigarette smoke exposure and before and 24 h after smoking three cigarettes. RNA from these samples was profiled on Affymetrix Human Gene 1.0 ST microarrays. RESULTS: We identified 91 genes differentially expressed 24 h after ASE (false discovery rate < 0.25). ASE induced genes involved in xenobiotic metabolism, oxidative stress, and inflammation and repressed genes related to cilium morphogenesis and cell cycle. While many genes altered by ASE are altered similarly in chronic smokers, metallothionein genes are induced by ASE and suppressed in chronic smokers. Metallothioneins are also suppressed in current and former smokers with lung cancer relative to those without lung cancer. CONCLUSIONS: Acute exposure to as little as three cigarettes and chronic smoking induce largely concordant changes in airway epithelial gene expression. Differences in short-term and long-term effects of smoking on metallothionein expression and their relationship to lung cancer requires further study given these enzymes' role in the oxidative stress response.

Inactivation of AMPK alters gene expression and promotes growth of prostate cancer cells.

AMP-activated protein kinase (AMPK) serves as a fuel-sensing enzyme that is activated by binding of AMP and subsequent phophorylation by upstream kinases such as the tumor suppressor LKB1, when cells sense an increase in the ratio of AMP to ATP. Acute activation of AMPK stimulates fatty acid oxidation to generate more ATP and simultaneously inhibits ATP-consuming processes including fatty acid and protein syntheses, thereby preserving energy for acute cell-surviving program, whereas chronic activation leads to inhibition of cell growth. The goal of the present study is to explore the mechanisms by which AMPK regulates cell growth. Toward this end, we established stable cell lines by introducing a dominant-negative mutant of AMPK alpha1 subunit or its shRNA into the prostate cancer C4-2 cells and other cells, or wild type LKB1 into the lung adenocarcinoma A549 and breast MB-MDA-231 cancer cells, both of which lack functional LKB1. Our results showed that the inhibition of AMPK accelerated cell proliferation and promoted malignant behavior such as increased cell migration and anchorage-independent growth. This was associated with decreased G1 population, downregulation of p53 and p21, and upregulation of S6K, IGF-1 and IGF1R. Conversely, treatment of the C4-2 cells with 5-aminoimidazole-4-carboxamide 1-D-ribonucleoside (AICAR), a prototypical AMPK activator, caused opposite changes. In addition, our study using microarray and RT-PCR revealed that AMPK regulated gene expression involved in tumor cell growth and survival. Thus, our study provides novel insights into the mechanisms of AMPK action in cancer cells and presents AMPK as an ideal drug target for cancer therapy.

Epithelial mesenchymal transition traits in human breast cancer cell lines.

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

Genetic evidence for a morphogenetic function of the Saccharomyces cerevisiae Pho85 cyclin-dependent kinase.

The Saccharomyces cerevisiae PHO85 gene encodes a nonessential cyclin-dependent kinase that associates with 10 cyclin subunits. To survey the functions provided by Pho85, we identified mutants that require PHO85 for viability. We identified mutations that define seven Pho Eighty-Five Requiring or Efr loci, six of which are previously identified genes-BEM2 (YER155C), SPT7 (YBR081C), GCR1 (YPL075W), SRB5 (YGR104C), HFI1 (YPL254W), and BCK1 (YJL095W)-with one novel gene (YMR212C). We found that mutations in the EFR genes involved in morphogenesis are specifically inviable when the Pho85-associated G1 cyclins encoded by PCL1 and PCL2 are absent. pcl1 Delta bem2, pcl1 Delta pcl2 Delta cla4 Delta, and pcl1 Delta pcl2 Delta cdc42-1 strains are inviable. pcl1 Delta pcl2 Delta mpk1 Delta, pcl1 Delta pcl2 Delta bck1, and pcl1 Delta pcl2 Delta cln1 Delta cln2 Delta strains are also inviable, but are rescued by osmotic stabilization with 1 m sorbitol. We propose that the G1 cyclins encoded by PCL1 and PCL2 positively regulate CDC42 or another morphogenesis promoting function.

Signaling phosphate starvation.

Phosphate starvation induces the transcription of several genes involved in phosphate metabolism in the budding yeast Saccharomyces cerevisiae. The signal transduction pathway that mediates this response consists of components that resemble those used to regulate the eukaryotic cell cycle; these include a cyclin-dependent kinase or CDK (Pho85), a cyclin (Pho80) and a CDK inhibitor (Pho81). The possibility that this pathway mediates cell-cycle responses to phosphate starvation is discussed.

Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain.

CD4 is crucial for antigen-driven helper T cell signaling and is used as receptor by the human immunodeficiency virus (HIV). The HIV early protein Nef causes a loss of CD4 from cell surfaces through a previously undefined posttranscriptional mechanism. Here, we demonstrate that Nef acts by inducing CD4 endocytosis, resulting in its degradation in lysosomes. CD4 down-regulation is strongly enhanced by the association of Nef with cell membranes through myristoylation. The study of chimeric molecules reveals that 20 membrane-proximal residues of the CD4 cytoplasmic domain are sufficient to confer Nef sensitivity. Within this region, a dileucine motif, reminiscent of an endocytosis and lysosomal targeting signal found in the CD3 gamma and delta chains, is crucial for CD4 response to Nef.

Vpu-induced degradation of CD4: requirement for specific amino acid residues in the cytoplasmic domain of CD4.

Two functions have been attributed to the product of the human immunodeficiency virus type 1 vpu open reading frame: it increases virion release from infected cells and induces rapid degradation of CD4 shortly after its synthesis. In the absence of Vpu, newly synthesized gp160 and CD4 associate in the endoplasmic reticulum (ER), forming a complex whose further maturation is blocked and which is eventually degraded. In studies using NL4-3-based expression vectors, it has been previously shown that Vpu induces the release of gp160 from the complex that it forms with CD4 in the ER. This release, which appears to be due to the rapid degradation of CD4 induced by Vpu, allows gp160 to transit to the Golgi, where it matures further. We investigated which regions of CD4 are important for its susceptibility to Vpu-induced degradation by transfecting HeLa cells with isogenic vpu-positive and vpu-negative proviruses and vectors expressing various truncated or mutated CD4 molecules. The results suggested that the cytoplasmic domain of CD4 contains a determinant lying within amino acids 418 to 425 that is critical for susceptibility to Vpu-induced degradation. Neither the phosphorylation sites in the cytoplasmic domain nor the Lck interaction region was required for the effect. Vpu-induced degradation was specific for CD4, since CD8, even when retained in the ER, was not degraded. In addition, under conditions of high-level Vpu expression, CD4 degradation could be observed in the absence of gp160 or other means of retaining CD4 in the ER.

Disruption of the cytoskeleton-extracellular matrix linkage promotes the accumulation of plasminogen activators in F9 derived parietal endoderm.

When F9 teratocarcinoma cells are treated with retinoic acid plus cyclic AMP (RACF9) they differentiate into parietal endoderm. Differentiation is accompanied by the acquisition of substrate adhesion sites and a change in the pattern of gene expression, including the synthesis of tissue-type plasminogen activator (tPA). We demonstrate here that dihydrocytochalasin B (DHCB) treatment of differentiating F9 cells prevents the assembly of a structured actin cytoskeleton and generates a more rounded and stellate cell morphology. This morphological change is accompanied by the accumulation of the usually visceral endoderm-specific marker urokinase-type plasminogen activator (uPA) and an increase in tPA levels in comparison to untreated RACF9 controls. The increase in tPA accumulation is preceded by an increase in tPA mRNA levels. These effects are reversible, with a lag, when DHCB is removed, and PA accumulation can be stimulated within 24 h when differentiated cells are exposed to DHCB. Exposure to the microtubule disrupting agent colchicine has no effect on uPA or tPA accumulation. In addition, antibody directed against the beta 1 integrin subunit can also specifically elicit increased PA production. Thus disturbing the cytoskeleton and cytoskeleton associated substrate adhesions promotes PA accumulation.