Chromosomal instability determines taxane response.

Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these "CIN-survival" genes is associated with poor outcome in estrogen receptor-positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats.

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCbeta2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

Age-dependent changes of gene expression in the Drosophila head.

Previous gene expression profiling studies in Drosophila have provided clues for understanding the aging process at the gene expression level. For a detailed understanding, studies of specific regions of the body are necessary. We therefore employed microarray analysis to examine gene expression changes in the Drosophila head during aging. Six hundred and eighty-four of the 5405 genes present in the microarray showed significant age-dependent changes as determined by significance analysis of microarray (SAM) (q < 0.05). The biological significance of the changes was analyzed using the gene annotations provided by the Gene Ontology Consortium. Major changes involved genes affecting energy metabolism (proton transport, energy pathways, oxidative phosphorylation) and neuronal function, especially responses to light. Genes involved in protein catabolism and several other metabolic processes also showed age-dependent changes. Most of the changes were reductions in gene expression and occurred before day 13 of adult life. After day 13, the age-dependent gene expression changes were relatively smaller than earlier life. Interestingly, the two biological processes of major gene expression changes are related to the two known environmental changes that increase life span in Drosophila: caloric restriction and light reduction. Our findings suggest that light signaling and energy metabolism may be important biological processes affected by aging and be interesting targets for the further investigation related to the longevity in Drosophila.

Downregulation of lipopolysaccharide response in Drosophila by negative crosstalk between the AP1 and NF-kappaB signaling modules.

IkappaB kinase (IKK) and Jun N-terminal kinase (Jnk) signaling modules are important in the synthesis of immune effector molecules during innate immune responses against lipopolysaccharide and peptidoglycan. However, the regulatory mechanisms required for specificity and termination of these immune responses are unclear. We show here that crosstalk occurred between the drosophila Jnk and IKK pathways, which led to downregulation of each other's activity. The inhibitory action of Jnk was mediated by binding of drosophila activator protein 1 (AP1) to promoters activated by the transcription factor NF-kappaB. This binding led to recruitment of the histone deacetylase dHDAC1 to the promoter of the gene encoding the antibacterial protein Attacin-A and to local modification of histone acetylation content. Thus, AP1 acts as a repressor by recruiting the deacetylase complex to terminate activation of a group of NF-kappaB target genes.

MED16 and MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced transcriptional activators.

Transcriptional activators interact with diverse proteins and recruit transcriptional machinery to the activated promoter. Recruitment of the Mediator complex by transcriptional activators is usually the key step in transcriptional activation. However, it is unclear how Mediator recognizes different types of activator proteins. To systematically identify the subunits responsible for the signal- and activator-specific functions of Mediator in Drosophila melanogaster, each Mediator subunit was depleted by RNA interference, and its effect on transcriptional activation of endogenous as well as synthetic promoters was examined. The depletion of some Mediator gene products caused general transcriptional defects, whereas depletion of others caused defects specifically related to activation. In particular, MED16 and MED23 were required for lipopolysaccharide- and heat-shock-specific gene expression, respectively, and their activator-specific functions appeared to result from interaction with specific activators. The corequirement of MED16 for other forms of differentiation-inducing factor-induced transcription was confirmed by microarray analysis of differentiation-inducing factor (DIF)- and MED16-depleted cells individually. These results suggest that distinct Mediator subunits interact with specific activators to coordinate and transfer activator-specific signals to the transcriptional machinery.

A molecular basis for embryo apposition at the luminal epithelium.

To obtain a gene expression profile during embryo apposition to the luminal epithelium, we isolated mouse luminal epithelium from implantation (IM) and interimplantation (INTER) sites using laser capture microdissection (LCM), and analyzed their gene expression by microarray analysis. IM and INTER sites were sampled on day 4.5 after mating of female mice with fertile males (day 0.5 = vaginal plug). RNA was extracted, amplified, labeled, and hybridized to microarrays and results were analyzed using the significance analysis of microarrays (SAM) method. Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes most highly expressed at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were ESTs or were of unknown function, and the remain 53 genes had known functions mainly relating to cellular structuring and others such as cell cycling, gene/protein expression, immune responses, invasion, metabolism, oxidative stress, or signal transduction. Specifically, of the 24 structural genes, 14 were implicated in extracellular matrix and tissue remodeling. Meanwhile, of the 13 genes that were highly expressed at INTER, eight were ESTs or of unknown function, and the remaining five were implicated in metabolism, signal transduction, and gene/protein expression. Among these 58 (53 + 5) genes with known functions, 13 genes (22.4%) were associated with Ca2+ for their function. Results of the present study suggest that (1) at IM sites, active tissue remodeling is occurring for embryo invasion while the INTER sites are relatively quiescent and (2) Ca2+ may be a vital regulatory factor in the apposition process. Investigations of human homologues of those genes expressed in the mouse luminal epithelium during apposition may help to understand the implantation process and/or implantation failure in humans.

cDNA microarray gene expression profiling of hydroxyurea, paclitaxel, and p-anisidine, genotoxic compounds with differing tumorigenicity results.

The potential application of toxicogenomics to predictive toxicology has been discussed widely, but the utility of the approach remains largely unproven. Using cDNA microarrays, we compared the gene expression profiles produced in mouse lymphoma cells by three genotoxic compounds, hydroxyurea (a carcinogen), p-anisidine (a noncarcinogen), and paclitaxel (carcinogenicity unknown). To minimize the effect of biological variability and technological limitations, quadruplicate observations were made for each compound and a subset of genes yielding reproducible induction/repression was selected for comparison. A method was applied to attach normalized expression data to genes with a low false-discovery rate (<0.1) to yield more confidence regarding differential expression. This analysis identified genotoxicity-specific gene expression. Seven genes were consistently upregulated and 12 downregulated more than 2-fold by the three genotoxic compounds. Using additional genes, the expression pattern induced by the genotoxic noncarcinogen, p-anisidine, was readily distinguished from that associated with the genotoxic carcinogen, hydroxyurea. Comparison of paclitaxel-induced expression data to data for p-anisidine and hydroxyurea suggested that paclitaxel's profile is more similar to the genotoxic noncarcinogen. With further supporting evidence it may be possible to perform large-scale monitoring of gene expression during drug and chemical development that can provide an early warning of potential toxicological responses.

Signal-induced transcriptional activation by Dif requires the dTRAP80 mediator module.

The Mediator complex is the major multiprotein transcriptional coactivator complex in Drosophila melanogaster. Mediator components interact with diverse sets of transcriptional activator proteins to elicit the sophisticated regulation of gene expression. The distinct phenotypes associated with certain mutations in some of the Mediator genes and the specific in vitro interactions of Mediator gene products with transcriptional activator proteins suggest the presence of activator-specific binding subunits within the Mediator complex. However, the physiological relevance of these selective in vitro interactions has not been addressed. Therefore, we analyzed dTRAP80, one of the putative activator-binding subunits of the Mediator, for specificity of binding to a number of natural transcriptional activators from Drosophila. Among the group of activator proteins that requires the Mediator complex for transcriptional activation, only a subset of these proteins interacted with dTRAP80 in vitro and only these dTRAP80-interacting activators were defective for activation under dTRAP80-deficient in vivo conditions. In particular, activation of Drosophila antimicrobial peptide drosomycin gene expression by the NF-kappa B-like transcription factor Dif during induction of the Toll signaling pathway was dependent on the dTRAP80 module. These results, and the indirect support from the dTRAP80 artificial recruitment assay, indicate that dTRAP80 serves as a genuine activator-binding target responsible for a distinct group of activators.