Patricia Mcloughlin

Transcriptional responses to epigallocatechin-3 gallate in HT 29 colon carcinoma spheroids

Catechins have been reported to possess anti‐cancer activity in vitro and in vivo. To identify target genes that may be involved in the anti‐tumorigenic effect of catechins, gene expression profiles in adherent human HT 29 colon carcinoma cells, in HT 29 spheroids and in epigallocatechin‐3 gallate (EGCG)‐treated HT 29 cells have been analysed by high‐density oligonucleotide microarrays. Treatment of HT 29 cells with EGCG (2.5–50 µm) resulted in a dose‐dependent inhibition of spheroid formation of HT 29 cells. Forty transcripts were induced at least twofold in 3‐day‐old spheroids relative to normal adherent cells using three replicates. Oncogenes like c‐fos and c‐jun are significantly up‐regulated in spheroids. We identified several signal transduction and proliferation genes which are down‐regulated in response to EGCG treatment. Increase in the mRNA expression profile of c‐Fos correlated well with protein levels in HT 29 spheroids whereas EGCG did not affect protein formation. In agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids but protein formation was totally blocked in EGCG‐treated cells. Interestingly, no change in expression of cytotoxic or apoptotic related genes has been observed in EGCG‐treated cells. Our findings suggest that EGCG may exert its anti‐cancer activity through modulation of expression of a number of genes that are involved in cell proliferation, cell‐cell contacts and cell‐matrix interactions.

A longitudinal study of gene expression in healthy individuals

BackgroundThe use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22–55 years and > 55 years) and gender.MethodsEleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays.ResultsGene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with ~2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF), 95% of all samples profiled fell within 1.5–2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers.ConclusionThis study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated expression in a subject with iron deficiency anemia and another subject being treated for lung cancer.

Preclinical biomarkers for a cyclin-dependent kinase inhibitor translate to candidate pharmacodynamic biomarkers in phase I patients

A genomics-based approach to identify pharmacodynamic biomarkers was used for a cyclin-dependent kinase inhibitory drug. R547 is a potent cyclin-dependent kinase inhibitor with a potent antiproliferative effect at pharmacologically relevant doses and is currently in phase I clinical trials. Using preclinical data derived from microarray experiments, we identified pharmacodynamic biomarkers to test in blood samples from patients in clinical trials. These candidate biomarkers were chosen based on several criteria: relevance to the mechanism of action of R547, dose responsiveness in preclinical models, and measurable expression in blood samples. We identified 26 potential biomarkers of R547 action and tested their clinical validity in patient blood samples by quantitative real-time PCR analysis. Based on the results, eight genes (FLJ44342, CD86, EGR1, MKI67, CCNB1, JUN, HEXIM1, and PFAAP5) were selected as dose-responsive pharmacodynamic biomarkers for phase II clinical trials. [Mol Cancer Ther 2009;8(9):2517–25]

Gene expression profiling on pre- and post-erlotinib tumors from patients with head and neck squamous cell carcinoma†‡

The purpose of our work was to identify genomic predictive markers of erlotinib response in patients with head and neck squamous cell carcinoma (HNSCC).