and Keene,J

and Keene,J.D. were repressed following T cell activation and were either unstable in the resting state or destabilized following cellular activation. Thus, rapid mRNA degradation RQ-00203078 appears to be an important mechanism for turning gene expression off in an RQ-00203078 activation-dependent manner. INTRODUCTION In diverse eukaryotic organisms ranging from yeast to humans, control of mRNA turnover plays a key role in regulating cellular responses to environmental stimuli (1,2). Following transcriptional activation, for example, the regulated decay of mammalian immediate early response gene transcripts, including c-fos, c-jun and c-myc, is crucial for normal cellular functions such as cell cycle progression, proliferation and apoptosis (3). Aberrant regulation of decay leads to oncogenic activation and malignancy (3C7). In T lymphocytes, T cell receptor (TCR) stimulation induces the expression of numerous early response genes. Many of these genes, including cytokine genes and proto-oncogenes, produce mRNA transcripts that exhibit rapid degradation, but subsets of these short-lived transcripts can undergo differential regulation. For example, CD28 co-stimulation of TCR-activated T lymphocytes leads RQ-00203078 to specific stabilization of cytokine transcripts, including interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)- and interferon (IFN)-, while proto-oncogene transcripts such as c-myc remain unstable (8). Thus, the decay of an individual mRNA transcript can exhibit gene-specific, stimulus-dependent regulation that impacts the overall expression of the gene. Although increasing information suggests that mRNA degradation is an important control point Rabbit Polyclonal to DHRS4 for regulating T lymphocyte gene expression, mRNA decay rates have been measured for only a small number of T lymphocyte mRNA transcripts. Recently developed microarray technology has revolutionized gene expression research, allowing the expression of thousands of genes to be simultaneously profiled in different cell types or different treatment conditions. The vast majority of experiments involving microarray technology have evaluated only steady-state mRNA levels. Recent work, however, suggested that microarray technology can be used to categorize mRNA transcripts based on their mRNA decay rates (9,10). In the present study, microarray technology was used to quantitatively measure on a genome-wide basis the decay rates of mRNA transcripts in resting and activated primary human T lymphocytes following transcriptional arrest. The half-life and 95% confidence interval (CI) was determined for each of approximately 6000 transcripts expressed in T lymphocytes. This approach allowed the identification of hundreds of T lymphocyte genes that are regulated at the level of mRNA degradation. MATERIALS AND METHODS Purification of human T lymphocytes Human T lymphocytes were purified as described previously (11). Briefly, human peripheral blood mononuclear cells were isolated through a Ficoll-Hypaque (Amersham Biosciences) RQ-00203078 cushion from buffy coat white blood cell packs (American Red Cross) and were then passed through T cell enrichment columns (R&D Systems). Purified cells consisted of 90C95% CD3+ T lymphocytes based on flow cytometry analysis. T lymphocyte stimulation and RNA isolation following actinomycin D treatment Purified T lymphocytes were cultured overnight in RPMI 1640 (Life Technologies Inc.) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin G and 100 g/ml streptomycin. Cells (25 000 000C50 000 000 cells/ group) were then stimulated for 3 h with medium alone or with immobilized monoclonal antibodies (1.0 g/ml) directed against the CD3 component of the TCR complex (CD3) (R&D Systems) or a combination of CD3 and a monoclonal antibody directed against the CD28 co-stimulatory molecule (CD28) (R&D Systems).