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DNA array analysis of interleukin-2-regulated immediate/early genes.

Beadling C, Smith KA - Med Immunol (2002)

Bottom Line: In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs.CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved.This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, Department of Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA. kasmith@med.cornell.edu

ABSTRACT
BACKGROUND: Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. RESULTS: To identify interleukin-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of >/= 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs. CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.

No MeSH data available.


Related in: MedlinePlus

IL-2-regulated genes in PBMCs. Pre-activated, rested human PBMCs were left untreated or restimulated for 4 hr with IL-2, and RNA probes were prepared for hybridization with Affymetrix U95Av2 arrays. A total of 84 probe sets were identified that showed fold change ≥ 2 (columns, left axis), subtracted difference ≥ 100 (diamonds, right axis), and a significance value of the difference between the means of p < 0.05. The probe sets represent 72 unique genes, and can be segregated into 8 functional groups.
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Figure 1: IL-2-regulated genes in PBMCs. Pre-activated, rested human PBMCs were left untreated or restimulated for 4 hr with IL-2, and RNA probes were prepared for hybridization with Affymetrix U95Av2 arrays. A total of 84 probe sets were identified that showed fold change ≥ 2 (columns, left axis), subtracted difference ≥ 100 (diamonds, right axis), and a significance value of the difference between the means of p < 0.05. The probe sets represent 72 unique genes, and can be segregated into 8 functional groups.

Mentions: These 84 probe sets represent 72 unique genes (60 IL-2-induced genes and 12 IL-2-repressed genes) that can be segregated into 8 functional groups. Figure 1 shows the fold induction and subtracted differences in expression levels of these probe sets. Among the 4 apoptosis-regulating genes, bcl2, caspase 3 and DRAK2 are all induced by IL-2, while the expression of Toso is suppressed. It is noteworthy that the IL-2 induction of bcl2 would be expected to promote cellular survival, while the IL-2 regulation of the other three genes would be expected to promote apoptosis. Additional IL-2-responsive genes include those encoding 6 cell surface proteins, 11 cytokine and chemokine receptors, as well as 15 signal transduction mediators, 6 genes involved in cellular metabolism and biosynthesis, 3 cell cycle regulators and 12 transcription factors. IL-2-suppressed genes include lymphocyte antigen 9, inositol 1,4,5-trisphosphate 3-kinase B, and cdc25B, as well as transcriptional regulators MAX-interacting protein 1, CBP/p300-interacting transactivator, inhibitor of DNA binding 3 (Id3), glucocorticoid-induced leucine zipper (GILZ), and zinc finger protein 36/ERF-2. All of the 19 already known IL-2-regulated genes (Table 2) were also identified in this 72-gene cohort, thereby yielding 53 newly identified IL-2-regulated genes expressed by PBMCs after 4 hours of IL-2 stimulation.


DNA array analysis of interleukin-2-regulated immediate/early genes.

Beadling C, Smith KA - Med Immunol (2002)

IL-2-regulated genes in PBMCs. Pre-activated, rested human PBMCs were left untreated or restimulated for 4 hr with IL-2, and RNA probes were prepared for hybridization with Affymetrix U95Av2 arrays. A total of 84 probe sets were identified that showed fold change ≥ 2 (columns, left axis), subtracted difference ≥ 100 (diamonds, right axis), and a significance value of the difference between the means of p < 0.05. The probe sets represent 72 unique genes, and can be segregated into 8 functional groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC149405&req=5

Figure 1: IL-2-regulated genes in PBMCs. Pre-activated, rested human PBMCs were left untreated or restimulated for 4 hr with IL-2, and RNA probes were prepared for hybridization with Affymetrix U95Av2 arrays. A total of 84 probe sets were identified that showed fold change ≥ 2 (columns, left axis), subtracted difference ≥ 100 (diamonds, right axis), and a significance value of the difference between the means of p < 0.05. The probe sets represent 72 unique genes, and can be segregated into 8 functional groups.
Mentions: These 84 probe sets represent 72 unique genes (60 IL-2-induced genes and 12 IL-2-repressed genes) that can be segregated into 8 functional groups. Figure 1 shows the fold induction and subtracted differences in expression levels of these probe sets. Among the 4 apoptosis-regulating genes, bcl2, caspase 3 and DRAK2 are all induced by IL-2, while the expression of Toso is suppressed. It is noteworthy that the IL-2 induction of bcl2 would be expected to promote cellular survival, while the IL-2 regulation of the other three genes would be expected to promote apoptosis. Additional IL-2-responsive genes include those encoding 6 cell surface proteins, 11 cytokine and chemokine receptors, as well as 15 signal transduction mediators, 6 genes involved in cellular metabolism and biosynthesis, 3 cell cycle regulators and 12 transcription factors. IL-2-suppressed genes include lymphocyte antigen 9, inositol 1,4,5-trisphosphate 3-kinase B, and cdc25B, as well as transcriptional regulators MAX-interacting protein 1, CBP/p300-interacting transactivator, inhibitor of DNA binding 3 (Id3), glucocorticoid-induced leucine zipper (GILZ), and zinc finger protein 36/ERF-2. All of the 19 already known IL-2-regulated genes (Table 2) were also identified in this 72-gene cohort, thereby yielding 53 newly identified IL-2-regulated genes expressed by PBMCs after 4 hours of IL-2 stimulation.

Bottom Line: In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs.CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved.This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, Department of Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA. kasmith@med.cornell.edu

ABSTRACT
BACKGROUND: Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. RESULTS: To identify interleukin-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of >/= 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs. CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.

No MeSH data available.


Related in: MedlinePlus