In many cases, silencing of gene expression by CpG methylation is causally involved in carcinogenesis. Furthermore, cancer-specific CpG methylation may serve as a tumor marker. In order to identify candidate genes for inactivation by CpG methylation in prostate cancer, the prostate cancer cell lines LNCaP, PC3, and Du-145 were treated with 5-aza-2V deoxycytidine and trichostatin A, which leads to reversion of epigenetic silencing. By microarray analysis of 18,400 individual transcripts, several hundred genes were found to be induced when compared with cells treated with trichostatin A. Fifty re-expressed genes were selected for further analysis based on their known function, which implied a possible involvement in tumor suppression. Twelve of these genes showed a significant degree of CpG methylation in their promoters. Six genes were silenced by CpG methylation in the majority of five analyzed prostate cancer cell lines, although they displayed robust mRNA expression in normal prostate epithelial cells obtained from four different donors. In primary prostate cancer samples derived from 41 patients, the frequencies of CpG methylation detected in the promoter regions of these genes were: GPX3, 93%; SFRP1, 83%; COX2, 78%; DKK3, 68%; GSTM1, 58%; and KIP2/p57, 56%. Ectopic expression of SFRP1 or DKK3 resulted in decreased proliferation. The expression of DKK3 was accompanied by attenuation of the mitogen-activated protein kinase pathway. The high frequency of CpG methylation detected in the promoters of the identified genes suggests a potential causal involvement in prostate cancer and may prove useful for diagnostic purposes. (Cancer Res 2005; 65(10): 4218-27)

The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

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The results indicate that this DNA stool test of hypermethylation of the secreted frizzled-related protein-1 promoter is a sensitive and specific method. It has the potential of a clinically useful test for the early detection of colorectal cancer.

Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: human diploid fibroblast (HDF); 6-anilino-5,8-quinolinequinone (LY83583); LY83583 (LY); mouse embryo fibroblasts (MEFs); elongation factor 1α (ELF1α); soluble guanylate cyclase β3 (sGC-β3); quantitative real-time PCR (qPCR); crossing point (CP); propidium iodide (PI); sodium nitroprusside (SNP); senescence-associated β-galactosidase (SA-β-gal); bromodeoxyuridine (BrdU); soluble guanylate cyclase α3 (sGC-α3); nitric oxide (NO); cyclin-dependent kinase (Cdk); human papilloma virus (HPV); reactive oxygen species (ROS).numerous unwanted side effects are observed during chemotherapy (14). Furthermore, these agents generate mutations in precancerous cells, which may result in secondary cancer. In this study, we aimed to identify substances that activate cellular senescence without inducing DNA damage. We hypothesized that pharmacological inhibition of signaling pathways that are specifically downregulated during replicative senescence may result in the reactivation of the senescence program in tumor cells. The ideal drug target for this strategy would be an enzyme encoded by a gene that is repressed during senescence. Inhibition of such an enzyme by a small, membrane-permeable drug molecule in early-passage or tumor cells should theoretically be sufficient to induce cellular senescence. In order to detect genes and pathways repressed during replicative senescence, the gene-expression pattern of senescent HDFs was compared with the expression signature of confluent early-passage cells. We thereby identified a pharmacological substance that induces cellular senescence. MethodsCell culture and drug treatments. Neonatal skin HDFs were obtained from Clonetics (San Diego, California, USA) and cultivated in DMEM (Invitrogen Corp., Carlsbad, California, USA) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, Missouri, USA). To obtain senescent HDFs, the cells were diluted every 3 days in a ratio of 1:10 (equal to 1 passage) until they ceased to proliferate.HCT116 cells were cultured in McCoy's medium (Invitrogen Corp.) supplemented with 10% FBS. A-375, HeLa, HEK293, p16 -/-mouse embryo fibroblasts (MEFs) and NIH3T3-L1 derivatives were kept in DMEM containing 10% FBS. 6-Anilino-5,8-quinolinedione (LY83583, referred to as LY hereafter; Calbiochem, San Diego, California, USA) was dissolved in DMSO (Sigma-Aldrich) at a concentration of 300 µM (∼300× solution). As a control, cells were treated with equal volumes of DMSO (<1%). The LY concentration was restored at intervals of 24 hours by media exchange.Microarray analysis of gene expression. RNA was isolated using RNAgents reagents (Promega Corp., Madison, Wisconsin, USA). After mRNA isolation, integrity and enrichment was ensured using Northern blot analysis. Six hundred nanograms of poly-A mRNA was converted to cDNA with incorporation of Cy3-or Cy5-labeled deoxynucleotide-triphosphates (dNTPs). Hybridization to arrays coated on glass, quality control, and normali...

Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: human diploid fibroblast (HDF); 6-anilino-5,8-quinolinequinone (LY83583); LY83583 (LY); mouse embryo fibroblasts (MEFs); elongation factor 1α (ELF1α); soluble guanylate cyclase β3 (sGC-β3); quantitative real-time PCR (qPCR); crossing point (CP); propidium iodide (PI); sodium nitroprusside (SNP); senescence-associated β-galactosidase (SA-β-gal); bromodeoxyuridine (BrdU); soluble guanylate cyclase α3 (sGC-α3); nitric oxide (NO); cyclin-dependent kinase (Cdk); human papilloma virus (HPV); reactive oxygen species (ROS).numerous unwanted side effects are observed during chemotherapy (14). Furthermore, these agents generate mutations in precancerous cells, which may result in secondary cancer. In this study, we aimed to identify substances that activate cellular senescence without inducing DNA damage. We hypothesized that pharmacological inhibition of signaling pathways that are specifically downregulated during replicative senescence may result in the reactivation of the senescence program in tumor cells. The ideal drug target for this strategy would be an enzyme encoded by a gene that is repressed during senescence. Inhibition of such an enzyme by a small, membrane-permeable drug molecule in early-passage or tumor cells should theoretically be sufficient to induce cellular senescence. In order to detect genes and pathways repressed during replicative senescence, the gene-expression pattern of senescent HDFs was compared with the expression signature of confluent early-passage cells. We thereby identified a pharmacological substance that induces cellular senescence. MethodsCell culture and drug treatments. Neonatal skin HDFs were obtained from Clonetics (San Diego, California, USA) and cultivated in DMEM (Invitrogen Corp., Carlsbad, California, USA) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, Missouri, USA). To obtain senescent HDFs, the cells were diluted every 3 days in a ratio of 1:10 (equal to 1 passage) until they ceased to proliferate.HCT116 cells were cultured in McCoy's medium (Invitrogen Corp.) supplemented with 10% FBS. A-375, HeLa, HEK293, p16 -/-mouse embryo fibroblasts (MEFs) and NIH3T3-L1 derivatives were kept in DMEM containing 10% FBS. 6-Anilino-5,8-quinolinedione (LY83583, referred to as LY hereafter; Calbiochem, San Diego, California, USA) was dissolved in DMSO (Sigma-Aldrich) at a concentration of 300 µM (∼300× solution). As a control, cells were treated with equal volumes of DMSO (<1%). The LY concentration was restored at intervals of 24 hours by media exchange.Microarray analysis of gene expression. RNA was isolated using RNAgents reagents (Promega Corp., Madison, Wisconsin, USA). After mRNA isolation, integrity and enrichment was ensured using Northern blot analysis. Six hundred nanograms of poly-A mRNA was converted to cDNA with incorporation of Cy3-or Cy5-labeled deoxynucleotide-triphosphates (dNTPs). Hybridization to arrays coated on glass, quality control, and normali...