Induction of the Cdk inhibitor p21 by LY83583 inhibits tumor cell proliferation in a p53-independent manner

Abstract

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...

Upregulation of Akt/NF-&kappa;B-regulated inflammation and Akt/Bad-related apoptosis signaling pathway involved in hepatic carcinoma process: suppression by carnosic acid nanoparticle

“…However, P21, cyclin-dependent inhibitor (CKI), has been suggested to lead to the G1 arrest and proliferation inhibition of breast cancer cells. 25 In our study, P21 was found to be upregulated for CA treatment in both two liver cancer cell lines, which was consistent with a previous study 26 ( Figure 8C and D). And conversely, Cyclin-D1 and Cyclin-D3, as two essential regulators for cell cycle, were also determined.…”

Prostate cancer is characterized by epigenetic silencing of 14-3-3σ expression

“…RNA was converted to biotinylated cRNA and hybridized to oligonucleotide arrays representing B22 000 individual transcripts. A large number of genes were found to be induced after treatment with 5Aza-2 0 dC plus TSA vs TSA alone (Lodygin et al, 2002). GSTP1, a gene known to be hypermethylated in the majority of PCa (Singal et al, 2001), was induced 1.87-fold in LNCaP cells after combined treatment with 5Aza-2 0 dC and TSA.…”

“…At the indicated time points, cells (both adherent and floating) were harvested by trypsinization, washed once with PBS and fixed in 70% ethanol for 2 h on ice, washed once with PBS and incubated for 30 min at room temperature in PBS solution containing 50 mg/ml propidium iodide, 0.2 mg/ml RNase A and 0.1% Triton X-100 v/v (Lodygin et al, 2002). For each sample, 10 000 events were analysed with a FACScan unit (Becton Dickinson).…”

Functional Epigenomics Identifies Genes Frequently Silenced in Prostate Cancer

“…Western blotting analysis was done as described previously (15). Antibodies used were directed against a-tubulin (Santa Cruz), vesicular stomatitis virus (Sigma), and phospho-ERK1/2 and ERK1/2 (Cell Signaling Technology, Frankfurt, Germany).…”