Monks, Anne

Abstract CHEK2 codes for a serine/threonine kinase (Chk2) activated by ATM in response to DNA double-strand breaks (DSB). CHEK2 has been described as a tumor suppressor with pro-apoptotic, cell cycle checkpoint and mitotic functions. However, Chk2 is also commonly activated (phosphorylated at T68) in cancers and precancerous lesions. In the present report, we characterized CHEK2 across the panel of 60 established cancer cell lines from the NCI Anticancer Tumor Cell Line panel (the NCI-60) using genomic and proteomic analyses including exon-specific mRNA expression, DNA copy number variation (CNV) by aCGH, exome sequencing, as well as Western blotting analyses for total and activated (pT68-Chk2) Chk2. We demonstrate that the high heterogeneity of Chk2 levels in the NCI-60 is primarily due to its inactivation (owing to low gene expression, alternative splicing, point mutations, copy number alterations and premature truncation) or reduction of protein levels. Notably, we also found that a significant percentage of cancer cells (12% of the NCI-60 and the HeLa cervix cell line) display high endogenous Chk2 activation, always associated with p53 loss of function. Notably, all the 12 cancer cell lines characterized as being p53 wild-type showed almost undetectable levels of pT68-Chk2. The potential impact of endogenous Chk2 activation was analyzed by transcriptome profiling of the five highest pT68-Chk2 cell lines, comparing them with five, tissue-matched low pT68-Chk2 and p53-matched deficient cell lines. Two-sided t tests revealed 176 genes differentially expressed with statistical significance (P < 0.01, uncorrected) in the two groups (high vs. low pT68-Chk2). Among the downregulated expressed genes, a significant number codes for proteins involved in DNA recombination, replication and repair (FANCA, FANCG, FANCL, BRCA2, RAD51C, EME1, and RECQL5 being the most relevant), and centromere organization. Several of these downregulated genes were parts of the Fanconi Anemia pathway or related to homologous recombination (HR, P < 0.05 for statistically significant enrichment using Ingenuity Pathway Analysis®). In conclusion, the biology of Chk2 remains complex, multifaceted and fast evolving. Our study provides information on the relationship between different genomic and proteomic parameters related to Chk2 in cancer, and suggests a possible role for activated Chk2 in p53-deficient or HR/Fanconi Anemia-deficient cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2116. doi:1538-7445.AM2012-2116

Abstract Purpose: According to some studies, susceptibility of cells to anticancer drug-induced apoptosis is markedly inhibited by targeted deletion of genes encoding apoptotic protease activating factor 1 (Apaf-1) or certain caspases. Information about levels of these polypeptides in common cancer cell types and any possible correlation with drug sensitivity in the absence of gene deletion is currently fragmentary. Experimental Design: Immunoblotting was used to estimate levels of Apaf-1 as well as procaspase-2, -3, -6, -7, -8, and -9 in the 60-cell-line panel used for drug screening by the National Cancer Institute. Sensitivity of the same lines to >80,000 compounds was determined with 48-hour sulforhodamine B binding assays. Additional 6-day assays were performed for selected agents. Results: Levels of Apaf-1 and procaspases varied widely. Apaf-1 and procaspase-9, which are implicated in caspase activation after treatment of cells with various anticancer drugs, were detectable in all of the cell lines, with levels of Apaf-1 ranging from ∼1 × 105 to 2 × 106 molecules per cell and procaspase-9 from ∼5 × 103 to ∼1.6 × 105 molecules per cell. Procaspase-8 levels ranged from 1.7 × 105 to 8 × 106 molecules per cell. Procaspase-3, a major effector caspase, varied from undetectable to ∼1.6 × 106 molecules per cell. Correlations between levels of these polypeptides and sensitivity to any of a variety of experimental or conventional antineoplastic agents in either 2-day or 6-day cytotoxicity assays were weak at best. Conclusions: With the exception of caspase-3, all of the components of the core cell-death machinery are expressed in all of the cell lines examined. Despite variations in expression, levels of any one component are not a major determinant of drug sensitivity in these cells in vitro.