Integrative genome-wide gene expression profiling of clear cell renal cell carcinoma in Czech Republic and in the United States.

Gene expression microarray and next generation sequencing efforts on conventional, clear cell renal cell carcinoma (ccRCC) have been mostly performed in North American and Western European populations, while the highest incidence rates are found in Central/Eastern Europe. We conducted whole-genome expression profiling on 101 pairs of ccRCC tumours and adjacent non-tumour renal tissue from Czech patients recruited within the "K2 Study", using the Illumina HumanHT-12 v4 Expression BeadChips to explore the molecular variations underlying the biological and clinical heterogeneity of this cancer. Differential expression analysis identified 1650 significant probes (fold change ≥2 and false discovery rate <0.05) mapping to 630 up- and 720 down-regulated unique genes. We performed similar statistical analysis on the RNA sequencing data of 65 ccRCC cases from the Cancer Genome Atlas (TCGA) project and identified 60% (402) of the downregulated and 74% (469) of the upregulated genes found in the K2 series. The biological characterization of the significantly deregulated genes demonstrated involvement of downregulated genes in metabolic and catabolic processes, excretion, oxidation reduction, ion transport and response to chemical stimulus, while simultaneously upregulated genes were associated with immune and inflammatory responses, response to hypoxia, stress, wounding, vasculature development and cell activation. Furthermore, genome-wide DNA methylation analysis of 317 TCGA ccRCC/adjacent non-tumour renal tissue pairs indicated that deregulation of approximately 7% of genes could be explained by epigenetic changes. Finally, survival analysis conducted on 89 K2 and 464 TCGA cases identified 8 genes associated with differential prognostic outcomes. In conclusion, a large proportion of ccRCC molecular characteristics were common to the two populations and several may have clinical implications when validated further through large clinical cohorts.

Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2.

BACKGROUND: The gene CHEK2 encodes a checkpoint kinase playing a key role in the DNA damage pathway. Though CHEK2 has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE) represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whether CHEK2 was subject to DAE in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified. METHODS: We implemented an assay based on high-resolution melting (HRM) curve analysis and developed an analysis tool for DAE assessment. RESULTS: We observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element of CHEK2 that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs. CONCLUSIONS: Our results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms.

Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks.

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.

The Fanconi anemia group A protein modulates homologous repair of DNA double-strand breaks in mammalian cells.

Fanconi anemia (FA) cells exhibit hypersensitivity to DNA interstrand cross-links (ICLs) and high levels of chromosome instability. FA gene products have been shown to functionally or physically interact with BRCA1, RAD51 and the MRE11/RAD50/NBS1 complex, suggesting that the FA complex may be involved in the repair of DNA double-strand breaks (DSBs). Here, we have investigated specifically the function of the FA group A protein (FANCA) in the repair of DSBs in mammalian cells. We show that the targeted deletion of Fanca exons 37-39 generates a null for Fanca in mice and abolishes ubiquitination of Fancd2, the downstream effector of the FA complex. Cells lacking Fanca exhibit increased chromosomal aberrations and attenuated accumulation of Brca1 and Rad51 foci in response to DNA damage. The absence of Fanca greatly reduces gene-targeting efficiency in mouse embryonic stem (ES) cells and compromises the survival of fibroblast cells in response to ICL agent treatment. Fanca-null cells exhibit compromised homology-directed repair (HDR) of DSBs, particularly affecting the single-strand annealing pathway. These data identify the Fanca protein as an integral component in the early step of HDR of DSBs and thereby minimizing the genomic instability.

IkappaB kinase beta phosphorylates Dok1 serines in response to TNF, IL-1, or gamma radiation.

Dok1 is an abundant Ras-GTPase-activating protein-associated tyrosine kinase substrate that negatively regulates cell growth and promotes migration. We now find that IkappaB kinase beta (IKKbeta) associated with and phosphorylated Dok1 in human epithelial cells and B lymphocytes. IKKbeta phosphorylation of Dok1 depended on Dok1 S(439), S(443), S(446), and S(450). Recombinant IKKbeta also phosphorylated Dok1 or Dok1 amino acids 430-481 in vitro. TNF-alpha, IL-1, gamma radiation, or IKKbeta overexpression phosphorylated Dok1 S(443), S(446), and S(450) in vivo, as detected with Dok1 phospho-S site-specific antisera. Moreover, Dok1 with S(439), S(443), S(446), and S(450) mutated to A was not phosphorylated by IKKbeta in vivo. Surprisingly, mutant Dok1 A(439), A(443), A(446), and A(450) differed from wild-type Dok1 in not inhibiting platelet-derived growth factor-induced extracellular signal-regulated kinase 1/2 phosphorylation or cell growth. Mutant Dok1 A(439), A(443), A(446), and A(450) also did not promote cell motility, whereas wild-type Dok1 promoted cell motility, and Dok1 E(439), E(443), E(446), and E(450) further enhanced cell motility. These data indicate that IKKbeta phosphorylates Dok1 S(439)S(443) and S(446)S(450) after TNF-alpha, IL-1, or gamma-radiation and implicate the critical Dok1 serines in Dok1 effects after tyrosine kinase activation.

Frameshift mutation in the Dok1 gene in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is a malignant disease characterized by an accumulation of monoclonal CD5+ mature B cells, with a high percentage of cells arrested in the G0/G1 phase of the cell cycle, and a particular resistance toward apoptosis-inducing agents. Dok1 (downstream of tyrosine kinases) is an abundant Ras-GTPase-activating protein (Ras-GAP)-associated tyrosine kinase substrate, which negatively regulates cell proliferation, downregulates MAP kinase activation and promotes cell migration. The gene encoding Dok1 maps to human chromosome 2p13, a region previously found to be rearranged in B-CLL. We have screened the Dok1 gene for mutations from 46 individuals with B-CLL using heteroduplex analysis. A four-nucleotide GGCC deletion in the coding region was found in the leukemia cells from one patient. This mutation causes a frameshift leading to protein truncation at the carboxyl-terminus, with the acquisition of a novel amino-acid sequence. In contrast to the wild-type Dok1 protein, which has cytoplasmic/membrane localization, the mutant Dok1 is a nuclear protein containing a functional bipartite nuclear localization signal. Whereas overexpression of wild-type Dok1 inhibited PDGF-induced MAP kinase activation, this inhibition was not observed with the mutant Dok1. Furthermore the mutant Dok1 forms heterodimers with Dok1 wild type and the association can be enhanced by Lck-mediated tyrosine-phosphorylation. This is the first example of a Dok1 mutation in B-CLL and the data suggest that Dok1 might play a role in leukemogenesis.