Characterisation of an oesophageal adenocarcinoma cell line (The MFD-1)
ABSTRACT: A cell line (MFD-1) was derived from a 55-year old male with oesophageal adenocarcinoma. Using different sources of genetic material from normal and tumour tissue surgically resected, peripheral blood and the derived cell line a high concordance of genotypes calls across the whole genome confirms MFD-1 was derived from parent tumour. The SNP6 array contained 906,000 probes for the genotyping of SNPs and 946,000 probes for the genotyping of non-polymorphic copy number. Affymetrix CEL files were analysed using the tool PICNIC2 (predicting absolute allele copy number variation with microarray cancer data).
Project description:A cell line (MFD-1) was derived from a 55-year old male with oesophageal adenocarcinoma. Using different sources of genetic material from normal and tumour tissue surgically resected, peripheral blood and the derived cell line a high concordance of genotypes calls across the whole genome confirms MFD-1 was derived from parent tumour. The SNP6 array contained 906,000 probes for the genotyping of SNPs and 946,000 probes for the genotyping of non-polymorphic copy number. Affymetrix CEL files were analysed using the tool PICNIC2 (predicting absolute allele copy number variation with microarray cancer data).
Project description:New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.
Project description:Intra-tumour heterogeneity (ITH) foster tumour adaptation and hamper the efficiency of personalised medicine approaches. We investigated the extent of ITH within individual clear cell renal cell carcinomas (ccRCC) by multi-region sampling and copy number analysis. We analyzed 63 tumour regions and 8 normal samples from eight clear cell renal cell carcinomas using Affymetrix SNP6 arrays. All individual tumours were subjected to multi-region sampling and copy-number analysis using Affymetrix SNP6 arrays.
Project description:Mfd-dependent transcription termination plays an important role in transcription-coupled DNA repair, transcription-replication conflict resolution, and antimicrobial resistance development. Despite extensive studies, the molecular mechanism of Mfd-dependent transcription termination in bacteria remains unclear, with several long-standing puzzles. How Mfd is activated by stalled RNA polymerase (RNAP) and how activated Mfd translocates along the DNA are unknown. Here, we report the single-particle cryo-electron microscopy structures of T. thermophilus Mfd-RNAP complex with and without ATP?S. The structures reveal that Mfd undergoes profound conformational changes upon activation, contacts the RNAP ?1 domain and its clamp, and pries open the RNAP clamp. These structures provide a foundation for future studies aimed at dissecting the precise mechanism of Mfd-dependent transcription termination and pave the way for rational drug design targeting Mfd for the purpose of tackling the antimicrobial resistance crisis.
Project description:ATP-dependent nucleic acid helicases and translocases play essential roles in many aspects of DNA and RNA biology. In order to ensure that these proteins act only in specific contexts, their activity is often regulated by intramolecular contacts and interaction with partner proteins. We have studied the bacterial Mfd protein, which is an ATP-dependent DNA translocase that relocates or displaces transcription ECs in a variety of cellular contexts. When bound to RNAP, Mfd exhibits robust ATPase and DNA translocase activities, but when released from its substrate these activities are repressed by autoinhibitory interdomain contacts. In this work, we have identified an interface within the Mfd protein that is important for regulating the activity of the protein, and whose disruption permits Mfd to act indiscriminately at transcription complexes that lack the usual determinants of Mfd specificity. Our results indicate that regulation of Mfd occurs through multiple nodes, and that activation of Mfd may be a multi-stage process.
Project description:Genome-wide DNA copy number profiling of gastric tumors and matched non-maligant samples. The affymetrix SNP6 array was used to obtain DNA copy number profiles in 193 gastric tumors and 98 matched gastric non-malignant samples. Genomic DNA extracted from 193 gastric tumors and 98 matched gastric non-malignant (normal) tissues was hybridized on Affymetrix SNP6 genotyping arrays.
Project description:Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (ECs). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.
Project description:Whole genome sequencing (WGS) from snap-frozen oesophageal tumour tissue and germline nucleic acids isolated from peripheral blood mononuclear cells (PBMC) was performed as part of the International Cancer Genome Consortium project and OCCAMS consortium (1,2). Filtered read sequences were mapped to the human reference genome (GRCh37) using Burrows-Wheeler Alignment (BWA). In the matched tumour/germline samples, somatic acquired mutation identification was performed using a Bayesian algorithm implemented in the tool Seurat (3). Functional annotation of identified somatic mutations was performed with the tool SnpEff (4). CNV detection was performed with the tool Control-FREEC (5) 1) Weaver, J. M. et al. Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis. Nat.Genet. 46, 837-843 (2014). 2) Weaver, J. M., Ross-Innes, C. S. & Fitzgerald, R. C. The '-omics' revolution and oesophageal adenocarcinoma. Nature reviews. Gastroenterology & hepatology 11, 19-27 (2014) 3) Christoforides, A. et al. Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs. BMC.Genomics 14, 302 (2013). 4) Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly.(Austin.) 6, 80-92 (2012). 5) Boeva V, Popova T, Bleakley K, Chiche P, Cappo J, Schleiermacher G, Janoueix-Lerosey I, Delattre O, Barillot E. (2011) Control-FREEC: a tool for assessing copy number and allelic content using next generation sequencing data. Bioinformatics. 2011 Dec 6