Functional investigation of tumor and angiogenesis suppressive candidate tumor suppressor, cysteine-rich intestinal protein 2 in nasopharyngeal carcinoma

Session 8 - Epithelial cells, Infection, Carcinoma: abstract no. 55postprintThe 14th Biennial Symposium of the International Association for Research on Epstein-Barr Virus and Associated Diseases (EBV 2010), Birmingham, U.K., 4-7 September 2010

Allelic losses on chromosome 3p are accumulated in relation to morphological changes of lung adenocarcinoma

We performed allelotyping analysis at nine regions on chromosome 3p using 56 microdissected samples from 23 primary lung adenocarcinomas to examine the process of progression within individual lung adenocarcinoma with various grades of differentiation. Identical allelic patterns among various grades of differentiation were found in eight cases. Accumulation of allelic losses from high to lower differentiated portions was found in seven cases and accumulation of allelic losses from low to higher differentiated portions was found in five cases. Various allelic patterns among various grades of differentiation were found in three cases. These results suggested that allelic losses on 3p play an important role in morphological changes of lung adenocarcinomas. We also investigated the relationship between allelic losses on 3p and histological subtypes of lung adenocarcinoma. The frequencies of allelic losses at 3p14.2 and telomeric region of 3p21.3 were higher in papillary type tumour (nine out of 14, 64% and 11 out of 15, 73%) than in bronchioloalveolar carcinoma-type tumour (one out of 8, 13%; P=0.031 and four out of 12, 33%; P = 0.057). These results indicated that allelic losses at 3p14.2 and telomeric region of 3p21.3 are related to pattern of the proliferation of lung adenocarcinoma

High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer

BACKGROUND:Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS:We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE:This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread

Revised Selection Criteria for Candidate Restriction Enzymes in Genome Walking

A new method to improve the efficiency of flanking sequence identification by genome walking was developed based on an expanded, sequential list of criteria for selecting candidate enzymes, plus several other optimization steps. These criteria include: step (1) initially choosing the most appropriate restriction enzyme according to the average fragment size produced by each enzyme determined using in silico digestion of genomic DNA, step (2) evaluating the in silico frequency of fragment size distribution between individual chromosomes, step (3) selecting those enzymes that generate fragments with the majority between 100 bp and 3,000 bp, step (4) weighing the advantages and disadvantages of blunt-end sites vs. cohesive-end sites, step (5) elimination of methylation sensitive enzymes with methylation-insensitive isoschizomers, and step (6) elimination of enzymes with recognition sites within the binary vector sequence (T-DNA and plasmid backbone). Step (7) includes the selection of a second restriction enzyme with highest number of recognition sites within regions not covered by the first restriction enzyme. Step (8) considers primer and adapter sequence optimization, selecting the best adapter-primer pairs according to their hairpin/dimers and secondary structure. In step (9), the efficiency of genomic library development was improved by column-filtration of digested DNA to remove restriction enzyme and phosphatase enzyme, and most important, to remove small genomic fragments (<100 bp) lacking the T-DNA insertion, hence improving the chance of ligation between adapters and fragments harbouring a T-DNA. Two enzymes, NsiI and NdeI, fit these criteria for the Arabidopsis thaliana genome. Their efficiency was assessed using 54 T3 lines from an Arabidopsis SK enhancer population. Over 70% success rate was achieved in amplifying the flanking sequences of these lines. This strategy was also tested with Brachypodium distachyon to demonstrate its applicability to other larger genomes

Mandatory chromosomal segment balance in aneuploid tumor cells

Copyright: Copyright 2013 Elsevier B.V., All rights reserved.Background: Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost. Methods: Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids. Results: In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region. Conclusion: The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.publishersversionPeer reviewe

DLEC1 is a functional 3p22.3 tumour suppressor silenced by promoter CpG methylation in colon and gastric cancers

Promoter CpG methylation of tumour suppressor genes (TSGs) is an epigenetic biomarker for TSG identification and molecular diagnosis. We screened genome wide for novel methylated genes through methylation subtraction of a genetic demethylation model of colon cancer (double knockout of DNMT1 and DNMT3B in HCT116) and identified DLEC1 (Deleted in lung and oesophageal cancer 1), a major 3p22.3 TSG, as one of the methylated targets. We further found that DLEC1 was downregulated or silenced in most colorectal and gastric cell lines due to promoter methylation, whereas broadly expressed in normal tissues including colon and stomach, and unmethylated in expressing cell lines and immortalised normal colon epithelial cells. DLEC1 expression was reactivated through pharmacologic or genetic demethylation, indicating a DNMT1/DNMT3B-mediated methylation silencing. Aberrant methylation was further detected in primary colorectal (10 out of 34, 29%) and gastric tumours (30 out of 89, 34%), but seldom in paired normal colon (0 out of 17) and gastric (1 out of 20, 5%) samples. No correlation between DLEC1 methylation and clinical parameters of gastric cancers was found. Ectopic expression of DLEC1 in silenced HCT116 and MKN45 cells strongly inhibited their clonogenicity. Thus, DLEC1 is a functional tumour suppressor, being frequently silenced by epigenetic mechanism in gastrointestinal tumours

DLEC1 and MLH1 promoter methylation are associated with poor prognosis in non-small cell lung carcinoma

The significance of chromosome 3p gene alterations in lung cancer is poorly understood. This study set out to investigate promoter methylation in the deleted in lung and oesophageal cancer 1 (DLEC1), MLH1 and other 3p genes in 239 non-small cell lung carcinomas (NSCLC). DLEC1 was methylated in 38.7%, MLH1 in 35.7%, RARβ in 51.7%, RASSF1A in 32.4% and BLU in 35.3% of tumours. Any two of the gene alterations were associated with each other except RARβ. DLEC1 methylation was an independent marker of poor survival in the whole cohort (P=0.025) and in squamous cell carcinoma (P=0.041). MLH1 methylation was also prognostic, particularly in large cell cancer (P=0.006). Concordant methylation of DLEC1/MLH1 was the strongest independent indicator of poor prognosis in the whole cohort (P=0.009). However, microsatellite instability and loss of MLH1 expression was rare, suggesting that MLH1 promoter methylation does not usually lead to gene silencing in lung cancer. This is the first study describing the prognostic value of DLEC1 and MLH1 methylation in NSCLC. The concordant methylation is possibly a consequence of a long-range epigenetic effect in this region of chromosome 3p, which has recently been described in other cancers