Single nucleotide polymorphisms 5′ upstream the coding region of the NEIL2 gene influence gene transcription levels and alter levels of genetic damageKinslow, Carla J.; El‐Zein, Randa A.; Hill, Courtney E.; Wickliffe, Jeffrey K.; Abdel‐Rahman, Sherif Z.
doi: 10.1002/gcc.20594pmid: 18651651
NEIL2 (EC 4.2.99.18), a mammalian DNA glycosylase and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single‐stranded structures, suggesting its involvement in transcription‐coupled DNA repair. Because base excision repair (BER) proteins act collectively and in a progressive fashion, their proper balance is essential for optimal repair. Thus, inter‐individual variability in transcription levels of NEIL2 may predispose to compromised DNA repair capacity and genomic instability by altering the balance of critical BER proteins. In a study of lymphocytes of 129 healthy subjects, using absolute quantitative reverse transcription PCR, we found that NEIL2 transcription varied significantly (up to 63 fold) and that this variability was influenced by certain single nucleotide polymorphisms (SNPs) located 5′ of the start site. Using the mutagen sensitivity assay to characterize the biological significance of these SNPs, we observed a significant increase in mutagen‐induced genetic damage associated with two SNPs in the promoter region of the NEIL2 gene. To characterize the functional significance of these SNPs, we engineered luciferase‐reporter constructs of the NEIL2 promotor with mutations corresponding to these SNPs. We transfected these constructs into MRC‐5 cells and evaluated their impact on NEIL2 expression levels. Our results indicate that NEIL2 expression was significantly reduced by over 50% (P < 0.01) in the presence of two SNPs, ss74800505 and rs8191518, located near the NEIL2 start site, which were in significant linkage disequilibrium (D′ = 73%; P < 0.05). This first report on in vivo variability in NEIL2 expression in humans identifies SNPs in the NEIL2 promoter region that have functional effects. © 2008 Wiley‐Liss, Inc.
High‐resolution copy number arrays in cancer and the problem of normal genome copy number variationGorringe, Kylie L.; Campbell, Ian G.
doi: 10.1002/gcc.20595pmid: 18663746
High‐resolution techniques for analysis of genome copy number (CN) enable the analysis of complex cancer somatic genetics. However, the analysis of these data is difficult, and failure to consider a number of issues in depth may result in false leads or unnecessary rejection of true positives. First, segmental duplications may falsely generate CN breakpoints in aneuploid samples. Second, even when tumor data were each normalized to matching lymphocyte DNA, we still observed copy number polymorphisms masquerading as somatic alterations due to allelic imbalance. We investigated a number of different solutions and determined that evaluating matching normal DNA, or at least using locally derived normal baseline data, were preferable to relying on current online databases because of poor cross‐platform compatibility and the likelihood of excluding genuine small somatic alterations. © 2008 Wiley‐Liss, Inc.
The noncoding RNA, miR‐126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3‐kinase signaling and is frequently lost in colon cancersGuo, Chunguang; Sah, Jerome F.; Beard, Lydia; Willson, James K. V.; Markowitz, Sanford D.; Guda, Kishore
doi: 10.1002/gcc.20596pmid: 18663744
MicroRNAs (miRNA/miR) are a class of small noncoding RNAs implicated in the pathogenesis of various malignancies. In the current study, using micro(RNA) arrays, we found a ubiquitous loss of miR‐126 expression in colon cancer lines when compared to normal human colon epithelia. Reconstitution of miR‐126 in colon cancer cells resulted in a significant growth reduction as evidenced in clonogenic assays. A search for miR‐126 gene targets revealed p85β, a regulatory subunit involved in stabilizing and propagating the phosphatidylinositol 3‐kinase (PI3K) signal, as one of the potential substrates. Restoration of miR‐126 in cancer cells induced a ≥3‐fold reduction in p85β protein levels, with no concomitant change in p85α, a gene that is functionally related to p85β but not a supposed target of miR‐126. Additionally, using reporter constructs, we show that the p85β‐3′ untranslated region is directly targeted by miR‐126. Furthermore, this miR‐126 mediated reduction of p85β was accompanied by a substantial reduction in phosphorylated AKT levels in the cancer cells, suggesting an impairment in PI3K signaling. Finally, in a panel of matched normal colon and primary colon tumors, each of the tumors demonstrated miR‐126 down‐regulation together with an increase in the p85β protein level. Taken together, we propose that miR‐126 regulates PI3K signaling partly by targeting p85β, and that the loss of miR‐126 may provide a selective growth advantage during colon carcinogenesis. © 2008 Wiley‐Liss, Inc.
Different telomere maintenance mechanisms in alveolar and embryonal rhabdomyosarcomaOhali, Anat; Avigad, Smadar; Naumov, Inna; Goshen, Yacov; Ash, Shifra; Yaniv, Isaac
doi: 10.1002/gcc.20600pmid: 18663749
The activation of a telomere maintenance mechanism (TMM) is crucial for the immortalization of tumor cells. Most human cancers apply telomerase‐dependent TMM but some use a mechanism called alternative lengthening of telomeres (ALT). The latter was suggested to be mainly characterizing sarcomas with nonspecific complex karyotypes, whereas telomerase activation is typical of sarcomas generated by specific translocations. In this study, we investigated the TMM and its association with survival in rhabdomyosarcoma (RMS), which is characterized by two major subtypes: one that is harboring a specific translocation (alveolar) and one that has a nonspecific karyotype (embryonal). Telomerase activity (TA), using telomerase repeat amplification protocol (TRAP) assay, and telomere length (TRF), using Southern blotting, were analyzed in tumor samples from 31 patients (16 embryonal and 15 alveolar). Alveolar RMS tumors exhibited no ALT phenotype and the majority presented TA. Some embryonal tumors exhibited an ALT or “ALT‐like” phenotype which lacked TA, whereas others expressed telomerase‐dependent TMM, and neither TA nor ALT correlated with outcome. The average TRF length of the embryonal tumors was significantly higher than that of the alveolar tumors (10.8 vs. 7.2 kb, P = 0.003). Interestingly, some tumors of both subtypes presented no TMM. These observations suggest that alveolar RMS predominantly use telomerase‐dependent TMM, whereas in embryonal tumors both telomerase and ALT may play a role. These findings have important implications for understanding the role of TMM in the development of RMS tumors, and for future designing adapted treatment strategies. © 2008 Wiley‐Liss, Inc.
NFIB rearrangement in superficial, retroperitoneal, and colonic lipomas with aberrations involving chromosome band 9p22Italiano, Antoine; Ebran, Nathalie; Attias, Rita; Chevallier, Anne; Monticelli, Isabelle; Mainguené, Claire; Benchimol, Daniel; Pedeutour, Florence
doi: 10.1002/gcc.20602pmid: 18663748
Lipomas are frequently characterized by rearrangements resulting in the fusion of the HMGA2 gene (12q14.3) with a variety of partners. Chromosome band 9p22 rearrangements occur in about 1% of lipomas. We report here the molecular cytogenetic analysis of five cases of lipoma with a 9p22 aberration, including the first cytogenetic analysis of a colonic lipoma. Three out of the five cases showed a rearrangement of NFIB at 9p22.3. The NFIB rearrangement involved a fusion with HMGA2 in two cases. We have identified an in‐frame fusion of the first three exons of HMGA2 with exon 6 of MSRB3 (12q14.3) and exons 8 and 9 of NFIB by using 3′RACE‐PCR in a case of superficial lipoma. In a case of retroperitoneal lipoma we found a fusion of HMGA2 with NFIB by fluorescence in situ hybridization analysis. The colonic lipoma was characterized by a t(9;16;19)(p22;q21;q13) with a rearrangement of NFIB and no rearrangement of HMGA2. NFIB belongs to the nuclear factor I transcription family. It has been previously shown to be fused with HMGA2 in one case of lipoma and to be a recurrent partner of HMGA2 in pleormorphic adenoma of salivary glands. We here demonstrate that NFIB can also be rearranged independently from HMGA2, indicating a potentially important role in lipoma pathobiology. Our findings suggest that the rearrangement of NFIB might be associated with deep‐seated lipomas, such as retroperitoneal or gastro‐intestinal lipomas. © 2008 Wiley‐Liss, Inc.
Allelic methylation bias of the RARB2 tumor suppressor gene promoter in cancerPappas, Jane J.; Toulouse, André; Hébert, Josée; Fetni, Raouf; Bradley, W. E. C.
doi: 10.1002/gcc.20603pmid: 18663751
Retinoic acid receptor B2 (RARB2) is frequently inactivated in cancer. Methylation in the 5′‐untranslated region and first exon is known to play a role; however, few studies have analyzed the detailed methylation pattern of the promoter region. We show that hypo‐ and hypermethylated alleles coexist in 5/11 cell lines in which RARB2 is inactivated. We present evidence supporting the mitotic transmission of these divergent methylation patterns and find a correlation between methylation divergence and heterozygosity at the 3p24 loci, suggesting an allelic methylation bias in these lines. Using a newly devised strategy based on allelic identification via methylation‐sensitive restriction enzyme digestion combined with the use of a single nucleotide polymorphism, rs755661, we demonstrate that such a bias exists in three cancer cell specimens heterozygous at rs755661 and therefore amenable to this study. This previously unreported phenomenon of allelic methylation bias suggests that a promoter methylation‐independent mechanism may be responsible for inactivation at the hypomethylated allele and this inactivation is reminiscent of an aberrant form of de novo imprinting. Approaches to interpreting methylation data should incorporate the notion of allelic methylation bias. © 2008 Wiley‐Liss, Inc.
Across array comparative genomic hybridization: A strategy to reduce reference channel hybridizationsBuffart, Tineke E.; Israeli, Daniëlle; Tijssen, Marianne; Vosse, Sjoerd J.; Mršić, Alan; Meijer, Gerrit A.; Ylstra, Bauke
doi: 10.1002/gcc.20605pmid: 18663753
Array comparative genomic hybridization (array CGH) is widely used for studying chromosomal copy number aberrations (CNAs) on a genome‐wide and high‐resolution scale in heritable disorders and cancers. The aim of this study was to test if the separate channels of dual channel arrays can be interchanged (across array) to either make array CGH more sensitive and cost effective and/or to generate profiles of CNAs and copy number variations (CNVs). Therefore the BT474 breast cancer cell line was compared with a mix of normal reference DNAs hybridized on different arrays and days and DNA copy number profiles were evaluated. Quality was assessed, using regular dual channel array CGH as a standard, using four quality measures, i.e., the median absolute deviation value of chromosome 2, the amplitude of the ERBB2 gene amplification, a deletion on chromosome 9, and the deflection on chromosome 8. The quality of the across array CGH profiles matched or even surpassed the quality of regular dual channel array CGH. In addition, this across array approach was tested for genomic DNA derived from formalin‐fixed paraffin‐embedded tumors tissue samples, resulting in high‐quality copy number profiles, comparable to regular dual channel arrays. Finally, we demonstrated this approach to obtain both CNA and CNV profiles. In summary, across array CGH avoids redundant hybridizations of the same reference material in every experiment either allowing hybridization of two test samples on one array or producing both CNA and CNV profiles simultaneously. © 2008 Wiley‐Liss, Inc.