Molecular cytogenetic characterization of chromosome 9-derived material in a human thyroid cancer cell line

Abstract

The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the eight years following the accident, the average incidence of childhood PTCs (chPTC) increased 70-fold in Belarus, 200-fold in Gomel, 10-fold in the Ukraine and 50-fold in Tschnigov, Kiev, Rovno, Shitomyr and Tscherkassy compared to the rate of about 1 tumor incidence per 106 children per year prior to 1986 (Likhtarev et al., 1995; Sobolev et al., 1997; Jacob et al., 1998). To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed ‘BAC-FISH’ to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.

BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer

“…The DNA concentrations were determined using a TKO100 instrument and Hoechst 33342 fluorometry (Hoefer, San Francisco, CA) [23]. The BAC-derived DNA (typically 1-2 μl of DNA in a 10 μl reaction) was labeled via random priming following the instruction of the kit manufacturer (BioPrime Kit, Invitrogen) [35] incorporating biotin-14-dCTP (part of the BioPrime kit), digoxigenin-11-dUTP (dig-11-dUTP, Roche Molecular Biochemicals, Indianapolis, IN), fluorescein-12-dUTP (Roche Molecular Biochemicals), Cy5-dUTP (Amersham, Arlington Heights, IN) or Cy5.5-dCTP (Perkin Elmer, Wellesley, MA) [10,34,35].…”

Chromosomal aberrations in thyroid follicular-cell neoplasia: in the search of novel oncogenes and tumour suppressor genes

“…In primary cultures of radiation-associated thyroid tumours, novel breakpoints on chromosomes 4q, 5q, 6p, 12q, 13q and 14q were described by comprehensive analysis using M a n u s c r i p t 12 conventional and spectral karyotyping (Zitzelsberger, et al 1999). Spectral karyotyping also uncovered complex rearrangements in some tumours (Lu, et al 2009;Weier, et al 2006)that have been investigated further by a BAC-FISH approach in order to delineate chromosomal breakpoints. In Figure 2 an example for fine-mapping of chromosomal breakpoints by FISH is shown for a case of a post-Chernobyl PTC.…”

“…One BAC-FISH-based approach allows cloning of aberrations by using pools of uniformly labelled BAC clones covering a relatively large candidate region in the first place followed by a hybridisation with individual BAC probes labelled with individual dyes (Lu et al 2009). This approach has been applied to a primary culture of post-Chernobyl thyroid tumours PTC in order to characterise chromosomal alterations on chromosome 9 (Weier et al 2006). In…”

DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

“…Gross chromosomal changes in the S48TK6 metaphases were characterized by means of G banding, comparative genomic hybridization (CGH), and spectral karyotyping (SKY) (Zitzelsberger et al 1999;Weier et al 2006).…”