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, 92 (2), 382-8

Multicolour-banding Fluorescence in Situ Hybridisation (mbanding-FISH) to Identify Recurrent Chromosomal Alterations in Breast Tumour Cell Lines

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Multicolour-banding Fluorescence in Situ Hybridisation (mbanding-FISH) to Identify Recurrent Chromosomal Alterations in Breast Tumour Cell Lines

A Letessier et al. Br J Cancer.

Abstract

Recurrent chromosome breakpoints in tumour cells may point to cancer genes, but not many have been molecularly characterised. We have used multicolour-banding fluorescence in situ hybridisation (mbanding-FISH) on breast tumour cell lines to identify regions of chromosome break created by inversions, duplications, insertions and translocations on chromosomes 1, 5, 8, 12 and 17. We delineate a total of 136 regions of break, some of them occurring with high frequency. We further describe two examples of dual-colour FISH characterisation of breakpoints, which target the 1p36 and 5p11-12 regions. Both breaks involve genes whose function is unknown to date. The mbanding-FISH strategy constitutes an efficient first step in the search for potential cancer genes.

Figures

Figure 1
Figure 1
Examples of mbanding-FISH in breast tumour cell lines. MDA-MB-231 (A), MDA-MB-157 (B and C) and ZR-75-1 (D) were analysed by FISH with specific mband cocktail probes for chromosome 1 (A), 5 (B), 12 (C) and 17 (D), respectively. M-FISH images of normal (save for chromosome 12 in MDA-MB-157 (C)), and derivative chromosomes 1, 5, 12 and 17 previously characterised in these cell lines (Popovici et al, 2002) are shown on the left hand of the corresponding mband images (pseudocolour profile) defined by colour spectra (on the right). Regional locations of breakpoints targeting der(1)t(1;14)(p34;q?) (A), I(5)(p10) (B), der(12)t(1;12)(?;p13.3) (C upper part), der(12;16)(q10;?q10) (C middle part) and der(11)(t(11;17)(?;q21.3–22) (D) present in MDA-MB-231 (A), MDA-MB-157 (B and C) and ZR-75-1 (D), respectively, were assigned by comparison with the normal profile. They are indicated by arrowheads on the corresponding ideograms (bottom part), which exhibit on their right hand the sequence of microdissected region-specific PCP labelled using a unique fluorochrome combination defining then the normal colour spectrum. Each PCP was labelled and partly overlaps with the neighbouring one.
Figure 2
Figure 2
(A) Regional distribution of breakpoints in breast tumour cell lines. From mbanding-FISH analysis on breast tumour cell lines, a total of 136 breakpoints were described (Supplementary Table 1). Each coloured circle corresponds to a cell line as defined in the inset in upper right corner. These circles are positioned on ideograms of normal 1, 5, 8, 12 and 17 chromosomes in the region affected by breakpoints characterised in cell lines derivatives reported in the supplementary table. Each coloured circle represents a breakpoint observed in one derivative. The occurrence order of coloured circles follows the order of breakpoints characterised in the corresponding cell lines reported in the supplementary table, for example, two circles with the same colour on the same region mean that this region is involved in two different derivatives present in the same cell line. Black arrows show 1p36 and 5p12 breakpoints of the t(1;6)(p36;p21) and t(5;12)(p12;p11) present in IPC-BC-116 and UACC-812, respectively. (B and C) Dual-colour FISH refinement of two target regions. Dual-colour FISH experiment carried out on metaphase chromosomes from IPC-BC-116 (B) with digoxigenin-labelled DNA of RP11-164A22 (revealed in red, TRITC) in combination with biotinylated DNA of RP11-99F3 and centromeric probe specific for the chromosome 6 (revealed in green, FITC). The telomeric position of RP11-164A22 allowed the identification of normal chromosome 1 and derivative chromosome 6. The concomitant presence of RP11-99F3 on both derivatives and on chromosome 1 suggested that this clone spans the 1p36 breakpoint. Similarly, dual-colour FISH experiment carried out on metaphase chromosomes from UACC-812 (C) using biotinylated DNA of RP11-190J8 BAC (revealed in green, FITC) and digoxigenin-labelled centromeric probe specific for chromosome 12 (revealed in red, TRITC) shows that RP11-190J8 spans the 5p12 breakpoint.

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