Precise breakpoint characterization of the colon adenocarcinoma cell line HT‐29 clone 19A by means of 24‐color fluorescence in situ hybridization and multicolor banding

In a previous issue of this journal, we read with interest the article of Kawai et al. (2002) regarding the karyotype analysis of the colon adenocarcinoma cell line HT-29. To study this karyotype, the authors combined G-banding, fluorescence in situ hybridization (FISH), and spectral karyotyping (SKY). As mentioned by Kawai et al. (2002), Gbanding analysis for HT-29 had been carried out before by Chen et al. (1987) and Bertrand et al. (1999). In addition, however, there are two other previous attempts to determine the karyotype of HT-29, through use of a combination of comparative genomic hybridization (CGH) and SKY (Ghadimi et al., 2000; Abdel-Rahman et al., 2001). As demonstrated recently, the multicolor banding (MCB; according to Liehr et al., 2002a) method allows the characterization of exactly the breakpoints and the orientation of chromosomal rearrangements (Weise et al., 2002), even in highly rearranged karyotypes (Mrasek et al., 2001). Thus, a detailed analysis of aberrations and characterization of breakpoints in HT-29 clone 19A, which shares the main chromosomal aberrations with HT-29, was performed by MCB. HT-29 clone 19A (terminally differentiated and described by Augeron and Laboisse, 1984) differs according to our data presented below from the HT-29 cell line described by Kawai et al. (2002) in 10 chromosomal aberrations. Furthermore, the HT-29 clone described by Abdel-Rahman et al. (2001) differs from the one investigated by Kawai et al. (2002) by 10 rearrangements. After an orienting classification of chromosomal rearrangements by 24-color FISH as described by Kuechler et al. (2002), MCB was performed on all chromosomes involved in rearrangements in HT-29 clone 19A (Fig. 1). When necessary for exact characterization of rearrangements and/or origin of derivative chromosomes, MCB analysis was complemented by centromere-specific probes (chromosomes 1, 2, 4, 6, 7, 8, 9, 12, 13, 17, 18, 19, and 22), locus-specific probes (MYC and TP53), partial (chromosome arms 3p, 3q, 12p, 17q, and 18p), and whole chromosome painting probes (chromosomes 2, 3, 4, 5, 6, 11, 16, 17, 18, 19, and 22). At least 10 metaphases were evaluated per hybridized probe or probe set. On the basis of the combination of 24-color FISH, MCB, and the additional FISH probes, a composite karyotype could be described (Table 1). A survey of hybridization results of all aberrant and corresponding normal chromosomes is given in Figure 1. The composite karyotype after MCB contained 20 different aberrant chromosomes, with a total number of 36 breakpoints, 33 of which could be characterized in detail. The der(22)t(17;22;17), with high probability identical to marker M9 described by Kawai et al. (2002), was the only marker that could not be fully characterized. In concordance with the literature, all detected structural aberrations were unbalanced and almost all human chromosomes were involved, apart from chromosomes 10, 14, 15, and 21. However, Kawai et al. (2002) and Ghadimi et al. (2000) reported a t(6;14) and a der(6)t(6;14), respectively. The latter reflects the heterogeneity of this very common cell line. Our HT-29 clone 19A and the HT-29 cell line studied by Kawai et al. (2002) presented with a similar copy number for most of the chromosomes. Differences were that HT-29 contained the marker chromosomes M1, M10, M11, M13, M15, M17, and M18, which were not present in clone 19A. However, our clone showed an additional marker similar to M12 (Chen et al., 1987), a der(5)t(5;19), and a der(11)t(11; 20). A der(2)t(1;2), also described in HT-29 by AbdelRahman et al. (2001), a der(3)ins(3;12), also detected in HT-29 by Ghadimi et al. (2000), and a der(4)t(2;4) were present instead of a normal chromosome 2, M1, or M17, respectively. Applying MCB, breakpoints could be confirmed for M2, M4, and M14 and further characterized for M6 and M16. Additionally, the huge derivative (8q)-isochromosome (M6) was demonstrated by MCB to include a homogeneously staining region (HSR) derived from 8q24, which was subsequently verified to consist of a MYC amplicon (Fig. 1).