Identification of novel breakpoints for locus- and region-specific translocations in 293 cells by molecular cytogenetics before and after irradiation

Abstract

The human kidney embryonic 293 cell line (293 cells) is extensively used in biomedical and pharmaceutical research. These cells exhibit a number of numerical and structural chromosomal anomalies. However, the breakpoints responsible for these structural chromosomal rearrangements have not been comprehensively characterized. In addition, it is not known whether chromosomes with structural rearrangement are more sensitive to external toxic agents, such as ionizing radiation. We used G-banding, spectral karyotyping (SKY), and locus- and region-specific fluorescence in situ hybridization (FISH) probes designed in our lab or obtained from commercial vendor to address this gap. Our G-banding analysis revealed that the chromosome number varies from 66 to 71, with multiple rearrangements and partial additions and deletions. SKY analysis confirmed 3 consistent rearrangements, two simple and one complex in nature. Multicolor FISH analysis identified an array of breakpoints responsible for locus- and region-specific translocations. Finally, SKY analysis revealed that radio-sensitivity of structurally rearranged chromosomes is dependent on radiation dose. These findings will advance our knowledge in 293 cell biology and will enrich the understanding of radiation biology studies.

Figure 1

A representative G-band karyogram of a HEK-293 cell showing 71 chromosomes. Arrows indicate rearrangements, additions, and deletions.

Figure 2

A representative (A) spectral and (B) classified images of a HEK-293 cell showing 70 chromosomes. Arrows indicate rearrangements, additions, and deletions.

Figure 3

Two sequential hybridizations showing that (A) one copy of X chromosome has two peri-centromere regions (aqua) with missing Xpter (arrows), while the derivative X has interstitial Xpter signal (yellow; arrow head/inset), the FISH “cocktail” also contains probes for 1p (green) and 1q (red) and (B) re-hybridization confirming the derivative X has Xqter signal (yellow) on either end (arrows and arrow head in the inset), the FISH “cocktail” also containing probes for 2p (green) and 2q (red). (C) Translocation of PML locus (15q22; aqua) and the sub-telomere region (15qter; yellow) from chromosome 15 (green box; arrow heads/inset) to two copies of chromosome 1 (red circle; arrows/inset). The FISH “cocktail” also contains probes for the sub-telomere regions of chromosome 10 (red and green). (D) 15q21 locus is present on chromosome 15 (green box; arrows/inset), not on chromosome 1. (E) Confirmation of 1q31 (green) and 1q32 (red) regions are present on three copies of chromosome 1 (red circles; arrows/inset). (F) The same metaphase after two sequential hybridizations showing insertion of 19qter into another chromosome (red circle; arrow/inset); (G) re-hybridization of the same metaphase confirming the insertion is distal to 11pter as evident from sub-telomere hybridization of p (green) and q (red) arm of three copies chromosome 11 (red circles; arrows/inset); the FISH “cocktail” also contains probes for the sub-telomere (yellow) and centromere (aqua) regions of chromosome 18, and (H) absence of 3p21 on the derivative chromosome 11 (green box; arrow/inset), but present on the two copies of chromosome 3 (red circles; arrow heads/inset). (I) The same metaphase after two sequential hybridizations showing translocation of 9qter (red) from chromosomes 9 (red circles; arrow heads/inset) to a group D chromosome (arrow); the FISH cocktail also contains positive hybridization for sub-telomere (17qter; yellow) and centromere (aqua) regions of chromosome 17 and (J) re-hybridization of the same metaphase confirming the receptor chromosome is chromosome 13 as detected by 13qter (yellow) and 13q14 (aqua) and indicated by arrows in the inset; the FISH “cocktail” also contains probes for 6p (green) and 6q (orange).

Figure 4

Distribution of acentric fragments in 293 cells by SKY analysis after (A) 2 Gy and (B) 4 Gy of radiation exposure. A total of 10 metaphase spreads were scored from each treatment group. Each row represents distribution of acentric fragments per metaphase spread.

Figure 5

Karyogram of 293 cells showing copy number of individual chromosome, centromere location (as indicated by horizontal dashed line), the break points (as indicated by white arrows), and inter chromosomal rearrangements (by different color junctions) as detected by G-banding, SKY analysis, and locus- and region-specific FISH hybridization.