A der(8)t(8;11) chromosome in the Karpas-620 myeloma cell line expresses only cyclin D1: yet both cyclin D1 and MYC are repositioned in close proximity to the 3'IGH enhancer

Abstract

The Karpas-620 human myeloma cell line (HMCL) expresses high levels of Cyclin D1 (CCND1), but has a der(8)t(8;11) and a der(14)t(8;14), and not a conventional t(11;14). Fluorescent in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) studies suggest that der(14)t(11;14) from a primary translocation underwent a secondary translocation with chromosome 8 to generate der(8)t(8;[14];11) and der(14)t(8;[11];14). Both secondary derivatives share extensive identical sequences from chromosomes 8, 11, and 14, including MYC and the 3' IgH enhancers. Der(14), with MYC located approximately 700 kb telomeric to the 3'IGH enhancer, expresses MYC. By contrast, der(8), with both CCND1 and MYC repositioned near a 3'IGH enhancer, expresses CCND1, which is telomeric of the enhancer, but not MYC, which is centromeric to the enhancer. The secondary translocation that dysregulated MYC resulted in extensive regions from both donor chromosomes being transmitted to both derivative chromosomes, suggesting a defect in DNA recombination or repair in the myeloma tumor cell.

Fig. 1

Identification and sequence of t(11;14) translocation breakpoint in Karpas-620. (A) Map of probes used to detect translocations. Circles and boxes indicate structural elements; thin vertical arrows indicate Bam HI (B) and SphI (S) restriction sites. Thick vertical arrow indicates site of translocation breakpoint. (B, C) Southern blots of Karpas-620 genomic DNA digested with SphI (B) or BamHI (C) were hybridized with probes that flank the mu or gamma switch regions, as described previously [21]. The sizes of fragments, in kb, is shown. Arrows in (B) indicate SμSγ legitimate switch recombination fragments. (D, E) the structure of the t(11;14) translocation breakpoint (vertical arrow) is shown, with the telomeric end at the right. The horizontal arrow indicates the direction of transcription of CCND1. The breakpoint includes an overlapping AAG sequence. For Chr 14, the asterisk follows position 105,398,505; and for Chr 11, the asterisk precedes position 68,821,862 (NCBI build 35).

Fig. 2

An RNA FISH analysis shows that CCND1 is expressed only from der(8)t(8;11). The left panel shows an RNA FISH experiment in which a CCND1 probe identifies two sites of CCND1 transcription in a Karpas-620 cell. The right panel shows a DNA FISH experiment on the same cell with a CH probe, demonstrating that the two sites of CCND1 transcription are coincident with two of the six CH sequences, both of which are coincident with the CCND1 gene on der(8)t(8;11) (not shown).

Fig. 3

Array comparative genomic hybridization identifies duplicated sequences and breakpoints. Copy number of Karpas-620 genomicDNA sequences normalized to healthy male DNA was determined by segmental analysis on Agilent custom arrays (A, B), or an Agilent 244K array (C). MYC locus (A) results include position of MYC, and approximate extent of duplicated sequences centromeric and telomeric to the 5′ end of the MYC. CCND1 locus (B) results include position of MYEOV and CCND1 genes, approximate region of increased copy number, and position of cloned 11;14 breakpoint (vertical arrow) relative to 5′ end of CCND1 gene. IGH locus results (C) include positions of Eα1&2 3′ IGH enhancers, IGH gamma coding regions, five genes centromeric to Eα2 enhancer, and approximate region of increased DNA copy number. Diagram is not to scale. See text for additional details.

Fig. 4

Primary and secondary translocations that dysregulate CCND1 and MYC (A) Proposed pathways by which primary and secondary translocations generate der(14)t(8;14), der(8)t(8;11), and der(11)t(11;13) in Karpas-620. (B) Duplicated regions (cross-hatched) and location of breakpoints are shown for der(8)t(8;[14];11) der(14)t(14;[11];8). The reference locations (0) are the centromeric (5′) ends of MYC and CCND1 on chromosomes 8 and 11, respectively; and the telomeric end of the Eα1 3′ IGH enhancer on chromosome 14. Centromeric and telomeric sites are negative and positive, respectively, with numbers in kb. Boxed chromosomes represent chromosomal precursors and proposed intermediates that are no longer present in the Karpas-620 cell line; arrowheads indicate possible sites of single stranded breaks. The horizontal arrows over the two Eα 3′ IGH enhancers indicate that their activity may be directed exclusively in a telomeric direction, and the horizontal arrows over MYC and CCND1 indicate their 5′−3′ orientations.