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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4442-4444
CORRESPONDENCE
Jumping Translocation Breakpoint Regions Lead to Amplification of
Rearranged Myc
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LETTER |
To the Editor:
The amplification of cellular proto-oncogenes, a common feature of
malignant tumors, results in distinct cytogenetic alterations in
carcinomas and hematopoietic neoplasms. In carcinomas, it mainly produces elongation of chromosomes by homogeneously staining regions or
extrachromosomal elements referred to as double minutes. In hematopoietic malignancies, in which homogeneously staining regions and
double minutes are rare, it gives rise to jumping translocations of
chromosomal segments containing oncogenes, ie, segmental jumping translocations (SJTs). SJTs were detected recently in treatment-related leukemias in humans.1 They have been shown to relocate
chromosomal regions harboring the ABL, MLL, INT-2, and MYC oncogenes to
one or more recipient chromosomes, creating, thereby, structurally abnormal chromosomes, unidentifiable marker chromosomes, and partial polysomy of the amplified chromosomal segment. Thus far, SJTs have been
demonstrated to lead only to the amplification of unrearranged oncogenes in normal genomic configuration. We describe here a new type
of SJT that appears to lead to the multiplication of rearranged and
activated (ie, constitutively transcribed) oncogenes. The new SJT was
observed in the BALB/c mouse plasmacytoma, MOPC 315, in which it
effected the transposition onto two marker chromosomes of a chromosomal
segment that contained the same clonotypic MOPC 315-typical T(12;15)
translocation breakpoint region. This region is known to harbor a
recombined and transcriptionally deregulated Myc gene.
Spectral karyotyping (SKY)2,3 was used to analyze the
chromosome complement of a subline of the inflammation-induced mouse
plasmacytoma, MOPC 315. It readily identified the Chr T(12;15), the
hallmark chromosome of BALB/c plasmacytomas, which is thought to be
essential for these tumors, because it contains the transcriptionally deregulated Myc.4 In addition, SKY showed the
presence of two chimeric marker chromosomes that contained small hybrid
segments of Chr 12- and Chr 15-derived material, the Chrs
T(12;15;16;12;15;16) and T(17;15;16;12;15;16). The marker chromosomes
were of particular interest, because they could be considered as
tripartite chromosomes that were produced by joining Chrs 12, 15, and
16, and Chrs 17, 15, and 16, respectively, and then inserting a
chromosomal segment that harbored a T(12;15) breakpoint region (Fig
1A). To test this hypothesis, the Chr
T(12;15) and the two marker chromosomes were separated by flow
sorting.5 The genomic DNA obtained from flow-sorted chromosomes was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)6 to
prepare fluorescence in situ hybridization (FISH) probes for the
reverse painting of normal mouse chromosomes. Reverse painting was used
to determine the purity of the flow-sorted chromosomes and to confirm
and map their composite nature as seen by SKY (Fig 1B). To demonstrate that all three flow-sorted tumor chromosomes contained the MOPC 315-typical translocation breakpoint region, a clonotypic junction fragment between Myc and the switch region of the Ig
heavy-chain  locus was generated. This was accomplished by PCR using
DNA obtained from the flow-sorted chromosomes as template. The DNA sequence analysis of the PCR fragments showed the presence on all three
chromosomes of the same T(12;15) translocation breakpoint region
previously determined to be unique for MOPC 3157 (Fig 1C).
These findings were interpreted to mean that in MOPC 315 the identical
Myc rearrangement was relocated by SJTs to two marker
chromosomes.
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| Fig 1.
Amplification of T(12;15) translocation breakpoint
regions by segmental jumping translocation (SJT) in BALB/c mouse
plasmacytoma, MOPC 315. (A) SKY display image of MOPC 315. Rearrangements between Chrs 12 and 15 (indicated by arrows) were
observed consistently in 30 images on both the plasmacytoma-specific
chromosome T(12;15) and the marker chromosomes, T(12;15;16;12;15;16)
and T(17;15;16;12;15;16). The chromosomes are numbered I, II, and III,
respectively. (B) Reverse painting of flow-sorted tumor chromosomes on
normal mouse chromosomes stained with DAPI. The normal DAPI-stained
chromosomes are shown in the white insets to facilitate the
interpretation of the reverse painting results. Three distinct flow
peaks designated I, II, and III were identified (not shown). They must
have contained the translocated chromosomes I, II, and III (shown in
[A]) for the following reasons. Peak "I" contained Chr T(12;15)
because the translocation juxtaposed the Ig heavy-chain gene cluster,
located on Chr 12F2, to the Myc locus, residing on Chr 15D2.
Therefore, upon reverse painting, Chr 12 should be labeled completely
in red (with the exception of the small telomeric cap distal to band F2
that is not discernible in the image due to its small size), whereas
Chr 15 should be labeled from band D2 to the telomere. The observed
reverse-painting pattern depicted at the top matched this expectation.
Peak "II" contained Chr T(12;15;16;12;15;16), because the FISH
probe derived from it stained the distal half of Chr 16, but not Chr
17. Peak "III" contained Chr T(17;15;16;12;15;16), because the
FISH probe obtained from it stained both Chr 16 and Chr 17. Thus, the
reverse painting pattern of the three flow peaks corresponded to the
structure of the translocated chromosomes as predicted by SKY. (C)
Detection of the same clonotypic junction fragment between the switch
region (S ) of the Ig heavy chain gene (C) and intron 1 of
Myc by direct, two-round PCR amplification with nested primer
pairs (arrowheads). The identical hybrid fragment (indicated by the
two-colored horizontal bar) was obtained when DNA samples prepared from
the flow-sorted marker chromosomes I, II, and III were used as
templates in three different PCR reactions. DNA sequence analysis
confirmed the identity of the Myc/S breaksite and its
flanking regions on all three chromosomes. Twenty basepairs of
Myc and S are shown at the bottom to left and right of the
breakpoint, respectively. Exons 2 and 3 of Myc, exon 1 of C,
and S are depicted as labeled boxes. The T(12;15) translocation
breaksite is indicated by an arrow.
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The mechanism of the SJT that amplified the T(12;15) breakpoint region
in MOPC 315 is not known. Possibilities include, first, the involvement
of extrachromosomal precursors, such as episomes or double minutes
containing T(12;15) translocation segments, before a postulated
chromosomal reintegration event; second, the occurrence of
illegitimate, nonreciprocal, trans-chromosomal recombinations between
hyperreplicative or fragile sites; and third, the involvement of
recombinogenic repetitive sequences at the breaksites. The latter
explanation is supported by findings that breakpoints of jumping
translocations, the next close relatives of SJT, are usually found at
sites of repetitive DNA, eg, in centromeres or pericentromeric heterochromatin,8 telomeres, subtelomeric regions, variant telomeric repeats or interstitial telomeric sequences,9-11
or constitutive heterochromatin12,13; however, this has not
been shown for MOPC 315. Furthermore, it is conceivable that the
Myc gene facilitates its own amplification via SJTs as a
consequence of a Myc-induced mutator phenotype. This hypothesis
is based on the proposal that Myc acts as a mutator gene in
plasmacytomas14 and the finding that chromosomal
translocations were induced by another oncogene, the SV40 large
T-antigen.15
In conclusion, it is suggested that SJTs may be not only a mechanism
for increasing the copy number of unrearranged oncogenes, but also a
tumor progression mechanism that leads to the amplification of
rearranged, transcriptionally active oncogenes. Additional studies are
warranted to determine the prevalence of SJTs in mouse plasmacytomas
and to explore if recombined oncogenes can jump in human leukemias and
lymphomas, too.
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ACKNOWLEDGMENTS |
The authors thank Dr J.F. Mushinski for reading the letter and making
helpful editorial suggestions.
Allen E. Coleman
Alexander
L. Kovalchuk
Siegfried Janz
Laboratory of Genetics
Division of Basic Sciences
National Cancer Institute
National
Institutes of Health
Bethesda, MD
Alessio Palini
FAST
Systems
Gaithersburg, MD
Thomas Ried
Genome Technology
Branch
National Center for Human Genome Research
National
Institutes of Health
Bethesda, MD
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