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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2691-2698
NEOPLASIA
Concurrent activation of a novel putative transforming gene,
myeov, and cyclin D1 in a subset of multiple
myeloma cell lines with t(11;14)(q13;q32)
Johannes W. G. Janssen,
Jan-Willem Vaandrager,
Tanja Heuser,
Anna Jauch,
Philip M. Kluin,
Erik Geelen,
P. Leif Bergsagel,
W. Michael Kuehl,
Hans G. Drexler,
Takemi Otsuki,
Claus
R. Bartram, and
Ed Schuuring
From the Institute of Human Genetics, University of Heidelberg,
Heidelberg, Germany; the Department of Pathology, Leiden University
Medical Center, Leiden, The Netherlands; the Division of Hematology and
Oncology, Department of Medicine, Weill Medical College of Cornell
University, New York, NY; the Genetics Department, Medicine Branch,
National Cancer Institute, Bethesda, MD; the DSMZ-German Collection of
Microorganisms and Cell Cultures, Department of Human and Animal Cell
Cultures, Braunschweig, Germany; and the Department of
Hygiene, Kawasaki Medical School, Kurashiki, Okayama, Japan.
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Abstract |
Through the application of the NIH/3T3 tumorigenicity assay to DNA
from a gastric carcinoma, we have identified a novel transforming gene,
designated myeov (myeloma overexpressed gene in a subset of
t[11;14]-positive multiple myelomas). Sequence analyses did not
reveal any homology with sequences present in the GenBank, except the
deduced protein structure predicts a transmembrane localization.
Myeov was mapped to chromosome 11q13 and localized by DNA fiber
fluorescence in situ hybridization (FISH) 360-kilobase (kb) centromeric
of cyclin D1. In 3 of 7 multiple myeloma (MM) cell lines
with a t(11;14)(q13;q32) and cyclin-D1 overexpression, Northern
blot analysis revealed overexpression of myeov as well. In all
7 cell lines, the translocation breakpoint was mapped within the 360-kb
region between myeov and cyclin D1. DNA fiber FISH with
a contig of probes covering the constant region of the immunoglobulin heavy chain (IgH) revealed that exclusively in the 3 myeov-overexpressing cell lines (KMS-12, KMS-21, and XG-5),
either the 5' Eµ enhancer or the most telomeric 3' E
enhancer was juxtaposed to myeov. Although cyclin D1
overexpression represents a characteristic feature of all MM cell lines
with t(11;14), our results demonstrate aberrant expression of a second
putative oncogene in a subset of these cases, due to juxtaposition to
IgH enhancers. The clinical relevance of this dual activation remains
to be elucidated.
(Blood. 2000;95:2691-2698)
© 2000 by The American Society of Hematology.
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Introduction |
Illegitimate activity of the recombination machinery is
a frequent cause for chromosomal aberrations associated with various B-
and T-cell leukemias.1 Translocations involving the
immunoglobulin heavy chain (IgH) locus at 14q32.3 are mediated by
errors during VDJ-recombination that occur early in development or
during class-switch recombination at later stages in B-cell
development. The IgH locus contains 2 major types of enhancers: (1) the
5'-IgH intronic µ enhancer (Eµ) that is located between the
JH and switch µ sequences, and (2) the 3' enhancers that are
located downstream of each constant- (C) region, E1 and
E2.2,3 In mantle cell lymphomas and follicular lymphomas
carrying the t(11;14)(q13;q32) and t(14;18)(q32;q21), respectively, the
corresponding oncogenes cyclin D1 and bcl-2 are
deregulated because of the juxtaposition to the IgH-5' Eµ enhancer.4 In sporadic Burkitt's lymphomas, the
t(8;14)(q24;q32) leads to deregulation of the myc oncogene
because of an illegitimate switch recombination and joining onto the
3'-IgH E enhancers.5,6 Translocations involving
band 14q32 occur in approximately 20% (karyotype analysis), up to 70%
(fluorescence in situ hybridization [FISH]) of multiple myelomas (MM)
and plasma cell leukemias (PCL).7-16 The translocations to
the IgH locus at 14q32 primarily involve IgH switch regions and
various translocation partners.17 Four loci are most
frequently involved: cyclin D1/BCL1 on 11q13 in about 30% of the cases,7-13,15,16 FGFR3 on
4p16,14,16,18 MUM/IRF4 on 6p25,19 and
c-maf on 16q23.13,16,20 As a consequence of the
translocation to the switch regions, these genes are brought in the
proximity of the 3' E enhancers of the IgH locus and, consequently, are up-regulated. Translocations to any of the switch regions separate the IgH-5' Eµ and the IgH-3' E
enhancers and, theoretically, can simultaneously activate 2 different
genes on the 2 reciprocal chromosomes. Indeed, in case of t(4;14), 2 genes, FGFR3 and MMSET/WHSC1, are simultaneously
dysregulated by the E enhancers on der(14) and the Eµ enhancer on
der(4), respectively.21,22
Recently, we reported the identification of a novel transforming gene
that was isolated by the NIH/3T3 tumorigenicity assay with DNA from a
gastric carcinoma.23 Here we describe the isolation and
characterization of the full-length complementary DNA (cDNA) of this
gene designated myeov, its deduced 313-amino acid protein sequence, and its chromosomal location. Because myeov maps in the vicinity of cyclin D1 on 11q13, we evaluated expression of myeov in MM cell lines with t(11;14)(q13;q32). Although
cyclin D1 was overexpressed in all t(11;14)-positive
cell lines, we observed additional myeov overexpression in a
subset of the t(11;14)-positive MM cell lines. This was caused by an
aberrant class-switch event joining both myeov and cyclin
D1 to separate IgH enhancers.
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Materials and methods |
Cell culture
The MM cell lines ANBL-6, ARK, FLAM-76, H1112, JIM3, JJN3, KMM-1,
KMS-11, MM.1, MM-S1, OCI-My-5, OPM-2, SK-MM-1, SK-MM-2, U266,17 and EJM19 were described previously.
XG-1, XG-2, and XG-5 were obtained from Dr B. Klein (Montpellier,
France) and Dr S. Raynaud (Nice, France), KMS-12 and KMS-21 from Dr T. Otsuki (Okayama, Japan), FR4 from Dr S. Tagawa (Osaka, Japan), KHM-1 and KHM-11 from Dr H. Matsuzaki (Kumamoto, Japan), and LB84-1 from Dr
I. van Riet (Brussels, Belgium). The other MM cell lines (HL407, HL461,
L363, LP-1, MM-S4, MOLP2, MOLP3, NCI-H929, RPMI-8226, UTMC2), as well
as the non-MM cell lines in this study (Figure 1), were obtained from the German
Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).
MM and leukemia cell lines were grown in RPMI 1640, supplemented with
10% to 20% fetal calf serum as indicated by the supplier. The 5 mantle cell lymphomas (p11, p14, p26, p29, and p252) were described
previously.24-26
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| Fig 1.
Northern blot analysis of myeov gene expression
in various tumor cell lines of human malignancy, a third-cycle mouse
tumor (MA-T1A1) and NIH/3T3 recipient cells.
The purported or actual malignancy of the tumor cell lines are as
follows: MCF-7, breast adenocarcinoma; HT-29, colon adenocarcinoma;
HeLa, cervical carcinoma; THP-1, acute myeloid leukemia (M5); CCRF-CEM,
T-cell acute lymphoblastic leukemia; HEL, acute myeloid leukemia (M6);
Raji, Burkitt`s lymphoma; CTV-2, acute myeloid leukemia (M5); 5637, bladder carcinoma; BT474, breast adenocarcinoma; COLO-206F, colon
adenocarcinoma; COLO-680N, esophagus squamous cell carcinoma; A498,
renal carcinoma; HEP-3B, hepatocellular carcinoma; A-549, lung
adenocarcinoma; COLO-800, melanoma; SK-N-MC, neuroblastoma; MHH-ES-1,
Ewing's sarcoma; COLO-704, ovarian adenocarcinoma. Ten micrograms of
total RNA isolated from the indicated cell lines, MA-T1A1 tumor cells
(lane 9 in A) and NIH/3T3 cells (lane 10 in A), was submitted to
Northern transfer. (A) Filters were hybridized simultaneously with a
32P-labeled myeov cDNA insert and a murine
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe. Both
myeov transcripts of 2.8 and 3.5 kb and the GAPDH transcript
are indicated at the left side. Almost equal amounts of RNA were loaded
in each lane as indicated by the GAPDH hybridization. The stronger
hybridization of the 2 murine RNAs (MA-T1A1 and NIH/3T3, lanes 9 and
10) can be explained by the weaker hybridization of the murine GAPDH
probe to the human homolog. (B) Filters were hybridized with the same
myeov cDNA insert; 28S and 18S ribosomal RNA were used as
molecular weight markers. The lower panel shows the ethidium
bromide-stained gel as a control for the amount of RNA loaded in each
lane.
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Karyotypes
The following cell lines with a t(11;14)(q13;q32) were selected for
this study: XG-1 and XG-5,27 KMS-12,28 SK-MM-2,
H1112, and FLAM-7617 and KMS-21 (Otsuki et al; manuscript
in preparation). The MM cell line XG-2 was originally reported with a
complex t(5;11;14)(q31;q13;q32), but FISH with BCL1/11q13
probes revealed a breakpoint far away from the BCL1/11q13
region27; this is consistent with our finding that its
translocation breakpoint maps outside the 750-kilobase (kb) region
surrounding cyclin D1 (data not shown). All other MM cell lines
were not reported to harbor translocations involving the 11q13 region.
Isolation of a full-length myeov cDNA
A lambda gt10 cDNA library was constructed from polyA+
RNA of a gastric carcinoma (MA)-induced tertiary MA-transfectant
(MA-T1A1) and screened with the 400-base pair (bp) exon-trap
fragment.23 Normal human MA cDNA clones were isolated from
a human HeLa cervix carcinoma cell line cDNA library (Clontech,
Heidelberg, Germany). Respective clones were plaque purified and the
insert DNA was cloned into the EcoR I site of the pT7T3 plasmid vector
(Amersham Pharmacia Biotech, Freiburg, Germany). The nucleotide
sequence was determined by the dideoxy chain termination method using
an ALF-Express (Amersham).
Computer search and programs
Sequence analysis was evaluated with the University of Wisconsin
Genetics Computer Group (GCG) sequence analysis package (version 10.0) and with various server facilities; (BLAST;
http://www.ncbi.nlm.nih.gov/), (PSORT, http://psort.nibb.ac.jp), BLOCKS
(http://www.blocks.fhrc.org), PRODOM
(http://www.toulouse.inra.fr/prodom.html), PRINTS
(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/), SMART
(http://coot.EMBL-Heidelberg.de/SMART), PFAM
(http://genome.wustl.edu/pfam), SOSUI
(http://www.tuat.ac.jp/~mitaku/adv_sosui/), TMPRED, PROFILESCAN and
PROSITESCAN (http://ulrec3.unil.ch/software/), and PREDICTPROTEIN (http://www.embl-heidelberg.de/predictprotein/).
Northern blot analysis
Total RNA was isolated and purified using urea/LiCl as described
previously25 or with TriZol (purchased from GIBCO BRL, Gaithersburg, MD). Northern blot analysis and stripping of the filters
were performed as described previously.23,25 Briefly, 10 µg of total RNA was loaded on a 1% to 1.5% agarose gel and blotted
onto Nytran 13N membranes (Schleicher & Schuell, Dassel, Germany). RNA
filters were hybridized in 3 × SSC (0.45- mol/L sodium chloride, 0.045-mol/L sodium citrate), 5 × Denhardt's, 200 µg/mL 1 denatured salmon sperm DNA, 1% sodium
dodecylsulfate (SDS), and 10% dextran sulfate with
32P-dCTP-labeled probes at 63°C for 16 hours. Filters
were extensively washed in 3 × SSC, 0.1% SDS, and once in
0.1 × SSC, 0.1% SDS at 63°C. Filters were exposed to Kodak
X-Omat DS film at  70°C with Ilford intensifier screens. As
probes, we used a 1.1-kb 5' myeov cDNA EcoRI fragment, a
3'-end PRAD1/cyclin D1 probe,29 and for equal
RNA loading, a 400-bp murine glyceraldehyde 3-phosphate dehydrogenase
(GADPH) cDNA fragment.
RT-PCR analysis of myeov cDNA
Five micrograms of total RNA from a third cycle MA-transfectant were
transcribed into cDNA with random hexamers and the Superscript RT, as
recommended by the manufacturer (Gibco BRL, Karlsruhe, Germany). One
tenth of the cDNA reaction mixture was used in a 100-µL polymerase
chain reaction (PCR) reaction with 40-pmol primers and
1-unit Taq polymerase (Amplitaq DNA Polymerase, Perkin Elmer Applied
Biosystems, Weiterstadt, Germany). Amplifications were performed in an
automated PCR processor (BioMed, Theres, Germany) as follows: 35 cycles
comprising denaturation at 92°C for 30 seconds; annealing at
56°C for 60 seconds; primer extension at 72°C for 90 seconds,
with an initial denaturation step at 92°C for 3 minutes; and a
final extension step at 72°C for 10 minutes. PCR products were
electrophoresed in a 2.5% agarose gel and stained with ethidium bromide. For the detection of the alternatively spliced myeov mRNAs, the following primers were used: MA-14BU1
(5'-CCAGTGCTTTCACCAGC-3') and MA-mg2
(5'-GCGCCCACATAATTTCC-3').
Chromosomal in situ hybridization
For FISH, we used a 15-kb genomic fragment harboring the
myeov gene. Phage DNA was labeled by nick-translation with
biotin-16-dUTP. Hybridization was performed on human metaphase
chromosome preparations as described previously by Lichter and
Cremer.30 The biotinylated  phage clone was detected
using avidin conjugated to fluorescein, and signals were amplified
once.31 Chromosomes were counterstained with
4'-6'-diamidino-2-phenylindole (DAPI) and propidium
iodide. Chromosomes were analyzed by digital fluorescence
microscopy using a Zeiss Axiophot microscope, coupled to a cooled
charge-coupled device (CCD) camera (Photometrics, Munich, Germany)
equipped with a Kodak 1400 chip.
Interphase FISH
Interphase FISH for detection of t(11;14)(q13;q32) was performed
with 2 combinations of cosmid probes: (1) a centromeric
BCL1/11q13 cosmid (cos6.7) in combination with cosmid cosH1.5
located just telomeric of cyclin D1,32 and (2)
cos6.22 carrying the cyclin D1 gene with cosIg6 (from T.H.
Rabbitts, MRC, Cambridge, UK) covering the IgH constant gene
region.33 Cell lines that show both segregation of the
cos6.7/cosH1.5 signals and colocalization of the cos6.22/cosIg6 signals
in the majority of 200 evaluated nuclei were considered to carry a
BCL1/11q13-IgH breakpoint. Interphase nuclei were prepared and
FISH was performed as described elsewhere (Vaandrager et al, submitted
for publication). Probes were labeled with biotin-16-dUTP or
digoxygenin-11-dUTP (Roche, Mannheim, Germany) by standard nick-translation. Hybridization, immunodetection, and
fluorescence microscopy were performed as described
previously.26,33
Fiber FISH
DNA fibers were prepared as described previously.26,33
Immunodetection and fluorescence microscopy were the same as for interphase FISH. To generate a physical map of the BCL1/11q13 region using DNA fiber FISH, various combinations of DNA clones labeled
with either biotin or digoxygenin were hybridized and mapped relative
to our previously described set of probes, consisting of P1 clones
ICRF700B1587 and ICRF700J0777 and cosmid cos6.22.26,34 The
hybridization signal of cos6.22, cos3.62, and cos3.91 with a total
length of 113.4 kb, based on restriction mapping35 was used
as an internal standard. For the location of myeov, 3 genomic fragments subcloned from the original alu-positive genomic lambda phage
clone (genome/18935T1A1 harboring myeov coding sequences) were
used: a 2.1-kb EcoRI/SalI fragment (15RS2.1/4600, most 5'-clone), a 1.8-kb EcoRI fragment (10RR1.8/4599), and a 4.5-kb EcoRI fragment (19RR4.5/4598, most 3'-clone) (all in pT7T3). To generate a
contig of overlapping clones of the centromeric BCL1/11q13
region, several available and newly generated plasmid, cosmid, P1, and
YAC clones reported to map in this region were used: plasmids p11EH and
MTC-BCL1a24; cosmids cosH1.5, cos6.31, cos3.3, cos6.7, and
cos3.5135; plasmid BCL1/P1.7 (Brookes and Peters,
unpublished results); cosmids cCL11-44 and cCL11-505 (JBRC, Tokyo,
Japan); YAC yA7D7 (from St Louis Library) and cosmid
cCL-GW5536; and 3 P1 clones (9105/11q13, 9106/11q13, and
9107/11q13) obtained by screening a P1-library with 11q13 sequences
linked to the S breakpoint in SK-MM-2.37
For mapping of t(11;14) breakpoints in MM, fiber preparations were
hybridized with a combination of 11q13 probes and IgH probes. The
BCL1/11q13 probe set consisted of cosmid cCL11-505, a pool of
the 3 myeov genomic plasmid subprobes, (19RR4.5/4598,
10RR1.8/4599, and 15RS2.1/4600) P1 clones ICRF700B1587 and
ICRF700J0777, and cosmids cos6.22. The IgH probe set (cosmid clones
cosU2-2, cos3/64, and cosIg6 and plasmid probes for C and C4) was
described in detail recently.33 For detection of the
µ-enhancer, a 2.7-kb probe Eµ was made by PCR with primers
5'-GTAAGAATGGCCACTCTAGG-3' and
5'-CTAAAGCCATCTCATTGCCG-3'. This probe was hybridized
together with combinations of 11q13 and IgH probes.
GenBank/EMBL accession number
The sequence of the myeov cDNA has been deposited at the
EMBL-database under the accession number AJ223366.
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Results |
Identification and characterization of myeov
Application of the NIH/3T3-transformation/tumorigenicity assay to
DNA of a gastric carcinoma (MA) induced tumors in nude
mice.23 DNA of a tertiary transfectant was cloned into the
EMBL-3 phage vector and probed for human sequences using human
alu-repetitive sequences. Exon-trap analysis of genomic subfragments of
alu-positive phages revealed a novel sequence with no homology with
sequences present in the GenBank.23 Consecutively this
400-bp exon-trap fragment was used for the Northern blot analysis. A
prominent 2.8-kb transcript and a weaker RNA species of 3.5 kb were
detected in RNA of the third cycle transfectant (MA-T1A1) but not in
normal NIH/3T3 cells (Figure 1A, lanes 9 and 10). Similarly sized
transcripts were detected in various human tumor cell lines (Figure 1A
and B). Subsequently, a cDNA library of the tertiary transfectant was
screened with this exon-trap fragment as a probe and it enabled us to
identify various positive plaques. Nucleotide sequencing of different
cDNA clones representing the 2.8-kb transcript revealed minor splice
variants (50- to 200-bp difference), which explains the relatively
broad 2.8-kb band after Northern blot analysis. The presence of
these minor splice variants was also observed by RT-PCR analysis in
human cell lines expressing myeov (data not shown). Because
this novel putative transforming gene might be implicated in a
subset of human multiple myelomas with a typical t(11;14), the gene was
assigned the name myeov (HUGO/GDB Nomenclature Committee) for
MYEloma OVerexpressed (in a subset of
t[11;14]-positive multiple myelomas).
Sequence analysis of several MA-T1A1 cDNA clones revealed the presence
of 2 potential open reading frames, 1 of 313 amino acids and a shorter
product of 255 amino acids starting with an suboptimal
(CTCATGG) and an imperfect (CTCATGT) Kozak
sequence, respectively (Figure
2).38 The numerous minor splice
variants encode either the longer or the shorter product. A sequence
homology search for other sequences in the DNA databank, using the
full-length 2483-bp cDNA nucleotide (accession number AJ223366) and the deduced protein sequence, was negative, except for 6 expressed sequence
tags (ESTs) that are identical to the 3' end of our cDNA. Extended computer searches for homology with functional domains or
protein motifs were negative, except for the detection of an RNP-1
motif present in various RNA-binding proteins39 and some relatively short hydrophobic regions that might function as
transmembrane helices (Figure 2). Furthermore, the leucine-isoleucine
tail of the protein shows similarities with a class of cytoplasmically exposed membrane proteins with a C-terminal membrane
anchor.40
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| Fig 2.
The deduced myeov protein sequence.
Start sites of the 2 possible translation products are marked with an
arrow in front of the methionine start codon. An RNP-1 motif is
indicated by a dotted line. The 6 regions that might function as a
transmembrane domain are underlined.
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Localization of myeov at chromosome 11q13 in the vicinity of
the cyclin D1 oncogene
The original alu-positive genomic lambda phage clone
(genome/18935T1A1) harboring myeov coding sequences was used as
a probe to determine the chromosomal localization using FISH on banded metaphase chromosomes. Myeov was mapped on chromosome 11 band q13.1 (data not shown). Different regions within this band are involved
in a variety of disorders, including (1) translocations in mantle cell
lymphoma, multiple myeloma, and renal oncocytoma, (2) deletions in
multiple endocrine neoplasia type I, and (3) DNA amplification in
carcinoma of the head/neck, lung, esophagus, bladder, and
breast.41,42 To determine the localization of the
myeov gene in more detail, we used DNA fiber FISH with the 3 genomic myeov subfragments (clones 19RR4.5/4598, 10RR1.8/4599, and 15RS2.1/4600). In addition, we extended our previously reported 200-kb BCL1/11q13 fiber-FISH contig26 to 1100 kb
using DNA fiber FISH with available and newly generated cosmid, P1, and
YAC clones (Figure 3, upper panel). This
fiber-FISH map is in good agreement with mapping data obtained with
pulse-field gel electrophoresis.35 In this
BCL1/11q13 map, we located myeov 14-kb telomeric of
cosmid cCL11-505 (Figure 3), implicating that myeov and
cyclin D1 are approximately 360 kb apart.
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| Fig 3.
Map of the myeov-cyclin D1 region at 11q13,
constructed by fiber FISH.
The map is an extended version of the previously reported fiber-FISH
map.26 The scale was based on restriction mapping of the
cosmids cos 6.22, cos 3.62, and cos 3.91, which together were used as
internal standard of 113.4 kilobase (kb).35 All available
probes in the region are shown in the top part of the figure (see
"Materials and methods"). Myeov was detected using a pool
of the 3 subclones. The transcriptional orientation of the
myeov and cyclin D1 genes is indicated with horizontal
arrows. Hybridization of YAC A7D7* revealed an internal deletion that
is apparent as 2 separate, discontinuous signals. The bottom part shows
the localization of translocation breakpoints in 7 MM cell lines and 5 mantle cell lymphomas (p11, p14, p26, p29, and p252) as determined by
fiber FISH. Mapping of the mantle cell lymphoma breakpoints has also
been described previously.26
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To determine the transcriptional orientation of the myeov gene,
we performed fiber-FISH mapping with the 5' and 3' genomic subclones (respectively, 15RS2.1/4600 and 19RR4.5/4598) in different colors, combined with cosmid clone cCL11-505. The 5' subclone (15RS2.1/4600) was closest to cCL11-505 and also overlapped with probe
BCL1/p1.7 by Southern blot analysis. BCL1/p1.7 was
originally identified from a NotI-jumping library by screening with the
MTC/BCL1a probe and was considered to represent a CpG island
located 5' of a putative gene, approximately 350-kb centromeric
of cyclin D1 (Brookes and Peters, unpublished results). All
these data together suggest that myeov has the same
transcriptional orientation as the cyclin D1 gene.
Overexpression of myeov in a subset of
t(11;14)-positive multiple myelomas
Northern blot analysis of RNAs from various human cell lines
revealed expression of 2.8- and 3.5-kb myeov transcripts in
numerous tumor cell lines of divergent cellular origin (eg, Figure 1A
and B). A mouse myeov transcript was not detected (Figure 1A,
lane 10), either because the gene is normally not transcribed in mouse NIH/3T3 cells or, more likely, because of its lack of sequence conservation during evolution, as revealed by Zoo blot analyses under
low-stringency hybridization conditions (data not shown). We hybridized
Northern blots containing poly-A+ RNAs from 23 different
human tissue samples (Clontech) with an myeov cDNA probe. The
gene is expressed in various tissues, albeit at very low levels (data
not shown).
As reported previously,43 the breakpoint on 11q13 in the MM
cell line KMS-12 was mapped 330 kb centromeric from the cyclin D1 gene and immediately telomeric of cosmid cCL11-505. Using DNA fiber FISH, we were able to map myeov approximately 10-kb
centromeric of the breakpoint in the KMS-12 cell line (Figure 3, lower
panel). In this cell line, an illegitimate recombination of the
IgH-S2 switch region juxtaposes JH sequences, including the 5'
Eµ enhancer to the translocation allele harboring
cCL11-505.37,43 The combined mapping data suggested that
myeov becomes activated because of its juxtaposition to the
Eµ enhancer. As compared with non-MM cell lines, KMS-12 shows very
high levels of myeov expression. To evaluate expression levels
of myeov in other MM cell lines and its relation to the
presence of the t(11;14), we performed Northern blot analysis on a
series of 35 MM cell lines, including 7 cases with t(11;14)(q13;q32)
(FLAM-76, H1112, KMS-12, KMS-21, SK-MM-2, XG-1, and XG-5) and 28 cases
without this translocation (ANBL-6, ARK, EJM, FR4, HL407, HL461, JIM3,
JJN3, KHM-1, KHM-11, KMM-1, KMS-11, L363, LB84-1, LP-1, MM.1, MM-S1,
MM-S4, MOLP2, MOLP3, NCI-H929, OCI-My-5, OPM-2, RPMI-8226, SK-MM-1,
U266, UTMC2, and XG-2). Among the 7 cell lines carrying a t(11;14),
overexpression of myeov was observed in 3 MM cell lines,
namely, KMS-12, XG-5, and KMS-21 (Figure
4A, upper panel). Cyclin D1
expression was detected in all of them (Figure 4A, middle panel). Most
MM cell lines without t(11;14) showed no detectable or very low
myeov expression levels, and in 3 MM cell lines (NCI-H929,
L363, and KHM-11), moderate expression levels were detected (Figure 4B, upper panel). In the latter cell lines, cyclin D1 was not
expressed (Figure 4B, middle panel). Interphase FISH with various
probes, spanning a region of 550-kb centromeric of myeov and
250-kb telomeric of cyclin D1 flanked by markers cCL-GW55 and
cosH1.5 (Figure 3, upper panel), revealed no breakpoint in the NCI-H929
and L363 cell lines.
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| Fig 4.
Northern blot analysis of the 3 MM cell lines that show
high expression of both myeov and cyclin D1 (A) and
other MM cell lines (B).
Ten micrograms of total RNA isolated from the indicated cell lines was
submitted to Northern transfer. The filters were independently
hybridized to a 32P-labeled myeov cDNA insert
(upper panel), and a cyclin D1 cDNA insert (middle panel). 28S
and 18S ribosomal RNA were used as molecular weight markers. An
ethidium bromide-stained gel indicates the amount of RNA loaded into
each lane (lower panel).
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Overexpression of myeov caused by juxtaposition to an
IgH-enhancer
To resolve why only 3 of 7 MM cell lines with a t(11;14)(q13;q32)
showed myeov expression, whereas all overexpressed the
cyclin D1 gene, we determined the localization of the 11q13 and
14q32 breakpoint and especially the position of the various IgH
enhancers relative to myeov and cyclin D1. First,
interphase FISH was performed with the 2 cosmids flanking the
myeov-cyclin D1 region (respectively, cos6.7 and cos3.62;
Figure 3, upper panel). In 6 cases, segregation of these 2 cosmids was
observed, indicative of a translocation in this region. In the SK-MM-2
cell line, cosmid cos3.62 (localized just telomeric of cyclin
D1) was present twice, whereas the centromeric cosmid (cos6.7) was
present only once. Because cosmid cos6.7 colocalized with 1 of the
cos3.62 signals and thus represents the normal allele, this observation
suggests that the breakpoint occurred between the 2 cosmids with
simultaneous loss of the centromeric allele. By using DNA fiber FISH
with a combination of BCL1/11q13 and IgH probes,26,34 the position of the breakpoints within the
360-kb myeov-cyclin D1 region was fine mapped (Figure 3, lower
panel). Location of PAC clones (9105/11q13, 9106/11q13, and 9107/11q13) that were isolated by hybridization with a probe representing 11q13
sequences linked to the S breakpoint in SK-MM-237 by DNA
fiber FISH, confirmed the location of the SK-MM-2 breakpoint in the
BCL1/11q13 region (Figure 3). The use of a contig of clones covering the JH/constant region of the IgH locus enabled us to determine exactly what part of the IgH locus was joined to each respective translocation allele. At the same time, it allowed us to
study the class-switch recombinations within the IgH locus linked to
the BCL1/11q13 allele. An example of such an analysis for the
XG-5 cell line is illustrated in Figure 5.
To find out whether the Eµ enhancer is juxtaposed either to
myeov or to cyclin D1, we performed independent
fiber-FISH experiments using a PCR-generated Eµ probe in combination
with 11q13 and other IgH probes (Figure 5 and Figure
6, upper panel). A summary of these
fiber-FISH experiments for all 7 cell lines with t(11;14) is shown in
Figure 6 (lower panel).
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| Fig 5.
Example of t(11;14) breakpoint analysis by DNA fiber FISH
of the XG-5 cell line.
Red and green bars represent probes detected with Texas Red and FITC,
respectively. Overlapping areas of Texas Red- and FITC-stained probes
turn into yellow. From top to bottom, the following DNA fibers are
shown: (A) a normal 14q32/IgH locus, (B) a normal BCL1/11q13
locus, (C) the 14q+ translocation product containing the cyclin
D1 gene as observed in the XG-5 cell line, and (D and E) the
11q-product containing myeov in XG-5. Fibers A through D show
hybridization patterns obtained with the standard IgH and 11q13 probe
sets as described in "Materials and methods." Fiber E,
representing the 11q-product, shows the hybridization pattern of a
combination of IgH and 11q13 probes optimized for visualization of the
Eµ-enhancer probe in this particular cell line. For XG-5, this probe
set consisted of the 2.7-kb Eµ probe, 11q13 P1 B1587, and IgH cosmid
cosIg6.
|
|
View larger version (22K):
[in this window]
[in a new window]
| Fig 6.
Overview of the results of fiber-FISH mapping of t(11;14)
breakpoints in 7 MM cell lines.
Red and green bars represent probes detected with Texas Red and FITC,
respectively; yellow indicates areas of overlapping Texas Red- and
FITC-stained probes. The top 2 color bar codes show the normal
IgH/14q32 and BCL-1/11q13 loci. For each cell line, both 14q+
and 11q-fusion products are shown, and the myeov and
cyclin D1 genes are indicated with a small arrow and a larger
arrowhead, respectively. The position of the Eµ enhancer, as
determined using an Eµ-specific probe in separate experiments, is
indicated with a black circle. The position of the 3' E
enhancers was not determined by hybridization with a specific probe,
but was derived from the presence of the S/C plasmid probe. Where
2 S/C probe signals are present, the enhancers associated with
upstream signals and downstream signals are labeled E1 and E2,
respectively.
|
|
With respect to the 3 cell lines showing myeov overexpression,
the following conclusions from the translocation pattern can be drawn:
In KMS-21, IgH-C/ signals were detected on both translocation alleles, indicating a switch breakpoint (probably at S2) without concurrent class-switch deletion. Consequently, this results in juxtaposition of the E1 enhancer sequences to myeov and the
E2 enhancer to cyclin D1. In XG-5, the hybridization pattern
suggested a break at C1/S1 juxtaposing E enhancer sequences to
cyclin D1 (Figures 5 and 6). Hybridization with the Eµ
enhancer probe revealed its localization on the myeov
translocation allele. As previously reported,43 fiber-FISH
analysis revealed a breakpoint at switch gamma sequences in KMS-12,
resulting in a juxtaposition of the E enhancer to cyclin D1.
Using radiation-reduced hybrids, we previously reported that switch
gamma sequences were linked to 11q13 sequences.37
Additional hybridizations with the Eµ enhancer probe showed that this
enhancer was indeed joined to myeov. Taken together, the
simultaneous overexpression of both myeov and cyclin D1
in the 3 MM cell lines (KMS-21, XG-5, and KMS-12), corroborates the
identification of IgH-enhancer sequences on both translocation alleles.
On the contrary, in the H1112 and FLAM-76 cell lines, a rearrangement
in the JH region or its immediate vicinity was observed resulting in
juxtaposition of the two 3' E and the 5' Eµ enhancers to cyclin D1, whereas no enhancers were brought in the vicinity of myeov. As expected from our interphase FISH results, only 1 translocation allele harboring cyclin D1 and IgH sequences was identified in the SK-MM-2 cell line. Fiber FISH suggested that, because
of a S1 break, cyclin D1 was juxtaposed to both 3'
E enhancers. Hybridization with the Eµ enhancer probe revealed no signal. These results corroborate the previously determined switch gamma (S1) breakpoint to 11q13 sequences.37 In the XG-1
cell line, IgH-constant sequences with 1 of the 3' E enhancers
were juxtaposed to the cyclin D1 gene. On the 11q derivative
containing myeov, no IgH signal was visible, nor was it visible
after hybridization with the Eµ probe. Taken together, the
juxtaposition of the IgH enhancers to the cyclin D1 allele and
the concomitant lack of enhancer sequences linked to myeov in
SK-MM-2 (whole myeov allele is lost), XG-1, H1112, and FLAM-76
is in accordance with the lack of myeov expression and the
activation of cyclin D1. In other B-cell malignancies, ie, 5 mantle cell lymphomas with a breakpoint in the BCL1/11q13
region (Figure 3, lower panel) and juxtaposition to the JH locus, a
similar situation was observed, ie, linking of all IgH enhancers (Eµ,
E1, and E2) to cyclin D1 and expression of cyclin
D1. In accordance with these data, Northern blot analysis showed no
expression of myeov (data not shown).
| |
Discussion |
The application of the NIH/3T3 tumorigenicity assay with DNA from a
human gastric carcinoma resulted in the identification of a novel
putative transforming gene, designated myeov. Chromosomal mapping experiments located myeov within the 11q13 region. This chromosomal region has been implicated in a variety of disorders, including lymphomas, myelomas, renal oncocytomas, and a variety of carcinomas.
We observed numerous alternatively spliced transcripts of
myeov, and their respective role in tumorigenesis is presently
under investigation. Sequence homology searches revealed that
myeov lacks homology to any known genes. Extended computer
searches for functional domains resulted in the identification of an
RNP-1 motif that has been observed in various RNA-binding
proteins.39,44 Furthermore, some relatively short
hydrophobic regions and a C-terminal leucine/isoleucine tail were
observed that might function as transmembrane helices, whereas the
leucine/isoleucine tail of myeov shows similarities with a
class of cytoplasmically exposed membrane proteins with a C-terminal
membrane anchor.40 Some of these tail-anchored proteins are
endoplasmic reticulum-bound enzymes such as cytochrome b5,
heme oxygenase, or microsomal aldehyde dehydrogenase, but also comprise
certain viral proteins such as the middle T antigen. The presence of
these domains suggests that myeov will be directed to membranes
or more specifically to the endoplasmic reticulum. Preliminary
intracellular localization experiments with a myeov-GFP protein support this view (data not shown).
DNA fiber FISH enabled us to map myeov 360-kb centromeric
of the cyclin D1 oncogene. Thus far, all breakpoints in mantle
cell lymphomas26,45,46 and in MM cell
lines27,43,47,48 were restricted to this 360-kb
BCL1/11q13 region between cyclin D1 and myeov,
and correlate with overexpression of cyclin D1. However, in 3 (KMS-21, XG-5, and KMS-12) of the 7 MM cell lines carrying t(11;14)
investigated in our current study, overexpression of the oncogene
myeov was also observed. In these 3 cases, myeov and
cyclin D1 came under the separate control of different IgH enhancers (3' E1 or E2 and Eµ), respectively. A similar
situation has been described for the t(4;14)(p16;q32) translocation in
MM patients, where the FGFR3 and MMSET/WHSC1 genes are
controlled by 2 IgH enhancers, E and Eµ,
respectively.21,22
In the other MM cell lines with a breakpoint on chromosome 11q between
myeov and cyclin D1 and the JH or switch-class
recombination sites of the IgH locus, no myeov expression could
be detected. In these cases either the myeov-translocation
allele was lost (in SK-MM-2) or no IgH-enhancer sequences were
juxtaposed to this oncogene. The latter situation is similar to mantle
cell lymphomas with t(11;14) arising through an aberrant
VDJ-recombination. Consequently, myeov is not activated in
mantle cell lymphoma and is restricted to a subset of MM.
In MM patients, the t(11;14) accounts for approximately 30% of the
cases with 14q+,7-13,15,16 and the presence of this
breakpoint was reported to correlate with a worse
prognosis,15,49,50 although others16 could not
confirm this correlation. We found simultaneous activation of
myeov and cyclin D1 in only 3 of 7 t(11;14)-positive MM
cell lines, suggesting that activation of cyclin D1 is a more
essential step than activation of myeov. However, the fact that
high myeov expression in these cell lines was correlated with
the juxtaposition of IgH enhancers to the myeov allele suggests that myeov expression can function independently of the
activation of cyclin D1. In accordance with this hypothesis, we
also detected moderate myeov expression in 3 MM cell lines
without t(11;14) and without cyclin D1 expression.
The clustering of the t(11;14)(q13;q32) breakpoints within the 360-kb
BCL1/11q13 region between the cyclin D1 and
myeov gene may be of clinical relevance for the diagnosis of
B-cell NHL, in particular the diagnosis of mantle cell
lymphoma. As we previously reported, an almost perfect (33 of 34 cases)
correlation exists between cyclin D1 overexpression and the
presence of a breakpoint in the BCL1/11q13 locus centromeric of
the cyclin D1 gene.26,45 Assuming that enhancers
act on proximal promoter sequences,51 the identification,
mapping, and expression pattern of myeov suggest that no other
genes are located between cyclin D1 and myeov.
Consequently, the BCL-1 breakpoint region is restricted to 360 kb flanked by the cyclin D1 and myeov genes. Therefore,
the previously proposed set of probes to determine the presence of an
BCL-1/11q13 breakpoint using interphase FISH is indeed suitable
for diagnostic purposes.26,32 It might well be that in
native myelomas with t(11;14)(q13;q32), the breakpoints are located
within the same region. Mapped breakpoints in 4 patient samples with
t(11;14) have been reported to fall within the same 360-kb
BCL-1/11q13 region identified in the cell lines.47,52
In conclusion, we identified a novel putative transforming gene
involved in a subset of myeloma cell lines carrying t(11;14). Whether
breakpoints in the BCL-1/11q13 region with concomittant activation of the myeov gene is also observed in native
myelomas and whether it has an impact on the clinical behavior will be the subject of an ongoing study.
| |
Acknowledgments |
Excellent technical assistance of B. Gschwendt, A. Steenvoorden, A. Wunderlich, U. Spadinger, E. Schuuring-Scholtes, and M. Schmidberger is
greatly acknowledged. We thank T. Gibson for help with database
searching. We also thank S. Raynaud, B. Klein, C. Theillet, P. Gauddray, G. Peters, T. Rabbitts, S. Tagawa, H. Matsuzaki, and I. van
Riet for providing cell lines and DNA clones. We dedicate this article
to Professor F. Vogel on the occasion of his 75th birthday.
| |
Footnotes |
Submitted August 12, 1999; accepted December 22, 1999.
Part of this work is the subject of the PhD thesis of T.H.
Supported by grants of the Deutsche Forschungsgemeinschaft to J.W.G.J.
(Sonderforschungsbereich 322 "Lympho-Hämopoese") and the Dr
Mildred Scheel Stiftung für Krebsforschung (10-1253) to J.W.G.J.
and of the Dutch Cancer Society (NKB-RUL96-1647) to E.S.
Reprints: Johannes W.G. Janssen, Institut für
Humangenetik, Ruprecht-Karls-Universität Heidelberg, Im
Neuenheimer Feld 328, D-69120 Heidelberg, Germany; e-mail:
hans_janssen{at}med.uni-heidelberg.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction
