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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2930-2936
NEOPLASIA
MUC1 dysregulation as the consequence of a
t(1;14)(q21;q32) translocation in an extranodal lymphoma
Frédéric Gilles,
André Goy,
Yvonne Remache,
Peter Shue, and
Andrew D. Zelenetz
From the Laboratory of Molecular Hemato-Oncology, the Lymphoma
Service, and the Department of Medicine, Memorial Hospital, Memorial
Sloan-Kettering Cancer Center, New York, NY.
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Abstract |
Cytogenetic abnormalities at chromosome 1q21 are among the most
common lesions in diffuse large-cell lymphoma and have been associated
with a poor prognosis. A novel cell line, SKI-DLCL-1, was established
from ascitic fluid that carries a t(1;14)(q21;q32) chromosomal
translocation. Using pulsed-field gel electrophoresis, the breakpoint
on the IgH locus mapped to a gamma locus between C 1 and C2. A cosmid library was prepared
from SKI-DLCL-1, and C -positive clones spanning the breakpoint were
identified by screening with fluorescence in situ hybridization. The
breakpoint occurs 860 bp downstream of the 3' UTR of the MUC1
gene. The break appears to be a staggered double-strand break
consistent with an error in immunoglobulin class switching. The MUC1
gene is highly transcribed and translated, and the protein is highly
glycosylated. It is postulated that MUC1 expression is brought under
the control of the 3'E enhancer. MUC1 lies in a region of
chromosome 1 characterized by an unusually high density of genes, with
7 known genes in a region of approximately 85 kb. To determine whether
there was a pleiotropic effect of the expression of genes in the region as a consequence of the translocation, the expression of 6 additional genes was assessed. None of the other genes in this region (CLK2, propin, COTE1, GBA, metaxin, and thrombospondin 3) are overexpressed in
SKI-DLCL-1. Thus, the translocation t(1;14)(q21;q32) seen in both the
primary tumor and the derived cell line results in the marked
overexpression of MUC1 without affecting the expression of other genes
in the region.
(Blood. 2000;95:2930-2936)
© 2000 by The American Society of Hematology.
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Introduction |
Nonrandom chromosomal translocations in malignancy have
served as signposts identifying genes critical to the molecular
pathogenesis of the disease. In the lymphomas, these nonrandom
chromosomal translocations have frequently involved the immunoglobulin
and T-cell receptor loci. It has been speculated that these nonrandom chromosomal translocations arise as errors in the process of
immunoglobulin gene and T-cell receptor gene rearrangement or during
immunoglobulin class switching. The genes identified at these
translocation breakpoints have included those involved in critical
cellular processes regulating cell growth, cell cycle, and apoptosis.
Translocations involving the BCL1, BCL2, BCL6, and
c-myc genes account for approximately 45% of cases of
malignant lymphoma.1,2 However, there are a number of less
common, nonrandom chromosomal translocations that have also been
identified and have led to the identification of additional genes and
that may well be critical to the pathogenesis of malignant lymphoma.
One of the most common such genetic abnormalities observed in malignant
lymphoma occurs at chromosome 1q21.1,3 Abnormalities of
this region have not been associated with a particular subtype of
non-Hodgkin lymphoma, though in diffuse large-cell lymphoma (DLCL) they
are associated with poor prognosis.1,4 Abnormalities include the recurrent translocations t(1;6)(q21;q23-q26),
t(1;11)(q21;p15), and t(1;12)(q21;q24) together with the duplication
dup(1)(q21;q32).3,4 Several genes have been identified at
1q21 and are thought to be involved in the pathogenesis of some cases
of lymphoma, including BCL9 and genes homologous to Fc,
MUM2, and MUM3.3,5
We derived a cell line from a patient with malignant ascites that had a
complex karyotype including a translocation t(1;14)(q21;q32). We cloned
the translocation breakpoint from this cell line and found it to lie
0.86 kb downstream of the MUC1 gene. The MUC1 gene translocated to
der(14) chromosome is dramatically overexpressed in comparison to
normal lymphoid tissue and other lymphoma cell lines. Interestingly,
this dysregulated expression is confined to the MUC1 gene and does not
affect the expression of 6 other genes that lie in a 75-kb region
upstream of MUC1. The dysregulated expression of MUC1 may account for
the unusual extranodal presentation of the index case.
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Materials and methods |
Cells and cell lines
After informed consent, abdominal ascites was obtained from a
patient to establish the diagnosis of malignant lymphoma. The material
was designated case 2385 by the cytogenetics laboratory. Excess
material was frozen, and some was placed in culture in RPMI 1640 (Gibco-BRL, Bethesda, MD) supplemented with 20% fetal calf serum (FCS;
HyClone, Logan, UT). After 24 days in culture, these cells appeared to
be quiescent. However, in the fourth week, there was evidence of
cellular proliferation. Subsequently, the cells proliferated at a brisk
rate. Cytogenetic and flow cytometric analyses demonstrated that the
cells were derived from the tumor clone in the malignant ascites.
Cell lines OCI-Ly8, JP-DLCL-1, FL318, and FL18 demonstrate cells
derived from the transformation of low-grade lymphoma.6-8 Cell lines Daudi and Raji represent Burkitt lymphomas.9
MOLT-4 is derived from a T-cell lymphoma.9 They were
routinely maintained in RPMI 1640 supplemented with 10% FCS,
penicillin, streptomycin, and glutamine. Cells were grown
at 37°C in humidified incubators supplemented with 5%
CO2. MCF7 and HeLa were obtained from ATCC (American Type
Culture Collection, Rockville, MD) and were maintained in Iscove's
modified Dulbecco's medium supplemented with 10% FCS.
Cytogenetics and fluorescence in situ hybridization
Cytogenetics was performed by standard G-banding techniques as
previously described.10 Fluorescence in situ hybridization (FISH) was performed with fluorescein isothiocyanate (FITC)-labeled probes and phycoerythrin-labeled, chromosome-specific centromeric probes. The conditions have been described.11
Pulsed-field gel electrophoresis and Southern blotting
Megabase DNA was embedded in InCert agarose (FMC, Rockland, ME) as
previously described.12 DNA was resolved in a CHEF Mapper (Bio-Rad, Richmond, CA) using the auto-algorithm method or manually set
pulse sequences as indicated in the figure legends. Gels were run in
0.5 × TBE buffer with a field strength of 6 V/cm at 14°C. DNA was transferred to nylon membranes (Hybond, Amersham, MA) as
previously described with the following modification. Rather than
performing vacuum transfer as previously described, DNA was transferred
to immobilized membranes by traditional capillary action. DNA probes
were prepared with random hexamer priming as recommended by the
manufacturer (Stratagene, La Jolla, CA). Restriction endonuclease
digestion was performed on agarose-embedded DNA as previously
described.12 Blots were hybridized in conditions as
previously described.12 Initial exposures of blots were
performed on Fuji BAS PhosphorImager (Fuji Bio Imaging,
Tokyo, Japan) to determine the appropriate length of
exposure to film. Blots were subsequently exposed to film at
 70°C using Lighting Plus intensifying screens (Dupont,
Wilmington, DE).
Cosmid DNA cloning
DNA was extracted from the cell line SKI-DLCL-1 using standard
techniques. Conditions were established for partial MboI
digestion to result in approximately 40-kb DNA. This DNA was then
ligated to the SuperCos cosmid vector (Stratagene) and packaged with
GigaPak Gold (Stratagene). The library was plated at a density of
approximately 50 000 clones per plate, replicated and screened as
previously described.13 Cosmids were purified with multiple
rounds of screening until they were homogenous.
Northern blot hybridization
Total RNA was extracted using TRIZOL (Gibco-BRL). Polyadenylated RNA
was purified using the Fast Track RNA kit (Promega, Madison, WI). RNA
was resolved in a 1% GTG agarose gel (FMC) containing 0.22 mol/L
formaldehyde and 1 × MOPS. Gels were resolved at 9 V/cm and
transferred to nylon membranes as described above for Southern blots.
Blots were hybridized as for pulsed-field gel electrophoresis (PFGE).
Western blot hybridization
Cell lysates were prepared by adding lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 20 mmol/L EGTA, pH 8.0, 80 mmol/L
 -glycerophosphate, 1 mmol/L sodium vanadate, 50 mmol/L sodium
fluoride, 15 mmol/L MgCl2, 1 mmol/L adenosine triphosphate,
25 µg/mL leupeptin, 25 µg/mL soybean trypsin inhibitor, and 1 mmol/L phenylmethylsulfonyl fluoride) to the cell pellets,
followed by 8- to 10-second sonication (Sonic Disruptor; Tekmar,
Cincinnati, OH). Cell debris was removed by centrifugation at 10 000
rpm for 3 minutes. All procedures were performed at 4°C or on ice.
Protein concentration was determined spectrophotometrically using the
Protein Assay Reagent (Bio-Rad). Protein extracts were denatured by
boiling in sodium dodecyl sulphate (SDS) sample buffer (1.7% SDS, 5%
2-ME). Proteins were resolved in standard SDS-polyacrylamide gel
electrophoresis.14
The protein was transferred by electroblotting to nitrocellulose
membranes (Schleicher & Schuell, Keene, NH) as previously described.15 Nitrocellulose membranes were blocked with
blocking buffer (5% low-fat powdered milk in
1 × PBS [140 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L
Na2HPO4, 1.8 mmol/L
KH2PO4, pH 7.3], 0.1% Tween-20) for 6 to 8 hours at 4°C and were washed with 1 × PBS with 0.1%
Tween-20 (PBS-T). Protein was detected by overnight incubation with
antibody in blocking buffer at 4°C. Blots were rinsed twice with
PBS-T followed by washes of 1 × 15 minutes and 2 × 5
minutes in PBS-T. The second-step reagent, conjugated with horseradish
peroxidase (HRP), was incubated at room temperature for 60 minutes.
They were washed as above. Detection was accomplished with the ECL kit
(Amersham) and exposure to film.
MUC1 was detected using the HMFG1 and HMFG2 monoclonal antibodies,
which recognized the repeat region of MUC1, at a dilution of 1:600. The
specificity of HMFG2 is for the repeat region of MUC1, and HMFG1 is
dependent on proper glycosylation. The second step was a goat antimouse
IgG conjugated with HRP (Promega) at a dilution of 1:5000.
DNA sequencing
DNA sequencing was performed on an ABI automated sequencer (ABI373;
Applied Biosystems, Foster City, CA) and analyzed using Factura and
Autoassembler software. BLAST searches were performed on the web-based
application.16 Further DNA analysis was performed in
MacVector (Oxford Molecular, Campbell, CA).
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Results |
Derivation of SKI-DLCL-1
A patient had abdominal ascites. Cytologic analysis of the material
suggested a large-cell lymphoma, which was confirmed by flow cytometry
demonstrating a monoclonal IgM CD19+ and
CD20+ population of cells. The cells were negative for CD5,
CD10, and CD23. The fluid was placed in culture with RPMI 1640 supplemented with 20% FCS in a humidified incubator supplemented with
5% CO2. After a short period of quiescence, the cells
demonstrated spontaneous growth, and the resultant cell line was named
SKI-DLCL-1. The cells propagated in vitro demonstrated identical
cytologic features to the original ascitic fluid. Of note, there were
numerous cytoplasmic vacuoles (Figure 1).
Cytogenetic analysis was performed on both the initial ascitic fluid
and the cell line, which demonstrated identical complex cytogenetic
abnormalities (Figure 1). It was also noteworthy that there was a
reciprocal t(1;14)(q21;q32) chromosomal translocation. In addition, the
cell line contained cytogenetically normal chromosomes 1 and 14.
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| Fig 1.
Characteristics of malignant ascites and resultant
SKI-DLCL-1 cell line.
(A) Cells were fixed and stained with May-Grünwald-Giemsa
(magnification, ×100). SKI-DLCL-1 cells and cells from the
original ascitic fluid demonstrate identical cytologic features
consistent with a diagnosis of large-cell lymphoma. (B) Flow cytometric
analysis of the ascites and the SKI-DLCL-1 with a panel of monoclonal
antibodies. (C) Cytogenetic analysis of the diagnostic ascites and the
resultant cell line demonstrates similar though nonidentical findings.
This likely represents clonal selection by the process of establishing
the cell line.
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Cloning of the t(1;14) chromosomal breakpoint
To identify the position of the chromosomal breakpoint, megabase DNA
was digested with the restriction endonuclease NotI. This
enzyme was selected because the entire immunoglobulin heavy chain locus
(IGH) is subtended on a 750-kb NotI fragment. IGH gene rearrangements and chromosome breaks are identified as nongermline NotI fragments.12 DNA from polyclonal peripheral
blood lymphocytes (PBL) and the SKI-DLCL-1 cell line were sequentially
hybridized with probes derived from the IGH locus. A probe from the
joining region (JH) identified a germline 750-kb fragment
in PBL, which was absent from this cell line. A single 600-kb band was
detected with the JH probe (Figure
2A). This was surprising because we expected a productively rearranged allele (the cell expresses a surface
IgM) and a second allele involved in either a nonproductive rearrangement or in a germline configuration. This suggested that 1 IGH
allele was at least partially deleted. The identical pattern was seen
with a probe for the mu constant region (Cµ) (Figure 2B).
Hybridization with a probe for the gamma constant region (C) and
alpha constant region (C ) identified 3 fragments in the cell line
that measured 140 kb, 600 kb, and 800 kb (Figures 2C, 2D). The 600-kb
band comigrated with the band identified by the JH and Cµ
probes. Therefore, this must have represented the productive allele
from which the IgM transcript was expressed in this cell line. The
identification of 2 additional bands with the C probe suggests that
the deletion of the nonproductive allele does not extend through the
entire IGH locus. Furthermore, the chromosome breakpoint likely
occurred between the C1 and C2 genes
(Figure 2D). The additional finding of an identical pattern with the
C probe suggested that the break would be upstream of C4.
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| Fig 2.
Characterization of the IGH rearrangements in SKI-DLCL-1
by PFGE.
High molecular weight DNA from peripheral blood lymphocytes (lane 1)
and SKI-DLCL-1 cells (lane 2) was digested with NotI and
resolved through PFGE with a window of resolution of 50 kb to 1500 kb
(A-E) or 25 to 500 kb (F, G). The identical blot was sequentially
hybridized with a series of probes, as indicated in A to E. A second
blot was used (F, G) for hybridization by the indicated probes. The
samples are as follows: lane 1, PBL; lane 2, ascitic fluid; lane 3, SKI-DLCL-1. Complete removal of the different probes after stripping
was confirmed by exposure to film. ZNR, zone of no resolution.
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We prepared a cosmid library derived from an MboI partial
digestion. The library was screened for C-positive clones. We
expected to obtain clones derived from all the C loci, both on the
normal and on the translocated chromosomes. Thirteen clones were
obtained that formed 3 restriction patterns. Two of them, S7D and S5F, represented overlapping cosmids on the basis of shared restriction fragments. This mini-contig hybridized with probes for C, S, and
C (Figure 3); therefore, these cosmids
were candidates to span the break between C2 and
C4 derived from der(14). Two additional clones, S5A and
S7A, represented a second mini-contig based on the restriction pattern
and hybridized to both the C and the S probes. The lack of C
hybridization and the difference in restriction map from the S7D/S5F
contig made them candidates to be derived from the reciprocal der(1)
chromosome.
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| Fig 3.
Megabase map of the t(1;14)(q21;q32) and the location of
the cosmid and P1 clones.
Clones S7D (C+, C) and S5F (C+, C+) span the
breakpoint on der(14). The restriction maps overlap, creating a
mini-contig. Clones S5A and S7A (C+) also comprise a mini-contig,
and they span the breakpoint on the der(1) chromosome. The sizes of the
translocated bands detected by PFGE are 140 kb for the derivative
chromosome 14 and 600 kb for the derivative chromosome 1. PI-PAC clones 35(A12) and 35(A12) span a 100-kb region
centered around the MUC1 gene.
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Clone S7D, a candidate to span the breakpoint on the der(14)
chromosome, was screened by FISH on normal cells. Clone S7D identified signals at 14q32 and 1q21 (Figure 4A),
confirming that it must span the breakpoint on chromosome 1. The cosmid
was then subcloned as a BamHI sublibrary. Clones representative
of each of the BamHI fragments were selected from the libraries
and were screened for homology to a switch gamma (S) probe. Clone
B109 hybridized with S and therefore likely carried the
translocation breakpoint. Clones B101, B22, and B61 failed to show
hybridization with C and were presumed to be derived from chromosome
1 (Figure 5).
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| Fig 4.
Characterization of genomic clones by FISH.
(A) FISH analysis of normal cells using cosmid clone S7D, a candidate
to span the t(1;14) breakpoint. Biotinylated probes for the chromosome
14 centromere-specific probe and the heterochromatic region of
chromosome 1 were cohybridized with digoxigenin dUTP-labeled clone S7D.
The centromeric and heterochromatic regions were identified with
phycoerythrin-SA (red) and the breakpoint probe with
FITC-antidigoxigenin (green). The result confirmed that clone S7D split
the breakpoint. (B) FISH analysis of SKI-DLCL-1 cells using P1
PAC clone 35(A12) derived from the normal chromosome 1. The P1 PAC clone 35(A12) probe was labeled with
digoxigenin dUTP and detected by FITC-antidigoxigenin. The finding of
signals on the normal chromosome 1 and on the der(1) and der(14)
chromosomes confirms that P1 spans the region on
chromosome 1 involved with the breakpoint.
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| Fig 5.
Restriction map of cosmid S7D.
Cosmid S7D was subcloned as a BamHI sublibrary. Sequence of the
ends of clone B61 was found to be colinear with YAC MNG1 and was
homologous to the thrombospondin 3 gene. MUC1 gene extends from clone
B22 to clone B109, which spans the breakpoint. Two boxes indicate the
position of the fragments used as probes (RsaI 3.5-kb and
BstxI 1.1-kb fragment). Restriction sites are indicated: N,
NotI; B, BamHI; E, EcoRI; M,
MluI; Bss, BssHII.
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Localization of chromosome 1-derived sequences
The BamHI fragments subcloned from cosmid S7D were sequenced
from the ends, using primers homologous to the flanking T3 and T7
promoters. These sequences were screened by BLAST
searching.16 Clone B61 was found to be colinear with a
previously sequenced region from YAC MNG1.17 The sequence
of the YAC in this region spans a 75,000-bp region extending from the
CLK2 kinase at the centromeric end, and it spans a cluster of genes
near the Gaucher disease locus ending in the thrombospondin 3 gene. B61
was colinear with a 6-kb BamH1 fragment derived from a region
corresponding to thrombospondin 3. Thus, we were able to establish that
the cloned material was in fact derived from chromosome 1, and we had
additional information regarding the physical map in this region. The
T7 end of clone B22 corresponded to the 3' end of the
thrombospondin cDNA, consistent with this fragment's downstream location from the YAC MNG1. The T3 end of this clone was homologous to
the MUC1 gene, which is known to lie immediately downstream of
thrombospondin 3.18 Clone B101 proved to be colinear with the MUC1 gene. The T3 end of clone B109 was colinear with exon 7 of
MUC1 and a portion of the 3' untranslated region of the MUC1 gene. The T7 terminus was homologous to C. Thus, the chromosomal breakpoint occurred within the B109 clone, as expected. A 1.1-kb BstXI fragment derived from the T3 end of B109 was used as a
probe on a Southern blot of DNA extracted from SKI-DLCL-1 and PBL DNA from a healthy subject and was digested with BamHI
and EcoRI. As seen in Figure 6, the
probe identified a 9-kb BamHI fragment in the tumor that
corresponded to B109. In addition, a second 5.1-kb BamHI
fragment was identified in normal DNA and in the tumor. This
corresponded to the nontranslocated chromosome 1 allele. In the
EcoRI digestion, the germline fragment measured 5.6 kb, and the
translocated fragment measured 15 kb.
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| Fig 6.
Southern blot analysis confirming that B109 spanned the
t(1;14) breakpoint.
DNA from PBL (lane 1) and SKI-DLCL-1 (lane 2) was digested with
BamHI or EcoRI, and the blot was successively
hybridized with a C probe and the 1.1-kb BstXI fragment from
clone B109. The BstXI probe identified a
nongermline-rearranged band for each enzyme (9-kb band for
BamHI and 15-kb band for EcoRI). The nongermline bands
comigrated with nongermline bands detected with a C probe.
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To identify precisely the chromosomal breakpoint, the B109 fragment was
sequenced. To obtain the sequence of the normal chromosome 1 allele, a
P1 artificial chromosome (PAC) library was screened with a 3.5-kb
RsaI fragment derived from clone B101, identifying 2 clones PAC 35(A12) and PAC 280(09) (Figure 3). PAC 35(A12) was used as
a FISH probe on the SKI-DLCL-1 cell line (Figure 4B). In addition to
the normal chromosome 1, the der(1) and the der(14) chromosomes were
identified, confirming that this P1 spans the chromosomal breakpoint.
These PAC clones overlap, and each has a 5.1-kb BamHI fragment
that hybridizes to the 1.1-kb BstXI probe derived from B109
(data not shown). The 5.1-kb BamHI fragment was subcloned as
clone B5 and sequenced. Because clone B5 spanned the breakpoint on
chromosome 1, it would be expected to identify clones derived from both
the der(1) and the der(14) chromosomes. It was used as a probe to
screen the C-positive cosmids and hybridized to clones S5A and S7A,
which confirmed that they were derived from the der(1) chromosome as
expected. The breakpoint was contained within a 5-kb BamHI
fragment. This region, adjacent to the breakpoint, was sequenced. The
sequences of the der(1), der(14), normal chromosome 1, and switch gamma
4 sequences were aligned (Figure 7). The
break occurred 0.86 kb downstream of the final MUC1 exon on chromosome 1 and within the S4 region on chromosome 14. The normal
chromosome 1, der(1), and der(14) all share the sequence CATT at the
breakpoint. The der(1) allele has a 7-bp deletion of chromosome 14 sequences just 5' of the CATT, and the der(14) has a 5-bp
deletion just 3' of the CATT. This is consistent with a staggered
double-strand break with 3' to 5' exonuclease removal of
some bases from the free ends. The differences between the S4
sequence and the der(1) and der(14) sequences likely are allelic
variations between the cell line and the switch region sequence
reported in GenBank.
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| Fig 7.
Sequence of the der(1) and der(14) breakpoints aligned
with S4 and the normal sequence of chromosome 1.
Clone B109 was derived from cosmid S7D (Figure 3) and corresponds to
the der(14) chromosome. Clone B10 is derived from cosmid S5A (Figure 3)
and corresponds to the der(1) chromosome. P48-B5 is derived from the
normal chromosome 1 P1. The switch gamma 4 sequence was obtained from
GenBank. It is uncertain whether the minor differences between the
switch gamma 4 sequence and the B10 sequence represent polymorphism,
sequencing errors, or differences arising during the translocation.
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The productive VDJ allele is translocated to
chromosome 1
The PFGE mapping of the IGH locus in SKI-DLCL-1 (see above)
demonstrated that 1 of the JH loci was deleted and that the
productive allele was subtended on a 600-kb NotI fragment. A
fragment derived from clone B5, corresponding to the der(1) chromosome,
was used as a hybridization probe on the blot of PBL and SKI-DLCL-1 DNA digested with NotI (Figure 2E). A 140-kb doublet band was seen in the cell line, and a single 140-kb band was seen in PBL (the doublet
is best seen in the alternative window of resolution shown in Figure
2F). The band seen in both the PBL and the cell line must correspond to
the germline fragment. The second band at 140 kb in the cell line
comigrated with the band detected with the C and C probes
(Figures 2C, 2D, and 2E) thus represented a rearranged fragment. There
was an additional 600-kb band present only in the cell line. The
fragment comigrated with the band detected with the JH and
Cµ probes (Figures 2A and 2B). Because the cell line expresses an
sIgM and there is only 1 JH allele, this 600-kb NotI fragment represents the productive allele. Thus, in this cell line, the productive IGH locus is on the der(1) chromosome and
the normal 14 must have a deletion involving a portion of the
IGH locus.
MUC1 is highly expressed in this cell line
Given that MUC1 was adjacent to the chromosomal breakpoint, a 3.5-kb
RsaI fragment was derived from clone B101 and used to evaluate
the steady-state level of transcription of this gene. MUC1 mRNA was
highly expressed in the SKI-DLCL-1 cell line, though it was not
expressed in a number of other lymphoma cell lines (Figure
8A). Two transcripts were detected: a
4.4-kb transcript was strongly expressed, and a 6-kb transcript was
weakly expressed. It is likely that these represented alternative
splice variants.19 Expression of protein was demonstrated
by Western blot hybridization (Figure 8B). The HMGF2 antibody
recognizes the repeat region of MUC1. The level of expression is
significantly greater than in the breast carcinoma cell line MCF7. In
addition, it was expressed in the original ascites fluid and in the
SKI-DLCL-1 cell line (lanes 3 and 4). An identical pattern was observed
with the HMGF1 antibody that recognizes the glycosylated form of MUC1.
Cell surface expression was confirmed by immunofluorescence using an
MUC1-specific monoclonal antibody that demonstrated a strong expression
of MUC1 (Figure 8C).
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| Fig 8.
Expression of the MUC1 gene in SKI-DLCL-1.
(A) Expression of mRNA was evaluated by Northern blot hybridization.
One microgram of poly-A+ RNA was electrophoresed on a 1% formaldehyde
agarose gel. The blot was hybridized successively with an MUC1 probe
and a -actin probe. Length of exposure on film is 1 hour for both probes. Lanes 1 to 6 represent different
cell lines: lane 1, SKI-DLCL-1; lane 2, OCI-Ly8; lane 3, Daudi; lane 4, FL-318; lane 5, Raji; lane 6, Molt-4. (B) Protein
expression was evaluated by Western blot hybridization using the
MUC1-specific monoclonal antibodies HMFG1 (glycosylation
sensitive) and HMFG2 (specific for the repeat motif). Lane 1, Raji; lane 2, MCF-7; lane 3, primary ascites; lane 4, SKI-DLCL-1. (C)
Immunofluorescence was used to assess cell-surface expression. Staining
of SKI-DLCL-1 cells and MCF-7 cells with HMFG2 antibody demonstrated a
strong expression of MUC1. OCI-LY8 cells were used as a negative
control in this experiment.
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MUC1 alone is overexpressed among a cluster of genes
within 90 kb of the breakpoint
The MUC1 gene lies in a region of chromosome 1q21 that has an
unusually high density of genes18 (Figure
9A). Approximately 80 kb upstream of MUC1
is the CLK2 gene. Between CLK2 and MUC1 there are 5 additional
genespropin, COTE1, GBA, metaxin, and thrombospondin 3, which ends
less than 1 kb upstream of MUC1.17 To determine whether the
translocation resulted in the dysregulation of any other loci, we
examined the expression of the genes upstream of MUC1 in the SKI-DLCL-1
cell line and compared this with a number of other cell lines. The
results are shown in Figure 9B. Expression of the upstream genes is not
significantly augmented in the SKI-DLCL-1 cell line in comparison with
the other cell lines. Thus, the translocation resulted in the
dysregulated expression only of the MUC1 gene. The basis for this
narrow effect is under continued investigation.
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| Fig 9.
Expression of loci within 90 kb of the t(1;14)
translocation breakpoint.
(A) Physical map of the cluster of genes identified near the Gaucher
disease locus based on sequence data (map based on previously published
data in Adolph et al18). (B) Expression of the cluster of
genes upstream of the breakpoint by Northern blot analysis
included the following loci: CLK2, propin, cote1, GBA, metaxin, and
thrombospondin 3. Total RNA (10 µg) was electrophoresed on a 1%
formaldehyde agarose gel. 28S was used as a control for RNA loading.
The size of the expected transcripts is indicated. Lanes 1 to 8 represent different cell lines: lane 1, Hela; lane 2, UC726/G; lane 3, SKI-DLCL-1; lane 4, Daudi; lane 5, OCI-LY8; lane 6, FL318; lane 7, Raji; lane 8, Molt-4.
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Discussion |
We described the cloning and characterization of the chromosomal
breakpoint of a reciprocal translocation involving chromosomes 1q21 and
14q32. The breakpoint is not crossed by probes for either the MUM2 or
the BCL9 gene (data not shown).3,5 The MUC1 gene, immediately adjacent to the breakpoint, is markedly dysregulated. Expression of 6 other genes in proximity to the breakpoint are not
dysregulated as a consequence of the translocation.
The overexpression of mucins by adenocarcinoma tumors have been
associated with high metastatic potential and poor
prognosis.20-22 Expression of MUC1 is associated with
immunosuppression because it inhibits T-cell
proliferation.23 MUC1 expression has been described in
multiple myeloma (MM).24,25 Of note, 1q21 abnormalities are
seen in multiple myeloma, and some may occur near the breakpoint described in this article.26 This is a subject for further investigation.
The consequence of the expression of the MUC1 gene in this case is
uncertain. However, it is notable that the patient had lymphoma in the
form of abdominal ascites. It is possible that the expression of mucin
in this case contributed to the extranodal pattern of growth of this
lymphoma. MUC1 is an antiadhesive protein that interrupts cell-cell
and cell-matrix interactions.27,28 There is a report of
expression of EMA (which is equivalent to MUC1) in body cavity lymphoma
infected by human herpesvirus 8 (HHV8)29 and of anaplastic
large-cell lymphoma that frequently is accompanied by extranodal,
particularly cutaneous, disease.30-32 EMA is also expressed
in lymphocyte-predominant Hodgkin disease (75%), plasmacytoma (75%),
and T-cell lymphoma (50%).33 The tumor cells in this case
were tested and were found to be HHV8 negative (data not
shown). It is possible that a consequence of HHV8 expression is the
dysregulation of the MUC1 gene. This is under investigation.
The level of expression of MUC1 in the original tumor material and in
the derived cell line is remarkable and exceeds that of the MCF7 cell
line. As a consequence of the translocation, the MUC1 gene is brought
into proximity with the C4 and C2 loci. Downstream of C2 is an enhancer element homologous
to the well-characterized murine and rat
3'IgH-E(hs1,2).34 In the mouse, this enhancer is
active late in B-cell differentiation,35 and, though the activity of the human homolog has not been fully defined, it is active
in mature B cell lines in transient transfection assays.34 It is likely that the overexpression of the gene is driven by the
juxtaposition of the E enhancer to the MUC1 gene. The human 3'IgH enhancer has an octamer binding motif and an ETS/AP-1
motif. Regulatory elements have been identified within the MUC1
promoter, and the regulation is complex. It remains to be determined
whether and how the E enhancer alters the expression from the MUC1 promoter.
Interestingly, the translocation appears to result in the dysregulation
of only MUC1 in a cluster of 7 genes within 80 to 90 kb of the
translocation breakpoint. In other translocations involving the IGH or
IGL loci, the gene dysregulated can be remote from the breakpoint. In
the t(14;18) translocation characteristic of follicular
lymphoma,12 the breaks occur in 2 major cluster regions on
chromosome 18. The MBR is within the 3' untranslated region of
exon 3 of BCL2, and the MCR is approximately 30 kb downstream of BCL2.
The BCL2 gene has an unusually large second intron that measures
approximately 270 kb in length; thus the breakpoint is 270 kb to 300 kb
away from the BCL2 promoter region.36 Because the
translocation alters the rate of transcription of the BCL2 gene and
does not affect the message half-life,36 it is clear that
the translocation affects transcription at a significant distance.
Similarly, the dysregulation of cyclin D1, which is a consequence of
the t(1;14) translocation, can result from chromosome breaks as far as
150 kb downstream of the gene.37,38 In some patients with
Burkitt lymphoma with the variant t(2;8) chromosomal translocation, the
c-myc locus is up to 280 kb away from the breakpoint but is
nonetheless dysregulated.39-41 In the present case, the consequence of the translocation is the dysregulation only of the gene
immediately 5' to the breakpoint. The possibility remains that
this t(1;14)(q21;q32) translocation results in the dysregulation of
other loci more than 90 kb upstream of MUC1 or of loci on the der(1)
allele, which translocates the Eµ enhancer to
chromosome 1.
| |
Acknowledgments |
We thank Dr Philip Livingston for the gift of the HMFG1 and HMFG2
monoclonal antibodies, Dr Lloyd Old and Marc Fediricci for performing
the MUC1 immunofluorescence, and Dr Stephen Nimer for helpful
discussions. We also thank Letha Menon for secretarial assistance in
the preparation of this manuscript.
| |
Footnotes |
Submitted October 5, 1999; accepted January 6, 2000.
Supported in part by the Lymphoma Research Fund and by National
Institutes of Health grant R01-CA-61429 (A.D.Z.).
F.G. and A.G. contributed equally to this study.
Reprints: Andrew D. Zelenetz, Memorial Sloan-Kettering Cancer
Center, 1275 York Avenue, Box 330, New York, NY 10021; e-mail:
a-zelenetz{at}ski.mskcc.org.
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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Abstract
Introduction