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The characterisation of the lymphoma cell line U937, using comparative
genomic hybridisation and multi-plex FISH
Article in Cytogenetics and Cell Genetics · February 2001
DOI: 10.1159/000048774 · Source: PubMed
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Original Article
Cytogenet Cell Genet 94:9–14 (2001)
The characterisation of the lymphoma cell line
U937, using comparative genomic
hybridisation and multi-plex FISH
J.C. Strefford,a,c N.J. Foot,a T. Chaplin,a M.J. Neat,a R.T.D. Oliver,b,c
B.D. Younga and L.K. Jonesa
a ICRF
Medical Oncology Unit, Queen Mary and Westfield College, London;
of Medical Oncology and c The Orchid Cancer Appeal, St Bartholomew’s Hospital, London (UK)
b Department
Abstract. The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the
t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias
AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia).
Apart from this translocation, very little is known about the
other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to charac-
terise fully all of the cytogenetic alterations present in the U937
cell line. M-FISH analysis confirmed the presence of the
t(10;11) and an apparently normal copy of both chromosomes
10 and 11. A t(1;5) translocation was observed as well as several
unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes
could result in fusion gene products involved in carcinogenesis
or the positions of putative oncogenes and tumour suppressor
genes. A good correlation between conventional G-banding,
CGH and M-FISH was observed.
The U937 cell line was originally established from a patient
with diffuse histiocytic lymphoma (Sundstrom and Nilsson,
1976). This cell line has been used extensively in myeloid differentiation studies (Ralph et al., 1976; 1983) and in the characterisation of the t(10;11)(p13;q14), a fusion between the
MLLT10 gene and the Ap-3-like clathrin assembly protein,
PICALM (Dreyling et al., 1996). Apart from the PICALM:
MLLT10 gene fusion, very little is known about the genetic
abnormalities of U937. Its karyotype is complex (Shipley et
al., 1988) and the true identities of many rearrangements
have not yet been resolved. Although the t(10;11) and its
genetic role in leukaemogenesis has been well characterised
(Dreyling et al., 1996), little is known about other significant
genetic events in this cell line and their potential synergistic
effects.
Recent advances in molecular technology have had considerable impact on cytogenetic analysis, allowing greater resolution and accuracy. Significant advances in fluorescence technology include the development of comparative genomic hybridisation (CGH) (Kallioniemi et al., 1994) and multiplex
FISH (M-FISH) (Speicher et al., 1996). In essence, CGH provides information on those regions gained or lost in the DNA of
a tumour specimen. This method has been used to investigate
genetic changes in a wide spectrum of human cancer (Visakorpi
et al., 1995; Bergamo et al., 2000; Loveday et al., 2000; Schleger
et al., 2000) including lymphoma (Ohshima et al., 1999; Arranz
et al., 2000; Peters et al., 2000). M-FISH is a combinatorial
technique that allows the identification of human chromosomes by “painting” them with a spectrum of DNA probes
labelled with a unique combination of five fluorochromes.
Supported by the Orchid Cancer Appeal and by the Imperial Cancer Research Fund.
Received 10 January 2001; accepted 10 May 2001.
Request reprints from Jon C. Strefford, ICRF Medical Oncology Unit,
Queen Mary and Westfield College, Charterhouse Square,
London, EC1M 6BQ (UK);
telephone: 0207 882 6003; fax: 0207 882 6004; email: J.Strefford@icrf.icnet.uk
ABC
Fax + 41 61 306 12 34
E-mail karger@karger.ch
www.karger.com
© 2001 S. Karger AG, Basel
0301–0171/01/0942–0009$17.50/0
Copyright © 2001 S. Karger AG, Basel
Accessible online at:
www.karger.com/journals/ccg
Fig. 1. Summary of chromosome imbalances
identified in U937 by comparative genomic hybridisation. The vertical lines on the left side of
the ideograms indicates losses, whereas the vertical lines on the right side correspond to gains of
chromosome material. High-level over-representation is indicated by thickened lines. Chromosome region 1pter → p32 and chromosomes 19
and Y are shaded in grey; they were excluded
from the evaluation for reasons indicated elsewhere (see Materials and methods).
Sophisticated image analysis applies the relevant pseudocolour to allow visualisation in 24 discrete colours. M-FISH has
been used to investigate haematological diseases (Tosi et al.,
1999; Harrison et al., 2000; Lindbjerg Andersen et al., 2000;
Naumann et al., 2001) and solid tumours (Speicher et al., 2000;
Aurich-Costa et al., 2001; Strefford et al., 2001).
The study reported here is the first attempt to characterise
the chromosome rearrangements and imbalances in the U937
cell line using CGH and M-FISH and correlate them with Gbanding data.
Table 1. CGH abnormalities detected in
U937a
Methods
Cell culture and harvest
The cell line U937 (passage number 25 since acquisition from American
Tissue Culture Centre, ATCC) was acquired from the ATCC and cultured in
RPMI containing 10 % FCS, 45 IU/ml penicillin and 45 Ìg/ml streptomycin
and incubated at 37 ° C in culture flasks with an atmosphere of 5 % CO2 in
air. Metaphase slide preparations were made after mitotic arrest with colcemid (0.015 Ìg/ml, 2–4 h) (Gibco), hypotonic treatment (0.075 M KCl,
10 min, 37 ° C), and fixation with methanol:acetic acid (3:1) before spreading
onto slides.
Cytogenetic analysis, CGH and M-FISH
Comparative genomic hybridisation (CGH) was performed as previously
described (Kallioniemi et al., 1994) with the following modifications. 1 Ìg of
U937 DNA was labelled with Spectrum Green dUTP (Vysis, UK) in a standard nick translation reaction. Normal female DNA pre-labelled with Spectrum Red dUTP (Vysis, UK) was used as reference DNA. 500 ng of tumour
and 250 ng of female reference DNA were mixed together with 25 Ìg of
human Cot-1 DNA (Gibco-BRL, Life Technologies, UK) and hybridised to a
slide of freshly prepared metaphase spreads for 48–72 h. A normal female
lymphoblastoid cell line, confirmed to be normal with high-resolution Gbanding and M-FISH, was used as a source of template metaphase chromo-
10
Cytogenet Cell Genet 94:9–14 (2001)
somes, as the quality and quantity of metaphase templates was high. After
post-hybridisation washes, chromosomes were counter-stained with 125 ng/
ml of 4,6-diamidino-2-phenylindole (DAPI) prior to detection. CGH metaphases were captured through a fluorescent microscope (Leica) and highresolution digital camera (Cohu) then analysed using the MacProbe 4.1
image analysis software (all items supplied by Perceptive Scientific Instruments, UK). The CGH profile for each chromosome was calculated as a
mean of 20 metaphase chromosomes. Chromosome imbalances were detected on the basis of the ratio profile deviating from the balanced value 1.0,
with values 1.25 and 0.75 serving as diagnostic cut-off levels for over- and
under representation of chromosome material, respectively. These thresholds had been thoroughly tested in numerous studies and provide a robust
diagnostic criterion. Over-representations were considered high-level when
the green:red CGH ration exceeded 2.0. Problematic profiling regions were
eliminated by the analysis of non-overlapping chromosomes. Telomeric and
centromeric regions, as well as 1pter → p32, and chromosomes 19 and Y were
excluded from the CGH analysis for reasons specified elsewhere (SolinasToldo et al., 1996). Confidence intervals were chosen at 95 %.
M-FISH was carried out according to standard manufacturer’s instructions. Following ethanol dehydration, 10 Ìl of SpectraVysion M-FISH probe
Table 2. Current G-banded ISCN karyotype for the lymphoma-derived cell line, U937 prior to M-FISH analysis
Table 3. M-FISH abnormalities detected in U937. Further characterisation of G-banded abnormalities with M-FISH
(Vysis Inc, UK) was added to the metaphase slide, sealed and co-denatured
(5 min, 70 ° C) before an overnight hybridisation (37 ° C). After post-hybridisation washes, slides were dehydrated and mounted in DAPI prior to visualisation. For each of 10 M-FISH metaphases analysed, the Spectrum Gold, FarRed, Red, Aqua, Green and DAPI channels were captured with a Sensys
Camera (Photometric) fitted with filters compatible with SpectraVysion
probes. The five fluorescent Spectrum channels were combined and a pseudo-colour was applied using M-FISH 1.1 software (Perspective Scientific
Instruments, UK). G-banding and conventional chromosome painting were
used to supplement cell line characterisation with CGH and M-FISH.
Results
CGH analysis
CGH analysis revealed several regions of amplification and
deletion. Gains were observed more frequently than losses
(Fig. 1). The copy number imbalances identified in U937 are
detailed in Table 1.
G-banded and M-FISH analysis
G-banded analysis revealed several rearrangements previously described (Shipley et al., 1988). The marker chromosomes reported by Shipley et al. (1989) were identified using
G-banded analysis from three individual sources. The consistent aberrations detected in all three cell lines, were 3p–, 11q–,
16p+ and 17p– (Shipley et al., 1988). The ISCN nomenclature
(ISCN 1995) for the G-banded karyotype from this present
study is shown in Table 2.
U937 exhibited a hyperdiploid chromosome complement
(51–57 chromosomes per cell). M-FISH was used to clarify the
nature of the structural rearrangements.
M-FISH analysis of the U937 cell line showed a number of
clonal rearrangements and identified several abnormal chromosomes that could not be completely described by conventional cytogenetics. Significant cell to cell variation was observed, but only clonal chromosome aberrations are described
(seen in two or more metaphase cells). The M-FISH karyotype
and the clonal abnormalities are shown in Fig. 2 and Table 3
respectively. U937 contained 17 structural rearrangements.
Reciprocal translocations included the t(10;11) previously described as well as a t(1;5)(p22;q33). A normal copy of both
chromosomes 10 and 11 was also observed. Six unbalanced
rearrangements were detected involving chromosomes 1, 2, 3,
4, 6, 12, 16, 19 and 20.
Comparison of conventional G-banding, CGH and M-FISH
The parallel use of CGH, M-FISH and conventional Gbanding was a powerful combination for the characterisation of
such complex chromosome rearrangements. Examples of this
parallel approach are shown in Fig. 3. The copy number
changes detected by CGH for chromosome 1, 2, and 6 can be
explained by the derivative chromosomes detected by M-FISH
(Fig. 2). The origin of the two G-banded marker chromosomes
were further classified with M-FISH and shown to be a rearranged chromosome 2 and a der(16)t(16;20). CGH analysis
demonstrated enhancement of a relatively small region of the q
Cytogenet Cell Genet 94:9–14 (2001)
11
Fig. 2. M-FISH karyotype for U937. The corresponding G-banded image is shown to the right
of each abnormal M-FISH chromosome.
Fig. 3. The parallel use of CGH, M-FISH and
conventional banding to characterise fully the
chromosome alterations observed in the cell line,
U937. M-FISH, CGH and G-banded images for
chromosomes 1, 2, 6 and 13 are shown. The MFISH image with chromosome designation is at
the left. The G-banded image is next. On the right
is the CGH profile and picture for each of the
chromosomes.
arm of chromosome 2. This suggests that this marker chromosome 2 may have arisen from multiple copies of the region
2q31 → q32.
The CGH amplification of 13q reflects a duplication of a
region of its long arm, demonstrated by the G-banded and MFISH analysis (Table 1 and Fig. 2 respectively). Partial amplifications of chromosomes 15, 19 and 20 reflect a trisomy complement for those chromosomes (Fig. 2). CGH analysis demonstrated partial amplification of chromosome 19, which was
consistent with the der(19)t(1;19) detected by M-FISH. MFISH also showed chromosome 6 material in three other abnormal chromosomes. These rearrangements were not identified
by conventional G-banded analysis. The add(2)(q33),
add(6)(p23) and add(12)(p11.2) were all shown to involve rearrangement with chromosome 6 (Fig. 3). Several discrepancies
were highlighted, including a trisomy 21 detected by G-banding, but not CGH, extra material of chromosome 4 origin, with-
12
Cytogenet Cell Genet 94:9–14 (2001)
out CGH amplification, and CGH losses of chromosome 5,
without consequent changes to the G-banded or M-FISH
karyotype at this region. Discrepancies of this nature are likely
to be due to low level clonality of the chromosome marker in
question, or the restricted sampling of these aberrant chromosomes with G-banding and M-FISH.
Discussion
The simultaneous visualisation of each chromosome in a
discrete colour represents a valuable tool for metaphase analysis. Conventional banding data of U937 showed many of the
same marker chromosomes previously described (Shipley et al.,
1988). Karyotypic analysis with conventional banding techniques often leaves complex derivative chromosomes unclassified. Using a combination of conventional banding, CGH and
M-FISH, the origin of complex marker chromosomes can be
readily identified.
In this study, M-FISH and CGH were used to characterise
further the histiocytic lymphoma-derived cell line, U937. MFISH detected the t(10;11), resulting in a fusion between the
previously described MLLT10 gene (Chaplin et al., 1995) and
PICALM (Dreyling et al., 1996), This translocation consistently results in a fusion of the putative clathrin binding domain in
PICALM to a region containing the leucine zipper of MLLT10.
It is now clear that the PICALM:MLLT10 fusion plays a significant role in leukaemogenesis. This is determined by the presence of this translocation in T-cell acute lymphoblastic leukaemias, acute myeloblastic leukaemias (AML M0, AML M1),
acute monoblastic leukaemias (AML M4–M5) and lymphoma
(Dreyling et al., 1996, 1998; Silliman et al., 1998; Narita et al.,
1999). With regard to clinical outcome, a number of studies
have correlated the presence of the t(10;11) with poor prognosis
(Dreyling et al., 1998; Kumon et al., 1999). The range of haematological malignancy observed suggests that the translocation resulting in the PICALM:MLLT10 fusion occurs in a pluripotent stem cell or early haematopoietic progenitor cell, and
that secondary changes may also influence disease pathogenesis
and clinical outcome. (Dreyling et al., 1996).
In this study there was some correlation between the conventional banding, CGH and M-FISH results. A reciprocal
translocation, t(1;5)(p22;q33), was detected which was first
observed by Shipley et al, 1988. During this present study, the
derivative chromosome 1 was classified as der(1)t(1;5)(p22;
q33)add(1)(q25). Unbalanced abnormalities such as these may
result in loss or gain of putative tumour suppressor genes or
oncogenes, but may also reflect general genetic instability in
this cell line due to loss of cell cycle control or in vitro karyotypic evolution.
Several previous reports have included secondary karyotypic information from patients with the t(10;11) translocation
(Kumon et al., 1999; Narita et al., 1999; Carlson et al., 2000).
Certain chromosome abnormalities previously described are
similar to those exhibited by U937. Kumon and colleagues
showed a patient with a t(1;3) translocation where the breakpoint on chromosome 3 was in a similar region to the breakpoint in U937 (3q26). This study also demonstrated loss of 12p
in two patients through the formation of a derivative chromosome (chromosome 12 was translocated to chromosomes 15
and 18) (Kumon et al., 1999), similar to the der(12)t(6:12)
detected by M-FISH. Several of the previous studies showed
gain of chromosome 20 (Kumon et al., 1999; Carlson et al.,
2000). Over-representation of regions of chromosome 13 has
been demonstrated in this present M-FISH study and a previous report of a patient with multiple copies of chromosome
13 (Narita et al., 1999). In previous studies, deletions of chromosome 2 and 5 as well as several markers were also identified.
Any of these changes may reflect important events in malignant
progression, but also suggest that several of the M-FISH
changes observed in U937 were present in the tumour from
which the cell line was derived. Since this cell line has been
used so much in the study of myeloid differentiation, it is
important to establish the ways in which these secondary
changes may influence this process. Further molecular investigation will be needed to establish the role of these abnormalities.
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