Journal of Virological Methods 163 (2010) 344–352
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Journal of Virological Methods
journal homepage: www.elsevier.com/locate/jviromet
Molecular cytogenetic analysis of feline leukemia virus insertions in cat
lymphoid tumor cells
Yasuhito Fujino a,∗ , Hitoshi Satoh b , Koichi Ohno a , Hajime Tsujimoto a
a
b
Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
a b s t r a c t
Article history:
Received 27 April 2009
Received in revised form 15 October 2009
Accepted 20 October 2009
Available online 29 October 2009
Keywords:
FeLV
Insertional mutagenesis
Chromosome integration
FISH
HQ-banded karyotyping
This study was conducted to map the acquired proviral insertions in the chromosomal genome of
feline lymphoid tumors induced by feline leukemia virus (FeLV). Chromosome specimens of the lymphoid tumor-derived cell lines and normal cat lymphocytes were subjected to fluorescence in situ
hybridization and tyramide signal amplification, using an exogenous FeLV-A genome as a probe. Specific
hybridization signals were detected only on the metaphase chromosomes of the tumor cells. Poisson’s
distribution-based statistics indicated that 6 chromosomal loci in each cell line showed FeLV integration.
In the examination of metaphase chromosomes of FL-74, FT-1 and KO-1 cells, significant signals were
detected on B2p15-p14, B2q11, D1p14, E1p14-p13, E1q12 and F2q16; A2p23-p22, B2p15-p14, B4p15p14, D4q23-q24, E1p14-p13 and E2p13-p12; and A2p22, A3q22, B1p13, B1q13, D1p13 and D3p15-p14,
respectively. Consistently, Southern blot hybridization using an FeLV LTR-U3 probe specific for exogenous FeLV revealed the presence of at least 6 copies of exogenous FeLV proviruses at different integration
sites in each cell line. These results indicate that there may be common FeLV integration sites at least in
A2p22 and B2p15-p14. The cytogenetic analysis used in this study can promptly screen FeLV insertions
and provide tags for identifying the novel common integration site.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Feline leukemia virus (FeLV) is a type-C retrovirus associated
with hematopoietic and lymphoid malignancies in cats. Acquired
insertional mutagenesis is thought to be a major pathogenic
mechanism underlying the malignancies induced by oncogenic
retroviruses, including FeLV (Fujino et al., 2008). Identification of
clonal proviral insertions of oncogenic retroviruses has led to the
discovery of many tumor-associated genes (Jonkers and Berns,
1996; Li et al., 1999). Previous studies on FeLV-associated tumors
have suggested that the integrated proviruses induce activation of
cell growth-related genes leading to oncogenesis (Neil et al., 1984;
Forrest et al., 1987; Miura et al., 1987, 1989; Levesque et al., 1990,
1991; Levy et al., 1993a,b; Tsujimoto et al., 1993; Tsatsanis et al.,
1994; Fujino et al., 2009). Such insertional mutagenesis can contribute to the generation of a cell clone with growth advantage and
an eventual malignant phenotype.
Thus far, only 6 common integration sites for FeLV proviruses
have been identified in naturally occurring and experimentally
induced feline lymphomas. Pioneering studies have reported
insertional mutagenesis and over-expression of the c-myc proto-
∗ Corresponding author. Tel.: +81 3 5841 5413; fax: +81 3 5841 8178.
E-mail address: [email protected] (Y. Fujino).
0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2009.10.021
oncogene (Neil et al., 1984; Forrest et al., 1987; Miura et al., 1987,
1989; Levy et al., 1993b; Tsatsanis et al., 1994). Further, common
FeLV-insertion loci were identified adjacent to proto-oncogenes,
pim-1 (Tsatsanis et al., 1994) and flvi-2 encoding feline homolog of
bmi-1 (Levy and Lobelle-Rich, 1992; Levy et al., 1993a,b; Tsatsanis
et al., 1994). Several other unique common integration sites for
FeLV have been identified as flvi-1 (Levesque et al., 1990, 1991),
fit-1 (Tsujimoto et al., 1993) and flit-1 (Fujino et al., 2009). The fit-1
locus has been shown to be linked to the c-myb proto-oncogene
(Tsujimoto et al., 1993; Tsatsanis et al., 1994; Barr et al., 1999).
Further, the flit-1 locus is believed to be close to the ACVRL1/ALK1,
activin A receptor type II-like 1 gene, and proviral insertion at this
locus may promote the expression of the gene (Fujino et al., 2009).
Hence, acquired proviral insertions in FeLV-induced neoplasms can
be used as tags to identify tumor-associated genes.
Advanced cytogenetic analysis employing fluorescence in situ
hybridization (FISH) has made it possible to detect chromosomal proviral insertions of oncogenic retroviruses such as murine
leukemia virus (MuLV) (Acar et al., 2000) and human T-cell
leukemia/lymphoma virus type-I (Uemura et al., 1997; Ohshima
et al., 1998). According to these studies, it is considered that cytogenetic detection of FeLV proviral insertions can lead to prompt
determination of the map loci of proviruses in neoplastic cells with
FeLV-induced neoplasms (Fujino et al., 2003). Because of extensive
conserved synteny between human and cat chromosomes (Lyons et
Y. Fujino et al. / Journal of Virological Methods 163 (2010) 344–352
345
Table 1
Summary of chromosome aberrations and FeLV provirus integrations in 3 cell lines.
Cell line (chromosome
number, sex chromosome)
Chromosome aberration
Provirus insertion
FL-74 (2n = 39, XY)
B2p15-p14, B2q11, D1p14, E1p14-p13, E1q12, F2q16
FT-1 (2n = 39, X)
del(B1)(p13-p12), del(B1)(q24-q35), add(B2)(p15),
add(D1)(p14), add(D2)(p13), +D3, +D4, −F1, −F1, dup(F2)
+B2, dup(F1), +mar
KO-1 (2n = 41, XXX)
+B2, +F2
al., 1997; O’Brien et al., 1999; Murphy et al., 2000), FeLV integration
loci can be compared with the map loci in human chromosomes.
The present study was conducted to map the loci of somatically acquired FeLV proviral insertions in cells with FeLV-induced
neoplasms by employing FISH analysis in order to investigate insertional mutagenesis.
2. Materials and methods
2.1. Cells
In this study, the following 3 feline lymphoid tumor cell lines
were used; FL-74 (Theilen et al., 1969), FT-1 (Miura et al., 1987,
1989) and KO-1 (Fujino et al., 2004). FL-74 was derived from lymphoid leukemia and FT-1 and KO-1 were derived from thymic
lymphomas occurring naturally in FeLV-infected cats. These cells
were grown in RPMI-1640 medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and
antibiotics at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. Normal cat peripheral blood mononuclear cells (PBMCs) were collected
by centrifugation of peripheral blood samples on Ficoll-Hypaque
density gradient (Lymphoprep, Nycomed AS, Oslo, Norway) and
then stimulated with 0.01 mg/ml of concanavalin A (ConA) (Sigma)
for 3 days.
A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24,
E1p14-p13, E2p13-p12
A2p22, A3q22, B1p13, B1q13, D1p13, D3p15-p14
hybridization mixture, which was composed of 50% formamide,
10% dextran sulfate, 2× SSC and 0.01% bovine serum albumin
(BSA), and then denatured at 75 ◦ C for 10 min. Hybridization was
performed at 37 ◦ C for 42 h in a moist chamber. After hybridization, the samples on the slides were first washed with a solution
containing 50% formamide and 2× SSC, and then with that containing 1× SSC. Subsequently, tyramide signal amplification was
performed as described previously (Acar et al., 2000) by using the
TSATM Biotin System (NENTM Life Science Products, Boston, MA).
These samples were incubated further with fluorescein isothiocyanate (FITC)-conjugated avidin (Roche Molecular Biochemicals)
at 37 ◦ C for 45 min. The incubated samples were rinsed with 2× SSC,
and stained concurrently with antifade solutions (Intergen, Purchase, NY) with 4 ,6-diamidino-2-phenylindole (DAPI) (Sigma) to
identify individual chromosomes. The stained samples were then
observed under a fluorescence microscope. The chromosome loci
were determined on the basis of the nomenclature given for feline
Q-banded chromosomes (Cho et al., 1997c). Fluorescence signals
of each locus were counted from at least 10 metaphase chromosomes and significance of the hybridization signals was analyzed
statistically by Poisson’s distribution along with the 270-band stage
feline karyotype (O’Brien and Nash, 1982). p-Value for each locus
was calculated from the numbers of signals, observed metaphase
chromosomes and band stage (270) by Poisson’s law. A p-value of
<0.05 was considered significant.
2.2. Chromosome preparations
2.4. Southern blot hybridization
Metaphase chromosomes were obtained from FL-74, FT-1 and
KO-1 cell lines, and normal cat ConA-stimulated PBMCs. These cells
maintained in RPMI-1640 medium containing 10% FBS were treated
with 0.01 mg/ml of colcemid (Demecolcin, Wako, Osaka, Japan) for
2 h before harvest. The treated cells were resuspended in 0.075 M
potassium chloride at 37 ◦ C for 20 min and fixed in a 3:1 mixture
of methanol and acetic acid on ice. After repeating the fixation process more than 3 times, the cell suspension was placed on glass
slides and air-dried. To analyze the karyotype, chromosomes were
stained with a combined solution of Hoechst 33258 and quinacrine
mustard for HQ-banding essentially as described previously (Cho
et al., 1997c).
2.3. Fluorescence in situ hybridizaiton (FISH)
A plasmid clone of pFGA-2 containing an entire proviral genome
of FeLV-A/Glasgow-1 (Stewart et al., 1986) was biotinylated by nick
translation (Roche Molecular Biochemicals, Mannheim, Germany)
for use as a probe. FISH was performed essentially as described
previously (Fujino et al., 2001a,b, 2003).
In brief, the chromosome preparations were pretreated with
0.1 mg/ml of RNase A (Sigma) in 2× saline sodium citrate (SSC, 1×
SSC contains 0.15 M sodium chloride and 15 mM sodium citrate) for
1 h, and then with 0.05 mg/ml of pepsin (Sigma) at 37 ◦ C for 10 min.
Target chromosomes were denatured in a solution containing 70%
formamide and 2× SSC (pH 7.0) at 72 ◦ C for 3 min, and immediately
followed by ice-cold ethanol dehydration.
One milligram of the labeled DNA probe was mixed with 5 mg
of salmon sperm DNA (Invitrogen, Carlsbad, CA) in 0.01 ml of the
High-molecular-weight genomic DNA samples were extracted
from FL-74, FT-1 and KO-1 cells, and the liver of a feline fetus. The
samples of cultured cells and homogenized tissue were treated
with a lysis buffer containing 0.02 mg/ml of proteinase K, 0.01 M
Tris–hydrochloride (pH 8.0), 1 mM EDTA, 0.5% SDS and 0.01 mg/ml
of RNase A at 37 ◦ C for 24 h. Subsequently, DNAs were extracted
with phenol and chloroform, and precipitated with ethanol. The
DNAs (0.015 mg) were digested with 100 U of restriction enzymes,
electrophoresed on 0.8% agarose gels, and then transferred onto
nylon membranes. A probe specific for the exogenous FeLV genome
was prepared from the long terminal repeat (LTR) U3 region of
an exogenous FeLV clone, pJ7E2 (Miura et al., 1987). The DNA
samples on the membrane were hybridized with the 32 P-labelled
probe in a hybridization solution containing 5× SSC, 1% SDS, 0.05 M
Tris–hydrochloride (pH 7.6), 0.1 mg/ml of salmon sperm DNA, and
5× Denhardt’s solution (1× Denhardt’s solution contains 0.02%
each of Ficoll type 400, polyvinyl pyrrolidone and BSA fraction V) at
62 ◦ C for 16 h. After hybridization, the filters were washed 3 times
with a solution containing 1× SSC and 0.1% SDS at 57 ◦ C for 20 min
and then subjected to autoradiogaphy.
3. Results
3.1. Chromosome karyotypes
Twelve well-banded metaphase chromosomes of each cell line
were analyzed in detail by HQ-banded cat karyotyping (Felis catus,
2n = 38). All the 3 cell lines showed chromosomal abnormalities
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Y. Fujino et al. / Journal of Virological Methods 163 (2010) 344–352
Fig. 1. A representative HQ-banded karyotype of the FL-74 cell line. The karyotype is interpreted as [39, XY, del(B1) (p13-p12), del(B1) (q24-q35), add(B2) (p15), add(D1)
(p14), add(D2) (p13), +D3, +D4, −F1, −F1, dup(F2)]. Arrows indicate chromosomal aberrations of deletions of a distal region of the short arm p13-p12 and an interstitial
region of the long arm q24-q35 of each B1, additional unknown fragments attached to D1, D2 and an extra copy of B2, and duplication of F2.
including numerical changes (Table 1). Structural abnormalities
were found in the FL-74 and FT-1 cell lines.
All the metaphases of the FL-74 cells had an abnormal
chromosome number (2n = 39) with additional B2, D3 and D4
chromosomes, and loss of both F1 homologs (Fig. 1). Structural
abnormalities were found in B1, B2, D1, D2 and F2 chromosomes.
One B1 homolog showed a deletion at the distal short arm (p13p12), and the other B1 homolog showed a deletion of an interstitial
region of the long arm (q24-q35). Each homolog of B2, D1 and D2
chromosomes had unidentified fragments at its short-arm end. One
F2 homolog showed duplication, which was possibly due to centromeric fusion. Therefore, the representative karyotype of FL-74
cell line was designated as [39, XY, del(B1) (p13-p12), del(B1) (q24-
q35), add(B2) (p15), add(D1) (p14), add(D2) (p13), +D3, +D4, −F1,
−F1, dup(F2)].
All the metaphases of the FT-1 cells showed an abnormal chromosome number (2n = 39) with an additional B2 chromosome and
marker chromosome and a monosomy of sex chromosomes (Fig. 2).
An F1 homolog showed a structural abnormality of duplication,
which occurred because of binding with the apex each other.
Therefore, the representative karyotype of the FT-1 cell line was
designated as [39, X, +B2, dup(F1), +mar].
All the metaphases of the KO-1 cells showed hyperdiploidy
(2n = 41) because of the trisomies of B2, F2 and X chromosomes
(Fig. 3). In contrast to the 2 earlier described cell lines, structural abnormality was not observed in this cell line. Therefore, the
Fig. 2. A representative HQ-banded karyotype of the FT-1 cell line. The karyotype is interpreted as [39, X, +B2, dup(F1), +mar]. Arrows indicate chromosomal aberrations of
an extra copy of B2, and duplication of F1.
Y. Fujino et al. / Journal of Virological Methods 163 (2010) 344–352
347
Fig. 3. A representative HQ-banded karyotype of the KO-1 cell line. The karyotype is interpreted as [41, XXX, +B2, +F2]. Arrows indicate chromosomal aberrations of extra
copies of B2, F2 and X.
representative karyotype of the KO-1 cell line was designated as
[41, XXX, +B2, +F2].
3.2. Detection of FeLV proviruses in chromosomes by FISH
Significant hybridization delineating FeLV provirus integration
sites was detected in the metaphase preparations from the FL-74,
FT-1 and KO-1 cells (summarized in Table 1), but not in those from
normal cat PBMCs. Chromosomal regions positive for FeLV integration could be identified in these cell lines on the basis of Poisson’s
distribution (p-value in parentheses indicated the probability that
the signal was not significant).
In the examination of 25 metaphase chromosomes of the FL74 cells, significant signals were detected on B2p15-p14 (p < 10−4 ),
B2q11 (p < 10−8 ), D1p14 (p < 10−5 ), E1p14-p13 (p < 10−4 ), E1q12
(p < 10−4 ) and F2q16 (p < 10−5 ) (Fig. 4). The signals on D1p14 were
detected at the abnormal region corresponding to the junction of
the apex of the short arm and an additional unidentified fragment.
The signals on F2q16 were detected on both the authentic and
duplicate chromosomes.
In the examination of 160 metaphase chromosomes of the
FT-1 cells, significant signals were detected on A2p23-p22
(p < 10−28 ), B2p15-p14 (p < 10−22 ), B4p15-p14 (p < 10−17 ), D4q23q24 (p < 10−38 ), E1p14-p13 (p < 10−23 ) and E2p13-p12 (p < 10−28 )
(Fig. 5).
In the examination of 10 metaphase chromosomes of the KO-1
cells, significant signals were detected on A2p22 (p < 10−3 ), A3q22
(p < 10−3 ), B1p13 (p < 10−5 ), B1q13 (p < 10−3 ), D1p13 (p < 10−3 ) and
D3p15-p14 (p < 10−4 ) (Fig. 6).
3.3. Southern blot analysis for exogenous FeLV proviral genome
The copy number of integrated exogenous FeLV proviral genome
was examined by Southern blot hybridization using a probe specific for exogenous FeLV (Fig. 7). The FL-74, FT-1 and KO-1 DNAs
digested with BamHI, which has a cutting site in the pol gene of
most exogenous FeLV isolates (Mullins et al., 1980; Casey et al.,
1981; Stewart et al., 1986), gave 12 discrete bands corresponding to the DNA fragments containing the 5 or 3 LTR U3 region
and adjunctive cellular flanking DNA sequences. Normal cat liver
DNA did not give any detectable band. The Southern blot analysis
results showed that these 3 cell lines comprised clonal cell populations with exogenous FeLV proviral integrations and revealed
the copy numbers of the proviruses. The BamHI digest of FL-74
DNA gave 12 discrete bands of 18, 9.4, 8.0, 5.9, 4.8, 4.4, 4.2, 3.6,
3.4, 2.4, 2.2 and 1.9 kb. The FT-1 DNA gave 12 discrete bands of 15,
9.2, 7.1, 6.2, 4.9, 4.6, 4.0, 3.5, 3.0, 2.6, 2.3 and 2.1 kb. The KO-1 cell
DNA gave 12 discrete bands of 6.6, 6.0, 5.0, 4.1, 2.7, 2.3, 2.1, 1.7,
1.6, 1.5, 1.4 and 1.3 kb. The Southern blot analysis results of the
BamHI-digested DNA revealed the presence of at least 6 copies of
exogenous FeLV proviral genomes at different integration sites in
each of the 3 cell lines, since each band conceivably corresponded
to a DNA fragment containing a 5 or 3 LTR region plus their
flanking sequences. These results regarding the number of integrated FeLV proviruses could be consistent with those of the FISH
analysis.
4. Discussion
The present FISH analysis appeared to generate positive signals
only for exogenous FeLV proviral sequences since no significant
hybridization signal was observed on normal cat chromosomes. In
addition, the number of significant hybridization signals observed
in the FISH analysis could be almost equal to the copy number
of exogenous FeLV proviral sequences detected by Southern blot
analysis in the 3 cell lines. Although the feline genome contains
endogenous FeLV-like elements, the FeLV-A proviral sequence is
less homologous to these elements as compared to most FeLV-B
proviral sequences containing recombined endogenous FeLV env
(Roy-Burman, 1995). Use of FeLV-B isolates as probes might enable
detection of both endogenous and exogenous FeLV insertions. The
use of FeLV-A provirus probe in the analysis could only enable
detection of exogenous FeLV insertions. There have been several
reports on the detection of integrated oncogenic retroviruses by
FISH analysis (Uemura et al., 1997; Ohshima et al., 1998; Acar et
al., 2000). Detection of integrated FeLV provirus by FISH analysis in this study was considered to be useful for identifying the
FeLV-integrated loci on cat chromosomes.
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Y. Fujino et al. / Journal of Virological Methods 163 (2010) 344–352
Fig. 4. Partial metaphases of FL-74 cells showing FeLV proviral integration sites on the distal region of the short arm and the proximal region of the long arm of chromosome B2
in the panel a, the junction between an apex of the short arm of chromosome D1 and an additional unknown fragment in the panel b, the short and long arms of chromosome
E1 in the panel c, and the middle of chromosomes F2 and a duplicate F2 in the panels d and e. Arrow heads indicate FITC signals detecting FeLV provirus genomes. The same
metaphases are stained with DAPI for identification of individual chromosomes (the panels a –e ). Ideograms of chromosomes B2, D1, E1 and F2 are aligned on the right with
the integration sites shown in this study depicted by thick bars.
Exogenous FeLV proviral sequences were found in at least 6
chromosomes in each cell line examined in this study. On these
chromosomes, some tumor-associated genes and common retroviral integration sites have been mapped (O’Brien et al., 1993,
1997a,b, 1999; Murphy et al., 2000). On the short arm of chromosome A2 where FeLV insertions have been observed in FT-1 and
KO-1 cell lines, RAF1 has been mapped (O’Brien et al., 1993). On
chromosome B1 where FeLV insertions have been detected on two
loci in KO-1 cell line, KIT and IL2 have been mapped (O’Brien et al.,
1993; Murphy et al., 2000). On B2 chromosome where FeLV insertions have been detected in FL-74 and FT-1 cell lines (two loci in the
FL-74 cell line), FIT1, MOS, CDKN1A, PIM1, ROS1, MYB and FYN have
been mapped (O’Brien et al., 1997a,b; Murphy et al., 2000). On chromosome D1 where FeLV insertions have been detected in FL-74 and
Y. Fujino et al. / Journal of Virological Methods 163 (2010) 344–352
349
Fig. 5. Partial metaphases of FT-1 cells showing FeLV proviral integration sites on the short arms of chromosomes A2 and E2 in the panel a, the short arm of chromosome
B4 in the panel b, the short arms of chromosomes B2 and E1 in the panel c, and on the distal region of the long arm of chromosome D4 in the panel d. Arrow heads indicate
FITC signals detecting FeLV provirus genomes. The same metaphases are stained with DAPI for identification of individual chromosomes (the panels a –d ). Ideograms of
chromosomes A2, B4, E2, B2, D4 and E1 are aligned on the right with the integration sites shown in this study depicted by thick bars.
KO-1 cell lines, HRAS, FGF3 and ETS1 have been mapped (O’Brien et
al., 1993, 1997a,b). On chromosome E1 where FeLV insertions have
been observed in FL-74 and FT-1 cell lines (two loci in the FL-74 cell
line), TP53 and HIC1 have been mapped (Cho et al., 1997a; O’Brien
et al., 1997a,b; Murphy et al., 2000). Since TP53 has been mapped
regionally on E1p14-p13 (Cho et al., 1997a) which is one of the
loci of integrated FeLV provirus in the FL-74 and FT-1 cells, it can
be indicated that the FeLV provirus may have disrupted the tumor
suppressor gene. Integrated FeLV proviruses may activate known or
unknown tumor-associated genes adjacent to the integration sites
since LTR of FeLV has a potential to induce transcriptional activation of certain cellular genes (Ghosh and Faller, 1999; Ghosh et al.,
2000). The mapped positions of A2p22 and B2p15-p14 that are the
overlapping FeLV insertion loci among the cell lines might contain
the common FeLV integration sites.
In a previous study, FT-1 cells were shown to have an FeLV
insertion upstream of c-myc (Miura et al., 1989). However, in this
study FeLV integration was not found on the c-myc locus, F2q21.2
(Cho et al., 1997b). Such a contradictory result may be derived
from a certain chromosomal translocation, failure of the detection
by FISH analysis, or change in cell population during long-term
cultivation. Similarly, slight discrepancy was observed between
the present and previous results (Miura et al., 1989) of Southern
blot hybridization performed using the probe specific for exogenous FeLV. The genomic status of FT-1 and FL-74 cells may be
altered since they produce consistently infectious FeLV particles.
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Y. Fujino et al. / Journal of Virological Methods 163 (2010) 344–352
Fig. 6. Partial metaphases of KO-1 cells showing FeLV proviral integration sites on the short arms of chromosomes A2 and D3 in the panel a, the short arm of chromosome D1
in the panel b, and the long arm of chromosome A3 and the short and long arms of chromosome B1 in the panel c. Arrow heads indicate FITC signals detecting FeLV provirus
genomes. The same metaphases are stained with DAPI for identification of individual chromosomes (the panels a –c ). Ideograms of chromosomes A2, D1, D3, A3 and B1 are
aligned on the right with the integration sites shown in this study depicted by thick bars.
Meanwhile, the genomic status of KO-1 cells can remain undisturbed by cultivation since the cells are non-producers of FeLV
particles and the results of Southern blot hybridization analysis and
chromosome karyotyping performed in the present study are similar to those of the original study (Fujino et al., 2004). Hence, it
can be recommended to apply the analysis to clinical cases of neoplasms or FeLV non-producer tumor cell lines for understanding
FeLV-related spontaneous alterations.
In the present study, chromosome karyotypings showed some
abnormalities in all the 3 cell lines. Hyperdiploidy of chromosome B2 was observed among all the 3 cell lines. Since several
tumor-associated genes have been mapped on chromosome B2 as
described above, aberrations of this chromosome in the 3 cell lines
might be related to tumor development. In a previous cytogenetic
study, FL-74 cell line had monosomies of A2, E2 and F2, trisomy of
E1, loss of E3 and F1 chromosome pairs, an additional X chromo-
Y. Fujino et al. / Journal of Virological Methods 163 (2010) 344–352
351
present study (the analysis of FT-1 cells) was summarized as the
conference proceedings of Advances in Canine and Feline Genomics
2002, St. Louis, MO, USA. This work was supported in part by grants
from the Ministry of Education, Science, Sports and Culture in Japan.
References
Fig. 7. Detection of exogenous FeLV proviral genomes by Southern blot analysis.
DNA samples from FL-74 cells (lane 1), FT-1 cells (lane 2), KO-1 cells (lane 3) and a
feline fetal liver tissue as a negative control (lane 4) digested with BamHI are subjected to Southern blot analysis using an exogenous FeLV LTR-U3 probe. Molecular
size marker on the left side is HindIII fragments of lambda DNA (kilobase).
some, and several marker chromosomes (Gulino, 1992). However,
such aberrations in the FL-74 cell line were not observed in this
study. Possible explanations of this inconsistency are that the cell
population changed during long-term cultivation, or the improved
quality of karyotype analysis of Q-banded cat chromosomes (Cho et
al., 1997c) made it possible to identify the chromosomal abnormalities. Interestingly, a hybridization signal of possible FeLV provirus
insertion was observed at the locus of D1p14 in the FL-74 cells; this
locus was the junction of the apex of the short arm and an additional unidentified fragment. The aberration of D1 chromosome in
the FL-74 cells might be related to FeLV provirus insertion.
A previous study reported a large-scale analysis of proviral
insertion sites in MuLV-induced leukemia cells by long-template
inverse polymerase chain reaction to clone and sequence a large
numbers of proviral insertion sites (Li et al., 1999). Although this
method can be applicable to the analysis of FeLV insertions, it will be
difficult to identify the sequences adjacent to the proviral insertion
sites until the whole cat genome is successfully sequenced. FeLVinduced spontaneous malignancies have been frequently observed
in veterinary medicine and only 6 genomic common integration
sites associated with oncogenesis have been identified for FeLV.
The FISH technique employed in this study will provide valuable
molecular tags to reveal unidentified oncogenes and/or tumorsuppressor genes through the identification of common integration
loci in multiple FeLV-associated tumor cells as well as in clinical
cases.
Acknowledgements
The authors are grateful to Dr. James C. Neil for providing pFGA2 clone and Mrs. Akane Fujino for helpful discussions. A part of the
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