Journal of Heredity 2003:94(3)
© 2003 The American Genetic Association 94:251-255
Detection of the Integrated Feline Leukemia Viruses in a Cat Lymphoid Tumor Cell Line by Fluorescence In Situ Hybridization
From the Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan (Fujino, Masuda, Ohno, and Tsujimoto); Department of Cancer Research, Division of Pathology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan (Satoh); and Laboratory of Internal Medicine II, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara-shi Kanagawa, 229-8501, Japan (Hisasue).
Address reprint requests to H. Tsujimoto at the address above, or e-mail: atsuji{at}mail.ecc.u-tokyo.ac.jp.
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Abstract |
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Feline leukemia virus (FeLV) is a type-C retrovirus associated with lymphoid and hematopoietic malignancies in cats. The FeLV-induced tumors are thought to be caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. This study was undertaken to enumerate and map the acquired proviral insertions in the genome of a feline thymic lymphoma cell line (FT-1) infected with FeLV. Fluorescence in situ hybridization (FISH) combined with tyramide signal amplification was applied on the chromosome specimen of FT-1 cells and normal cat lymphocytes, with an entire FeLV-A genome used as a probe. Specific hybridization signals were detected from only the metaphases of the FT-1 cells, not from those of normal cat lymphocytes. Statistically based on the Poisson's distribution, at least six loci of chromosomal regions, A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13, and E2p13-p12, appeared to be positive for FeLV integration. Consistently, Southern blot hybridization analysis using an FeLV LTR-U3 probe specific for exogenous FeLV showed the integration of at least six FeLV proviral genomes in FT-1 cells. The cytogenetic technique employed here will provide valuable molecular tags to reveal unidentified tumor-associated genes in FeLV-associated tumor cells.
Identification of clonal proviral insertions by oncogenic retroviruses has led to the isolation of novel tumor-associated genes (Jonkers and Berns 1996; Li et al. 1999). Feline leukemia virus (FeLV) is an oncogenic type-C retrovirus associated with lymphoid and hematopoietic malignancies in cats (Linenberger and Abkowitz 1995). The FeLV-induced tumors are thought to be caused, at least in part, by acquired insertional mutagenesis. In some FeLV-associated tumors the integrated proviruses were shown to induce the activation of proto-oncogenes leading to tumorigenesis. Such insertional mutagenesis may produce a cell clone with growth advantage and eventually malignant phenotype.
Several common integration sites for FeLV proviruses have been identified in naturally occurring and experimentally induced lymphomas in cats. In such cases insertional mutagenesis and overexpression of the c-myc proto-oncogene have been reported (Forrest et al. 1987; Levy et al. 1993b; Miura et al. 1987; Neil et al. 1984; Tsatsanis et al. 1994). FeLV-insertion loci were also identified adjacent to proto-oncogenes, pim-1 and flvi-2, which encodes feline homolog of bmi-1 (Levy and Lobelle-Rich 1992; Levy et al. 1993a,b; Tsatsanis et al. 1994). Additional unique insertion loci for FeLV have been identified as flvi-1 (Levesque et al. 1990) and fit-1, which was shown to be linked to myb (Barr et al. 1999; Tsatsanis et al. 1994; Tsujimoto et al. 1993). Thus, the acquired proviral insertions in the FeLV-induced tumors can be used as molecular tags for the identification of tumor-associated genes.
Recent advances in the technique of chromosome analysis employing fluorescence in situ hybridization (FISH) have made it possible to detect chromosomal proviral insertions of oncogenic retroviruses such as murine leukemia virus (Acar et al. 2000) and human T-cell leukemia virus type I (Ohshima et al. 1998; Uemura et al. 1997). Now, detection of FeLV insertions can be used to rapidly determine the approximate map loci of proviruses in FeLV-induced tumor cells in the same manner. Moreover, considering extensive conserved synteny between human and cat chromosomes (O'Brien et al. 1997, 1999), the FeLV-integration sites can be compared with the map position in humans.
In order to understand the pathogenesis and tumor-associated genes in FeLV-induced malignancies, this study was undertaken to show the utilization of FISH analysis to enumerate and map somatically acquired FeLV proviral insertions in FeLV-associated tumor cells.
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Materials and Methods |
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Chromosome Preparations
Metaphase chromosomes were obtained from FT-1 cell line (Miura et al. 1987) and normal cat peripheral blood mononuclear cells (PBMC) stimulated with 0.02% concanavalin A. Both of the cells maintained in RPMI-1640 medium containing 10% fetal bovine serum were treated with 0.05 µg of colcemide per ml for 3 h before harvest. The cells were treated with 0.075 M potassium chloride at 37°C for 20 min and fixed with a 3:1 mixture of methanol and acetic acid. After repeating the fixation process more than three times, the cell suspension was dropped onto slides and air-dried.
FISH
A plasmid clone of pFGA-2 containing an entire proviral genome of FeLV-A/Glasgow-1 (Stewart et al. 1986) was biotinylated with a Nick Translation Kit (Roche Diagnostics, Mannheim, Germany) for use as a probe. FISH was performed essentially as described previously (Fujino et al. 2001a,b) and was combined with tyramide signal amplification (Acar et al. 2000) by means of a TSATM Biotin System (NENTM Life Science Products, Boston, MA). The chromosome locus was determined, based on the nomenclature for feline G- and Q-banded chromosomes (Cho et al. 1997c). The significance of hybridization signals was statistically analyzed by the Poisson's distribution, based on the feline 270-band stage karyotype.
Southern Blot Hybridization
High-molecular-weight genomic DNA samples were extracted from FT-1 cells and liver of a feline fetus. The samples of cultured cells and homogenized tissues were treated with lysing buffer containing 0.02 mg of proteinase K per ml, 0.01 M Tris-hydrochloride (pH 8.0), 1 mM ehthylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl-sulfate (SDS), and 0.01 mg of RNase A per ml at 37°C, overnight. The DNAs were then extracted with phenol and chloroform, and precipitated with ethanol. Fifteen µg of the DNAs were digested with 100 units of restriction enzymes, electrophoresed on 0.8% agarose gels, and 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 32P-labeled probe in a hybridization solution containing 5' SSC (1' SSC consists of 0.15 M sodium chloride and 0.015 M sodium citrate), 1% SDS, 0.05 M Tris-hydrochloride (pH 7.6), 0.1 mg of salmon testis DNA per ml, and 5' Denhardt solution (1' Denhardt solution consists of 0.02% each of ficoll type 400, polyvinyl pyrrolidone, and bovine serum albumine fraction V) at 62°C for 16 hours. After hybridization the filters were washed three times with 1' SSC and 0.1% SDS at 57°C for 30 min and subjected to autoradiogaphy.
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Results and Discussion |
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Significant hybridization signals by FISH were detected on the metaphases from the FT-1 cells (Figures 1 and 2) but not on those from normal cat PBMC. Statistically based on the Poisson's distribution in the examination of 160 metaphase chromosomes of FT-1 cells, at least six loci of chromosomal regions, A2p23-p22 (P < 10-28, 30 cells were positive for signals), B2p15-p14 (P < 10-22, 25 cells were positive for signals), B4p15-p14 (P < 10-17, 21 cells were positive for signals), D4q23-q24 (P < 10-38, 37 cells were positive for signals), E1p14-p13 (P < 10-23, 26 cells were positive for signals), and E2p13-p12 (P < 10-28, 30 cells were positive for signals), appeared to be positive for FeLV integration. An entire FeLV-A proviral genome was used as a probe for FISH in this study, enabling the specific detection of exogenous FeLV proviral sequences. FeLV-A proviral sequence is less homologous with endogenous FeLV elements than most of FeLV-B proviral sequences containing recombined env with endogenous FeLV env (Roy-Burman 1995).
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The copy number of integrated exogenous FeLV proviral genome was examined by Southern blot analysis using a probe specific for exogenous FeLV, which was prepared from the LTR-U3 region (Figure 3). The FT-1 DNA digested with KpnI, which has cutting sites in the LTR-R region and pol gene of most exogenous FeLV isolates (Casey et al. 1981), gave a nearly 1.0-kb strong band and three bands of larger sizes that were hybridized with exogenous FeLV LTR-U3 probe. The FT-1 DNA digested with BamHI, which has one or several cutting sites in the proviral genome and cannot generate the viral internal fragment in most FeLV isolates, gave twelve discrete bands of similar intensity. Normal cat liver DNA did not give any detectable band hybridized with the FeLV LTR-U3 probe in both of the KpnI and BamHI digests. The result of BamHI digest of FT-1 DNA revealed the presence of at least six copies of exogenous FeLV proviral genomes at different integration sites in the cellular DNA, because the bands conceivably corresponded to 5' and 3' LTR plus their flanking sequences. This result was consistent with the result by FISH analysis. In the result of the KpnI digest of FT-1 DNA, the intense band of nearly 1.0 kb and the other three bands were considered to correspond to 3' viral internal fragment containing 3' LTR and 5' flanking fragments containing 5' LTR. Because the results of the BamHI digest clearly indicated the presence of at least six copies of FeLV proviral DNA, it was suspected that the nearly 1.0-kb intense band might contain DNA derived from 5' flanking sequences containing 5' LTR.
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Before the present experiment, the FISH analysis using the entire genome of FeLV-A as a probe in this study was considered to detect endogenous FeLV elements, as well as exogenous FeLV genomes. However, it appeared to generate positive signals for only the exogenous FeLV proviral sequences, because no significant hybridization signal was observed on normal cat chromosome, and the number of significant hybridization signals in the FISH analysis agreed with the copy number of exogenous FeLV proviral sequences detected by Southern blot analysis. Moreover, the env sequence of the FeLV/FT-1 provirus integrated in FT-1 chromosomal DNA had been shown to be closely homologous to that of FeLV-A/Glasgow-1 in our previous report (Miura et al. 1989). There have been a few reports to detect integrated oncogenic retroviruses by FISH analysis (Acar et al. 2000; Ohshima et al. 1998; Uemura et al. 1997). Detection of integrated FeLV provirus by the FISH analysis carried out in this study will provide a novel strategy for tagging tumor-associated genes.
Exogenous FeLV proviral sequences were found to be located on six chromosomes in FT-1 cells. On these chromosomes some tumor-associated genes and retroviral common integration sites have been mapped, such as RAF1 on A2; FIT1, MOS, CDKN1A, PIM1, ROS1, MYB, and FYN on B2; CDKN1B, KRAS2, GLI, and WNT1 on B4; ABL1 on D4; and TP53 on E1 (Cho et al. 1997a; O'Brien et al. 1997, 1999). Because LTR of FeLV has a potential to induce transcriptional activation of certain cellular genes (Ghosh et al. 2000), integrated FeLV proviruses may activate known or unknown tumor-associated genes adjacent to the integration sites. Especially because TP53 has been regionally mapped on E1p14-p13 (Cho et al. 1997a), which is one of the loci of integrated FeLV provirus in FT-1 cells, the FeLV provirus may have disrupted the tumor suppressor gene. In a previous report 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 locus of c-myc, F2q21.2 (Cho et al. 1997b). Such contradictory results may derive from a certain chromosomal translocation, failure of the detection by FISH analysis, or change of the cell population during the long-term cultivation.
A recently published paper reported a large-scale analysis of proviral insertion sites in MuLV-induced leukemia, by long-template inverse polymerase chain reaction, to clone and sequence many numbers of proviral insertion sites (Li et al. 1999). Though this method will be applicable to the analysis of FeLV insertions, it will be difficult to identify the sequences adjacent to the proviral insertion site until a genome project on the whole cat genome is completed. The FISH technique employed here will provide valuable molecular tags to reveal unidentified oncogenes, tumor suppressor genes, or both through the identification of common integration loci in multiple FeLV-associated tumor cells.
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Acknowledgments |
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This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002. The authors are grateful to J. C. Neil for providing pFGA-2 clone and A. Fujino for helpful discussions. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture in Japan.
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Footnotes |
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Corresponding Editor: Stephen J. O'Brien
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