1 Division of Biological Sciences, College of Natural Sciences, Pusan National University, Pusan 609-735, Korea
2 Laboratory of Biopotency Evaluation, Korea Research Institute of Bioscience and Biotechnology, Taejeon 305-600, Korea
Correspondence
H.-S. Kim
khs307{at}pusan.ac.kr
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ABSTRACT |
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The DDBJ/EMBL/GenBank accession nos of the nucleotide sequences of the HERV-W structural genes (gag, pol and env) reported in this paper are AB114916AB115010 (95 clones for env), AB115186AB115238 (53 clones for pol) and AB120044AB120060 (17 clones for gag) from 12 human normal tissues and AB115094AB115178 (85 clones for env) and AB115247AB110306 (60 clones for pol) from 18 human cancer cells.
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INTRODUCTION |
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HERV-W has been one of the most widely investigated HERV elements following the isolation of an HERV-W-related retrovirus (multiple sclerosis-associated retrovirus, MSRV) from retroviral particles produced in cell cultures from patients with multiple sclerosis (Perron et al., 1997). The new HERV-W family was identified by successive overlapping cDNA clones from the pol region of MSRV (Komurian-Pradel et al., 1999
). It has been shown that the 7q21-22 region of human chromosome 7, which has already been identified as potentially being involved in genetic susceptibility to multiple sclerosis (Charmley et al., 1991
), contains an RGH proviral copy (HERV-H/RGH) and a complete HERV-W proviral copy named HERV-W/7q. The env gene of HERV-W/7q encodes a protein expressed preferentially in foetal tissues and in the placenta (Alliel et al., 1998
). Recently, the HERV-W env protein, syncytin, was shown to be involved in placental morphogenesis (Mi et al., 2000
). The major sites of syncytin expression were placental syncytiotrophoblasts and multinucleated cells that originated from foetal trophoblasts. The env gene sequences of the HERV-W family from human monochromosomes and several human cancer cell lines were identified and analysed in our previous study (Kim & Lee, 2001
; Yi et al., 2002
). In this study, we investigated the expression, sequences and phylogenetic relationships of the structural genes (gag, pol and env) belonging to the HERV-W family in various human tissues and cancer cells.
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METHODS |
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RNA samples and RT-PCR analysis.
Total RNA from human cancer cells was extracted using a High Pure RNA isolation kit (Roche). Using pure mRNA only, at a concentration of 1 µg µl1, expression patterns of the HERV-W genes (gag, pol and env) were examined by the Titan One Tube RT-PCR System (Roche). As a standard control, the human glyceraldehyde-3-phosphate dehydogenase (G3PDH) gene (GenBank accession no. AC068657) was amplified by the primers GPH-S (5'-CAAAGTTGTCATGGATGACC-3', nt 3172131740) and GPH-AS (5'-CCATGGAGAAGGCTGGGG-3', nt 3189831915). Using the RT-PCR approach, the HERV-W family was identified from mRNA of human tissues and cancer cell lines. The HERV-W env fragments (GenBank accession no. AF072506) were amplified using the primer pair HS46 (5'-TCCCTGTACCTGAACAATGG-3', nt 13601379) and HY76 (5'-CTTTCAGCGGTTAGCAAGTC-3', nt 19201939). The HERV-W pol fragments (GenBank accession no. AF009668) were amplified by the primer pair HS48 (5'-ACTACCTGTGGCTACAAGGTT-3', nt 10301050) and HY78 (5'-AAGTGCGCAGTCTCAGCA-3', nt 17031720). The HERV-W gag fragments (GenBank accession no. AF072500) were amplified using the primer pair JM76 (5'-AAACGTTCCCCGCAAGACAA-3', nt 611630) and JM28 (5'-GGTGTTTTACAAGGGTTAGG-3', nt 11511170). The RT-PCR conditions followed the standard protocol of the Titan One Tube RT-PCR System (Roche) with an annealing temperature of 56 °C.
Molecular cloning of RT-PCR products.
RT-PCR products were separated on a 1·8 % agarose gel, purified with the QIAEX II gel extraction kit (Qiagen) and cloned into the pGEM T-easy vector (Promega). The cloned cDNA was isolated by the alkali lysis method using the High Pure plasmid isolation kit (Roche).
Sequencing and data analysis.
Individual plasmid DNA was screened for inserts by PCR. Positive samples were subjected to sequence analysis on both strands with T7 and M13 reverse primers using an automated DNA sequencer (model 373A) and the DyeDeoxy terminator kit (Applied Biosystems). Nucleotide sequence analysis was performed using GAP, PILEUP and PRETTY programs from the GCG package (University of Wisconsin). The neighbour-joining phylogenetic analysis was performed with the MEGA2 program (Kumar et al., 2001). Statistical significance evaluation of the branching pattern was performed with 100 replications.
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RESULTS |
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DISCUSSION |
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To date, BLAST searches for the HERV-W family have shown that 140 sequences, representing 39 HERV-W proviruses, 40 full-length HERV-W retroposons and 61 truncated HERV-W retrosequences, have been identified in DDBJ/EMBL/GenBank databases (Costas, 2002). The number of identified HERV-W-related fragments at least 70 copies for gag and 30 copies for env per haploid genome is correlated with the increase in complexity from env to gag and pro regions previously described (Voisset et al., 2000
). In our previous study, we identified 15 HERV-W family env genes from human chromosomes 1, 3, 4, 5, 6, 7, 12, 14, 17, 20 and X (Kim & Lee, 2001
). One of them, clone W-7-1 from chromosome 7, showed identical sequences to the clones from brain, testis, kidney, lung, placenta, skeletal muscle and uterus of normal tissues (WE1-5, WE3-8, WE5-4, WE7-2, WE8-1, WE9-3 and WE10-14), suggesting that they are actively expressed in various normal tissues (Fig. 3
, group W-1). In groups W-2, W-3 and W-4, identical sequences appeared among clones from monochromosomes 12, 14, 17 and X, normal tissues (uterus, spleen, skeletal muscle and prostate) and cancer cells (Jurkat, RT4, HCT-116, AZ521, MIA-PaCa-2 and LOX-IMVI), as shown in Fig. 3
. The data imply that at least the HERV-W family on human chromosomes 12, 14, 17 and X may be associated with human cancers.
With advances in functional studies, the product of the HERV-W env gene has been indicated as having a direct role in human trophoblast cell fusion and differentiation (Frendo et al., 2003). The HERV-W env gene product has also been shown to block infection by an exogenous retrovirus, suggesting that the expressed HERV-W env gene could have a beneficial function to the host (Ponferrada et al., 2003
). The relationship between HERV elements and human diseases has been much discussed in recent years, with reports of the detection of various HERV mRNAs, HERV proteins and even HERV particles in human diseases, especially cancer. We previously reported the characterization of HERV-W env gene sequences in some human cancer cell lines to examine the role of HERV in human cancer (Yi et al., 2002
) and we also investigated by comparative analysis the HERV-W families in humans and monkeys to understand better their tissue distribution, evolution and phylogenetic relationships (Yi et al., 2003
). In this study, we have identified structural genes (gag, pol and env) of the HERV-W family that were expressed in normal tissues and cancer cell lines using the RT-PCR approach and sequence analysis. In bioinformatic analysis, a BLASTN query on the EST (expressed sequence tag) database with HERV-W showed multiple transcripts in various human tissues such as bladder, brain, breast, colon, hypothalamus, kidney, liver, lung, ovary, parathyroid, placenta, prostate, skin, stomach, testis and uterus (data not shown), supporting the suggestion that the HERV-W families could have biological roles in many human tissues. Taken together, these data suggest that recently proliferated and expressed HERV-W families are active in the genomes of human tissues and cancer cells and could, therefore, play a functional role in human diseases including cancers.
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ACKNOWLEDGEMENTS |
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Received 10 November 2003;
accepted 29 December 2003.