| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Research Abstract |
Epstein-Barr virus (EBV) initially isolated from cultured Burkitt lymphoma (BL) cells, is one of well-known oncogenic virus. The Raji cell line was established from BL tissue and used for research worldwide. Previous study showed that each Raji cell contains an average of 50-60 EBV genome equivalents, and a significant proportion of the EBV genome is linearly integrated into host genome through BamHI-W close to the BamHI-Y fragment. However, a definitive EBV integration site in the chromosome has not been identified as yet. In this study, direct evidence that EBV DNA is integrated into the host genome was provided through cloning of the fragments containing nucleotide sequence of Raji integration sites. Integrated EBV DNA consisted of the BamHI-W fragment at one end and BamHI-D fragment at another end. Both junction sites were highly G/C-rich. The BamHI-W fragment and the adjacent part of chromosome 6 showed 70% homology, while no homology was found between the BamHI-D and adjacent hos
… More
t sequences. EBV is present at intron 1 of the BACH2 gene which is located on chromosome 6q15. BACH2 mRNA was not expressed in the Raji cell line. Immunohistochemistry confirmed the loss of BACH2 expression at protein level. Because BACH2 is a putative tumor suppressor gene, loss of its expression through EBV integration might contribute to lymphomagenesis. Present findings might indicate a new paradigm for EBV oncogenesis. The NAB-2 line, which was established from a North American Burkitt tumor, was indicated to contain one copy of EBV DNA as the integrated form into chromosome 2p13 of the host genome. To directly demonstrate the integration site of EBV, and to clarify the relation between the integration sites and the oncogenes, fragments containing the nucleotide sequence of NAB-2 integration sites were cloned. EBV were integrated via the terminal repeats. Integration sites located in the clone RP11-440P5 on chromosome 2, between two oncogenes, REL and BCL11A, which is apart from approximately 350 kbp from each other. Expression level of REL was increased in NAB-2 lines. The flanking region of chromosome 2 at the bilateral junction sites showed no homology to the junction sites of EBV. The integration site 2p13 overlaps with common fragile site, FRA2E. NAB-2 cells expressed almost all latent genes but LMP-2A that flanks the terminal repeats, indicating the type III of latent infection of EBV. Integration event in NAB-2 might alter the regulation of the oncogenes and provide advantage for continuous cell proliferation. IB4 is a lymphoblastoid cell line frequently used for the functional analysis of the latent genes of EBV. Previous study indicated that EBV whole genome is integrated tandemly as the linear viral genome into host genome of IB4, although sites of integration have not been determined. Through cloning of the junctional regions between EBV and host genomes, one of the integration sites was identified on the BamHI-C fragments around oriP sequences and another on the EcoRI-I fragment. Both of the integration sites were located on the clone RP11-119H12 of chromosome 4q25 and separated approximately 6.5 kbp from each other. The integration sites identified were apart from the genes of the host genome, indicating that both host gene and EBV latent genes are not altered by the integration event. Less
|
