| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
We have established two independent in vitro cell lines from patients of primary effusion lymphoma (PEL), which is characterized by proliferating the tumor cells in suspension at the body cavity. Karyotyping analyses based on the conventional Hoechst-quinacrine chromosome banding technique and fluorescence in situ hybridization (FISH) analyses using whole chromosome painting probes have revealed that both of cell lines contained extraordinary broad insersions of chromosomal segment originated from the long arm of chromosome 1. Gaidano, et al. (1999) have reported that the BCBL-1, which is one of PEL cell lines, contained an derivative chromosome 1, dup(1)(q21-q25). Taken together with their report, it seemed quite interest to know these inserted amplifications of somewhere in the long arm of chromosome 1. To clarify the region, I have applied the comparative genomic hybridization (CGH) technique for detection of differences in genome copy number between two target cells. The same amoun
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t of DNAs from tumor cells and normal cells were labeled with biotin-or digoxigenine-dUTP and hybridized with denatured normal metaphase chromosomes, then FITC-avidin and Anti-dig-rhodamine were overlayed to visualize the resulted signals of genomic DNA subtraction. As a result, normal human metaphase hybridized with DNA mixture of the Psu cells and normal cells has showed the narrow green belt of staining on the chromosome 1q25-q32.1 which is derived from an additional chromosomal segment of ins(17:1)(p11.3;1q?) in Psu cell line. On the other hand, metaphase hybridized with DNA mixture of the OS-1 cells and normal cells had the wide green zone of staining on the chromosome 1q21-q32.1 originated from an additional chromosome segment of ins(5:1)(p15;1q?) in OS-1 cell line. The common amplification region was deduced to 1q25-q32.1. To study the common region in detail, 10 BAC clones distributed from 1q21-q32 covering 55 Mb wide in physical map were used for FISH analysis. In conclusion, amplified region of the Psu cell line was constructed with a duplication unit(1q32←1q31→1q32) and that of the OS-1 cell line was a duplication unit(1q21→1q31←1q21), respectively. Therefore, segmental amplification starting point of each cell line was narrowed down into the chromosome region of 1q31.3-q32.1. They might contain target sequences that directly associates with the genetic alteration occurring in PEL cell lines. Further, Psu cell line showed at least the tripulicated signals of BAC clone 472J12 (190kb insert) on the inserted region of the derivative chromosome of ins(17:1)(p11.3;1q?).
I haven't yet cloned any genomic fragment which associates with the origin of the present common duplication unit, detail FISH analysis at the narrow area is on going. Less
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