YOKOYAMA Takashi HIROSHIMA UNIVERSITY, MEDICAL HOSPITAL, PROFESSOR, 医学部・附属病院, 教授 (60034104)
HIYAMA Eiso HIROSHIMA UNIVERSITY, MEDICAL HOSPITAL, ASSOCIATE PROFESSOR, 医学部・附属病院, 助教授 (00218744)
|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2001 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 2000 : ¥3,000,000 (Direct Cost : ¥3,000,000)
We examined telomere length, telomerase activity, and the expression levels of several human telomere binding proteins such as human telomeic repeat binding factor 1 (hTRF1), hTRF2, TIN2, tankylase, and hRap1 in 241 frozen neuroblastoma tissues by Northern hybridization and RT-PCR using fluorescent thermal cycler. The length of telomere is significantly correlated with the expression levels of TRF1 but not with those of other telomere binding proteins. Immunohistochemical analysis using anti-hTRF1 and hTRF2 antibodies revealed that the locations of these proteins were located at telomere signals. The analyses of TUNNEL methods and DNA fragmentation revealed that apoptotic cells were detectable in 5-54% of the tumor cells with low or undetectable telomerase activity and these ratios were significantly correlated with the shortened telomere. On the other hand, the ration of apoptotic cells were less than 5% in the tumors with high telomerase activity.
In 5 neuroblatoma cell lines, all showed high telomerase activity and 4 had shortened telomere. In these cells lines, the length of telomere was also correlated with the expression of hTER1. In these cell lines, the administration of anti-hTRF 1 and hTRF 2 led to the no change of proliferative activity, telomerase activity, or the number of apoptotic cells. However, shortening of telomere occurred in 3 of these cell lines. Then, we added ribozymes to cut human telomerase RNA component (hTR), which is one of the essential components of human telomerase, in these cell lines. In two cell lines, telomerase activity was significantly inhibited and the vast number of cells was killed. The analyses of TUNNEL methods and DNA fragmentation revealed the increase signals of apoptosis in these cell lines.
In conclusion, the inhibition of telomerase might induce the apoptotic signals in neuroblastoma cells.