| Project/Area Number |
12672255
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Tokyo women's Medical university |
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Principal Investigator |
YAMADA Osamu Tokyo women's Medical university, Medical Research Institute, Assistant professor, 医学部, 助教授 (30167712)
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| Project Period (FY) |
2000 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2003: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Telomere / Telomerase / in situ / Diagnosis / Cancer |
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| Research Abstract |
The introduction of the telomeric repeat amplification protocol assay means telomerase can be detected in small samples from almost all types of tumors. However it is not known whether all or only a small subset of tumor cells have telomerase activity. Activated lymphocytes in normal tissue may give false positive results. We used in cell telomeric repeat synthesis and PCR in situ with in situ hybridization to document telomerase activity. The intracellular amplification of telomere sequences was achieved with single primer pairs and the PCR products were detected by subsequent in situ hybridization using digoxigenin-labeled oligonucleotide probes specific for the amplification products.
The validity and specificity of our methods were confirmed as follows. (1)Without TS primer, no signal was detected. (2)Without Taq DNA polymerase, no signal was detected. (3)TRAP negative samples gave no signals. (4)Specific indirect in-situ PCR was done. (5)HL60 cells showed down-regulation of telomerase activity after differentiation by VD3 and PHA-stimulated lymphocytes showed up-regulation of telomerase activity. (6)In artificial cell mixtures, there was an approximate correlation between the expected and observed number of positive cells.
This indirect method greatly increases the specificity of in situ PCR and enables the simultaneous observation of cell morphology and telomerase activity and could be used to detect minimal residual diseases.
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