2003 Fiscal Year Final Research Report Summary
New method for sensitive in situ telomerase assay and it's application.
| 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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| 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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[Publications] Akiyama, M., Yamada, O, Hideshima, T., Yanagisawa, T., Yokoi, K., Fujisawa, K., Eto, Y., Yamada, H., Kenneth C.Anderson.: "TNFα induces rapid activation and nuclear translocation of telomerase in human lymphocytes"B.B.R.C.. 316. 528-532 (2004)
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[Publications] Yamada, O., Akiyama, M., Kawauchi, K., Adachi, T., Yamada, H., Kanda, N., Aikawa, E.: "Overexpression of telomerase confers a survival advantage through suppression of TRF1 gene expression while maintaining differentiation characteristics in K562 cells"Cell Transplant. 12. 365-377 (2003)
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[Publications] Kato, T., Kosaka, K., Kimura, M., Imamura, S., Yamada, O., Iwai, K., Ando, M., Joh, K., Kuroe, K., Ohtake, A., Takao, A., Momma, K., Matsuoka, R.: "Thrombocytopenia in patients with 22q11.2 deletion syndrome and its association with glycoprotein Ib-β."Genetics in Medicine. 5. 113-119 (2003)
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[Publications] Ujiyara, M., Nomura, K., Yamada, O., Shibata, N., Kobayashi, M., Takano, K.: "Granulocyte-Macrophage colony-stimulating factor ensures macrophage survival and generation of the superoxide anion : a study using a monocytic-differentiated HL60 subline."Free Radical Biology & Medicine. 31. 1396-1404 (2001)
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[Publications] Yamada, O., Ichikawa, M., Okamoto, T., Park, T., Motoji, T., Mizoguchi, H, Shibuya, A.: "Killer T cell induction in patients with blastic NK cell lymphoma/leukaemia : implications for successful treatment, and possible therapeutic strategies."Br.J.Haematol.. 113. 153-160 (2001)
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[Publications] Akiyama, M., Yamadam, O., Akita, S., Urashima, M., Yamada, J.H., Ohno, T., Mizoguchi, H., Eto, Y., Yamada, H.: "Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it."Leukemia. 14. 1260-1265 (2000)
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[Publications] Akiyama, M., Asai, O., Kuraishi, Y., Urashima, M., Hoshi, Y., Sakamaki, H., Yabe, H., Furukawa, T., Yamada, O., Mizoguchi, H., Yamada, H.: "Shortening of telomeres in recipients of both autologous and allogeneic hematopoietic stem cell transplantation."Bone Marrow Transplantation. 25. 441-447 (2000)
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