| Project/Area Number |
11670579
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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 |
Respiratory organ internal medicine
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
HIYAMA Keiko Hiroshima University Faculty of Medicine, Research Associate, 医学部, 助手 (60253069)
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| Co-Investigator(Kenkyū-buntansha) |
ISOBE Takeshi Hiroshima University, University Medical Hospital, Research Associate, 医学部・附属病院, 助手 (70284198)
ISHIOKA Shinichi Hiroshima University Faculty of Medicine, Asistant Professor, 医学部, 講師 (10191868)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | lung cancer / telomerase / hTERT / chemotherapy / cytology / prognosis / alternative splicing / p53 / 遺伝子 / 遺伝子変異 |
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| Research Abstract |
We speculated that the existence of telomerase activity and specific genetic aberrations would be the key factors that destine the lung cancer cells to respond or resist to chemotherapies. First, we tried to evaluated the telomerase activity of lung cancers by the TRAP assay using proteins extracted from cytology samples. However, the sensitivity was as low as 40%, and its level was mainly dependent on the percentage of cancer cells among non-cancerous cells rather than the telomerase activity level of the original tumor.
To establish more sensitive and accurate protocol to estimate the telomerase activity level of lung cancers, we next tried the quantative RT-PCR method combined with fragment analysis. By RT-PCR, hTERT mRNA, coding catalytic component of telomerase, was amplified showing the full-length fragment and the alternatively spliced fragments. While the amount of the full-length fragment was in relation to the telomerase activity in general, the sensitivity and the specificity of quantitative RT-PCR in consideration of alternative splicing were not satisfactory.
Then we tried immunohistochemical detection of hTERT expression using anti-hTERT antibody. By this method, even in cytology samples with massive non-cancerous cells, we could speculate the telomerase activity level of the original tumors by the ratio of the telomerase positive/negative cancer cells. When we compared the expression pattern of the tumors with high or low telomerase activity by DNA microarray system, several candidate genes have been shown to be responsible to the activation of telomerase.
DNA was also extracted from the cytology samples and aberrations of p53 were investigated by PCR-DGGE technique. We analyzed relationship between the prognosis of patients without surgical intervention and p53 aberrations. The p53 aberration was significantly associated with poor prognosis in all groups of chemotherapy, without any anti-cancer therapy, and overall patients.
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