2000 Fiscal Year Final Research Report Summary
Analysis of ACTH receptor in adrenal tumor and tumor-like lesion.
| Project/Area Number |
11670176
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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 |
Human pathology
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| Research Institution | Nagasaki University |
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Principal Investigator |
ABE Kuniko University Hospital, Nagasaki University Assistant, 医学部・附属病院, 助手 (00253641)
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| Co-Investigator(Kenkyū-buntansha) |
KOJI Takehiko School of Medicine, Nagasaki University, Professor, 医学部, 教授 (30170179)
HAYASHI Tomayoshi University Hospital, Nagasaki University Assistant professor, 医学部・附属病院, 助教授 (20253651)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Adrenocortical tumor / ACTH receptor / Proliferative activity / p53 / myc / ras / immunostainig / in situ hybridization |
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| Research Abstract |
We selected 15 amino acid sequences from the ACTH receptor protein. The symtthesized peptides were coupled to KLH as carrier protein and was immunized to rabbits. ELISA showed positive reaction to the peptide at the dilution of 1 : 25,600. We evaluated the immunohistochemical reactivity on the paraffin sections of human adrenal tissue. One of the antibody reacted with the cell membrane of adrenocortical cells and the Western blot analysis revealed one band at approximately 60 kDa. Both positive reactions were abolished by preabsorption with the immunizing peptide.
Immunohistochemical reactivity was evaluated on the paraffin sections of the adrenocortical carcinoma, adenoma and hyperplasia. The reactivity for the PCNA and Ki-67 were 3.43-20.08 and 0-20.9 in carcinoma, 0.14-1.33 and 0-1.03 in adenoma, 0.19-0.7 and 0-0.05 in hyperplasia, respectively. The correlation of the biological malignant grading and proliferative activity was significant. ACTHR expression in carcinoma was lower than
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those in adenoma and hyperplasia. p53 was positive in three of carcinoma and negative in all of adenoma and hyperplasia. The relation between the carcinogenesis and p53 was suggested, however, the other factors are still to be considered. Ras was negative in all cases. The myc antibody (9E10) reacted with nuclei of normal adrenal cortical cells, nuclei and some cytoplasm in hyperplasia and adenoma, and predominantly cytoplasm in carcinoma.
We synthesized the oligo-DNA of 60mer with a selected sequence from the ACTHR DNA with additional 5 repeater of ATT sequence. Dot blot hybridization detected l0pg. We examined the in situ hybridization (ISH) using TT dimerized probe, on the paraffin section of the human adrenal tissue that was confirmed RNA preservation by methylgreen-pyronin stain and ISH of 28s rRNA.Compact cells in zona glomerulosa and zona fasciculata showed weak positivity. For more reactivity, we tried 2 different oligo-DNAs by ISH with digoxygenin-probe. They showed similar result to TT dimerized probe. Neoplastic lesion have not showed specific reactivity yet. Less
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