1991 Fiscal Year Final Research Report Summary
Growth/differentiation modulator produced by prostate stromal cells
| Project/Area Number |
02454371
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Urology
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| Research Institution | Kagawa Medical School |
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Principal Investigator |
WADA Fumio Kagawa Medical School, Department of Endocrinology, Professor, 医学部, 教授 (20028385)
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| Co-Investigator(Kenkyū-buntansha) |
OYA Haruyo Kagawa Medical School, Department of Endocrinology, Research Associate, 医学部, 助手 (80223973)
NISHI Nozomu Kagawa Medical School, Department of Endocrinology, Research Associate, 医学部, 助手 (10145047)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Prostate / Epithelial-Stromal Interactions / Growth Factor / Primary Culture / Extracellular Matrix / Androgen |
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| Research Abstract |
The present study was designed to clarify the role of stroma-derived growth factor(s)in proliferation and differentiation of the prostate epithelial cells. The study included 1)analysis and purification of growth factors in rat prostate and cell lines derived from rat prostatic cancer, 2)optimization of the conditions for serum-free primary culture of prostate cells. 3)identification and characterization of factor(s)produced by the stromal cells in serum-free primary culture.
Four distinct forms of growth factor were found in the extract of rat dorsolateral prostate. One of the factors was a member of heparin-binding growth factor(HBGF)family judging from its high affinity for heparin-sepharose. The other three factors were capable of competing with ^<125>I-epidermal growth factor(EGF)for the cell surface receptor, and recognized by anti-rat EGF antiserum. These EGF-like factors(EGF1-EGF3)were purified. EGFI showed microheterogeneity on chromatographic and electrophoretic separation and
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N-terminal sequence analysis. EGF1 showed an average molecular weight of about 35, 000 on SDS-polyacrylamide gel electrophoresis under reducing conditions. These results indicated that EGF1 was a mixture of high molecular weight forms of EGF. The molecular weights of EGF2 and EGF3 were similar to that of rat submaxillary gland EGF(Mr=5, 400). The amino acid sequence'of EGF2 was identical with that of rat EGF except, for the N- and C-terminal amino acids : aspartic acid instead of asparagine was found at the N-terminal position and C-terminal arginine was missing in EGF2. Although the N-terminal sequence of EGF3(1-19)was identical with that of EGF2. the two factors were completely separated by gel filtration indicating a difference in the C-terminal structure. EGF1, EGF2 and EGF3 but not the HBGF stimulated proliferation of primary cultured rat dorsolateral prostate epithelial cells.
We have established serum-free primary culture of rat prostate epithelial and stromal cells. Bovine pituitary extract(BPE), however, is included in the culture medium as the only unidentified factor. Various protease inhibitors were examined on their ability to substitute for BPE. Among them, soybean trypsin inhibitor(STI), antipain, chymostatin, ovomucoid and bovine pancreatic trypsin inhibitor fully or partly stimulated the growth of the primary cultured cells in the absence of BPE. The optimum concentration for STI was 300 ng/ml. The use of STI in stead of BPE permitted us to culture the prostate cells under completely defined conditions.
The third step of the present study has not been accomplished yet. The search for stroma-derived factor(s)using serum-free primary culture under completely defined conditions is now under progress. Less
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