2004 Fiscal Year Final Research Report Summary
Establishment of an efficient gene transfer system to normal epithelial cells and its application for cancer research
| Project/Area Number |
15590358
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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 |
Experimental pathology
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| Research Institution | Osaka Bioscience Institute |
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Principal Investigator |
AKAGI Tsuyoshi Osaka Bioscience Institute, Director's Lab., Vice Head., 分子腫瘍学部門, 研究副部長 (90184077)
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| Project Period (FY) |
2003 – 2004
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| Keywords | normal human epithelial cells / retroviral vector / immortalization / transformation / epidermal keratinocytes / ヒト正常表皮角化細胞 |
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| Research Abstract |
Determination of optimal condition for retroviral vector infection of normal epithelial cells.
We found that culturing in Epilife medium (Cascade), serum free and low-calcium medium, under 3% Oxygen condition significantly improve the growth of normal human epithelial cells, and thereby the efficiency of infection with, retroviral vectors. Under this condition, we have achieved around 70-80% efficiency of infection. Using this condition, we first introduced ecotropic retrovirus receptor gene making human cells susceptible to ecotropic retrovirus whose host range is limited to rodent cells. This enabled us to introduce oncogenes using ecotropic retroviral vector into human epithelial cells, providing a good system in terms of biosafety. We succeeded in making such human epithelial cells expressing ecotropic receptor using epidermal keratinocytes, bronchial epithelial cells.
Establishment of immortalized human epidermal keratinocytes.
Using the system described above, we consecutively introduced hTERT(human catalytic subunit of telomerase), Cdk4, and dominant negative mutant of p53 in normal human epidermal keratinocytes. The resulting cells seemed to be immortalized but still retain normal function of differentiation in response to calcium and serum.
Expression of dominant acting oncogenes in human epithelial cells.
Next, we analyzed the effect of dominant acting oncogenes in these immortalized human epidermal keratinocytes. Expression of activated H-Ras mutant (H-Ras V12) resulted in the formation of many vacuolated cells which died subsequently. Expression of N-terminally truncated active mutant of b-catenin reduced the growth rate. Both of these oncogenes could not induce malignant transformation of the immortalized human epidermal keratinocytes.
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Research Products
(6 results)
