Establishment of an efficient gene transfer system to normal epithelial cells and its application for cancer research

正常上皮细胞逆转录病毒载体感染最佳条件的确定。我们发现，在3%氧气条件下，在Epilife培养基（Cascade）、无血清和低钙培养基中培养，显著提高了正常人上皮细胞的生长，从而提高了逆转录病毒载体的感染效率。在这种情况下，我们达到了大约70-80%的感染效率。在此条件下，我们首次引入了嗜生态逆转录病毒受体基因，使人类细胞对嗜生态逆转录病毒易感，其宿主范围仅限于啮齿动物细胞。这使我们能够利用亲生态逆转录病毒载体将致癌基因导入人上皮细胞，提供了一个良好的生物安全性系统。我们成功地利用表皮角质形成细胞、支气管上皮细胞制备了表达生态受体的人上皮细胞。永生化人表皮角质形成细胞的建立。利用上述系统，我们在正常人表皮角质形成细胞中连续引入hTERT（人端粒酶催化亚基）、Cdk4和p53显性阴性突变体。所得到的细胞似乎是不朽的，但仍保留对钙和血清的正常分化功能。显性癌基因在人上皮细胞中的表达。接下来，我们分析了显性致癌基因在这些永生化的人表皮角质形成细胞中的作用。激活的H-Ras突变体（H-Ras V12）的表达导致大量空泡细胞的形成，这些细胞随后死亡。表达n端截断的b-catenin活性突变体降低了生长速度。这两种致癌基因都不能诱导永生化的人表皮角质形成细胞的恶性转化。

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.