Annab L A, Terry L, Cable P L, Brady J, Stampfer M R, Barrett J C, Afshari C A
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Gynecol Oncol. 2000 Apr;77(1):121-8. doi: 10.1006/gyno.2000.5734.
The aim of this study was to examine whether cells containing the heterozygous form of a BRCA1 185delAG mutation would exhibit abnormal growth or an altered response to DNA damage.
A primary culture of human mammary epithelial cells (90P) was obtained from the nontumor breast tissue of a 35-year-old patient who had undergone a mastectomy for removal of a breast tumor. These cells were immortalized (90PE6E7) following retroviral infection with HPV-16 viral E6/E7. genes. Both the 90P cell strain and the cell line were characterized for their ability to grow in culture, form colonies in soft agar, and produce tumors in athymic nude mice compared to normal breast epithelial cells containing wild-type BRCA1. 90P cells were also analyzed for cellular response to gamma radiation and H(2)O(2).
These cells were confirmed to contain a frameshift mutation, 185delAG, of the BRCA1 gene. Despite being heterozygous for wild-type BRCA1, the 220-kDa full-size BRCA1 protein was abundantly expressed. 90P and 90PE6E7 cells grew at a similar rate and were anchorage dependent. 90PE6E7 also failed to form tumors in athymic nude mice. Finally, 90P cells exhibited a survival response similar to that of normal mammary epithelial cells to radiation damage and exposure to oxidative stress.
To our knowledge the 90P cells and the 90PE6E7 cells are the first characterized, non-tumor-derived breast epithelial cells that are heterozygous for the BRCA1 germline mutation 185delAG. Our conclusion is that these BRCA1 mutant cells appear to have growth and stress response characteristics similar to those of normal human breast cells, which is consistent with the hypothesis that loss of heterozygosity must occur to impair putative BRCA1 function.
本研究旨在检测携带BRCA1基因185delAG杂合突变形式的细胞是否会表现出异常生长或对DNA损伤的反应改变。
从一名35岁因乳腺肿瘤行乳房切除术的患者的非肿瘤乳腺组织中获取人乳腺上皮细胞原代培养物（90P）。用HPV - 16病毒E6/E7基因进行逆转录病毒感染后，这些细胞被永生化（90PE6E7）。与含有野生型BRCA1的正常乳腺上皮细胞相比，对90P细胞系和细胞株在培养中的生长能力、在软琼脂中形成集落的能力以及在无胸腺裸鼠中产生肿瘤的能力进行了表征。还分析了90P细胞对γ射线和H₂O₂的细胞反应。
证实这些细胞含有BRCA1基因的移码突变185delAG。尽管对野生型BRCA1是杂合的，但220 kDa的全长BRCA1蛋白大量表达。90P和90PE6E7细胞以相似的速率生长且依赖贴壁生长。90PE6E7在无胸腺裸鼠中也未能形成肿瘤。最后，90P细胞对辐射损伤和氧化应激暴露表现出与正常乳腺上皮细胞相似的存活反应。
据我们所知，90P细胞和90PE6E7细胞是首批被表征的、非肿瘤来源的乳腺上皮细胞，它们对BRCA1种系突变185delAG是杂合的。我们的结论是，这些BRCA1突变细胞似乎具有与正常人乳腺细胞相似的生长和应激反应特征，这与杂合性缺失必须发生才能损害假定的BRCA1功能这一假设一致。
