Bright R K, Vocke C D, Emmert-Buck M R, Duray P H, Solomon D, Fetsch P, Rhim J S, Linehan W M, Topalian S L
Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Cancer Res. 1997 Mar 1;57(5):995-1002.
Difficulty in establishing long-term human prostate epithelial cell lines has impeded efforts to understand prostate tumorigenesis and to develop alternative therapies for prostate cancer. In the current study, we describe a method that was successful in generating 14 immortal benign or malignant prostate epithelial cell cultures from primary adenocarcinomas of the prostate resected from six successive patients. Immortalization with the E6 and E7 transforming proteins of human papilloma virus serotype 16 was necessary to establish long-term cultures. Microscopic examination of fresh tumor specimens exhibited a variable mixture of benign and malignant epithelium. Thus, single-cell cloning of tumor-derived cell cultures was essential for defining tumor cell lines. Efforts to characterize these cultures using traditional criteria such as karyotype, growth in nude mice, and prostate-specific antigen expression were noninformative. However, allelic loss of heterozygosity (LOH) represents a powerful alternative method for characterizing tumor cell lines originating from primary adenocarcinomas of the prostate. Microdissected fresh tumors from four of six patients revealed LOH at multiple loci on chromosome 8p, as assessed by PCR. LOH on chromosome 8p matching the patterns found in microdissected tumors was also observed in a tumor-derived cell line and its clones, as well as in one clone from a tumor-derived cell line from a second patient. LOH was not observed in immortal lines generated from autologous benign prostatic epithelium, seminal vesicle epithelium, or fibroblasts. The multifocal nature of prostate cancer, as well as the presence of an entire spectrum of malignant transformation within individual prostate glands, necessitates this type of careful analysis of derivative cell cultures for their validation as in vitro models that accurately reflect the primary cancers from which they are derived.
建立长期的人前列腺上皮细胞系存在困难，这阻碍了人们对前列腺肿瘤发生机制的理解以及开发前列腺癌替代疗法的努力。在当前研究中，我们描述了一种方法，该方法成功地从连续6例患者切除的原发性前列腺腺癌中生成了14种永生化的良性或恶性前列腺上皮细胞培养物。用人乳头瘤病毒16型的E6和E7转化蛋白进行永生化处理是建立长期培养物所必需的。对新鲜肿瘤标本的显微镜检查显示良性和恶性上皮细胞的混合情况各不相同。因此，肿瘤来源细胞培养物的单细胞克隆对于确定肿瘤细胞系至关重要。使用传统标准（如核型、在裸鼠中的生长情况和前列腺特异性抗原表达）对这些培养物进行表征的努力并无信息价值。然而，杂合性等位基因缺失（LOH）是表征源自原发性前列腺腺癌的肿瘤细胞系的一种有力替代方法。通过PCR评估，6例患者中有4例的显微切割新鲜肿瘤显示8号染色体短臂上多个位点存在LOH。在一个肿瘤来源的细胞系及其克隆中，以及在另一名患者的肿瘤来源细胞系的一个克隆中，也观察到了与显微切割肿瘤中发现的模式相匹配的8号染色体短臂上的LOH。在源自自体良性前列腺上皮、精囊上皮或成纤维细胞的永生化细胞系中未观察到LOH。前列腺癌的多灶性性质以及单个前列腺腺体内存在的整个恶性转化谱，使得有必要对衍生细胞培养物进行这种仔细分析，以验证它们作为准确反映其来源原发性癌症的体外模型。
