Shen Z, Cen S, Shen J, Cai W, Xu J, Teng Z, Hu Z, Zeng Y
Department of Tumor Pathology, Medical College Shantou University, Guandong, PR China.
J Cancer Res Clin Oncol. 2000 Oct;126(10):589-94. doi: 10.1007/pl00008469.
In order to study the effect of viruses and tumor promoters on the tumorigenicity of the esophagus, human embryonic esophageal epithelial cells were infected with human papilloma virus HPV18 E6E7-AAV in synergy with 12-O-tetradecanoylphorbol 13-acetate (TPA) to observe their malignant transformation. The cultured esophageal epithelial cells incubated with HPV18 E6E7-AAV were divided into two groups: the SHEEC1 group was exposed to TPA (5 ng/ml) for 4 weeks at the 5th passage of the cells; the SHEE group served as the control and was cultured in the same medium without TPA. The morphological phenotype, the DNA content during the cell cycle and the chromosomes were analyzed. The tumorigenicity was assessed by colony formation after cultivation in soft agar and transplanting the cells into nude mice. HPV18 E6E7 DNA was assayed by fluorescent in situ hybridization (FISH) and the polymerase chain reaction (PCR). The SHEE group, at its 20th passage, grew as a monolayer with the cells showing anchorage dependence and contact inhibition. The chromosome analysis showed diploidy, and soft-agar cultivation and injection into nude mice showed the cells to be non-tumorigenic. They were therefore immortalized cells. In contrast, the SHEEC1 group (TPA group) showed increased DNA synthesis and a proliferative index that was higher (45%) than that of the SHEE group (34%). The number of large colonies of dense multilayer cells (positively transformed foci) in soft agar was high in SHEEC1 group (4.0%) but low in the SHEE group (0.1%). Tumors resulting from transplantation were observed in all six nude mice injected subcutaneously with cells of the SHEEC1 group but no tumor developed in mice receiving cells of the SHEE group. In both groups of cells, HPV18 E6E7 DNA was positively detected by FISH and PCR. The malignant transformation of human embryonic epithelial cells was induced in vitro by HPV18 E6E7 in synergy with TPA. This is a good evidence for the close relationship between HPV and the etiology and pathogenicity of esophageal carcinoma. It is also a reliable model for studying the cellular and molecular mechanisms of carcinogenesis of esophageal carcinoma.
为研究病毒和肿瘤启动子对食管致瘤性的影响，将人乳头瘤病毒HPV18 E6E7 - AAV与人胚胎食管上皮细胞协同感染，并联合12 - O - 十四烷酰佛波醇13 - 乙酸酯（TPA），观察其恶性转化情况。将用HPV18 E6E7 - AAV培养的食管上皮细胞分为两组：SHEEC1组在细胞第5代时暴露于TPA（5 ng/ml）4周；SHEE组作为对照，在不含TPA的相同培养基中培养。分析细胞的形态表型、细胞周期中的DNA含量及染色体。通过软琼脂培养后集落形成及将细胞移植到裸鼠体内来评估致瘤性。采用荧光原位杂交（FISH）和聚合酶链反应（PCR）检测HPV18 E6E7 DNA。SHEE组在第20代时呈单层生长，细胞表现出贴壁依赖性和接触抑制。染色体分析显示为二倍体，软琼脂培养及注射到裸鼠体内显示细胞无致瘤性。因此，它们是永生化细胞。相比之下，SHEEC1组（TPA组）显示DNA合成增加，增殖指数高于SHEE组（分别为45%和34%）。SHEEC1组在软琼脂中致密多层细胞的大集落（阳性转化灶）数量较高（4.0%），而SHEE组较低（0.1%）。皮下注射SHEEC1组细胞的所有6只裸鼠均观察到移植瘤，而接受SHEE组细胞的小鼠未发生肿瘤。两组细胞中，通过FISH和PCR均阳性检测到HPV18 E6E7 DNA。HPV18 E6E7与TPA协同作用在体外诱导人胚胎上皮细胞发生恶性转化。这充分证明了HPV与食管癌病因及致病性之间的密切关系。它也是研究食管癌发生的细胞和分子机制的可靠模型。
