Yasumoto S
Laboratory of Molecular and Cellular Biology, Kanagawa Cancer Center Research Institute.
Gan To Kagaku Ryoho. 1989 Mar;16(3 Pt 2):549-61.
Human papillomaviruses (HPVs) are known as etiologic agents of various diseases in regions of the human epithelium. Specific type HPV16 is most frequently found in association with human squamous cell carcinoma. To examine biological activity of HPV type 16 in human cells, primary foreskin epidermal cells and dermal fibroblasts were transfected by recombinant viral DNA containing neo gene with Ca2+-phosphate precipitation. Epidermal cells were maintained in 0.5% Chelex-treated fetal calf serum, low calcium medium supplemented with bovine pituitary extracts and hormone mix. Fibroblasts were cultured in DMEM plus 10% fetal calf serum. The transfected cells were then selected with G418-resistant phenotype. These cells were propagated to maintain in culture and subsequently became stable lines carrying HPV16 genomes, while mock transfected control cells died off at approximately 40-50 population doublings (PD) in a parallel experiment. We have established two independently immortalized human epidermal cell lines (PHK16-I and II) which harbor different copies of HPV16 genome and express HPV16 specific mRNA. Although younger populations of PHK16 lines were fairly sensitive to high Ca2+-level to be differentiating keratinocytes, progressive changes of the cellular phenotype were demonstrated in terms of changes in Ca2+-response and anchorage independent growth during over 300 PD. Altered Ca2+-regulation of growth and differentiation appeared to be common reliable phenotype associated with stable transformation of skin epidermal cells. In contrast, none of the HPV16-transfected fibroblast line immortalized but simply showed extended life span up to 100 PD in average. The result suggested that this biological activity of HPV16 could be reflected in HPV-tropism related to epithelial transformation. We then studied correlations between HPV16 gene expression and the regulation of growth and differentiation of PHK lines during the progressive transformation. Northern blot analysis of RNA from cells in earlier passages demonstrated that down-regulation of HPV16 E6/E7 transcription was associated with keratinocyte differentiation induced by added 1.0mM calcium. The p97 promoter for HPV 16 early genes covering E6/E7 was specifically responsible for this Ca2+-regulation. The eventual loss of Ca2+-regulation could be implicated in a process of progressive transformation of HPV16-epidermal cell system.
人乳头瘤病毒（HPV）是人类上皮组织区域多种疾病的病原体。特定的HPV16型最常与人鳞状细胞癌相关。为了检测HPV16型在人细胞中的生物学活性，用含新霉素基因的重组病毒DNA通过磷酸钙沉淀法转染原代包皮表皮细胞和真皮成纤维细胞。表皮细胞培养于经0.5%螯合剂处理的胎牛血清、添加牛垂体提取物和激素混合物的低钙培养基中。成纤维细胞培养于含10%胎牛血清的DMEM中。然后用对G418有抗性的表型筛选转染细胞。这些细胞经传代培养得以维持，随后成为携带HPV16基因组的稳定细胞系，而在平行实验中，mock转染的对照细胞在约40 - 50次群体倍增（PD）时死亡。我们建立了两个独立的永生化人表皮细胞系（PHK16 - I和II），它们含有不同拷贝数的HPV16基因组并表达HPV16特异性mRNA。尽管较年轻的PHK16细胞系对高钙水平相当敏感，会分化为角质形成细胞，但在超过300次PD过程中，细胞表型在钙反应和锚定非依赖性生长方面发生了渐进性变化。生长和分化过程中钙调节的改变似乎是皮肤表皮细胞稳定转化相关的常见可靠表型。相比之下，HPV16转染的成纤维细胞系均未永生化，只是平均寿命延长至100次PD。结果表明，HPV16的这种生物学活性可能反映在与上皮转化相关的HPV嗜性上。然后我们研究了HPV16基因表达与PHK细胞系在渐进性转化过程中生长和分化调节之间的相关性。对早期传代细胞的RNA进行Northern印迹分析表明，添加1.0 mM钙诱导角质形成细胞分化与HPV16 E6/E7转录下调有关。覆盖E6/E7的HPV 16早期基因的p97启动子对此钙调节起特定作用。钙调节的最终丧失可能与HPV16 - 表皮细胞系统的渐进性转化过程有关。
