Böhm S, Wilczynski S P, Pfister H, Iftner T
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Germany.
Int J Cancer. 1993 Nov 11;55(5):791-8. doi: 10.1002/ijc.2910550517.
Human papillomavirus (HPV) type 16 is strongly implicated in the development of progressive neoplasias of the uterine cervix. Its oncogenic potential is decisively determined by the activity of the early gene products E6 and E7. To look for changes in the expression of these genes during tumour progression we cloned subgenomic fragments of HPV16 into RNA expression vectors, which allowed the generation of 35S-labelled riboprobes specific for distinct mRNA classes. Four constructs were made to differentiate between transcripts starting upstream of the E6 ORF or the E1 ORF, and one probe was specific for unspliced E6/E7 region transcripts. Five other constructs were used to identify transcripts covering the E1, E2, E4, L1 and L2 regions. With the help of these constructs, we analyzed by in situ hybridization 2 low-grade intraepithelial neoplasias of the vulva, 1 high-grade neoplasia of the cervix as well as 4 vulvar and 3 cervical carcinomas. Transcripts from the E1, E2, E4, L1 and L2 region that were consistently detected in the differentiated layers of benign lesions were variably expressed in precancers and carcinomas. None of the investigated cases revealed detectable amounts of unspliced E6/E7 transcripts with a coding potential for a full-length E6 protein. In benign lesions, the E7 transcripts were confined to isolated nuclei of differentiated cells, whereas high-grade lesions and invasive cancers showed elevated levels of equally distributed E7-specific signals in the cytoplasm of all tumour cells. The most abundant transcripts observed in intraepithelial neoplasias and in invasive cancers appear to initiate within ORF E7 and therefore have no coding potential for full-length E6 and E7 proteins. Our data show that the actual level of E7-specific transcripts in cancers is lower than anticipated from earlier studies using an ORF E6/E7-specific probe that hybridizes with the 5'-ends of the abundant mRNA class.
16型人乳头瘤病毒（HPV）与子宫颈进行性肿瘤的发生密切相关。其致癌潜能主要由早期基因产物E6和E7的活性决定。为了探寻肿瘤进展过程中这些基因表达的变化，我们将HPV16的亚基因组片段克隆到RNA表达载体中，从而能够产生针对不同mRNA种类的35S标记核糖探针。构建了4种载体以区分起始于E6开放阅读框（ORF）上游或E1 ORF上游的转录本，还有一种探针针对未剪接的E6/E7区域转录本。另外5种载体用于鉴定覆盖E1、E2、E4、L1和L2区域的转录本。借助这些载体，我们通过原位杂交分析了2例外阴低度上皮内瘤变、1例宫颈高度瘤变以及4例外阴癌和3例宫颈癌。在良性病变分化层中持续检测到的E1、E2、E4、L1和L2区域的转录本在前癌和癌组织中表达各异。所有研究病例均未检测到具有全长E6蛋白编码潜能的未剪接E6/E7转录本。在良性病变中，E7转录本局限于分化细胞的单个细胞核内，而高度病变和浸润性癌在所有肿瘤细胞的细胞质中均显示E7特异性信号水平升高且分布均匀。在上皮内瘤变和浸润性癌中观察到的最丰富转录本似乎起始于ORF E7内，因此没有全长E6和E7蛋白的编码潜能。我们的数据表明，癌症中E7特异性转录本的实际水平低于早期使用与丰富mRNA类5′端杂交的ORF E6/E7特异性探针所做研究的预期水平。
