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In order to elucidate the role of HPV-16 in the development of genital cancer, NIH3T3 cells were transfected by HPV-16 whole genome and its two early genes, E6-E7. Besides ordinary calcium phosphate/DNA coprecipitation technique, a newly designed recombinant retrovirus containing the HPV-16 genome or subgenomes was used to infect cells for transfer of the target genes. The transforming activities have been demonstrated to be most efficient when a bioengineering technique of this kind is used. HPV-16 DNA was proved to have transforming potential for NIH3T3 cells, and the DNA of HPV-16 was proved to undergo multisite integration into transformed cells and nude mice tumour cells. The E6-E7 open reading frames are sufficient for transforming NIH3T3 cells independently in vitro, which implies that E6-E7 open reading frames are transforming genes or even viral oncogenes of HPV-16. The RNA transcribed by the E6-E7 of HPV-16 was expressed in transformed cells and in tumour cells of nude mice. The use of a recombinant retrovirus for gene transfer in this study is much more efficient than that of calcium phosphate/DNA coprecipitation. The lack of a tissue-culture system suitable for HPV replication in vitro makes HPV gene recombination into a specially engineered retrovirus for viral-mediated gene transfer of particular significance for the possible application of viral carcinogenesis, both in vitro and in vivo, for basic and clinical research.

There are more supports for the view that human papillomavirus (HPV) infection might be an etiological factor in the development of cervical cancer when the association of persistent condylomata is considered. Biopsies from 318 cases with squamous cell carcinoma of uterine cervix, 48 with cervical and vulvar condylomata, 14 with cervical intraepithelial neoplasia (CIN), 34 with chronic cervicitis and 24 normal cervical epithelium were collected from 5 geographic regions of China with different cervical cancer mortalities. All specimens were prepared for Dot blot, Southern blot and in situ DNA-DNA hybridizations by using HPV-11, 16, 18 DNA labelled with 32P and 3H as probes to detect viral homologous sequences in samples. Among them, 32 cases with cervical cancer, 27 with condyloma and 10 normal cervical epitheliums were randomly chosen for comparative EM observation. The results showed that: 1), 192 out of 318 (60.4%) cases of cervical cancer were positive for HPV-16 DNA probe (Table I)

Human papillomavirus(HPV) type 16 is the most prevalent viral type and its E6 and E7 early genes might play a key role in the carcinogenesis of cervical cancer in Chinese women. In order to elucidate the role of HPV-16 in the development of genital cancer, the present study was designed to transfect NIH3T3 cells with HPV-16 whole early genes and its E6, E7 genes separately. Besides the ordinary calcium phosphate/DNA coprecipitation technique, a newly designed recombinant retrovirus containing HPV-16 genes were used to infect cells for transfer the aim genes as shown in Fig.1.1). The cells transformed by recombinant retrovirus containing whole early gene and its E6-E7 respectively appeared more refractive, round shaped with rapid growth and loss of contact inhibition. Hundreds of cell colonies presented in cells transformed by the recombinant retrovirus after selected culture for one week, compared with the routine method, only a few colonies till 3 weeks. By using such a genomic engineering technique,the transforming activities have been shown to be most efficient. 2). The tumorigenicity of transformed cells: Transformed cells HZIP16-3T3 and HZIP16K-3T3, as well as negative controlled cells, NIH3T3 and pZIP-3T3 which was infected by recombinant retrovirus containing no HPV-16 gene, were transplanted into nude mice respectively. Tumours were formed in all mice within 15 days after inoculation of transformed cells (Fig.2). Whereas, no tumour was found in all controlled animals as long as 50 days. 3).