Identification of Human Papillomavirus Type 18 E6 Polypeptide in Cells Derived from Human Cervical Carcinomas

Abstract

SUMMARYWe recently reported the expression of human papillomavirus type 18 (HPV-18) E6 protein in bacteria and the production of anti-E6 polyclonal antibodies. This work has now been extended with the production of a panel ofmonoclonal antibodies against the HPV-18 E6 protein. These antibodies demonstrate that there is little antigenic conservation in the E6 protein between HPV-16 and HPV-18, with only one antibody recognizing a cross-reactive epitope. We have used both the monoclonal and the polyclonal antibodies to look for E6 expression in a number of HPV DNA-containing cell lines. These reagents specifically detected a 16.5K tool. wt. polypeptide in cells derived from a human cervical carcinoma. INTRODUCTIONHuman papillomaviruses (HPVs) induce epithelial or fibroepithelial proliferations of skin or mucosa (zur Hausen, 1977). Infection with specific HPV types correlates with specific diseases such as common warts, epidermodysplasia verruciformis and genital warts (Pfister, 1984). In addition infection with some HPV types has been closely linked with specific human cancers. In particular, HPV-16 and HPV-18 DNAs have been found in a large proportion of malignant lesions of the cervix (Durst et al., 1983 ;Boshart et al., 1984), as well as in a number of cell lines derived from human cervical carcinomas. In some cases these DNAs are transcribed (Schwarz et al., 1985;Yee et al., 1985) and HPV-coded proteins are expressed (Smotkin & Wettstein, 1986).Until recently, bovine papillomavirus (BPV-1) served as the prototype for the study of the molecular biology and transforming genes of papillomaviruses. These studies have shown that the BPV-1 E6 open reading frame (ORF) is necessary for efficient transformation of mouse C 127 cells (Schiller et aL, 1984;Yang et al., 1985; Androphy eta[., 1985), and in one case using a BPV-1 E6-specific antiserum, E6 protein has been demonstrated (Androphy et al., 1985). More recently, the direct transforming ability of HPV-16 D NA has been demonstrated in vitro, both in the form of molecularly cloned HPV-16 DNA (Yasumoto et al., 1986) and also HPV-16 DNA derived directly from a cervical tumour biopsy (Tsunokawa et al., 1986). In both cases the HPV-16 DNA was transcribed.We recently reported the synthesis of HPV-18 E6 polypeptide in bacteria (Matlashewski et al., 1986a). The present paper reports the isolation of a panel of monoclonal antibodies to the HPV-18 E6 polypeptide, and their use in the immunological analysis of the E6 protein derived from different HPVs. The use of these reagents for analysis of E6 expression in a variety of HPV DNA-containing transformed cells is also described.

Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth

“…CaSki cells contain HPV-16 DNA sequences and HeLa cells contain HPV-18 DNA sequences. Both lines continue to express E6 protein Banks et al, 1987), and have low levels of wild type, transcriptionally competent, p53 (Butz et al, 1995); although the level to which p53 can be activated is much lower in HeLa than in CaSki cells (Butz et al, 1995). To investigate the e ects of E6* expression on p53 transcriptional activity, CaSki and HeLa cells were transfected with a reporter construct containing the mdm2 promoter upstream of a CAT gene (Barak et al, 1994) together with either vector alone or plasmids containing full length HPV-18 E6 or HPV-18 E6*.…”

“…Of this subset some HPV types, such as 6 and 11, are found associated with benign lesions whereas other types, such as 16 and 18, are commonly associated with lesions that can progress to high-grade cervical intraepithelial neoplasia and ultimately to cervical cancer (zur Hausen and Schneider, 1987). The early viral proteins E6 and E7 are continually expressed in cell lines derived from cervical tumours (Smotkin and Wettstein, 1986;Androphy et al, 1987;Banks et al, 1987). They interact with key cellular tumour suppressors; E7 with pRb (Dyson et al, 1989) and E6 with p53 , and can cooperate to immortalise primary human keratinocytes (Barbosa and Schlegel, 1989;Hawley-Nelson et al, 1989;Munger et al, 1989).…”

Inhibition of E6 induced degradation of p53 is not sufficient for stabilization of p53 protein in cervical tumour derived cell lines

“…These viruses encode two principal oncoproteins, E6 and E7, both of which are continually expressed in cervical tumours and cell lines derived therefrom (Smotkin and Wettstein, 1986;Androphy et al, 1987;Banks et al, 1987). Inhibition of the expression of either protein results in a cessation of transformed cell growth (von Knebel Doeberitz et al, 1988;Crook et al, 1989;Storey et al, 1994); hence both proteins represent ideal therapeutic targets.…”

Mutations in the human papillomavirus type 16 E2 protein identify a region of the protein involved in binding to E1 protein

“…Integration of the HPV DNA into the host genome frequently results in the loss of part of the early region including the El, E2 and E5 genes. However, the upstream regulatory region (URR) and the two major viral oncogenes, E6 and E7, are always retained and expressed (Schwarz et al, 1985;Androphy et al, 1987a;Smotkin & Wettstein, 1986;Banks et al, 1987).…”