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The close association between HPV‐16 and cervical cancer implies some role for the virus in development of this cancer. Recent studies have shown that the HPV‐16 E7 gene encodes the major transforming activity of the virus in baby rat kidney (BRK) cell transformation assays. To investigate the requirement for continued E7 expression in BRK cells transformed by HPV‐16 E7 plus EJ‐ras, we have developed a system for inducible expression of the E7 gene. The studies reported here show that continued expression of the HPV‐16 E7 gene is required for maintenance of the transformed phenotype in these cells. The implications these observations bear on the role of the E7 gene in cervical carcinoma are discussed.

The association of certain human papillomavirus (HPV) types with the majority of human cervical carcinomas suggests a role for the virus in the development of this type of cancer. In this paper, we have examined the transforming properties of several HPV types where the early region genes of the virus are under the control of a strong heterologous promoter and show that major differences exist between the HPV types in their ability to transform primary rat kidney epithelial cells in conjunction with an activated ras oncogene. Those HPV types most commonly found in carcinomas–types 16, 18, 31 and 33–are capable of co‐operating with ras to transform primary cells, but those types most commonly found in benign lesions–types 6 and 11–are not. We further demonstrate that the E7 gene of HPV16 by itself is sufficient to co‐operate with activated ras to produce transformed cells which are tumorigenic in immunocompetent animals.

The p53 oncogene is thought to play a role in the proliferation of normal and transformed cells and its expression was postulated to be cell cycle dependent. Using flow cytofluorimetry sorting of populations of exponentially growing cells, coupled to a radioimmune assay, we have investigated the accumulation of p53 along the cell cycle in normal FR 3T3 rat cells as well as in two types of SV40‐transformed derivatives, one of which only expresses the large‐T protein during the G2 phase of the cell cycle. p53 was accumulated at a constant level throughout the cell cycle in FR 3T3 cells. Its level and stability increased to different extents in the two types of transformants. However, the formation of complexes between p53 and large‐T was modulated by the G2‐restricted accumulation of large‐T, thus leading to a differential increase in the levels of p53 both in exponentially growing cells and along the cell cycle. This increase in the levels of p53 appeared to be regulated at a post‐transcriptional level.

SV40 large T antigen associates with a cellular phosphoprotein, p53, in virus‐transformed cells. We have raised three new monoclonal antibodies, PAb1101, PAb1102 and PAb1103, to this cellular protein, derived from SV40‐transformed human fibroblasts. These define at least two non‐overlapping determinants on human p53 that are in different areas of the molecule from those recognised by previously available antibodies. Unlike those antibodies, PAb1102 and PAb1103 do not react with rodent p53. PAb1101 reacts far more weakly with rodent p53 than with primate p53. All three antibodies show a preference for binding to the large T‐associated form of p53, an effect that is particularly marked with PAb1102. The novel specificity of these antibodies allows further probing of the nature and function of the large T/p53 complex in human cells.

The cellular tumour antigen p53 is implicated in the transformation process. To compare p53 from transformed cells and their normal counterparts in detail, and so to identify any structural differences that might alter p53 function, requires information on the primary structure of the protein. By making use of immunochemical techniques we have been able to purify nanomole amounts of p53. This was sufficient, using high sensitivity automated gas‐phase sequencing techniques to determine the amino acid sequence of two tryptic peptides from p53. Their sequences agree completely with the predicted polypeptide sequence derived from a cloned cDNA for p53 mRNA and provide the first data on the amino acid sequence of p53. A combination of the high sensitivity amino acid sequencing procedures used here and cDNA sequence analysis should provide the complete amino acid sequence of p53.

Six monoclonal antibodies (mAbs) have been raised against the E4 proteins of HPV‐1. Five of these were found to recognize denaturation‐resistant epitopes as determined by Western blotting–and their binding sites were identified by determining their reactivity against a panel of bacterial E4–beta‐galactosidase fusion proteins which contained progressive deletions at the C‐terminal end of the E4 region. The five mAbs were found to bind to four distinct sites. By using these epitope‐defined mAbs, along with anti‐peptide antibodies raised against putative N‐ and C‐terminal E4 sequences, we have determined the relationships between the eight distinct polypeptides (mol. wt 10/11 kd, 16/17 kd, 21/23 kd and 32/34 kd) previously shown to be expressed from the E4 gene of HPV‐1 in productively infected papillomas. The 17 kd E4 polypeptide appears to be the product of a spliced mRNA encoding five amino acids from open reading frame (ORF) E1 joined onto 120 from the E4 ORF. The 16 kd and 10/11 kd proteins, which may be derived from this, lack sequences (approximately 15 and 70 amino acids respectively) encoded by the 5′ end of the E4 gene. The 32/34 kd proteins were detected by all antibodies which reacted with the 16/17 kd polypeptides, suggesting that they represent dimers of the latter species. The 21/23 kd polypeptides, however, do not appear to be simple dimers of the 10/11 kd protein as previously predicted, and reacted with antibodies whose epitopes mapped in the N‐terminal half of the E4 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV‐16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV‐16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6‐kb DNA fragment, containing the early genes of HPV‐16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV‐LTRs), cooperates with EJ‐ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV‐16 genome.

A cDNA clone for human p53 cellular tumor antigen has been isolated and characterized. This clone contains the complete 3′‐untranslated region and most of the open reading frame for the protein. Nucleotide sequence analysis revealed that p53 mRNA contains an Alu repeat in the 3′‐untranslated region. Hybridization selection experiments showed this clone was capable of selectively binding p53 mRNA. In vitro translation of SV80 mRNA resulted in the synthesis of two immunoreactive p53 polypeptide species. Northern blot analysis showed that human p53 mRNA was 2.8 kb in length and was present in cell lines containing high and low levels of p53 protein. There appears to be only a single p53 gene in human cells and Southern blot analysis demonstrated no major genomic rearrangements or amplification of the p53 gene in the transformed cell lines examined.

We have developed quantitative radioimmunological solid phase assays for the host protein p53 from mouse cells and from human cells. The first assay, for mouse p53, depends on having two monoclonal antibodies reacting with different determinants on the p53 molecule. With this assay we have shown that SV40‐transformed cells have approximately 100‐fold more p53 than untransformed mouse cells and that other transformed cells have intermediate levels. Embryonal carcinoma cell lines have approximately 50‐fold less p53 than SV40‐transformed cells. This is in contrast to the high levels of incorporation of [35S]methionine into p53 in these cells and indicates that metabolic labelling is not a valid approach for measuring p53 levels. The second assay, for human p53, required a different approach and made use of the anti‐p53 antibodies detected in the sera of some breast cancer patients. Human tumour cell lines contained amounts of p53 varying from the high level seen in SV40‐transformed human fibroblasts down to less than one hundredth of this amount. Normal human cells showed low levels of p53. The data confirm that many, but not all, human tumour cell lines contain more p53 than normal cells.