E7 proteins from oncogenic human papillomavirus types transactivate p73: role in cervical intraepithelial neoplasia

Abstract

In common with other E2F1 responsive genes such as p14(ARF) and B-myb, the promoter of p73 is shown to be positively regulated in cell lines and primary human keratinocytes by E7 proteins from oncogenic human papillomavirus (HPV) types 16, 18, 31 and 33, but not HPV 6. Mutational analysis revealed that transactivation of the p73 promoter by HPV 16E7 requires association with pRb. Expression of p73 in normal cervical epithelium is confined to the basal and supra-basal layers. In contrast, expression in neoplastic lesions is detected throughout the epithelium and increases with grade of neoplasia, being maximal in squamous cell cancers (SCC). Deregulation of expression of the N-terminal splice variant p73Delta2 was observed in a significant proportion of cancers, but not in normal epithelium. The frequent over-expression of p73Delta2, which has recognized transdominant properties, in malignant and pre-malignant lesions suggests a role in the oncogenic process in cervical epithelium.

Figure 1

The p73 promoter is activated by HPV E7 proteins in H1299 cells. (A) Cells were transfected with p73 luciferase, and the indicated amounts of either pcDNA E2F1 or pcDNA3 16E7. Luciferase activity was determined after 48 h as described in Materials and Methods. Data shown are means of luciferase activity relative to cells receiving only empty vector ±s.d. of at least eight experiments. (B) Activation of the p73, p14^ARF and B-myb promoters by HPV 16E7. H1299 cells were transfected with the indicated luciferase reporter plasmids, and the indicated, increasing amounts of pcDNA3 16E7. Luciferase activity was determined after 48 h as described in Materials and Methods. Data shown are means of relative luciferase activity ±s.d. of at least six experiments. (C) Activation of the p73 promoter by E7 proteins from ‘oncogenic’ HPV types. Cells were transfected with p73 luciferase and pcDNA E7 plasmids from the indicated HPV types or empty vector alone (V). Luciferase activity was determined after 48 h as described in Materials and Methods. Data shown are means of relative luciferase activity ±s.d. of at least six experiments. (D) Activation of p73 is abrogated by mutants lacking binding to pRb. Cells were transfected with p73 luciferease and the indicated HPV E7 expression plasmids or empty vector only (V). Luciferase activity was determined after 48 h as described in Materials and Methods. Data shown are means ±s.d. of at least six experiments

Figure 2

Expression of p73 is activated by HPV 16E7 in human keratinocytes. (A) Primary human keratinocytes were transfected with p73 luciferease and the indicated pcDNA HPV E7 expression plasmids. Luciferase activity was determined after 48 h as described in Materials and Methods. Data shown are means±s.d. of at least three experiments. (B) Steady-state levels of p14^ARF and 73 mRNA in keratinocyte cell lines expressing HPV 16 E7. Lanes are: 1⁰=primary keratinocytes, HK1 and HK2=keratinocyte cell lines immortalized by HPV16 E6 and E7. RT–PCR and hybridization was performed as described in Materials and Methods.

Figure 3

Expression of p14^ARFand p73 in cervical epithelium. RT–PCR was performed as described in Materials and Methods. (A) p73 is over-expressed in HPV 16 positive cervical SCC. A single variant of p73 (p73α) is predominantly expressed in both normal (N) and malignant (T) cervical epithelium. The presence of equivalent amounts of cDNA in each sample was verified by amplification of β-actin as shown. (B) Cervical SCC express both full-length and Δ2 forms of p73. RT–PCR for p73Δ2 was performed as described in Materials and Methods. Amplification products were resolved on 2% agarose gels, then hybridized with oligonucleotide probes derived from exon 2 (upper blot) or exon 3 (lower blot). Hybridization with the probe from exon 2 detects only the full-length transcript, whereas the probe derived from exon 3 detects both the full-length and Δ2 forms of p73. In all cases N=normal tissue, T=tumour. Lanes N6 to T9 are four matched cases of normal and tumour. Lanes 10–13 are four examples of cervical SCC, shown without matched normal tissue. The presence of equivalent amounts of cDNA in each reaction was confirmed by amplification of β-actin as shown in the lower panel.

Figure 4

Immunocytochemical analysis of p73 and p14^ARF in cervical epithelium. Sections were prepared from formalin-fixed, parrafin- embedded tissue samples as described in Materials and Methods. (A) Expression of p73 is in the basal layer of normal cervical epithelium. RT–PCR analysis revealed that this was full-length, p73α in all cases studied. (B) p73 protein is over-expressed in cervical SCC. RT–PCR analysis revealed expression of both full-length and p73 Δ2 in the majority of cases. (C) p14^ARF protein expression is not detectable in normal cervical epithelium. (D) Over-expression of p14^ARF in cervical SCC. Note the prominent nucleolar expression and sparing of normal tissue.