Abstract
In common with other E2F1 responsive genes such as p14ARF 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 p73Δ2 was observed in a significant proportion of cancers, but not in normal epithelium. The frequent over-expression of p73Δ2, which has recognized transdominant properties, in malignant and pre-malignant lesions suggests a role in the oncogenic process in cervical epithelium.British Journal of Cancer (2002) 86, 263–268. DOI: 10.1038/sj/bjc/6600033 www.bjcancer.com© 2002 The Cancer Research Campaign
Highlights
Squamous cell carcinoma of the cervix is the second most common female cancer
In this work we present data suggesting that functional inactivation of pRb via interaction with HPV 16E7 results in p73 over-expression in transient assays and in cell lines expressing E7 from oncogenic HPV types
We extend these in vitro observations by demonstrating, at mRNA and protein levels, deregulated expression of p73 in a high proportion of cervical cancers and pre-malignant lesions
Summary
H1299 cells were maintained in DMEM with 10% foetal bovine serum. Primary human keratinocytes were purchased from Clonetics and grown according to the manufacturer’s instructions. In each case the diagnosis was checked by histopathological analysis, and a majority of neoplastic cells in the tissue for analysis was confirmed. Analysis of expression was performed by RT – PCR as described previously for p14ARF (Gazzeri et al, 1998), and p73 (De Laurenzi et al, 1998). For semi-quantitative analysis of gene expression, PCR was performed using the primers and thermal cycling conditions described and was for 22 cycles for p14ARF, and 28 cycles for p73. Analysis of N-terminal splice variants of p73 was performed using the primers described (Ng et al, 2000) Identity of these was confirmed by cloning and sequencing and by hybridization analysis of amplified products with oligonucleotide probes specific for exon 2 and exon 3 of p73.
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