Expression of p53 isoforms in squamous cell carcinoma of the head and neck

Abstract

Recent data indicate that, similar to p63 and p73, several different p53 isoforms can be produced in humans through alternative initiation of translation, usage of an internal promoter and alternative splicing. These isoforms are reported to have varying functions and expressions. In squamous cell carcinoma of the head and neck (SCCHN), disruption of the p53 pathway is one of the most common genetic alterations. However, to our knowledge, no studies regarding the expression of different p53 isoforms in SCCHN have so far been performed. We screened for the expression of different p53 isoforms in SCCHN and clinically normal oral epithelia using nested RT-PCR. p53 mRNA was expressed in all tumours, all matched clinically normal tissue adjacent to the tumour and in buccal mucosa from healthy volunteers. Of the novel isoforms, p53beta was detected in the majority of samples analysed, and all of the recently described isoforms were also detected in at least some tumour and normal epithelium samples, with the exception of Deltap53 isoforms. We conclude that p53 variant mRNAs are expressed in both normal oral stratified epithelium and SCCHN. Improvements in methodologies and reagents to detect and quantify p53 isoform expression in clinical material will be required to correlate p53 status with clinical outcomes.

Fig. 1

Structure of theoretically encoded p53 proteins. For detection of RNA of the different p53 isoforms, nested PCR was used. The primers used in the first round of PCR are illustrated with black arrows and the primers used for the second round of PCR are illustrated with grey arrows.

Fig. 2

Expression of p53 isoforms in tumour tissue (T), clinically normal tumour adjacent tissue (N) in patients with squamous cell carcinoma of the head and neck, and healthy non-smoking controls (NS) detected by nested RT-PCR. (a) Detection of p53, size of the amplified fragment was - 1250 bp. (b) Detection of p53β, size of the fragment was - 1050 bp. (c) Actin is shown as control. X indicates samples showing variable results in duplicate analysis that could not be classified as either positive or negative. M indicates molecular weight marker. p53, p53β and actin are amplified from the same cDNA samples, T5 has lower actin in this run but in the duplicate all samples have the same actin level.

Fig. 3

Summary of RT-PCR results. Bars show percent of positive (black bars) and negative (white bars) samples. In total 45 samples were analysed, 21 tumours (T), 16 clinically normal tumour adjacent tissue (N) and eight normal control samples (non-smokers) (NS). (a) Results from nested RT-PCR. (b) Results from PCR using only the inner primer pair.

Fig. 4

Detection of in vitro translated p53 isoforms and p53 protein in FaDu cell line expressing p53. (a) DO-12 antibody recognising all nine isoforms, p53, p53β, p53γ, Δ40p53 (lower band in lane p53), Δ40p53β (lower band in lane p53β), Δ40p53γ (lower band in lane p53γ), Δ133p53, Δ133p53β and Δ133p53γ. (b) DO-1 antibody, recognising p53, p53β and p53γ. (c) 1801 antibody recognises p53 and Δ40p53 isoforms. (d) Polyclonal Sapu serum recognising all p53 isoforms, although with different sensitivities.

Fig. 5

Western blot of 14 pairs of tumours (T) and clinically normal tumour adjacent tissue (N) from patients with squamous cell carcinoma of the head and neck. Detection of p53 using the DO-1 antibody. First three lanes show HeLa cell extract at different concentrations (10, 30 and 50 μg/μl) as control. The following lanes show the 14 pairs of tumours and clinically normal tumour adjacent tissue.