p63 – from expression to function studies of normal oral mucosa and squamous cell carcinoma of the head and neck
Abstract: The human p63 gene discovered in 1997 encodes a series of protein isoforms that differ in their N- and/or C-terminal sequences. These isoforms have widely differing properties in promoting or repressing p53-related functions such as growth arrest and apoptosis. In addition, p63 appears to play important roles in the maintenance and differentiation of epithelial cell populations. Many studies have shown that p63, particularly ?Np63?, is expressed in normal epithelium and also highly expressed in squamous cell carcinomas of surface epithelia.Methods. We have refined the analysis of the expression patterns of p63 isoforms by the use of a quantitative RT-PCR technique applied to micro-dissected normal oral mucosal samples. We have also studied p63 expression in squamous cell carcinoma of the head and neck (SCCHN) compared to normal oral mucosa taken from the same patient. Furthermore, tobacco-exposed buccal mucosa was compared to age and gender matched non exposed controls. RT-PCR for telomerase and immunohistochemical analysis for detection of p53 and Ki-67 proteins was further performed.We also explored the function of p63 in SCCHN cells by using small interfering RNA (siRNA) to silence the expression of different p63 isoforms in cell lines originating from SCCHN. The effect of p63 knockdown was studied using a Fluorescein Diacetate based cytotoxicity assay and immunohistochemistry looking at expression of differentiation markers. The response of the siRNA treated cells to radiation and cytostatic drug was also investigated.We have further studied normal oral wound healing using immunohistochemistry and quantitative PCR.By the use of a macro array comparing siRNA treated cells with non-treated cells a possible connection between the BRCA1 gene and p63 expression was shown and further studied with the use of cHIP and luciferase reporter assays.Results. The p63 isoforms are expressed in normal epithelium, with the highest levels consistently found in basal and parabasal layers. Extensive use of tobacco had no effect on p63. The quantitative PCR showed statistically increased levels of the ?Np63 and p63??isoforms. No correlation was found between p63-isoform expression patterns and proliferation.The exploration of the function of p63 in SCCHN cells by the use of small interfering RNA (siRNA) showed a statistically significant decreased survival for tumour cells when all p63 isoforms, the N-terminal truncated or the ? isoforms were inhibited. No effect on cell proliferation or expression of epithelial differentiation markers was observed. We also demonstrated that inhibition of p63 expression sensitizes cells to the effects of ionizing radiation and cisplatin.The study of normal oral wound healing using immunohistochemistry and quantitative PCR showed significant changes in p63 isoform expression. The ?Np63 isoform was mainly expressed in the basal layer in the non proliferating and migrating cells while TAp63 was almost absent.The BRCA1 study showed p63 to bind to the BRCA1 promoter and activate the expression of BRCA1 protein.Summary. The p63 proteins have different functions and the balance between the isoforms and their localisation within the epithelium seems to be important for normal wound healing as well as cancer cell survival.
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