RESUMO
Previously, we reported that pterygial epithelial cells show positive p53 staining by immunohistochemistry, and that they do not demonstrate apoptosis. We wished to determine whether the accumulation of p53 protein was caused by missense mutations in exons 5-8 of the TP53 gene, as is frequently the case in malignant tumours that contain high levels of abnormal p53. From 11 pterygia, epithelial cells were isolated by laser capture microdissection, or manually, in order to reduce the contribution of TP53 from normal cells. DNA from pterygial epithelial cells was amplified across exons 5-8 in 10 pterygia and across exons 5,7 and 8 in another pterygium. In 2 pterygia, all translated exons (2-11) were sequenced. No mutations were found, although normal polymorphisms in codon 72 were readily detected in 2 pterygia. RT-PCR was used to compare amounts of TP53 mRNA isolated from normal conjunctiva and pterygia from eight additional patients. We detected an approximate two-fold increase of TP53 RNA in pterygia compared to that in normal conjunctiva. Western blotting was used to compare amounts of p53 protein in pterygia and normal conjunctiva. Consistent with our previous immunohistochemical studies, amounts of p53 protein in pterygia, detected by the western blotting, were elevated compared to those detected in normal conjunctiva and corneal limbal epithelium. However, the TP53 gene in pterygia is not mutated, and therefore, the elevated levels of p53 protein must result from a different mechanism than that seen in malignant tumours containing TP53 missense mutations. The increased amount of p53 protein in pterygial cells does not cause apoptosis or block cell proliferation, suggesting that these normal p53 functions are inactivated in pterygia.
