Relative expression of TAp73 and ΔNp73 isoforms

Abstract

The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative ΔNp73 isoform. Interestingly, we detected higher levels of ΔNp73 in some mouse tissues, suggesting that ΔNp73 may have a physiological role in these tissues.

Figure 1. Expression of p73 isoforms

(A) Total RNA was isolated from different cell lines as described before [5] and TAp73 and ΔNp73 expression were evaluated by real-time PCR with the TAp73 and ΔNp73 specific primers. (B-C) Validation of p73 antibody using either specific siRNA against p73 or induction of its expression by etoposide. Endogenous p73 was silenced in indicated cell lines and 50 ug protein was used to detect p73. Specificity of the antibody was also verified by detecting up-regulated TAp73 in H1299 cells following treatment with 20-50 uM etoposide for 24 h. (D) Western blot analysis of p73 isoforms in different cell lines. 50 ug protein was used to detect endogenous p73 protein. (E) Total RNA was isolated from different tissues and TAp73 and ΔNp73 expression were evaluated by real-time PCR as in panel A.