Differential Expression of TAp63 Gene in Normal and Tumor Cell Lines

Introduction

P63 gene, a member of p53 superfamily, located at 3q27. As a homologue of P53, P63 has 6 isoforms (Kaghad et al., 1997; Yang et al., 1998; Benard et al., 2003). The transactivation (TA) isoforms consist of 3 domains: an acidic transactivation N-terminal domain, a central DNA binding domain and a C-terminal oligomerization domain (Figure 1). The DNA binding domains of P63 and P53 share over 60% identical primary amino acid sequences. And they share 25% identity in TA domain. Some P53 target genes including Bax, MDM2 and p21 can be transactivated by P63 TA isoforms (Jost et al., 1997; Yang and Kaghad, 2002).

The ¦¤N isoforms are produced from an intronic promoter, contain the same DNA binding and D-terminal oligomerization domains as the TA isoforms but lack a transactivation domain. Without the transactivation activity, ¦¤N isoforms function as a dominant-negative manner, inhibit TAp63 and other p53 family members. Different splicing in C-terminal of both TA and ¦¤N isoforms yields TAp63¦Á,-¦Â,-¦Ã and ¦¤Np63¦Á,-¦Â,-¦Ã 6 isoforms, whose detailed functions are not well understood yet. (van Bokhoven and Brunner, 2002)

P53 tumor suppressor gene properties is due to its DNA binding and transactivation of target genes which specify cell cycle and apoptosis. TAp63 protein, when over-expressed in human cells, also binds to p53 target gene, and induces cell cycle arrest, differentiation and apoptosis in a p53-like manner. (Jacobs et al., 2005; Mills, 2006)And ¦¤Np63 isoforms, unlike TAp63 isoforms which acts as tumor suppressors, act as oncogenes.( Lee, et al., 2006)

In this study TAp63 expression is tested in normal and tumor cell lines by RT-PCR using isoform-specific primers.

Figures

Figure 1-Figure1 Structure of P63 isoforms. TAp63 isoforms and ¦¤Np63 isoforms uses different transcription initiation sites (arrows) and alternative splicing, to generate different mRNAs. Several protein domains can be distinguished; of these, the TA domains(Transactivation Domain), the DBD(DNA Binding Domain), and the ISO domain (Tetramerization Domain) are highly homologous to the corresponding domains in p53.(van Bokhoven and Brunner, 2002)

Figure 2-Figure2 RT-PCR analysis of TAp63 expression. The mRNA level of TAp63 in two cell lines, Hela and Wi38, were tested by RT-PCR in this experiment using transactivation isoform specific p63 primers. GAPDH was used as the control. Compared to Wi38, Hela have a higher TAp63 expression.

Figure 3-Figure 3 Sequence alignment of the RT-PCR product in reference with the p63 NCBI reference sequence. The DNA sequence of the RT-PCR product, which has greater than 99% homology with part of TAp63 gene (AF075432), was determined by dideoxy method of sequencing.

The mRNA level of TAp63 in two cell lines, Hela and Wi38, were tested by RT-PCR in this experiment using transactivation isoform specific p63 primers. GAPDH was used as the control (Figure 2). The mRNA level of TAp63 in Hela was higher than in Wi38, suggesting more expression of this gene in Hela.

RT-PCR product was confirmed to be part of TAp63 by sequencing (Figure 3). The DNA sequence of the RT-PCR product was determined by dideoxy method of sequencing. Analyses in Genbank revealed that the 349bp product has greater than 99% homology with part of TAp63 gene (AF075432).

Full Paper

Acknowledgments

I would like to thank to thank Jinsong Qiu and Lisa Sarran for helping me finish this project, thank all my classmates and thank Dr. Rubin for his guidance and providing the opportunity and his lab to do this project.

This document was last modified 05/15/2006.
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