The p53-induced Wig-1 protein : studies of interaction partners and expression in tumor cells

Sammanfattning: The tumor suppressor p53 is a critical regulator of life and death in cells. The p53 protein is present at very low levels in cells under normal conditions but accumulates in response to different stress stimuli, such as DNA damage, hypoxia, and oncogene activation. By acting on its targets p53 can lead the cell into various responses ranging from reversible cell cycle arrest to irreversible cell death or senescence. The Wig-1 gene (for wild type p53-induced gene 1) was first discovered as a p53-induced gene in J3D mouse T lymphoma cells carrying temperature-sensitive p53. The wig-1 gene encodes a zinc finger protein containing three Cys2His2-type zinc fingers and a nuclear localisation signal between the second and the third zinc finger. Human Wig-1 is mapped it to chromosome 3q26.3-27. Wig-1 is highly conserved between man, mouse, rat, chicken, frog and fish, particularly the zinc fingers, which are almost perfectly conserved even between man and fish. Gel shift assay revealed that human Wig-1 binds a 100 bp dsRNA probe with high affinity compared to ssRNA and DNA-RNA hybrids. We were able to immunoprecipitated dsRNA from Saos-2 cells expressing Tet-regulated Wig-1 using a dsRNA-specific J2 antibody, demonstrating that Wig-1 binds endogenous dsRNA in living cells. However, Wig-1 harboring mutations in either the first or second zinc finger were not able to bind dsRNA. Thus, both the first and the second zinc finger are necessary for binding in living cells. A colony formation assay show that the first and second zinc finger are important for Wig-1-mediated growth inhibition. Interestingly, Wig-1 binds a 22-mer dsRNA with siRNA-like features, but not a control probe with 5 overhangs. We generated a Saos-2 cell line expressing Flag-tagged human Wig-1 under the control of tetracycline, and showed that Wig-1 does not induce significant cell cycle arrest or apoptosis in these cells. Using these cells we also identified two Wig-1-binding proteins: RNA Helicase A (RHA) and hnRNP A2/B1, both of which are involved in RNA processing at different levels. RNase treatment of the cell extracts abolished the binding between Wig-1, RHA and hnRNP A2/B1, demonstrating that these interactions are dependent on RNA. Wig-1 harbouring mutations in the first or second zinc finger did not immunoprecipitate RHA or hnRNP A2/B1, confirming that the interaction occurs via RNA. Finally, knockdown of Wig-1, hnRNP A2/B1 or both of these simultaneously had similar growth inhibitory effect on cells in a WST-1 proliferation assay, suggesting that they are involved in the same pathway. Gain within the chromosomal region 3q, where Wig-1 is located, is a common feature of cervical carcinoma, and is also linked to the transition from an in situ tumor to invasive carcinoma. To address wether a 3q gain in cervical cancers involves the Wig-1 gene at 3q26, we evaluated eight established cervical cancer cell lines. We could show that Wig-1 is not a main target for the frequent gains and amplifications in 3q seen in cervical cancer cells. However, Wig-1 mRNA and protein levels were found to be relatively lower in HPV positive cervical carcinoma cells independently of p53 protein levels. This suggests that Wig-1 expression at lest in part independent of p53, and raise the interesting possibility that HPV somehow influences Wig-1 expression, directly or indirectly. In conclusion, this thesis provides important clues to the cellular function of Wig-1, by demonstrating dsRNA binding in cells, identification of the protein partners RHA and hnRNP A2/B1 and revealing a possible correlation between Wig-1 expression and HPV in cervical carcinoma.