TP63, an important epithelial developmental gene, has significant homology to p53. Unlike p53, the expression of p63 is regulated by two different promoters resulting in proteins with opposite functions: the full-length transcriptionally active TAp63 and the dominant-negative DNp63. We investigated the downstream mechanisms by which TAp63a elicits apoptosis. TAp63a directly transactivates the CD95 gene via the p53 binding site in the first intron resulting in upregulation of a functional CD95 death receptor. Stimulation and blocking experiments of the CD95, TNF-R and TRAIL-R death receptor systems revealed that TAp63a can trigger expression of each of these death receptors. Furthermore, our findings demonstrate a link between TAp63a and the mitochondrial apoptosis pathway. TAp63a upregulates expression of proapoptotic Bcl-2 family members like Bax and BCL2L11 and the expression of RAD9, DAP3 and APAF1. Of clinical relevance is the fact that TAp63a is induced by many chemotherapeutic drugs and that inhibiting TAp63 function leads to chemoresistance. Thus, beyond its importance in development and differentiation, we describe an important role for TAp63a in the induction of apoptosis and chemosensitivity.

Purpose: Tumor necrosis factor-related apoptosis^inducing ligand (TRAIL/Apo2L) exhibits potent antitumor activity on systemic administration in nonhuman primates without deleterious side effects for normal tissue. However, there is a controversy about the potential toxicity of TRAIL on human hepatocytes. The use of different recombinant TRAIL forms only partially explains the contradicting reports onTRAIL sensitivity in primary human hepatocytes (PHH). Experimental Design: To clarify this issue, we comprehensively tested four different recombinant forms of TRAIL for their apoptosis-inducing capacity on PHH obtained from a total of 55 human livers between day 1and day 8 of in vitro culture. Results: One day after single-cell isolation, all but one recombinant form of TRAIL [i.e., an untagged form of TRAIL (TRAIL.0)] induced apoptosis in PHH. Apoptosis induction byTRAIL in these cells could only be fully inhibited by concomitant blockade of TRAIL receptor 1and TRAIL receptor 2. At day 4 of in vitro culture, when surrogate markers indicated optimal hepatocyte in vitro function, only high doses of cross-linked FLAG-TRAIL killed PHH whereas the other three recombinant TRAIL forms did not. Strikingly, cotreatment of day 4 PHH with cisplatin sensitized for TRAIL-induced apoptosis whereas 5-fluorouracil, etoposide, gemcitabine, irinotecan, or oxaliplatin, which are commonly used in the treatment of gastrointestinal cancers, did not. Conclusion: Our data show that whereas TRAIL alone or together with selected chemotherapeutic drugs seems to be safe, the combination of TRAIL with cisplatin is toxic to PHH.

We investigated the mechanisms by which TAp73b and dominant-negative p73 (DNp73) regulate apoptosis. TAp73b transactivated the CD95 gene via the p53-binding site in the first intron. In addition, TAp73b induced expression of proapoptotic Bcl-2 family members and led to apoptosis via the mitochondrial pathway. Endogenous TAp73 was upregulated in response to DNA damage by chemotherapeutic drugs. On the contrary, DNp73 conferred resistance to chemotherapy. Inhibition of CD95 gene transactivation was one mechanism by which DNp73 functionally inactivated the tumor suppressor action of p53 and TAp73b. Concomitantly, DNp73 inhibited apoptosis emanating from mitochondria. Thus, DNp73 expression in tumors selects against both the death receptor and the mitochondrial apoptosis activity of TAp73b. The importance of these data is evidenced by our finding that upregulation of DNp73 in hepatocellular carcinoma patients correlates with reduced survival. Our data indicate that DNp73 is an important gene in hepatocarcinogenesis and a relevant prognostic factor.