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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.

Porcine aortic valves can be almost completely acellularized by a non-tanning detergent extraction procedure. The xenogenic matrix was reseeded with human endothelial cells. This approach may eventually lead to the engineering of tissue heart valves repopulated with the patients own autologous cells.

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.

Inhibition of cyclooxygenase (COX)-2 elicits chemopreventive and therapeutic effects in solid tumors that are coupled with the induction of apoptosis in tumor cells. We investigated the mechanisms by which COX-2 inhibition induces apoptosis in hepatocellular carcinoma (HCC) cells. COX-2 inhibition triggered expression of the CD95, tumor necrosis factor (TNF)-R, and TNF-related apoptosis-inducing ligand (TRAIL)-R1 and TRAIL-R2 death receptors. Addition of the respective specific ligands further increased apoptosis, indicating that COX-2 inhibition induced the expression of functional death receptors. Overexpression of a dominant-negative Fas-associated death domain mutant reduced COX-2 inhibitor-mediated apoptosis. Furthermore, our findings showed a link between COX-2 inhibition and the mitochondrial apoptosis pathway. COX-2 inhibition led to a rapid down-regulation of myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, followed by translocation of Bax to mitochondria and cytochrome c release from mitochondria. Consequently, overexpression of Mcl-1 led to inhibition of COX-2 inhibitormediated apoptosis. Furthermore, blocking endogenous Mcl-1 function using a small-interfering RNA approach enhanced COX-2 inhibitor-mediated apoptosis. It is of clinical importance that celecoxib acted synergistically with chemotherapeutic drugs in the induction of apoptosis in HCC cells. The clinical relevance of these results is further substantiated by the finding that COX-2 inhibitors did not sensitize primary human hepatocytes toward chemotherapy-induced apoptosis. In conclusion, COX-2 inhibition engages different apoptosis pathways in HCC cells stimulating death receptor signaling, activation of caspases, and apoptosis originating from mitochondria. (Cancer Res 2006; 66(14): 7059-66)

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We investigated the downstream mechanisms by which chemotherapeutic drugs elicit apoptosis in hepatocellular carcinoma (HCC). Genomic signatures of HCC cell lines treated with different chemotherapeutic drugs were obtained. Analyses of apoptosis pathways were performed and RNA interference was used to evaluate the role of the p53 family. Endogenous p53, p63 and p73 were upregulated in response to DNA damage by chemotherapeutic drugs. Blocking p53 family function led to chemoresistance in HCC. Stimulation and blocking experiments of the CD95-, the TNF-and the TRAIL-receptor systems revealed that cytotoxic drugs, via the p53 family members as transactivators, can trigger expression of each of these death receptors and consequently sensitize HCC cells toward apoptosis. Furthermore, our findings demonstrate a link between chemotherapy, the p53 family and the mitochondrial apoptosis pathway in HCC. Chemotherapeutic treatment induces expression of proapoptotic Bcl-2 family members like Bax and BCL2L11 and the expression of Apaf1, BNIP1, Pdcd8 and RAD. Thus, upon DNA damage, p53, p63 and p73 promote apoptosis via the extrinsic and the intrinsic signaling pathway. In addition, not only proapoptotic genes were upregulated, but also genes known to exert antiapoptotic functions. Bleomycininduced upregulation of BCL-XL/BCLXL1 and MDM2 suggests that it is the ratio of proapoptotic and antiapoptotic proteins that regulates the apoptosis response of HCC cells toward chemotherapy, thereby playing a decisive role between treatment sensitivity vs. drug resistance. The clinical importance of these data is evidenced by our finding that the bleomycin target gene signature can predict the prognosis of patients suffering from HCC.Inactivation of tumor suppressor genes or activation of protooncogenes can lead to clonal outgrowth and tumor progression. These oncogenic events evolve as important determinants in the response of human tumors to commonly used DNA damaging agents. 1 Many anticancer agents induce DNA damage as part of their mechanism of tumor cytotoxicity. DNA damage activates p53, which in turn induces the expression of proteins that halt the cell-division cycle to allow for DNA repair. 2,3 Activation of p53 can also initiate programs of cell death (apoptosis) or permanent growth arrest (senescence) if the DNA damage is severe. [4][5][6][7][8][9][10][11][12][13] Furthermore, the recent identification and characterization of the p53/p63/p73 network provides evidence of a tight link between developmental processes and tumorigenesis. 14-16 p63 and p73 are not only important for normal development and differentiation, but are also implicated in tumorigenesis and the response to chemo-or radiotherapies. [17][18][19][20] The p53 family genes, p53, p63 and p73, produce multiple isoforms that vary in composition of the NH 2 -and C-termini. Isoforms of the p53 family can interact with each other and form a complicated network. The dominant-negative (DN) isoforms can oppose the transactivation capabilities of the full length (TA) prot...