The oligonucleotide primers used in this study were as follows: humanTLP, 5-CCTCTTCCCACGGATGTGAT-3 (sense) and 5-GAGTCCAATGTGCAGCAGT-3 (reverse); mouseTLP, 5-GCCATTTGAACTTAAGGA-3 (forward) and 5-TGTAAATTCTGGCAA-3 (reverse); humanTAp63, 5-GTCCCAGAGCACACAGACAA-3 (forward) and 5-GAGGAGCCGTTCTGAATCTG-3 (reverse); chickenTAp63, 5-GAAACAGCCATGCCCAGTAT-3 (forward) and 5-CAAATGCGAGCTTCAAAACA-3 (reverse); humanTAp73, 5-CGGGACGGACGCCGATG-3 (forward) and 5-GAAGGTCGAAGTAGGTGCTGTCTGG-3 (reverse); humanp53, 5- ATTTGATGCTGTCCCCGGACGATATTGAAC-3 (forward) and 5-ACCCTTTTTGGACTTCCGGACGATATTGAAC-3 (reverse); human p21waf1, 5-GACACCACTGGAGGGTGACT-3 (forward) and 5-CCCTAGGCTGTGCTCACTTC-3 (reverse); human 14-3-3, 5-AGAGCGAAACCTGCTCTCAG-3 (forward) and 5-CTCCTTGATGAGGTGGCTGT-3 (reverse); humanLamin A/C, 5-CCGAGTCTGAAGAGGTGGTC-3 (forward) and 5-AGGTCACCCTCCTTCTTGGT-3 (reverse); humanBAX, 5-TCTGACGCAACTTCAACAC-3 (forward) and 5-GAGGAGTCTCACCCAACCAC-3 (reverse); humanPUMA, 5-GCCCAGACTGTGAATCCTGT-3 (forward) and 5-TCCTCCCTCTTCCGAGATTT-3 (reverse); humanNOXA, 5-GCAAGAATGGAAGACCCTTG-3 (forward) and 5-GTGCTGAGTTGGCACTGAAA-3 (reverse); humanGAPDH, 5-ACCTGACCTGCCGTCTAGAA-3 (forward) and 5-TCCACCACCCTGTTGCTGTA-3 (reverse). 487 to 29, where +1 represents the transcriptional initiation site ofTAp63, is required for TLP-dependent transcriptional activation ofTAp63and also TLP is usually efficiently recruited onto this region. Additionally, cells treated with anti-cancer drug etoposide underwent apoptosis in association with the transcriptional enhancement ofTAp63in a p53-impartial manner, and the knockdown of the endogenous TLP reduced etoposide-induced apoptosis through repression ofTAp63expression. Taken together, our present study identifies a TLP-TAp63 pathway that is further implicated in stress-induced apoptosis. == Introduction == Transcriptional regulation involves the functional integration of diverse factors and is a critical regulatory step for cellular events that include growth, differentiation, and death. These cellular activities often occur simultaneously due to the action of regulatory factors with broad targets. A representative example of such a factor is the tumor suppressor p53 and its family members, including p63 and p73, which contribute to tumor suppression, cell cycle checkpoint, DNA repair, and apoptosis (1). alpha-hederin p63 acts as a pro-apoptotic transcription factor (2,3) and, like p53 and p73, is usually expressed alpha-hederin as multiple isoforms (4). They include thetrans-activating (TA)3isoform of p63, termed alpha-hederin TAp63, and an NH2-terminal activation domain-deficient isoform, Np63, that acts as a dominant negative factor over p53, TAp63, and TAp73 (2). p63 has been clearly implicated in a variety of developmental processes (5), whereas its anticipated role as a tumor suppressor is usually unclear, mainly because of its low frequency of the somatic mutations in human tumors (6,7). However, a study focused on long term effects ofp63mutations in mice showed that mice bearing mutations in bothp63andp53develop a more aggressive tumor, indicating the presence of a tumor suppressive activity of p63 (8). This is consistent with earlier studies indicating that p63 is required for p53-dependent apoptotic response (9) and that, in response to certain DNA damage insults, p63 activates an overlapping set of p53-target genes implicated in cell cycle arrest and apoptosis (10). Although extensive studies of p63 in human tumors have suggested that deregulated expression of TAp63 and Np63 contributes to tumor development and progression (4), the precise molecular mechanisms behind the transcriptional regulation ofTAp63remain to be unclear. TATA-binding protein (TBP) is usually a general transcription factor that plays a central role in the regulation of pre-initiation complex formation by eukaryotic RNA polymerases (11,12). Eukaryotic cells also contain multiple TBP paralogs implicated in transcriptional regulation during cell growth, differentiation, and development (11,12). TBP-like protein (TLP) (13), also known as TBP-related factor 2 (14,15), TLF (16), or TRP (17), is one of the TBP paralogs common to Metazoa and has been implicated by genetic studies in various developmental processes, including spermiogenesis in mice (11,12). Although TLP fails to bind to TATA box (11,12), it stimulates transcription from several TATA-less promoters (18). Mammalian TLP, unlike TBP, does not associate with TAFs to form a transcription factor IID-type complex but, instead, associates with TFIIA in cells (14,19). It was reported earlier (20) that mammalian TLP activates transcription from the TATA-less neurofibromatosis type 1 (NF1) promoter through site-specific binding, but represses the TATA-containing c-fospromoter, thus leading to the prediction of an anti-oncogenic ability of TLP as well as the potential for direct binding to other target genes. Shimadaet al.(21) reported that chicken TLP Mouse monoclonal to KLHL11 represses the G2/M transition and, through its nuclear translocation, mediates apoptosis induction in a p53-impartial manner. Hence, TLP is usually proposed to have both checkpoint and anti-oncogenic functions, although its physiological role and also the precise molecular mechanisms behind TLP-mediated apoptosis as well as cell cycle checkpoint remain to be elusive. Here, we have analyzed mammalian TLP function in relation to TAp63 expression and show that TLP enhances the promoter activity ofTAp63and thus leads to apoptosis. alpha-hederin Further observations suggest that this novel TLP-TAp63 pathway increases the sensitivity to anti-cancer drug etoposide. == EXPERIMENTAL PROCEDURES == == == == == == Cell Culture and Transfection == Chicken DT40 cells were produced in RPMI 1640 medium (Invitrogen) as previously described (21). DT40-TLP/cells derived from parental DT40 cells lackTLP(21). Human cervical carcinoma-derived HeLa and human hepatocellular carcinoma-derived HepG2 cells were cultured in Dulbecco’s altered Eagle’s medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), penicillin (100 IU/ml), and streptomycin (100 g/ml). Human hepatocellular carcinoma-derived Hep3B cells were produced in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. HepG2 and HeLa cells carry wild-typep53. Hep3B and DT40 cells lackp53. Where indicated, cells were exposed to etoposide (at a final concentration.