Supplementary Figure 1 from The Human Orthologue of <i>Drosophila</i> Ecdysoneless Protein Interacts with p53 and Regulates Its Function

Supplementary Material, Methods and Figure Legend from The Human Orthologue of Drosophila Ecdysoneless Protein Interacts with p53 and Regulates Its Function

Calcineurin inhibitors [CI] provide effective immunosuppression after liver transplantation [LT], but is often complicated by nephrotoxicity. To evaluate the outcomes of LT patients converted from maintenance calcineurin inhibitor immunosuppression to a combination of mycophenolate mofetil [MMF] and sirolimus. Using patient database, medical records of patients with post LT renal dysfunction transitioned to MMF and sirolimus from CIs were reviewed. Baseline characteristics, immunosuppression, renal function, patient and graft survival, adverse events and tolerability were analyzed. Eleven LT patients were converted to MMF and sirolimus after a median duration of 60 [32–168] months on CI. Nine patients were on tacrolimus and two were on cyclosporine pre conversion. Post conversion median follow up on MMF and sirolimus was 18 months. Serum Cr improved from median of 1.85 mg/dl [SD 0.88] before conversion to 1.24 mg/dl [SD 0.15] at last follow up of 18 months [P ≤ .01]. GFR based on MDRD improved from 31.5 ml/min/1.73m2[SD 9.45] pre conversion to 53 ml/min/1.73m2 [SD 13.87] at last follow up [P ≤ .01]. One patient [9%] required hemodialysis after 16 months of MMF and sirolimus and one patient [9%] died because of complications of renal failure. There were no episodes of rejection. Median wbc count was 6600 /cu mm [SD 3.3] before and 5900/cu mm [SD 2.6] after conversion. Median hemoglobin value was 13.1 g/dl [SD 1.32] before and 13 g/dl at last follow up [SD 1.3]. Median LDL and triglyceride pre conversion were 193 mg/dl [SD 212.58] and 208 mg/dl [SD 359.97] while post conversion values were 118 mg/dl [SD 25.58] and 121.5 mg/dl [SD 22.58] respectively. Improvement in renal function was associated with conversion to MMF and sirolimus combination. No rejection was noted even in patients with prior history of rejection on CI. There was no significant anemia or leukopenia requiring blood transfusions or growth factors. One patient had significant hyperlipidemia needing treatment. There were no drug discontinuations. Combination MMF and sirolimus appears to be safe and effective in LT recipients with renal dysfunction. Larger studies are needed to substantiate these results.

Members of the evolutionarily conserved Mastermind (MAM) protein family, including the three related mammalian Mastermind-like (MAML) proteins MAML1-3, function as crucial coactivators of Notch-mediated transcriptional activation. Given the recent evidence of cross-talk between the p53 and Notch signal transduction pathways, we have investigated whether MAML1 may also be a transcriptional coactivator of p53. Indeed, we show here that MAML1 is able to interact with p53. We show that MAML1-p53 interaction involves the N-terminal region of MAML1 and the DNA-binding domain of p53, and we use a chromatin immunoprecipitation assay to show that MAML1 is part of the activator complex that binds to native p53-response elements within the promoter of the p53 target genes. Overexpression of wild-type MAML1 as well as a mutant, defective in Notch signaling, enhanced the p53-dependent gene induction in mammalian cells, whereas MAML1 knockdown reduced the p53-dependent gene expression. MAML1 increases the half-life of p53 protein and enhances its phosphorylation/acetylation upon DNA damage of cells. Finally, RNA interference-mediated knockdown of the single Caenorhabditis elegans MAML homolog, Lag-3, led to substantial abrogation of p53-mediated germ-cell apoptotic response to DNA damage and markedly reduced the expression of Ced-13 and Egl-1, downstream pro-apoptotic targets of the C. elegans p53 homolog Cep-1. Thus, we present evidence for a novel coactivator function of MAML1 for p53, independent of its function as a coactivator of Notch signaling pathway.

Abstract Biochemical mechanisms that control the levels and function of key tumor suppressor proteins are of great interest as their alterations can lead to oncogenic transformation. Here, we identify the human orthologue of Drosophila melanogaster ecdysoneless (hEcd) as a novel p53-interacting protein. Overexpression of hEcd increases the levels of p53 and enhances p53 target gene transcription whereas hEcd knockdown has the opposite effects on p53 levels and target gene expression. Furthermore, hEcd interacts with murine double minute-2 and stabilizes p53 by inhibiting murine double minute-2–mediated degradation of p53. Thus, hEcd protein represents a novel regulator of p53 stability and function. Our studies also represent the first demonstration of a biochemical function for hEcd protein and raise the possibility that altered hEcd levels and/or function may contribute to oncogenesis. (Cancer Res 2006; 66(14): 7167-75)

Our laboratory is involved in identifying genes that can be used as early diagnostic or prognostic markers in breast cancer. We previously identified a gene (NES1) that is expressed in normal but not in transformed mammary epithelial cells (MECs). NES1 is located on chromosome 19q13.4 within the kallikrein locus and thus was designated as human kallikrein 10 (hK10), although we have been unable to detect any protease activity. Importantly, hK10 expression is decreased in a majority of breast cancer cell lines. Transfection of hK10 into hK10-negative breast cancer cells reduces the tumorigenicity. Using methylation-specific PCR and subsequent sequencing, we demonstrate a strong correlation between hypermethylation of hK10 and loss of mRNA expression. Further analysis showed that essentially 100% of normal breast specimens had hK10 expression, whereas 46% of ductal carcinoma in situ (DCIS) and the majority of infiltrating ductal carcinoma (IDC) samples lacked the hK10 mRNA. Importantly, hK10-negative DCIS diagnosed at the time of biopsy were subsequently diagnosed as IDC at the time of definitive surgery. It has been shown that hK10 protein expression is regulated by steroids. In addition to breast cancers, hK10 is downregulated in cervical cancer, prostate cancer and acute lymphocytic leukemia, whereas it is upregulated in ovarian cancers. These results point to the paradoxical role of hK10 in human cancers and underscore the importance of further studies of this kallikrein.

Human kallikrein 10 (hK10) protein is expressed in normal breast but is significantly downregulated in a majority of invasive breast cancers. Thus, understanding how hK10 expression is regulated is of substantial significance. In this study, we analyzed the promoter region of hK10 using a website software (TRANSFAC 3.0), which predicted three possible retinoic acid response elements (RAREs), RARE1 at -1041 (TGACCTCGTGATCC), RARE2 at -859 (TGACCTCCTATGA) and RARE3 at -765 (TGACCTCCTGTGA), each with a half-site of a canonical sequence (TGACCT; reverse complement AGGTCA). Using electrophoretic mobility shift assays and nucleotide competition analysis, as well as chromatin immunoprecipitation of the native hK10 promoter, we demonstrated specific binding of RXR only to RARE1. The functional importance of RARE in the hK10 promoter was demonstrated by retinoid induction of hk10 promoter-reporters; furthermore, mutation of RARE1 but not of RARE2 or RARE3 abolished the induction of the reporter. Finally, we demonstrated the induction of hK10 mRNA and protein expression upon retinoid treatment of cells. In view of the correlation of the downregulation of hK10 mRNA and protein with breast cancer progression, these findings suggest a potential approach to restore hK10 expression in cancer patients.