Daxx represses expression of a subset of antiapoptotic genes regulated by nuclear factor-kappaB.

Daxx is a nuclear protein that localizes to PML oncogenic domains, sensitizes cells to apoptosis, and functions as a transcriptional repressor. We found that Daxx represses the expression of several antiapoptotic genes regulated by nuclear factor-kappaB, including cIAP2, in human tumor cell lines. Daxx interacts with RelB and inhibits RelB-mediated transcriptional activation of the human cIAP2 gene promoter. Daxx also forms complexes with RelB while bound to its target sites in the cIAP2 promoter, as shown by electrophoretic mobility shift assays and chromatin immunoprecipitation experiments. Using cells from daxx-/- mouse embryos, we observed that levels of the corresponding murine c-IAP mRNA and protein are increased in cells lacking Daxx. Conversely, c-IAP mRNA and protein levels were reduced in relB-/- cells. Taken together, these observations provide a mechanism that links two previously ascribed functions of Daxx: transcriptional repression and sensitization to apoptosis.

Synthetic triterpenoids cooperate with tumor necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.

Differences in DNA binding properties between E2F1 and E2F4 specify repression of the Mcl-1 promoter.

E2F1 is a potent inducer of apoptosis whereas its relative, E2F4, generally does not promote cell death. Other work from our laboratory has demonstrated that E2F1 can directly bind and represss the Mcl-1 promoter - contributing to E2F1-mediated apoptosis. Here we show that while E2F1 can repress the Mcl-1 promoter, other members of the E2F family (such as E2F4) cannot. Characterization of the Mcl-1 promoter demonstrates that the -143/+10 region is critical for E2F1-mediated downregulation. We demonstrate that the ability of E2F1 to repress the Mcl-1 promoter correlates with its ability to bind within the required -143/+10 region of this promoter. In contrast, E2F4 is unable to bind to the -143/+10 region of the Mcl-1 promoter. We propose that E2F4 is unable to repress the Mcl-1 promoter primarily as a result of insufficient binding to the essential regulatory region. This is the first evidence of DNA binding specificity among E2F family members that results in differential regulation of a naturally occurring promoter.

Direct repression of the Mcl-1 promoter by E2F1.

E2F1 induces apoptosis via both p53-dependent and p53-independent mechanisms. The direct targets in the p53-independent pathway remain enigmatic; however, the induction of this pathway does not require the transactivation domain of E2F1. Using cells that are defective in p53 activation, we show that E2F1 potently represses the expression of Mcl-1--an anti-apoptotic Bcl-2 family member whose depletion results in apoptosis. We also show that this transcriptional repression is direct and dependent upon E2F1's DNA-binding domain, but does not require the transactivation domain of E2F1. Consistent with this DNA binding requirement of E2F1 to repress Mcl-1, we show that E2F1 binds to the Mcl-1 promoter both in vitro and in vivo, and have identified the DNA element (-143/-117) within this promoter that is required for E2F1 binding and repression. Additionally, cell lines constitutively expressing Mcl-1 are resistant to E2F1-mediated apoptosis--suggesting that Mcl-1 downregulation is a necessary event in the p53-independent apoptotic process. Thus, we identify a p53 family-independent mechanism of E2F1-induced apoptosis in which E2F1 directly represses Mcl-1 expression.

Cytoprotective gene bi-1 is required for intrinsic protection from endoplasmic reticulum stress and ischemia-reperfusion injury.

Ischemia-reperfusion (IR) injury induces endoplasmic reticulum (ER) stress and cell death. Bax Inhibitor-1 (BI-1) is an evolutionarily conserved ER protein that suppresses cell death and that is abundantly expressed in both liver and kidney. We explored the role of BI-1 in protection from ER stress and IR injury by using bi-1 knockout mice, employing models of transient hepatic or renal artery occlusion. Compared to wild-type bi-1 mice, bi-1 knockout mice subjected to hepatic IR injury exhibited these characteristics: (i) increased histological injury; (ii) increased serum transaminases, indicative of more hepatocyte death; (iii) increased percentages of TUNEL-positive hepatocytes; (iv) greater elevations in caspase activity; and (v) more activation of ER stress proteins inositol-requiring enzyme 1 and activating transcription factor 6 and greater increases in expression of ER stress proteins C/EBP homologous protein and spliced XBP-1 protein. Moreover, hepatic IR injury induced elevations in bi-1 mRNA in wild-type liver, suggesting a need for bi-1 gene induction to limit tissue injury. Similar sensitization of kidney to ER stress and IR injury was observed in bi-1(-/-) mice. We conclude that bi-1 provides endogenous protection of liver and kidney from ER stress and IR injury. Analysis of components of the bi-1-dependent pathway for protection from IR injury may therefore reveal new strategies for organ preservation.

Identification of novel E2F1-regulated genes by microarray.

The E2F pathway has been proposed to regulate genes involved in the transition from quiescence into DNA synthesis. However, this hypothesis has not been rigorously tested on a genomic scale. Toward this end, we have infected quiescent mouse fibroblasts, which do not express E2F1, with an E2F1-expressing adenovirus and examined the expression of more than 6000 genes using high-density microarrays. Microarray results clearly support the current paradigm; however, they suggest that E2F1 may also regulate unanticipated cellular functions including pathways involved in apoptosis, signal transduction, transcriptional control, and membrane biology. Most surprisingly, we identified a number of genes that are repressed by E2F1 expression, suggesting that E2F1 may have the potential to repress transcription of numerous genes through an unknown mechanism.