[Abstract unavailable

No abstract

The sphingomyelin pathway, which is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide, signals apoptosis for tumor necrosis factor alpha, Fas, and ionizing radiation. In the present studies, the anticancer drug daunorubicin also stimulated ceramide elevation and apoptosis in P388 and U937 cells. Cell-permeable analogs of ceramide, but not other lipid second messengers, mimicked daunorubicin in inducing apoptosis. Daunorubicin-stimulated ceramide elevation, however, did not result from sphingomyelin hydrolysis, but rather from de novo synthesis via activation of the enzyme ceramide synthase. An obligatory role for ceramide synthase was defined, since its natural specific inhibitor, fumonisin B1, blocked daunorubicin-induced ceramide elevation and apoptosis. These studies demonstrate that ceramide synthase activity can be regulated in eukaryotes and constitute definitive evidence for a requirement for ceramide elevation in the induction of apoptosis.

No abstract

Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoproteinencoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level of MDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10-to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1 promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.Transcriptional control is mediated by a hierarchy of regulatory components. Although the interplay between DNA elements and transcription factors occurs within the presence of a complex chromosomal architecture, the contribution of chromatin to transcriptional regulation is not fully understood. However, a heightened interest in this area has been spurred by the recent cloning of the histone-modifying enzymes histone acetyltransferases (HATs) and histone deacetylases (HDACs) (5,29,39,45), enzymes with opposing effects on chromatin organization. HATs specifically catalyze the acetylation of the ε-amino group of lysine residues at the N-terminal domain of histones, weakening histone-DNA interactions and leading to a destabilization of nucleosome structure (open chromatin), while HDACs remove the acetyl group, leading to a more closed chromatin configuration. It has been proposed that this restructuring of chromatin regulates accessibility of transcription factors to their DNA targets, whereby open chromatin allows for factor binding and closed chromatin does not (43,44). While this is likely to be an oversimplified model for the role that these enzymes play in transcriptional regulation,

No abstract

The tumor suppressor protein p53 has been well documented as a transcriptional activator involved in the regulation of a number of critical genes involved in the cell cycle, response to DNA damage, and apoptosis. Activation by p53 requires the interaction of the protein with a consensus binding site consisting of two halfsites, each comprising two copies of the sequence PuPuPuC(A/T) arranged head-to-head and separated by 0 -13 base pairs. In addition to activation, p53 has been shown to be a potent repressor of transcription. However, the basis for p53-mediated repression is not well understood and has been proposed to occur indirectly through interactions with other promoter-bound transcription factors. In the present study, we show that p53 can repress transcription directly by binding to a novel head-to-tail (HT) site within the MDR1 promoter. A mutation that disrupted p53 binding to the MDR1 HT site blocked p53-mediated repression of the MDR1 promoter in transfection assays. Replacement of the HT site with a head-to-head (HH) site converted the activity of p53 from repression to activation, indicating that simple recruitment of p53 to the promoter is not sufficient for repression and that the orientation of the binding element determines the fate of p53-regulated promoters.The tumor suppressor protein p53 is mutated in over half of all human cancers, implicating the loss of this inducible phosphoprotein in the destabilization of the genome and the malignant transformation that follows (1). It is clear that a fundamental mechanism by which p53 regulates cell growth and death decisions is through its role as a transcriptional activator. However, p53 can also repress the transcription of a number of genes including those involved in regulatory cascades mediating cell proliferation and tumorigenesis (2). Indeed, analysis of transactivation-competent p53 mutant proteins that have lost repressor activity suggests that transcriptional repression by p53 plays a critical role in the execution of the apoptotic program (3).p53 activates transcription by binding DNA in a sequencespecific manner through a highly conserved DNA-binding domain. The consensus p53 binding site consists of two half-sites, each comprising two copies of the sequence PuPuPuC(A/T) arranged head-to-head (HH) 1 and separated by 0 -13 nucleotides (4). Each half-site binds a dimer of p53, resulting in the formation of a functional p53 tetramer-DNA activator complex. The majority of p53 mutations found in human tumors occur within the DNA binding domain, emphasizing the importance of DNA-protein interactions in p53 function (5).In contrast to the wealth of information with respect to p53 as an activator, the mechanism by which repression occurs is relatively unknown. This is due in large part to the lack of a p53 consensus binding site within the vast majority of repressed promoters (1). Genes repressed by p53 fall into two general categories. In the first class of promoters, repression is mediated through upstream activators. In almost all of these ca...

Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A >500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.Sorcin was initially identified as a 22-kDa protein in cultured cells selected for resistance to natural product cancer drugs, such as vincristine, adriamycin, and actinomycin D, i.e. multidrug-resistant cells (1-5). One of the major mechanisms of resistance in these cells is mediated by overexpression of the membrane-bound drug transporter, P-glycoprotein (6). Molecular cloning studies demonstrated that the sorcin and P-glycoprotein genes are tightly linked and that both may be amplified during the acquisition of multidrug resistance. However, while P-glycoprotein overexpression correlates with resistance development, increased sorcin expression is not obligatory, and its abundance does not correlate with the degree of resistance (4 -9). Complementary DNA for sorcin has been isolated from hamster (2) and human (10) multidrug-resistant cells, which amplify the sorcin gene. The highly conserved sequence, with 95% homology between hamster and human sorcin, predicts a 22-kDa protein with four putative Ca 2ϩ