ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.ATF5 (activating transcription factor 5; also known as ATFx) is a member of the ATF/cAMP response element-binding protein (CREB) 2 family of bZIP (basic zipper) proteins (1). It can bind to several transcription regulatory DNA elements that include cAMP response elements (CRE), an amino acid response element, and an ATF5-specific response element (ARE) and regulate gene expression in different types of cells (1-4). ATF5 has been identified as a factor whose down-regulation is essential for differentiation of rat neural progenitor cells and PC12 pheochromocytoma cells (5-7). ATF5 is expressed in a number of cancer cells and down-regulated in those cells following growth factor deprivation; exogenous expression of ATF5 suppresses apoptosis in HeLa cells induced by serum withdrawal and in FL5.12 cells, an IL-3-dependent cell line, by IL-3 deprivation (8). Conversely, dominant-negative ATF5 induces apoptosis of HeLa and FL5.12 cells and a number of glioma and breast cancer cell lines cultured in the presence of growth factors (8 -10). Similar interference of ATF5 function in non-neoplastic breast cells or in non-tumor brain cells, such as mature neurons and glial cells, does not affect their survival (9, 10). The mechanism of such selective survival requirement for ATF5 in different cell types is not understood.The BCL-2 family of proteins includes both anti-apoptotic (BCL-2, BCL-X L , and MCL-1) and apoptotic (BAK, BAX, BID, and BIM) proteins. The regulation and balance of this BCL-2 family of proteins in a particular cell result in inhibition or induction of the apoptotic signaling pathways (11,12). Notably, the expression pattern ...

Drug overdose now exceeds car accidents as the leading cause of accidental death in the U.S. Of those drug overdoses, a large percentage of the deaths are due to heroin and/or pharmaceutical overdose, specifically misuse of prescription opioid analgesics. It is imperative, then, that we understand the mechanisms that lead to opioid abuse and addiction. The rewarding actions of opioids are mediated largely by the mu opioid receptor (MOR), and signaling by this receptor is modulated by various interacting proteins. The neurotransmitter dopamine also contributes to opioid reward, and opioid addiction has been linked to reduced expression of dopamine D2 receptors (D2R) in brain. That said, it is not known if alterations in the expression of these proteins relates to drug exposure and/or to the “addiction-like” behavior exhibited for drug. Here, we held total drug self-administration constant across acquisition and showed that reduced expression of the D2R and the MOR interacting protein, Wntless, in the medial prefrontal cortex was associated with greater “addiction-like” behavior for heroin, in general, and with a greater willingness to work for drug, in particular. In contrast, reduced expression of the D2R in the nucleus accumbens and hippocampus was correlated with greater seeking during signaled non-availability of drug. Taken together, these data link reduced expression of both the D2R and Wntless to the explicit motivation for drug, rather than to differences in total drug intake, per se.

Gaucher’s disease is a sphingolipidosis characterized by a specific deficiency in an acidic glucocerebrosidase, which results in aberrant accumulation of glucosylceramide primarily within the lysosome. Gaucher’s disease has been correlated with cases of myeloma, leukemia, glioblastoma, lung cancer, and hepatocellular carcinoma, although the reasons for the correlation are currently being debated. Some suggest that the effects of Gaucher’s disease may be linked to cancer, while others implicate the therapies used to treat Gaucher’s disease. This debate is not entirely surprising, as the speculations linking Gaucher’s disease with cancer fail to address the roles of ceramide and glucosylceramide in cancer biology. In this review, we will discuss, in the context of cancer biology, ceramide metabolism to glucosylceramide, the roles of glucosylceramide in multidrug-resistance, and the role of ceramide as an anticancer lipid. This review should reveal that it is most practical to associate elevated glucosylceramide, which accompanies Gaucher’s disease, with the progression of cancer. Furthermore, this review proposes that the therapies used to treat Gaucher’s disease, which augment ceramide accumulation, are likely not linked to correlations with cancer.

Neuronal calcium sensor-1 (NCS-1) is a small calcium binding protein that plays a key role in the internalization and desensitization of activated D2 dopamine receptors (D2Rs). Here, we have used fluorescence anisotropy (FA) and a panel of NCS-1 EF hand variants to interrogate the interaction between the D2R and NCS-1. Our data is consistent with the following conclusions: 1) FA titration experiments indicate that at low D2R peptide concentrations calcium-loaded NCS-1 binds to the D2R peptide in a monomeric form. At high D2R peptide concentrations, the FA titration data is best fit by a model in which the D2R peptide binds two NCS-1 monomers sequentially in a cooperative fashion. 2) Competition FA experiments in which unlabeled D2R peptide was used to compete with labeled peptide for binding to NCS-1 shifted titration curves to higher NCS-1 concentrations, suggesting that the binding of NCS-1 to the D2R is highly specific and that binding occurs in a cooperative fashion. 3) N-terminally myristoylated NCS-1 dimerizes in a calcium-dependent manner. 4) Co-immunoprecipitation experiments in HEK293 confirm that NCS-1 can oligomerize in cell lysates, and that oligomerization is dependent on calcium binding and requires functionally intact EF hand domains. 5) Ca2+/Mg2+ FA titration experiments revealed that NCS-1 EF-hands 2-4 contributed to binding with the D2R peptide. EF-2 appears to have the highest affinity for Ca2+ and occupancy of this site is sufficient to promote high-affinity binding of the NCS-1 monomer to the D2R peptide. Magnesium ions may serve as a physiological co-factor with calcium for NCS-1/D2R binding. Finally, we propose a structural model that predicts that the D2R peptide binds to the first 60 residues of NCS-1. Together, our results support the possibility of using FA to screen for small molecule drugs that can specifically block the interaction between the D2R and NCS-1.