Alejo A. Morales

STING Recognition of Cytoplasmic DNA Instigates Cellular Defense

How the cell recognizes cytosolic DNA including DNA-based microbes to trigger host-defense-related gene activation remains to be fully resolved. Here, we demonstrate that STING (stimulator of interferon genes), an endoplasmic reticulum translocon-associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self- (apoptotic, necrotic) or pathogen-related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and proinflammatory genes in addition to type I IFN. Our data indicate that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.

Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1-expressing myeloma cells.

Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-x(L)/Bcl-2 dependence. Surprisingly, analysis of Mcl-1-dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-x(L) in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.

BH3-only proteins Noxa, Bmf, and Bim are necessary for arsenic trioxide-induced cell death in myeloma

The use of arsenic trioxide (ATO) to treat multiple myeloma (MM) is supported by preclinical studies as well as several phase 2 studies, but the precise mechanism(s) of action of ATO has not been completely elucidated. We used gene expression profiling to determine the regulation of apoptosis-related genes by ATO in 4 MM cell lines and then focused on Bcl-2 family genes. ATO induced up-regulation of 3 proapoptotic BH3-only proteins (Noxa, Bmf, and Puma) and down-regulation of 2 antiapoptotic proteins Mcl-1 and Bcl-X(L). Coimmunoprecipitation demonstrated that Noxa and Puma bind Mcl-1 to release Bak and Bim within 6 hours of ATO addition. Bak and Bim are also released from Bcl-X(L). Silencing of Bmf, Noxa, and Bim significantly protected cells from ATO-induced apoptosis, while Puma silencing had no effect. Consistent with a role for Noxa inhibition of Mcl-1, the Bad-mimetic ABT-737 synergized with ATO in the killing of 2 MM lines. Finally, Noxa expression was enhanced by GSH depletion and inhibited by increasing GSH levels in the cells. Understanding the pattern of BH3-only protein response should aid in the rational design of arsenic-containing regimens.

Acquisition of a Multidrug Resistant Phenotype with a Proteasome Inhibitor in Multiple Myeloma

Acquisition of a multidrug-resistant phenotype with a proteasome inhibitor in multiple myeloma

Reactive Oxygen Species Are Not Required for an Arsenic Trioxide-induced Antioxidant Response or Apoptosis

Arsenicals are both environmental carcinogens as well as therapeutic agents for the treatment of trypanosomiasis and more recently cancer. Arsenic trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has activity in multiple myeloma (MM). While signaling events associated with carcinogenesis have been well studied, it still remains to be determined which of these events are involved in anti-cancer signaling. To better define this response, gene expression profiling following ATO treatment of four MM cell lines was performed. The pattern was consistent with a strong antioxidative response, particularly of genes activated by Nrf2. While Nrf2 is expressed constitutively at the mRNA level, the protein is not detected in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized and present in the nucleus within 6 h of ATO treatment. Despite the activation of this antioxidative response, ROS may not be important in ATO-induced death. Inhibition of ATO-induced ROS with butylated hydroxyanisole (BHA) does not affect Nrf2 activation or cell death. Moreover, silencing Nrf2 had no effect on ATO-induced apoptosis. Together these data suggest that ROS is not important in the induction of the antioxidative response or cellular death by ATO.

Darinaparsin Induces a Unique Cellular Response and is Active in an Arsenic Trioxide-Resistant Myeloma Cell Line

Here, we report on the organic arsenical darinaparsin (ZIO-101, S-dimethylarsino-glutathione) and its anti-myeloma activity compared with inorganic arsenic trioxide. Darinaparsin induced apoptosis in multiple myeloma cell lines in a dose-dependent manner, and the addition of N-acetylcysteine, which increases intracellular glutathione (GSH), blocked cytotoxicity of both darinaparsin and arsenic trioxide. In contrast to arsenic trioxide, intracellular GSH does not appear to be important for darinaparsin metabolism, as an inhibitor of GSH synthesis, buthionine sulfoximine, had little effect on drug activity. This discrepancy was resolved when we determined the effects of thiols on drug uptake. The addition of exogenous GSH, l-cysteine, or d-cysteine prevented darinaparsin cellular uptake and cell death but had no effect on the uptake or activity of arsenic trioxide, suggesting a difference in the transport mechanism of these two drugs. In addition, gene expression profiling revealed differences in the signaling of protective responses between darinaparsin and arsenic trioxide. Although both arsenicals induced a transient heat shock response, only arsenic trioxide treatment induced transcription of metal response genes and anti-oxidant genes related to the Nrf2-Keap1 pathway. In contrast to the protective responses, both arsenicals induced up-regulation of BH3-only proteins. Moreover, silencing of BH3-only proteins Noxa, Bim, and Bmf protected myeloma cells from darinaparsin-induced cell death. Finally, treatment of an arsenic trioxide-resistant myeloma cell line with darinaparsin resulted in dose-dependent apoptosis, indicating that cross-resistance does not necessarily develop between these two forms of arsenic in multiple myeloma cell lines. These results suggest darinaparsin may be useful as an alternative treatment in arsenic trioxide-resistant hematologic cancers.[Mol Cancer Ther 2009;8(5):OF1–10]

Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria

Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system. DOI: http://dx.doi.org/10.7554/eLife.06508.001

Alterations in Glutathione Levels and Apoptotic Regulators Are Associated with Acquisition of Arsenic Trioxide Resistance in Multiple Myeloma

Arsenic trioxide (ATO) has been tested in relapsed/refractory multiple myeloma with limited success. In order to better understand drug mechanism and resistance pathways in myeloma we generated an ATO-resistant cell line, 8226/S-ATOR05, with an IC50 that is 2–3-fold higher than control cell lines and significantly higher than clinically achievable concentrations. Interestingly we found two parallel pathways governing resistance to ATO in 8226/S-ATOR05, and the relevance of these pathways appears to be linked to the concentration of ATO used. We found changes in the expression of Bcl-2 family proteins Bfl-1 and Noxa as well as an increase in cellular glutathione (GSH) levels. At low, clinically achievable concentrations, resistance was primarily associated with an increase in expression of the anti-apoptotic protein Bfl-1 and a decrease in expression of the pro-apoptotic protein Noxa. However, as the concentration of ATO increased, elevated levels of intracellular GSH in 8226/S-ATOR05 became the primary mechanism of ATO resistance. Removal of arsenic selection resulted in a loss of the resistance phenotype, with cells becoming sensitive to high concentrations of ATO within 7 days following drug removal, indicating changes associated with high level resistance (elevated GSH) are dependent upon the presence of arsenic. Conversely, not until 50 days without arsenic did cells once again become sensitive to clinically relevant doses of ATO, coinciding with a decrease in the expression of Bfl-1. In addition we found cross-resistance to melphalan and doxorubicin in 8226/S-ATOR05, suggesting ATO-resistance pathways may also be involved in resistance to other chemotherapeutic agents used in the treatment of multiple myeloma.

The Comparative Utility of Viromer RED and Lipofectamine for Transient Gene Introduction into Glial Cells.

The introduction of genes into glial cells for mechanistic studies of cell function and as a therapeutic for gene delivery is an expanding field. Though viral vector based systems do exhibit good delivery efficiency and long-term production of the transgene, the need for transient gene expression, broad and rapid gene setup methodologies, and safety concerns regarding in vivo application still incentivize research into the use of nonviral gene delivery methods. In the current study, aviral gene delivery vectors based upon cationic lipid (Lipofectamine 3000) lipoplex or polyethylenimine (Viromer RED) polyplex technologies were examined in cell lines and primary glial cells for their transfection efficiencies, gene expression levels, and toxicity. The transfection efficiencies of polyplex and lipoplex agents were found to be comparable in a limited, yet similar, transfection setting, with or without serum across a number of cell types. However, differential effects on cell-specific transgene expression and reduced viability with cargo loaded polyplex were observed. Overall, our data suggests that polyplex technology could perform comparably to the market dominant lipoplex technology in transfecting various cells lines including glial cells but also stress a need for further refinement of polyplex reagents to minimize their effects on cell viability.