Background: TRPM2 channels play an essential role in cell death following oxidative stress. Results: Dominant negative TRPM2-S decreases growth of neuroblastoma xenografts and increases doxorubicin sensitivity through modulation of HIF-1/2␣ expression, mitophagy, and mitochondrial function. Conclusion: TRPM2 is important for neuroblastoma growth and viability through modulation of HIF-1/2␣. Significance: Modulation of TRPM2 may be a novel approach in cancer therapeutics.

Background: TRPM2 channels are present in the heart, but their function is unknown. Results: Genetic ablation of TRPM2 results in cardiac mitochondrial dysfunction, enhanced ROS production, and exacerbated cardiac ischemic injury. Conclusion: TRPM2 channels preserve cardiac mitochondrial bioenergetics and protect cardiac myocytes from ischemic injury. Significance: TRPM2 is a rational target for treatment of ischemic heart disease.

TRPM2 is a Ca(2+)-permeable channel activated by oxidative stress or TNF-alpha, and TRPM2 activation confers susceptibility to cell death. The mechanisms were examined here in human monocytic U937-ecoR cells. This cell line expresses full-length TRPM2 (TRPM2-L) and several isoforms including a short splice variant lacking the Ca(2+)-permeable pore region (TRPM2-S), which functions as a dominant negative. Treatment with H(2)O(2), a model of oxidative stress, or TNF-alpha results in reduced cell viability. Expression of TRPM2-L and TRPM2-S was modulated by retroviral infection. U937-ecoR cells expressing increased levels of TRPM2-L were treated with H(2)O(2) or TNF-alpha, and these cells exhibited significantly increased intracellular calcium concentration ([Ca(2+)](i)), decreased viability, and increased apoptosis. A dramatic increase in cleavage of caspases-8, -9, -3, and -7 and poly(ADP-ribose)polymerase (PARP) was observed, demonstrating a downstream mechanism through which cell death is mediated. Bcl-2 levels were unchanged. Inhibition of the [Ca(2+)](i) rise with the intracellular Ca(2+) chelator BAPTA blocked caspase/PARP cleavage and cell death induced after activation of TRPM2-L, demonstrating the critical role of [Ca(2+)](i) in mediating these effects. Downregulation of endogenous TRPM2 by RNA interference or increased expression of TRPM2-S inhibited the rise in [Ca(2+)](i), enhanced cell viability, and reduced numbers of apoptotic cells after exposure to oxidative stress or TNF-alpha, demonstrating the physiological importance of TRPM2. Our data show that one mechanism through which oxidative stress or TNF-alpha mediates cell death is activation of TRPM2, resulting in increased [Ca(2+)](i), followed by caspase activation and PARP cleavage. Inhibition of TRPM2-L function by reduction in TRPM2 levels, interaction with TRPM2-S, or Ca(2+) chelation antagonizes this important cell death pathway.

Edited by Xiao-Fan WangTransient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2␣ (HIF-1/2␣) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.

Cycloheximide inhibits ribosomal DNA (rDNA) transcription in vivo. The mouse homologue of yeast Rrn3, a polymerase-associated transcription initiation factor, can complement extracts from cycloheximide-treated mammalian cells. Cycloheximide inhibits the phosphorylation of Rrn3 and causes its dissociation from RNA polymerase I. Rrn3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a Rrn3⅐rpa43 complex in vivo. Rrn3 produced in Sf9 cells but not in bacteria interacts with rpa43 in vitro, and such interaction is dependent upon the phosphorylation state of Rrn3. Significantly, neither dephosphorylated Rrn3 nor Rrn3 produced in Escherichia coli can restore transcription by extracts from cycloheximide-treated cells. These results suggest that the phosphorylation state of Rrn3 regulates rDNA transcription by determining the steady-state concentration of the Rrn3⅐RNA polymerase I complex within the nucleolus.In the early 1970s Feigelson and colleagues (1-3) reported that cycloheximide caused a rapid cessation of nucleolar RNA synthesis (ribosomal DNA transcription) and concluded that a rapidly turning over protein was required for RNA polymerase I (pol I) 1 activity in vivo. Subsequent studies have demonstrated that transcription by RNA polymerase I is subject to regulation at many levels (4, 5). At least three, and possibly more, polymerase-associated proteins, TIF-IA, Factor C*, and TFIC (6 -8), have been demonstrated to contribute to the regulation of rDNA transcription. TIF-IA and Factor C* were identified as factors that were required for the complementation of extracts of quiescent or cycloheximide-treated cells. TFIC was identified as that activity required to reconstitute transcription by extracts of glucocorticoid-treated P1798 cells.This lymphosarcoma cell line exits the cell cycle in response to the synthetic glucocorticoid dexamethasone (DEX) (6). Interestingly, TIF-IA, Factor C*, and TFIC shared several properties, including a tight association with the core polymerase (8 -10). TIF-IA and TFIC were purified and consisted of different polypeptides (10, 11). However, the lack of immunological and molecular tools precluded a definitive statement that TIF-IA and TFIC were the same or different proteins (reviewed in Refs. 4 and 5).The formation of the stable preinitiation complex in yeast requires an interaction between the upstream activating factor bound to the upstream promoter element and core factor, bound to the core promoter element. This complex then recruits transcriptionally competent RNA polymerase I to the transcription initiation site (Ref. 12 and references therein). Mechanistically, Rrn3 appears to "bridge" the polymerase and transcription initiation complexes (13-15). Thus, only pol I molecules in complex with Rrn3 are able to recognize the preinitiation complex and initiate transcription.Studies comparing the state of RNA polymerase I in growing and stationary yeast cells demonstrated that ϳ2% of the pol I in whole cell extracts was...

RNA polymerase I (PolI) transcription is activated by the HMG box architectural factor UBF, which loops approximately 140 bp of DNA into the enhancesome, necessitating major chromatin remodeling. Here we show that the acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation "flip-flop." Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

Five days post-injection, gKO-GFP heart slices had higher reactive oxygen species (ROS) levels but lower oxygen consumption rate (OCR) than WT-GFP heart slices. Trpm2 but not E960D decreased ROS and restored OCR in gKO hearts back to normal levels. In gKO myocytes expressing Trpm2 or its mutants, Trpm2 but not E960D reduced the elevated mitochondrial superoxide (O 2 .Ϫ ) levels in gKO myocytes. After hypoxia-reoxygenation (H/R), Trpm2 but not E906D or P1018L (inactivates Trpm2 current) lowered O 2 .Ϫ levels in gKO myocytes and only in the presence of extracellular Ca 2ϩ , indicating sustained Ca 2ϩ entry is necessary for Trpm2-mediated preservation of mitochondrial function. After ischemic-reperfusion (I/R), cardiac-specific Trpm2 KO hearts exhibited lower maximal first time derivative of LV pressure rise (ϩdP/dt) than WT hearts in vivo. After doxorubicin treatment, Trpm2 KO mice had worse survival and lower ϩdP/dt. We conclude 1) cardiac Trpm2-mediated Ca 2ϩ influx is necessary to maintain mitochondrial function and protect against H/R injury; 2) Ca 2ϩ influx via cardiac Trpm2 confers protection against H/R and I/R injury by reducing mitochondrial oxidants; and 3) Trpm2 confers protection in doxorubicin cardiomyopathy. ischemic cardiomyopathy; doxorubicin cardiomyopathy; voltage-independent Ca 2ϩ channels; cardiac Trpm2 currents; mitochondrial superoxide; hypoxia-reoxygenation IN MAMMALIAN CELLS, THE TRANSIENT receptor potential (Trp) protein superfamily is a diverse group of voltage-independent, cation-permeable channels organized into six subfamilies based on amino acid sequence homology (9, 30). Monomeric Trp proteins have six putative transmembrane (TM) domains and intracellular NH 2 and COOH termini. To form a functional channel, Trp proteins assemble into either homo-or heterotetramers, with the putative pore formed by loops between the fifth and sixth TM domains (21, 23). The Trp-melastatin (Trpm) subfamily consists of eight mammalian members (Trpm1-Trpm8)(30), of which Trpm2 (27), -m4, -m5, and -m6 (49) are expressed in the heart. Although Trpm4 is associated with conduction abnormalities and cardiac arrhythmias (1, 36), there is little information on the physiological and pathophysiological function of Trpm2 in the heart.