Monitoring of transglutaminase2 under different oxidative stress conditions

Transglutaminase 2 (TG2) is a multifunctional calcium-dependent enzyme which catalyzes the post-translational protein crosslinking with formation of intra- or inter-molecular epsilon(gamma-glutamyl)lysine bonds or polyamine incorporation. The up-regulation and activation of TG2 have been reported in a variety of physiological events, including cell differentiation, signal transduction, apoptosis, and wound healing, as well as in cell response to stress evoked by different internal and external stimuli. Here we review TG2 role in cell response to redox state imbalance both under physiological and pathological conditions, such as neurodegenerative disorders, inflammation, autoimmune diseases and cataractogenesis, in which oxidative stress plays a pathogenetic role and also accelerates disease progression. The increase in TG activity together with mitochondrial impairment and collapse of antioxidant enzymatic cell defences have been reported to be the prominent biochemical alterations becoming evident prior to neurodegeneration. Moreover, oxidative stress-induced TG2 pathway is involved in autophagy inhibition and aggresome formation, and TG2 has been suggested to function as a link between oxidative stress and inflammation by driving the decision as to whether a protein should undergo SUMO-mediated regulation or proteasomal degradation. Literature data suggest a strong association between oxidative stress and TG2 up-regulation, which in turn may result in cell survival or apoptosis, depending on cell type, kind of stressor, duration of insult, as well as TG2 intracellular localization and activity state. In particular, it may be suggested that TG2 plays a pro-survival role when the alteration of cell redox state homeostasis is not associated with intracellular calcium increase triggering TG2 transamidation activity.     

The same drug but a different mechanism of action: comparison of free doxorubicin with two different N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugates in EL-4 cancer cell line

Doxorubicin is one of the most potent anti-tumor drugs with a broad spectrum of use. To reduce its toxic effect and improve its pharmacokinetics, we conjugated it to an HPMA copolymer carrier that enhances its passive accumulation within solid tumors via the EPR effect and decreases its cytotoxicity to normal, noncancer cells. In this study, we compared the antiproliferative, pro-survival, and death signals triggered in EL-4 cancer cells exposed to free doxorubicin and doxorubicin conjugated to a HPMA copolymer carrier via either enzymatically (PK1) or hydrolytically (HYD) degradable bonds. We have previously shown that the intracellular distribution of free doxorubicin, HYD, and PK1 is markedly different. Here, we demonstrated that these three agents greatly differ also in the antiproliferative effect and cell death signals they trigger. JNK phosphorylation sharply increased in cells treated with HYD, while treatment with free doxorubicin moderately decreased and treatment with PK1 even strongly decreased it. On the other hand, treatment with free doxorubicin greatly increased p38 phosphorylation, while PK1 and HYD increased it slightly. PK1 also significantly increased ERK phosphorylation, while both the free doxorubicin and HYD conjugate slightly decreased it. Long-term inhibition of JNK significantly increased both proliferation and viability of EL-4 cells treated with free doxorubicin, showing that the JNK signaling pathway could be critical for mediating cell death in EL-4 cells exposed to free doxorubicin. Both activation of caspase 3 and decreased binding activity of the p50 subunit of NFkappaB were observed in cells treated with free doxorubicin and HYD, while no such effects were seen in cells incubated with PK1. Analysis of the expression of genes involved in apoptosis and regulation of the cell cycle demonstrated that free doxorubicin and HYD have very similar mechanisms of action, while PK1 has very different characteristics.     

NADPH oxidases participate to doxorubicin-induced cardiac myocyte apoptosis

Cumulative doses of doxorubicin, a potent anticancer drug, lead to serious myocardial dysfunction. Numerous mechanisms including apoptosis have been proposed to account for its cardiotoxicity. Cardiac apoptosis induced by doxorubicin has been related to excessive reactive oxygen species production by the mitochondrial NADH dehydrogenase. Here, we explored whether doxorubicin treatment activates other superoxide anion generating systems such as the NADPH oxidases, membrane-embedded flavin-containing enzymes, and whether the subsequent oxidative stress contributes to apoptosis. We showed that doxorubicin treatment of rat cardiomyoblasts H9c2 triggers increases in caspase-3 like activity and hypoploid cells, both common features of apoptosis. Doxorubicin exposure also leads to a rapid superoxide production through NADPH oxidase activation. Inhibition of these enzymes using diphenyliodonium and apocynin reduces doxorubicin-induced reactive oxygen species production, caspase-3 like activity and sub-G1 cell population. In conclusion, NADPH oxidases participate to doxorubicin-induced cardiac apoptosis.     

层粘连蛋白对晚G1期人胃癌BGC-823细胞生长的促进作用

为研究层粘连蛋白（laminin,LN）促进肿瘤细胞生长作用,采用脉冲标记计数有丝分裂百分率(percentage labeling mitosis,PLM)法测得体外培养人胃癌 (BGC 823) 细胞周期时间为41 h,其中G1期时间为24.5 h. 分裂细胞脱离法获取分裂期细胞,继续培养23 h,在细胞运行进入G1晚期时,将其置于LN 0、0.11、0.55、1.10 μmol/L基质上孵育4 h; 细胞荧光光度计检测晚G1期细胞内Ca2+浓度、钙调蛋白、DNA含量. 结果显示,LN与其膜上受体结合后引起细胞内Ca2+浓度、钙调蛋白、DNA含量增加,尤以在0.55 μmol/L LN作用显著（P<0. 001）.蛋白质免疫印迹分析证明,cPKC α呈现表达,提示 LN与其受体结合可增强其细胞cPKC-α的活性;分析G1期细胞周期蛋白E(cyclinE)、细胞周期蛋白依赖性激酶CDK2表达水平,呈现逐渐增强的趋势; LN可诱导c-Myc蛋白呈现高表达,提示 LN与其受体结合增强与细胞增殖密切相关的基因表达;在LN作用前后的BGC 823细胞均未检测到Bax蛋白表达.结果提示,在人胃癌 (BGC 823)细胞G1/S期交界处,层粘连蛋白与其膜上受体结合引起细胞内Ca2+浓度升高,诱导钙调蛋白的释放,其含量增加,增强蛋白激酶C的活化,导致细胞内DNA含量增加、G1/S期细胞周期蛋白与CDK表达增强、诱导原癌基因c-Myc呈持续表达状态,而凋亡基因Bax不表达.     

Type 2 transglutaminase differentially modulates striatal cell death in the presence of wild type or mutant huntingtin

Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt), is characterized by extensive loss of striatal neurons. The dysregulation of type 2 transglutaminase (TG2) has been proposed to contribute to the pathogenesis in HD as TG2 is up-regulated in HD brain and knocking out TG2 in mouse models of HD ameliorates the disease process. To understand the role of TG2 in the pathogenesis of HD, immortalized striatal cells established from mice in which mutant htt with a polyglutamine stretch of 111 Gln had been knocked-in and wild type (WT) littermates, were stably transfected with human TG2 in a tetracycline inducible vector. Overexpression of TG2 in the WT striatal cells resulted in significantly greater cell death under basal conditions as well as in response to thapsigargin treatment, which causes increased intracellular calcium concentrations. Furthermore, in WT striatal cells TG2 overexpression potentiated mitochondrial membrane depolarization, intracellular reactive oxygen species production, and apoptotic cell death in response to thapsigargin. In contrast, in mutant striatal cells, TG2 overexpression did not increase cell death, nor did it potentiate thapsigargin-induced mitochondrial membrane depolarization or intracellular reactive oxygen species production. Instead, TG2 overexpression in mutant striatal cells attenuated the thapsigargin-activated apoptosis. When in situ transglutaminase activity was quantitatively analyzed in these cell lines, we found that in response to thapsigargin treatment TG2 was activated in WT, but not mutant striatal cells. These data suggest that mutant htt alters the activation of TG2 in response to certain stimuli and therefore differentially modulates how TG2 contributes to cell death processes.     

Using FLIM-FRET to Measure Conformational Changes of Transglutaminase Type 2 in Live Cells

Transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET), as measured by fluorescence lifetime imaging microscopy (FLIM). Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.     

Involvement of Yes‐associated protein 1 (YAP1) in doxorubicin‐induced cytotoxicity in H9c2 cardiac cells

Cardiac cell death is one of the major events implicated in doxorubicin‐induced cardiotoxicity, which leads to heart failure. We recently reported that Yes‐associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin‐induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin‐induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin‐treated mice with increased caspase‐3 activation. Doxorubicin also caused cell death by increasing caspase‐3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase‐3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1–5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin‐induced cell death by decreasing caspase‐3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin‐ and YAP1 short interfering RNA (siRNA)‐treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin‐induced cardiac cell death is mediated in part by down‐regulation of YAP1 and YAP1‐targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin‐induced cardiotoxicity.     

Opposing roles for ERK1/2 in neuronal oxidative toxicity: distinct mechanisms of ERK1/2 action at early versus late phases of oxidative stress

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress.     

TG2 protects neuroblastoma cells against DNA-damage-induced stress, suppresses p53 activation

Tissue transglutaminase (TG2) is a multifunctional member of the transglutaminase (TGase) family (E.C.2.3.2.13), which catalyzes in a calcium-dependent reaction the formation of covalent bonds between the γ-carboxamide groups of peptide-bound glutamine residues and various primary amines. Here, we investigated the role of TG2 in a response of the neuroblastoma SH-SY5Y cells to topoisomerase II inhibitor etoposide, known to trigger DNA-damage cell response. We found an early and transient (~2 h) increase of the TG2 protein in SH-SY5Y cells treated with etoposide, along with the increase of phosphorylated and total levels of the p53 protein. Next, we showed that SH-SY5Y cells, which overexpress wild-type TG2 were significantly protected against etoposide-induced cell death. The TG2 protective effect was associated only with the transamidation active form of TG2, because overexpression the wild-type TG2, but not its transamidation inactive C277S form, resulted in a pronounced suppression of caspase-3 activity as well as p53 phosphorylation during the etoposide-induced stress. In addition, exacerbation of cell death with a significant increase in caspase-3 and p53 activation was observed in SH/anti-TG2 cells, in which expression of the endogenous TG2 protein has been greatly reduced by the antisense cDNA construct. Though the cell signaling and molecular mechanisms of the TG2-driven suppression of the cell death machinery remain to be investigated, our findings strongly suggest that TG2 plays an active role in the response of neuroblastoma cells to DNA-damage-induced stress by exerting a strong protective effect, likely by the suppression of p53 activation and p53-driven cell signaling events.     

Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.     

Mechanism underlying oxidative stress-mediated lipotoxicity: exposure of J774.2 macrophages to triacylglycerols facilitates mitochondrial reactive oxygen species production and cellular necrosis

The aim of this study was to elucidate death pathways in macrophages resulting from exposure to triacylglycerols (TG), mechanisms which may be relevant to the development of atherosclerosis. A commercial TG emulsion (lipid emulsion, LE; 0.1-1.5 mg lipids/ml) was added to J774.2 cells in culture. Within the first 24 h after TG treatment, cellular reactive oxygen species (ROS) levels were strongly elevated and basal caspase-3 activity was attenuated. In contrast, after 48 h, ROS production was arrested. TG-mediated ROS production was demonstrated to be via mitochondrial complex 1 of the electron-transfer chain since the inhibitor of complex 1 rotenone significantly attenuated the cellular ROS levels in TG-treated cells. The TG effect culminated in cell death, with no caspase-3 activation. We therefore evaluated the effect of TG on apoptotic cells showing high caspase activity. TG induced elevated ROS levels and suppressed caspase-3 in apoptotic cells pretreated for 24 h with cycloheximide. Dual staining with propidium iodide and Annexin V followed by flow cytometric analysis showed that TG facilitated cell death with clear necrotic characteristics. To elucidate whether the necrotic cell death process is indeed oxidant dependent, antioxidant protection was studied. Treatment with N-acetylcysteine (NAC) (0.5 mM), ascorbic acid (0.5 mM), and resveratrol (0.2 mM) protected against the TG lipotoxic effect, while, surprisingly, lipophilic antioxidants did not. The combination of NAC, ascorbic acid, and resveratrol, each at much lower concentrations, had a synergistic protective effect. In conclusion, we show here for the first time that exposure to TG can directly regulate lipotoxicity in macrophages by inducing mitochondria-mediated prolonged oxidative stress; this, in turn, can inactivate the apoptotic caspase system, resulting in necrotic cell death which can be prevented by specific antioxidants.     

Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a neuronal cell line and primary cortical neuron cultures

Oxidative stress can trigger neuronal cell death and has been implicated in several chronic neurological diseases and in acute neurological injury. Oxidative toxicity can be induced by glutamate treatment in cells that lack ionotrophic glutamate receptors, such as the immortalized HT22 hippocampal cell line and immature primary cortical neurons. Previously, we found that neuroprotective effects of geldanamycin, a benzoquinone ansamycin, in HT22 cells were associated with a down-regulation of c-Raf-1, an upstream activator of the extracellular signal-regulated protein kinases (ERKs). ERK activation, although often attributed strictly to neuronal cell survival and proliferation, can also be associated with neuronal cell death that occurs in response to specific insults. In this report we show that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures. Furthermore, we find that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22 cells and immature primary cortical neuron cultures from glutamate toxicity. Glutamate-induced ERK activation requires the production of specific arachidonic acid metabolites and appears to be downstream of a burst of reactive oxygen species (ROS) accumulation characteristic of oxidative stress in HT22 cells. However, inhibition of ERK activation reduces glutamate-induced intracellular Ca(2+) accumulation. We hypothesize that the precise kinetics and duration of ERK activation may determine whether downstream targets are mobilized to enhance neuronal cell survival or ensure cellular demise.     

Mechanisms Underlying Ectopic Persistent Tooth-Pulp Pain following Pulpal Inflammation

In order to clarify the peripheral mechanisms of ectopic persistent pain in a tooth pulp following pulpal inflammation of an adjacent tooth, masseter muscle activity, phosphorylated extracellular signal-regulated protein kinase (pERK) and TRPV1 immunohistochemistries and satellite cell activation using glial fibrillary acidic protein (GFAP) immunohistochemistry in the trigeminal ganglion (TG) were studied in the rats with molar tooth-pulp inflammation. And, Fluorogold (FG) and DiI were also used in a neuronal tracing study to analyze if some TG neurons innervate more than one tooth pulp. Complete Freund’s adjuvant (CFA) or saline was applied into the upper first molar tooth pulp (M1) in pentobarbital-anesthetized rats, and capsaicin was applied into the upper second molar tooth pulp (M2) on day 3 after the CFA or saline application. Mean EMG activity elicited in the masseter muscle by capsaicin application to M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. The mean number of pERK-immunoreactive (IR) TG cells was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. Application of the satellite cell inhibitor fluorocitrate (FC) into TG caused a significant depression of capsaicin-induced masseter muscle activity and a significant reduction of satellite cell activation. The number of TRPV1-IR TG cells innervating M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats, and that was decreased following FC injection into TG. Furthermore, 6% of TG neurons innervating M1 and/or M2 innervated both M1 and M2. These findings suggest that satellite cell activation following tooth pulp inflammation and innervation of multiple tooth pulps by single TG neurons may be involved in the enhancement of the activity of TG neurons innervating adjacent non-inflamed teeth that also show enhancement of TRPV1 expression in TG neurons, resulting in the ectopic persistent tooth-pulp pain following pulpal inflammation of adjacent teeth.     

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.     

DNA cleavage function of seryl-histidine dipeptide and its application

Summary. Previously published evidences highlighted the effect of transglutaminase (TG, EC 2.3.2.13) activation on the reduction of the in vitro adhesive and invasive behaviour of murine B16-F10 melanoma cells, as well as in vivo. Here, we investigated the influence of spermidine (SPD) incorporation by TG into basement membrane components i.e. laminin (LN) or Matrigel (MG), on the adhesion and invasion of B16-F10 melanoma cells by these TG/SPD-modified substrates. The adhesion assays showed that cell binding to the TG/SPD-modified LN was reduced by 30%, when compared to untreated LN, whereas the reduction obtained using TG/SPD-modified MG was 35%. Similarly, tumor cell invasion by the Boyden chamber system through TG/SPD modified LN or MG was respectively reduced by 45%, and by 69%. Evaluation of matrix metalloproteinase (gelatinases MMP-2 and MMP-9) activities by gel-zymography showed that MMP-2 activity was unaffected, while MMP-9 activity was reduced by about 32% using TG/SPD-modified substrate. These results strongly suggest that the observed antiinvasive effect of TG activation in the host may be ascribed to the covalent incorporation of polyamines, which led to the post-translational modification of some components of the cell basement membrane. This modification may interfere with the metastatic property of melanoma cells, affecting the proteolytic activity necessary for their migration and invasion activities. Authors’ address: Simone Beninati, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, I-00133 Rome, Italy     

SERCA activity is required for timely progression through G1/S

Changes in intracellular Ca2+ correlate with specific events in the cell cycle. Here we investigated the role of Ca2+ in the G1 phase. HEK 293 cells were arrested in mitosis and subjected to short-term treatments that alter Ca2+ homeostasis prior to their release into G1. Treatment with thapsigargin (TG), an irreversible inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) lengthened the G1 phase. Moreover, TG treatment also resulted in a dramatic alteration in cellular morphology and attachment and in the reduction of MAPK activity and lower levels of cyclin D1 and cyclin E proteins. Treatments with reagents that transiently increase or decrease cytosolic Ca2+ or that temporarily inactivate SERCA did not alter any of the above parameters. Cells expressing a TG-resistant form of SERCA progressed normally through the G1/S transition after TG treatment. These results suggest that long-term SERCA inactivation affects cell cycle-dependent events and compromises progression through G1/S.     

Inhibition of AMP-activated protein kinase α (AMPKα) by doxorubicin accentuates genotoxic stress and cell death in mouse embryonic fibroblasts and cardiomyocytes: role of p53 and SIRT1

Doxorubicin, an anthracycline antibiotic, is widely used in cancer treatment. Doxorubicin produces genotoxic stress and p53 activation in both carcinoma and non-carcinoma cells. Although its side effects in non-carcinoma cells, especially in heart tissue, are well known, the molecular targets of doxorubicin are poorly characterized. Here, we report that doxorubicin inhibits AMP-activated protein kinase (AMPK) resulting in SIRT1 dysfunction and p53 accumulation. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) or H9C2 cardiomyocyte were exposed to doxorubicin at different doses and durations. Cell death and p53, SIRT1, and AMPK levels were examined by Western blot. In MEFs, doxorubicin inhibited AMPK activation, increased cell death, and induced robust p53 accumulation. Genetic deletion of AMPKα1 reduced NAD(+) levels and SIRT1 activity and significantly increased the levels of p53 and cell death. Pre-activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside or transfection with an adenovirus encoding a constitutively active AMPK (AMPK-CA) markedly reduced the effects of doxorubicin in MEFs from Ampkα1 knock-out mice. Conversely, pre-inhibition of Ampk further sensitized MEFs to doxorubicin-induced cell death. Genetic knockdown of p53 protected both wild-type and Ampkα1(-/-) MEFs from doxorubicin-induced cell death. p53 accumulation in Ampkα1(-/-) MEFs was reversed by SIRT1 activation by resveratrol. Taken together, these data suggest that AMPK inhibition by doxorubicin causes p53 accumulation and SIRT1 dysfunction in MEFs and further suggest that pharmacological activation of AMPK might alleviate the side effects of doxorubicin.