Anwarul A. Akhand

Transgenic mouse model for skin malignant melanoma

We report here on a novel metallothionein-I (MT)/ret transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma metastasizing to distant organs develop stepwise. The process of tumor development and its malignant transformation in this line may resemble that of the human giant congenital melanocytic nevus that is present at birth and that frequently gives rise to malignant melanoma during aging. We observed an increase in the expression level and activity of the ret transgene during the disease progression. That increase in transgene expression accompanied an activation of mitogen-activated protein kinases (MAPKs) and c-Jun as well as matrix metalloproteinases. These results suggest that progressive dysregulation of the expression level of the ret transgene might play a crucial role in the malignant transformation of melanocytic tumors developed in the MT/ret transgenic mouse line.

Nitric Oxide Controls Src Kinase Activity through a Sulfhydryl Group Modification-mediated Tyr-527-independent and Tyr-416-linked Mechanism

c-Src kinase was activated when either murine NIH3T3 fibroblast cells or immunoprecipitated c-Src proteins were treated with nitric oxide generator,S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside. Nitric oxide (NO) scavenger hemoglobin and N2O3 scavenger homocysteine abolished the SNAP-mediated c-Src kinase activation. Phosphoamino acid analysis and peptide mapping of in vitro labeled phospho-c-Src proteins revealed that SNAP promoted the autophosphorylation at tyrosine, which preferentially took place at Tyr-416. Peptide mapping of in vivo labeled c-Src kinase excluded the involvement of phospho-Tyr-527 dephosphorylation in the SNAP-mediated activation mechanism. Correspondingly, protein-tyrosine phosphatase inhibitor Na3VO4 did not abolish the SNAP-mediated activation of Src kinase, and the constitutively activated v-Src kinase was also further up-regulated in activity by SNAP. SNAP, however, failed to up-regulate the kinase activity of Phe-416 mutant v-Src. 2-Mercaptoethanol or dithiothreitol, which should disrupt N2O3-mediated S-nitrosylation and subsequent formation of the S-S bond, abolished the up-regulated catalytic activity, and the activity was regained after re-exposing the enzyme to SNAP. Exposure of Src kinase to SNAP promoted both autophosphorylation and S-S bond-mediated aggregation of the kinase molecules, demonstrating a linkage between the two events. These results suggest that the NO/N2O3-provokedS-nitrosylation/S-S bond formation destabilizes the Src structure for Tyr-416 autophosphorylation-associated activation bypassing the Tyr-527-linked regulation.

Superoxide-mediated early oxidation and activation of ASK1 are important for initiating methylglyoxal-induced apoptosis process

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress and causing apoptosis. Our previous studies demonstrated that MG induced apoptosis in Jurkat cells by activating the c-Jun N-terminal kinase (JNK) signal transduction pathway, which induced an obvious decrease in mitochondrial membrane potential, followed by caspase-3 activation. Here, we observed that MG-induced apoptosis was associated with both rapid production of superoxide anion (O(2)(-)) followed by a marked increase in ROS and striking and temporal activation of ASK1. Overexpression of wild-type ASK1 could enhance the rate of apoptosis induced by MG, whereas the expression of the kinase-inactive form of ASK1 notably prevented cells from MG-induced death. NAC and PDTC blocked the activation of ASK1 and MG-induced apoptosis completely. Moreover, nonthiol antioxidants SOD-mimic MnTBAP and catalase together obviously inhibited MG-induced ASK1 activation and apoptosis induction. Correspondingly, MG-mediated ASK1 activation was enhanced by diethyldithiocarbamate (DDC). Addition of antioxidant into the culture of cells at a later stage (4-8 h after the initial MG treatment) failed to prevent their death. These results suggest that activating ASK1 at the early stage linking to production of O(2)(-) is crucial for subsequent progression of apoptosis in MG-treated Jurkat cells.

Glyoxal and methylglyoxal trigger distinct signals for map family kinases and caspase activation in human endothelial cells

Carbonyl compounds with diverse carbon skeletons may be differentially related to the pathogenesis of vascular diseases. In this study, we compared intracellular signals delivered into cultured human umbilical vein endothelial cells (HUVECs) by glyoxal (GO) and methylglyoxal (MGO), which differ only by a methyl group. Depending on their concentrations, GO and MGO promoted phosphorylations of ERK1 and ERK2, which were blocked by the protein-tyrosine kinase (PTK) inhibitors herbimycin A and staurosporine, thereby being PTK-dependent. GO and MGO also induced phosphorylations of JNK, p38 MAPK, and c-Jun, either PTK-dependently (GO) or -independently (MGO). Next, we found that MGO, but not GO, induced degradation of poly(ADP-ribose) polymerase (PARP) as the intracellular substrate of caspase-3. Curcumin and SB203580, which inhibit JNK and p38 MAPK signaling pathways, but not herbimycin A/staurosporine, prevented the MGO-induced PARP degradation. We then found that MGO, but not GO, reduced the intracellular glutathione level, and that cysteine, but not cystine, inhibited the MGO-mediated activation of ERK, JNK, p38 MAPK, or c-Jun more extensively than did lysine or arginine. In addition, all the signals triggered by GO and MGO were blocked by amino guanidine (AG), which traps carbonyls. These results demonstrated that GO and MGO triggered two distinct signal cascades, one for PTK-dependent control of ERK and another for PTK-independent redox-linked activation of JNK/p38 MAPK and caspases in HUVECs, depending on the structure of the carbon skeleton of the chemicals.

Arsenite Induces Apoptosis of Murine T Lymphocytes Through Membrane Raft-Linked Signaling for Activation of c-Jun Amino-Terminal Kinase

Because of its dual roles in acute toxicity and in therapeutic application in cancer treatment, arsenic has recently attracted a renewed attention. In this study, we report NaAsO2-induced signal cascades from the cell surface to the nucleus of murine thymic T lymphocytes that involve membrane rafts as an initial signal transducer. NaAsO2 induced apoptosis through fragmentation of DNA, activation of caspase, and reciprocal regulation of Bcl-2/Bax with the concomitant reduction of membrane potential. We demonstrated that NaAsO2-induced caspase activation is dependent on curcumin-sensitive c-Jun amino-terminal kinase and barely dependent on SB203580-sensitive p38 kinase or PD98059-sensitive extracellular signal-regulated kinase. Additionally, staurosporine, which severely inhibited the activation of mitogen-activated protein (MAP) family kinases and c-Jun, partially blocked the NaAsO2-mediated signal for poly(ADP-ribose) polymerase (PARP) degradation. Potentially as the initial cell surface event for intracellular signaling, NaAsO2 induced aggregation of GPI-anchored protein Thy-1 and superoxide production. This Thy-1 aggregation and subsequent activation of MAP family kinase and c-Jun and the degradation of PARP induced by NaAsO2 were all inhibited by DTT, suggesting the requirement of interaction between arsenic and protein sulfhydryl groups for those effects. β cyclodextrin, which sequestrates cholesterol from the membrane rafts, inhibited NaAsO2-induced activation of protein tyrosine kinases and MAP family kinases, degradation of PARP, and production of superoxide. In addition, β cyclodextrin dispersed NaAsO2-induced Thy-1 clustering. These results suggest that a membrane raft integrity-dependent cell surface event is a prerequisite for NaAsO2-induced protein tyrosine kinase/c-Jun amino-terminal kinase activation, superoxide production, and downstream caspase activation.

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c‐Jun N‐Terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG‐induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT‐4, HeLa, or COS‐7 cells. A caspase inhibitor, Z‐DEVD‐fmk, completely blocked MG‐induced poly(ADP‐ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG‐induced caspase‐3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential. J. Cell. Biochem. 77:333–344, 2000.

Protein phosphatase 2A-linked and -unlinked caspase-dependent pathways for downregulation of Akt kinase triggered by 4-hydroxynonenal.

AbstractWe studied the signal pathways for regulation of serine/threonine protein kinase Akt in Jurkat cells that had been treated with 4-hydroxynonenal (HNE) for caspase-dependent apoptosis induction. Treatment of cells with HNE led to a decrease in the level of Akt activity due to the dephosphorylation at Ser473, a major regulatory phosphorylation site. HNE-mediated dephosphorylation of Akt was prevented by a protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and by a caspase-3 inhibitor, DEVD-CHO. HNE treatment resulted in an increase in the total level of PP2A activity, release of active tyrosine-dephosphorylated PP2A from the cytoskeleton and PP2A-Akt association, which were all dependent on caspase-3 activation. These results suggest that the level of PP2A activity is at least in part determined by its tyrosine phosphorylation, which is dually controlled by okadaic acid-sensitive phosphatases and protein-tyrosine kinases. Possibly underlying the mechanism of caspase-mediated activation of PP2A, HNE treatment resulted in downregulation of the activity of Src kinase, as a representative caspase-sensitive kinase to phosphorylate PP2A at tyrosine. In addition, activated caspase-3 partially cleaved Akt at a late stage of the apoptosis. These results indicate the existence of two distinct caspase-dependent signal pathways for downregulation of Akt that works as a mechanism of positive feedback regulation for HNE-triggered apoptotic signals.

4-hydroxynonenal triggers multistep signal transduction cascades for suppression of cellular functions.

4-hydroxynonenal (HNE), an aldehyde product of membrane lipid peroxidation, has been suggested to mediate a number of oxidative stress-linked pathological events in humans, including cellular growth inhibition and apoptosis induction. Because HNE is potentially reactive to a number of both cell surface and intracellular proteins bearing sulfhydryl, amino and imidazole groups, it seems that there are multiple signal transduction cascades. Here we briefly review the HNE-triggered signal transduction cascades that lead to suppression of cellular functions and to cell death, based mainly on our own recent study results. We first showed that formation of HNE-cell surface protein adducts, which mimicked ligand-cell surface receptor binding, induced activation of receptor-type protein tyrosine kinases such as epithelial growth factor receptor (EGFR) and that this caused growth inhibition through a cascade of activation of EGFR, Shc and ERK. Next, we showed that HNE-mediated scavenging of cellular glutathione led to activation of caspases and to DNA fragmentation through a Fas-independent and mitochondria-linked pro-apoptotic signal pathway. More recently, we have obtained evidence that the HNE-triggered signal cascade for caspase activation encounters complex positive feedback regulatory mechanisms that are linked to the inhibition of anti-apoptotic signals and are dependent on caspase activity. Underlying multiple regulatory mechanisms, including mechanisms of activation of Akt-dephosphorylating PP2A activity, activities of protein tyrosine kinases have been shown to be biphasically controlled by HNE. In addition, we have obtained results suggesting that HNE inhibits phosphorylation of IkappaB, possibly by targeting some elements upstream of IkappaB, which might downregulate the NF-kappaB-mediated cellular responses, including serum deprivation-induced iNOS expression and generation of anti-apoptotic signals. These results suggest that HNE reacts with multiple cell surface and intracellular sites for triggering a network of signal transduction that is ultimately focused on suppression of cellular functions.

Paeoniflorin induces apoptosis of lymphocytes through a redox‐linked mechanism

Paeoniflorin (PF), isolated from paeony root, has been used as a herbal medicine for more than 1,200 years in China, Korea, and Japan for its anti‐allergic, anti‐inflamatory, and immunoregulatory effects. In this study, we found that PF induces apoptosis in both murine T‐lineage cells and human T‐cell leukemia Jurkat cells. This apoptosis was mediated through the reduction of mitochondrial membrane potential, activation of caspase, and fragmentation of DNA. Interestingly, PF induced generation of reactive oxygen species (ROS) and a reducing agent, dithiothreitol (DTT), and a ROS scavenger, N‐acetyl cysteine (NAC), successfully attenuated the PF‐induced apoptosis. Additionally, PF induced the phosphorylation of three mitogen‐activated protein (MAP) family kinases, extracellular signal‐regulated kinase, c‐Jun amino‐terminal kinase (JNK), and p38 MAP kinase. Curcumin, an anti‐oxidant and JNK inhibitor, inhibited PF‐induced apoptosis, suggesting the possible involvement of curcumin‐sensitive JNK or other redox‐sensitive elements in PF‐induced apoptosis. These results partially explain the action mechanism of PF‐containing paeony root as a herbal medicine.

Carbonyl compounds cross-link cellular proteins and activate protein-tyrosine kinase p60c-Src.

Glyoxal, a dicarbonyl compound, is produced under oxidative stress by the autoxidation of glucose and reacts with the protein amino group to form Schiff base. In vitro treatment of murine thymocytes and fibroblasts with glyoxal induced extensive tyrosine phosphorylation of multiple proteins, which was drastically inhibited by the addition of OPB‐9195, an inhibitor of the carbonyl reaction with proteins. Glyoxal induced cross‐linking of a number of cellular proteins, including glycosylphosphatidylinositol (GPI)‐anchored cell surface Thy‐1. We then demonstrated that treatment of cells with glyoxal promptly induced activation of non‐receptor protein‐tyrosine kinase c‐Src, which was partially inhibited by OPB‐9195. It is suggested from these results that carbonyl amine reaction quickly activates c‐Src, possibly through cross‐linkage of GPI‐anchored proteins or putative specific receptors. J. Cell. Biochem. 72:1–7, 1999.