Jianghong Wu

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.

Distinct involvement of NF-κB and p38 mitogen-activated protein kinase pathways in serum deprivation-mediated stimulation of inducible nitric oxide synthase and its inhibition by 4-hydroxynonenal

Cytokine‐induced expression of inducible nitric oxide synthase (iNOS) and concomitant production of nitric oxide (NO) involve activation of mitogen‐activated protein (MAP) kinases and are in most cases mediated by the transcription factor NF‐κB. We investigated the role of p38 MAP kinase activation and IκB phosphorylation in iNOS expression in a novel iNOS‐inducing model in mouse macrophages. Deprivation of serum from the culture medium of RAW 264.7 cells up‐regulated iNOS and NO production, which were inhibited by 4‐hydroxy‐2‐nonenal (HNE), a component of oxidatively modified low‐density lipoprotein (oxLDL). Serum withdrawal induced phosphorylation of Akt, IκB, and p38 MAP kinase. Pretreatment with the potent PI3 kinase inhibitor wortmannin, the NF‐κB inhibitor PDTC or the specific p38 MAP kinase inhibitor SB203580 each partially attenuated the induction of iNOS and NO production, demonstrating that both p38 activation and IκB phosphorylation are required for iNOS expression. SB203580, however, did not prevent the phosphorylation of Akt and IκB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an IκB‐phosphorylation‐independent pathway. HNE, which markedly inhibited iNOS expression and NO production, prevented the serum withdrawal‐triggered IκB phosphorylation but not that of Akt or p38 MAP kinase. A high concentration of HNE stimulated dephosphorylation of IκB but promoted activation of p38 MAP kinase. Taken together, these results suggest that NF‐κB and p38 MAP kinase lie in separate signal pathways for serum deprivation‐stimulated iNOS expression and NO production. HNE selectively suppresses the former pathway, targeting a site downstream of Akt. J. Cell. Biochem. 83: 271–280, 2001.

Cepharanthine Activates Caspases and Induces Apoptosis in Jurkat and K562 Human Leukemia Cell Lines

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy‐provoked leukopenia. We here report that apoptosis was induced by low concentrations (1–5 μM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5–10 μM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr‐Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas‐independent pathway. CEP treatment induced activation of caspase‐9 and ‐3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase‐8 activation and Akt cleavage were observed only in Jurkat cells. The CEP‐induced apoptosis was completely blocked by zVAD‐fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr‐Abl protein in K562 cells, and this degradation was prevented partially by zVAD‐fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2–5 μM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr‐Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy. J. Cell. Biochem. 82: 200–214, 2001.

Acrolein induces activation of the epidermal growth factor receptor of human keratinocytes for cell death

Acrolein, which is a highly reactive formaldehyde generated by lipid peroxidation, can affect skin and cause various disorders. The effect of exposure of human keratinocytes to acrolein on cell surface‐oriented signal transduction into cells was examined. Incubation of human keratinocytes with a relatively low concentration (50 μM) of acrolein caused a prompt and selective induction of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) as a 180‐kDa molecule during the period from 5–30 min after the start of incubation. This early event was followed by an increase in the density and number of phosphotyrosine‐containing proteins during the period from 60–120 min after the start of incubation. The catalytic activity of EGFR as measured by the levels of autophorphorylation and phosphorylation of an exogenously added substrate, casein, in in vitro kinase assay, greatly increased as early as 1 min after the start of incubation and then decreased gradually 30 min later. MAP family kinases, including ERK, JNK, and p38 kinase, and the potentially downstream transcription factor c‐Jun were all promoted for phosphorylation/activation during a period of 5–30 min. Selective prompt phosphorylation/activation of EGFR followed by phosphorylation of MAP family kinases and c‐Jun and their blockade by a specific EGFR inhibitor, AG1478, suggested that activation of EGFR is the major, and possibly single, cell surface element for intracellular signal transduction in acrolein‐treated cells. Incubation of human keratinocytes with 50 μM of acrolein induced atypical apoptosis with morphologic apoptotic features with low‐grade oligonucleoside‐sized DNA fragmentation. Partial inhibition of such a cytopathic effect of acrolein on human keratinocytes by preincubation with AG1478 suggests the involvement of an EGFR‐mediated signal pathway for atypical apoptosis. These results provide new information on acrolein‐induced cell surface‐oriented signal transduction to human keratinocytes, and this information may be useful for understanding the pathogenesis of a number of skin diseases in response to environmental acrolein and acrolein‐generating ultraviolet irradiation. J. Cell. Biochem. 81: 679–688, 2001.

Caspase activation is accelerated by the inhibition of arsenite-induced, membrane rafts-dependent Akt activation

Renewed interest in arsenic has been shown recently due to its dual nature of being a potent toxin and a drug for treatment of acute promyelocytic leukemia (APL) because of its ability to trigger caspase activation. Here, we found that sodium arsenite (NaAsO(2)) also triggers the signal for activation of Akt and downstream glycogen synthase 3beta (GSK3beta). Such Akt/GSK3beta activation was abrogated completely by wortmannin, an inhibitor of PI-3 kinase, and greatly by pertussis toxin, a G-protein inhibitor. Arsenite-induced Akt phosphorylation also was inhibited by sequestrating membrane cholesterol with beta cyclodextrin. Reducing reagents/reactive oxygen species (ROS) scavengers reduced arsenite-induced Akt phosphorylation and beta cyclodextrin reduced arsenite-mediated ROS production, suggesting that arsenite-induced G-protein/Akt/GSK3beta pathway is membrane raft dependent and redox linked. We also found that a combination of a low concentration (1 microM) of arsenite and wortmannin triggers the signal for caspase activation, whereas neither of these elements alone did so. These results suggested that selective blockade of the arsenite-provoked PI-3 kinase/Akt pathway can promote the arsenite-triggered pathway for caspase activation, and this may open a new study area for wider applications of arsenic as a drug for treating various kinds of leukemia.

Modes of activation of mitogen-activated protein kinases and their roles in cepharanthine-induced apoptosis in human leukemia cells.

We previously showed that cepharanthine (CEP), a biscoclaurine alkaloid, induces caspase-dependent and Fas-independent apoptosis in Jurkat and K562 human leukemia cells. In the present study, we investigated the effect of CEP on three groups of human mitogen-activated protein kinases (MAPKs) in relation to CEP-induced apoptosis. CEP, at the concentration required for and at the time of induction of apoptosis, activated MAPKs p38 in both Jurkat and K562 cells and activated extracellular signal-regulated kinases (ERKs) only in K562 cells. However, CEP treatment did not trigger c-Jun NH(2)-terminal kinases (JNKs) activation. CEP increased the expression and phosphorylation levels of c-Jun and ATF-2 transcription factors. zVAD-fmk, a general caspase inhibitor, did not inhibit CEP-triggered p38 activation in Jurkat and K562 cells or ERK activation in K562 cells. Unexpectedly, pretreatment with a specific p38 inhibitor, SB203580, promoted CEP-induced apoptosis and caspase activation in Jurkat and K562 cells, whereas pretreatment with an MEK-1 inhibitor PD98059 inhibited CEP-induced apoptosis and caspase activation in K562 cells. A selective tyrosine kinase inhibitor, herbimycin A, which completely inhibited CEP-triggered ERKs activation, clearly promoted CEP-induced c-Jun expression and phosphorylation. Our results suggest that each of the three groups of MAP family members is uniquely involved in the CEP-mediated signal cascades in two different leukemia cell lines for inducing/regulating caspase activation and DNA fragmentation.

Activation of caspase-8 is critical for sensitivity to cytotoxic anti-Fas antibody-induced apoptosis in human ovarian cancer cells

Two ovarian cancer cell lines named NOS4 and SKOV-3 have been shown to have different sensitivities to a cytotoxic anti-Fas antibody, CH-11. Although both cell lines express Fas molecules on the cell surfaces at the same intensities, apoptosis is induced by CH-11 in NOS4 cells but not in SKOV-3 cells. In this study, the different apoptosis-sensitivities of these cells were assessed. Both cell lines express almost the same levels of FADD, RIP, c-FLIP, FAP-1, Bax, Bcl-2 and Bcl-XL. Evidence of caspase-8, caspase-9 and caspase-3 activation and of cleavage of PARP and Bid was obtained in NOS4 cells but not in SKOV-3 cells. When triggered by FasL protein, DNA fragmentation and caspase-8 activation were observed in SKOV-3 cells, though they were not as clear as in NOS4 cells. All the anti-Fas antibody-mediated signals for apoptosis induction in NOS4 cells were completely blocked by a caspase-8-specific inhibitor, Z-IETD-FMK. These results indicate that the different sensitivities to the anti-Fas antibody are solely dependent on the activation of caspase-8, which could be influenced by yet unknown qualitative or quantitative abnormalities in molecules involved in DISC formation.

Osmotic Stress-Mediated Activation of RET Kinases Involves Intracellular Disulfide-Bonded Dimer Formation

We showed that osmotic stress induces activation of c-RET and second-set activation of constitutively activated RET-MEN2B. A few percentage of RET proteins normally formed disulfide-bonded dimers in the cell, and osmotic stress promoted formation of these dimers. The disulfide-bonded dimers displayed higher levels of autophosphorylation and catalytic activity per molecule than did monomers. Osmotic stress also promoted activation and disulfide-bonded dimerization of the extracellular domain-depleted mutant RET (RET-PTC-1), suggesting that the target amino acid(s) for dimerization is located intracellularly rather than in the cysteine-rich region of the extracellular domain. In the mutant c-RET and RET-PTC-1 in which Cys987 of c-RET or Cys376 of RET-PTC-1 was replaced with Ala, the levels of intrinsic kinase activity were greatly reduced and barely increased in response to osmotic stress. Correspondingly, the Cys376-defective RET-PTC-1 did not form any demonstrable levels of dimers even after exposure to osmotic stress. In contrast, another RET-PTC-1 mutant that had a replacement of Cys365 with Ala mostly behaved like parental RET-PTC-1. These results suggest that Cys987 of c-RET or Cys376 of RET-PTC-1 plays a crucial role in maintenance and promotion of dimerization and activation of the RET kinases.

Murine lymph node‐derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

Little is known about the homeostatic mechanisms by which the levels of peripheral lymphocytes are maintained. The survival of naïve T cells in vivo must be maintained by some factors that have not been characterized in an in vitro culture system. In this study, we established a culture system of stromal cells derived from murine lymph nodes and investigated the action of the stromal cells in supporting the survival of resting T cells in vitro. Most of the T cells cocultured with the stromal cells did not die, and the supernatant of cultured stromal cells increase the viability of T cells. This T‐cell survival‐supporting activity was maintained for more than 7 days. Although interleukin (IL)‐4, IL‐6, IL‐7, and interferon‐β also rescued peripheral T cells from spontaneous cell death, medium‐soluble and heat‐sensitive factor(s) derived from the stromal cells supported the survival of T cells more effectively and for a longer time than did these cytokines. T cells maintained in the culture system with the stromal cells appeared to remain in a resting G0/G1 state and did not show remarkable DNA synthesis. From these results, it is presumed that some soluble factor(s) other than the tested cytokines that have been identified as supporting T‐cell survival are produced from lymph node stromal cells. These factor(s) play an important role in maintenance of resting T cells.

Involvement of MKK6 in TCRαβintCD69lo: a target population for apoptotic cell death in thymocytes

By analyzing real‐time caspase activity and DNA fragmentation in live thymocytes, we found that apoptosis occurs predominantly in a TCRαβint/hiCD69lo pulation. The number of caspase‐active cells and DNA‐fragmented cells in MKK6‐deficient mice, which were originally generated in our laboratory by gene targeting, was decreased in the TCRαβintCD69lo population but not in the TCRαβhiCD69lo population. The percentage of caspase‐active cells in the H‐Y‐specific TCRint population was more clearly decreased in male MKK6‐deficient H‐Y TCR‐transgenic mice. Furthermore, the absolute number of TCRhiCD4loCD8lo cells, which are developmentally next to TCRintCD4hiCD8hi cells, was increased in MKK6‐deficient H‐Y TCR‐transgenic mice. Deletion of TCRαβintCD4hiCD8hi cells by injecting antigenic lymphocytic chorio‐meningitis virus (LCMV) peptide into LCMV‐specific TCR‐transgenic mice was incomplete in MKK6‐deficient mice. Cellular death of TCRαβint fetal thymocytes induced by adding an antigenic peptide into an in vitro fetal thymic organ culture system was also diminished in MKK6‐deficient TCR‐transgenic thymi. These results indicate that MKK6 plays a role in the developing thymocytes, especially in the population of TCRαβintCD69lo cells, which possibly undergo negative selection.