Myoung Woo Lee

The involvement of oxidative stress in tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HeLa cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves as an extracellular signal triggering apoptosis in tumor cells. However, the molecular mechanisms leading to the apoptosis are largely unknown. To characterize the molecular events involved in TRAIL-induced apoptosis, we examined the association of reactive oxygen species (ROS) in human adenocarcinoma HeLa cells. In this study, we show strong ROS accumulation upon TRAIL induction, with activation of caspases, followed by apoptosis. The pre-treatment with gamma-glutamylcysteinylglycine or estrogen, both effective antioxidants, significantly attenuated TRAIL-induced apoptosis through the reduction of ROS accumulation and diminished caspases activity. Furthermore, zVAD-fmk, an inhibitor of pan-caspase, effectively inhibited the activation of caspases and prevented apoptosis by TRAIL, although TRAIL-induced ROS generation was not attenuated. These data indicate that ROS may play a role as an upstream mediator of caspases. Taken together, our results suggest that oxidative stress mediates TRAIL-induced apoptosis in HeLa cells.

C-terminal fragment of amyloid precursor protein induces astrocytosis

One of the pathophysiological features of Alzheimers disease is astrocytosis around senile plaques. Reactive astrocytes may produce proinflammatory mediators, nitric oxide, and subsequent reactive oxygen intermediates such as peroxynitrites. In the present study, we investigated the possible role of the C‐terminal fragment of amyloid precursor protein (CT‐APP), which is another constituent of amyloid senile plaque and an abnormal product of APP metabolism, as an inducer of astrocytosis. We report that 100 nm recombinant C‐terminal 105 amino acid fragment (CT105) of APP induced astrocytosis morphologically and immunologically. CT105 exposure resulted in activation of mitogen‐activated protein kinase (MAPK) pathways as well as transcription factor NF‐κB. Pretreatment with PD098059 and/or SB203580 decreased nitric oxide (NO) production and nuclear factor‐kappa B (NF‐κB) activation. But inhibitors of NF‐κB activation did not affect MAPKs activation whereas they abolished NO production and attenuated astrocytosis. Furthermore, conditioned media derived from CT105‐treated astrocytes enhanced neurotoxicity and pretreatment with NO and peroxynitrite scavengers attenuated its toxicity. These suggest that CT‐APP may participate in Alzheimers pathogenesis through MAPKs‐ and NF‐κB‐dependent astrocytosis and iNOS induction.

P53 mediates ceramide-induced apoptosis in SKN-SH cells

Ceramide induces apoptotic cell death in a dose- and time-dependent manner in neuroblastoma SKN-SH cells. Pretreatment with caspase inhibitors blocks cell death, suggesting that a set of caspase activities including caspase 1, as well as caspase 3, are involved in ceramide-induced apoptosis in SKN-SH cells. Treatment with a caspase inhibitor 3 h after ceramide addition did not inhibit cell death, although caspase activity was substantially reduced. Ceramide-induced apoptosis is accompanied by accumulation of p53 followed by an increase of Bax and decrease of Bcl-2 levels. Inhibition of p53 expression with p53 antisense oligonucleotides inhibits apoptosis and prevents the increase in Bax and decrease in Bcl-2. Furthermore, pretreatment with p53 antisense oligonucleotides markedly inhibits the induction of caspase activity. These results suggest that p53 regulates the ratio Bcl-2/Bax and the expression/activation of caspases during ceramide-induced apoptosis in SKN-SH cells. Caspase inhibition did not alter the expression of p53, Bcl-2 and Bax. Thus ceramide-induced reduction in the Bcl-2/Bax ratio, increase in caspase activity, and apoptosis is dependent upon increases in cellular p53 levels which play a critical role in the regulation of apoptotic cell death.

The activation of ERK1/2 via a tyrosine kinase pathway attenuates trail-induced apoptosis in HeLa cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves as an extracellular signal that triggers apoptosis in tumor cells. To characterize the molecular events involved in TRAIL-induced apoptotic signaling, we investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in HeLa cell death. Here we show that TRAIL-activated ERK1/2 through a tyrosine kinase-dependent pathway, subsequently elevated anti-apoptotic Bcl-2 protein levels. ERK1/2 inhibition with PD98059 promoted apoptotic cell death through the downregulation of ERK1/2 activity and Bcl-2 protein levels. Moreover, tyrosine kinase inhibition with Genistein in TRAIL-induced apoptosis effectively attenuated ERK1/2 activity and enhanced apoptotic cell death. Taken together, our results indicate that ERK1/2 activation via tyrosine kinase pathway plays a protective role as the cellular defense mechanism through the upregulation of Bcl-2 protein levels in TRAIL-induced apoptosis.

Hepatic differentiation of cord blood-derived multipotent progenitor cells (MPCs) in vitro.

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells that possesses practical and ethical advantages. We previously reported a novel UCB‐derived adult stem cells which we termed umbilical cord blood‐derived multipotent progenitor cells’ (MPCs). MPCs were capable of differentiating into functional neuronal cells. Under appropriate conditions lasting several days or weeks, we now show that the MPCs differentiate into hepatocyte‐like cells in vitro; their properties were verified using reverse transcription‐polymerase chain reaction (RT‐PCR), Western blot, immunofluorescence, periodic acid‐Schiff (PAS) staining of accumulated glycogen and an enzyme‐linked immunosorbent assay (ELISA). We also found that hepatic differentiated cells expressed hepatocyte specific markers, such as albumin, hepatocyte nuclear factor (HNF)‐1α, HNF4, cytokeratin (CK)‐8, CK‐18, tyrosine amino transferase (TAT), and CYP2B6. Moreover, albumin was secreted, which suggests that MPCs from UCB possess multi‐differentiation potential and have the capacity to differentiate into functional cells of hepatic lineage in vitro.

Effect of Ex Vivo Culture Conditions on Immunosuppression by Human Mesenchymal Stem Cells

A microarray analysis was performed to investigate whether ex vivo culture conditions affect the characteristics of MSCs. Gene expression profiles were mainly influenced by the level of cell confluence rather than initial seeding density. The analysis showed that 276 genes were upregulated and 230 genes downregulated in MSCs harvested at ~90% versus ~50% confluence (P < 0.05, FC > 2). The genes that were highly expressed in MSCs largely corresponded to chemotaxis, inflammation, and immune responses, indicating direct or indirect involvement in immunomodulatory functions. Specifically, PTGES and ULBP1 were up-regulated in MSCs harvested at high density. Treatment of MSCs with PTGES or ULBP1 siRNA reversed their inhibition of T-cell proliferation in vitro. The culture conditions such as cell confluence at harvest seem to be important for gene expression profile of MSCs; therefore, the results of this study may provide useful guidelines for the harvest of MSCs that can appropriately suppress the immune response.

Effect of Everolimus (RAD001) on Acute Graft-Versus-Host Disease in Mice.

To the Editor: Despite advances in donor HLAtyping methods and posttransplant immune suppression, acute graftversus-host disease (aGVHD) often occurs after allogeneic hematopoietic stem cell transplantation. Although aGVHD is usually treated with steroidbased immunosuppressive therapy as well as second-line therapies such as antithymocyte globulin, the risk of morbidity and mortality remains high1; therefore, alternative approaches are required. Themammalian target of rapamycin inhibitor everolimus (RAD001), which targets the causes of allograft dysfunction, offers the prospect of improved immunosuppressive management of organ transplant patients.2–4 In addition, several studies report that everolimus ameliorates chronic GVHD.5,6 However, it is unclear whether everolimus alleviates aGVHD in vivo. Here, we examined the effect of everolimus in a mouse model of aGVHD. The mouse xenograft model for assessment of aGVHD was established as follows: irradiated (3.0Gy) NOD/ SCID mice were intravenously injected with human peripheral blood mononuclear cells (2 10 cells) followed by 16 daily oral administration of phosphate buffered saline (PBS). All showed symptoms of GVHD, including weight loss, alopecia, a hunched back posture, and reduced physical activity, and all died within 46 days (median, 21 d [Fig. 1A] or 24 d [Fig. 1B]). To elucidate whether everolimus alleviates aGVHD, mice received oral everolimus (1 or 2mg/kg) daily for 16 days after transplantation of human peripheral blood mononuclear cells and survival rates were examined. Symptoms and survival of aGVHD mice that received everolimus were not significantly different from those that received PBS. Indeed, mice receiving 1 and 2mg/kg of everolimus died within 45 and 50 days, respectively (median survival, 20 and 28d, respectively; Figs. 1A and B, respectively). There was no significant difference in symptoms and survival rates between groups that received different doses of everolimus. Most cases of aGVHD result from proliferation/activation of transplanted immune cells, including T cells, which is triggered by recognition of histoincompatible antigens expressed by the recipient; this causes a variety of injuries to host tissues.1,7 Thus, strong immune suppression should be critical for alleviating GVHD. However, the results presented herein suggest that everolimus did not significantly increase survival rates, implying that its immunosuppressive properties are limited in this respect. Indeed, the mechanism underlying the immunosuppressive effects of everolimus in clinical trials and in vivo studies of immune disease is unclear. In addition, finding the correct dose may be difficult because the drug can cause significant toxicity, including sinusoidal obstruction syndrome and transplant-associated microangiopathy.3,8–10 To avoid this, everolimus could be combined with other drugs to prevent rejection of solid organ transplants; however, the narrow set of conditions examined in this study mean that we cannot rule out that different doses and dosing frequencies may yield different results. Therefore, further investigations incorporating a wide range of conditions are needed to confirm our conclusions.