Alternative Fas-mediated cell death pathway is dependent on the different cleavage patterns of procaspase-8.

It has been reported that mitochondria-independent or mitochondria-dependent (type I/II) Fas signaling pathways in leukemia cells depend on the amount of active caspase-8. However, Bid molecules, which could not be cleaved in type I cells, could be effectively cleaved by recombinant active caspase-8 in vitro. The cleavage of recombinant Bid by recombinant active caspase-8 could be blocked by anti-p10 and anti-p18 specific antibodies. Fas receptors could be similarly internalized into cytoplasm in type I and type II cells. Interestingly, p10 subunit of active caspase-8 could be detected in both type I and II cells, while p18 subunit of active caspase-8 could be detected only in type II cells but not in type I cells. These results demonstrated that p18 subunit was necessary for Bid cleavage and the mitochondria pathway might be dependent on the release of p18 subunit from active caspase-8.

Resistance to TRAIL-induced apoptosis caused by constitutional phosphorylation of Akt and PTEN in acute lymphoblastic leukemia cells.

OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, which induces apoptosis in cancer cells but not in normal cells. Akt/protein kinase B, when phosphorylated to its active form, promotes cell survival and blocks apoptosis. The aim of this study was to investigate the role of Akt pathway in acquired TRAIL resistance of acute lymphoblastic leukemia cells. MATERIALS AND METHODS: MB-IT and NALM-24 cells that developed resistance to TRAIL, i.e., TRAIL-resistant cells (MB-IT R and NALM-24 R) were established from TRAIL-sensitive acute lymphoblastic leukemia cell lines (MB-IT S and NALM-24 S), respectively, through application of TRAIL and repetitive limiting dilution. Apoptosis was measured by flow cytometry using propidium iodide/Annexin-V fluorescein isothiocyanate staining. TRAIL receptor cell surface expression of MB-IT and NALM-24 were analyzed by flow cytometry. Protein levels were analyzed by Western blot analysis. RESULTS: The obtained resistant cell lines presented the same pattern of receptor expression as sensitive parent cells, and the internalization of DR5 after TRAIL treatment was similar. Caspase-8/3, FLIP, BID, XIAP were cleaved/downregulated in sensitive cells after treatment with TRAIL, but not in the resistant cells. We also observed that phosphoinositide-3-kinase (PI3K)/Akt pathway was constitutively active in resistant clones, and was not downregulated upon TRAIL treatment. Phosphate and tensin homologue deleted on chromosome 10 (PTEN) level was the same in both sensitive cells and resistant cells, but was quickly downregulated in sensitive cells after TRAIL treatment. Also, resistant cells expressed a high level of phosphorylated inactive form of PTEN than the sensitive cells. Expression levels of PH domain leucine-rich repeat protein phosphatase were slightly higher in sensitive than resistant cells. When resistant cells were treated with LY 294002 (a PI3K inhibitor), the expression level of phosphorylated Akt was distinctly downregulated, and there was induction of apoptosis when these cells were treated with a combination of TRAIL and LY 294002. When MB-IT-sensitive cells were treated with okadaic acid, a phosphatase inhibitor, TRAIL-induced apoptosis was significantly reduced. CONCLUSION: These results suggest that cellular resistance to TRAIL could be developed through phosphorylation (activation) of Akt and phosphorylation (inactivation) of PTEN.

A nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate ameliorates pulmonary hypertension in rats.

BACKGROUND: Pulmonary hypertension (PH) is a fatal disorder that is associated with structural changes and inflammatory responses in the pulmonary vasculature. Nuclear factor (NF)-kappaB is a key transcription factor that is involved in the tissue remodeling mediated by inflammatory and fibroproliferative responses. However, the contribution of NF-kappaB-mediated inflammatory pathways to the development of PH is unknown. METHODS: We therefore investigated whether NF-kappaB activation and the expression of a downstream product vascular cell adhesion molecule (VCAM)-1 is associated with pulmonary vascular diseases in rats that have been injected with the toxin monocrotaline (MCT), and whether a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), ameliorates such diseases in rats. RESULTS: VCAM-1 expression and the nuclear localization of the p65 subunit of NF-kappaB, as analyzed immunohistochemically, was significantly up-regulated in the endothelium of diseased vessels on the days 8 to 22 (p < 0.05). Next, 39 rats were divided into three groups (rats injected with MCT and treated with saline solution or PDTC, and controls similarly treated with saline solution). Compared to controls, MCT treatment increased the mean (+/- SE) pulmonary artery pressure (31.2 +/- 1.4 mm Hg [p < 0.05] vs 22.8 +/- 0.9 mm Hg, respectively), which was reduced by PDTC treatment (24.3 +/- 1.2 mm Hg; p < 0.05). Indexes of right ventricular hypertrophy and pulmonary vascular diseases induced by MCT were similarly inhibited (p < 0.05), which was associated with the suppression of VCAM-1 expression and macrophage infiltration. CONCLUSIONS: We concluded that the NF-kappaB nuclear localization and VCAM-1 expression is temporally and spatially associated with the development of MCT-induced PH in rats, which was ameliorated by administering a NF-kappaB inhibitor, PDTC.

Discrimination of acute graft-versus-host disease from infections by enumeration of peripheral blood interferon-gamma spot-forming cells.

Infections may coexist and in certain circumstances aggravate acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation. Early detection of aGVHD is often difficult in patients with concurrent infections. Using an enzyme-linked immunospot assay that reflects ongoing immune status in vivo, we enumerated spot-forming cells (SFCs) for interferon (IFN)-gamma, interleukin (IL)-4, and IL-12 in peripheral blood from 56 patients with hematological disorders. Eleven patients had viral, fungal, or bacterial systemic infections during first 10 weeks posttransplant. Of these, six patients with grade 0-I aGVHD showed normal levels of IFN-gamma SFCs. On the other hand, IFN-gamma SFCs were elevated in five patients with grade II-IV aGVHD. These data indicate that increased IFN-gamma SFCs seemed to be correlated with clinically significant aGVHD, but not with infection itself. IL-4 and IL-12 SFCs increased in some patients with infections, irrespective of the presence of aGVHD. Thus, IFN-gamma SFCs may be used to distinguish systemic infections from aGVHD.

Development of a dendritic cell vaccine against measles for patients following hematopoietic cell transplantation.

Most patients who have undergone hematopoietic cell transplantation (HCT) lose specific immunity to measles. However, due to its immunosuppressive potential, it has been recommended that a live attenuated measles vaccination be administered two years following HCT. Measles virus (MV) glycoproteins including hemagglutinin (HA) are expressed on MV-infected dendritic cells (DCs), and they impair efficient antigen presentation between the DC and T cell. We produced a DC-based vaccine against MV by loading DCs with MV-infected autologous DCs. MV in the infected DCs was inactivated using ultraviolet-B. The DC-based vaccine neither expressed HA nor inhibited allogeneic T cell proliferation, while it induced the production of interferon-gamma (IFN-gamma) by autologous CD4 and CD8 naive T cells ex vivo. Importantly, the vaccine derived from patients who had undergone HCT also efficiently induced IFN-gamma producing cells. These findings indicate that our DC-based MV vaccine induces MV-specific immunity even in post-HCT patients without causing immunosuppression.

Cell cycle dependency of caspase activation in Fas-induced apoptosis in leukemia cells.

The relationship between apoptosis and the cell cycle remains unclear. In the present study we have investigated the relationship between cell cycle progression and the activation of caspases (caspase-3 and caspase-8) in Fas (CD95)-mediated apoptosis in asynchronously growing leukemia cells. We found that cells expressing the active form of caspase-3 were cyclin A/B1 and Ki-67 negative but cyclin E positive, whereas expression of the active form of caspase-8 was detected in cyclin A/B1/E-negative and Ki-67-negative cells. In addition, both the activation of caspases and Fas-mediated apoptosis were completely abolished when leukemia cells were arrested in early G1 phase. Using post-sorting western blot analysis, we demonstrated that caspase-3 and caspase-8 were activated in p27-negative cells. These results suggest that caspase-3 would be activated in cells entering into late G1 or early S phase, and caspase-8 would be activated in middle or late G1 phase. The speed of cell cycle progression from G1 to S phase might be influential in the speed of caspase activation and induction of Fas-mediated apoptosis.