In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34؉ cells using a three-phase culture system. We first cultured 500 CB CD34

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We have identified TNFSF10 (TRAIL) as a p53-transcriptional target gene. There are two p53 DNA-binding sites in the human TNFSF10 promoter region, at 346 and 625 bp upstream of the transcription start site. A human p53-expressing adenovirus (Ad-p53) induced TRAIL mRNA and protein expression in HCT116 p53-/- human colon cancer cells. A human TRAIL-promoter reporter assay showed increased luciferase activity with the promoter vector that contains two p53 DNA-binding motifs,following Ad-p53 infection, compared to the control adenovirus infection. Using HCT116 cells, gene silencing of TNFSF10 by siRNA suppressed caspase 3 and 7 activity, even after treatment with the DNA-damaging chemotherapeutic agent adriamycin. TRAIL protein expression was elevated in adriamycin-treated breast cancer cells. In vivo, TRAIL expression was induced in mouse natural killer cells at 24 hours after systemic treatment with 5-Fluorouracil. p53-dependent TRAIL induction in natural killer cells after chemotherapy exposure provides a link between the tumor suppressor p53 and the host immune response during cancer therapy as well as a paracrine-mediated cell-extrinsic death response. Our findings provide new mechanistic insights into the signaling of p53-dependent cell death and tumor suppression, including the involvement of the host immune system and natural killer cells in vivo in the anti-tumor efficacy of chemotherapy.

Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-l-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.

The p53 pathway is targeted for inactivation in most human cancers either directly or indirectly, highlighting its critical function as a tumor suppressor gene. p53 is normally activated by cellular stress and mediates a growth-suppressive response that involves cell cycle arrest and apoptosis. In the case of cell cycle arrest, p21 appears sufficient to block cell cycle progression out of G1 until repair has occurred or the cellular stress has been resolved. The p53-dependent apoptotic response is more complex and involves transcriptional activation of multiple proapoptotic target genes, tissue, and signal specificity, as well as additional events that are less well understood. In this chapter, we summarize the apoptosis pathway regulated by p53 and include some open questions in this field.

Abstract. Few target molecules have been identified that enable the diagnosis of lung cancer with high sensitivity and specificity, especially in the early clinical stages. Herein, we present the first evidence for mRNA overexpression of SALL4, a transcription factor essential for embryonic development and the self-renewal of embryonic stem cells, in lung cancer. Analysis using cancerous and noncancerous tissues revealed that the sensitivity and specificity of SALL4 mRNA were 85.1 and 92.9%, respectively, estimated using the cutoff value obtained from analyzing the receiver operating characteristic curve. Furthermore, comparison of paired tissues from the same patient revealed elevated SALL4 mRNA levels that were greater than two-fold in 93% of the specimens. SALL4 mRNA was highly expressed even in the early clinical stages and there was no difference in the positivity rate between stage IA and other stages. An siRNA approach to determine the significance of SALL4 expression revealed catastrophic growth inhibition of SBC-1 lung cancer cells that was induced by cell cycle arrest at the G1/early S phase. Therefore, SALL4 mRNA may be a candidate for use as support in the diagnosis of lung cancer, and may also represent a therapeutic target.

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Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we performed a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds we identified were a structurally-related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and CDK1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator Hif-1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.