No abstract

Etoposide is a widely used anticancer drug successfully used for the treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia involving the mixed-lineage leukemia (MLL) gene (11q23) translocations. Previous studies demonstrated that the phenoxyl radical of etoposide can be produced by action of myeloperoxidase (MPO), an enzyme found in developing myeloid progenitor cells, the likely origin for myeloid leukemias. We hypothesized, therefore, that one-electron oxidation of etoposide by MPO to its phenoxyl radical is important for converting this anticancer drug to genotoxic and carcinogenic species in human CD34ϩ myeloid progenitor cells. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34 ϩ cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In separate studies, etoposide-induced DNA damage and MLL gene rearrangements were demonstrated to be dependent in part on MPO activity in CD34 ϩ cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34 ϩ cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia.

IntroductionStudies were conducted to determine if y8 T (9). Toxoplasma gondii is an intracellular parasite that has become a major opportunistic pathogen in patients with defects in cell-mediated immunity (10, 11). These patients are at risk of developing reactivation of a chronic (latent) T. gondii infection which is usually manifested as toxoplasmic encephalitis (10, 11). T cells have been shown to play a critical role in protection against T. gondii in mice (12, 13) and the relatively high incidence of toxoplasmosis in patients with a defect in T cell function (e.g., patients with Hodgkin's disease and AIDS) provides indirect evidence for the role of T cells in resistance against this parasite in humans ( 10,11).The fact that the natural route of infection with T. gondii is through oral ingestion of the parasite would suggest that y6 T cells, which are known to be present in the intestinal mucosa, may be one of the first immune cells that would interact with the parasite. Therefore, we were interested to determine whether y6 T cells play a role in the immune response against T. gondii.In the present study, we show that human peripheral blood y6 T cells from either T. gondii-seropositive or-seronegative subjects are directly activated by incubation with cells that contain intracellular tachyzoites of T. gondii. This

MicroRNAs (miRNAs) inhibit expression of target genes by binding to their RNA transcripts. It has been recently shown that RNA transcripts targeted by the same miRNA could “compete” for the miRNA molecules and thereby indirectly regulate each other. Experimental evidence has suggested that the aberration of such miRNA-mediated interaction between RNAs—called competing endogenous RNA (ceRNA) interaction—can play important roles in tumorigenesis. Given the difficulty of deciphering context-specific miRNA binding, and the existence of various gene regulatory factors such as DNA methylation and copy number alteration, inferring context-specific ceRNA interactions accurately is a computationally challenging task. Here we propose a computational method called Cancerin to identify cancer-associated ceRNA interactions. Cancerin incorporates DNA methylation, copy number alteration, gene and miRNA expression datasets to construct cancer-specific ceRNA networks. We applied Cancerin to three cancer datasets from the Cancer Genome Atlas (TCGA) project. Our results indicated that ceRNAs were enriched with cancer-related genes, and ceRNA modules in the inferred ceRNA networks were involved in cancer-associated biological processes. Using LINCS-L1000 shRNA-mediated gene knockdown experiment in breast cancer cell line to assess accuracy, Cancerin was able to predict expression outcome of ceRNA genes with high accuracy.

Studies to determine if Toxoplasma gondii-specific human T cells lyse parasite-infected cells have yielded conflicting results. Furthermore, attempts to obtain human cytotoxic CD8 ؉ T lymphocytes have been difficult because of the lack of a reproducible system for their generation. By using paraformaldehyde-fixed, T. gondii-infected peripheral blood mononuclear cells as antigen-presenting cells, we developed a method whereby T. gondii-specific T-cell lines can be reproducibly generated. Six T. gondii-specific T-cell lines were generated from an individual chronically infected with T. gondii. Cytofluorometric analysis of these lines revealed >99% CD3 ؉ , 85 to 95% CD3 ؉ ␣␤ T-cell-receptor-positive (TCR ؉), 5 to 9% CD3 ؉ ␥␦ TCR ؉ , 50 to 70% CD4 ؉ , and 20 to 40% CD8 ؉ cells when cells were examined during the first 3 weeks of stimulation and >99% CD3 ؉ , >99% CD3 ؉ ␣␤ TCR ؉ , <1% CD3 ؉ ␥␦ TCR ؉ , 20 to 40% CD4 ؉ , and 60 to 80% CD8 ؉ cells when cells were examined between 5 and 11 weeks. Both CD4 ؉ and CD8 ؉ T cells had remarkable cytotoxic activity against T. gondiiinfected target cells (30 to 50% specific Cr release at an effector-to-target ratio of 30:1) but not against uninfected target cells (<10% at an effector-to-target ratio of 30:1). Cytotoxic activity by the whole T-cell lines was not T. gondii strain specific. Whole T-cell lines were cytotoxic for target cells infected with the C56 and ME49 strains and the RH strain (which was used to infect peripheral blood mononuclear cells). T. gondiispecific T-cell lines displayed the predominant expression of V␤7 TCR. The CDR3 regions of the V␤7 TCRs of these T-cell lines showed a striking degree of sequence identity (oligoclonality). T-cell lines obtained by the method reported here can be used to characterize functional activity of T-lymphocyte subsets in humans infected with T. gondii.