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Background: Methylation-mediated silencing of genes is one epigenetic mechanism implicated in cancer. Studies regarding the role of modulation of gene expression utilizing inhibitors of DNA methylation, such as decitabine, in osteosarcoma (OS) have been limited. A biological understanding of the overall effects of decitabine in OS is important because this particular agent is currently undergoing clinical trials. The objective of this study was to measure the response of the OS cell line, U2OS, to decitabine treatment both in vitro and in vivo. Results: Microarray expression profiling was used to distinguish decitabine-dependent changes in gene expression in U2OS cells, and to identify responsive loci with demethylated CpG promoter regions. U2OS xenografts were established under the sub-renal capsule of immune-deficient mice to study the effect of decitabine in vivo on tumor growth and differentiation. Reduced nuclear methylation levels could be detected in xenografts derived from treated mice by immunohistochemistry utilizing a 5-methylcytidine antibody. Decitabine treatment reduced tumor xenograft size significantly (p < 0.05). Histological analysis of treated U2OS xenograft sections revealed a lower mitotic activity (p < 0.0001), increased bone matrix production (p < 0.0001), and a higher number of apoptotic cells (p = 0.0329). Microarray expression profiling of U2OS cultured cells showed that decitabine treatment caused a significant induction (p < 0.0025) in the expression of 88 genes. Thirteen had a ≥2-fold change, 11 of which had CpG-island associated promoters. Interestingly, 6 of these 11 were pro-apoptotic genes and decitabine resulted in a significant induction of cell death in U2OS cells in vitro (p < 0.05). The 6 pro-apoptotic genes (GADD45A, HSPA9B, PAWR, PDCD5, NFKBIA, and TNFAIP3) were also induced to ≥2-fold in vivo. Quantitative methylation pyrosequencing confirmed that the tested pro-apoptotic genes had CpG-island DNA demethylationas a result of U2OS decitabine treatment both in vitro and in xenografts Conclusion: These data provide new insights regarding the use of epigenetic modifiers in OS, and have important implications for therapeutic trials involving demethylation drugs. Collectively, these data have provided biological evidence that one mode of action of decitabine may be the induction of apoptosis utilizing promoter-CpG demethylation of specific effectors in cell death pathways in OS. [ABSTRACT FROM AUTHOR]

Background: Advances made in the area of microarray comparative genomic hybridization (aCGH) have enabled the interrogation of the entire genome at a previously unattainable resolution. This has lead to the discovery of a novel class of alternative entities called large-scale copy number variations (CNVs). These CNVs are often found in regions of closely linked sequence homology called duplicons that are thought to facilitate genomic rearrangements in some classes of neoplasia. Recently, it was proposed that duplicons located near the recurrent translocation break points on chromosomes 9 and 22 in chronic myeloid leukemia (CML) may facilitate this tumor-specific translocation. Furthermore, ~15— 20% of CML patients also carry a microdeletion on the derivative 9 chromosome (der(9)) and these patients have a poor prognosis. It has been hypothesised that der(9) deletion patients have increased levels of chromosomal instability. Results: In this study aCGH was performed and identified a CNV (RP11-125A5, hereafter called CNV14q12) that was present as a genomic gain or loss in 10% of control DNA samples derived from cytogenetically normal individuals. CNV14q12 was the same clone identified by Iafrate et al. as a CNV. Real-time polymerase chain reaction (Q-PCR) was used to determine the relative frequency of this CNV in DNA from a series of 16 CML patients (both with and without a der(9) deletion) together with DNA derived from 36 paediatric solid tumors in comparison to the incidence of CNV in control DNA. CNV14q12 was present in ~50% of both tumor and CML DNA, but was found in 72% of CML bearing a der(9) microdeletion. Chi square analysis found a statistically significant difference (p ≤ 0.001) between the incidence of this CNV in cancer and normal DNA and a slightly increased incidence in CML with deletions in comparison to those CML without a detectable deletion. Conclusion: The increased incidence of CNV14q12 in tumor samples suggests that either acquired or inherited genomic variation of this new class of variation may be associated with onset or progression of neoplasia. [ABSTRACT FROM AUTHOR]

Background While T-cell immune responses are recognized as important determinants of acute hepatitis C virus (HCV) infection outcome in non-pregnant adults, little is known about HCV-specific T-cell immunity during pregnancy or its impact on vertical transmission. Here we describe the temporal course of T-cell responses and HCV replication in a patient with acute HCV infection during pregnancy. Materials and methods A 34 year old pregnant woman was diagnosed with acute genotype 1a HCV infection when she presented with jaundice at 24 weeks gestation. She delivered at 34 weeks gestation. T-cell immunity, plasma viremia, and liver function (ALT) were assessed serially from week 28 gestation through week 26 post-partum. HCV-specific T-cell activity in peripheral blood was quantified by ex vivo interferon-gamma ELISpot using overlapping peptides spanning the entire HCV polyprotein. Results During pregnancy, HCV viral load (IU/mL) rose from 1.3 million at 24 weeks gestation to 25.4 million at 34 weeks gestation (delivery). High viremia during pregnancy was associated with low frequency T-cell responses restricted to HCV NS3 and NS5b proteins. At week 8 post-partum the viral load fell sharply to 11,400, but rebounded to 16.6 million at week 26. The transient 1000-fold drop in viremia in the early post-partum period was accompanied by a transient 5-fold increase in T-cell frequency with broadening of the response to most viral proteins. T-cell activity returned to low levels by week 26 post-partum. Failing T-cell immunity accompanied by a climb in viral load in the months after delivery may predict a persistent course of infection for the mother. Preliminary data also indicate that the baby was infected in the perinatal period. Comparisons of HCV evolution and immune responses in the mother and child are ongoing. Conclusions In this case of acute HCV infection during pregnancy, T-cell responses increased prominently in frequency and breadth in the early post-partum period, and were associated with a sharp fall in viral load. This surge occurred several months after the onset of symptoms, suggesting that it was not the initial primary T-cell immune response to HCV, but perhaps related to a post-partum phenomenon. This finding needs to be corroborated in further studies of HCV infection during pregnancy, as it may afford new insights into T-cell control of HCV and maternal influences on vertical HCV transmission. [ABSTRACT FROM AUTHOR]