Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines.

BACKGROUND: Epigenetic alterations are inherent to cancer cells, and epigenetic drugs are currently primarily used to treat hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and also exhibit epigenetic alterations involving e.g. apoptotic pathways, which suggests that these tumors may also be sensitive to epigenetic drugs. This notion prompted us to assess molecular and functional effects of low dosage epigenetic drugs in neuro-ectodermal tumor-derived cell lines of pediatric origin. RESULTS: In 17 neuroblastoma (NBL) and 5 peripheral primitive neuro-ectodermal tumor (PNET) cell lines a combination treatment of 5-aza-2'-deoxycytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages was found to reduce proliferation and to induce wide-spread DNA demethylation, accompanied by major changes in gene expression profiles. Approximately half of the genes that were significantly up-regulated upon treatment exhibited a significant demethylation in their promoter regions. In the NBL cell lines, almost every cellular pathway (193/200) investigated showed expression alterations after treatment, especially a marked up-regulation of genes in the p53 pathway. The combination treatment also resulted in up-regulation of known epigenetically regulated genes such as X-chromosomal genes, tissue-specific genes and a limited number of imprinted genes, as well as known tumor suppressor genes and oncogenes. CONCLUSIONS: Nanomolar dosages of epigenetic drugs have a dramatic impact on the genomes of neuro-ectodermal tumor-derived cell lines, including alterations in DNA methylation and concomitant alterations in gene expression.

A novel and fast normalization method for high-density arrays.

BACKGROUND: Among the most commonly applied microarray normalization methods are intensity-dependent normalization methods such as lowess or loess algorithms. Their computational complexity makes them slow and thus less suitable for normalization of large datasets. Current implementations try to circumvent this problem by using a random subset of the data for normalization, but the impact of this modification has not been previously assessed. We developed a novel intensity-dependent normalization method for microarrays that is fast, simple and can include weighing of observations. RESULTS: Our normalization method is based on the P-spline scatterplot smoother using all data points for normalization. We show that using a random subset of the data for normalization should be avoided as unstable results can be produced. However, in certain cases normalization based on an invariant subset is desirable, for example, when groups of samples before and after intervention are compared. We show in the context of DNA methylation arrays that a constant weighted P-spline normalization yields a more reliable normalization curve than the one obtained by normalization on the invariant subset only. CONCLUSIONS: Our novel intensity-dependent normalization method is simpler and faster than current loess algorithms, and can be applied to one- and two-colour array data, similar to normalization based on loess. AVAILABILITY: An implementation of the method is currently available as an R package called TurboNorm from www.bioconductor.org.

High anaplastic lymphoma kinase immunohistochemical staining in neuroblastoma and ganglioneuroblastoma is an independent predictor of poor outcome.

Anaplastic lymphoma kinase (ALK) mutations occur in 3% to 11% of neuroblastoma (NBL) cases and are associated with high ALK levels. However, high ALK levels appear to be a mutation-independent hallmark of NBL. Evidence about the prognostic relevance of ALK mutations and ALK tumor positivity in patients with NBL has been inconclusive. In this study, we investigated the prognostic relevance of ALK positivity by IHC and ALK mutation status by PCR sequencing in 71 NBL, 12 ganglioneuroblastoma (GNBL), and 20 ganglioneuroma samples in a multivariate model. ALK mutations were present in 2 of 72 NBL and 2 of 12 GNBL samples, which all contained many ALK-positive cells (>50%). In addition, half of all NBL samples showed ALK positivity in most (>50%) of tumor cells, whereas half of the GNBL showed staining in <20% of the tumor cells. In most ganglioneuroma samples, a low percentage of tumor cells stained positive for ALK, which mainly involved ganglion cells. Higher percentages of ALK-positive cells in NBL and GNBL patient samples correlated with inferior survival in univariate and multivariate analyses with established prognostic factors, such as stage, age, and MYCN status. In conclusion, ALK positivity by IHC is an independent, poor prognostic factor in patients with GNBL and NBL. ALK IHC is an easy test suitable for future risk stratification in patients with NBL and GNBL.

Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels.

BACKGROUND: In pediatric neuroblastoma (NBL), high anaplastic lymphoma kinase (ALK) levels appear to be correlated with an unfavorable prognosis, regardless of ALK mutation status. This suggests a therapeutic role for ALK inhibitors in NBL patients. We examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated and wild type (WT) NBL cell lines. METHODS: We measured protein levels by western blot and ALK inhibitor sensitivity (TAE684) by viability assays in 19 NBL cell lines of which 6 had a point mutation and 4 an amplification of the ALK gene. RESULTS: ALK 220 kDa (p = 0.01) and ALK 140 kDa (p = 0.03) protein levels were higher in ALK mutant than WT cell lines. Response to ALK inhibition was significantly correlated with ALK protein levels (p < 0.01). ALK mutant cell lines (n = 4) were 14,9 fold (p < 0,01) more sensitive to ALK inhibition than eight WT cell lines. CONCLUSION: NBL cell lines often express ALK at high levels and are responsive to ALK inhibitors. Mutated cell lines express ALK at higher levels, which may define their superior response to ALK inhibition.

Bovine herpesvirus 1 UL49.5 protein inhibits the transporter associated with antigen processing despite complex formation with glycoprotein M.

Bovine herpesvirus 1 (BHV-1) interferes with peptide translocation by the transporter associated with antigen processing (TAP). Recently, the UL49.5 gene product of BHV-1 was identified as the protein responsible for the observed inhibition of TAP. In BHV-1-infected cells and virions, the UL49.5 protein forms a complex with glycoprotein M (gM). Hence, it was investigated whether UL49.5 can combine the interactions with gM and the TAP complex. In cell lines constitutively expressing both UL49.5 and gM, UL49.5 appears to be required for functional processing of gM. Immunofluorescence-confocal laser scanning microscopy demonstrated that both proteins are interdependent for their redistribution from the endoplasmic reticulum to the trans-Golgi network. Remarkably, expression of cloned gM results in the abrogation of the UL49.5-mediated inhibition of TAP and prevents the degradation of the transporter. However, in BHV-1-infected cells, differences in UL49.5 and gM expression kinetics were seen to create a window of opportunity at the early stages of infection, during which time the UL49.5 protein can act on TAP without gM interference. Moreover, in later periods, non-gM-associated UL49.5 can be detected in addition to the UL49.5/gM complex. Thus, it has been deduced that different functions of UL49.5, editing of gM processing and inhibition of TAP, can be combined during BHV-1 infection.

Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing.

Detection and elimination of virus-infected cells by cytotoxic T lymphocytes depends on recognition of virus-derived peptides presented by MHC class I molecules. A critical step in this process is the translocation of peptides from the cytoplasm into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Here, we identified the bovine herpesvirus 1-encoded UL49.5 protein as a potent inhibitor of TAP. The expression of UL49.5 results in down-regulation of MHC class I molecules at the cell surface and inhibits detection and lysis of the cells by cytotoxic T lymphocytes. UL49.5 homologs encoded by two other varicelloviruses, pseudorabies-virus and equine herpesvirus 1, also block TAP. Homologs of UL49.5 are widely present in herpesviruses, acting as interaction partners for glycoprotein M, but in several varicelloviruses UL49.5 has uniquely evolved additional functions that mediate its participation in TAP inhibition. Inactivation of TAP by UL49.5 involves two events: inhibition of peptide transport through a conformational arrest of the transporter and degradation of TAP by proteasomes. UL49.5 is degraded along with TAP via a reaction that requires the cytoplasmic tail of UL49.5. Thus, UL49.5 represents a unique immune evasion protein that inactivates TAP through a unique two-tiered process.

Auxin distribution in Lotus japonicus during root nodule development.

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.