A multi-objective based clustering for inferring BCR clonal lineages from high-throughput B cell repertoire data.

The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clonal lineages. A high number of clonal lineages in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity reflect antigen selective pressure. Identifying clonal lineages is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking, and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose MobiLLe, a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial groups and iteratively applies two objective functions that optimize cohesion and separation within clonal lineages simultaneously. We show that our method greatly improves clonal lineage grouping on simulated benchmarks with varied mutation rates compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools and can reproduce the results of previous publications. The method based on multi-objective clustering can accurately identify clonally-related antibody sequences and presents the lowest running time among state-of-art tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context. MobiLLe can potentially help unravel the mechanisms involved in developing and evolving B cell malignancies.

Comparative Transcriptomics Highlights New Features of the Iron Starvation Response in the Human Pathogen Candida glabrata.

In this work, we used comparative transcriptomics to identify regulatory outliers (ROs) in the human pathogen Candida glabrata. ROs are genes that have very different expression patterns compared to their orthologs in other species. From comparative transcriptome analyses of the response of eight yeast species to toxic doses of selenite, a pleiotropic stress inducer, we identified 38 ROs in C. glabrata. Using transcriptome analyses of C. glabrata response to five different stresses, we pointed out five ROs which were more particularly responsive to iron starvation, a process which is very important for C. glabrata virulence. Global chromatin Immunoprecipitation and gene profiling analyses showed that four of these genes are actually new targets of the iron starvation responsive Aft2 transcription factor in C. glabrata. Two of them (HBS1 and DOM34b) are required for C. glabrata optimal growth in iron limited conditions. In S. cerevisiae, the orthologs of these two genes are involved in ribosome rescue by the NO GO decay (NGD) pathway. Hence, our results suggest a specific contribution of NGD co-factors to the C. glabrata adaptation to iron starvation.

JET2 Viewer: a database of predicted multiple, possibly overlapping, protein-protein interaction sites for PDB structures.

The database JET2 Viewer, openly accessible at http://www.jet2viewer.upmc.fr/, reports putative protein binding sites for all three-dimensional (3D) structures available in the Protein Data Bank (PDB). This knowledge base was generated by applying the computational method JET2 at large-scale on more than 20 000 chains. JET2 strategy yields very precise predictions of interacting surfaces and unravels their evolutionary process and complexity. JET2 Viewer provides an online intelligent display, including interactive 3D visualization of the binding sites mapped onto PDB structures and suitable files recording JET2 analyses. Predictions were evaluated on more than 15 000 experimentally characterized protein interfaces. This is, to our knowledge, the largest evaluation of a protein binding site prediction method. The overall performance of JET2 on all interfaces are: Sen = 52.52, PPV = 51.24, Spe = 80.05, Acc = 75.89. The data can be used to foster new strategies for protein-protein interactions modulation and interaction surface redesign.

Integrated molecular portrait of non-small cell lung cancers.

BACKGROUND: Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. METHODS: Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. RESULTS: At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1 and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. CONCLUSIONS: Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

Independent transcriptional reprogramming and apoptosis induction by cisplatin.

Neither the molecular mechanisms whereby cancer cells intrinsically are or become resistant to the DNA-damaging agent cisplatin nor the signaling pathways that account for cisplatin cytotoxicity have thus far been characterized in detail. In an attempt to gain further insights into the molecular cascades elicited by cisplatin (leading to resistance or underpinning its antineoplastic properties), we comparatively investigated the ability of cisplatin, C2-ceramide and cadmium dichloride, alone or in the presence of an array of mitochondrion-protective agents, to trigger the permeabilization of purified mitochondria. In addition, we compared the transcriptional response triggered by cisplatin, C2-ceramide and cadmium dichloride in non-small cell lung carcinoma A549 cells. Finally, we assessed the capacity of cisplatin, C2-ceramide and cadmium dichloride to reduce the clonogenic potential of a battery of yeast strains lacking proteins involved in the regulation of cell death, DNA damage signaling and stress management. This multipronged experimental approach revealed that cisplatin elicits signaling pathways that are for the most part "private," i.e., that manifest limited overlap with the molecular cascades ignited by other inducers of mitochondrial apoptosis, and triggers apoptosis mainly in a transcription-independent fashion. Indeed, bona fide cisplatin-response modifiers that we have recently identified by a functional genome-wide siRNA screen are either not transcriptionally regulated during cisplatin-induced cell death or their transcriptional modulation reflects the activation of an adaptive response promoting cisplatin resistance.

Prognostic impact of vitamin B6 metabolism in lung cancer.

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.

Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia.

RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34(+)CD38(-) progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.

Expression analysis of mitotic spindle checkpoint genes in breast carcinoma: role of NDC80/HEC1 in early breast tumorigenicity, and a two-gene signature for aneuploidy.

BACKGROUND: Aneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes, but little is known about somatic alterations of mitotic spindle checkpoint genes in breast cancer. METHODS: To obtain further insight into the molecular mechanisms underlying aneuploidy in breast cancer, we used real-time quantitative RT-PCR to quantify the mRNA expression of 76 selected mitotic spindle checkpoint genes in a large panel of breast tumor samples. RESULTS: The expression of 49 (64.5%) of the 76 genes was significantly dysregulated in breast tumors compared to normal breast tissues: 40 genes were upregulated and 9 were downregulated. Most of these changes in gene expression during malignant transformation were observed in epithelial cells.Alterations of nine of these genes, and particularly NDC80, were also detected in benign breast tumors, indicating that they may be involved in pre-neoplastic processes.We also identified a two-gene expression signature (PLK1 + AURKA) which discriminated between DNA aneuploid and DNA diploid breast tumor samples. Interestingly, some DNA tetraploid tumor samples failed to cluster with DNA aneuploid breast tumors. CONCLUSION: This study confirms the importance of previously characterized genes and identifies novel candidate genes that could be activated for aneuploidy to occur. Further functional analyses are required to clearly confirm the role of these new identified genes in the molecular mechanisms involved in breast cancer aneuploidy. The novel genes identified here, and/or the two-gene expression signature, might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies.

Involvement of p38alpha in the mitotic progression of p53(-/-) tetraploid cells.

The tumor suppressor protein p53 plays a major role in preserving genomic stability. p53 suppresses a pathway leading from normal diploidy to neoplastic aneuploidy (via an intermediate metastable stage of tetraploidy) at two levels: first by preventing the generation/survival of tetraploid cells, and second by repressing their aberrant multipolar division. Here, we report the characterization of p53(-/-) tetraploid cells, which-at difference with both their p53(-/-) diploid and their p53(+/+) tetraploid counterparts-manifest a marked hyperphosporylation of the mitogen-activated protein kinase MAPK14 (best known as p38alpha) that is particularly strong during mitosis. In p53(-/-) tetraploid cells, phosphorylated p38alpha accumulated at centrosomes during the metaphase and at midbodies during the telophase. Selective knockdown or pharmacological inhibition of p38alpha had a dramatic effect on p53(-/-) (but not p53(+/+)) tetraploids, causing the activation of the spindle assembly checkpoint, an arrest during the metaphase, a major increase in abnormal bipolar and monopolar mitoses, as well as an increment in the generation of multinuclear cells. We conclude that the mitotic progression of p53(-/-) (but not p53(+/+)) tetraploids heavily relies on p38alpha, revealing a novel function for this protein in the context of aneuploidizing cell divisions.

Effects of silencing RET/PTC1 junction oncogene in human papillary thyroid carcinoma cells.

BACKGROUND: RET/PTC1 rearrangement is the most common genetic alteration identified to date in papillary thyroid carcinomas (PTC) and represents an interesting target for small interfering RNA (siRNA) strategies because it is present only in the tumor cells and not in the normal cells. Our aims were (i) to target the RET/PTC1 oncogene by siRNAs, (ii) to assess the knockdown effects on cell growth and cell cycle regulation, and (iii) to identify genes affected by the RET/PTC1 silencing. METHODS: Three efficient siRNAs previously designed in our laboratory in a model of murine PTC (RP-1 cells) were used to knockdown RET/PTC1 in the TPC-1 cells. By reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (Q-RT-PCR) they were found unable to silence RET/PTC1. After sequencing, we redesigned an siRNA against RET/PTC1 (siRNARET/PTC1) and compared it for its efficiency and specificity with an siRNA against RET (siRNARET) in the TPC-1 cells, in human cell lines that expressed RET (MCF-7 and BT-474 cells), and in the murine RP-1 cells. The effects on cell cycle growth (MTT tests), cell cycle (flow cytometry), and apoptosis (TUNEL method) were studied. Genes affected by the RET/PTC1 knockdown were identified by microarray analysis followed by Q-RT-PCR validation. RESULTS: A mutation was found by sequencing within the H4 part of the RET/PTC1 junction leading to a ²97T→G substitution. The redesigned siRNARET/PTC1 inhibits about 85% of the oncogene expression in the human TCP-1 cells. The specificity of the siRNARET/PTC1 was confirmed by the absence of a silencing effect on the human breast MCF-7 and BT-474 cells without RET/PTC1 and the murine RP-1 with ²97G→T mutation. The downregulation of RET/PTC1 modified the cell cycle and induced an apoptotic response. Microarray analysis revealed an inhibition of E2F2 transcription factor known to be involved in the cell cycle regulation. CONCLUSIONS: This study shows the impact of a point mutation within a junction oncogene on the siRNA design. In the case of a therapeutic approach by siRNA, the junction oncogene must be systematically sequenced. The E2F2 gene regulation would have a biological significance and seems to be directly mediated by RET/PTC1.

miR-181a and miR-630 regulate cisplatin-induced cancer cell death.

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non-small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27(Kip1) as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC.

PTCH1 +/- dermal fibroblasts isolated from healthy skin of Gorlin syndrome patients exhibit features of carcinoma associated fibroblasts.

Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS) causes predisposition to basal cell carcinoma (BCC), the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3) and NBCCS fibroblasts bearing either nonsense (n = 3) or missense (n = 3) PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1(+/-) genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients.

Recurrent overexpression of c-IAP2 in EBV-associated nasopharyngeal carcinomas: critical role in resistance to Toll-like receptor 3-mediated apoptosis.

The oncogenic process leading to nasopharyngeal carcinoma (NPC) requires the combination of genetic and epigenetic alterations, latent infection by the Epstein-Barr virus and local inflammation. A transcriptome analysis of NPC xenografts identified the gene encoding the cellular inhibitor of apoptosis protein 2 (c-IAP2) among the top five most intensely expressed. Consistently, the very high levels of the c-IAP2 protein were detected in 11 of 13 NPC biopsies. RMT 5265, a structural analog of second mitochondria-derived activator of caspase (SMAC), induced the rapid degradation of c-IAP2 in nasopharyngeal epithelial cells, whether malignant or not, but blocked clonal cell growth in NPC cells only. In short-term experiments, RMT 5265 induced apoptosis in a fraction of NPC cells, and this apoptosis was dramatically enhanced when RMT 5265 was combined with Toll-like receptor 3 (TLR3) stimulation. By contrast, the cooperative effect with tumor necrosis factor alpha was only marginal. The apoptosis induced by the combination of RMT 5265 and TLR3 stimulation was mediated by caspase-8 and associated with a decrease in the cellular content of the long isoform of FLICE-like inhibitory protein. Similar caspase-8 activation was obtained when siRNA knockdown of c-IAP2 was combined with TLR3 stimulation. In conclusion, c-IAP2 has a specific protective function in NPC cells challenged by TLR3 agonists. This protective function is probably important to make NPC cells tolerant to their own production of small viral RNAs, which are potential agonists of TLR3. Our data will help to design a rational use of IAP inhibitors in NPC patients.

MYCN-non-amplified metastatic neuroblastoma with good prognosis and spontaneous regression: a molecular portrait of stage 4S.

Stage 4 neuroblastoma (NB) are heterogeneous regarding their clinical presentations and behavior. Indeed infants (stage 4S and non-stage 4S of age <365days at diagnosis) show regression contrasting with progression in children (>365days). Our study aimed at: (i) identifying age-based genomic and gene expression profiles of stage 4 NB supporting this clinical stratification; and (ii) finding a stage 4S NB signature. Differential genome and transcriptome analyses of a learning set of MYCN-non amplified stage 4 NB tumors at diagnosis (n=29 tumors including 12 stage 4S) were performed using 1Mb BAC microarrays and Agilent 22K probes oligo-microarrays. mRNA chips data following filtering yielded informative genes before supervised hierarchical clustering to identify relationship among tumor samples. After confirmation by quantitative RT-PCR, a stage 4S NB's gene cluster was obtained and submitted to a validation set (n=22 tumors). Genomic abnormalities of infant's tumors (whole chromosomes gains or loss) differ radically from that of children (intra-chromosomal rearrangements) but could not discriminate infants with 4S from those without this presentation. In contrast, differential gene expression by looking at both individual genes and whole biological pathways leads to a molecular stage 4S NB portrait which provides new biological clues about this fascinating entity.

A novel epidermal growth factor receptor inhibitor promotes apoptosis in non-small cell lung cancer cells resistant to erlotinib.

Non-small cell lung cancer (NSCLC) with activating mutations in the epidermal growth factor receptor (EGFR) responds to EGFR tyrosine kinase inhibitors such as erlotinib. However, secondary somatic EGFR mutations (e.g., T790M) confer resistance to erlotinib. BMS-690514, a novel panHER/vascular endothelial growth factor receptor (VEGFR) inhibitor described here, exerted antiproliferative and proapoptotic effects on NSCLC cell lines, with prominent efficacy on H1975 cells expressing the T790M mutation. In this model, BMS-690514 induced a G(1) cell cycle arrest, as well as ultrastructural hallmarks of apoptosis, mitochondrial release of cytochrome c, and activation of caspases involved in the intrinsic (e.g., caspase-2, caspase-3, caspase-7, and caspase-9), but not in the extrinsic (e.g., caspase-8), pathway. Caspase inhibition conferred partial protection against BMS-690514 cytotoxicity, pointing to the involvement of both caspase-dependent and caspase-independent effector mechanisms. Transcriptome analyses revealed the up-regulation of proapoptotic (e.g., Bim, Puma) and cell cycle inhibitory (e.g., p27(Kip1), p57(Kip2)) factors, as well as the down-regulation of antiapoptotic (e.g., Mcl1), heat shock (e.g., HSP40, HSP70, HSP90), and cell cycle promoting [e.g., cyclins B1, D1, and D3; cyclin-dependent kinase 1 (CDK1); MCM family proteins; proliferating cell nuclear antigen (PCNA)] proteins. BMS-690514-induced death of H1975 cells was modified in a unique fashion by a panel of small interfering RNAs targeting apoptosis modulators. Down-regulation of components of the nuclear factor-kappaB survival pathway (e.g., p65, Nemo/IKK gamma, TAB2) sensitized cells to BMS-690514, whereas knockdown of proapoptotic factors (e.g., Puma, Bax, Bak, caspase-2, etc.) and DNA damage-related proteins (e.g., ERCC1, hTERT) exerted cytoprotective effects. BMS-690514 is a new pan-HER/VEGFR inhibitor that may become an alternative to erlotinib for the treatment of NSCLC.

Robust interlaboratory reproducibility of a gene expression signature measurement consistent with the needs of a new generation of diagnostic tools.

BACKGROUND: The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. While the reproducibility of microarray gene expression measurements has been the subject of several recent reports, there is still a need for systematic investigation into what factors most contribute to variability of measured expression levels observed among different laboratories and different experimenters. RESULTS: We report the results of an interlaboratory comparison of gene expression array measurements on the same microarray platform, in which the RNA amplification and labeling, hybridization and wash, and slide scanning were each individually varied. Identical input RNA was used for all experiments. While some sources of variation have measurable influence on individual microarray signals, they showed very low influence on sample-to-reference ratios based on averaged triplicate measurements in the two-color experiments. RNA labeling was the largest contributor to interlaboratory variation. CONCLUSION: Despite this variation, measurement of one particular breast cancer gene expression signature in three different laboratories was found to be highly robust, showing a high intralaboratory and interlaboratory reproducibility when using strictly controlled standard operating procedures.

Microarray-based identification of tenascin C and tenascin XB, genes possibly involved in tumorigenesis associated with neurofibromatosis type 1.

PURPOSE: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies. EXPERIMENTAL DESIGN: To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR. RESULTS: We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes. CONCLUSION: TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.

Interrelation between polyploidization and megakaryocyte differentiation: a gene profiling approach.

Polyploidization is a part of the normal developmental process leading to platelet production during megakaryocyte (MK) differentiation. Ploidization is mainly involved in cell enlargement, but it is not clear whether gene expression is modified during MK ploidization. In this study, human MKs were grown from CD34(+) cells in the presence of thrombopoietin and sorted according to their ploidy level. A pangenomic microarray technique was applied to compare gene expression in 2N-, 4N-, 8N-, and 16N-sorted MKs. Using hierarchical clustering, we demonstrated that 2N and 4N MKs or 8N and 16N MKs are 2 different close populations with 105 discriminating genes. In the second approach, we determined the profile of genes that were continuously down- and up-regulated during polyploidization. Among the 100 down-regulated genes, 24 corresponded to genes involved in DNA replication and repair. The great majority of up-regulated genes corresponded to genes directly involved in platelet functions, such as genes encoding specific platelet glycoproteins and alpha-granule proteins, actin and microtubule cytoskeleton, factors involved in signaling, and transport proteins. Together, these results suggest that MK polyploidization per se does not regulate gene expression but is intrinsically included in the differentiation process.

ppGalNAc-T13: a new molecular marker of bone marrow involvement in neuroblastoma.

BACKGROUND: To identify new molecular markers of bone marrow dissemination in human neuroblastoma (NB), we studied the transcriptome profiles of malignant neuroblasts established from the human MYCN-amplified IGR-N-91 model. METHODS: This experimental model includes human neuroblastoma cells derived from a subcutaneous stage 4 disease, myocardium (Myoc) and bone marrow (BM) metastatic cells. RESULTS: Gene expression profiles obtained with Agilent oligo microarrays revealed a set of 107 differentially expressed genes in the metastatic neuroblasts. This set included up-regulated genes involved in chemoresistance, cell motility, neuronal structure/signaling, and the recently characterized GALNT13 gene encoding a glycosyltransferase that initiates mucin-type O-glycosylation. Because the glycosylation process is involved in the progression of primary tumor to metastatic disease, we investigated whether the most strongly up-regulated gene, GALNT13, might be a marker of bone marrow involvement in stage 4 NB patients. Importantly, in the BM of healthy adults no GALNT13 transcript was detected with analysis by quantitative (n = 3) and nested reverse transcription-PCR (n = 4) assays. In contrast, GALNT13 transcripts were detected in 23/23 cytologically involved BM samples obtained at diagnosis of stage 4 NB patients and in 5/27 cytologically noninvolved BM samples obtained from patients with stage 1-4 and 4S and treated stage 4 NB. The quantitative measurements of tyrosine hydroxylase (TH), ganglioside D2 synthase, dopa decarboxylase, and GALNT13 transcript values were compared in the same NB patients, and the results showed that GALNT13 expression was most highly correlated to poor clinical outcome at diagnosis. CONCLUSION: We propose ppGalNAc-T13 as a new informative marker for the molecular diagnosis of BM involvement and the follow-up of minimal residual disease in NB patients.