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Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.

Refinement of IKZF1 status in pediatric Philadelphia-positive acute lymphoblastic leukemia

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

Cell states in hematopoiesis are controlled by master regulators and by complex circuits of a growing family of RNA species impacting cell phenotype maintenance and plasticity. Circular RNAs (circRNAs) are rapidly gaining the status of particularly stable transcriptome members with distinctive qualities. RNA-seq identified thousands of circRNAs with developmental stage- and tissue-specific expression corroborating earlier suggestions that circular isoforms are a natural feature of the cell expression program. CircRNAs are abundantly expressed also in the hematopoietic compartment. There are a number of studies on circRNAs in blood cells, a specific overview is however lacking. In this review we first present current insight in circRNA biogenesis discussing the relevance for hematopoiesis of the highly interleaved processes of splicing and circRNA biogenesis. Regarding molecular functions circRNAs modulate host gene expression, but also compete for binding of microRNAs, RNA-binding proteins or translation initiation and participate in regulatory circuits. We examine circRNA expression in the hematopoietic compartment and in hematologic malignancies and review the recent breakthrough study that identified pathogenic circRNAs derived from leukemia fusion genes. CircRNA high and regulated expression in blood cell types indicate that further studies are warranted to inform the position of these regulators in normal and malignant hematopoiesis.

Precursor B-acute lymphoblastic leukemia (pB-ALL) is a heterogeneous disease and multiparameter flow cytometry, molecular genetics, and cytogenetic studies have all contributed to classification of subgroups with prognostic significance. Recently, gene expression microarray technology has been used to investigate lymphoblastic leukemias, demonstrating that known and novel pB-ALL subclasses can be separated on the basis of gene expression profiles. The strength of microarray technique lays in part in the multivariate nature of the expression data. We propose a parallel multiparametric approach based on immunophenotypic flow-cytometry expression data for the analysis of leukemia patients. Specifically, we tested the potential of this approach on a data set of 145 samples of pediatric pB-ALL that included 46 samples positive for mixed lineage leukemia (MLL) translocations (MLL+) and 99 control pB-ALLs, negative for this translocation (MLL-). The expression levels of 16 marker proteins have been monitored by four-color flow cytometry using a standardized diagnostic panel of antibodies. The protein expression database has been then analyzed using those univariate and multivariate computational techniques normally applied to mine and model large microarray data sets. Marker protein expression profiling not only allowed separating pB-ALL cases with an MLL rearrangement from other ALLs, but also demonstrates that MLL+ leukemias constitute a heterogeneous group in which MLL/AF4 leukemias represent a homogenous subclass described by a specific expression fingerprint.

The interaction of self-propelled biological molecular-motors and cytoskeletal filaments holds relevance for a variety of applications such as biosensing, drug screening, diagnostics and biocomputation. The use of these systems for lab-on-a-chip biotechnology applications shows potential for replacement of microfluidic flow by active, molecular-motor driven transport of filaments. The ability to control, confine and detect motile objects in such a system is possible by development of nanostructured surfaces for on-chip applications and fundamental studies of molecular-motors. Here we describe the localized detection (Lard et al., Sci Rep 3:1092, 2013) and fast transport of actin filaments by myosin molecular-motors (Lard et al., Biosens Biolectron 48(0):145–152, 2013), inserted within nanostructures, as a method for biocomputation and molecular concentration. These results include extensive myosin driven concentration of actin filaments on a miniaturized detector, of relevance for use of molecular-motors in a diagnostics platform. Also, we discuss the local enhancement of the fluorescence signal of filaments, relevant for use in a biocomputation device where tracking of potentially thousands of motile objects is of primary significance.

Next to a prominent nutritional role during larval growth the teleost yolk cell may function during early developmental processes as a source of inducing signals. In Xenopus mesoderm-inducing signals reside in the vegetal cells of the blastula and exert their function on the more animal located cells in the margin. Because of its position reminiscent of the vegetal cells in Xenopus, the yolk cells of teleost fishes may have a similar inductive capacity. Removal of the blastoderm from the yolk cell (YC) offers an experimental approach to study to what extent blastoderm differentiation and morphogenesis depend on inductive interaction with the YC. Expression of marker genes for dorsal-ventral and anterior/posterior determination such as no tail, goosecoid and caudal was examined in blastoderm explants, which were incubated until sibling controls reached the tail bud stage. Blastoderms removed at 3 h post fertilisation expressed rudimentary dorsal-ventral polarity. Apparently signals leading to this expression had reached the margin of 3-h blastoderms. Blastoderms removed at 4 h showed in addition to dorsal-ventral polarity notochord-like structures and also expression patterns of goosecoid and caudal suggesting that gastrulation movements had occurred.

This paper describes an ultrastructural study of the cell movements during the gastrulation of the common carp, Cyprinus carpio, using scanning electron microscopy. The morphology of the deep cells was studied in several consecutive stages ranging from 0-100% epiboly. Furthermore, the formation of the embryonic shield was followed from its earliest appearance at 50% epiboly onwards. This paper gives morphological evidence for the existence of two different pathways for involving and convergent movements. Firstly, cells may move along the inner surface of the not (yet) involuted cells. Secondly, a much smaller group may use the YSL as their substrate. These results are discussed in the light of the hypothesis that the two migrating cell populations may be differently induced, subsequently leading to the formation of mesoderm and endoderm.

1. Force measurements in isolated myofibrils (15 degrees C; sarcomere length, 2.10 microns) were used in this study to determine whether sarcomeric proteins are responsible for the large differences in the amounts of active and passive tension of cardiac versus skeletal muscle. Single myofibrils and bundles of two to four myofibrils were prepared from glycerinated tibialis anterior and sartorius muscles of the frog. Skinned frog atrial myocytes were used as a model for cardiac myofibrils. 2. Electron microscope analysis of the preparations showed that: (i) frog atrial myocytes contained a small and variable number of individual myofibrils (from 1 to 7); (ii) the mean cross-sectional area and mean number of myosin filaments of individual cardiac myofibrils did not differ significantly from those of single skeletal myofibrils; and (iii) the total myofibril cross-sectional area of atrial myocytes was on average comparable to that of bundles of two to four skeletal myofibrils. 3. In maximally activated skeletal preparations, values of active force ranged from 0.45 +/- 0.03 microN for the single myofibrils (mean +/- S.E.M.; n = 16) to 1.44 +/- 0.24 microN for the bundles of two to four myofibrils (n = 9). Maximum active force values of forty-five cardiac myocytes averaged 1.47 +/- 0.10 microN and exhibited a non-continuous distribution with peaks at intervals of about 0.5 microN. The results suggest that variation in active force among cardiac preparations mainly reflects variability in the number of myofibrils inside the myocytes and that individual cardiac myofibrils develop the same average amount of force as single skeletal myofibrils. 4. The mean sarcomere length-resting force relation of atrial myocytes could be superimposed on that of bundles of two to four skeletal myofibrils. This suggests that, for any given amount of strain, individual cardiac and skeletal sarcomeres bear essentially the same passive force. 5. The length-passive tension data of all preparations could be fitted by an exponential equation. Equation parameters obtained for both types of myofibrils were in reasonable agreement with those reported for larger preparations of frog skeletal muscle but were very different from those estimated for multicellular frog atrial preparations. It is concluded that myofibrils are the major determinant of resting tension in skeletal muscle; structures other than the myofibrils are responsible for the high passive stiffness of frog cardiac muscle.

One of the biggest challenges in tumour research is the possibility to reprogram cancer cells towards less aggressive phenotypes. In this study, we reprogrammed primary Glioblastoma multiforme (GBM)-derived cells towards a more differentiated and less oncogenic phenotype by activating the Wnt pathway in a hypoxic microenvironment. Hypoxia usually correlates with malignant behaviours in cancer cells, but it has been recently involved, together with Wnt signalling, in the differentiation of embryonic and neural stem cells. Here, we demonstrate that treatment with Wnt ligands, or overexpression of β-catenin, mediate neuronal differentiation and halt proliferation in primary GBM cells. An hypoxic environment cooperates with Wnt-induced differentiation, in line with our finding that hypoxia inducible factor-1α (HIF-1α) is instrumental and required to sustain the expression of β-catenin transcriptional partners TCF-1 and LEF-1. In addition, we also found that Wnt-induced GBM cell differentiation inhibits Notch signalling, and thus gain of Wnt and loss of Notch cooperate in the activation of a pro-neuronal differentiation program. Intriguingly, the GBM sub-population enriched of cancer stem cells (CD133(+) fraction) is the primary target of the pro-differentiating effects mediated by the crosstalk between HIF-1α, Wnt, and Notch signalling. By using zebrafish transgenics and mutants as model systems to visualize and manipulate in vivo the Wnt pathway, we confirm that Wnt pathway activation is able to promote neuronal differentiation and inhibit Notch signalling of primary human GBM cells also in this in vivo set-up. In conclusion, these findings shed light on an unsuspected crosstalk between hypoxia, Wnt and Notch signalling in GBM, and suggest the potential to manipulate these microenvironmental signals to blunt GBM malignancy.

PAX5/ETV6 alters the gene expression profile of precursor B cells with opposite dominant effect on endogenous PAX5

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59-100%) and a specificity of 87% (CI 70-96%). Positive and negative predictive values were 71% (CI 42-92%) and 96% (CI 82-100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria. © 2013 Macmillan Publishers Limited. All rights reserved.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has achieved an 80% cure rate as a result of a risk-adapted therapy largely based on minimal residual disease (MRD) monitoring. However, relapse is still the most frequent adverse event, occurring mainly in the patients with intermediate MRD levels (intermediate risk, IR), emphasizing the need for new prognostic markers. We analyzed the prognostic impact of cytokine receptor-like factor 2 (CRLF2) over-expression and P2RY8-CRLF2 fusion in 464 BCP-ALL patients (not affected by Down syndrome and BCR-ABL negative) enrolled in the AIEOP-BFM ALL2000 study in Italy. In 22/464 (4.7%) samples, RQ-PCR showed CRLF2 over-expression (≥20 times higher than the overall median). P2RY8-CRLF2 fusion was detected in 22/365 (6%) cases, with 10/22 cases also showing CRLF2 over-expression. P2RY8-CRLF2 fusion was the most relevant prognostic factor independent of CRLF2 over-expression with a threefold increase in risk of relapse. Significantly, the cumulative incidence of relapse of the P2RY8-CRLF2 + patients in the IR group was high (61.1% ± 12.9 vs 17.6% ± 2.6, P

Shwachman-Diamond syndrome (SDS) is a rare inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufficiency. SDS patients have an increased risk for myelodisplastic syndrome and acute myeloid leukemia. Mesenchymal stem cells (MSCs) are the key component of the hematopoietic microenvironment and are relevant in inducing genetic mutations leading to leukemia. However, their role in SDS is still unexplored. We demonstrated that morphology, growth kinetics and expression of surface markers of MSCs from SDS patients (SDS-MSCs) were similar to normal MSCs. Moreover, SDS-MSCs were able to differentiate into mesengenic lineages and to inhibit the proliferation of mitogen-activated lymphocytes. We demonstrated in an in vitro coculture system that SDS-MSCs, significantly inhibited neutrophil apoptosis probably through interleukin-6 production. In a long-term coculture with CD34+-sorted cells, SDS-MSCs were able to sustain CD34+ cells survival and to preserve their stemness. Finally, SDS-MSCs had normal karyotype and did not show any chromosomal abnormality observed in the hematological components of the BM of SDS patients. Despite their pivotal role in the hematopoietic stem cell niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients. © 2012 Macmillan Publishers Limited All rights reserved.

Gene expression signatures of pediatric myelodysplastic syndromes are associated with risk of evolution into acute myeloid leukemia

Acute lymphoblastic leukemia (ALL) is a malignant disease of the white blood cells. The etiology of ALL is believed to be multifactorial and likely to involve an interplay of environmental and genetic variables. We performed a genome-wide association study of 355 750 single-nucleotide polymorphisms (SNPs) in 474 controls and 419 childhood ALL cases characterized by a t(12;21)(p13;q22) - the most common chromosomal translocation observed in childhood ALL - which leads to an ETV6-RUNX1 gene fusion. The eight most strongly associated SNPs were followed-up in 951 ETV6-RUNX1-positive cases and 3061 controls from Germany/Austria and Italy, respectively. We identified a novel, genome-wide significant risk locus at 3q28 (TP63, rs17505102, P(CMH)=8.94 × 10(-9), OR=0.65). The separate analysis of the combined German/Austrian sample only, revealed additional genome-wide significant associations at 11q11 (OR8U8, rs1945213, P=9.14 × 10(-11), OR=0.69) and 8p21.3 (near INTS10, rs920590, P=6.12 × 10(-9), OR=1.36). These associations and another association at 11p11.2 (PTPRJ, rs3942852, P=4.95 × 10(-7), OR=0.72) remained significant in the German/Austrian replication panel after correction for multiple testing. Our findings demonstrate that germline genetic variation can specifically contribute to the risk of ETV6-RUNX1-positive childhood ALL. The identification of TP63 and PTPRJ as susceptibility genes emphasize the role of the TP53 gene family and the importance of proteins regulating cellular processes in connection with tumorigenesis.Leukemia advance online publication, 11 November 2011; doi:10.1038/leu.2011.302.

MLL partner genes drive distinct gene expression profiles and genomic alterations in pediatric acute myeloid leukemia: an AIEOP study

PTPN11 mutations in childhood acute lymphoblastic leukemia occur as a secondary event associated with high hyperdiploidy

Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia.

Validation of NG2 antigen in identifying BP-ALL patients with MLL rearrangements using qualitative and quantitative flow cytometry: a prospective study

Acute myeloid leukemia (AML) is a heterogeneous disease group morphologically classified, based on the French–American–British (FAB) classification, into eight main subgroups defined as subtypes M0–M7. Besides morphologic differences, genetic abnormalities have been recognized; cytogenetics and molecular analyses are currently used to identify subgroups of AML with different clinical prognosis. However, in spite of available prognostic factors, accurate prediction of risk for treatment failure or relapse is not completely satisfactory. In order to improve risk assignment and develop new therapeutic strategies, gene expression profiling and proteomic analysis seem to offer important improvements in leukemia classification.

Non-invasive prenatal diagnosis (NIPD) is becoming increasingly important and its application in prenatal diagnosis is reaching consensus in the scientific research community. We discuss the opportunities and ethics of non-invasive prenatal testing for non-medical purposes, including forensic genetics. A number of ethical issues arise from non-medical applications of NIPD, such as sex determination and paternity testing in earlier gestational age and subsequent offspring selection. NIPD provides a source of information about the genetic make-up of the foetus, avoiding the small but significant risk of pregnancy loss related to invasive testing such as amniocentesis or chorionic villi sampling. NIPD is characterized by: safety, early detection and easy sampling. These features of NIPD increase the opportunity of prenatal testing also for non-medical reasons. Even if NIPD can be qualified as a good practice prenatal diagnosis tout court remains a topic of ethical judgements. The non-medical use of NIPD will benefit from an informed and open debate involving both pregnant women and physicians.

The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.

The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation.

The homeobox gene Carp-Ovx1 shows similarity to vertebrate and invertebrate Ovx genes and to Drosophila unplugged. Its expression pattern was studied by in situ hybridization in carp embryos and juveniles. During segmentation, expression becomes gradually limited to the neural tube. In juveniles up to 9 weeks old, cells in the ventral telencephalon, the facial lobe and the vagal lobe show Ovx1 expression, confining expression to parts with chemosensory projections.

Correction to: Cell Death and Disease (2013) 4, e500; doi:10.1038/cddis.2013.32; published online 21 February 2013 Since the publication of this article the authors have noticed that the Authors Contribution section was omitted. The error has now been rectified. The article with the Authors Contribution appears online together with this corrigendum.

This report describes the cDNA sequence and embryonic RNA expression pattern of carp Hoxb-1. Carp Hoxb-1 is a labial-like, homeobox-containing gene of the 3' end of the Hox gene cluster. The expression pattern in carp is compared to that of homologs in other vertebrates. As holds for other Hox genes, carp Hoxb-1 is expressed with highest intensity at a sharp anterior boundary, and expression fades out towards posterior. At later stages, gaps were found in the domain. The gene is expressed from late gastrulation onwards, first mainly in the hypoblast but later in all germ layers. Its most prominent expression area is rhombomere 4 (r4) of the hindbrain. Transcripts were also found in the neural tube, mesoderm (lateral, head and presomite), epidermis and neural crest. At 30 hours post fertilization, Hoxb-1 was still expressed in r4, in the anterior trunk neural tube and in the branchial arches posterior to r4. Hox genes are thought to be involved in the specification of positional values along the embryonic anterior-posterior axis, and Hoxb-1 expression in r4 is supposed to be important for specifying the unique identity of this hindbrain segment. The conserved expression in r4 suggests that this is also true for carp Hoxb-1.

Early development of cyprinid teleosts is summarized with special attention to gastrulation. Making use of comparison with Xenopus, functions are suggested for the uncleaved yolk cell, concerning induction and patterning processes before and during gastrulation. It is concluded that cyprinid development has a number of very specific aspects, involving morphogenesis and, very likely, gene functions. This should be realized when gene expression patterns during development are studied in this group of teleosts as a model of development in higher vertebrates.

The effects of growth and limb immobilization on muscle mass, total physiological cross-section (PC), the number of sarcomeres in series and the length of sarcomere components were investigated in the soleus muscle (SOL) and compared to previously obtained data on gastrocnemius (GM) muscles of rats between age 10 and 16 weeks. For SOL this period of growth was reflected in an increased muscle mass and PC. No such increases were found for GM. In contrast, immobilization caused severe atrophy of fibres of both muscles. Compared to the value at the start of the immobilization, it was found that the fast twitch muscle (GM) atrophied more than the typically slow twitch one (SOL). The number of sarcomeres in series within fibres increased after growth and decreased after immobilization of SOL. For fibres of GM no such changes were observed. Muscle architecture is proposed as an important factor for the explanation of the results concerning the number of sarcomeres in series and those arranged in parallel. Due to the difference in muscle architecture, GM being more pennate than SOL, during growth, it is thought that increases in bone length affect the length of fibres of SOL more than those of GM. During immobilization, atrophy of fibres of GM was sufficient for the muscle length adaptation to meet the muscle length change induced by immobilization but in SOL, atrophy had to be accompanied by decreases in the number of sarcomeres in series to achieve adequate muscle length adaptation.

The mammalian blastocyst consists of an inner cell mass (ICM) enclosed by the trophectoderm. The origin of these two cell populations lies in the segregation of inner and outer cells in the early morula. In the present study, the segregation of inner and outer cells has been studied in porcine embryos and is compared with segregation in mouse embryos. For this, nuclei of inner and outer cells were differentially labelled with two fluorochromes after partial complement-mediated lysis of the outer cells. In porcine and mouse embryos compaction and the first appearance of inner cells occur at different stages of development. In porcine embryos compaction was observed as early as the 4-cell stage, while in mouse embryos compaction occurred in the 8-cell stage. The first inner cells segregated in porcine embryos which were in the transition from four to eight cells and inner cells were added during two subsequent cell cycles. In mouse embryos inner cells segregated predominantly during the fourth cleavage division. From the results obtained we conclude that the segregation of inner and outer cells follows a different pattern in mouse and in porcine embryos.

The aim of this study was to isolate probes to repetitive sequences of porcine (Great Yorkshire × Landrace) genomic DNA. The production of transgenic animals involves the isolation of stem cell lines and the understanding of DNA methylation modifications. Probes to repetitive sequences enable the analysis of DNA methylation in the tissues of various embryonic stages of the pig. A primary library of porcine genomic DNA was screened with labeled fragments of porcine DNA, and 6 clones containing repetitive DNA were isolated and analyzed for the presence of potential methylation moieties (CCGG sites). Probes of all 6 clones were tested in a hybridization analysis of HpaII and MspI digests of porcine sperm DNA, and it was found that methylation was not present in the methylation moieties of the repetitive sequences.

Preimplantation cleaving-stage embryos were recovered from Dutch Landrace (DL) and F1 Dutch Landrace x Great Yorkshire (DL x GY) gilts for which the time of insemination and ovulation were known. Embryonic cell counts were performed, usually after brief in vitro culture to estimate DNA synthesis. Special attention was given to the 4-cell stage. Beyond this stage, the mean cell cycle time was 14 hours for DL and 17 hours for F1 gilts. Generally, high indices of DNA synthesis were obtained (more than 60% of nuclei). There was prominent within and between gilt variability with regard to embryonic cell numbers. Gilts are especially heterogeneous with respect to the length of the 4-cell stage. The G 2 M phase of the 4-cell stage takes approximately 3.5 hours. Especially for F1 gilts, the age of the spermatozoa at the moment of ovulation was not related to the rate of cleavage and/or embryonic death. It is postulated that variability in the length of the 4-cell stage is reflected in genetic activation of the embryos at this stage and subsequently influences embryonic survival.

Carp embryos were dechorionated and their early development was studied in the presence or absence of a-amanitin. Cleavage and the formation of the enveloping layer and yolk syncytial layer were not influenced by the drug. However, a-amanitin largely blocked epiboly which started 6 h after fertilization in controls. Involution of deep cells, taking place during gastrulation movements, appeared to be blocked to a lesser degree. This might reflect differences in the degree to which maternal transcripts influence these developmental steps.

10042 Background: Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Although advances in therapy have led to improved long term survival, relapse remains a major challenge. In a recent study we transplanted pediatric leukemia samples from newly diagnosed BCP-ALL patients into NOD/SCID mice. Time to leukemia (TTL) was analyzed for each patient sample as time from transplant to overt leukemia in the recipients. Patients whose leukemia cells engrafted rapidly showed a clearly inferior relapse free survival in contrast to patient samples with prolonged in vivo growth. Multivariate analysis showed an almost 45- fold increased risk for relapse in patients with short TTL. Methods: Gene expression profiles of ALL samples (N = 14) with short versus long TTL in the xenograft model were analyzed using a human whole genome array (Affymetrix U133 Plus 2.0) correlating gene expression values (relative expression) to the time from transplant to manifestation of leukemia in the NOD/SCID mice (TTL, in weeks) by quantitative traits analysis (QTA). Results: Among 5 genes significantly correlated (Spearman correlation, P < .0001) XIAP-associated factor 1 (XAF1) was found to be up-regulated in patients with long TTL. XAF1 abrogates the inhibitory effect of XIAP on caspase-3 thereby sensitizing for apoptosis. In accordance to this caspase-3 activation was also found to be up-regulated in patients with long TTL. Patient samples exhibiting a short time to overt leukemia in the xenotransplant model associated with poor relapse free survival showed down-regulated XAF1 and impaired caspase-3 activation leading to decreased apoptosis of the leukemia cells. Conclusions: Taken together, we used a novel approach directly correlating gene expression values to time from transplant to overt leukemia (TTL) identifying the apoptosis regulator XAF1 to be associated with poor outcome of patients. Small XIAP-inhibiting molecules can be used to substitute the lacking inhibitory effect of down-regulated XAF1 in these poor responding pediatric ALL patients. No significant financial relationships to disclose.

e13031 It has been suggested that oxygen tension is a crucial component of the brain tumor niche, as hypoxia positively correlates with tumor aggressiveness and over-activity of hypoxia inducible factor-1α (HIF-1α) reinforces tumor progression. Furthermore, hypoxia has been implicated in the regulation of several signaling pathways (Notch, BMP), angiogenesis and importantly, glucose metabolism. Here we investigate the effects mediated by in vitro glycolysis inhibition by using 2-deoxyglucose (2-DG) in glioblastoma (GBM) derived cells maintained under two different oxygen tensions, a lowered oxygen tension (2%) versus a higher non-physiological (20%). GBM account for 50% of all gliomas and arise after age 50 in most patients and it has been seen that younger patients tend to have a better prognosis than the elderly. Our results show that adult GBM displaying a highly immature phenotype manifested the highest resistance to glucose deprivation. Furthermore, increase of multi-drug resistant cell fraction, described as side population, occurred following 2-DG treatment, but only under hypoxia. Neuronal committed precursors were selected by 2-DG, but these effects were mitigated by hypoxia. Also, hypoxia inhibits the mitochondria-controlled apoptosis induced by 2-DG, by conferring cell resistance through progressive activation of pro-survival NF-kB and induction of tumor cell autophagy. Importantly, HIF-1α level reduction and proline hydroxylase 2 (PHD2) upregulation occurred following 2-DG treatment even under hypoxia, and this may depend on reactive oxygen species reduction. These results indicate differences in tumor cells behavior that may be predictive of cell response to therapy aiming to limit glucose uptake or glucose metabolism. No significant financial relationships to disclose.

10001 Background: While the outcome for children with Acute Lymphoblastic Leukemia (ALL) has improved under current therapies, 30% do not have a favourable outcome. Infants with MLL rearrangements form the most striking example of ALL patients who have not benefited from the improved treatment regimens developed over recent decades. Our study aims to better map the biology of the MLL disease and to identify aberrantly activated phosphoproteins specific for MLL rearranged patients using phosphoproteomic measurement of signal transduction pathways (STPs). This will offer possibilities to identify new drug targets for more effective and less toxic targeted therapies. Methods: Reverse Phase Protein Arrays (RPPA) were employed to profile the working state of cellular STPs in 120 pediatric B-ALL specimens collected prior to treatment. The phosphorylation status of 92 key signalling proteins was analyzed. We compared 9 MLL rearranged patients vs 42 patients without known genomic aberrancies to find differentiall...

The embryonic ectoderm of the pig differentiated and became part of the outer barrier of the blastocyst (earlier formed by the trophectoderm alone) before shedding of the overlying polar trophectoderm around Day 10, thus securing the integrity of the rapidly expanding blastocyst. Ferritin, added to the medium of the blastocyst, was taken up rapidly by trophectoderm cells, but did not reach the blastocoele, and consequently no tracer was found within hypoblast cells. Embryonic ectoderm cells did not absorb the macromolecule, before or after loss of the polar trophectoderm. When ferritin was injected into the blastocoele, trophectoderm, hypoblast and embryoblast cells all absorbed the tracer. At Day 11, blastocyst diameter and embryoblast cell number varied widely and were hardly correlated. We suggest that embryoblast development may be a more reliable indicator for the developmental stage of a blastocyst than its diameter, which may merely be an indication of the viability of the trophoblast.