Tenascin-C is a novel RBPJkappa-induced target gene for Notch signaling in gliomas.

Tenascin-C (TNC) expression is known to correlate with malignancy in glioblastoma (GBM), a highly invasive and aggressive brain tumor that shows limited response to conventional therapies. In these malignant gliomas as well as in GBM cell lines, we found Notch2 protein to be strongly expressed. In a GBM tumor tissue microarray, RBPJk protein, a Notch2 cofactor for transcription, was found to be significantly coexpressed with TNC. We show that the TNC gene is transactivated by Notch2 in an RBPJk-dependent manner mediated by an RBPJk binding element in the TNC promoter. The transactivation is abrogated by a Notch2 mutation, which we detected in the glioma cell line Hs683 that does not express TNC. This L1711M mutation resides in the RAM domain, the site of interaction between Notch2 and RBPJk. In addition, transfection of constructs encoding activated Notch2 or Notch1 increased endogenous TNC expression identifying TNC as a novel Notch target gene. Overexpression of a dominant negative form of the transcriptional coactivator MAML1 or knocking down RBPJk in LN319 cells led to a dramatic decrease in TNC protein levels accompanied by a significant reduction of cell migration. Because addition of purified TNC stimulated glioma cell migration, this represents a mechanism for the invasive properties of glioma cells controlled by Notch signaling and defines a novel oncogenic pathway in gliomagenesis that may be targeted for therapeutic intervention in GBM patients.

Impact of fetal-maternal microchimerism on women's health--a review.

Microchimerism is defined by the presence of circulating cells, bi-directionally transferred from one genetically distinct individual to another. It occurs either physiologically during pregnancy, or iatrogenically after blood transfusion and organ transplants. The migrated cells may persist for decades. Much controversy exists around the role of microchimeric cells in the pathogenesis of various diseases and around their role in tissue repair. Microchimerism has been investigated in different autoimmune disorders, such as systemic sclerosis, systemic lupus erythematosus, autoimmune thyroid diseases, primary biliary cirrhosis and juvenile inflammatory myopathies. Recent data have demonstrated the promising role of microchimeric cells in the maternal response to tissue injuries by differentiating into many lineages. Therefore, further understanding of fetal-maternal microchimerism may help in anticipating its implications in disease as well as in more general women's health issues.

Direct detection of fetal cells in maternal blood: a reappraisal using a combination of two different Y chromosome-specific FISH probes and a single X chromosome-specific probe.

BACKGROUND: We have recently explored the detection of circulatory male fetal cells directly in maternal whole blood samples by fluorescence in-situ hybridization (FISH). In order to improve the efficacy of fetal cell detection, we have now examined whether this could be enhanced by the use of two different Y chromosome-specific FISH probes (alpha-satellite and classical satellite III regions) in combination with an X chromosome-specific FISH probe. METHODS: Nineteen maternal blood samples (median gestational age = 28 weeks, range = 12-37 weeks) were examined in a blinded manner. No enrichment procedure was performed. Following hypotonic treatment and Carnoy's fixation, total nucleated cells were examined by two color FISH with a single X and two Y chromosome-specific probes. Nine cases were examined in parallel by conventional XY-FISH. RESULTS: Fetal cell detection was superior when using two Y chromosome-specific probes (specificity = 75%; sensitivity = 91%) when compared to the conventional XY-FISH approach (specificity = 50%; sensitivity = 60%). CONCLUSIONS: Male fetal cells can be detected in most maternal blood samples examined. Specificity and sensitivity is improved when using a combination of single X and two Y chromosome-specific probes when compared to a conventional XY-FISH protocol.

Nucleic acid based biosensors: the desires of the user.

The need for nucleic acid based diagnostic tests has increased enormously in the last few years. On the one hand, this has been stimulated by the discovery of new hereditary genetic disease loci following the completion of the Human Genome Project, but also by the presence of new rapidly spreading viral threats, such as that of the SARS epidemic, or even micro-organisms released for the purpose of biological warfare. As in many instances rapid diagnoses of specific target genetic loci is required, new strategies have to be developed, which will allow this to be achieved directly at the point-of-care setting. One of these avenues being explored is that of biosensors. In this review, we provide an overview of the current state of the art concerning the high-throughput analysis of nucleic acids, and address future requirements, which will hopefully be met by new biosensor-based developments.

Numerous erythroblasts in maternal blood are impervious to fluorescent in situ hybridization analysis, a feature related to a dense compact nucleus with apoptotic character.

BACKGROUND AND OBJECTIVES: The analysis by fluorescence in situ hybridization (FISH) of fetal erythroblasts enriched from maternal blood remains an attractive alternative for risk-free prenatal diagnosis of aneuploidies. However, current results are discouraging because of the low levels of sensitivity or the inability to detect fetal erythroblasts by FISH. DESIGN AND METHODS: Erythroblasts were enriched from 35 maternal blood samples by magnetic cell sorting (MACS), identified morphologically following May-Grünwald Giemsa staining and examined by FISH for chromosomes X, Y and 18. RESULTS: We observed that circulating erythroblasts comprised two distinct groups: one was clearly of maternal origin and could be reliably analyzed by FISH, whereas the other, which appeared to be of fetal origin, was largely impervious to FISH analysis. This latter feature seemed to be related to an abnormally dense nucleus with an apoptotic character. Since the oxygen tension in the maternal circulation is higher than that in the fetus, we cultured fetal cord blood erythroblasts in conditions mimicking this difference in oxygen concentrations and found that high oxygen concentrations rapidly induced shrinkage of the erythroblast nucleus, rendering it impervious to FISH analysis. INTERPRETATION AND CONCLUSIONS: Our data show that circulating erythroblasts of presumed fetal origin cannot be reliably analyzed by FISH because of an abnormally dense nucleus. This nuclear phenotype appears to be induced by the higher oxygen tension present in the maternal circulation than in fetal blood.

FISH analysis of all fetal nucleated cells in maternal whole blood: improved specificity by the use of two Y-chromosome probes.

Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12-20 male fetal cells/ml of maternal blood were identified by XY- and YY-FISH, respectively.

Evaluation of a soybean lectin-based method for the enrichment of erythroblasts.

We performed a comparative study of the enrichment of erythroblasts by a soybean agglutinin galactose-specific lectin method and a standardized magnetic cell-sorting (MACS) protocol. Blood samples, obtained from 11 pregnant women at between 11 and 40 weeks of gestation, were split and examined by each method in parallel. The number of erythroblasts recovered by the lectin method was approximately eightfold higher than the number obtained by MACS. Our data suggest that the lectin-based method may provide a better approach for the enrichment of rare fetal erythroblasts from maternal blood.

Characterization of genomic variants in CSH1 and GH2, two candidate genes for Silver-Russell syndrome in 17q24-q25.

Silver-Russell syndrome (SRS) is a syndrome of severe pre- and postnatal growth retardation and typical dysmorphic features. Rare chromosomal aberrations have been reported in SRS; among these are two balanced translocations involving 17q24-q25. Recently, we described a patient with a paternally inherited heterozygous deletion of the chorionic somatomammotropin hormone 1 (CSH1) gene. The CSH1 gene is member of the growth hormone (GH) gene cluster on 17q, which consists of two growth hormone genes and three CSH genes. Genomic alterations in the GH cluster are well known, causing different phenotypes depending on the size of the deletion and the genes involved. By screening 63 SRS cases with marker D17S254, we have detected 2 further patients with a heterozygous deletion in the GH cluster. Quantitative analysis using restriction assays confirmed these findings. Additionally, in a cohort of 17 patients with isolated intrauterine and postnatal growth retardation, we detected a further patient to be carrier of a CSH1 deletion. Screening of 141 unrelated controls revealed hemizygosity in one person for which data on growth were not available. We additionally analyzed our cohort of SRS patients for mutations in CSH1 and its 3' neighbour GH2. However, analyses failed to reveal any pathogenic mutation. While the central role of GH1 in human growth is well established, the physiological roles of CSH1 and other components of the cluster are unclear. The increased prevalence of hemizygosity of CSH1 in our population in comparison to controls indicates a role for CSH1 haploinsufficiency in the etiology of growth retardation. Investigation of CSH1 deletions in further SRS and growth retarded patients will enable us to establish under which circumstances haploinsufficiency of CSH1 is likely to result in clinical changes.

Correlation of protein expression and gene expression in acute leukemia.

BACKGROUND: Flow cytometry (FC) is a standard method for diagnosing and subclassifying acute myeloid (AML) and acute lymphoblastic (ALL) leukemias and allows the analysis of cell surface and intracellular proteins. In the future, diagnostic procedures may include oligonucleotide microarray analysis (MA) to detect expression patterns of large numbers of specific genes. METHODS: For comparison between methods, we performed FC and MA by using the Affymetrix GeneChip HG-U133A microarray in parallel and correlated protein expression levels and mRNA abundance of 39 relevant genes in 113 patients with newly diagnosed AML and ALL and four normal bone marrow samples. RESULTS: In 1,512 of 2,187 (69.1%) comparisons between methods, congruent results were obtained with regard to positivity or negativity of expression, respectively. Specifically, there was a significant correlation between protein expression and mRNA abundance for genes essential for diagnosing and subclassifying AML and ALL with regard to positivity and expression. CONCLUSIONS: These data suggest that protein expression is highly correlated to mRNA abundance in AML and ALL. Further, expression patterns of specific genes provide important information at diagnosis for patients with AML and ALL that may be used for the discrimination from other leukemias.

Acute myeloid leukemias with reciprocal rearrangements can be distinguished by specific gene expression profiles.

Acute myeloid leukemia (AML) is a heterogeneous group of genetically defined diseases. Their classification is important with regard to prognosis and treatment. We performed microarray analyses for gene expression profiling on bone marrow samples of 37 patients with newly diagnosed AML. All cases had either of the distinct subtypes AML M2 with t(8;21), AML M3 or M3v with t(15;17), or AML M4eo with inv(16). Diagnosis was established by cytomorphology, cytogenetics, fluorescence in situ hybridization, and reverse transcriptase-PCR in every sample. By using two different strategies for microarray data analyses, this study revealed a unique correlation between AML-specific cytogenetic aberrations and gene expression profiles.