Biomedical subjects

David Givol

Publications and source records attributed to David Givol.

12 recordsLinked to original sources

PubMed2006

Nanog transforms NIH3T3 cells and targets cell-type restricted genes.

The transcription factor Nanog is uniquely expressed in embryonic stem (ES) cells and in germ cell tumors and is important for self-renewal. To understand the relation between this and cell transformation, we expressed Nanog in NIH3T3 cells, and these cells showed an increased growth rate and a transformed phenotype as demonstrated by foci formation and colony growth in soft agar. This suggests that Nanog possesses an oncogenic potential that may be related to the role it plays in germ cell tumors and to its function in self renewal of ES cells. We studied the transcription targets of Nanog using microarrays to identify Nanog regulated genes. The list of genes regulated by Nanog was unique for each cell type and more than 10% of the Nanog regulated genes, including transcription factors, are primary Nanog targets since their promoters bind Nanog in ES cells. Some of these target genes can explain the transformation of NIH3T3.

Animals↗

PubMed2005

BCL6 is regulated by p53 through a response element frequently disrupted in B-cell non-Hodgkin lymphoma.

The BCL6 transcriptional repressor mediates survival, proliferation, and differentiation blockade of B cells during the germinal-center reaction and is frequently misregulated in B-cell non-Hodgkin lymphoma (BNHL). The p53 tumor-suppressor gene is central to tumorigenesis. Microarray analysis identified BCL6 as a primary target of p53. The BCL6 intron 1 contains a region in which 3 types of genetic alterations are frequent in BNHL: chromosomal translocations, point mutations, and internal deletions. We therefore defined it as TMDR (translocations, mutations, and deletions region). The BCL6 gene contains a p53 response element (p53RE) residing within the TMDR. This p53RE contains a motif known to be preferentially targeted by somatic hypermutation. This p53RE is evolutionarily conserved only in primates. The p53 protein binds to this RE in vitro and in vivo. Reporter assays revealed that the BCL6 p53RE can confer p53-dependent transcriptional activation. BCL6 mRNA and protein levels increased after chemotherapy/radiotherapy in human but not in murine tissues. The increase in BCL6 mRNA levels was attenuated by the p53 inhibitor PFT-alpha. Thus, we define the BCL6 gene as a new p53 target, regulated through a RE frequently disrupted in BNHL.

Animals↗

PubMed2005

Vascular gene expression and phenotypic correlation during differentiation of human embryonic stem cells.

The study of the cascade of events of induction and sequential gene activation that takes place during human embryonic development is hindered by the unavailability of postimplantation embryos at different stages of development. Spontaneous differentiation of human embryonic stem cells (hESCs) can occur by means of the formation of embryoid bodies (EBs), which resemble certain aspects of early embryos to some extent. Embryonic vascular formation, vasculogenesis, is a sequential process that involves complex regulatory cascades. In this study, changes of gene expression along the development of human EBs for 4 weeks were studied by large-scale gene screening. Two main clusters were identified-one of down-regulated genes such as POU5, NANOG, TDGF1/Cripto (TDGF, teratocarcinoma-derived growth factor-1), LIN28, CD24, TERF1 (telomeric repeat binding factor-1), LEFTB (left-right determination, factor B), and a second of up-regulated genes such as TWIST, WNT5A, WT1, AFP, ALB, NCAM1. Focusing on the vascular system development, genes known to be involved in vasculogenesis and angiogenesis were explored. Up-regulated genes include vasculogenic growth factors such as VEGFA, VEGFC, FIGF (VEGFD), ANG1, ANG2, TGFbeta3, and PDGFB, as well as the related receptors FLT1, FLT4, PDGFRB, TGFbetaR2, and TGFbetaR3, other markers such as CD34, VCAM1, PECAM1, VE-CAD, and transcription factors TAL1, GATA2, and GATA3. The reproducibility of the array data was verified independently and illustrated that many genes known to be involved in vascular development are activated during the differentiation of hESCs in culture. Hence, the analysis of the vascular system can be extended to other differentiation pathways, allocating human EBs as an in vitro model to study early human development.

Antigens, CD34↗

PubMed2005

CD133-positive hematopoietic stem cell "stemness" genes contain many genes mutated or abnormally expressed in leukemia.

Affymetrix human Hu133A oligonucleotide arrays were used to study the expression profile of CD133+ cord blood (CB) and peripheral blood (PB) using CD133 cell-surface marker. An unsupervised hierarchical clustering of 14,025 valid probe sets showed a clear distinction between the CD133+ cells representing the hematopoietic stem cell (HSC) population and CD133-differentiated cells. Two hundred forty-four genes were found to be upregulated by at least twofold in the CD133-positive cells of both CB and PB compared with the CD133-negative cells. These genes represent the hematopoietic "stemness," whereas the 218 and 304 upregulated genes exclusively in PB and CB, respectively, represent tissue specificity. Some of the stemness genes were also common to HSC genes found to be upregulated in several recently published studies. Among these common stemness genes, we identified several groups of genes that have an important role in hematopoiesis: growth factor receptors, transcription factors, genes that have an important role in development, and genes involved in cell growth. Sixteen selected stemness genes are known to be mutated or abnormally regulated in acute leukemias. It can be suggested that key hematopoietic stemness machinery genes may lead to abnormal proliferation and leukemia upon mutation or change of their expression.

AC133 Antigen↗

PubMed2004

Design principle of gene expression used by human stem cells: implication for pluripotency.

Human embryonic stem cells (ESC) are undifferentiated and are endowed with the capacities of self-renewal and pluripotential differentiation. Adult stem cells renew their own tissue, but whether they can transdifferentiate to other tissues is still controversial. To understand the genetic program that underlies the pluripotency of stem cells, we compared the transcription profile of ESC with that of progenitor/stem cells of human hematopoietic and keratinocytic origins, along with their mature cells to be viewed as snapshots along tissue differentiation. ESC gene profiles show higher complexity with significantly more highly expressed genes than adult cells. We hypothesize that ESC use a strategy of expressing genes that represent various differentiation pathways and selection of only a few for continuous expression upon differentiation to a particular target. Such a strategy may be necessary for the pluripotency of ESC. The progenitors of either hematopoietic or keratinocytic cells also follow the same design principle. Using advanced clustering, we show that many of the ESC expressed genes are turned off in the progenitors/stem cells followed by a further down-regulation in adult tissues. Concomitantly, genes specific to the target tissue are up-regulated toward mature cells of skin or blood.

Cell Differentiation↗

PubMed2004

Outcome signature genes in breast cancer: is there a unique set?

MOTIVATION: Predicting the metastatic potential of primary malignant tissues has direct bearing on the choice of therapy. Several microarray studies yielded gene sets whose expression profiles successfully predicted survival. Nevertheless, the overlap between these gene sets is almost zero. Such small overlaps were observed also in other complex diseases, and the variables that could account for the differences had evoked a wide interest. One of the main open questions in this context is whether the disparity can be attributed only to trivial reasons such as different technologies, different patients and different types of analyses. RESULTS: To answer this question, we concentrated on a single breast cancer dataset, and analyzed it by a single method, the one which was used by van't Veer et al. to produce a set of outcome-predictive genes. We showed that, in fact, the resulting set of genes is not unique; it is strongly influenced by the subset of patients used for gene selection. Many equally predictive lists could have been produced from the same analysis. Three main properties of the data explain this sensitivity: (1) many genes are correlated with survival; (2) the differences between these correlations are small; (3) the correlations fluctuate strongly when measured over different subsets of patients. A possible biological explanation for these properties is discussed. CONTACT: eytan.domany@weizmann.ac.il SUPPLEMENTARY INFORMATION: http://www.weizmann.ac.il/physics/complex/compphys/downloads/liate/

Biomarkers, Tumor↗

PubMed2004

Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype.

We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. These observations indicate that the MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. These observations suggest a model in which Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1.

Achondroplasia↗

PubMed2003

Hair growth induction by the Tellurium immunomodulator AS101: association with delayed terminal differentiation of follicular keratinocytes and ras-dependent up-regulation of KGF expression.

The synthetic immunomodulator AS101[ammonium trichloro(dioxoethylene-o,o')tellurate] was previously found to protect cancer patients from chemotherapy-induced bone marrow toxicity and alopecia. Here we show that AS101 induces hair growth in nude and normal mice. AS101 possesses the dual ability to both induce anagen and retard spontaneous catagen in the C57BL/6 mouse model. Anagen induced by AS101 is mediated by keratinocyte growth factor (KGF), as it is abrogated both in nude mice co-treated with AS101 plus neutralizing anti KGF antibodies and in AS101-treated transgenic mice expressing a dominant-negative KGF receptor transgene in basal keratinocytes. AS101 up-regulates KGF expression by activating the ras signaling pathway in cultured fibroblasts. AS101-induced delayed catagen is associated with inhibition of terminal differentiation marker expression both in nude and C57BL/6 mice epidermal follicular keratinocytes and in cultures of primary mouse follicular keratinocytes induced to differentiate. This activity is associated with relatively sustained elevation of p21waf. Delayed expression of terminal differentiation markers was not induced by AS101 in follicular keratinocytes from p21waf knockout mice. Because similar results were obtained with cultures of primary human keratinocytes and fibroblasts, preliminary case report studies revealed substantial hair growth when AS101 was topically applied on three adolescents who had remained alopeciac 1-2 years after chemotherapy. The results emphasize the unique mode of action of AS101 and highlight its potential clinical use for treating certain types of alopecia.

Adjuvants, Immunologic↗

PubMed2003

Genome-wide comparison of human keratinocyte and squamous cell carcinoma responses to UVB irradiation: implications for skin and epithelial cancer.

To gain insight into the transformation of epidermal cells into squamous carcinoma cells (SCC), we compared the response to ultraviolet B radiation (UVB) of normal human epidermal keratinocytes (NHEK) versus their transformed counterpart, SCC, using biological and molecular profiling. DNA microarray analyses (Affymetrix), approximately 12000 genes) indicated that the major group of upregulated genes in keratinocytes fall into three categories: (i). antiapoptotic and cell survival factors, including chemokines of the CXC/CC subfamilies (e.g. IL-8, GRO-1, -2, -3, SCYA20), growth factors (e.g. HB-EGF, CTGF, INSL-4), and proinflammatory mediators (e.g. COX-2, S100A9), (ii). DNA repair-related genes (e.g. GADD45, ERCC, BTG-1, Histones), and (iii). ECM proteases (MMP-1, -10). The major downregulated genes are DeltaNp63 and PUMILIO, two potential markers for the maintenance of keratinocyte stem cells. NHEK were found to be more resistant than SCC to UVB-induced apoptosis and this resistance was mainly because of the protection from cell death by secreted survival factors, since it can be transferred from NHEK to SCC cultures by the conditioned medium. Whereas the response of keratinocytes to UVB involved regulation of key checkpoint genes (p53, MDM2, p21(Cip1), DeltaNp63), as well as antiapoptotic and DNA repair-related genes - no or little regulation of these genes was observed in SCC. The effect of UVB on NHEK and SCC resulted in upregulation of 251 and 127 genes, respectively, and downregulation of 322 genes in NHEK and 117 genes in SCC. To further analyse these changes, we used a novel unsupervised coupled two-way clustering method that allowed the identification of groups of genes that clearly partitioned keratinocytes from SCC, including a group of genes whose constitutive expression levels were similar before UVB. This allowed the identification of discriminating genes not otherwise revealed by simple static comparison in the absence of UVB irradiation. The implication of the changes in gene profile in keratinocytes for epithelial cancer is discussed.

Apoptosis↗

PubMed2003

Inhibition of p53-induced apoptosis without affecting expression of p53-regulated genes.

Using DNA microarray and clustering of expressed genes we have analyzed the mechanism of inhibition of wild-type p53-induced apoptosis by the cytokine interleukin 6 (IL-6) and the calcium mobilizer thapsigargin (TG). Clustering analysis of 1,786 genes, the expression level of which changed after activation of wild-type p53 in the absence or presence of IL-6 or TG, showed that these compounds did not cause a general inhibition of the ability of p53 to up-regulate or down-regulate gene expression. Expression of various p53 targets implicated as mediators of p53-induced apoptosis was also not affected by IL-6 or TG. These compounds thus can bypass the effect of wild-type p53 on gene expression and inhibit apoptosis. IL-6 and TG activated different p53-independent pathways of gene expression that include up-regulation of antiapoptotic genes. IL-6 and TG also activated different differentiation-associated genes. The ability of compounds such as cytokines and calcium mobilizers to inhibit p53-mediated apoptosis without generally inhibiting gene expression regulated by p53 can facilitate tumor development and tumor resistance to radiation and chemotherapy in cells that retain wild-type p53.

Apoptosis↗

PubMed2002

Identification of direct p73 target genes combining DNA microarray and chromatin immunoprecipitation analyses.

The newly discovered p53 family member, p73, has a striking homology to p53 in both sequence and modular structure. Ectopic expression of p73 promotes transcription of p53 target genes and recapitulates the most characterized p53 biological effects such as growth arrest, apoptosis, and differentiation. Unlike p53-deficient mice that develop normally but are subject to spontaneous tumor formation, p73-deficient mice exhibit severe defects in the development of central nervous system and suffer from inflammation but are not prone to tumor development. These phenotypes suggest different biological activities mediated by p53 and p73 that might reflect activation of specific sets of target genes. Here, we have analyzed the gene expression profile of H1299 cells after p73alpha or p53 activation using oligonucleotide microarrays capable of detecting approximately 11,000 mRNA species. Our results indicate that p73alpha and p53 activate both common and distinct groups of genes. We found 141 and 320 genes whose expression is modulated by p73alpha and p53, respectively. p73alpha up-regulates 85 genes, whereas p53 induces 153 genes, of which 27 are in common with p73alpha. Functional classification of these genes reveals that they are involved in many aspects of cell function ranging from cell cycle and apoptosis to DNA repair. Furthermore, we report that some of the up-regulated genes are directly activated by p73alpha or p53.

Base Sequence↗

PubMed2002

A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1.

p53 exerts its tumor suppressor effects by activating genes involved in cell growth arrest and programmed cell death. The p53 target genes inducing growth arrest are well defined whereas those inducing apoptosis are not fully characterized. Proapoptotic activity of p53 was shown to involve several genes like Bax, Noxa and Puma, which may function in the release of cytochrome c from the mitochondria. Cytochrome c associates with Apaf-1 and caspase 9 to form the apoptosome. Genetic and cellular data indicate that Apaf-1 deficiency abrogates the apoptotic effect of p53 and substitutes for p53 loss in promoting tumor formation. Here we show that Apaf-1, the mammalian homologue of C. elegans CED4, is a direct target of p53 as demonstrated by gel shift analysis of the target site sequence in the presence of p53 and by Apaf-1 promoter-luciferase assays. We also show that the p53 activation of the Apaf-1 luciferase construct can be enhanced by the putative tumor suppressor gene product, Zac-1, a transcription factor that has previously been shown to inhibit cell proliferation. Furthermore, we demonstrate that Zac-1 is a possible direct target of p53 since the sequence upstream to the first coding exon of Zac-1 contains a p53 recognition site and the luciferase construct containing this region is activated by p53. These results suggests the existence of a tightly controlled self amplifying mechanism of transcriptional activation leading to apoptosis by p53.

Animals↗