NORMAL GENOMIC

• Veeck J et al.
• Prognostic relevance of Wnt-inhibitory factor-1 (WIF1) and Dickkopf-3(DKK3) promoter methylation in human breast cancer.
• BMC Cancer. 2009; 9: 217-217
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• BACKGROUND: Secreted Wnt signaling antagonists have recently beendescribed as frequent targets of epigenetic inactivation in human tumorentities. Since gene silencing of certain Wnt antagonists was found to becorrelated with adverse patient survival in cancer, we aimed atinvestigating a potential prognostic impact of the two Wnt antagonizingmolecules WIF1 and DKK3 in breast cancer, which are frequently silenced bypromoter methylation in this disease. METHODS: WIF1 and DKK3 promotermethylation were assessed by methylation-specific PCR withbisulfite-converted DNA from 19 normal breast tissues and 150 primarybreast carcinomas. Promoter methylation was interpreted in a qualitative,binary fashion. Statistical evaluations included two-sided Fisher's exacttests, univariate log-rank tests of Kaplan-Meier curves as well asmultivariate Cox regression analyses. RESULTS: WIF1 and DKK3 promotermethylation were detected in 63.3% (95/150) and 61.3% (92/150) of breastcarcinoma samples, respectively. In normal breast tissues, WIF1methylation was present in 0% (0/19) and DKK3 methylation in 5.3% (1/19)of samples. In breast carcinomas, WIF1 methylation was significantlyassociated with methylation of DKK3 (p = 0.009). Methylation of eithergene was not associated with clinicopathological parameters, except forDKK3 methylation being associated with patient age (p = 0.007). Inunivariate analysis, WIF1 methylation was not associated with clinicalpatient outcome. In contrast, DKK3 methylation was a prognostic factor inpatient overall survival (OS) and disease-free survival (DFS). EstimatedOS rates after 10 years were 54% for patients with DKK3-methylated tumors,in contrast to patients without DKK3 methylation in the tumor, who had afavorable 97% OS after 10 years (p < 0.001). Likewise, DFS at 10 years forpatients harboring DKK3 methylation in the tumor was 58%, compared with78% for patients with unmethylated DKK3 (p = 0.037). Multivariate analysesrevealed that DKK3 methylation was an independent prognostic factorpredicting poor OS (hazard ratio (HR): 14.4; 95% confidence interval (CI):1.9-111.6; p = 0.011), and short DFS (HR: 2.5; 95% CI: 1.0-6.0; p = 0.047)in breast cancer. CONCLUSION: Although the Wnt antagonist genes WIF1 andDKK3 show a very similar frequency of promoter methylation in human breastcancer, only DKK3 methylation proves as a novel prognostic markerpotentially useful in the clinical management of this disease.

• Davidson G, Mao B, del Barco Barrantes I, Niehrs C
• Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNSpatterning.
• Development. 2002; 129: 5587-96
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• A gradient of Wnt/beta-catenin signalling formed by posteriorising Wntsand anteriorising Wnt antagonists regulates anteroposterior (AP)patterning of the central nervous system (CNS) during Xenopusgastrulation. In this process, the secreted Wnt antagonist Dkk1 functionsin the Spemann organiser and its anterior derivatives by blocking Wntreceptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class.In addition to LRP6, Dkk1 interacts with another recently identifiedreceptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2(Krm2) to synergistically inhibit LRP6. We have investigated the role ofKrm1 and Krm2 during early Xenopus embryogenesis. Consistent with a rolein zygotic Wnt inhibition, overexpressed Krm anteriorises embryos andrescues embryos posteriorised by Wnt8. Antisense morpholinooligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency ofanterior neural development. In this process, Krm proteins functionallyinteract with Dkk1: (1) in axis duplication assays krm2 synergises withdkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2 rescues microcephalicembryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3)injection of krm1/2 antisense Mo enhances microcephaly induced byinhibitory anti-Dkk1 antibodies. The results indicate that Krm proteinsfunction in a Wnt inhibition pathway regulating early AP patterning of theCNS.

• Pandur P, Maurus D, Kuhl M
• Increasingly complex: new players enter the Wnt signaling network.
• Bioessays. 2002; 24: 881-4
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• Wnt proteins can activate different intracellular signaling cascades invarious organisms by interacting with receptors of the Frizzled family.The first identified Wnt signaling pathway, the Wnt/beta-catenin pathway,has been studied in much detail and is highly conserved among species. Asto non-canonical Wnt pathways, the current situation is more nebulouspartly because the intracellular mediators of this pathway are not yetfully understood and, in some cases, even identified. However, there areincreasing data that prove the existence of non-canonical Wnt signalingand demonstrate its involvement in different developmental processes. Invertebrates, Wnt-11 and Wnt-5A can activate the Wnt/JNK pathway, whichresembles the planar cell polarity pathway in Drosophila. TheWnt/Ca(2+)-pathway has only been described in Xenopus and zebrafish so farand it is unclear whether it also exists in other organisms. Two recentpapers provide us with new insight into non-canonical Wnt signaling by (1)presenting a new intracellular mediator of non-canonical signaling inXenopus1 and (2) implicating the existence of an additional non-canonicalWnt signaling pathway in flies.

• Hikasa H, Shibata M, Hiratani I, Taira M
• The Xenopus receptor tyrosine kinase Xror2 modulates morphogeneticmovements of the axial mesoderm and neuroectoderm via Wnt signaling.
• Development. 2002; 129: 5227-39
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• The Spemann organizer plays a central role in neural induction, patterningof the neuroectoderm and mesoderm, and morphogenetic movements duringearly embryogenesis. By seeking genes whose expression is activated by theorganizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, weisolated the receptor tyrosine kinase Xror2. Xror2 is expressed initiallyin the dorsal marginal zone, then in the notochord and the neuroectodermposterior to the midbrain-hindbrain boundary. mRNA injection experimentsrevealed that overexpression of Xror2 inhibits convergent extension of thedorsal mesoderm and neuroectoderm in whole embryos, as well as theelongation of animal caps treated with activin, whereas it does not appearto affect cell differentiation of neural tissue and notochord.Interestingly, mutant constructs in which the kinase domain waspoint-mutated or deleted (named Xror2-TM) also inhibited convergentextension, and did not counteract the wild-type, suggesting that theectodomain of Xror2 per se has activities that may be modulated by theintracellular domain. In relation to Wnt signaling for planar cellpolarity, we observed: (1) the Frizzled-like domain in the ectodomain isrequired for the activity of wild-type Xror2 and Xror2-TM; (2)co-expression of Xror2 with Xwnt11, Xfz7, or both, synergisticallyinhibits convergent extension in embryos; (3) inhibition of elongation byXror2 in activin-treated animal caps is reversed by co-expression of adominant negative form of Cdc42 that has been suggested to mediate theplanar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2interacts with Xwnts in co-immunoprecipitation experiments. These resultssuggest that Xror2 cooperates with Wnts to regulate convergent extensionof the axial mesoderm and neuroectoderm by modulating the planar cellpolarity pathway of Wnt.

• Pohl BS, Knochel W
• Overexpression of the transcriptional repressor FoxD3 prevents neuralcrest formation in Xenopus embryos.
• Mech Dev. 2001; 103: 93-106
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• Xenopus FoxD3 (XFD-6) is an intron-less gene initially expressed withinthe Spemann organizer and later in premigratory neural crest cells. Basedupon sequence and expression pattern comparisons, it represents theXenopus orthologue to zebrafish fkd6, chicken CWH-3 and mammalian HFH-2(genesis). Early expression of FoxD3 is activated by the Wnt-pathway andinhibited by BMP signalling. Ectopic overexpression of FoxD3 leads to anenlargement of the neural plate concomitant with a failure in neural crestformation, loss of anterior structures, lack of closure of the neural tubeand severe defects in somitogenesis. Phenotypic variation is accompaniedby down-regulation of neural crest markers, including Xslug, Xtwist andXcadherin-11. FoxD3 also inhibits its own expression, thereby acting in anegative autoregulatory loop. By injections of VP16 and engrailed fusionswe can demonstrate that FoxD3 acts as a negative transcriptionalregulator; this repressive function strictly requires the presence of thewinged helix domain. Transplantation experiments show that FoxD3overexpressing cells from the prospective neural crest do neitherdifferentiate nor migrate.

• Jiang LI, Sternberg PW
• An HMG1-like protein facilitates Wnt signaling in Caenorhabditis elegans.
• Genes Dev. 1999; 13: 877-89
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• We show that during Caenorhabditis elegans male spicule development, thespecification of a glial versus neuronal cell fate in a canonicalneurogenic sublineage is dependent on Wnt signaling. Inactivation of a Wntsignaling pathway mediated by the Wnt receptor LIN-17 transforms the SPDsheath cell into its sister, the SPD neuron. We discovered a new mutant,son-1, that displays this same cell fate transformation. The son-1mutation enhances the phenotypes of reduction-of-function lin-17 mutantsin several developmental processes, including vulva development, somaticgonad development, and male tail patterning. son-1 encodes an HMG1/2-likeDNA-binding protein and is localized in all cell nuclei throughdevelopment as revealed by a GFP reporter construct. Disruption of son-1function by RNA-mediated interference results in the same spicule defectas caused by overexpression of POP-1, a TCF/LEF class HMG protein known toact downstream of the Wnt signaling pathway. Our results provide in vivoevidence for the functional involvement of an HMG1/2-like protein, SON-1,in Wnt signaling. The sequence nonspecific HMG protein SON-1 and thesequence specific HMG protein POP-1 might both act in the Wnt respondingcells to regulate gene transcription in opposite directions.

• Gumbiner BM
• Propagation and localization of Wnt signaling.
• Curr Opin Genet Dev. 1998; 8: 430-5
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• Cellular mechanisms for the transport and localization of Wnt signalingcomponents are important for the propagation, distribution, andpolarization of Wnt signals in embryonic tissues. Wnt signals aredistributed through tissues by vesicular transport of Wnt proteins,localized in embryos by directed transport of cytoplasmic Wnt-signalingcomponents, and propagated asymmetrically during cell division.

• Iwama A, Okano K, Sudo T, Matsuda Y, Suda T
• Molecular cloning of a novel receptor tyrosine kinase gene, STK, derivedfrom enriched hematopoietic stem cells.
• Blood. 1994; 83: 3160-9
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• To identify the novel receptor tyrosine kinases (RTKs) critical to theproliferation of hematopoietic stem cells, we performed polymerase chainreaction-based cloning from highly purified murine hematopoietic stemcells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- orSca-1-positive (Lin-c-KIT+Sca-1+) cells were sorted by afluorescence-activated cell sorter. Two sets of degenerate oligonucleotideprimers were directed to the conserved sequences of the catalytic domain,and were used to amplify cDNAs that encode protein tyrosine kinases(PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified,as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs wereidentical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK,FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK).Six novel cDNA sequences (stk series) were identified. However, three ofthem turned out to be BPK, RYK, and TEK. The remaining three showed highhomology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively.Characterization of full-length cDNA sequence of the v-SEA/cMET-relatedgene showed that this was a novel RTK gene and we named this gene STK(stem cell-derived tyrosine kinase). We identified two distinct forms ofSTK cDNA; the short one encoded a putative truncated protein that lackedmost of the extracellular domain. STK was expressed at various stages ofhematopoietic cells, including stem cells, but we could not detect anyapparent expression in other adult tissues. The expression of thetruncated form of mRNA was more predominant than that of the completeform. STK was assigned by fluorescent in situ hybridization to theR-positive F1 band of chromosome 9, the same region to which hepaticgrowth factor-like protein has been assigned. Characterization of thesePTKs, including STK, will be helpful to elucidate the molecular mechanismof the growth regulation of hematopoietic stem cells.

• Bohme B et al.
• PCR mediated detection of a new human receptor-tyrosine-kinase, HEK 2.
• Oncogene. 1993; 8: 2857-62
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• We have previously amplified cDNA subfragments of protein-tyrosine-kinases(PTKs) by using the polymerase chain reaction (PCR) and specific sets ofoligonucleotide primers derived from nucleotide sequences of their kinasedomain. In this study we have used a more directed approach to identifynew members of the EPH/elk-family by PCR of human embryonic cDNA: weutilized oligonucleotide primers specifically designed to a highlyconserved N-terminal motif and the kinase region of EPH/elk-PTKs inRNA-PCRs. The 5' and 3' elongation of the primary PCR product was achievedby the RACE (rapid amplification of cDNA ends)-technique. Sequenceanalysis of 3.8 kb of overlapping PCR products allowed to identify a novelreceptor-PTK, HEK 2 (human embryo kinase 2), as an additional member ofthis family, without the need to screen a cDNA library. This approachshould be useful for the rapid isolation of other PTK-genes as well.Analysis of genomic DNA placed HEK 2 on chromosome 3. Northern blotanalysis demonstrated the expression of a 4.6 kb HEK 2-mRNA in lung,brain, pancreas, liver, placenta, kidney, skeletal muscle, heart andseveral human cells. In a protein kinase assay with HEK 2-specificimmunoprecipitates from the human epidermoid carcinoma cell line A431, aprotein of 130 kDa was found phosphorylated.

• Potts JD, Harocopos GJ, Beebe DC
• Identification of receptor tyrosine kinases in the embryonic chicken lens.
• Curr Eye Res. 1993; 12: 759-63
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• Protein phosphorylation plays a critical role in the control of growth andregulation of many eukaryotic cells. Members of the protein tyrosinekinase (PTK) family of peptides function as growth factor receptors andoncoproteins. A common feature of members of the PTK family is a highlyconserved intracellular catalytic domain. We analyzed the chicken lensepithelium, which responds to several known growth factors, for thepresence of receptor PTK's. Using reverse transcription polymerase chainreaction (rtPCR) and degenerate primers made to conserved regions withinkinase domains, we amplified RNA from embryonic day 6 (E6) lens epitheliumand sequenced 135 cDNA clones. Sixteen distinct kinase sequences wereobtained. Eight of these sequences represented kinase domains of knownmammalian growth factor receptors, and six represented intercellularkinases. Two sequences appeared to code for new kinases. The amino acididentity of the chicken homologs ranged from 80-100% when compared totheir mammalian counterparts.

• Aroian RV, Sternberg PW
• Multiple functions of let-23, a Caenorhabditis elegans receptor tyrosinekinase gene required for vulval induction.
• Genetics. 1991; 128: 251-67
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• The let-23 gene, which encodes a putative tyrosine kinase of the epidermalgrowth factor (EGF) receptor subfamily, has multiple functions duringCaenorhabditis elegans development. We show that let-23 function isrequired for vulval precursor cells (VPCs) to respond to the signal thatinduces vulval differentiation: a complete loss of let-23 function resultsin no induction. However, some let-23 mutations that genetically reducebut do not eliminate let-23 function result in VPCs apparentlyhypersensitive to inductive signal: as many as five of six VPCs can adoptvulval fates, in contrast to the three that normally do. These resultssuggest that the let-23 receptor tyrosine kinase controls two opposingpathways, one that stimulates vulval differentiation and another thatnegatively regulates vulval differentiation. Furthermore, analysis of 16new let-23 mutations indicates that the let-23 kinase functions in atleast five tissues. Since various let-23 mutant phenotypes can be obtainedindependently, the let-23 gene is likely to have tissue-specificfunctions.

• Horvitz HR, Sulston JE
• "Joy of the worm".
• Genetics. 1990; 126: 287-92

• Wilks AF, Kurban RR, Hovens CM, Ralph SJ
• The application of the polymerase chain reaction to cloning members of theprotein tyrosine kinase family.
• Gene. 1989; 85: 67-74
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• Degenerate oligodeoxyribonucleotide (oligo) primers derived from aminoacid (aa) sequence motifs held in common between all members of theprotein tyrosine kinase (PTK) family were used to prime the amplificationof PTK-related sequences from a variety of murine cDNA sources, includingthe haemopoietic cell lines, FDC-P1 and WEHI-3B D+, peritoneal macrophagesand whole brain. Several parameters, such as the length (short, i.e., lessthan 20 nucleotides (nt) vs. long, i.e., greater than 30 nt) anddegeneracy (i.e., moderately degenerate vs. highly degenerate) of theoligo primers and the temperature of the extension phase of the reaction,were examined. The data from these analyses suggest that the mosteffective type of primer in this application of the polymerase chainreaction is a short, moderately degenerate oligo such as that which mightbe derived from the small patches of aa sequence homology that arefrequently found to be held in common among members of protein families.In addition to a number of previously described PTK sequences, a novelmammalian PTK-related sequence was uncovered.

• Finney M, Ruvkun G, Horvitz HR
• The C. elegans cell lineage and differentiation gene unc-86 encodes aprotein with a homeodomain and extended similarity to transcriptionfactors.
• Cell. 1988; 55: 757-69
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• Mutations in the gene unc-86 affect development of the nematode C. elegansby altering cell lineages and cell differentiation. We molecularly clonedunc-86 by chromosomal walking from linked polymorphic genetic loci, andidentified the gene by locating polymorphisms specific for unc-86 alleles.A transcript containing a 467 amino acid open reading frame was inferredfrom the DNA sequence of a genomic clone. The unc-86 transcript encodes aprotein containing a 158 amino acid sequence, referred to as the pou("pow") domain, which is strikingly similar to sequences found in threemammalian transcription factors. Within this conserved region, there is ahomeodomain related to but distinct from homeodomains previouslyidentified in Drosophila and other organisms. These findings suggest thatunc-86 encodes a transcription factor, and that the related mammaliantranscription factors may function to control cell fates and celldifferentiation.

• Kemphues KJ, Priess JR, Morton DG, Cheng NS
• Identification of genes required for cytoplasmic localization in early C.elegans embryos.
• Cell. 1988; 52: 311-20
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• We have isolated and analyzed eight strict maternal effect mutationsidentifying four genes, par-1, par-2, par-3, and par-4, required forcytoplasmic localization in early embryos of the nematode C. elegans.Mutations in these genes lead to defects in cleavage patterns, timing ofcleavages, and localization of germ line-specific P granules. Fourmutations in par-1 and par-4 are fully expressed maternal effect lethalmutations; all embryos from mothers homozygous for these mutations arrestas amorphous masses of differentiated cells but are specifically lackingintestinal cells. Four mutations in par-2, par-3, and par-4 areincompletely expressed maternal effect lethal mutations and are alsograndchildless; some embryos from homozygous mothers survive and grow tobecome infertile adults due to absence of functional germ cells. Wepropose that all of these defects result from the failure of a maternallyencoded system for intracellular localization in early embryos.

• Davis RJ, Czech MP
• Tumor-promoting phorbol diesters cause the phosphorylation of epidermalgrowth factor receptors in normal human fibroblasts at threonine-654.
• Proc Natl Acad Sci U S A. 1985; 82: 1974-8
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• The effect of tumor-promoting phorbol diesters to potentiate the action ofepidermal growth factor (EGF) on cell proliferation is associated withphosphorylation of EGF receptors, acute depression of EGF binding, andinhibition of EGF receptor tyrosine kinase activity. In the presentstudies, normal human fibroblasts and A431 carcinoma cells were labeledwith [32P]phosphate and treated with and without 10 nM 4 beta-phorbol 12beta-myristate 13 alpha-acetate (PMA). The EGF receptors then wereisolated by immunoprecipitation and digested with trypsin. Analysis of thelabeled receptor phosphopeptides by reversed-phase HPLC revealed that PMAinduces the phosphorylation of a unique phosphopeptide containing[32P]phosphothreonine. Comparison of several chemical and physicalproperties of the 32P-labeled phosphopeptide with the primary structure ofthe EGF receptor suggested the identify Lys-Arg-Thr(P)-Leu-Arg. This wasconfirmed by direct demonstration that a synthetic peptide of thisstructure comigrates during HPLC and electrophoresis with the 32P-labeledphosphopeptide isolated from the EGF receptors of normal humanfibroblasts. The phosphorylated site on the peptide corresponds tothreonine-654 of the EGF receptor, which is located on the cytoplasmicside of the plasma membrane nine residues distant from the transmembranedomain. These data indicate that phosphorylation of the EGF receptor inhuman fibroblasts and A431 cells at threonine-654 may regulate the EGFreceptor tyrosine kinase activity and the binding of EGF.