Ilse Wieland

Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome.

Craniofrontonasal syndrome (CFNS) is an X-linked craniofacial disorder with an unusual manifestation pattern, in which affected females show multiple skeletal malformations, whereas the genetic defect causes no or only mild abnormalities in male carriers. Recently, we have mapped a gene for CFNS in the pericentromeric region of the X chromosome that contains the EFNB1 gene, which encodes the ephrin-B1 ligand for Eph receptors. Since Efnb1 mutant mice display a spectrum of malformations and an unusual inheritance reminiscent of CFNS, we analyzed the EFNB1 gene in three families with CFNS. In one family, a deletion of exons 2-5 was identified in an obligate carrier male, his mildly affected brother, and in the affected females. In the two other families, missense mutations in EFNB1 were detected that lead to amino acid exchanges P54L and T111I. Both mutations are located in multimerization and receptor-interaction motifs found within the ephrin-B1 extracellular domain. In all cases, mutations were found consistently in obligate male carriers, clinically affected males, and affected heterozygous females. We conclude that mutations in EFNB1 cause CFNS.

Differences of E-cadherin expression levels and patterns in primary and metastatic human lung cancer

Normal lung epithelium and 52 lung carcinomas obtained at surgical resection were examined by immunofluorescence for their expression levels and patterns of the calcium-dependent intercellular adhesion molecule E-cadherin. In dysplastic lung tissue and in well-differentiated squamous cell and adenocarcinomas, expression of E-cadherin was confined to the lateral cell border, similar to the expression level and pattern of normal lung tissue. The E-cadherin level was reduced and expression pattern was spotty or diffuse in moderately and poorly differentiated squamous cell and in small cell carcinomas of the lung. Most metastases resected also had a reduced level and an altered pattern of E-cadherin expression. In contrast, no such correlation was found in adenocarcinomas of the lung. This indicates that different cellular mechanisms are responsible in the progression of squamous cell carcinomas and adenocarcinomas of the lung.

Monoclonal antibodies NORM-1 and NORM-2 induce more normal behavior of tumor cells in vitro and reduce tumor growth in vivo

In this study, large numbers of hybridomas (produced by syngeneic immunization with B16 mouse melanoma and fusion with NS-1 myeloma cells) were screened for the production of antibodies that affected morphology and growth of animal and human tumor cells in vitro. Two such antibodies, NORM-1 and NORM-2 (both IgG2a), inhibited the growth of B16 melanoma cells in soft agar and increased the serum requirements of tumor cells in tissue culture. Antibody NORM-2 also inhibited the growth of SV40-transformed 3T3 cells in agar and caused them to deposit more fibronectin into extracellular matrix. These antibodies thus seem to induce a more normal behavior of tumor cells in vitro. In vivo both antibodies reduced the number of growing lung tumors of B16 melanoma in C57BL/6 mice by 70%-90% when injected 3 days after the tumor cells. By immunoprecipitation of 35S-methionine-labeled cell extracts, NORM-2 antibody recognized a 59 kd protein in B16 mouse and in A375 human melanoma cells but not in 3T3 fibroblasts.

Refinement of the deletion in 7q21.3 associated with split hand/foot malformation type 1 and Mondini dysplasia.

Split hand/foot malformation type I (SHFM1, OMIM *183600) is an autosomal dominant developmental disorder of limb formation that results in the absence of the central digital rays, deep median clefts, and syndactyly of the remaining digits. Patients with SHFM1 harbour deletions, translocations, and inversions in chromosomal region 7q21–q22.1 The deletions at 7q21–q22 encompass different genomic regions and probably result in a contiguous gene syndrome that includes growth impairment, microcephaly, craniofacial manifestations, hernias, hearing loss, and mental retardation.2,3 Cases with translocations do not show this broad pattern of abnormalities but are associated with hearing loss in most cases.4,5 Split hand/foot malformation type I is the only form of split hand/foot malformation associated with sensorineural hearing loss, and it has been designated SHFM1D (OMIM *605617).5,6 Recently, SHFM1D was shown to result from Mondini dysplasia in a boy with a de novo deletion of about 8.9–17 cM of the paternal chromosome 7q21.1–q21.3.7 So far, microdeletions at 7q21.3 have been described in only two cases: one in a boy with split hand/foot malformation plus mild mental retardation, growth retardation of post-natal onset, and hypotonia and another in a patient with ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome.8,9 Mapping of the deletion and translocation breakpoints in several patients showed a critical interval of about 1 Mb for the SHFM1 locus at 7q21.3.9 This interval included a 500 kb region that spanned five of seven known translocation breakpoints. In this region, the candidate genes DLX5 and DLX6 (human homologues of the Drosophila distal-less homeobox gene family) and DSS1 (deleted in the split hand/split foot SHFM1 region) were identified. No mutations were detected in patients with sporadic split hand/foot malformation with translocations or two families with split hand/foot malformation, sensorineural deafness, and normal chromosomes who …

Deletion analysis at the DEL-27, APC and MTS1 loci in bladder cancer: LOH at the DEL-27 locus on 5p13-12 is a prognostic marker of tumor progression

Inactivation of relevant tumor‐suppressor genes by allelic or homozygous deletion is a characteristic event in tumor cells. Here, the prognostic value of allelic deletions on 5p13‐12 at the putative del‐27 tumor‐suppressor locus and in the APC tumor‐suppressor gene on 5q21, as well as homozygous deletions of the MTS1 (p16INK4, CDKN 2) tumor‐suppressor gene on 9p21 was assessed in 87 bladder cancers using microdissection and PCR‐based assays. Tumor‐specific LOH was detected in 10 of 38 (26%, del‐27), and 15 of 30 (50%, APC) informative specimens. Homozygous deletion of the MTS1 gene was detected in 33% of 84 tumors investigated. These deletion frequencies implicate the 3 tumor‐suppressor regions in the genesis of transitional‐cell carcinoma. In contrast to deletions of the APC or MTS1 genes, LOH at the del‐27 locus correlated with tumor progression. This suggests that loss of the putative tumor‐suppressor gene DEL‐27 is involved in an aggressive behavior of the tumor cells and appears to be a prognostic marker for the clinical outcome of patients with transitional‐cell carcinoma. Int. J. Cancer 74:291‐295, 1997.

Molecular characterization of the DICE1 (DDX26) tumor suppressor gene in lung carcinoma cells.

We have determined the genomic structure of the candidate tumor suppressor gene DICE1 (DDX26). The DICE1 gene colocalizes with microsatellite marker D13S284 telomeric to the RB1 gene in chromosomal region 13q14.3. The DICE1 gene encodes 18 exons that are preceded by a GC-rich promoter region. CpG sites flanking a predicted TATA box were found to be hypermethylated in tumor cells that exhibited decreased DICE1 expression. This suggests tumor-specific transcriptional silencing of the DICE1 gene may occur. Aberrantly spliced products were detected in two of three DICE1 expressing cell lines. The predicted DICE1 amino acid sequence is evolutionarily conserved in mouse, fruit fly (D. melanogaster), and nematode (C. elegans). A DEAD box characteristic of ATP-dependent helicases is the predominant motif found in DICE1 and its mouse and fruit fly homologues. Motifs other than the DEAD box are reminiscent of members of the helicase superfamily II but there is considerable variation from the typical DEAD box helicases. Expression of DICE1 green fluorescent fusion protein showed a preferential localization of DICE1 in the nucleus. This suggests that DICE1 is involved in nuclear processes such as DNA repair, transcription, or RNA splicing.

INTS6/DICE1 inhibits growth of human androgen-independent prostate cancer cells by altering the cell cycle profile and Wnt signaling

BackgroundThe gene encoding integrator complex subunit 6 (INTS6), previously known as deleted in cancer cells 1 (DICE1, OMIM 604331) was found to be frequently affected by allelic deletion and promoter hypermethylation in prostate cancer specimens and cell lines. A missense mutation has been detected in prostate cancer cell line LNCaP. Together, these results suggest INTS6/DICE1 as a putative tumor suppressor gene in prostate cancer. In this study, we examined the growth inhibitory effects of INTS6/DICE1 on prostate cancer cells.ResultsMarkedly decreased INTS6/DICE1 mRNA levels were detected in prostate cancer cell lines LNCaP, DU145 and PC3 as well as CPTX1532 as compared to a cell line derived from normal prostate tissue, NPTX1532. Exogenous re-expression of INTS6/DICE1 cDNA in androgen-independent PC3 and DU145 cell lines substantially suppressed their ability to form colonies in vitro. This growth inhibition was not due to immediate induction of apoptosis. Rather, prostate cancer cells arrested in G1 phase of the cell cycle. Expression profiling of members of the Wnt signaling pathway revealed up-regulation of several genes including disheveled inhibitor CXXC finger 4 (CXXC4), frizzled homologue 7 (FZD7), transcription factor 7-like 1 (TCF7L1), and down-regulation of cyclin D1.ConclusionThese results show for the first time a link between INTS6/DICE1 function, cell cycle regulation and cell-cell communication involving members of the Wnt signaling pathway.

Promoter CpG hypermethylation and downregulation of DICE1 expression in prostate cancer

A critical region of loss of heterozygosity on human chromosome 13q14 harbors the tumor suppressor gene DICE1 (DDX26). To elucidate the reduced DICE1 expression in tumor cells, the putative promoter sequence upstream of the DICE1 gene was analysed. This sequence shows a high GC content and is rich in CpG sites and binding sites of transcriptional factors. Promoter activity was identified within three overlapping fragments of the 800 bp sequence upstream of the DICE1 gene. A 13 bp deletion polymorphism detected in the DICE1 promoter region showed a decreased activity compared with the undeleted variant. However, this 13 bp deletion was seen in male control samples and patients with prostate cancer or benign prostatic hyperplasia at similar rates. A reduced DICE1 expression was observed in prostate cancer cell lines DU145 and LNCaP. This downregulation is associated with hypermethylation of the DICE1 promoter. Treatment of both prostate cancer cell lines with 5-azacytidine leads to upregulation of DICE1 expression. Hypermethylation of CpG sites of the DICE1 promoter was observed in four of eight analysed prostate cancers. This study suggests that transcriptional repression of DICE1 is caused by hypermethylation of the DICE1 promoter region in prostate cancer cells.

Dissecting the molecular mechanisms in craniofrontonasal syndrome : differential mRNA expression of mutant EFNB1 and the cellular mosaic

Craniofrontonasal syndrome (CFNS) is an X-linked malformation syndrome with variable phenotype that is caused by mutations in the ephrin-B1 gene (EFNB1). Over 50% of EFNB1 mutations result in premature termination codons that may elicit mRNA degradation by the nonsense-mediated decay pathway. To assess the effects of various mutations at the transcript level, expression of EFNB1 mRNA was studied by RT-PCR in fibroblast cultures established from CFNS female patients. Compared to the wild-type and two missense mutation alleles, severe depletion of transcripts was observed for mutant alleles harbouring either splice site mutation c.407-2A>T at the exon 2/3 boundary or frameshift mutation c.377_384delTCAAGAAG in exon 2. In contrast, escape from mRNA decay was observed for mutation c.614_615delCT, which generates a premature termination codon close to the 3′-end of the penultimate exon 4 disobeying the ‘50–55 bp’ rule. These results suggest differential degradation of mutant EFNB1 transcripts by the nonsense-mediated mRNA decay pathway. Although the clinical phenotypes of the patients were not highly suggestive of a phenotype–genotype correlation, the two female patients were diagnosed with diaphragmatic hernia harbouring putative ephrin-B1 truncating mutations. Previously, disease manifestation in heterozygous females had been attributed mainly to cellular interference of divergent cell populations expressing wild-type or mutant EFNB1, depending on the pattern of X-inactivation. Upon clonal expansion of patient cells with either the wild-type or mutant EFNB1 on the active X-chromosome, we were able to separate mutant and wild-type EFNB1-expressing cells in vitro, further supporting the concept of cellular interference in CFNS.

Molecular interactions of B-CAM (basal-cell adhesion molecule) and laminin in epithelial skin cancer

Molecular events underlying the progression of malignant tumors through the surrounding tissue are largely mediated by membrane-bound adhesion molecules. Basal-cell adhesion molecule (B-CAM), a 90-kDa laminin receptor of the immunoglobulin superfamily, is induced in some epithelial malignancies. Its function in these tumors, however, still remains obscure. We demonstrated that expression of B-CAM is very weak, if detectable at all, in normal epidermis but is strongly induced in both basal cell carcinomas and squamous cell carcinomas of the skin, and most pronounced at the basal surface of the tumor nests. Interestingly, the only known B-CAM ligand, laminin, was markedly upregulated within corresponding microanatomical sites surrounding the tumor nests, suggesting that both molecules may interact there. Consistent with this hypothesis, we were able to directly demonstrate binding of a B-CAM/Fc chimeric molecule to the peritumoral stroma in situ. Finally, in proof-of-principle experiments, human B-CAM was overexpressed both in murine and in human fibroblasts. The haptotactic migration of these novel B-CAM+ cell populations on a laminin matrix was significantly increased (P=0.02) as compared to mock-transfected cells when integrin-mediated adhesion was blocked by chelation of divalent cations. Thus, our findings provide the first direct experimental evidence that interactions of B-CAM and laminin may be involved in progression of epithelial skin tumors.