Recurrent rearrangements in the high mobility group protein gene, HMGI-C, in benign mesenchymal tumours.

We recently showed that the 1.7 megabase multiple aberration region (MAR) on human chromosome 12q15 harbours recurrent breakpoints frequently found in a variety of benign solid tumours. We now report a candidate gene within MAR suspected to be of pathogenetical relevance. Using positional cloning, we have identified the high mobility group protein gene HMGI-C within a 175 kilobase segment of MAR and characterized its genomic organization. By FISH analysis, we show the majority of the breakpoints of eight different benign solid tumour types fall within this gene. By Southern blot and 3'-RACE analysis, we demonstrate consistent rearrangements in HMGI-C and/or expression of altered HMGI-C transcripts. These results suggest a link between a member of the HMG gene family and benign solid tumour development.

Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican.

The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1) (Aviezer, D., D. Hecht, M. Safran, M. Eisinger, G. David, and A. Yayon. 1994. Cell 79:1005-1013). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan showed a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhanced the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that were not cotransfected with proteoglycan, but had no effect on this binding and decreased the yield of bFGFR cross-links in cells that were cotransfected with proteoglycan. Receptor-transfectant cells showed a decrease in glycophorin A expression when exposed to bFGF. This suppression was dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Finally, complementary cell-free binding assays indicated that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain was increased threefold when the syndecan-4 ectodomain was coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, had no effect on this binding. We conclude that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness.

Study of the synthesis and secretion of normal and artificial mutants of murine amyloid precursor protein (APP): cleavage of APP occurs in a late compartment of the default secretion pathway.

Amyloid precursor protein (APP) secretase plays a pivotal role in the processing of APP since its activity precludes the formation of amyloid peptide in Alzheimer's Disease. The identity and the subcellular localization of this enzyme are at this moment unknown. It is also unclear how APP escapes the activity of this enzyme when amyloid is formed. We have previously shown that APP-secretase activity is not inhibited by exogenously added proteinase inhibitors of different specificity (De Strooper, B., F. Van Leuven, and H. Van Den Berghe. 1992. FEBS (Fed. Eur. Biochem. Soc.) Lett. 308:50-53). We show here that the primary amine methylamine inhibits the secretion of APP into the medium. Furthermore, we show that a truncated form of APP, devoid of the cytoplasmic domain, is more efficiently cleaved and secreted than wild-type APP, which together with the methylamine block, shows that APP-secretase is located in a late compartment of the default constitutional secretion pathway. The sorting signals in the cytoplasmic domain of APP are therefore important in the deviation of APP from the secretase pathway. Finally we show that mutation of Arg609 to Asp in combination with Lys612 to Glu makes APP a less efficiently cleaved substrate for APP-secretase. The results are discussed in the context of recent findings on the targeting of APP and a parallel is drawn with some lysosomal glycoproteins that follow similar pathways.

Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content.

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains.

Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells.

We have synthesized an antisense oligonucleotide primer that matches a supposedly conserved sequence in messages for heparan sulfate proteoglycans with transmembrane orientations. With the aid of this primer we have amplified partial and selected full-length copies of a message from human lung fibroblasts that codes for a novel integral membrane heparan sulfate proteoglycan. The encoded protein is 198 amino-acids long, with discrete cytoplasmic, transmembrane, and amino-terminal extracellular domains. Except for the sequences that represent putative heparan sulfate chain attachment sites, the extracellular domain of this protein has a unique structure. The transmembrane and cytoplasmic domains, in contrast, are highly similar to the corresponding domains of fibroglycan and syndecan, the two cell surface proteoglycans that figured as models for the design of the antisense primer. This similarity includes the conservation of four tyrosine residues, one immediately in front of the stop transfer sequence and three in the cytoplasmic segment, and of the most proximal and most distal cytoplasmic sequences. The cDNA detects a single 2.6-kb message in cultured human lung fibroblasts and in a variety of human epithelial and fibroblastic cell lines. Polyclonal and monoclonal antibodies raised against the encoded peptide after expression as a beta-galactosidase fusion protein react with the 35-kD coreprotein of a cell surface heparan sulfate proteoglycan of human lung fibroblasts and decorate the surface of many cell types. We propose to name this proteoglycan "amphiglycan" (from the Greek words amphi, "around, on both sides of" and amphoo, "both") referring to its domain structure which extends on both sides of the plasmamembrane, and to its localization around cells of both epithelial and fibroblastic origin.

Developmental changes in heparan sulfate expression: in situ detection with mAbs.

Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated.

Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes.

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.

Molecular cloning of a phosphatidylinositol-anchored membrane heparan sulfate proteoglycan from human lung fibroblasts.

Two mAbs raised against the 64-kD core protein of a membrane heparan sulfate proteoglycan from human lung fibroblasts also recognize a nonhydrophobic proteoglycan which accumulates in the culture medium of the cells. Pulse-chase studies suggest that the hydrophobic cell-associated forms act as precursors for the nonhydrophobic medium-released species. The core proteins of the medium-released proteoglycans are slightly smaller than those of the hydrophobic cell-associated species, but the NH2-terminal amino acid sequences of both forms are identical. The characterization of human lung fibroblast cDNAs that encode the message for these core proteins and the effect of bacterial phosphatidylinositol-specific phospholipase C suggest that the hydrophobic proteoglycan is membrane-anchored through a phospholipid tail. These data identify a novel membrane proteoglycan in human lung fibroblasts and imply that the shedding of this proteoglycan may be related to the presence of the phospholipid anchor.

Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts.

Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.

Nuclear sexing in a population of Congolese metropolitan newborns.

In a study of 5631 Congolese newborn children, the incidence of numerical X-chromosomal anomalies was the same as was previously reported in Europe and in North America.

Philadelphia chromosome in human multiple myeloma.

Four patients with multiple myeloma in whom a Ph1 chromosome was found were described; 1 patient had a (9;22) translocation, 2 had no evidence of a translocation, and 1 had a complex translocation (3;8;22). Ph1 chromosomes with standard (9;22) or with unusual translocations were recently found in various myeloproliferative disorders (other than chronic myelogenous leukemia) and in acute lymphoblastic leukemia. These findings point to the genesis of a Ph1 chromosome in diseases other than chronic myelogenous leukemia and other myeloproliferative disorders.

Translocation t(12;16)(q13;p11) in myxoid liposarcoma and round cell liposarcoma: molecular and cytogenetic analysis.

Translocation t(12;16)(q13;p11) is regarded as a diagnostic marker for myxoid liposarcoma. Cytogenetic data on round cell liposarcomas and combined myxoid and round cell tumors is scarce, and the genetic basis of progression of myxoid tumors to high grade, round cell lesions is unknown. We have accumulated six round cell, four combined myxoid and round cell, and three myxoid liposarcomas for analysis. t(12;16)(q13;p11) was present in three round cell lesions and was detectable in all of the tumors by DNA analysis. In each tumor type, the CHOP gene in 12q13 was rearranged and fused to the TLS gene in 16p11. A variant TLS-CHOP RNA transcript was detected by polymerase chain reaction but did not correlate with clinicopathological data. No distinguishing cytogenetic or molecular markers for round cell or mixed lesions were found. The histogenic and genetic relatedness of myxoid and round cell liposarcomas is apparent from these data.

Four cytogenetic subgroups can be identified in endometrial polyps.

We have cytogenetically investigated a total of 33 simple benign endometrial polyps, 7 of which have been reported previously. Clonal chromosome rearrangements are found in 19 of 33 lesions (57%). Three major cytogenetically abnormal subgroups can be distinguished: (a) those with rearrangements in the 6p21-p22 region; (b) those with rearrangements of the 12q13-15 region; (c) those with rearrangements of the 7q22 region. A normal karyotype is found in a fourth subgroup. Recombinations of the 6p21-22 region with 2q35 and 10q22, as well as rearrangements of 7q22, have not been described before. It can be concluded that endometrial polyps, like several other types of benign mesenchymal tumors, present several cytogenetically different subgroups despite a seemingly identical clinical and morphological appearance. It is mandatory, therefore, to look for a common denominator of these tumors at the molecular level.

Cytogenetic characterization of tenosynovial giant cell tumors (nodular tenosynovitis).

Chromosome investigation in six localized forms of tenosynovial giant cell tumors, also known as modular tenosynovitis, revealed an identical translocation between chromosomes 1 and 2, t(1;2)(p11;q35-36) in three tumors, a variant translocation t(1;5)(p11;q22) in a fourth case, and a t(2;16)(q33;q24) in a fifth case. One case showed a normal karyotype. Although morphologically rather uniform, these benign tumors appear to be cytogenetically heterogeneous, but the chromosome changes seem to cluster in 2 regions, 1p11 and 16q24.

Fusion of ETV6 to MDS1/EVI1 as a result of t(3;12)(q26;p13) in myeloproliferative disorders.

We identified a fusion between ETV6 on 12p13 and MDS1/EVI1 on 3q26 in a t(3;12)(q26;p13) found in two cases of myeloproliferative disorder. The resulting chimeric transcript consists of the first two exons of ETV6 fused to MDS1 sequences, which in turn is fused to the second exon of the EVI1 gene. It has recently been reported that MDS1 can be expressed in normal tissues both as a single gene and fused to EVI1. ETV6 does not contribute any known functional domain to the predicted fusion protein. Association with blast crisis and myelodysplastic syndrome-derived leukemia, bad prognosis, and relative complex karyotype are in agreement with observations made in other cases of t(3;12)(q26;p13). Furthermore, a comparison can be made with the formation of an AML1/MDS1/EVI1 fusion gene in translocations (3;21)(q26;q22).

Biallelic alterations of both ETV6 and CDKN1B genes in a t(12;21) childhood acute lymphoblastic leukemia case.

Recently, a new recurrent t(12;21)(pl3;q22) has been identified in a B-cell lineage childhood acute lymphoblastic leukemia (ALL). The translocation results in a fusion of two known genes, ETV6/TEL (12p13) and AML1 (21q22), previously shown to be involved in the pathogenesis of myeloid disorders. We report results of cytogenetic fluorescence in situ hybridization and molecular studies of a B-cell childhood common ALL with a cryptic 12;21 translocation. Aberrations identified in this case involve both chromosomes 12 and include not only the ETV6-AML1 gene fusion and two different microdeletions of ETV6 but also the hemizygous loss of CDKN1B, D12S119, and KRAS2 loci and a putative rearrangement of the second CDKN1B allele as a result of an inv(12)(p13q24). Moreover, it was shown that the AML1-ETV6 reciprocal chimeric transcript was not present in the malignant cells, and hence may not play a major role in leukemogenesis. In addition, the putative loss of wild-type function of CDKN1B and ETV6 could indicate a synergistic effect of both genes in the pathogenesis of this leukemia case.

Genomic changes in endometrial polyps associated with tamoxifen show no evidence for its action as an external carcinogen.

Eighty-eight endometrial specimens from 36 postmenopausal breast cancer patients treated with tamoxifen were investigated cytogenetically and molecularly using fluorescence in situ hybridization with appropriate probes for the HMGIC and HMGIY genes. Twenty control specimens, 10 endometrial polyps, and 10 endometrial biopsy specimens were investigated in the same way. Of the 88 specimens, 44 were from endometrial polyps; 3 were from endocervical polyps; 7 were from cystic endometrium; 30 were from normal or atrophic endometrium, normal endocervix, or myometrium; and 4 were from endometrial carcinomas. Chromosome investigation of the endometrial polyps showed the nature of the chromosome changes in tamoxifen-induced polyps to be the same as that in the controls and in sporadic endometrial polyps described in the literature. HMGIC and HMGIY gene rearrangements in both groups were identical as shown by fluorescence in situ hybridization, which also allowed for the detection of seven hidden paracentric inversions involving 12q15, one of which occurred in a cystic endometrium. The carcinomas did not exhibit any of these changes. Because abnormal expression of HMGIC or HMGIY as a consequence of structural chromosome changes in 12q15 or 6p21, respectively, is invariably associated with benign neoplasia, tamoxifen-associated endometrial polyps are unlikely to undergo further malignant transformation, and a mode of action of tamoxifen as an external carcinogen is unlikely.

A novel gene, AF-1p, fused to HRX in t(1;11)(p32;q23), is not related to AF-4, AF-9 nor ENL.

Most of the translocations affecting the chromosome band 11q23, frequently seen in human acute leukemias, involve a restricted area of the HRX gene. We have characterized two t(1;11)(p32;q11) translocations which fuse the HRX gene to a novel gene, AF-1p on chromosome 1p32, in two myeloid leukemias. The der (11) chromosome expresses the 1368 N-terminal amino acids of HRX, including the AT-hook, snRNP and methyltransferase similarities, fused to almost all the AF-1p product. The predicted wild type AF-1p product is a 98 kDa acidic protein which does not exhibit similarity to the AF-4, AF-9 and ENL gene products. It is highly similar to the murine eps 15 gene product, which encodes a cytoplasmic phosphoprotein. Our data indicate that AF-1p defines another class of genes fused to HRX in 11q23 abnormalities.

A variant translocation t(2;18) in follicular lymphoma involves the 5' end of bcl-2 and Ig kappa light chain gene.

We have examined three cases of human lymphoma bearing a t(2;18)(p11;q21) chromosome translocation. The bcl-2 gene appeared to be rearranged in all three cases and breakpoints were clustered in the 5' flanking region of the gene. In all three cases, bcl-2 was juxtaposed to J segments of the Ig kappa gene. This juxtaposition of the bcl-2 and Ig kappa genes is very similar to the variant chromosome translocations of Burkitt lymphoma that juxtapose the c-myc locus to IgL genes.

The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25.

Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identified a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To define the interval containing the Xp11.2 break, we first performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identified non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Amplification of the 5' portion of cDNAs containing the 3' portion of TFE3 in two different ASPS cases identified a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically significant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBX-like domain that shows significant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocation-associated sarcomas. Oncogene (2001) 20, 48 - 57.