Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-β to TGF-β

The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-β) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR⁺). a human prouPA cDNA (LhuPA), or a control neomycinresistance cDNA (Lneo). When LhuPAR⁺ cells were co-cultured with LhuPA cells, the plasmin-dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co-cultures containing LhuPA and Lneo cells instead of the LhuPAR⁺ cells. The preferential activation of TGF-β by co-cultures with the greatest plasmin-generation potential, LhuPAR⁺ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF-β production, was observed with BAE cells treated with conditioned medium (CM) from co-cultures of LhuPA and LhuPAR⁺ cells. Inclusion of neutralizing antibodies to TGF-β abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-β. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co-cultures blocked both the fibrinolytic activity and the generation of TGF-β activity in the CM. In the second assay, CM from co-cultures of LhuPA and LhuPAR⁺ cells inhibited the migration of BAE cells in a wound assay. Controls with anti-TGF-β IgG indicated that the inhibition was due to TGF-β. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co-cultures of LhuPA and LhuPAR⁺ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co-culture of LhuPA with LhuPAR^? cells. Excess mannose-6-phosphate (M6P) blocked the generation of TGF-β as assayed by both the BAE migration and PA assays, presumably because it interfered with cellsurface localization of LTGF-β. Additionally, small numbers of LhuPA and LhuPAR⁺ cells co-cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF-β. These results support the conclusion that plasmindependent activation LTGF-β by LB6 cells is promoted by the surface localization of uPA by its receptor. © 1994 Wiley-Liss, Inc.     

Localisation régionale des gènes humains IDHS,MDHS, PGK, α-GAL,G6PD par l'hybridation cellulaire interspécifique

Summary 22 independent man-hamster (HGPRT^–) hybrids using male human cells with balanced reciprocal translocation t(X;2)(p22;q32) were analysed for human genes localized on chromosome 2 (IDH_S, MDH_S), on chromosome X (PGK, GAL, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.2, chr.2q^–, chr.Xp⁺).The following results were obtained:The chromosomes 2 and 2q^– are absent in the 22 hybrids.In 9 hybrids, the absence of MDH_S in spite of the presence of the chromosome Xp⁺ indicates that the gene for MDH_S is not localized on this chromosome (or that the gene for MDH_S is not on the segment 2q32-2qter translocated on X).In 14 hybrids, the three markers of X (PGK, GAL, G6PD) and IDH_S are expressed in the presence of the chromosome Xp⁺. This result indicates that the genes for these markers are on Xp⁺ or that the genes PGK, GAL, G6PD are on X without the Xp22-Xter segment, translocated on the chr.2, and that the gene for IDH_S is on the 2q32-2qter segment translocated on X.In 8 hybrids, in the absence of the intact chromosome Xp⁺, the higher percentage of the presence of G6PD (7 hybrids) and the lower percentage of the presence of IDH_S (3 hybrids) are explained by the fact that these hybrids selected in HAT medium had to retain a segment of Xp⁺ bearing the human gene HGPRT. G6PD appeared very close to HGPRT and IDH_S very distant from HGPRT.The study of the different correlations between the presence and the absence of these four markers on Xp⁺ in the different hybrids indicates the following order on the chromosome Xp⁺ from p to q: IDH_s — PGK — GAL — G6PD.

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Groupe INSERM: Directeur J. Frézal

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Groupe CNRS, ER, 149: Directeur J. de Grouchy     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Microsomal epoxide hydrolase activity in human x mouse hybrid cells

Microsomal epoxide hydrolase (E.C.3.3.2.3) activity has been measured in human x mouse hybrid cells prepared from human cells expressing 6-7 x the activity of the mouse cells. Rabbit antihuman and antimouse antisera raised against purified enzymes were used to discriminate between human and mouse enzymes. All twenty five clones examined did not express human enzyme and this correlated with the loss of human chromosome 6 from each cell line. Four hybrids expressed 2-3 x the activity expressed by the mouse cell parent and these all retained more human chromosomes, specifically chromosome 19, than those with low activity. It is concluded that the human gene for epoxide hydrolase may be on chromosome 6 and that other gene products can affect the level of activity expressed by a cell.     

Assignment of the gene for cystathionine β-synthase to human chromosome 21 in somatic cell hybrids

Summary Among several established mouse, rat, and Chinese hamster cell lines that were screened for cystathionine -synthase (CBS) activity, mouse 3T3 and Chinese hamster Don fibroblasts were found to contain no detectable activity. Somatic cell hybrids between human fibroblasts KG-7 with normal CBS activity and Don/a23TK^- cells (series XXI) were examined for CBS activity and for human chromosome content. Only chromosome 21 cosegregated with CBS activity. Because the activities measured could represent either Chinese hamster or human gene products, we have prepared a new series of hybrids between Don/a23TK^- cells and mutant human fibroblasts from a patient with homocystinuria due to deficiency of functional CBS mRNA. None of these (series XXV) hybrids contained detectable CBS activity, although collectively all human chromosomes were represented. Our results suggest that the human gene for CBS, called CBS, and thus for the most common form of homocystinuria, is located on chromosome 21.     

pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen

Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K⁺ analogue, ⁸⁶Rb⁺, from prelabelled cells, and the influx of ²²Na⁺. The permeabilizing activity of PA was limited to a proteolytically activated form (PA_N) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PA_N facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PA_N permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, γ-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

Assignment of the gene governing cellular ouabain resistance to Mus musculus chromosome 3 using human/mouse microcell hybrids

Human/mouse microcell hybrids were used to establish the assignment of the gene governing resistance to the cardiac glycoside ouabain (Oua-1) to Mus musculus chromosome 3. Microcells were prepared from primary mouse embryo fibroblasts and fused with HeLa S3 cells, and microcell hybrids were isolated and maintained in medium containing 10^–6 m ouabain. Resistance to ouabain was not expressed concordantly with any of 26 murine isozyme markers. Karyotypic analysis of five primary clones showed that one to five murine chromosomes had been transferred from donor to recipient in these experiments. Only mouse chromosome 3 was common to all ouabain-resistant primary clones. Both ouabain-resistant and -sensitive subclones were isolated from hybrids grown in the absence of selective pressure, and karyotyping showed that loss of resistance to ouabain was concordant with the loss of murine chromosome 3.These studies were supported by Grant GM9966 from the National Institutes of Health.     

X-chromosome activity in heterokaryons and hybrids between mouse fibroblasts and teratocarcinoma stem cells

To determine whether 2X-active cells contain factors capable of reactivating the inactive mammalian X chromosome, fibroblast lines, having a cytologically or genetically marked inactive X, were fused with 2X-active mouse embryos or ovarian teratocarcinoma stem cells. Fusions with 2–16 cell embryos were uninformative because no mitosis occurred in heterokaryons. Fusions with 2X-active teratocarcinoma cells, and screening for re-expression of alleles on the inactive X showed that reactivation did not occur with detectable frequency in heterokaryon. Hybridization of HPRT^?M. musculus × M. caroli cells with XO HPRT^? teratocarcinoma cells yielded hybrids with a frequency of >10^?6; these hybrids all expressed the Hpt allele on the inactive M. caroli X, but not the M. caroliGpd or Pgk. Late replication-banding studies of hybrids and 6-thioguanine-resistant revertants showed that the reactivated Hp⁺ allele was still located on the late replicating X. Similar results were obtained with hybridization of this line to 1X-active (male-derived) fibroblast lines, indicating that hybridization per se, rather than a specific factor contributed by the teratocarcinoma cell partner, was reponsible for the frequent localized derepression of the Hpt⁺ allele on the inactive X.     

Suppression of Immunoglobulin Synthesis by Cellular Hybridization

WHEN two cells which show differentiated characteristics are fused, the hybrids generally do not show these characteristics. Ephrussi et al.¹ have interpreted such trans dominant loss to show that the cells contain a diffusible repressor and this idea is supported by the finding that some mouse/human hybrids can spontaneously redifferentiate when the C10 human chromosome is lost². We now report another example of redifferentiation in hybrid cells; when IT mouse fibroblasts are fused with MPC 11 mouse myeloma cells which produce immunoglobulin, the hybrids neither produce nor excrete detectable amounts of immunoglobulin.     

Assignment of ecto-5′-nucleotidase to human chromosome 6

Summary Ecto-5-nucleotidase activity (5NT) was measured on whole cells of 26 human x Chinese hamster hybrids. Concordance analysis showed 100% correlation between enzyme activity and inheritance of human chromosome 6. This observation was confirmed by a segregation analysis in which cells of a hybrid containing chromosome 6 were stained by indirect immunofluorescence for HLA Class 1 antigen and sorted by a fluorescence-activated cell sorter (FACS). Cells in the HLA^- compartment were cloned and expression of HLA and 5NT was determined. Of nine clones, three were HLA^-, 5NT^- and six were HLA⁺, 5NT⁺, supporting the linkage of 5NT to chromosome 6.     

Recognition of chromosome 6-associated target structures by human lymphokine-activated killer cells

Different populations of unstimulated and IL-2-activated PBL were used in binding and killing assays against somatic mouse/human lymphocyte cell hybrids containing different human chromosomes. Unstimulated PBL effector cells showed low binding and killing activity to both cell hybrids and mouse parental cell lines. However, IL-2-activated killer (LAK) cells bound strongly to, and effectively killed, cell hybrids carrying human chromosome 6, but were inefficient in both assays to mouse parental cells and to cell hybrids not carrying human chromosome 6. These results show that human LAK cells but not endogenous NK cells bind and kill mouse/human lymphocyte hybrids containing human chromosome 6. We thus suggest that LAK cells recognize ligands encoded by genes on chromosome 6.     

Extinction of Albumin Gene Expression in a Panel of Human Chromosome 2 Microcell Hybrids

Expression of the serum albumin gene is extinguished in rat hepatoma microcell hybrids that retain mouse chromosome 1. These data define atrans-dominant extinguisher locus,Tse-2,on mouse chromosome 1. To localize the human TSE2 locus, we prepared and characterized rat/human microcell hybrids that contained either human chromosome 1 or chromosome 2, the genetic homologues of mouse chromosome 1. Rat hepatoma microcell hybrids retaining a derivative human chromosome 1 [der 1 t(1;17)(p34.3;q11.2)] expressed their serum albumin genes at levels similar to those of parental hepatoma cells. In contrast, microcell transfer of human chromosome 2 into rat hepatoma recipients produced karyotypically heterogeneous collections of hybrid clones, some of which displayed dramatic albumin extinction phenotypes. For example, albumin mRNA levels in several extinguished microcell hybrids were reduced at least 500-fold, similar to albumin mRNA levels in hepatoma × fibroblast whole-cell hybrids. Expression of several other liver genes, including α1-antitrypsin, aldolase B, alcohol dehydrogenase, and phosphoenolpyruvate carboxykinase, was also affected in some of the microcell hybrids, but expression of these genes was not concordant with expression of albumin. Hybrid segregants were prepared from the albumin-extinguished hybrids, and reexpression of albumin mRNA and protein was observed in sublines that had lost or fragmented human chromosome 2. Finally, expression of mRNAs encoding the liver-enrichedtransactivators HNF-1, HNF-4, HNF-3α, and HNF-3β was not affected in any of the chromosome 2-containing hybrids. These data define and map a genetic locus on human chromosome 2 that extinguishes albumin gene expression intrans,and they suggest that TSE2-mediated extinction is independent of HNF-1, -4, -3α, and -3β expression.     

Requirement of human chromosomes 19, 6 and possibly 3 for infection of hamster x human hybrid cells with baboon M7 type C virus.

The replication pattern of the endogenous baboon type C virus M7 was studied in 29 primary Chinese hamster × human hybrid clones generated with leukemic cells from two different patients with acute lymphoblastic or myeloblastic leukemia. There was no evidence of viral particulate RDDP or M7 antigen before viral infection. M7 virus replicated in human and some hybrid cells but not in Chinese hamster cells, indicating that M7 requires dominantly expressed human gene(s) for replication. Enzyme and cytogenetic analyses show that a gene(s) coded for by human chromosome 19 is necessary for M7 infection of these hybrids. Detailed cytogenetic correlations revealed, however, that the chromosome 19+/M7 + hybrid clones with intact chromosomes also had copies of chromosomes 3 and 6. Previously, Bevi, the putative integration site for M7 virus, has been assigned to human chromosome 6. Many clones with various combinations of chromosomes 3 and 6 lacked chromosome 19, however, and failed to replicate exogenously applied M7 virus, while tests of 27 secondary clones showed that M7 markers co-segregated with chromosome 19 markers. These findings all confirm the need for a chromosome 19-coded function in Chinese hamster × human hybrids. In addition, the yield of viral particulate RDDP produced into the culture fluid was 50–100 fold less per viral antigen-positive cell in the hybrids compared with human cells. Thus some form of regulation of viral components exists in the hybrid cells. When the virus replicating in hybrid cells was transferred back to human cells, this regulation was relaxed and the yield of RDDP per FA(+) cell greatly increased. We conclude that human chromosomes 6 and 19 code for functions involved in M7 virus metabolism, and we cannot exclude a function coded for by chromosome 3.     

Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome

Microcell-mediated chromosome transfer is a useful technique for the study of gene function, gene regulation, gene mapping, and functional cloning in mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have been developed. These donor cell lines contain a single human chromosome marked with a dominant selectable gene in a rodent cell background. However, a similar panel does not exist for murine chromosomes. To produce mouse monochromosomal donor hybrids, we have utilized embryonic stem (ES) cells with targeted gene disruptions of known chromosomal location as starting material. ES cells with mutations in aprt, fyn, and myc were utilized to generate monochromosomal hybrids with neomycin phosphotransferase-marked murine Chr 8, 10, or 15 respectively in a hamster or rat background. This same methodology can be used to generate a complete panel of marked mouse chromosomes for somatic cell genetic experimentaion. Received: 28 July 1998 / Accepted: 15 December 1998     

Phytohemagglutinin-enhanced hybrid colony formation by cells from aged mice: Expression of th 4mod-1 mouse locus in human-mouse hybrids

Summary Cells from a continuous human line and freshly isolated cells from old adult mice heterozygous at theMod-1 locus were fused in the presence of polyethylene glycol (PEG). The production of hybrid cells, as a function of PEG concentration in the presence and absence of phytohemagglutining (PHA), was measured by cell survival and proliferation on selective medium. The incorporation of PHA into the fusion mixture allowed cell fusion to take place at nontoxic concentrations of PEG. PHA increased the frequency of cell fusion and increased the production of viable hybrid cells from 138- to over 2800-fold depending on cell type. The results suggest that the procedure may have broad application in promoting the fusion of cells sensitive to PEG. Clones were analyzed for isozymes of malic enzyme and glucose-6-phosphate dehydrogenase. The expression of the gene encoding X-linked mouse glucose-6-phosphate dehydrogenase confirmed that the cells were hybrids. These cells lost other mouse isozymes rapidly. In those clones in which the mouse malic enzyme gene was expressed, the product ofMod-1 ^α was detected significantly more frequently than that ofMod-1 ^b.     

Localisation régionale des gènes humains LDHB,TPI, ENO2, PepB,PGK, αGALA,G6PD et HGPRT par l'hybridation cellulaire interspécifique

Summary Twenty-two independent man-mouse (A9, HGPRT^-) and manhamster (CH, HGPRT)-hybrids using male human with balanced reciprocal translocation XY t(X;12)(q2,3;q1,2) were analysed for human genes localized on chromosome 12 (LDH_B, PepB, ENO₂, TPI), on chromosome X (PGK, GAL_A, G6PD) and for human chromosomes in relation with the balanced reciprocal translocation (chromosome 12, 12q^-, Xq⁺). The human chromosome 12q^- was not analysed because of its morphology is similar to some rodent chromosomes.The following results were obtained:1.Man-mouse hybrids (12 independent hybrids):The chromosome 12 is absent. A positive correlation is observed between Xq⁺, PGK, and PepB. Four hybrids are Xq⁺+PGK+PepB+ and eight hybrids are Xq⁺-PGK-PepB-This result indicate that the genes for human PGK, PepB are on the chromosome Xq+. 2.Man-hamster hybrids (10 independent hybrids):A positive correlation is observed between Xq⁺, PGK, GAL_A. Two hybrids are Xq⁺+PGK+GAL_A+ and eight hybrids are Xq⁺-PGK-GAL_A-.A positive correlation is observed between Xq⁺, PGK, GAL_A, PepB with the seven hybrids missing the normal human chromosome 12. One hybrid is Xq⁺+PGK+GAL_A+PepB+ and six hybrids are Xq⁺-PGK-GAL_A-PepB-. These results indicate that the genes for human PGK, GAL_A, PepB are on the chromosome Xq⁺. 3.Man-mouse and man-hamster hybrids:The highest percentage of presence in the hybrids is observed for the following markers: G6PD (100%), LDH_B, TPI, ENO₂ (90%). This is explained by the fact that these hybrids selected in HAT medium, had to retain a segment of X (Xq⁺ or 12q^-) bearing the human HGPRT gene. The different results indicated that the segment of X retained in these hybrids must be on the 12q^- and not on the Xq⁺, for Xq⁺ is rarely present in man rodent hybrids.The different results implicate finally the following localisations:LDH_B, TPI, ENO₂ on 12 q12 12pter,PepB on 12 q12 12qter;PGK, GAL_A on X q23 Xpter;HGPRT, G6PD on X q23 Xqter.     

Assignment of the gene for carboxypeptidase A to human chromosome 7q22→qter and to mouse chromosome 6

Summary A rat cDNA probe for preprocarboxypeptidase A was used to follow the segregation of the human gene for carboxypeptidase A (CPA) in 49 human x mouse somatic cell hybrids using Southern filter hybridization techniques. CPA was assigned to human chromosome 7q22qter. Similarly, the probe was used to follow the segregation of the mouse gene for carboxypeptidase A (Cpa) in 19 mouse x Chinese hamster somatic cell hybrids. Cpa was assigned to mouse chromosome 6. The gene for carboxypeptidase A forms part of a syntenic group that is conserved in man and mouse.Preliminary chromosomal assignments of carboxypeptidase A in man and mouse have been made in abstract (Honey et al. 1983a, b)     

The UPS locus encoding uroporphyrinogen I synthase is located on human chromosome 11

The expression of the UPS locus encoding uroporphyrinogen I synthase has been investigated in human/mouse somatic cell hybrids. Human and mouse uroporphyrinogen I synthase can be readily distinguished by their isoelectric points. In hybrid cells, both human and mouse isozymes are detected. The multiple human uroporphyrinogen I synthase isozymes segregate as a single unit, as expected if they are the products of a single locus. The absence of new heteropolymers in hybrid cells supports the biochemical evidence that the active enzyme is a monomer. The presence of human uroporphyrinogen I synthase in hybrid clones was correlated with the presence of human chromosome 11, or its enzymatic marker, without exception in 44 independent hybrid lines. All other chromosomes could be eliminated as possible locations for this locus, due to their independent segregation. This report represents the first gene assignment for an enzyme in the heme biosynthesis pathway.     

Chromosomal localization of the type-I 15-PGDH gene to 4q34–q35

The gene encoding the human NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase, designated type-I 15-PGDH, was mapped to chromosome 4 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This gene was further localized to bands 4q34–q35 by in situ hybridization on human chromosomes. Received: 7 October 1996