Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction.

A broad spectrum of biological activities has been proposed for transforming growth factor-beta 3 (TGF-beta 3). To study TGF-beta 3 function in development, TGF-beta 3 null mutant mice were generated by gene-targeting. Within 20 hours of birth, homozygous TGF-beta 3-/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis. Unlike other null mutants with cleft palate, TGF-beta 3-/- mice lack other concomitant craniofacial abnormalities. This study demonstrates an essential function for TGF-beta 3 in the normal morphogenesis of palate and lung, and directly implicates this cytokine in mechanisms of epithelial-mesenchymal interaction.

A mouse model for the human lysosomal disease aspartylglycosaminuria.

Aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation in humans, is caused by mutations in the gene encoding the lysosomal enzyme glycosylasparaginase (Aga). The resulting enzyme deficiency allows aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines to accumulate in tissues and body fluids, from early fetal life onward. The clinical course is characterized by normal early development, slowly progressing to severe mental and motor retardation in early adulthood. The exact pathogenesis of AGU in humans is unknown and neither therapy nor an animal model for this debilitating and ultimately fatal disease exists. Through targeted disruption of the mouse Aga gene in embryonic stem cells, we generated mice that completely lack Aga activity. At the age of 5-10 months a massive accumulation of GlcNAc-Asn was detected along with lysosomal vacuolization, axonal swelling in the gracile nucleus and impaired neuromotor coordination. A significant number of older male mice had massively swollen bladders, which was not caused by obstruction, but most likely related to the impaired function of the nervous system. These findings are consistent with the pathogenesis of AGU and provide further data explaining the impaired neurological function in AGU patients.

c-sis is translocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia.

By analysis of a series of somatic cell hybrids derived by fusion of either mouse or Chinese hamster cells with leukocytes from different chronic myelocytic leukemia (CML) patients or from normal donors, we have localized the human oncogene, c-sis, on the q11 to qter segment of chromosome 22 and demonstrated its translocation from chromosome 22 to chromosome 9 (q34) in CML.

Isolation of human oncogene sequences (v-fes homolog) from a cosmid library.

To define the human homolog (or homologs) of transforming sequences (v-fes gene) common to Gardner (GA) and Snyder Theilen (ST) isolates of feline sarcoma virus (FeSV), a representative library of human lung carcinoma DNA in a cosmid vector system was constructed. Three cosmid clones were isolated containing GA/ST FeSV v-fes homologous cellular sequences, within 32- to 42-kilobase cellular inserts representing 56 kilobases of contiguous human cellular DNA. Sequences both homologous to, and colinear with, GA or ST FeSV v-fes are distributed discontinuously over a region of up to 9.5 kilobases and contain a minimum of three regions of nonhomology representing probable introns. A thymidine kinase selection system was used to show that, upon transfection to RAT-2 cells, the human c-fes sequence lacked detectable transforming activity.

Increased resistance to a farnesyltransferase inhibitor by N-cadherin expression in Bcr/Abl-P190 lymphoblastic leukemia cells.

In leukemia patients, resistance to drug treatment develops while the malignant cells can interact with and derive support from their microenvironment, such as bone marrow stroma. To model this process, lymphoblastic leukemia cells from BCR/ABL transgenic mice were treated with the farnesyltransferase inhibitor (FTI) SCH66336 while in coculture with primary mouse embryonic fibroblasts. Coculture with fibroblasts allowed the outgrowth of a subpopulation of drug-resistant lymphoblasts that expressed N-cadherin, a cell-cell adhesion protein that normally is only expressed on specific cell types, including hematopoietic stem cells and fibroblasts. N-cadherin expression promoted increased adhesion of the lymphoblasts to the fibroblasts. Importantly, de novo expression of N-cadherin in parental nonexpressing lymphoblasts using lentiviral transduction increased the ability of the cells to survive FTI treatment. We conclude that FTI drug treatment of Bcr/Abl-positive lymphoblastic leukemia cells that are in contact with a defined microenvironment induces the selective survival of a more primitive subpopulation of leukemia cells that expresses N-cadherin. Experimental drug treatment of cancer cells in model systems that include a microenvironment may reveal novel molecules that contribute to drug resistance and may aid in the design of specific therapies to eradicate more primitive cells.

Isolation and sequence analysis of a novel human tyrosine kinase gene.

Using v-abl probes, we have identified and cloned a novel fes/fps-homologous human cDNA, which we have designated FER (pronounced "fair"). This apparently full-length cDNA of 3.0 kilobases has an open reading frame of 2,466 base pairs and the capacity to encode a protein of 94,000 molecular weight. The cDNA contains regions homologous to the highly conserved tyrosine protein kinase domain of other oncogenes and growth factor receptors but lacks a clear transmembrane region, indicating that it encodes a tyrosine kinase of the nonreceptor type. The deduced amino acid sequence of FER resembles that of c-fes/fps. Our data indicate that the protein product of FER, p94FER, corresponds to a previously reported cellular phosphoprotein, NCP94, detected with a v-fps-specific antipeptide antiserum.

Evidence that the phl gene encodes a 160,000-dalton phosphoprotein with associated kinase activity.

In chronic myelocytic leukemia, the human c-abl oncogene is translocated from chromosome 9 to a region on chromosome 22 designated as the breakpoint cluster region (bcr) (A. de Klein, A. Guerts van Kessel, G. Grosveld, C. R. Bartram, A. Hagemeyer, D. Bootsma, N. K. Spurr, N. Heisterkamp, J. Groffen, and J. R. Stephenson, Nature (London) 300:765-767, 1982; J. Groffen, J. R. Stephenson, N. Heisterkamp, A. de Klein, C. R. Bartram, and G. Grosveld, Cell 36:93-99.) Abnormal c-abl homologous mRNA and protein have been detected in the leukemic cells of patients with chronic myelocytic leukemia (E. Canaani, D. Stein-Saltz, E. Aghai, R. P. Gale, A. Berrebi, and E. Januszewicz, Lancet 1:593-595, 1984; S. J. Collins and M. T. Groudine, Proc. Natl. Acad. Sci. USA 80:4813-4817, 1983; R. P. Gale and E. Canaani, Proc. Natl. Acad. Sci. USA 81:5648-5652, 1984; J. B. Konopka, S. M. Watanabe, J. W. Singer, S. J. Collins, and O. N. Witte, Proc. Natl. Acad. Sci. USA 82:1810-1814, 1985). The abnormal mRNA represents a chimeric transcript consisting of 5' bcr and 3' c-abl sequences (G. Grosveld, J. Verwoerd, T. van Agthoven, A. de Klein, K. L. Ramachandran, N. Heisterkamp, K. Stam, and J. Groffen, Mol. Cell. Biol. 6:607-616, 1986; E. Shtivelman, B. Lifshitz, R. B. Gale, and E. Canaani, Nature (London) 315:550-554, 1985; K. Stam, N. Heisterkamp, G. Grosveld, A. de Klein, R. S. Verma, M. Coleman, H. Dosik, and J. Groffen, N. Engl. J. Med. 313:1429-1433, 1985). In the present study, we demonstrated that the abnormal c-abl protein is a fusion protein. In addition, the normal gene encompassing bcr sequences was shown to encode a 160,000-dalton phosphoprotein with an associated serine or threonine kinase activity. We propose that this gene be designated phl, reserving the term bcr for the region within the phl gene encompassing the Ph' translocation breakpoints.

Involvement of the tyrosine kinase fer in cell adhesion.

The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120(cas) and elevated tyrosine phosphorylation of p120(cas). beta-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and beta-catenin were found to be in complex. Significantly, although the levels of alpha-catenin, beta-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of alpha-catenin and beta-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion.

Nuclear and cytoplasmic location of the FER tyrosine kinase.

The location of the FER protein within the cell was investigated by using subcellular fractionation and immunofluorescence. FER was found in the cytoplasm and in the nucleus, where it was associated with the chromatin fraction. Its ubiquitous expression and its subcellular location indicate that it may be involved in key regulatory processes.

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.

Inhibition of Bcr serine kinase by tyrosine phosphorylation.

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.

In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.

TGF-beta3-induced palatogenesis requires matrix metalloproteinases.

Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membrane-associated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-beta3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-beta3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-beta3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion.

Restricted oncogenicity of BCR/ABL p190 in transgenic mice.

A chimeric BCR/ABL oncogene encoding the p190 protein has been introduced into the mouse germline using microinjection of one-cell fertilized eggs. Founder and progeny transgenic animals, when becoming ill, were found to develop lymphoblastic leukemia/lymphoma which was transplantable to compatible recipients. Lymphoblasts were arrested at the pre-B stage of development. Expression of BCR/ABL was not detected in peripheral blood during the early stages of leukemia but became evident as the disease progressed. However, the transgene was expressed early in development in bone marrow and was also transcribed in nonhematopoietic tissues although this did not result in tumorigenesis. These results strongly suggest that the oncogenicity of BCR/ABL is limited to hematopoietic cells, including pre-B cells or their progenitors.

Deletion within the D17S34 locus in a primitive neuroectodermal tumor.

Loss of heterozygosity on chromosome 17p13.3 is frequently observed in solid tumors, and the presence of a tumor suppressor gene has been predicted in this region of chromosome 17. We have analyzed a primitive neuroectodermal tumor sample exhibiting loss of heterozygosity at the D17S34 locus, a commonly used telomeric marker on the short arm of chromosome 17. The remaining allele showed a rearrangement. Cosmids spanning the D17S34 locus and probes from that region were used to demonstrate a 9-kb deletion within the D17S34 locus and were found to contain evolutionary, conserved sequences. Genetic alterations in this region may also affect expression of immediately adjacent genes, such as ABR, and could be a common mechanism in the causation of primitive neuroectodermal tumors.

Rearrangement of the human ABL oncogene in a glioblastoma.

A number of protooncogenes have been implicated in human tumorigenesis. The ABL oncogene is consistently rearranged and activated as a consequence of the translocation t(9;22) that gives rise to the Philadelphia chromosome in chronic myeloid leukemia and in some cases of acute lymphoblastic leukemia. Here we describe rearrangement of ABL in a different type of malignancy. The glioblastoma cell line A172 lacks germline alleles of ABL. A recombination event, presumably followed by a duplication, has created two ABL alleles in which exon 11 is joined to chromosome 16 sequences. Although the main body of ABL exons was still present, two considerably shortened ABL mRNAs of 3.8 and 2.8 kilobases were detected; the 3.8-kilobase mRNA hybridized exclusively to an exon IB probe. Neither mRNA hybridized to an ABL probe encompassing part of the tyrosine kinase domain. Thus, the cell line A172 is able to survive in the absence of a functional ABL gene product, indicating that the role of ABL is unlikely to be "housekeeping."

Cellular interactions of CRKL, and SH2-SH3 adaptor protein.

Chronic myelogenous leukemia is characterized by a specific chromosomal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chimeric BCR/ABL gene produces a P210 fusion protein with deregulated tyrosine kinase activity. We have recently isolated a complementary DNA, CRKL, which could code for an adaptor protein consisting of one SH2 and two SH3 domains and lacking any catalytic domain. In the current study, we show that CRKL is highly phosphorylated in the chronic myelogenous leukemia cell line K562 and that it is a substrate for the p210 BCR/ABL and p145 ABL kinases. BCR/ABL and ABL are coimmunoprecipitated with CRKL in vivo, demonstrating that relatively stable complexes are formed. In addition, the nucleotide exchange factor mSOS1 was found to be coimmunoprecipitated with CRKL. These findings establish a putative signal transduction pathway way through which BCR/ABL mediates its oncogenic activity.

Crkl enhances leukemogenesis in BCR/ABL P190 transgenic mice.

The adapter protein Crkl has been implicated in the abnormal signal transduction pathways activated by the Bcr/Abl oncoprotein, which causes Philadelphia-positive leukemias in humans. To investigate the role of Crkl in tumorigenesis, we have generated transgenic mice that express human Crkl from the CRKL promoter. Western blot analysis showed a 4-6-fold overexpression of transgenic Crkl above endogenous crkl in two lines and increased constitutive complex formation between Crkl and C3G, an exchange factor for the small GTPase Rap1. This was associated with a significant increase in integrin-based motility of transgenic macrophages. Overexpression of Crkl was associated with increased incidence of tumor formation, and Rap1 was activated in a metastatic mammary carcinoma. The coexpression of Crkl and Bcr/Abl in mice transgenic for P190 BCR/ABL and CRKL markedly increased the rapidity of development of leukemia/lymphoma, decreasing the average survival by 3.8 months. These results provide direct evidence that Crkl plays a role in tumor development and is important in the leukemogenesis caused by Bcr/Abl.

Expression of the chronic myelogenous leukemia-associated p210bcr/abl oncoprotein in a murine IL-3 dependent myeloid cell line.

We have studied the effect of a replication-defective murine retroviral vector expressing the chronic myelogenous leukemia associated oncoprotein p210bcr/abl in murine IL-3 dependent myeloid 32D C13(G) cells. This cell line can be induced to differentiate along either the granulocytic or monocytic lineages thus permitting an independent assessment of the effect of p210bcr/abl on growth and differentiation. Cells expressing p210bcr/abl displayed a complete non-autocrine abrogation of IL-3 dependence and an enhanced response to an activity in FBS which is not IGF-I or IGF-II. During the first few generations following infection with the bcr/abl vector, cells became larger with an increased fraction of cells in G2/M and monocyte/macrophage markers were expressed. Four cytoplasmic proteins phosphorylated in response to IL-3 in the parental cell line with apparent molecular weights of 98, 70, 62, and 52 Kd were amongst those constitutively phosphorylated in p210bcr/abl expressing cells. These results suggest that the functional substitution of IL-3 by p210bcr/abl is due to constitutive activation of proteins involved in IL-3 signal transduction. Alterations of cell differentiation, cell cycle and growth which cannot be attributed to IL-3 like effects indicate that p210bcr/abl has pleiotropic effects involving several other pathways of cellular regulation.

Characterization of the human ABL promoter regions.

Gel retardation and DNAase 1 footprinting experiments have been performed to characterize the promoter sequences of exon 1a and 1b of the human ABL gene. Several Sp1 motifs and CCAAT boxes are found to be protected by nuclear proteins in the 1b promoter but none of the 7 reported Sp1 sites in 1a were found to bind protein. Multiple sets of initiation sites seem to exist in the 1b promoter region which may represent individual initiation sites, distributed over a DNA region of up to 700 bp. Starting with the most distal initiation site, 1a and 1b ABL promoter sequences show a high degree of homology, suggesting that one is derived from the other. However, multiple evolutionary changes in the 1a promoter sequence indicate that type 1a ABL expression may be differently regulated than 1b.