Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. (12). There are several mechanisms by which growth factor receptors can be rendered transforming. Retroviral transduction of protooncogenes can result in truncation and mutation of the normal version of the gene. This has been shown for the epidermal growth factor receptor (EGFR) (v-erbB) (18), colony-stimulating factor 1 receptor (CSF-1R) (v-fms) (68), and ros (v-ros) (52) genes. Overexpression of an otherwise normal receptor, with or without the concomitant application of ligand, can also result in neoplastic transformation, as shown for the insulinlike growth factor 1 receptor (38), EGFR (15, 77), CSF-1R (65), eph (51), and neu (16) HOS (10), has been demonstrated to be the cell surface receptor for hepatocyte growth factor, a potential growth factor for a broad spectrum of cell types as well as a mediator of liver regeneration (7). In addition, CSF-1R mediates the pleiotropic effects of its cognate ligand, CSF-1. Together, these two molecules stimulate the proliferation and differentiation of cells of the macrophage lineage (68).In an effort to determine genes involved in the progression of chronic myelogenous leukemia (CML) to acute-phase leukemia, we previously reported the identification of a transforming gene in the DNAs of two patients with CML (47). Molecular cloning and characterization indicate that this gene, which we term axl (from the Greek word anexelekto, or uncontrolled), is a receptor tyrosine kinase with a structure novel among tyrosine kinases. Our data indicate that the ax! protein has tyrosine kinase activity and is capable of transforming NIH 3T3 cells. Furthermore, axl's transforming capacity results from overexpression of axl mRNA rather than from structural mutation. MATERIALS AND METHODSIdentification of a transforming gene in CML cells. Transfections and nude mouse tumorigenicity assays were performed as described previously (47). The cell lines AF6295 and AF3642 were derived from secondary nude mouse tumors arising from transfection of DNA from blast crisis and chronic-phase CML patients, respectively. Tumors were isolated from nude mice and digested in the presence of trypsin-EDTA. A portion of these cells was then plated in plastic tissue culture flasks.Isolation of cosmid and cDNA clones. DNA fragments for cosmid cloning were generated by partial MboI digests of genomic DNA from a secondary nude mouse tumor cell line, AF6295. The restricted DNA fragment...

Loss of a whole chromosome S or a deletion of its long arm (5q) is a recurring abnormality in malignant myeloid neoplasms. To determine the location of genes on Sq that may be involved in leukemogenesis, we examined the deleted chromosome 5 homologs in a series of 135 patients with malignant myeloid diseases. By comparing the breakpoints, we identified a small segment of 5q, consisting of band 5q31, that was deleted in each patient. This segment has been termed the critical region. Distal Sq contains a number of genes encoding growth factors, hormone receptors, and proteins involved in signal transduction or transcriptional regulation. These include several genes that are good candidates for a tumorsuppressor gene, as well as the genes encoding five hematopoietic growth factors (CSF2, IL3, IL4, IL5, and IL9). By using fluorescence in situ hybridization, we have refrned the localization of these genes to 5q31.1 and have determined the order of these genes and of other markers within 5q31. By hybridizing probes to metaphase cells with overlapping deletions involving 5q31, we have narrowed the critical region to a smail segment of 5q31 containing the EGRI gene. The five hematopoietic growth factor genes and seven other genes are excluded from this region. The EGRI gene was not deleted in nine other patients with acute myeloid leukemia who did not have abnormalities of chromosome 5. By physical mapping, the minimum size of the critical region was estimated to be 2.8 megabases. This cytogenetic map of 5q31, together with the molecular characterization of the critical region, will facilitate the identification of a putative tumor-suppressor gene in this band.Recurring chromosomal rearrangements are characteristic of human malignant diseases, particularly the leukemias and lymphomas (1) (93%o) had a clonal chromosomal abnormality and 97 (75%) had loss or deletion of chromosome 5 and/or 7. Among these 97 patients, 21 had loss of chromosome 5, 26 had a del(5q), 8 had loss of 5q following unbalanced translocations, 51 had loss of chromosome 7, 11 had a del(7q), and 12 had loss of 7q as a result of an unbalanced translocation. Thirty-one patients had abnormalities of both chromosomes 5 and 7. Overall, 55 patients (43%) had abnormalities of chromosome 5. A del(5q) was the most common structural aberration in our series.In addition to t-MDS/t-AML, a -5/del(Sq) has also been observed in the malignant cells of 10%o ofpatients with AML de novo and in 15% ofpatients who have MDS arising de novo (8). Many of these patients have had significant occupational exposure to potential environmental carcinogens, suggesting that abnormalities of chromosome 5 or 7 may be a marker of mutagen-induced leukemia. A distinct clinical syndrome associated with a del(5q) is seen in a subset ofpatients with MDS de novo. Clinically, this disorder, termed the "5q-syndrome," is characterized by refractory anemia (RA). These patients having a del(Sq) as the sole abnormality tend to have a relatively mild course that usually does not progress to ...

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3) are the three members of the neurotrophin family known to exist in mammals. Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4), which shares all of the features found in the mammalian neurotrophins, has been identified in Xenopus and viper. We used sequences specific to the Xenopus/viiper NT-4 to isolate a neurotrophin from both human and rat genomic DNA that appears to represent the mammalian counterpart of Xenopus/viper NT-4. Human NT-4 as well as a human NT-4 pseudogene colocalize to chromosome 19 band q13.3. Mammalian NT-4 has many unusual features compared to the previously identified neurotrophins and is less conserved evolutionarily than the other neurotrophins. However, mammalian NT-4 displays bioactivity and trk receptor specificity similar to that of Xenopus NT-4.Nerve growth factor (NGF) is the prototypical member of a family of mammalian neuronal survival and differentiation factors (1), known as the neurotrophins, that also includes brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (2-10). These three neurotrophins are all initially synthesized as larger precursors that are proteolytically cleaved to release the mature neurotrophins. The mature regions of the three neurotrophins display 50-55% amino acid identity to each other, with the major regions of structural similarity bordering six invariant cysteine residues that, in active NGF, have been shown to form three intrachain disulfide bonds.The neurotrophins can be distinguished based on their distinct patterns of spatial and temporal expression (11-15) as well as their differing effects on neuronal targets (3-7). The ability of a cell to respond to a particular neurotrophin appears to be dependent on the presence of the appropriate trk receptor. The three known trk receptors (designated here as trkA, trkB, and trkC) are transmembrane tyrosine protein kinases that specifically bind to the neurotrophins; trkA binds and can be activated by , trkB binds and can mediate functional responses to , and trkC seems relatively specific for .Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4) has been molecularly cloned from Xenopus (26); a gene segment encoding part of the mature NT-4 from viper was also isolated. Xenopus NT-4 shares all the important features that characterize the mammalian neurotrophins. Here we describe the molecular cloning and characterization of a neurotrophin that apparently corresponds to the mammalian counterpart of Xenopus/viper NT-4. § (The D-number assignment for humNT-4 chromosomal location is D19S202E.)

Chromosomal translocations involving chromosome 3, band q27, are among the most common rearrangements in B-cell non-Hodgkin lymphoma. From a bacteriophage lambda library prepared from a lymphoma characterized by a t(3;14)(q27;q32), genomic clones were isolated using a probe from the immunoglobulin heavy chain locus (IGH) joining region. In addition to clones containing an apparently normal IGH rearrangement, others were found to contain one of the translocation breakpoint junctions. Normal chromosome 3 sequences and the reciprocal breakpoint junction were subsequently isolated. DNA probes on each side of the chromosome 3 breakpoint hybridized at high stringency to the DNA of various mammalian species, demonstrating evolutionary conservation. One such probe from the presumptive der(3) chromosome detected an 11-kilobase transcript when hybridized to RNA of B- and T-cell lines. A probe made from partial cDNA clones isolated from a T-cell line hybridized with genomic DNA from both sides of the chromosome 3 breakpoint, indicating that the t(3;14) is associated with a break within the gene on chromosome 3. In situ chromosomal hybridization revealed that the same gene is involved in the t(3;22)(q27;q11). Preliminary nucleotide sequencing shows no identity of the cDNA to gene sequences in available data banks. We propose the name BCL6 (B-cell lymphoma 6) for this gene, since it is likely to play a role in the pathogenesis of certain B-cell lymphomas.

Translocations involving chromosome 11, band q23, are frequent recurring abnormalities in human acute lymphoblastic and acute myeloid leukemia. We used 19 biotinlabeled probes derived from genes and anonymous cosmids for hybridization to metaphase chromosomes from leukemia cells that contained four translocations involving band 1 Iq23: t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;pl3). The location of the cosmid probes relative to the breakpoint in 11q23 was the same in all translocations. Of the cosmid clones containing known genes, CD3D was proximal and PBGD, THYI, SRPR, and ETSI were distal to the breakpoint on 11q23. Hybridization of genomic DNA from a yeast clone containing yeast artificial chromosomes (YACs), that carry 320 kilobases (kb) of human DNA including CD3D and CD3G genes, showed that the YACs were split in all four translocations. These results indicate that the breakpoint at 11q23 in each of these translocations occurs within the 320 kb encompassed by these YACs; whether the breakpoint within the YACs is precisely the same in the different translocations is presently unknown)