Fc receptor gene translocation in a t(1;19) pre-B ALL cell line

We have recently mapped the human FCGR2 gene to chromosome 1 bands q23-q24. In situ hybridization of FCGR2 cDNA with a cell line containing a t(1;19)(g23;p13) derived from a patient with pre-B ALL has allowed a more accurate localization of this gene to chromosome 1 band q23. Furthermore, this study indicated a splitting of the FCGR2 gene or gene cluster by the t(1;19). However, Southern analysis showed no genetic rearrangement when compared with a karyotypically normal Epstein-Barr virus (EBV)-transformed cell line from the same patient. This suggests that the translocation breakpoint does not occur within the coding region of this gene.     

Linkage relationships of the insulin receptor gene with the complement component 3, LDL receptor,apolipoprotein C2 and myotonic dystrophy loci on chromosome 19

Summary Myotonic dystrophy is associated with disturbances in the insulin response, possibly due to an abnormality of the insulin receptor. Both the myotonic dystrophy (DM) and insulin receptor (INSR) genes are on chromosome 19. Using a cloned gene probe for INSR, we have studied its linkage relationships with the DM locus and other chromosome 19 markers. The results show that INSR is not closely linked to DM, but is located very close to C3, in the region 19pter-19p13.2. This implies that the basic genetic defect which causes DM is not directly responsible for the disturbed insulin response in these patients.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Nerve Growth Factor Receptor TrkA,a New Receptor in Insulin Signaling Pathway in PC12 Cells

TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

The human VAV proto-oncogene maps to chromosome region 19p12→19p13.2

Summary A novel human oncogene, designated VAV, has been recently characterized. This oncogene was generated by a rearrangement within the 5 coding sequences of a normal cellular gene, the VAV proto-oncogene. The normal VAV gene is specifically expressed in hematopoietic cells regardless of their differentiation lineage. We now report that the VAV locus has been localized in the human genome at chromosome 19p12 19p13.2 by analysis of its segregation pattern in rodenthuman somatic cell hybrids and by chromosomal in situ hybridization. The VAV locus might be closely linked to the insulin receptor (INSR) locus, as suggested by comigration of INSR and VAV high-molecular-weight DNA fragments after pulsed-field gel electrophoresis. The VAV chromosomal assignment is of interest because chromosome region 19p13 is involved in different karyotypic abnormalities in a variety of malignancies including melanomas and leukemias. The identification of a novel proto-oncogene that maps to that region will enable us to define whether VAV is involved in any of the translocations observed.     

Molecular analysis of a t(7;14)(q35;q32) chromosome translocation in a T cell leukemia of a patient with ataxia telangiectasia

Molecular analysis of somatic cell hybrids derived from T cells carrying a t(7;14)(q35;q32) chromosomal translocation from a patient with ataxia telangiectasia and T cell leukemia indicates that the breakpoint on chromosome 14 is proximal to the IgH locus and to the D14S1 locus, while the breakpoint on chromosome 7 involves the T cell receptor beta chain locus immediately 5' to J beta 1.5 on chromosome 7. The separation of V beta and C beta observed in somatic cell hybrids defined the orientation of the T cell receptor beta chain locus on chromosome 7 where the V beta genes are centromeric and the C beta genes are telomeric. A novel chromosomal alteration, undetected cytogenetically, was revealed as being an inversion with duplication of the distal band of chromosome 14q32. The importance of the 14q32 region in the leukemogenic process is discussed.     

XBP1 promotes NRASG12D pre-B acute lymphoblastic leukaemia through IL-7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS

Activating point mutations of the RAS gene act as driver mutations for a subset of precursor-B cell acute lymphoblastic leukaemias (pre-B ALL) and represent an ambitious target for therapeutic approaches. The X box-binding protein 1 (XBP1), a key regulator of the unfolded protein response (UPR), is critical for pre-B ALL cell survival, and high expression of XBP1 confers poor prognosis in ALL patients. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. Here, we demonstrate that XBP1 acts as a downstream linchpin of the IL-7 receptor signalling pathway and that pharmacological inhibition or genetic ablation of XBP1 selectively abrogates IL-7 receptor signalling via inhibition of its downstream effectors, JAK1 and STAT5. We show that XBP1 supports malignant cell growth of pre-B NRAS^G12D ALL cells and that genetic loss of XBP1 consequently leads to cell cycle arrest and apoptosis. Our findings reveal that active XBP1 prevents the cytotoxic effects of a dual PI3K/mTOR pathway inhibitor (BEZ235) in pre-B NRAS^G12D ALL cells. This implies targeting XBP1 in combination with BEZ235 as a promising new targeted strategy against the oncogenic RAS in NRAS^G12D-mutated pre-B ALL.     

Human neonatal lymphocytes immortalized after microinjection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV) is a highly efficient acute transforming agent in human cells, provided that the intact virus is used. To investigate the ability of viral DNA alone to transform cells, we introduced the EBV genome into human lymphocytes. After microinjection of EBV DNA into neonatal B lymphocytes, we established a cell line that in early passages contained multiple viral fragments. This cell line retained sequences from the short, unique (Us) region of the EBV genome and sequences from EcoRI-E. The viral sequences were not expressed; however, the cells expressed a 2.3-kilobase polyadenylated message homologous to the c-fgr oncogene, a cellular locus believed to be activated by EBV infection [M. S. C. Cheah, T. J. Ley, S. R. Tronick, and K. C. Robbins, Nature (London) 319:238-240.]. The cell line was monoclonal with rearrangement at the immunoglobulin locus and had a reciprocal translocation t(1;7)(p34;q34) and a deletion of sequences within the locus for the beta chain of the T-cell receptor. The close proximity of the translocation to the chromosomal loci for c-fgr on chromosome 1 and the T-cell receptor beta chain on chromosome 7 suggests that structural alteration of these genes was critical to this transformation event.     

Association of a RFLP for the insulin receptor gene, but not insulin, with essential hypertension.

Insulin has cardiovascular actions and patients with essential hypertension display insulin resistance. A cross-sectional study of the R1 RFLP of the insulin receptor gene (INSR) was carried out in 67 hypertensive (HT) and 75 normotensive (NT) subjects whose parents had a similar blood pressure status at age greater than or equal to 50. The frequency of the minor (+) allele was 0.31 in HTs and 0.44 in NTs, and the difference between observed alleles in all subjects in each group was significant (chi 2 = 4.8, P less than 0.05). Allele frequencies of a BglI RFLP of the insulin gene, however, did not differ between the HT and NT groups. The data thus provide evidence in favour of an association of HT with a polymorphism at the INSR locus (19p13.3-13.2), so implicating this locus, and possibly a genetic variant of the insulin receptor itself, in HT.     

Immunological typing of blast cells. Cases with pre-B cell characteristics

Leukemic cells from 32 patients were examined by using conventional immunological markers (E and EAC rosettes, surface immunoglobulins). Additionally, the test for intracytoplasmic IgM, Fc IgG receptor and the presence of light chains were performed. Leukemic blasts of all patients were investigated according to morphological and cytochemical criteria. Lymphoblasts from 3 patients had pre-B cell phenotype: cIgM +, sIg-. Each of 3 patients with pre-B cell characteristics had different diagnosis and different morphological and cytochemical features of the leukemic cells (ALL, NHL and CML). In 24 ALL cases the diagnosis of non-T, non-B ALL, in 4 cases T-ALL and in one B-ALL was established. The correlation of cytochemical results with special reference to acid phosphatase and immunological subclasses of ALL was also analyzed. An important question is raised with regard to diagnostic classification and treatment by finding ALL phenotypes in lymphoproliferative disorders that are not diagnosed as ALL.     

Pre-B cell receptor signaling in acute lymphoblastic leukemia

B cell lineage ALL represents by far the most frequent malignancy in children and is also common in adults. Despite significant advances over the past four decades, cytotoxic treatment strategies have recently reached a plateau with cure rates at 80 percent for children and 55 percent for adults. Relapse after cytotoxic drug treatment, initial drug-resistance and dose-limiting toxicity are among the most frequent complications of current therapy approaches. For this reason, pathway-specific treatment strategies in addition to cytotoxic drug treatment seem promising to further improve therapy options for ALL patients. In a recent study on 111 cases of pre-B cell-derived human ALL, we found that ALL cells carrying a BCR-ABL1-gene rearrangement lack expression of a functional pre-B cell receptor in virtually all cases. In a proof-of-principle experiment, we studied pre-B cell receptor function during progressive leukemic transformation of pre-B cells in BCR-ABL1-transgenic mice: Interestingly, signaling from the pre-B cell receptor and the oncogenic BCR-ABL1 kinase are mutually exclusive and only "crippled" pre-B cells that fail to express a functional pre-B cell receptor are permissive to transformation by BCR-ABL1.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Closely linked loci on the long arm of chromosome 13 flank a specific 2;13 translocation breakpoint in childhood rhabdomyosarcoma

A specific chromosomal translocation, t(2;13)(q35;q14), is present in tumor cells from about one-half of children with alveolar rhabdomyosarcoma, who generally have widely disseminated disease at diagnosis. Using a series of six DNA probes from five loci previously assigned to bands 13q12----q14, we have localized the translocation breakpoint on chromosome 13 by in situ hybridization. Each probe was used to examine metaphase spreads from two or more rhabdomyosarcoma cell lines that have the t(2;13), as well as from control lymphoblastoid cell metaphases. All six probes bound to chromosome 13q12----q14 in the control cell line, but showed no appreciable hybridization to other sites. With rhabdomyosarcoma metaphases, cDNA clones of the retinoblastoma susceptibility gene (RB1) and the esterase D gene (ESD), as well as the arbitrary genomic fragment 7D2 (D13S10), showed specific hybridization to the normal chromosome 13 and the der(2) marker, but not to the der(13). By contrast, the genomic fragments HU10 (D13S6) and 7F12 (D13S1) hybridized specifically to the normal chromosome 13 and the der(13), but not to the der(2). Thus, the breakpoint of this translocation lies distal to D13S6 and D13S1 and proximal to ESD, RB1, and D13S10. Our data indicate that the locus affected by the translocation breakpoint on chromosome 13, which we have termed RMS, is physically distinct from the RB1 locus and is, in fact, proximal to ESD, which others have placed at least 10(6) bp proximal to RB1. The consistent presence of the der(2) marker chromosome, coupled with occasional loss of the der(13), suggests that the RMS gene, or at least a critical component, moves to chromosome 2 in tumors with this translocation.     

Novel nuclear localization and potential function of insulin-like growth factor-1 receptor/insulin receptor hybrid in corneal epithelial cells

Background

Type I insulin-like growth factor receptor (IGF-1R) and insulin receptor (INSR) are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R). The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium.

Methodology/Principle Findings

IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi) cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways.

Conclusion/Significance

In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic pathways. The development of novel therapeutic strategies designed to target the IGF-1/IGF-1R pathway must take into account the modulatory roles IGF-1R/INSR play in the epithelial cell nucleus.     

Synaptonemal complex analysis of a three-breakpoint translocation in a subfertile bull.

Somatic chromosome analysis of a subfertile Brown Swiss bull demonstrated a three-breakpoint translocation involving chromosomes 1, 8, and 9 in G- and R-banded karyotypes. Based on standard bovine chromosome nomenclature, the translocation was defined as t(1;8;9)(q43;q13;q26). Synaptonemal complex analysis of the chromosome aberration by electron microscopy revealed a hexavalent configuration in 52 of 53 pachytene cells. Twenty-seven cells (51%) had a completely paired hexavalent configuration showing distinctly nonhomologous pairings between normal and/or translocated chromosomes involved in the exchanges. Thirteen cells showed a hexavalent configuration with centrally unpaired chromosome segments but with completely paired terminal arms. In 13 cells (including one at zygotene) the translocation chromosomes formed an open hexavalent, and in one cell there were two completely paired trivalents. Thirty-two cells at diakinesis-MI demonstrated 28 configurations, including one large hexavalent. Testicular histology, testis size, and seminal characteristics were normal.     

Fibronectin increases the migration induced by stromal cell-derived factor-1 alpha (SDF-1 alpha) in pre-B acute lymphoblastic leukemia cells

The chemokine, stromal cell-derived factor-1 alpha (SDF-1 alpha) and its receptor CXCR-4 (fusin, LESTR) are thought to be involved in the trafficking of hematopoietic progenitors and stem cells, as suggested by the chemotactic effect of SDF-1 alpha on these cells. Gene inactivation studies have shown that both SDF-1 alpha and CXCR-4 are essential for B lymphopoiesis. Migration of leukemic cells may also be dependent on SDF-1 alpha and CXCR-4. Fibronectin (FN) is a component of the extracellular matrix (ECM), and one of the natural supports for cell movement in their bone hematopoietic environment. In the present study, we examined the influence of FN on the chemotactic effect of SDF-1 alpha and on the CXCR-4 expression and function on human precursor-B acute lymphoblastic leukemia (pre-B ALL) cells at sequential stages of development. Fourteen children with pre-B ALL were studied. Their immunophenotypes belonged to the first three stages of B cell differentiation. Despite relatively high levels of CXCR-4 expression at all stages, the responsiveness to SDF-1 alpha, measured as the percentage of migrating cells in the transwell culture system, varied with patients and seems to be less significant for pre-B3 (and pre-B1) than for pre-B2. There was no correlation (r = 0.2) between the SDF-1 alpha induced migration (range: 2.5-39%) and the cell surface density of CXCR-4 (range: 46.5-97.5%). The extracellular matrix protein FN, either coated on the filter (for more than 18 hours) or in soluble form, enhanced the SDF-1 alpha induced migration of pre-B ALL respectively (2 fold and 1.6 fold) without influencing CXCR-4 expression in short term cultures. Therefore, we analyzed the expression of the FN receptors, VLA-4 (CD49d) and VLA-5 (CD49e), by direct immunofluorescence, on these leukemic cells. VLA-4 was strongly expressed in all stages of pre-B ALL (range: 77-97%) while VLA-5 expression was more variable (range: 14-94%), but no correlation with the FN-dependent increased SDF-1 alpha chemotactic effect was noted. In conclusion, the migratory behavior of pre-B leukemic cells in response to SDF-1 alpha partly depends upon the stage of differentiation, and partly upon unexplained patient variability. Our results suggest that several molecules from the extracellular matrix, such as FN, may be implicated in this phenomenon.