Loss of Ubr1 promotes aneuploidy and accelerates B-cell lymphomagenesis in TLX1/HOX11-transgenic mice.

The TLX1/HOX11 homeobox gene was originally identified at the recurrent t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T-cell acute lymphoblastic leukemias (T-ALLs). Proviral insertional mutagenesis studies performed on transgenic mice ectopically expressing TLX1/HOX11 in B lymphocytes (IgHmu-HOX11(Tg) mice) revealed the Ubr1 gene locus as a frequent site of proviral insertion, concomitant with accelerated development of diffuse large B-cell lymphoma. Insertion into this genomic region was confirmed by Southern blotting and by the ability to generate a polymerase chain reaction (PCR) amplicon across the viral-genome junction. Western immunoblot and semiquantitative reverse transcriptase-PCR analysis revealed downregulated expression of the Ubr1 gene product subsequent to viral integration. Loss or reduced levels of Ubr1 expression was associated with 5/14 spontaneous B-cell lymphomas in IgHmu-HOX11(Tg) mice and one of nine primary human T-ALLs. To gain mechanistic insight into the cooperativity between TLX1/HOX11 and Ubr1, IgHmu-HOX11(Tg)/Ubr1(-/-) mice were generated. IgHmu-HOX11(Tg)/Ubr1(-/-) mice exhibited a modest but statistically significant acceleration of disease onset relative to IgHmu-HOX11(Tg)/Ubr1(+/-) mice. Moreover, micronucleus assays to detect for chromosome missegregation were conducted and revealed increased presence of micronuclei in IgHmu-HOX11(Tg)/Ubr1(-/-) primary B lymphocyte cultures, and in both TLX1/HOX11-overexpressing T cell lines and fibroblast cultures following transfection with short interfering RNAs (siRNAs) targeting Ubr1. Karyotyping of primary B lymphocyte cultures revealed increased incidences of hypodiploid karyotypes. Finally, mitotic figures analysed from Ubr1 siRNA-transfected fibroblast cultures revealed no defects in chromosome congression to the metaphase plate, but increased incidences of atypical anaphase figures, including the development of anaphase bridges and lagging chromosomes. Based on these findings, we identify a synergistic role between TLX1/HOX11 overexpression and Ubr1 inactivation in promoting chromosome missegregation, permitting the accrual of additional chromosome losses and cytogenic abnormalities en route to malignancy.

Dysregulated expression of mitotic regulators is associated with B-cell lymphomagenesis in HOX11-transgenic mice.

Dysregulated expression of the homeobox gene, HOX11 is a frequent etiologic event in T-cell acute lymphoblastic leukemias. HOX11-transgenic mice (IgHmu-HOX11Tg)-expressing HOX11 in the B-cell compartment develop B-cell lymphomas with extended latency. The latency suggests that additional genetic events are required prior to the onset of malignant lymphoma. We report the identification of 17 HOX11 collaborating genes, revealed through their propensity to be targeted in a proviral insertional mutagenesis screen. Seven integrations disrupted genes in mitotic spindle checkpoint control, suggesting that cells with elevated HOX11 expression are especially sensitive to dysregulation of chromosome segregation during mitosis. IgHmu-HOX11Tg primary B-lymphocyte cultures exposed to the aneugenic agents, colchicine and colcemid, exhibited increased incidences of chromosome missegregation as assessed by cytokinesis-block micronucleus assays. Additionally, IgHmu-HOX11Tg cultures were shown to exhibit aberrant bypass of spindle checkpoint arrest, as assessed by the increased presence of cycling cells determined by assessment of DNA content and by BrdU immunolabelling. Western immunoblotting revealed elevated expression of the mitotic effector molecules, cyclin A, cyclin B1 and cdc20 in IgHmu-HOX11Tg cultures. Moreover, spontaneously arising lymphoid neoplasms in IgHmu-HOX11Tg mice frequently exhibit aberrant expression of mitotic regulators, concomitant with increased development of micronuclei, abnormal mitotic checkpoint control and increased incidences of abnormal karyotypes when expanded in culture. Collectively, these findings indicate that abnormal regulation of spindle checkpoint control as a result of HOX11 overexpression leads to a heightened predisposition for development of aneuploidy, contributing to oncogenesis.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice.

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

A model for spontaneous B-lineage lymphomas in IgHmu-HOX11 transgenic mice.

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR alpha/delta regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHmu-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin's lymphoma of peripheral mature B cell origin.

Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia.

Multiple members of the A, B, and C clusters of Hox genes are expressed in hematopoietic cells. Several of these Hox genes have been found to display distinctive expression patterns, with genes located at the 3' side of the clusters being expressed at their highest levels in the most primitive subpopulation of human CD34+ bone marrow cells and genes located at the 5' end having a broader range of expression, with downregulation at later stages of hematopoietic differentiation. To explore if these patterns reflect different functional activities, we have retrovirally engineered the overexpression of a 5'-located gene, HOXA10, in murine bone marrow cells and demonstrate effects strikingly different from those induced by overexpression of a 3'-located gene, HOXB4. In contrast to HOXB4, which causes selective expansion of primitive hematopoietic cells without altering their differentiation, overexpression of HOXA10 profoundly perturbed myeloid and B-lymphoid differentiation. The bone marrow of mice reconstituted with HOXA10-transduced bone marrow cells contained in high frequency a unique progenitor cell with megakaryocytic colony-forming ability and was virtually devoid of unilineage macrophage and pre-B-lymphoid progenitor cells derived from the transduced cells. Moreover, and again in contrast to HOXB4, a significant proportion of HOXA10 mice developed a transplantable acute myeloid leukemia with a latency of 19 to 50 weeks. These results thus add to recognition of Hox genes as important regulators of hematopoiesis and provide important new evidence of Hox gene-specific functions that may correlate with their normal expression pattern.

Overexpression of HOXB3 in hematopoietic cells causes defective lymphoid development and progressive myeloproliferation.

HOXB3 mRNA levels are high in the earliest CD34+ lineage- bone marrow cells and low to undetectable in later CD34+/CD34- cells. To gain some insight into the role this gene may play in hematopoiesis, HOXB3 was overexpressed in murine bone marrow cells using retroviral gene transfer. Thymi of HOXB3 marrow recipients were reduced in size compared with control transplant recipients, with a 24-fold decrease in the absolute number of CD4+ CD8+ cells and a 3-fold increase in the number of CD4- CD8- thymocytes that contained a high proportion of gammadelta TCR+ cells. B cell differentiation was also perturbed in these mice, as indicated by the virtual absence of transduced IL-7-responsive pre-B clonogenic progenitors. Recipients of HOXB3-transduced cells also had elevated numbers of mature granulocyte macrophage colony-forming cells in their bone marrow and spleen. Together these results suggest roles for HOXB3 in proliferation and differentiation processes of both early myeloid and lymphoid developmental pathways.

Cross-linking the murine heat-stable antigen induces apoptosis in B cell precursors and suppresses the anti-CD40-induced proliferation of mature resting B lymphocytes.

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes, the co-stimulation of CD4+ T cells, and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells, but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together, these data implicate HSA as a potent negative regulator of B cell development and activation.

Reduction of early B lymphocyte precursors in transgenic mice overexpressing the murine heat-stable antigen.

To study the role of the murine heat-stable Ag (HSA) in lymphocyte maturation, we generated transgenic mice in which the HSA cDNA was under the transcriptional control of the TCR V beta promoter and Ig mu enhancer. The HSA transgene was expressed during all stages of B lymphocyte maturation. Expression was first detected in the earliest lymphoid-committed progenitors, which normally do not express HSA, and subsequently reached the highest levels in pro- and pre-B cells. In bone marrow, the number of IL-7-responsive clonogenic progenitors was < 4% of normal, whereas the frequency of earlier B lymphocyte-restricted precursors, detectable as Whitlock-Witte culture-initiating cells, was normal. Pro- and pre-B cells detected by flow cytometry were reduced by approximately 50% relative to controls. Mature splenic B cells were also reduced but to a lesser extent than in marrow, and their response to LPS stimulation was impaired. Reconstitution of SCID and BALB/c-nu/nu mice with HSA transgenic marrow indicated that the perturbations in B lymphopoiesis were not caused by a defective marrow microenvironment or by abnormal T cells. Our previous studies showed elevated HSA expression throughout thymocyte development, which resulted in a profound depletion of CD4+CD8+ double-positive and single-positive thymocytes. Together, these results indicate that HSA levels can determine the capacity of early T and B lymphoid progenitors to proliferate and survive. Therefore, HSA could serve as an important regulator during the early stages of B and T lymphopoiesis.

Mapping of CD24 and homologous sequences to multiple chromosomal loci.

The human cell surface antigen, CD24, is a glycosyl phosphatidylinositol (GPI)-linked glycoprotein that has been implicated in the differentiation and activation of granulocytes and B lymphocytes. Changes in expression of the antigen occur at critical times during B lineage development. CD24 was cloned by its homology to mouse heat-stable antigen. Southern blot analysis suggested the presence of multiple CD24 related sequences in the human genome. We have now mapped CD24 homologous sequences to chromosomes 6q21, 15q21-q22, and Yq11 by screening a panel of somatic cell hybrid DNAs and by in situ hybridization. At least two additional homologues, one located on chromosome 1 at band p36 and one tentatively mapped to chromosome 20, that are distantly related to CD24 were identified. Southern analysis of male and female DNA samples confirmed the presence of CD24 homologous sequences on the human Y chromosome. Sequencing of DNA fragments amplified from monochromosomal somatic cell hybrids showed that the CD24 cDNA was derived from a transcript originating from a gene on chromosome 6. The CD24 gene on the Y chromosome had many base changes compared to the cDNA but had retained an open reading frame, leaving open the question of whether this gene is functional. Both ATGs in the translation initiation region of CD24 homologous sequences on chromosome 15 were converted to GTGs, making it unlikely that this gene, if functional, encodes a protein similar to CD24. CD24, therefore, is a member of a multigene family, but it remains to be determined whether any of the related genes are functional.

Defective development of thymocytes overexpressing the costimulatory molecule, heat-stable antigen.

Heat-stable antigen (HSA) is a small, glycosyl phosphatidylinositol-anchored protein that can act as a costimulatory molecule for antigen-dependent activation of helper T cells. In addition to being expressed on antigen-presenting B cells, HSA is also expressed during the initial stages of T cell development in the thymus. HSA levels are very high on immature CD4-, CD8- double negative thymocytes, but are reduced on CD4+, CD8+ double positive cells undergoing selection in the thymus, and are entirely eliminated when these cells differentiate into immunologically competent CD4+ or CD8+ single positive T cells. To examine the potential roles of this molecule in T cell development and selection, we generated transgenic mice in which HSA was highly expressed on all classes of thymocytes. The consequence of deregulated HSA expression was a pronounced reduction in the numbers of double positive and single positive thymocytes, whereas the numbers of their double negative precursors were largely unaffected. These results demonstrate that downregulation of HSA expression at the double positive stage is a critical event in thymocyte development. The depletion of thymocytes resulting from HSA overexpression begins at the same time as the onset of negative selection, suggesting that HSA may provide signals that contribute to determining the efficiency of this process.

Relative tumorigenicities of hybrid cells with and without HSR-bearing chromosomes from a human melanoma cell line.

Some cell types within the human melanoma cell line MeWo contain homogeneously staining regions (HSRs) consisting of repetitive DNA sequences and ribosomal RNA (rRNA) genes derived from chromosome 15. To further examine the association between enhanced tumorigenicity and the presence of HSR-bearing chromosomes, hybrid cell lines were constructed by fusing X-HSR-containing MeWo cells with ouabain-resistant, HPRT-deficient Chinese hamster ovary cells and culturing in HAT medium containing ouabain. A hybrid containing the X-HSR chromosome and several MeWo chromosomes was more tumorigenic in BALB/c nude mice than derivative cells lacking the X-HSR and human chromosome 18. However, since this enhanced tumorigenicity could be due to sequences on either the X-HSR or chromosome 18, a second series of hybrids was constructed by micro-cell fusion. In this case, the tumorigenicity of hybrid cells containing 2 copies of the X-HSR as the only MeWo chromosome was similar to that of derivative cells lacking these chromosomes. Cytogenetic analysis revealed that the nucleolar organizer regions (NORs) on the HSR were inactive in the hybrid cells. Our data indicate that DNA sequences amplified on MeWo HSRs do not enhance tumorigenicity under experimental conditions in which rRNA genes are not expressed. As the only active NORs in MeWo HSR-containing cells are on the HSRs, we suggest that expression of these amplified rRNA genes is responsible for the selective growth advantage of these cell types in nude mice. Our data also indicate that the enhanced tumorigenicity of MeWo HSR-containing cells is not due to co-amplification of a dominant oncogene.

Tumorigenicity of ten karyotypically distinct cell types present in the human melanoma cell line MeWo-A.

The earliest passage of the human melanoma cell line, MeWo-A, consists of ten cell types that can be distinguished on the basis of chromosome markers. Two of these cell types have chromosomes with long homogeneously staining regions (HSR) containing sequences derived from the short arm of a chromosome #15. In one cell type the HSR is found on a chromosome #15 and in the other it is on a der(15;10)-HSR chromosome. Four other cell types were identifiable by morphologic differences of the short arm of chromosome #13, whereas, the four remaining cell types were identifiable by the presence of prominent satellites on other chromosomes. This study was directed at assessing the relative tumorigenic properties of the different cell types by injecting different numbers of cells intraperitoneally, subcutaneously, or intravenously into Balb/c nude mice. The primary tumors and nodules that developed in the peritoneal cavity and lungs were explanted into tissue culture. One hundred metaphase chromosome spreads from each established cell line were analyzed cytogenetically to detect changes in proportions of the different cell types. The cell type containing the der(15;10)-HSR chromosome was present in only 20% of the cells injected, but increased in proportion to between 28% and 98% after growth in nude mice. Although the degree of selection of the der(15;10)-HSR-containing cell type was influenced by the number of cells injected, the consistent selection of these cells strongly suggests that this cell type has a growth advantage. Because the 15-HSR-containing cell type rarely increased in proportion, it is likely that the HSR by itself can not confer the enhanced tumorigenic phenotype but requires the expression of other sequences present on other MeWo chromosomes to provide the selective growth advantage to the cells in which it is found.

Evidence for unequal crossing-over as the mechanism for amplification of some homogeneously staining regions.

The mechanism of DNA amplification in homogeneously staining regions (HSR) was studied in the human melanoma cell line MeWo. Three karyotypically distinguishable cell types within this cell line contain HSR on four different chromosomes, but all HSR contain the same amplified sequences derived from the short arm of chromosome #15. We examined metaphases of MeWo cells from different passages for changes in the length and location of the HSR. In addition, we examined the replication patterns of the HSR sequences and the organization of repeated sequences within these structures. We found that variation in the lengths of the HSR was due to fewer or more copies of a unit that consisted of a later-replicating, distamycin A/DAPI-positive block and active nucleolar organizing regions (NOR). Lateral asymmetry studies suggested that the satellite DNA sequences that are present within the HSR are organized in large inverted repeats. This organization would account for the pairing in both orientations with exchanges resulting in the types of derivative chromosomes observed. The frequency of sister chromatid exchanges (SCE) within the HSR was increased over other chromosomal regions and four examples of unequal SCE within the HSR, with prominent looping out of the longer chromatids, were seen. These results support a model of unequal SCE, rather than saltatory replication for the amplification of DNA sequences in these HSR.

Effect of salts on membrane binding and activity of adipocyte phosphatidate phosphohydrolase.

Isosmotic replacement of sucrose in a low ionic strength homogenizing buffer (0.25 M sucrose/1 mM EDTA/1 mM Tris-HCl, pH 7.4) with KCl increased the microsomal and decreased the soluble phosphatidate phosphohydrolase (EC 3.1.3.4) activity of isolated rat fat cells. At 54 mM KCl the microsomal specific activity was increased 6-fold and the soluble activity was decreased to less than one-third. Binding of enzyme was promoted by KCl and NaCl when the once isolated soluble and microsomal fractions were recombined and incubated at 37 degrees C. Half-maximal binding occurred at about 17 mM salt and maximal binding at about 50 mM. The pH optimum of binding was 7.8 in 15 mM Hepes. MgCl2, CaCl2 and spermine prevented desorption of microsomal enzyme at mu molar levels and maximal effects were observed at concentrations below the 1 mM level. At maximum, however, the prevention of desorption was less by these salts than it was by KCl. MgCl2 and spermine also interfered with the effect of KCl. Moderate salt-induced loading of microsomes with the phosphohydrolase (specific activity increased 3.5-fold) increased their ability to incorporate 14C into triacylglycerol from sn-[U-14C]glycerol 3-phosphate while a high loading (specific activity increased 6-fold) had no effect or even suppressed it. The results are discussed in relation to a role of translocation of phosphatidate phosphohydrolase in glyceride biosynthesis and its control.