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Biomedical subjects

T H Rabbitts

Publications and source records attributed to T H Rabbitts.

At least 19 recordsLinked to original sources
PubMed2006

A conditional model of MLL-AF4 B-cell tumourigenesis using invertor technology.

MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukaemia and is associated with a poor prognosis. MLL-AF4 is thought to be required in haematopoietic stem cells to elicit leukaemia and may be involved in tumour phenotype specification as it is only found in B-cell tumours in humans. We have employed the invertor conditional technology to create a model of MLL-AF4, in which a floxed AF4 cDNA was knocked into Mll in the opposite orientation for transcription. Cell-specific Cre expression was used to generate Mll-AF4 expression. The mice develop exclusively B-cell lineage neoplasias, whether the Cre gene was controlled by B- or T-cell promoters, but of a more mature phenotype than normally observed in childhood leukaemia. These findings show that the MLL-AF4 fusion protein does not have a mandatory role in multi-potent haematopoietic stem cells to cause cancer and indicates that MLL-AF4 has an instructive function in the phenotype of the tumour.

Animals↗
PubMed2005

The versatile mixed lineage leukaemia gene MLL and its many associations in leukaemogenesis.

The marked association of abnormalities of chromosome 11 long arm, band q23, with human leukaemia led to the identification of the 11q23 gene called MLL (or HTRX, HRX, TRX1, ALL-1). MLL can become fused with one of a remarkable panoply of genes from other chromosome locations in individual leukaemias, leading to either acute myeloid or lymphoid tumours (hence the name MLL for mixed lineage leukaemia). The unusual finding that a single protein could be involved in both myeloid and lymphoid malignancies and that the truncated protein could do so as a fusion with very disparate partners has prompted studies to define the molecular role of MLL-fusions in leukaemogenesis and to the development of MLL-controlled mouse models of leukaemogenesis. These studies have defined MLL-fusion proteins as regulators of gene expression, controlling such elements as HOX genes, and have indicated a variety of mechanisms by which MLL-fusion proteins contribute to leukaemogenesis.

Animals↗
PubMed2004

Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice.

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules.

The use of antibodies in medicine and research depends on their specificity and affinity in the recogniton and binding of individual molecules. However, these applications are limited to the extracellular targets. Advances in antibody engineering has allowed the manipulation of the antibody segments containing the antigen-binding regions and generation of small fragments that can be stably expressed in cells. These entities are called intracellular antibodies or intrabodies and have being successfully applied, mainly in the scFv format, to inhibit the function of intracellular target proteins in specific cellular compartments. As new techniques to select and isolate intrabody fragments have been developed, intrabodies are beginning to be used to interfere with the function of a greater number of relevant disease targets. Just as monoclonal antibodies are opening a new era in human therapeutics, intrabodies promise a new prospective for antibody tools for therapy and research. Their varied mode of action gives intrabodies great potential in different approaches in the treatment of human diseases, as well as in the area of functional genomics for characterisation of novel gene products and subsequent validation as potential drug targets. While techniques for identifying functional intrabodies have improved, there are still many significant problems to be overcome before intrabodies can actually be used in treatment of diseases such as cancer, AIDS or neuro-degenerative disorders.

Acquired Immunodeficiency Syndrome↗
PubMed2003

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene.

Chromosome 3 allele loss in preinvasive bronchial abnormalities and carcinogen-exposed, histologically normal bronchial epithelium indicates that it is an early, possibly the first, somatic genetic change in lung tumor development. Candidate tumor suppressor genes have been isolated from within distinct 3p regions implicated by heterozygous and homozygous allele loss. We have proposed that DUTT1, nested within homozygously deleted regions at 3p12-13, is the tumor suppressor gene that deletion-mapping and tumor suppression assays indicate is located in proximal 3p. The same gene, ROBO1 (accession number ), was independently isolated as the human homologue of the Drosophila gene, Roundabout. The gene, coding for a receptor with a domain structure of the neural-cell adhesion molecule family, is widely expressed and has been implicated in the guidance and migration of axons, myoblasts, and leukocytes in vertebrates. A deleted form of the gene, which mimics a naturally occurring, tumor-associated human homozygous deletion of exon 2 of DUTT1/ROBO1, was introduced into the mouse germ line. Mice homozygous for this targeted mutation, which eliminates the first Ig domain of Dutt1/Robo1, frequently die at birth of respiratory failure because of delayed lung maturation. Lungs from these mice have reduced air spaces and increased mesenchyme, features that are present some days before birth. Survivors acquire extensive bronchial epithelial abnormalities including hyperplasia, providing evidence of a functional relationship between a 3p gene and the development of bronchial abnormalities associated with early lung cancer.

Amino Acid Sequence↗
PubMed2001

Chromosomal translocation master genes, mouse models and experimental therapeutics.

Molecular biologists have elucidated general principles about chromosomal translocations by cloning oncogenes or fusion genes at chromosomal translocation junctions. These genes invariably encode intracellular proteins and in acute cancers, often involve transcription and developmental regulators, which are master regulators of cell fate (e.g. LMO2 which is involved in acute leukaemia). Chromosomal translocations are usually associated with specific cell types. The reason for this close association is under investigation using mouse models. We are trying to emulate the cell-specific consequences of chromosomal translocations in mice using homologous recombination in embryonic stem cells to generate de novo chromosomal translocations or to mimic the consequence of these translocations. In addition, chromosomal translocation genes and their products are important targets for therapy. We have designed new therapeutic strategies which include antigen-specific recruitment of endogenous cellular pathways to affect cellular viability and a novel structured form of antisense to ablate the function of fusion mRNAs. We will evaluate these procedures in the mouse models of chromosomal translocations and the long term aim is to perfect rapid procedures for characterizing patient-specific chromosomal translocations to tailor therapy to individual patients.

Animals↗
PubMed2001

T cell tumorigenesis in Lmo2 transgenic mice is independent of V-D-J recombinase activity.

The LMO2 gene is involved in T-cell acute leukaemia (T-ALL) in children with chromosomal translocations t(11;14)(p13;q11) or (7;11)(q35;p13). Transgenic expression of Lmo2 in T cells results in clonal tumours with long latency indicating that mutations in other genes are required for the development of overt tumours. RAG V-D-J recombinase can mediate genetic transposition and thus might create the secondary mutations necessary for T-ALL. Tumour development was compared in Lmo2 transgenic mice in the presence or absence of the Rag1 gene. No difference was observed in the rate of tumour formation nor in tumour histology in Lmo2-transgenic mice with or without Rag1. We conclude that, in this model, RAG recombinase is not a major mediator of mutations needed for T cell tumorigenesis and that antigen binding to alpha-beta or to gamma-delta T cell receptor does not play a role in tumorigenesis. The driving force behind the mutational process involved in this transgenic model remains obscure.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The T cell oncogene Tal2 is necessary for normal development of the mouse brain.

Transcription factors are commonly involved in leukemia by activation through chromosomal translocations and normally function in cell type(s) that differ from that of the tumor. TAL2 is a member of a basic helix-loop-helix gene family specifically involved in T cell leukemogenesis. Null mutations of Tal2 have been made in mice to determine its function during development. Tal2 null mutant mice show no obvious defects of hematopoiesis. During embryogenesis, Tal2 expression is restricted to the developing midbrain, dorsal diencephalon, and rostroventral diencephalic/telencephalic boundary, partly along presumptive developing fiber tracts. The null mutant mice are viable at birth but growth become progressively retarded and they do not survive to reproductive age. Tal2-deficient mice show a distinct dysgenesis of the midbrain tectum. Due to loss of superficial gray and optical layers, the superior colliculus is reduced in size and the inferior colliculus is abnormally rounded and protruding. Death is most likely due to progressive hydrocephalus which appears to be caused by obstruction of the foramen of Monro (the connection between the ventricles of the forebrain). Thus, in addition to its oncogenicity when ectopically expressed, Tal2 normally plays a pivotal role in brain development and without this gene, mice cannot survive to maturity.

Animals↗
PubMed2000

Intracellular antibody-caspase-mediated cell killing: an approach for application in cancer therapy.

Antibodies have been expressed inside cells in an attempt to ablate the function of oncogene products. To make intracellular antibodies more generally applicable and effective in cancer therapy, we have devised a method in which programmed cell death or apoptosis can be triggered by specific antibody-antigen interaction. When intracellular antibodies are linked to caspase 3, the "executioner" in the apoptosis pathway, and bind to the target antigen, the caspase 3 moieties are self-activated and thereby induce cell killing. We have used this strategy in a model system with two pairs of intracellular antibodies and antigens. In vivo coexpression of an antibody-caspase 3 fusion with its antigenic target induced apoptosis that was specific for antibody, antigen, and active caspase 3. Moreover, the antibody-caspase 3 fusion protein was not toxic to cells in the absence of antigen. Therefore, intracellular antibody-mediated apoptosis should be useful as a specific therapeutic approach for the treatment of cancers, a situation where target cell killing is required.

Animals↗
PubMed2000

Structural basis for the heterodimeric interaction between the acute leukaemia-associated transcription factors AML1 and CBFbeta.

Mutations in the genes encoding the interacting proteins AML1 and CBFbeta are the most common genetic abnormalities in acute leukaemia, and congenital mutations in the related AML3 gene are associated with disorders of osteogenesis. Furthermore, the interaction of AML1 with CBFbeta is essential for haematopoiesis. We report the 2.6 A resolution crystal structure of the complex between the AML1 Runt domain and CBFbeta, which represents a paradigm for the mode of interaction of this highly conserved family of transcription factors. The structure demonstrates that point mutations associated with cleidocranial dysplasia map to the conserved heterodimer interface, suggesting a role for CBFbeta in osteogenesis, and reveals a potential protein interaction platform composed of conserved negatively charged residues on the surface of CBFbeta.

Acute Disease↗
PubMed2000

Tumorigenesis in mice with a fusion of the leukaemia oncogene Mll and the bacterial lacZ gene.

Many different chromosomal translocations occur in man at chromosome 11q23 in acute leukaemias. Molecular analyses revealed that the MLL gene (also called ALL-1, HRX or HTRX) is broken by the translocations, causing fusion with genes from other chromosomes. The diversity of MLL fusion partners poses a dilemma about the function of the fusion proteins in tumour development. The consequence of MLL truncation and fusion has been analysed by joining exon 8 of Mll with the bacterial lacZ gene using homologous recombination in mouse embryonic stem cells. We show that this fusion is sufficient to cause embryonic stem cell-derived acute leukaemias in chimeric mice, and these tumours occur with long latency compared with those found in MLL-Af9 chimeric mice. These findings indicate that an MLL fusion protein can contribute to tumorigenesis, even if the fusion partner has no known pathogenic role. Thus, truncation and fusion of MLL can be sufficient for tumorigenesis, regardless of the fusion partner.

Amino Acid Sequence↗
PubMed2000

The oncogenic LIM-only transcription factor Lmo2 regulates angiogenesis but not vasculogenesis in mice.

The LMO2 gene is activated by chromosomal translocations in human T cell acute leukemias, but in mouse embryogenesis, Lmo2 is essential for initiation of yolk sac and definitive hematopoiesis. The LMO2 protein comprises two LIM-zinc-finger-like protein interaction modules and functions by interaction with specific partners in DNA-binding transcription complexes. We have now investigated the role of Lmo2-associated transcription complexes in the formation of the vascular system by following the fate of Lmo2-null embryonic stem (ES) cells in mouse chimeras. Lmo2 is expressed in vascular endothelium, and Lmo2-null ES cells contributed to the capillary network normally until around embryonic day 9. However, after this time, marked disorganization of the vascular system was observed in those chimeric mice that have a high contribution of Lmo2-null ES cells. Moreover, Lmo2-null ES cells do not contribute to endothelial cells of large vessel walls of surviving chimeric mice after embryonic day 10. These results show that Lmo2 is not needed for de novo capillary formation from mesoderm but is necessary for angiogenic remodeling of the existing capillary network into mature vasculature. Thus, Lmo2-mediated transcription complexes not only regulate distinct phases of hematopoiesis but also angiogenesis, presumably by Lmo2 interacting with distinct partners in the different settings.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Masked antisense: a molecular configuration for discriminating similar RNA targets.

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These 'masked' antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR-ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.

Autoradiography↗
PubMed2000

Inter-chromosomal recombination of Mll and Af9 genes mediated by cre-loxP in mouse development.

Chromosomal translocations are crucial events in the aetiology of many leukaemias, lymphomas and sarcomas, resulting in enforced oncogene expression or the creation of novel fusion genes. The study of the biological outcome of such events ideally requires recapitulation of the tissue specificity and timing of the chromosomal translocation itself. We have used the Cre-loxP system of phage P1 to induce de novo Mll-Af9 chromosomal recombination during mouse development. loxP sites were introduced into the Mll and Af9 genes on chromosomes 9 and 4, respectively, and mice carrying these alleles were crossed with mice expressing Cre recombinase. A resulting Mll-Af9 fusion gene was detected whose transcription and splicing were verified. Thus, programmed interchromosomal recombination can be achieved in mice. This approach should allow the design of mouse models of tumorigenesis with greater biological relevance than those available at present.

Animals↗
PubMed1999

Selection of antibodies for intracellular function using a two-hybrid in vivo system.

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.

Animals↗
PubMed1999

The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis.

The MLL gene from human chromosome 11q23 is involved in >30 different chromosomal translocations resulting in a plethora of different MLL fusion proteins. Each of these tends to associate with a specific leukaemia type, for example, MLL-AF9 is found mainly in acute myeloid leukaemia. We have studied the role of the Mll-AF9 gene fusion made in mouse embryonic stem cells by an homologous recombination knock-in. Acute leukaemias developed in heterozygous mice carrying this fusion as well as in chimeric mice. As with human chromosomal translocation t(9;11), the majority of cases were acute myeloid leukaemias (AMLs) involving immature myeloblasts, but a minority were acute lymphoblastic leukaemia. The AMLs were preceded by effects on haematopoietic differentiation involving a myeloproliferation resulting in accumulation of Mac-1/Gr-1 double-positive mature myeloid cells in bone marrow as early as 6 days after birth. Therefore, non-malignant expansion of myeloid precursors is the first stage of Mll-AF9-mediated leukaemia followed by accumulation of malignant cells in bone marrow and other tissues. Thus, the late onset of overt tumours suggests that secondary tumorigenic mutations are necessary for malignancy associated with MLL-AF9 gene fusion and that myeloproliferation provides the pool of cells in which such events can occur.

Animals↗