ABSTRACT
The LIM-domain protein LMO2 is a T-cell oncogenic protein first recognized by gene activation through chromosomal translocations, but it is also responsible for leukaemias arising as secondary, adverse effects in an X-SCID gene therapy trial. There are no specific reagents currently available to analyse the LMO2 multiprotein complex or to combat LMO2-dependent leukaemias. Accordingly, we have isolated an anti-LMO2 single chain Fv antibody fragment to determine if intracellular interference with LMO2-protein complexes can avert LMO2-dependent functions in normal and cancer settings. The anti-LMO2 single chain Fv, obtained using Intracellular Antibody Capture (IAC) technology, is specific for LMO2 among the LIM-only protein family and binds LMO2 through the third and fourth LIM fingers. Using vector-mediated expression of anti-LMO2 scFv, we show inhibition of Lmo2-dependent erythropoiesis but not endothelial development. We also demonstrate inhibition of Lmo2-dependent leukaemia in a mouse T-cell tumourigenesis transplantation assay with retroviral-mediated expression of anti-LMO2 scFv. Our studies establish that interference with the LMO2 multiprotein complex inhibits both normal and tumourigenic roles. The antibody fragment is a tool for dissecting LMO2 function in haematopoiesis and leukaemia and is a lead for development of therapeutics against LMO2-dependent T-ALL.
Subject(s)
Antibodies/immunology , DNA-Binding Proteins/antagonists & inhibitors , Leukemia, T-Cell/pathology , Metalloproteins/antagonists & inhibitors , Adaptor Proteins, Signal Transducing , Animals , CHO Cells , Cricetinae , Cricetulus , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Green Fluorescent Proteins/genetics , LIM Domain Proteins , Metalloproteins/genetics , Metalloproteins/metabolism , MiceABSTRACT
Mouse models of human cancers are important for understanding determinants of overt disease and for "preclinical" development of rational therapeutic strategies; for instance, based on macrodrugs. Chromosomal translocations underlie many human leukemias, sarcomas, and epithelial tumors. We have developed three technologies based on homologous recombination in mouse ES cells to mimic human chromosome translocations. The first, called the knockin method, allows creation of fusion genes like those typical of translocations of human leukemias and sarcomas. Two new conditional chromosomal translocation mimics have been developed. The first is a method for generating reciprocal chromosomal translocations de novo using Cre-loxP recombination (translocator mice). In some cases, there is incompatible gene orientation and the translocator model cannot be applied. We have developed a different model (invertor mice) for these situations. This method consists of introducing an inverted cDNA cassette into the intron of a target gene and bringing the cassette into the correct transcriptional orientation by Cre-loxP recombination. We describe experiments using the translocator model to generate MLL-mediated neoplasias and the invertor method to generate EWS-ERG-mediated cancer. These methods mimic the situation found in human chromosome translocations and provide the framework for design and study of human chromosomal translocations in mice.
Subject(s)
Neoplasms/genetics , Translocation, Genetic , Animals , Chromosome Inversion , Chromosomes, Human/genetics , Disease Models, Animal , Genetic Engineering , Humans , Mice , Mice, Mutant Strains , Models, Genetic , Neoplasms/etiology , Recombination, GeneticABSTRACT
Soft-x-ray emission from a cryogenically cooled Ne jet irradiated by intense, 25-fs laser pulses was measured. The Ne spectrum started to drastically change in emitting ions from Ne5+ to Ne7+ below the preexpansion temperature of -120 degrees C. The significant change in the spectrum is attributed to the collisional heating of small-sized Ne clusters formed in the cooled jet. The increase of the laser pulse length from 25 fs to 100 fs resulted in further increase of x-ray emission from Ne7+ states.
