Figure 1. Consequences of chromosomal translocations in human tumors. a, Chromosomal translocations (generally between non-homologous chromosomes but sometimes between homologous chromosomes, such as t(14;14) in T-cell leukemias) are common in leukemias and sarcomas where the molecular consequences have been widely studied (reviewed in ref. 1). Cytogenetic and molecular analysis of epithelial tumors shows that these, too, have chromosomal translocations, although the basic principles may differ in those situations. In the leukemias and sarcomas studied, 2 major outcomes have emerged, namely enforced oncogene expression that occurs when an oncogene is subjected to a new chromosomal environment, or gene fusion between exons for 2 genes. b, After the chromosomal translocation, the genes affected by the translocation are transcribed and translated into proteins. All of these molecules are intracellular, so therapeutics must be designed to work in the intracellular milieu.

Figure 2. Intracellular therapy strategies for products of chromosomal translocations. Intracellular targets can include chromosomal DNA, primary transcripts, mRNA and protein products. Strategies have been designed for each point in the flow of information from chromosomal translocation breakpoint region to effective oncogenic protein, including gene switches such as polyamide inhibitors and DNA-binding proteins (shown, a zinc-finger protein) to RNA-specific reagents such as antisense and RNAi (siRNA), and protein-specific reagents such as intracellular antibodies (intrabodies), peptide aptamers and small chemical entities. Met, methionine intiation codon; stop, translation stop codon of mRNA.

Figure 3. Mouse models of chromosomal translocation are essential for objective assessment of potential therapeutic reagents before clinical application. Several strategies are available. a, Transgenic mice can be made in which an expression plasmid is stably incorporated into the mouse genome. This results in chromosomal translocation-oncogene expression (gain-of-function), and can be tissue-specific if the cassette uses a specified promoter region or has a locus control region attached to it. Alternatively, an inducible system can be used. b, Retroviral vectors may be used to express a chromosomal translocation-oncogene of choice. Bone marrow cells are infected ex vivo with the vector and infected progenitors are transferred to recipient mice with subsequent re-population of the hematopoietic compartment. Oncogenic effects can be measured as alterations of differentiation or as overt tumorigenesis. The use of homologous recombination in embryonic stem cells in vitro offers another route for generation of chromosomal translocation mimics, as direct gene manipulation can be exploited to create fusion genes or even de novo chromosomal translocations. c, Fusion genes can be made by a homologous recombination knock-in strategy (knock-in HR) in which a targeting vector, comprising a relevant exon of the target locus fused with the cDNA sequence of a naturally occurring chromosomal translocation-fusion partner, is transfected into embryonic stem cells. Those cells with a knock-in within the target locus are selected (these cells have 1 targeted allele with a gene fusion configuration and 1 wild-type allele). Injecting the embryonic stem cells into blastocysts and transferring into recipients will yield chimeric mice with the translocation mimic; it is possible to obtain germ-line transmission of the knock-in allele. d, Conditional chromosomal translocation mimics have been obtained using the Cre-loxP recombination system. In one strategy, de novo chromosomal translocations have been generated in mice using homologous recombination in embryonic stem cells to introduce loxP recombination sites next to 2 target genes for chromosomal translocation. By making mice with 'floxed' alleles (alleles in which 2 loxP recombination sites are added to a single region) and Cre recombinase expressed from a transgene, chromosomal translocations can be made to occur by inter-chromosomal rearrangements. A different strategy uses a floxed transcription stop signal (a signal flanked by loxP recombination sites) downstream of a fusion gene knock-in. The transcription stop signal can be removed by Cre-mediated deletion, thereby activating the fusion gene.