Ectopic production of the EVI1 transcriptional repressor zinc finger protein is seen in 4 -6% of human acute myeloid leukemias. Overexpression also transforms Rat1 fibroblasts by an unknown mechanism, which is likely to be related to its role in leukemia and which depends upon its repressor activity. We show here that mutant murine Evi-1 proteins, lacking either the N-terminal zinc finger DNA binding domain or both DNA binding zinc finger clusters, function as dominant negative mutants by reverting the transformed phenotype of Evi-1 transformed Rat1 fibroblasts. The dominant negative activity of the non-DNA binding mutants suggests sequestration of transformation-specific cofactors and that recruitment of these cellular factors might mediate Evi-1 transforming activity. C-terminal binding protein (CtBP) co-repressor family proteins bind PLDLS-like motifs. We show that the murine Evi-1 repressor domain has two such sites, PFDLT (site a, amino acids 553-559) and PLDLS (site b, amino acids 584 -590), which independently can bind CtBP family co-repressor proteins, with site b binding with higher affinity than site a. Functional analysis of specific CtBP binding mutants show site b is absolutely required to mediate both transformation of Rat1 fibroblasts and transcriptional repressor activity. This is the first demonstration that the biological activity of a mammalian cellular transcriptional repressor protein is mediated by CtBPs. Furthermore, it suggests that CtBP proteins are involved in the development of some acute leukemias and that blocking their ability to specifically interact with EVI1 might provide a target for the development of pharmacological therapeutic agents.A small number of transcription factors are frequently targets for de-regulation by recurring chromosome translocations in acute leukemias, and these events play a pivotal role in disease progression (1). The EVI-1 gene encodes one of these transcription factors, which is activated in 4 -6% of acute myeloid leukemia (AML) 1 patients with various karyotypic abnormalities of chromosome 3q26 (2), which result in the ectopic production of intact or, occasionally, C-terminal-truncated EVI1 proteins (3-6). In addition, novel EVI1 fusion proteins are sometimes produced. For example, patients with karyotypes t(3;21) (q26;q22) or t(3;12) (q26;p13) express AML1/EVI1 (7) and ETV6(TEL)/EVI1 (8) chimeras, respectively, and similar fusions with a naturally occurring MDS1/EVI1 isoform (9).The precise contribution of ectopic EVI1 and EVI1 fusion protein production in leukemia progression is unknown, but a combination of enforced transgene expression and intervention studies shows a causative role, affecting both cell differentiation and proliferation. Expression of AML1/MDS1/EVI1 induces AML in mice, resulting in the accumulation of myeloid blast cells and immature differentiated myelocytic and monocytic lineages (10). EVI1 or AML1/EVI1 expression in either 32Dcl3 cells or murine primary bone marrow cells abrogates granulocyte colony-stimulating factor and eryth...

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The long terminal repeat U3 sequences were determined for multiple feline leukemia virus proviruses isolated from naturally occurring T-cell tumors. Heterogeneity was evident, even among proviruses cloned from individual tumors. Proviruses with one, two, or three repeats of the long terminal repeat enhancer sequences coexisted in one tumor, while two proviruses with distinct direct repeats were found in another. The enhancer repeats are characteristic of retrovirus variants with accelerated leukemogenic potential and occur between-155 and-244 base pairs relative to the RNA cap site. The termini of the repeats occur at or near sequence features which have been recognized at other retrovirus recombinational junctions. In vitro footprint analysis of the feline leukemia virus enhancer revealed three major nuclear protein binding sites, located at consensus sequences for the simian virus 40 core enhancer, the nuclear factor 1 binding site, and an indirect repeat which is homologous to the PEA2 binding site in the polyomavirus enhancer. Only the simian virus 40 core enhancer sequence is present in all of the enhancer repeats. Cell type differences in binding activities to the three motifs may underlie the selective process which leads to outgrowth of viruses with specific sequence duplications.

Support for multistage models of oncogenesis has been provided by several highly leukaemogenic retrovirus isolates that have transduced more than one host cell gene. Where functional studies have been performed, these retroviral oncogenes show synergy for in vitro transformation and leukaemogenesis. In naturally occurring feline leukaemias associated with feline leukaemia virus (FeLV), retroviral transduction of myc is a frequent oncogenic mechanism. But evidence suggesting that the FeLV v-myc genes might be insufficient for leukaemogenesis was provided by the latency (12 weeks) and clonality of FeLV/v-myc-induced tumours and the absence of demonstrable in vitro transformation by these viruses. In the search for secondary leukaemogenic events in FeLV/v-myc tumours, we have identified a case of FeLV transduction of a T-cell antigen receptor beta-chain gene. The proviruses carrying this gene (which we have named v-tcr) were a separate population from those carrying v-myc. In its normal role, the T-cell receptor beta-chain forms part of a multimeric complex involved in antigen recognition and T-cell activation. We suggest that v-tcr is a novel viral oncogene which assisted v-myc in the genesis of a naturally occurring case of thymic lymphosarcoma.

Five tumours, which arose in cats naturally or experimentally infected with feline immunodeficiency virus (FIV), were examined with molecular probes to establish tumour cell lineage and to screen for integrated viral sequences. Three of the tumours were classed as B-cell lymphomas on the basis of morphology, immunocytochemistry, rearrangement of immunoglobulin heavy chain genes and lack of rearrangement of T-cell receptor (TCR) beta-chain genes. Two of these B-cell tumours arose in specific pathogen-free (SPF) cats experimentally infected with FIV. One case of multi-centric lymphosarcoma came from a cat naturally infected with both FIV and feline leukaemia virus (FeLV). This tumour contained integrated FeLV proviral sequences and was judged to be of T-cell origin on the basis of TCR gene rearrangement. The fifth case was a mast cell tumour. Rearrangement of the c-myc locus was not found in any of the FIV-associated tumours but was shown to be present in a rare immunoblastic B-cell lymphoma which arose in an uninfected SPF cat. None of the FIV-associated tumours showed evidence of integrated FIV sequences by Southern blot hybridisation, despite isolation of infectious virus from in vitro cultures of tumour cells in I case. These results confirm that FIV-associated tumours can occur in the absence of FeLV and suggest that the role of FIV in lymphomagenesis is generally indirect.