Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-κB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered.

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The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosaassociated lymphoid tissue (MALT)-type lymphomas, and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence, constitutive nuclear factor-KB (NF-KB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis, EM-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless, API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IKB kinase ; (IKK;), indicative for enhanced NF-KB activation, was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition, we show that the API2-MALT1 fusion resided in the cholesterol-and sphingolipid-enriched membrane microdomains, termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts, which is mediated by the API2 portion, is sufficient to trigger NF-KB activation via enhanced polyubiquitination of IKK;. Taken together, these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-KB activation. (Cancer Res 2006; 66(10): 5270-7)

A murine model of minor histocompatibility antigen-mismatched bone marrow transplantation (BMT) was used to study the role of timing of donor lymphocyte infusion (DLI) in eliciting graft-versushost (GVH) and graft-versus-leukemia (GVL) reactivity. We gave DLI at weeks 3 and 12 after BMT and related its ability to induce a GVL effect with (1) evolution of T cell chimeric status and (2) the extent to which DLI could elicit lymphohematopoietic GVH (LHGVH) reactivity. All mice remained free of GVH disease, but only week 3 DLI chimeras exhibited a significant GVL response when challenged with host-type leukemia cells. In these week 3 DLI chimeras, host-reactive T cells were found to proliferate in vivo (5-[and-6]-carboxyfluorescein diacetate, succinimidyl esther [CFSE]-labeled DLI inocula, TCR-V␤6 ؉ T-cell frequency) and T-cell chimerism rapidly converted from mixed into complete donor type, indicating the occurrence of LHGVH reactivity. In week 12 chimeras, DLI elicited none of the activities noted at week 3. Yet, in both instances, splenocytes, recovered following DLI, generated an equally strong antihost proliferative response in a mixed lymphocyte reaction, thereby arguing against a decisive role of regulatory cells. The lack of in vivo LHGVH reactivity after week 12 DLI was associated with a substantially increased level of pre-existing host-type T-cell chimerism. We conclude that elicitation of a GVL effect may require LHGVH reactivity and that the reason why timing of DLI was critical for obtaining LHGVH reactivity and the desired GVL effect may lie in the evolution of chimeric status. A possible direct involvement of residual host-type antigen-presenting cells in eliciting LHGVH reactivity after DLI should be studied using models that allow chimerism analysis in non-T-cell

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