Interleukin 4 (IL-4) induces B-cell differentiation and survival of chronic lymphocytic leukemia (CLL) cells. MicroRNAs (miRNAs) regulate mRNA and protein expression, and several miRNAs, deregulated in CLL, might play roles as oncogenes or tumor suppressors. We have studied the miRNA profile of CLL, and its response to IL-4, by oligonucleotide microarrays, resulting in the detection of a set of 129 mature miRNAs consistently expressed in CLL, which included 41 differentially expressed compared to normal B cells (NBC), and 6 significantly underexpressed in ZAP-70 positive patients. IL-4 stimulation brought about up-regulation of the 5p and 3p mature variants of the miR-21 gene, which maps immediately downstream to the VMP1 gene, and of the mature forms generated from the miR-362 (3p and 5p), miR-500a (3p), miR-502 (3p), and miR-532 (3p and 5p) genes, which map within the third intron of the CLCN5 gene. Both genes are in turn regulated by IL-4, suggesting that these miRNAs were regulated by IL-4 as passengers from their carrier genes. Their levels of up-regulation by IL-4 significantly correlated with cytoprotection. MiR-21 has been reported to be leukemogenic, associated to bad prognosis in CLL, and the miRNA more frequently overexpressed in human cancer. Up-regulation by IL-4 of miR-21 and the miRNAs hosted in the CLCN5 locus may contribute to evasion of apoptosis of CLL cells. These findings indicate that the IL-4 pathway and the miRNAs induced by IL-4 are promising targets for the development of novel therapies in CLL.

Dock10 is one of the three members of the Dock-D family of Dock proteins, a class of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Its homologs Dock9 and Dock11 are Cdc42 GEFs. Dock10 is required for maintenance of rounded morphology and amoeboid-type movement. Full-length isoforms of Dock10 have been recently cloned. Here, we address GTPase specificity and GEF activity of Dock10. In order of decreasing intensity, Dock10 interacted with nucleotide-free Rac1, Cdc42, and Rac3, and more weakly with Rac2, RhoF, and RhoG. Inducible expression of Dock10 in HeLa epithelial cells promoted GEF activity on Cdc42 and Rac1, and a morphologic change in two-dimensional culture consisting in loss of cell elongation, increase of filopodia, and ruffles. Area in contact with the substrate of cells that spread with non-elongated morphology was larger in cells expressing Dock10. Inducible expression of constitutively active mutants of Cdc42 and Rac1 in HeLa cells also induced loss of elongation. However, Cdc42 induced filopodia and contraction, and Rac1 induced membrane ruffles and flattening. When co-expressed with Dock10, Cdc42 potentiated filopodia, and Rac1 potentiated ruffles. These results suggest that Dock10 functions as a dual GEF for Cdc42 and Rac1, affecting cell morphology, spreading and actin cytoskeleton protrusions of adherent HeLa cells.

The promyelocytic leukemia zinc finger ( PLZF ) gene, involved in rare cases of acute promyelocytic leukemia, encodes a Krüppel-type zinc finger transcription factor. It has been reported that PLZF affects myeloid cell growth, differentiation, and apoptosis. However, the function of PLZF in the lymphoid compartment, where PLZF is also expressed, remains largely unknown. To investigate a potential relationship between PLZF expression in lymphocytes and programmed cell death, an inducible model of stable clones of the lymphoid Jurkat cell line was created by using the tet-off system. Although induction of PLZF expression by itself did not produce changes in the basal levels of apoptosis, PLZF had a significant anti-apoptotic effect in Jurkat cells cultured in conditions of serum starvation, as measured by annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. In addition, retarded loss of mitochondrial transmembrane potential was observed in the PLZF-expressing clones, suggesting that PLZF protects from cell death through a mitochondrial-dependent mechanism. To identify apoptosis-related targets of PLZF, a screen for differential expression identified BID, a proapoptotic member of the Bcl2 family, as significantly down-regulated by PLZF. Furthermore, a high-affinity PLZF-binding site element was identified upstream of the BID transcriptional start site, as assessed by electrophoretic mobility-shift assays. These results suggest that BID is a target of PLZF repression and a candidate gene to mediate the PLZF-induced resistance to apoptosis.

Background The one-way mixed lymphocyte culture (MLC) is the classic culture used for studying the allogenic immunoresponse in vitro, but stimulator and responder cell identifications and quantification of apoptotic or proliferative responder cells are unreliable. Methods Peripheral blood mononuclear cells were labeled with 5- (and 6-) carboxy fluorescein diacetate succinimidyl ester (CFSE) and stimulated with allogenic unlabeled irradiated cells in unidirectional cultures. Apoptosis was determined by the 7-aminoactinomycin D technique, and the absolute number of each cell population was calculated by adding a fixed number of cells stained with propidium iodide as the reference standard for each test. Results CFSE labeling of cells under different cultures did not affect the results of proliferation or apoptosis. Data of apoptosis obtained with this method were comparable to those of the monoclonal antibody technique, and the proliferation level determined by [³H]-thymidine incorporation or counting the number of proliferative living cells, as proposed in this method, showed a good correlation. Conclusions The method presented in this report allows the simultaneous determination of apoptosis and proliferation in MLCs and the analysis of cell phenotype, thereby avoiding the use of radioactivity. This assay opens new perspectives for a better understanding of the mechanisms implied in the establishment or break of tolerance to the graft in solid organ transplants. Cytometry Part A 51A: 107–118, 2003.