The 11;22 chromosomal translocation specificaOly linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the aminoterminal-encoding portion of the EWS gene to the carboxylterminal DNA-binding domain encoded by the FLII gene. We have isolated a fourth EWS-FLII fusion cDNA that is structurally distinct from the three forms previously described. To determine the transforming activity of this gene, alternative forms of the EWS-FLI1 fusion were transduced into NIH 3T3 Structural alteration or aberrant expression of transcription factors is also common in human malignancies but usually results through somatic genomic mutation (for reviews, see refs. 2 and 3). Karyotypic analyses have revealed a tumor-specific t(11;22)(q24;ql2) chromosomal translocation in 86% of both Ewing sarcoma and primitive neuroectodermal tumor (PNET), suggesting that the product of this rearrangement is necessary for the formation of both these malignancies (4,5
MATERIALS AND METHODSPNET cDNA Library Construction and Isolation of EWS-FL!) Chimeras. TC-32, a PNET tumor cell line containing the 11;22 translocation, was grown in RPMI medium/10% fetal calf serum, as described (5). Total RNA was harvested by lysis with guanidine isothiocyanate and purified over cesium chloride (9). Poly(A)+ RNA was obtained by using columns packed with oligo(dT)-cellulose (Collaborative Research) and used for construction of cDNA libraries.A TC-32 cDNA library was made according to previously published procedures (10). Briefly, first-strand synthesis was accomplished using methyl mercury-denatured poly(A)+ RNA primed with oligo(dT) and murine leukemia virus reverse transcriptase (GIBCO/BRL). Second-strand synthesis was done by using RNase H and polymerase I (GIBCO/ BRL), and synthesized products were purified over a Sephadex G100 column (Pharmacia). cDNAs were blunted by using T4 polymerase (GIBCO/BRL) and ligated to a molar excess of EcoRI adaptors (Invitrogen, San Diego). The adaptor ends were phosphorylated with T4 polynucleotide kinase (United States Biochemical), and cDNAs were fractionated over a 6% acrylamide gel. DNA species >600 bp were recovered from gel slices by electroelution, purified over
EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewing's sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-I has been shown to be a potent transforming gene Aberrant expression and structural alteration of transcription factors are frequent, primary molecular mechanisms in oncogenesis (11). In lower mammals and avian species, these alterations are often mediated by retroviral insertion. In humans, deregulation or structural alteration of transcription factors is frequently the result of somatic genomic rearrangement.The 11;22 chromosomal translocation found in Ewing's sarcoma and primitive neuroectodermal tumor of childhood (PNET) juxtaposes the 5' sequences from a newly described gene, termed EWS, with the 3' sequences from FLI-1, which encodes a member of the Ets transcription factor family (6). Like most Ets family members (for a review, see reference 27), the carboxyl domain of FLI-1 mediates sequencespecific DNA binding. The FLI-1 amino terminus contains a putative transcription activation domain that may interact on a protein-protein level with other transcription factors. As with other Ets proteins, it is probably the combination of protein-DNA and protein-protein binding specificities that determines which genes are transcriptionally modulated by FLI-1. As a result of the 11;22 rearrangement, the aminoterminal domain of FLI-1 is replaced by a portion of EWS containing a series of degenerate, glutamine-rich repeats. The carboxyl terminus of EWS has amino acid similarity to proteins involved in RNA synthesis and processing (6). However, the function in tumor cells of the EWS aminoterminal domain that is fused to FLI-1 is unknown.We have recently demonstrated that the EWS/FLI-1 chi-
Information regarding the distribution and persistence of DNA encapsulated in poly-(lactide co-glycolide) microspheres was collected to provide additional information regarding the safety of DNA vaccines and to support the clinical testing of this new delivery system for DNA. Plasmid DNA was encapsulated in poly(lactide co-glycolide) microspheres and the distribution and persistence of plasmid in murine tissues resulting from parenteral administration were examined by a sensitive PCR assay. Encapsulated DNA delivered by intramuscular or subcutaneous injection can be detected for 100 days post-injection and is distributed primarily at the site of injection and the lymphoid organs. Intravenous administration results in more widespread dissemination with long term persistence limited to the lymphoid organs and those of the reticuloendothelial system. Specific cellular uptake of DNA by professional antigen presenting cells (APCs) following injection suggests the utility of microspheres as DNA delivery agents. Distribution and persistence studies support the safety of encapsulated DNA and the specific cellular uptake of DNA by professional APCs following injection suggests the utility of microspheres as DNA delivery agents.
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