Cancer cells are able to overproduce lactic acid aerobically, whereas normal cells undergo anaerobic glycolysis only when deprived of oxygen. Tumor aerobic glycolysis was recognized about seven decades ago; however, its molecular basis has remained elusive. The lactate dehydrogenase-A gene (LDH-A), whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, was identified as a c-Myc-responsive gene. Stably transfected Rat1a fibroblasts that overexpress LDH-

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