Mutational analysis of DBD*--a unique antileukemic gene sequence.
DBD* is a novel gene encoding an 89 amino acid peptide that is constitutively lethal to leukemic cells. DBD* was derived from the DNA binding domain of the human glucocorticoid receptor by a frameshift that replaces the final 21 C-terminal amino acids of the domain. Previous studies suggested that DBD* no longer acted as the natural DNA binding domain. To confirm and extend these results, we mutated DBD* in 29 single amino acid positions, critical for the function in the native domain or of possible functional significance in the novel 21 amino acid C-terminal sequence. Steroid-resistant leukemic ICR-27-4 cells were transiently transfected by electroporation with each of the 29 mutants. Cell kill was evaluated by trypan blue dye exclusion, a WST-1 tetrazolium-based assay for cell respiration, propidium iodide exclusion, and Hoechst 33258 staining of chromatin. Eleven of the 29 point mutants increased, whereas four decreased antileukemic activity. The remainder had no effect on activity. The nonconcordances between these effects and native DNA binding domain function strongly suggest that the lethality of DBD* is distinct from that of the glucocorticoid receptor. Transfections of fragments of DBD* showed that optimal activity localized to the sequence for its C-terminal 32 amino acids.
Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77555-0645, USA.
Yan-shan
YS
Ji
Betty H
BH
Johnson
M Scott
MS
Webb
E Brad
EB
Thompson
D000595:Amino Acid Sequence; D017209:Apoptosis; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D007942:Leukemia, Experimental / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000473:pathology
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid