Up-regulation of homeodomain genes, DLX1 and DLX2, by FLT3 signaling.

BACKGROUND: Activating mutations in fms-like tyrosine kinase-3 (FLT3) are frequent in acute myeloid leukemia and represent both a poor prognostic feature and a therapeutic target. We have identified a previously unrecognized downstream effect of FLT3 activation, namely up-regulation of the homeodomain genes, DLX1 and DLX2. DESIGN AND METHODS: MV4;11 cells with FLT3-internal tandem duplication mutation, RS4;11 cells with wild-type FLT3 and blasts from patients with acute myeloid leukemia were used to pursue the relation between FLT3, DLX1/2 and transforming growth factor-ß (TGFß). Real-time quantitative reverse transcriptase polymerase chain reaction, western blot and reverse-phase protein array were performed to detect changes in gene and protein expression. RNA interference and MTS assays were used to study the interaction of PKC412, FLT3 inhibitor and TGFß1. RESULTS: A direct relationship between FLT3 activity and DLX1/2 expression was revealed by both inhibition and up-regulation of FLT3 signaling in MV4;11 and RS4;11 cell lines, respectively, in isolated blast cells from patients with acute myeloid leukemia, and in reverse-phase protein array assays of samples from patients with acute myeloid leukemia. Mechanistically, the link between FLT3 and DLX1 expression appears to involve MAPK signaling through the ERK and JNK pathways. To determine whether elevated DLX1 had a functional consequence, we explored the reported inhibition by DLX1 on TGFß/Smad signaling. Indeed, TGFß responses were blunted by FLT3 activation in a DLX1-dependent manner and FLT3 inhibition resulted in a time-dependent increase in nuclear phospho-Smad2. CONCLUSIONS: These findings suggest that alterations in DLX1/2 contribute to the biological consequences of FLT3 activation.

Site-directed mutagenesis of residues involved in G Strand DNA binding by Escherichia coli DNA topoisomerase I.

Crystal structures of complexes between type IA DNA topoisomerases and single-stranded DNA suggest that the residues Ser-192, Arg-195, and Gln-197 in a conserved region of Escherichia coli topoisomerase I may be important for direct interactions with phosphates on the G strand of DNA, which is the substrate for DNA cleavage and religation (Changela A., DiGate, R. J., and Mondragón, A. (2001) Nature 411, 1077-1081; Perry, K., and Mondragón, A. (2003) Structure 11, 1349-1358). Site-directed mutagenesis experiments altering these residues to alanines and other amino acids were carried out to probe the relevance of these interactions in the catalytic activities of the enzyme. The results show that the side chains of Arg-195 and Gln-197 are required for DNA cleavage by the enzyme and are likely to be important for positioning of the G strand of DNA at the active site prior to DNA cleavage. Mutation of Ser-192 did not affect DNA binding and cleavage but nevertheless decreased the overall rate of relaxation of supercoiled DNA probably because of its participation in a later step of the reaction pathway.

Flexibility at Gly-194 is required for DNA cleavage and relaxation activity of Escherichia coli DNA topoisomerase I.

The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA. The shifting of an alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures. Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change.