Identification of t(5;10) translocation with H4/PDGFβR fusion genes and induction of apoptosis by imatinib treatment. (A) Left panel: FISH using chromosome-specific painting probes to chromosome 5 (green) and chromosome 10 (red). Right panel: representative karyotype of a metaphase from bone marrow aspirate at diagnosis of disease. Arrows point to derivative chromosomes resulting from t(5;10)(q33;q22). (B) Amplification of the flanking region implicated in the translocation t(5;10) by nested RT-PCR with 2 sets of primers, set I (lanes 1,3) and set II (lanes 2,4). Total RNA from bone marrow collected from the t(5;10) patient (lanes 1-2) as well as from a Philadelphia-positive patient (lanes 3-4) was used as negative control. A450-bp region from the housekeeping gene G3PDH was also amplified from t(5;10) (lane 5) and control (lane 6) RNAs. (C) Bone marrow aspirate from patient and a control donor was cultured for 24 hours in the presence of the indicated amounts of imatinib. Cells were then lysed and submitted to Western blot analysis using anticleaved PARP (poly-adenosine diphosphate ribose polymerase) antibodies to determine the ratio of apoptosis. (D) Recession of H4/PDGFβR expression after therapeutic treatment with imatinib. Isolated RNAfrom peripheral blood before (lanes 1,3) and after (lanes 2,4) imatinib treatment was used in a nested RT-PCR analysis. The G3PDH gene (lanes 3-4) was used as an internal control to normalize the expression of H4/PDGFβR gene product (lanes 1-2). Intensity for each of the bands was quantified with a BioRad GelDoc2000 analyzer (Hercules, CA) and the ratio between them determined.