Abnormal Mitosis and Genetic Instability Induced by Downregulation of Candidate Myeloid Tumor Suppressor Genes Isolated from the Long Arm of Chromosome 7.

To isolate myeloid tumor suppressor genes from 7q, we tried to detect microdeletions (< 100 Kb) that might be present in MDS/AML cells carrying apparently normal chromosome 7. For this purpose, we developed our original microarray-based CGH technology. In this system, instead of BAC clones generally used as probes, we applied short (3–5 Kb) genomic DNA fragments containing strictly no repetitive sequences. We made 235 probes in a region spanning 21.7 Mb within 7q21.3–7q31.1. Although we selected MDS/AML patients whose marrow did not show cytogenetically visible 7q deletions, gross copy number changes frequently observed in adult patients prevented us from identification of common microdeletions. By investigation of 21 childhood myeloid leukemia patients with normal karyotype, we successfully identified a common microdeletion spanning approximately 120 Kb. Eight (38%) patients shared this microdeletion, which was not detected in normal individuals. Database search revealed that this region contains three hypothetical genes. Only vertebrates have these genes that likely evolved from one common ancestral gene of fish. Real-time quantitative PCR revealed that 9 (29%) out of 31 adult MDS/AML harbors microdeletions in at least one of these three genes. None of these genes had been well characterized nor has known motifs that would suggest function of the gene products. We named them Miki, Titan and Kasumi. Immunoblot analysis revealed expression of all three genes at high levels in most lymphoid leukemia cell lines, while half of myeloid cell lines lacked at least one of their expression. In leukemia cells carrying monosomy 7, expression levels were generally low. Miki, a heavily glycosylated protein, co-localized with centrosomes and spindles in the mitotic phase. To test the function of Miki, we used si-RNAs to downregulate Miki expression in HeLa and K562 cells, both of which show basically normal metaphase and nuclear morphology. Cells expressing Miki at reduced levels showed small and fragmented centrosomes, loss of spindle tension, tripolar mitosis or even completely disturbed spindle formation. As a result, anaphase lagging, colchicine-mitosis (C-mitosis), premature chromosome decondensation and chromatid bridges were observed in virtually all cells in the mitotic phase. In the interphase, bi- or tri-nuclear or even multinuclear cells with micronuclei, all of which are characteristic to MDS, were frequently observed. On the other hand, proteomic analysis revealed that Titan and Kasumi bind to the DNA-PK complex, which plays critical roles in the non-homologous end joining (NHEJ) of double stranded DNA (dsDNA) breaks. Indeed, these proteins were translocated from cytoplasm to nucleus by ionizing radiation (IR) or by treatment of drugs that yield dsDNA breaks. Cells expressing Kasumi at reduced levels by si-RNA showed increased radiosensitivity, sister chromatid exchange, and number of background-level phosphorylated histone H2AX foci (i.e., foci formed without IR), which are co-localized with dsDNA breaks. These results indicate that the genes we isolated are promising candidates for anti-leukemic genes located in 7q, because downregulation of these gene products by 7q deletions would cause the abnormal morphology of MDS and genetic instability.