We previously reported a candidate myeloid tumor-suppressor gene Miki (mitotic kinetic regulator, LOC253012) isolated from a common microdeletion cluster in chromosome subband 7q21.2 that was identified by microarray-based CGH analyses of JMML (ASH Annual Meeting, 2006 and 2007). Deletion of one Miki gene was also detected in 28 % of adult MDS/AML patients by copy-number assessment using real-time quantitative PCR (qPCR). Miki encodes a centrosomal protein: downregulation of Miki by siRNA disturbs the maturation and positioning of centrosomes, as well as spindle formation in mitotic cells, resulting in severe mitotic defects, such as chromosome lagging and proanaphase arrest. This causes abnormal nuclear morphology. When Miki expression levels were constitutively reduced by short-hairpin (sh) RNA in K562 cells, morphology of cells changed drastically: bi- tri- or multiple-nuclear cells with or without micronuclei appeared frequently that strikingly resembled the bone marrow picture of MDS. In addition, FISH analysis revealed widely varying chromosome numbers in these cells, suggesting that Miki-downregulation induces chromosome instability. Six myeloid leukemia cell lines derived from MDS/AML patients with monosomy 7 generally expressed Miki protein at very low (but detectable) levels, suggesting that haploinsufficient effects and/or epigenetic mechanisms reduce Miki expression levels. These six lines harboring monosomy 7 showed severe abnormal mitosis and nuclear morphology, and induction of Miki to these cells using a retroviral vector restored normal mitosis. These findings suggested that loss of Miki gene contributes to myelodysplasia and chromosome instability, which are characteristic of -7/7q- MDS/AML. To elucidate molecular mechanism through which Miki plays roles in centrosomal maturation and spindle formation, we tried to identify proteins associated with Miki using yeast two hybrid assay or mass spectrometry and found that a centrosomal giant scaffold protein CG-NAP (also known as AKAP9/AKAP450/yotiao) binds to Miki in a G2/M specific manner. Intriguingly, CG-NAP gene locates to subband 7q21.2, 1.2 Mb centromeric to Miki gene. qPCR revealed that CG-NAP was also frequently deleted in adult MDS/AML (36 %) and protein expression levels were very low (but detectable) in six cell lines derived from MDS/AML with monosomy 7, raising a possibility that CG-NAP is another candidate for the responsible genes of 7q deletion. CG-NAP is known to promote microtubule nucleation in centrosomes. As expected, downregulation of CG-NAP by siRNA showed abnormal spindle formation and mitotic disturbance (proanaphase arrest and chromosome lagging) similar to those by Miki-downregulation. Moreover, constitutive downregulation of CG-NAP by shRNA transformed K562 cells to bi- tri- or multiple-nuclear with or without micronuclei, indicating that the Miki/CG-NAP protein complex is responsible for the mitotic disturbance and abnormal nuclear morphology. Finally, we found that CG-NAP does not localize to mitotic centrosomes when Miki expression is downregulated by siRNA, suggesting that Miki contributes to organized progression of mitosis by transporting and/or anchoring CG-NAP to mitotic centrosomes. Our data indicate that Miki and CG-NAP in subband 7q21.2 encode centrosomal proteins, which play critical roles in mitosis. Loss of one 7q allele would cause marked reduction of these two gene products, resulting in myelodysplasia and chromosome instability.

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