Kazutaka Ohta

Critical Role of Pcid2 in B Cell Survival through the Regulation of MAD2 Expression

The mitotic checkpoint is essential for maintaining genomic stability in differentiating B cells undergoing genetic alterations of the Ig gene. In this study, using real-time RT-PCR and in situ RNA hybridization, we demonstrated that MAD2 mRNA export is selectively regulated by Pcid2/Thp1. Pcid2 small interfering RNA induced a cell-cycle abnormality with increased apoptosis and polyploidy, as previously observed in MAD2-knockdown cells. Pcid2 small interfering RNA reduced MAD2 expression, but not the expression of other cell-cycle checkpoint proteins, such as MAD1 and BUBR1, or the cell-cycle–associated proteins, cyclin A, cyclin B1, and cyclin-dependent kinase 1. In mouse B lineage cells, Pcid2 transcripts appeared in a stage-dependent manner at high levels in bone marrow pre-B and immature B cells, and in spleen transitional 1 and follicular B cells, but at lower levels in pro-B, transitional 2, and marginal zone B cells, suggesting a stage-dependent requirement for MAD2 regulation. Cd19-cre–derived targeting of the Pcid2 gene induced a mature B cell deficiency in mice. These findings indicate that Pcid2 is essential for B cell survival through the regulation of MAD2 expression during B cell differentiation.

Germinal center-associated nuclear protein (GANP) is involved in mRNA export of Shugoshin-1 required for centromere cohesion and in sister-chromatid exchange

Germinal center‐associated nuclear protein (GANP) is a 210‐kDa protein that is upregulated in rapidly proliferating B cells. GANP contains regions for RNA‐primase and minichromosome maintenance 3 (MCM3)‐associated activities, as well as a Sac3‐homology region, which is associated with mRNA export in yeast. Here, we examined the role of GANP in mRNA export and cell proliferation in mammalian cells. The ganp small interfering RNA (siRNA) induced cell‐cycle arrest at the G2/M‐phase, but increased abnormal chromosome alignment of metaphase chromosomes and cell apoptosis in HeLa cells. These changes were not associated with either the abnormality of the spindle assembly checkpoint or the expression level of cohesin. ganp siRNA disrupted the assembly and localization of cohesin at the centromeres in metaphase cells, which is a quite similar phenotype caused by Shugoshin‐1 (Sgo1) siRNA‐treatment, which was reported previously. ganp siRNA did induce a selective decrease in Sgo1 transcript levels in the cytoplasm, resulting in a lack of cohesin at the centromeres in metaphase and premature separation of the sister chromatids at mitosis. GANP lacking the Sac3‐homology region caused the dominant‐negative effect with similar abnormalities and impaired mRNA export. Thus, human GANP is critically involved in cell proliferation at the mitotic phase through its selective support of Sgo1 mRNA export.

Decreased expression of germinal center-associated nuclear protein is involved in chromosomal instability in malignant gliomas

Malignant glioma (MG) is highly proliferative and invasive, with the malignant characteristics associated with aneuploidy and chromosomal instability (CIN). Here, we found that the level of germinal center–associated nuclear protein (GANP), a mammalian homologue of yeast Sac3, was markedly decreased in MGs with a poor prognosis; and thus we explored the effect of its decrease on cell‐cycle progression of MG cell lines. Glioblastomas showed a significantly lower level of ganp mRNA than anaplastic astrocytomas, as measured by real‐time reverse transcription‐PCR, in 101 cases of adult MG. MGs of ganpLow expression displayed more malignant characteristics, with loss of heterozygosity on chromosome 10, epidermal growth factor receptor gene amplification, and significantly poorer prognosis than the ganpHigh group. Human diploid fibroblasts depleted of ganp mRNA by the RNA interference (RNAi) method showed a decreased percentage of S‐phase cells and a cellular‐senescence phenotype. MG cell lines harboring abnormalities of various cell‐cycle checkpoint molecules displayed slippage of mitotic checkpoints and an increased proportion of hyperploid cells after ganp RNAi‐treatment. These results suggest that GANP protects cells from cellular senescence caused by DNA damage and that a significant decrease in GANP expression leads to malignancy by generating hyperploidy and CIN. (Cancer Sci 2009); 00: 000–000)

Selective cell death of p53-insufficient cancer cells is induced by knockdown of the mRNA export molecule GANP

Cancer cells often contain p53 abnormalities that impair cell-cycle checkpoint progression and cause resistance to various anti-cancer treatments. DNA damage occurs at actively transcribed genes during G1-phase in yeast cells that have a deficient mRNA export capacity. Here, we show that germinal center-associated nuclear protein (GANP), a homologue of yeast Sac3 that is involved in mRNA export, is indispensable for ensuring the stability of human genomic DNA and that GANP knockdown causes apoptosis and necrosis of p53-insufficient cancer cells. Ganp small interfering RNA (siGanp)-induced DNA damage, accompanied by a decrease in the number of cells in S-phase, caused late apoptosis and necrosis in p53-insufficient cancer cells through both caspase-dependent and -independent mechanisms. siGanp effectively induced DNA damage leading to cell death in p53-insufficient cancer cells in vitro and protect the growth of cancer cells transplanted into immunocompromized mice, suggesting that siGanp has potential as a selective treatment for p53-insufficient cancer cells.

Bilateral posterior fossa chronic subdural hematoma treated with craniectomy: Case report and review of the literature.

Background: Posterior chronic subdural hematomas (pCSHs) are rare. Their diagnosis and treatment are difficult. Description: A 69-year-old woman was admitted to our hospital with nausea, headache, and mild consciousness disturbance. Computed tomography and magnetic resonance imaging showed bilateral pCSH. To prevent further neurological deterioration, we performed surgery under general anesthesia by midline suboccipital craniectomy. Unexpected bleeding from a developed circuitous occipital sinus was stopped with hemoclips. After hematoma removal, she recovered and was transferred to a rehabilitation hospital. By the 19th postoperative day, she had developed no neurologic deficits. Conclusion: This experience demonstrates the risk of blind surgical therapy in patients with pCSH. In such patients, posterior fossa craniectomy may be preferable in terms of diagnosis and safe treatment.