Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

Side population (SP) cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11) express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2 gamma nul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3) express genes involved in the cell cycle and mitosis (e. g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM), polycomb (e. g., EZH2, EPC1) and ubiquitin-proteasome (e. g., UBE2D3, UBE3C, PSMA5) more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680) and a proteasome inhibitor (bortezomib) with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3) and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma

Control of Cortical Axon Elongation by a GABA-Driven Ca<sup style="margin: 0px; padding: 0px; border: 0px; outline-style: none; font-weight: inherit; font-style: inherit; font-size: 0.85em; font-family: inherit; line-height: 0; text-align: inherit; vertical-align: super;">2+/Calmodulin-Dependent Protein Kinase Cascade</sup>

Ca(2+) signaling plays important roles during both axonal and dendritic growth. Yet, whether and how Ca(2+) rises may trigger and contribute to the development of long range cortical connections remains largely unknown. Here we demonstrate that two separate limbs of CaMK kinase (CaMKK) - CaMKI cascades, CaMKK-CaMKIα and CaMKK-CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABA(A) receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK-CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIα significantly impaired the terminal axonal extension, and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK-CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process which may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the central nervous system

代謝型グルタミン酸受容体サブタイプ6欠損マウスを用いた網膜神経回路形成の形態学的解析

京都大学0048新制・論文博士博士(医学)乙第10197号論医博第1695号新制||医||723(附属図書館)UT51-99-S314(主査)教授 金子 武嗣, 教授 本田 孔士, 教授 中西 重忠学位規則第4条第2項該当Doctor of Medical ScienceKyoto UniversityDA

Dysfunction of KCNK potassium channels impairs neuronal migration in the developing mouse cerebral cortex.

Development of the cerebral cortex depends partly on neural activity, but the identity of the ion channels that might contribute to the activity-dependent cortical development is unknown. KCNK channels are critical determinants of neuronal excitability in the mature cerebral cortex, and a member of the KCNK family, KCNK9, is responsible for a maternally transmitted mental retardation syndrome. Here, we have investigated the roles of KCNK family potassium channels in cortical development. Knockdown of KCNK2, 9, or 10 by RNAi using in utero electroporation impaired the migration of late-born cortical excitatory neurons destined to become Layer II/III neurons. The migration defect caused by KCNK9 knockdown was rescued by coexpression of RNAi-resistant functional KCNK9 mutant. Furthermore, expression of dominant-negative mutant KCNK9, responsible for the disease, and electrophysiological experiments demonstrated that ion channel function was involved in the migration defect. Calcium imaging revealed that KCNK9 knockdown or expression of dominant-negative mutant KCNK9 increased the fraction of neurons showing calcium transients and the frequency of spontaneous calcium transients. Mislocated neurons seen after KCNK9 knockdown stayed in the deep cortical layers, showing delayed morphological maturation. Taken together, our results suggest that dysfunction of KCNK9 causes a migration defect in the cortex via an activity-dependent mechanism

Activity-Dependent Callosal Axon Projections in Neonatal Mouse Cerebral Cortex

Callosal axon projections are among the major long-range axonal projections in the mammalian brain. They are formed during the prenatal and early postnatal periods in the mouse, and their development relies on both activity-independent and -dependent mechanisms. In this paper, we review recent findings about the roles of neuronal activity in callosal axon projections. In addition to the well-documented role of sensory-driven neuronal activity, recent studies using in utero electroporation demonstrated an essential role of spontaneous neuronal activity generated in neonatal cortical circuits. Both presynaptic and postsynaptic neuronal activities are critically involved in the axon development. Studies have begun to reveal intracellular signaling pathway which works downstream of neuronal activity. We also review several distinct patterns of neuronal activity observed in the developing cerebral cortex, which might play roles in activity-dependent circuit construction. Such neuronal activity during the neonatal period can be disrupted by genetic factors, such as mutations in ion channels. It has been speculated that abnormal activity caused by such factors may affect activity-dependent circuit construction, leading to some developmental disorders. We discuss a possibility that genetic mutation in ion channels may impair callosal axon projections through an activity-dependent mechanism

Macular thickness changes in a patient with Leber's hereditary optic neuropathy

Background: Leber's hereditary optic neuropathy (LHON) refers to an optic nerve dysfunction due to mutations in the mitochondrial DNA, resulting in visual loss by apoptosis of retinal ganglion cells (RGC). In 20% of LHON cases, their fundus examination looks entirely normal at early stage. There are some reports regarding the circumpapillary retinal nerve fiber layer (cpRNFL) and the ganglion cell analysis around the macula in LHON patients and carriers by using optical coherence tomography. Case presentation: A 40-year-old female complained of acute visual loss in both eyes. Her best-corrected visual acuity was 0.3 in the right eye and 0.2 in the left eye at the initial visit. Goldmann perimetry revealed bilateral central scotomas. Fundus examination and fluorescein angiography findings were normal, but decreased retinal inner layer thickness was detected around the macular area on spectral domain optical coherence tomography (SD-OCT). One month later, her visual acuity deteriorated to counting fingers in both eyes, and the thinning area of retinal inner layer spread rapidly. Suspected progressive RGC loss led us to check the possibility of LHON, with which the patient was diagnosed due to a positive result for the mitochondrial DNA (mtDNA) 11778 mutation. The ganglion cell complex (GCC) and cpRNFL thicknesses were observed for 24 months by using SD-OCT. The GCC thickness plunged sharply within 3 months followed by gradual decline until 6 months, thereafter showing a plateau up to 24 months. On the cpRNFL map, the temporal quadrant also showed the earliest thinning as seen in the macular area of the GCC map. The thicknesses of the superior, nasal, and inferior quadrants decreased gradually, keeping their normal ranges up to 6 months. Conclusions: SD-OCT was a useful tool in the diagnosis and follow-up of LHON. The macular GCC thickness map may detect the earliest morphological changes in LHON, as well as the temporal area of cpRNFL, before funduscopic examination reveals optic nerve atrophy

Optical coherence tomography as a possible tool to monitor and predict disease progression in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes

Optical coherence tomography (OCT) is an imaging technique used to obtain three-dimensional information on the retina. In this article, we evaluated the structural neuro-retinal abnormalities, especially the thickness in the ganglion cell complex (GCC), in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The GCC thickness in MELAS patients was significantly thinner than that in normal controls even when they had no history of transient homonymous hemianopia. There was a negative correlation between GCC thickness and disease duration. In conclusion, OCT may be an effective tool to monitor and predict disease progression in MELAS patients

Evaluating the efficacy of epinastine ophthalmic solution using a conjunctivitis allergen challenge model in patients with birch pollen allergic conjunctivitis

Background: The efficacy of epinastine 0.05% ophthalmic solution for pollen allergic conjunctivitis has already been shown in a conjunctival allergen challenge (CAC) test using cedar pollen as a challenge. The present study investigated the efficacy of this solution against birch pollen conjunctivitis in a CAC test. Methods: Ten adult subjects (eight males and two females) with asymptomatic birch pollen conjunctivitis were enrolled in this study. The average age of the subjects was 41.1 years. This study was conducted during a period without birch pollen dispersion. In each subject, the epinastine 0.05% ophthalmic solution was instilled in one eye, and an artificial tear fluid was instilled in the fellow eye in a double-blind manner. Five minutes or 4 h after the drug instillation, both eyes were challenged with an optimal concentration of birch pollen, and ocular itching and conjunctival hyperemia were then graded. Tears were collected before the drug instillation and 20 min after the pollen challenge, and the histamine level was measured. Results: The ocular itching scores and palpebral conjunctival hyperemia scores of the epinastine-treated eyes were significantly lower than those of the contralateral control eyes when the eyes were pretreated with the drug 4 h before the CAC. There was a significant correlation between the tear histamine level and mean ocular itching score of three time points (3, 5 and 10 min) following the CAC in the control eyes but not the epinastine-treated eyes. Conclusions: Epinastine is effective in suppressing ocular itching and conjunctival hyperemia in birch pollen conjunctivitis