COUP-TFII plays a role in cAMP-induced Schwann cell differentiation and in vitro myelination by up-regulating Krox20.

Schwann cells (SCs) are known to produce myelin for saltatory nerve conduction in the peripheral nervous system (PNS). Schwann cell differentiation and myelination processes are controlled by several transcription factors including Sox10, Oct6/Pou3f1, and Krox20/Egr2. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan receptor that plays a role in the development and differentiation. However, the role of COUP-TFII in the transcriptional regulatory network of SC differentiation has not been fully identified yet. Thus, the objective of this study was to investigate the role and molecular hierarchy of COUP-TFII during cAMP-induced SC differentiation. Our results showed that dibutyryl-cAMP (db-cAMP) increased expression levels of COUP-TFII along with the expressions of Oct6, Krox20, and myelin-related genes known to be related to SC differentiation. Our mechanistic studies showed that COUP-TFII acted downstream of Hsp90/ErbB2/Gab1/ERK-AKT pathway during db-cAMP-induced SC differentiation. In addition, we found that COUP-TFII induced Krox20 expression by directly binding to Krox20-MSE8 as revealed by chromatin immunoprecipitation assay and promoter activity assay. In line with this, the expression of COUP-TFII was increased before up-regulation of Oct6, Krox20, and myelin-related genes in the sciatic nerves during early postnatal myelination period. Finally, COUP-TFII knockdown by COUP-TFII siRNA or via AAV-COUP-TFII shRNA in SCs inhibited db-cAMP-induced SC differentiation and in vitro myelination of sensory axons, respectively. Taken together, these findings indicate that COUP-TFII might be involved in postnatal myelination through induction of Krox20 in SCs. Our results present a new insight into the transcriptional regulatory mechanism in SC differentiation and myelination.

Dual role of ERK2/NF-κB signaling in TRAIL sensitivity.

Targeting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling is a promising approach in cancer treatment. Although ERK and/or NF-κB signaling is involved in the expression of TRAIL receptors (TRAIL-R), the exact underlying mechanisms remain unknown. In this study, we evaluated the role of ERK2 and NF-κB in the cytotoxicity of TRAIL during cisplatin treatment. Cisplatin treatment of neuroepithelioma cells (SK-N-MC) significantly induced ERK2 activation and increased TRAIL cytotoxicity via the upregulation of death receptor 5 (DR5) expression. In partial ERK2 knockdown cell lines that maintained only basal levels of ERK2 activity, cisplatin treatment did not increase ERK2 activity or DR5 expression. These findings indicate that induced (rather than basal) ERK2 activity enhances TRAIL susceptibility via DR5 expression. In complete ERK2 knockdown cell lines with no basal ERK2 activity, DR4, DR5, and DcRs expression levels were increased, and additional treatment with cisplatin did not further increase TRAIL-R expression. Chemical inhibition of ERK2 also enhanced TRAIL cytotoxicity by upregulating DR4 and DR5 expression. These findings indicate that basal ERK2 activity suppresses TRAIL-R expression. Both basal and inducible ERK2 activities regulate TRAIL-R expression via the NF-κB signaling pathway. Overall, our findings suggest that the ERK2/NF-κB signaling pathway has a dual role in TRAIL susceptibility by differentially regulating TRAIL-R expression in the same cellular system.

Phenotypic heterogeneity in patients with NEFL-related Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is the most common hereditary peripheral neuropathy. Mutations in the neurofilament light polypeptide (NEFL) gene produce diverse clinical phenotypes, including demyelinating (CMT1F), axonal (CMT2E), and intermediate (CMTDIG) neuropathies. From 2005 to 2020, 1,143 Korean CMT families underwent gene sequencing, and we investigated the clinical, genetic, and neuroimaging spectra of NEFL-related CMT patients. Ten NEFL mutations in 17 families (1.49%) were identified, of which three (p.L312P, p.Y443N, and p.K467N) were novel. Eight de novo cases were identified at a rate of 0.47 based on a cosegregation analysis. The age of onset was ≤3 years in five cases (13.5%). The patients revealed additional features including delayed walking, ataxia, dysphagia, dysarthria, dementia, ptosis, waddling gait, tremor, hearing loss, and abnormal visual evoked potential. Signs of ataxia were found in 26 patients (70.3%). In leg MRI analyses, various degrees of intramuscular fat infiltration were found. All compartments were evenly affected in CMT1F patients. The anterior and anterolateral compartments were affected in CMT2E, and the posterior compartment was affected in CMTDIG. Thus, NEFL-related CMT patients showed phenotypic heterogeneities. This study's clinical, genetic, and neuroimaging results could be helpful in the evaluation of novel NEFL variants and differential diagnosis against other CMT subtypes.

Doxycycline potentiates the anti-proliferation effects of gemcitabine in pancreatic cancer cells.

Gemcitabine is often recommended as a first-line treatment for patients with metastatic pancreatic cancer. However, gemcitabine resistance is a major challenge in the treatment of pancreatic ductal adenocarcinoma. Our group serendipitously identified the role of doxycycline as a potentiator of gemcitabine efficacy in pancreatic cancer cells. Doxycycline and gemcitabine co-treatment was significantly more cytotoxic to pancreatic cancer cells compared to gemcitabine alone. Interestingly, doxycycline only exerted synergistic effects when coupled with gemcitabine as opposed to other conventional chemotherapeutics including nucleoside analogs. The anti-clonogenic effects of gemcitabine on pancreatic cancer cells were also enhanced by doxycycline. According to cell cycle analyses, doxycycline prolonged gemcitabine-mediated S phase cell cycle arrest. Further, gene expression profiling analyses indicated that a small set of genes involved in cell cycle regulation were uniquely modulated by gemcitabine and doxycycline co-treatment compared to gemcitabine alone. Western blot analyses indicated that several cell cycle-related proteins, including cyclin D1, p21, and DNA damage inducible transcript 4 (DDIT4), were further modulated by doxycycline and gemcitabine co-treatment. Taken together, our findings indicate that doxycycline enhances the effects of gemcitabine on cell cycle progression, thus rendering pancreatic cancer cells more sensitive to gemcitabine. However, additional studies are required to assess the mechanisms of doxycycline and gemcitabine synergism, which might lead to novel treatment options for pancreatic cancer.

p53-mediated regulation of mitochondrial dynamics plays a pivotal role in the senescence of various normal cells as well as cancer cells.

The tumor suppressor p53 is known as a critical mediator of many cellular processes, including cellular senescence, but its role in mitochondrial dynamics is not fully understood. We have previously shown that p53 regulates mitochondrial dynamics via the PKA-Drp1 pathway to induce cellular senescence. In this study, to further understand the role of p53-dependent regulation of mitochondrial dynamics, the effect of p53 expression on mitochondrial morphology was examined in various cancer cell lines and normal human cells. We found that p53 induced remarkable mitochondrial elongation and cellular senescence in various cancer cells regardless of their p53 status. p53 also induced mitochondrial elongation in various human primary normal cells, suggesting that p53-mediated mitochondrial elongation is a general phenomenon. Moreover, we found that p53 plays an essential role in mitochondrial elongation in H-Ras-induced cellular senescence and in the replicative senescence of normal human cells. Treatment with the MDM-2 antagonist Nutlin-3a also induced mitochondrial elongation through the PKA-Drp1 pathway in IMR90 normal human cells. Furthermore, the inhibition of PKA activity in late-passage normal cells significantly reduced both mitochondrial elongation and cellular senescence, suggesting that the p53-PKA pathway is essential for maintaining the senescence phenotype in normal cells. Together, these results further confirm the direct regulation of mitochondrial dynamics by p53 and the important role of p53-mediated mitochondrial elongation in cellular senescence.

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy.

Paclitaxel is a representative anticancer drug that induces chemotherapy-induced peripheral neuropathy (CIPN), a common side effect that limits many anticancer chemotherapies. Although PINK1, a key mediator of mitochondrial quality control, has been shown to protect neuronal cells from various toxic treatments, the role of PINK1 in CIPN has not been investigated. Here, we examined the effect of PINK1 expression on CIPN using a recently established paclitaxel-induced peripheral neuropathy model in Drosophila larvae. We found that the class IV dendritic arborization (C4da) sensory neuron-specific expression of PINK1 significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. In contrast, knockdown of PINK1 resulted in an increase in thermal hypersensitivity, suggesting a critical role for PINK1 in sensory neuron-mediated thermal nociceptive sensitivity. Interestingly, analysis of the C4da neuron morphology suggests that PINK1 expression alleviates paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in sensory dendrites in C4da neurons. We found that paclitaxel induces mitochondrial dysfunction in C4da neurons and that PINK1 expression suppressed the paclitaxel-induced increase in mitophagy in C4da neurons. These results suggest that PINK1 mitigates paclitaxel-induced sensory dendrite alterations and restores mitochondrial homeostasis in C4da neurons and that improvement in mitochondrial quality control could be a promising strategy for the treatment of CIPN.

Application of differentiated human tonsil-derived stem cells to trembler-J mice.

INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into various cell types. METHODS: In this study we investigated the potential of human tonsil-derived MSCs (T-MSCs) for neuromuscular regeneration in trembler-J (Tr-J) mice, a model for Charcot-Marie-Tooth disease type 1A (CMT1A). RESULTS: T-MSCs differentiated toward skeletal myocytes with increased expression of skeletal muscle-related markers (including troponin I type 1, and myogenin), and the formation of myotubes in vitro. In-situ transplantation of T-MSC-derived myocytes (T-MSC myocytes) into the gastrocnemius muscle in Tr-J mice enhanced motor function, with recovery of compound muscle action potential amplitudes. Morphology of the sciatic nerve and skeletal muscle recovered without the formation of teratomas, and the expression levels of nerve growth factor and glial-cell-line-derived neurotrophic factor were increased significantly in T-MSC myocytes compared with T-MSCs in vitro. DISCUSSION: Transplantation of T-MSC myocytes could enable neuromuscular regeneration in patients with CMT1A. Muscle Nerve 57: 478-486, 2018.

Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration.

Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.

Recessive optic atrophy, sensorimotor neuropathy and cataract associated with novel compound heterozygous mutations in OPA1.

Mutations in the optic atrophy 1 gene (OPA1) are associated with autosomal dominant optic atrophy and 20% of patients demonstrate extra-ocular manifestations. In addition to these autosomal dominant cases, only a few syndromic cases have been reported thus far with compound heterozygous OPA1 mutations, suggestive of either recessive or semi­dominant patterns of inheritance. The majority of these patients were diagnosed with Behr syndrome, characterized by optic atrophy, ataxia and peripheral neuropathy. The present study describes a 10-year-old boy with Behr syndrome presenting with early­onset severe optic atrophy, sensorimotor neuropathy, ataxia and congenital cataracts. He had optic atrophy and was declared legally blind at six years old. Electrophysiological, radiological, and histopathological findings were compatible with axonal sensorimotor polyneuropathy. At birth, he presented with a congenital cataract, which has not been previously described in patients with OPA1 mutations. Whole exome sequencing indicated a pair of novel compound heterozygous mutations: p.L620fs*13 (c.1857­1858delinsT) and p.R905Q (c.G2714A). Neither mutation was observed in controls (n=300), and thus, they were predicted to be pathogenic by multiple in silico analyses. The mutation sites were highly conserved throughout different vertebrate species. The patients parents did not have any ophthalmic or neurologic symptoms and the results of electrophysiological studies were normal, suggestive of an autosomal recessive pattern of inheritance. The present study identified novel compound heterozygous OPA1 mutations in a patient with recessive optic atrophy, sensorimotor neuropathy and congenital cataracts, indicating an expansion of the clinical spectrum of pathologies associated with OPA1 mutations. Thus, OPA1 gene screening is advisable in the workup of patients with recessive optic atrophy, particularly with Behr syndrome and cataracts.

A Mutation in PMP2 Causes Dominant Demyelinating Charcot-Marie-Tooth Neuropathy.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of peripheral neuropathies with diverse genetic causes. In this study, we identified p.I43N mutation in PMP2 from a family exhibiting autosomal dominant demyelinating CMT neuropathy by whole exome sequencing and characterized the clinical features. The age at onset was the first to second decades and muscle atrophy started in the distal portion of the leg. Predominant fatty replacement in the anterior and lateral compartment was similar to that in CMT1A caused by PMP22 duplication. Sural nerve biopsy showed onion bulbs and degenerating fibers with various myelin abnormalities. The relevance of PMP2 mutation as a genetic cause of dominant CMT1 was assessed using transgenic mouse models. Transgenic mice expressing wild type or mutant (p.I43N) PMP2 exhibited abnormal motor function. Electrophysiological data revealed that both mice had reduced motor nerve conduction velocities (MNCV). Electron microscopy revealed that demyelinating fibers and internodal lengths were shortened in both transgenic mice. These data imply that overexpression of wild type as well as mutant PMP2 also causes the CMT1 phenotype, which has been documented in the PMP22. This report might expand the genetic and clinical features of CMT and a further mechanism study will enhance our understanding of PMP2-associated peripheral neuropathy.

A novel homozygous MPV17 mutation in two families with axonal sensorimotor polyneuropathy.

BACKGROUND: Mutations in MPV17 cause the autosomal recessive disorder mitochondrial DNA depletion syndrome 6 (MTDPS6), also called Navajo neurohepatopathy (NNH). Clinical features of MTDPS6 is infantile onset of progressive liver failure with seldom development of progressive neurologic involvement. METHODS: Whole exome sequencing (WES) was performed to isolate the causative gene of two unrelated neuropathy patients (9 and 13 years of age) with onset of the syndrome. Clinical assessments and biochemical analysis were performed. RESULTS: A novel homozygous mutation (p.R41Q) in MPV17 was found by WES in both patients. Both showed axonal sensorimotor polyneuropathy without liver and brain involvement, which is neurophysiologically similar to axonal Charcot-Marie-Tooth disease (CMT). A distal sural nerve biopsy showed an almost complete loss of the large and medium-sized myelinated fibers compatible with axonal neuropathy. An in vitro assay using mouse motor neuronal cells demonstrated that the abrogation of MPV17 significantly affected cell integrity. In addition, the expression of the mutant protein affected cell proliferation. These results imply that both the loss of normal function of MPV17 and the gain of detrimental effects of the mutant protein might affect neuronal function. CONCLUSION: We report a novel homozygous mutation in MPV17 from two unrelated patients harboring axonal sensorimotor polyneuropathy without hepatoencephalopathy. This report expands the clinical spectrum of diseases caused by mutations of MPV17, and we recommend MPV17 gene screening for axonal peripheral neuropathies.

Overexpression of mutant HSP27 causes axonal neuropathy in mice.

BACKGROUND: Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. RESULTS: We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. CONCLUSIONS: Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

A family with axonal sensorimotor polyneuropathy with TUBB3 mutation.

Mutations in the ß­tubulin isotype III (TUBB3) gene result in TUBB3 syndrome that includes congenital fibrosis of the extraocular muscle type 3 (CFEOM3), intellectual impairments and/or an axonal sensorimotor neuropathy. In the present study, a TUBB3 D417N mutation was identified in a family with axonal sensorimotor polyneuropathy by whole exome sequencing. The proband exhibited gait disturbance at the age of 12 years and was wheelchair bound at 40 years. However, the proband's cousin exhibited gait disabilities at 45 years of age and was still able to walk when he was 60 years old. Ophthalmoplegia and intellectual impairment were not observed in either patient. A sural nerve biopsy identified an absence of large myelinated fibers without demyelinating degeneration. Based on these clinical features, the two patients exhibited an axonal peripheral neuropathy without CFEOM3. These results therefore suggested that certain TUBB3 mutations may predominantly be associated with axonal peripheral neuropathy. Furthermore, the results also suggested that TUBB3 mutations may be implicated in modulating the inter­ and intra­familial heterogeneity of clinical phenotypes.

HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction.

Overexpression and/or activation of HER2 confers resistance of cancer cells to chemotherapeutic drugs. NRF2 also gives drug resistance of cancer cells through induction of detoxification and/or drug efflux proteins. Although several upstream effectors of NRF2 overlapped with the downstream molecules of HER2 pathway, no direct link between HER2 and NRF2 has ever been established. Here, we identified that co-expression of a constitutively active HER2 (HER2CA) and NRF2 increased the levels of NRF2 target proteins, HO-1 and MRP5. We also identified HER2CA activated the DNA-binding of NRF2 and the antioxidant response element (ARE)-mediated transcription in an NRF2-dependent manner. In addition, NRF2 and HER2CA cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes including GSTA2, GSTP1, CYP3A4, HO-1, MRP1, and MRP5. We also demonstrated that NRF2 binds to HER2 not only in transiently transfected HEK293T cells but also in HER2-amplified breast cancer cells. Functionally, overexpression of HER2CA gave resistance of MCF7 breast cancer cells to either paraquat or doxorubicin. Overexpression of dominant negative NRF2 (DN-NRF2) reduced the HER2CA-induced resistance of MCF7 cells to these agents. Taken together, these results suggest that active HER2 binds and regulates the NRF2-dependent transcriptional activation and induces drug resistance of cancer cells.

Recessive C10orf2 mutations in a family with infantile-onset spinocerebellar ataxia, sensorimotor polyneuropathy, and myopathy.

Recessive mutations in chromosome 10 open reading frame 2 (C10orf2) are relevant in infantile-onset spinocerebellar ataxia (IOSCA). In this study, we investigated the causative mutation in a Korean family with combined phenotypes of IOSCA, sensorimotor polyneuropathy, and myopathy. We investigated recessive mutations in a Korean family with two individuals affected by IOSCA. Causative mutations were investigated using whole exome sequencing. Electrophysiological analyses and muscle and nerve biopsies were performed, along with magnetic resonance imaging (MRI) of the brain and lower extremities. Compound heterozygous mutations c.1460C>T and c.1485-1G>A in C10orf2 were identified as causative of IOSCA. Skeletal muscle showed mitochondrial DNA (mtDNA) deletions. Both patients showed a period of normal development until 12-15 months, followed by ataxia, athetosis, hearing loss, and intellectual disability. Electrophysiological findings indicated motor and sensory polyneuropathies. Muscle biopsy revealed variations in the size and shape of myofibers with scattered, small, and angulated degenerating myofibers containing abnormal mitochondria; these observations are consistent with myopathy and may be the result of mtDNA deletions. Sural nerve biopsy revealed an axonal neuropathy. High-signal-intensity lesions in the middle cerebellar peduncles were correlated with clinical severity, and MRI of the lower legs was compatible with the hypothesis of length-dependent axonal degeneration. We identified novel compound heterozygous mutations of the C10orf2 gene as the cause of IOSCA with sensorimotor polyneuropathy and myopathy. Signs of motor neuropathy and myopathy were discovered for the first time in IOSCA patients with C10orf2 mutations. These results suggest that the clinical spectrum of IOSCA caused by C10orf2 mutations may be more variable than previously reported.

Inhibition of NRF2 by PIK-75 augments sensitivity of pancreatic cancer cells to gemcitabine.

We describe the potential benefit of PIK-75 in combination of gemcitabine to treat pancreatic cancer in a preclinical mouse model. The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses. Additionally, the combinatorial effect of PIK-75 and gemcitabine was evaluated in human pancreatic cancer cell lines and a xenograft model. PIK-75 reduced NRF2 protein levels and activity to regulate its target gene expression through proteasome-mediated degradation of NRF2 in human pancreatic cancer cell lines. PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5. Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo. Our present study provides a strong mechanistic rationale to evaluate NRF2 targeting agents in combination with gemcitabine to treat pancreatic cancers.

Exome Sequencing Reveals a Novel PRPS1 Mutation in a Family with CMTX5 without Optic Atrophy.

BACKGROUND: X-linked Charcot-Marie-Tooth disease type 5 (CMTX5) is caused by mutations in the gene encoding phosphoribosyl pyrophosphate synthetase I (PRPS1). There has been only one case report of CMTX5 patients. The aim of this study was to identify the causative gene in a family with CMTX with peripheral neuropathy and deafness. CASE REPORT: A Korean family with X-linked recessive CMT was enrolled. The age at the onset of hearing loss of the male proband was 5 months, and that of steppage gait was 6 years; he underwent cochlear surgery at the age of 12 years. In contrast to what was reported for the first patients with CMTX5, this patient did not exhibit optic atrophy. Furthermore, there was no cognitive impairment, respiratory dysfunction, or visual disturbance. Assessment of his family history revealed two male relatives with very similar clinical manifestations. Electrophysiological evaluations disclosed sensorineural hearing loss and peripheral neuropathy. Whole-exome sequencing identified a novel p.Ala121Gly (c.362C>G) PRPS1 mutation as the underlying genetic cause of the clinical phenotype. CONCLUSIONS: A novel mutation of PRPS1 was identified in a CMTX5 family in which the proband had a phenotype of peripheral neuropathy with early-onset hearing loss, but no optic atrophy. The findings of this study will expand the clinical spectrum of X-linked recessive CMT and will be useful for the molecular diagnosis of clinically heterogeneous peripheral neuropathies.

Inhibition of checkpoint kinase 2 (CHK2) enhances sensitivity of pancreatic adenocarcinoma cells to gemcitabine.

Checkpoint kinase 2 (CHK2) plays pivotal function as an effector of cell cycle checkpoint arrest following DNA damage. Recently, we found that co-treatment of NSC109555 (a potent and selective CHK2 inhibitor) potentiated the cytotoxic effect of gemcitabine (GEM) in pancreatic cancer MIA PaCa-2 cells. Here, we further examined whether NSC109555 could enhance the antitumour effect of GEM in pancreatic adenocarcinoma cell lines. In this study, the combination treatment of NSC109555 plus GEM demonstrated strong synergistic antitumour effect in four pancreatic cancer cells (MIA PaCa-2, CFPAC-1, Panc-1 and BxPC-3). In addition, the GEM/NSC109555 combination significantly increased the level of intracellular reactive oxygen species (ROS), accompanied by induction of apoptotic cell death. Inhibition of ROS generation by N-acetyl cysteine (NAC) significantly reversed the effect of GEM/NSC109555 in apoptosis and cytotoxicity. Furthermore, genetic knockdown of CHK2 by siRNA enhanced GEM-induced apoptotic cell death. These findings suggest that inhibition of CHK2 would be a beneficial therapeutic approach for pancreatic cancer therapy in clinical treatment.

Mutations in the PLEKHG5 gene is relevant with autosomal recessive intermediate Charcot-Marie-Tooth disease.

BACKGROUND: Mutations in the Pleckstrin homology domain-containing, family G member 5 (PLEKHG5) gene has been reported in a family harboring an autosomal recessive lower motor neuron disease (LMND). However, the PLEKHG5 mutation has not been described to cause Charcot-Marie-Tooth disease (CMT). METHODS: To identify the causative mutation in an autosomal recessive intermediate CMT (RI-CMT) family with childhood onset, whole exome sequencing (WES), histopathology, and lower leg MRIs were performed. Expression and activity of each mutant protein were analyzed. RESULTS: We identified novel compound heterozygous (p.Thr663Met and p.Gly820Arg) mutations in the PLEKHG5 gene in the present family. The patient revealed clinical manifestations of sensory neuropathy. Fatty replacements in the distal lower leg muscles were more severe than in the thigh muscles. Although the symptoms and signs of this patient harboring slow nerve conduction velocities suggested the possibility of demyelinating neuropathy, a distal sural nerve biopsy was compatible with axonal neuropathy. Immunohistochemical analysis revealed that the patient has a low level of PLEKHG5 in the distal sural nerve and an in vitro assay suggested that the mutant proteins have a defect in activating the NF-κB signaling pathway. CONCLUSIONS: This study identifies compound heterozygous PLEKHG5 mutations as the cause of RI-CMT. We suggest that PLEKHG5 might play a role in the peripheral motor and sensory nervous system. This study expands the phenotypic spectrum of PLEKHG5 mutations.

Correlations between BRCA1 defect and environmental factors in the risk of breast cancer.

The risk factors for breast cancer, the most common female malignant cancer, include environmental factors such as radiation, tobacco, a high-fat diet, and xenoestrogens as well as hormones. In addition, BRCA1 and BRCA2 are the most well-known genetic factors that increase risk for breast cancer. Coincidence of those environmental and genetic factors might augment the risk of tumorigenesis of breast. To verify this hypothesis, we briefly evaluated the carcinogenic potency of various environmental factors in the absence or presence of BRCA1 as a genetic factor in a normal mammary epithelial cell line, MCF10A. Many environmental factors tested increased cellular ROS level in the absence of other insult. In addition, TCDD, DMBA, 3MC, and BPA enhanced the BaP-induced ROS production. BRCA1 knockdown (BRCA1-KD) cells by siRNA significantly induced cellular accumulation of ROS compared to control cells. In this setting, the addition of paraquat, TCDD, DMBA, 2OHE2 or 4OHE2 significantly augmented ROS generation in BRCA1-KD MCF10A cells. Measurements of BaP-DNA adduct formation as a marker of DNA damage also revealed that BRCA1 deficiency leads increased DNA damage. In addition, TCDD and DMBA significantly increased BaP-DNA adduct formation in the absence of BRCA1. These results imply that elevated level of ROS is correlated with increase of DNA damage in BRCA1 defective cells. Taken together, our study suggests that several environmental factors might increase the risk of tumorigenesis in BRCA1 defective breast epithelial cells.