Nicaraven attenuates the acquired radioresistance of established tumors in mouse models via PARP inhibition.

Nicaraven has been reported to inhibit the activity of poly (ADP-ribose) polymerase (PARP). In this study, we investigated the probable ability of nicaraven to attenuate cancer radioresistance during fractionated radiotherapy. Tumor models were established in C57BL/6 mice and BALB/c nude mice by subcutaneous injection of Lewis mouse lung carcinoma cancer cells and A549 human lung cancer cells, respectively. When the tumors had grown to approximately 100 mm3, we initiated fractionated radiotherapy. Nicaraven or saline was administered immediately after each irradiation exposure. Compared to saline treatment, nicaraven administration significantly induced gamma-H2AX foci formation and cell apoptosis in tumors at 1 or 3 days after an additional challenge exposure to 10 Gy and inhibited tumor growth during the short-term follow-up period, suggesting increased radiosensitivity of cancer cells. Moreover, the expression of PARP in tumor tissue was decreased by nicaraven administration. Our data suggest that nicaraven likely attenuates the acquired radioresistance of cancers through PARP inhibition.

Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a first-line chemotherapeutic drug for various malignancies that causes cardiotoxicity. Plant-derived exosome-like nanovesicles (P-ELNs) are growing as novel therapeutic agents. Here, we investigated the protective effects in DOX cardiotoxicity of ELNs from Momordica charantia L. (MC-ELNs), a medicinal plant with antioxidant activity. RESULTS: We isolated MC-ELNs using ultracentrifugation and characterized them with canonical mammalian extracellular vesicles features. In vivo studies proved that MC-ELNs ameliorated DOX cardiotoxicity with enhanced cardiac function and myocardial structure. In vitro assays revealed that MC-ELNs promoted cell survival, diminished reactive oxygen species, and protected mitochondrial integrity in DOX-treated H9c2 cells. We found that DOX treatment decreased the protein level of p62 through ubiquitin-dependent degradation pathway in H9c2 and NRVM cells. However, MC-ELNs suppressed DOX-induced p62 ubiquitination degradation, and the recovered p62 bound with Keap1 promoting Nrf2 nuclear translocation and the expressions of downstream gene HO-1. Furthermore, both the knockdown of Nrf2 and the inhibition of p62-Keap1 interaction abrogated the cardioprotective effect of MC-ELNs. CONCLUSIONS: Our findings demonstrated the therapeutic beneficials of MC-ELNs via increasing p62 protein stability, shedding light on preventive approaches for DOX cardiotoxicity.

Negative pressure induces dedifferentiation of hepatocytes via RhoA/ROCK pathway.

Biomechanical forces are known to regulate the biological behaviors of cells. Although negative pressure has been used for wound healing, it is still unknown about its role in regulating cell plasticity. We investigated whether negative pressure could induce the dedifferentiation of hepatocytes. Using a commercial device, we found that the exposure of primary human hepatocytes to -50 mmHg quickly induced the formation of stress fibers and obviously changed cell morphology in 72 h. Moreover, the exposure of hepatocytes to -50 mmHg significantly upregulated RhoA, ROCK1, and ROCK2 in 1-6 h, and dramatically enhanced the expression of marker molecules on "stemness", such as OCT4, SOX2, KLF4, MYC, NANOG, and CD133 in 6-72 h. However, all these changes in hepatocytes induced by -50 mmHg stimulation were almost abrogated by ROCK inhibitor Y27623. Our data suggest that an appropriate force of negative pressure stimulation can effectively induce the dedifferentiation of hepatocytes via RhoA/ROCK pathway activation.

Ex vivo expansion of primary cells from limb tissue of Pleurodeles waltl.

Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin- and gelatin-coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non-coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2 O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good-quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment.

Losartan alleviates renal fibrosis by inhibiting the biomechanical stress-induced epithelial-mesenchymal transition of renal epithelial cells.

Angiotensin receptor blockers (ARBs) have been reported to be beneficial of renal fibrosis, but the molecular and cellular mechanisms are still unclear. In this study, we investigated the effectiveness and relevant mechanism of ARBs in alleviating renal fibrosis, especially by focusing on biomechanical stress-induced epithelial to mesenchymal transition (EMT) of renal epithelial cells. Unilateral ureteral obstruction (UUO) renal fibrosis model was established in mice by ligating the left ureter, and then randomly received losartan at a low dose (1 mg/kg) or a regular dose (3 mg/kg) for 2 weeks. Compared to the control, histological analysis showed that losartan treatment at either a low dose or a regular dose effectively attenuated renal fibrosis in the UUO model. To further understand the mechanism, we ex vivo loaded primary human renal epithelial cells to 50 mmHg hydrostatic pressure. Western blot and immunostaining analyses indicated that the loading to 50 mmHg hydrostatic pressure for 24 h significantly upregulated vimentin, ß-catenin and α-SMA, but downregulated E-cadherin in renal epithelial cells, suggesting the EMT. The addition of 10 or 100 nM losartan in medium effectively attenuated the EMT of renal epithelial cells induced by 50 mmHg hydrostatic pressure loading. Our in vivo and ex vivo experimental data suggest that losartan treatment, even at a low dose can effectively alleviate renal fibrosis in mouse UUO model, at least partly by inhibiting the biomechanical stress-induced EMT of renal epithelial cells. A low dose of ARBs may repurpose for renal fibrosis treatment.

Hyperoxia but not high tidal volume contributes to ventilator-induced lung injury in healthy mice.

BACKGROUND: Mechanical ventilation is a supportive therapy used to maintain respiratory function in several clinical and surgical cases but is always accompanied by lung injury risk due to improper treatment. We investigated how tidal volume and oxygen delivery would contribute independently or synergistically to ventilator-induced lung injury (VILI). METHODS: Under general anesthesia and tracheal intubation, healthy female C57BL/6 N mice (9 weeks old) were randomly ventilated for 2 h by standard (7 ml/kg) or high (14 ml/kg) tidal volume at positive end-expiratory pressure (PEEP) of 2 cmH2O, with room air, 50% O2 (moderate hyperoxia), or 100% O2 (severe hyperoxia); respectively. Mice were sacrificed 4 h after mechanical ventilation, and lung tissues were collected for experimental assessments on lung injury. RESULTS: Compared with the healthy control, severe hyperoxia ventilation by either standard or high tidal volume resulted in significantly higher wet-to-dry lung weight ratio and higher levels of IL-1ß and 8-OHdG in the lungs. However, moderate hyperoxia ventilation, even by high tidal volume did not significantly increase the levels of IL-1ß and 8-OHdG in the lungs. Western blot analysis showed that the expression of RhoA, ROCK1, MLC2, and p-MLC2 was not significantly induced in the ventilated lungs, even by high tidal volume at 2 cmH2O PEEP. CONCLUSION: Severe hyperoxia ventilation causes inflammatory response and oxidative damage in mechanically ventilated lungs, while high tidal volume ventilation at a reasonable PEEP possibly does not cause VILI.

Biphasic effect of mechanical stress on lymphocyte activation.

Mechanical forces can modulate the immune response, mostly described as promoting the activation of immune cells, but the role and mechanism of pathological levels of mechanical stress in lymphocyte activation have not been focused on before. By an ex vivo experimental approach, we observed that mechanical stressing of murine spleen lymphocytes with 50 mmHg for 3 h induced the nuclear localization of NFAT1, increased C-Jun, and increased the expression of early activation marker CD69 in resting CD8+ cells. Interestingly, 50 mmHg mechanical stressing induced the nuclear localization of NFAT1; but conversely decreased C-Jun and inhibited the expression of CD69 in lymphocytes under lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin stimulation. Additionally, we observed similar changes trends when comparing RNA-seq data of hypertensive and normotensive COVID-19 patients. Our results indicate a biphasic effect of mechanical stress on lymphocyte activation, which provides insight into the variety of immune responses in pathologies involving elevated mechanical stress.

Angiotensin receptor blocker alleviates liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.

Angiotensin receptor blockers have been reported to be beneficial to liver fibrosis, but the relevant molecular and cellular mechanisms remain unclear. We herein investigated whether low-dose angiotensin receptor blocker alleviated liver fibrosis through mechanotransduction regulation. Hydrostatic pressure-induced liver fibrosis model was established in mice by ligating partially the inferior vena cava, and then randomly received a very low dose of losartan (0.5 mg/kg) or placebo treatment for 8 weeks. We found that losartan administration interfered the expression of several mechanotransductive molecules, and effectively alleviated liver fibrosis. Using a commercial device, we further confirmed that ex vivo loading of hepatic stellate cells to 50 mmHg hydrostatic pressure for 24 h significantly upregulated RhoA, ROCK, AT1R, and p-MLC2, which was effectively attenuated by adding 10 nM losartan in medium. Our in vivo and ex vivo experimental data suggest that low-dose angiotensin receptor blockers may alleviate hydrostatic pressure-induced liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.NEW & NOTEWORTHY Our ex vivo and in vivo experiments clearly indicated that low-dose losartan alleviated liver fibrosis, likely by modulating the mechanotransduction properties of HSCs. Uncovering the biomechanical signaling pathway of ARB treatment on liver fibrosis will be helpful to develop novel molecular targeting therapy for liver diseases.

Smooth muscle cell fate decisions decipher a high-resolution heterogeneity within atherosclerosis molecular subtypes.

BACKGROUND: Mounting evidence has revealed the dynamic variations in the cellular status and phenotype of the smooth muscle cell (SMC) are vital for shaping the atherosclerotic plaque microenvironment and ultimately mapping onto heterogeneous clinical outcomes in coronary artery disease. Currently, the underlying clinical significance of SMC evolutions remains unexplored in atherosclerosis. METHODS: The dissociated cells from diseased segments within the right coronary artery of four cardiac transplant recipients and 1070 bulk samples with atherosclerosis from six bulk cohorts were retrieved. Following the SMC fate trajectory reconstruction, the MOVICS algorithm integrating the nearest template prediction was used to develop a stable and robust molecular classification. Subsequently, multi-dimensional potential biological implications, molecular features, and cell landscape heterogeneity among distinct clusters were decoded. RESULTS: We proposed an SMC cell fate decision signature (SCFDS)-based atherosclerosis stratification system and identified three SCFDS subtypes (C1-C3) with distinguishing features: (i) C1 (DNA-damage repair type), elevated base excision repair (BER), DNA replication, as well as oxidative phosphorylation status. (ii) C2 (immune-activated type), stronger immune activation, hyper-inflammatory state, the complex as well as varied lesion microenvironment, advanced stage, the most severe degree of coronary stenosis severity. (iii) C3 (stromal-rich type), abundant fibrous content, stronger ECM metabolism, immune-suppressed microenvironment. CONCLUSIONS: This study uncovered atherosclerosis complex cellular heterogeneity and a differentiated hierarchy of cell populations underlying SMC. The novel high-resolution stratification system could improve clinical outcomes and facilitate individualized management.

Ex Vivo Hydrostatic Pressure Loading of Atrial Tissues Activates Profibrotic Transcription via TGF-ß Signal Pathway.

Excessive mechanical stress causes fibrosis-related atrial arrhythmia. Herein, we tried to investigate the mechanism of atrial fibrogenesis in response to mechanical stress by ex vivo approach. We collected atrial tissues from mice and then cultured them as "explants" under atmospheric pressure (AP group) or 50 mmHg hydrostatic pressure loading (HP group) conditions. Pathway-specific PCR array analysis on the expression of fibrosis-related genes indicated that the loading of atrial tissues to 50 mmHg for 24 hours extensively upregulated a series of profibrotic genes. qRT-PCR data also showed that loading atrial tissues to 50 mmHg enhanced Rhoa, Rock2, and Thbs1 expression at different time points. Interestingly, the enhanced expression of Thbs1 at 1 hour declined at 6-24 hours and then increased again at 72 hours. In contrast, an enhanced expression of Tgfb1 was observed at 72 hours. In contrast, daily loading to 50 mmHg for 3 hours significantly accelerated the outgrowth of mesenchymal stem-like stromal cells from atrial tissues; however, we did not observe significant phenotypic changes in these outgrowing cells. Our ex vivo experimental data clearly show the induction of profibrotic transcription of atrial tissues by HP loading, which confirms the common pathological feature of atrial fibrosis following pressure overload.

Prolonged oxygen exposure causes the mobilization and functional damage of stem or progenitor cells and exacerbates cardiac ischemia or reperfusion injury in healthy mice.

Oxygen is often administered to patients and occasionally to healthy individuals as well; however, the cellular toxicity of oxygen, especially following prolonged exposure, is widely known. To evaluate the potential effect of oxygen exposure on circulating stem/progenitor cells and cardiac ischemia/reperfusion (I/R) injury, we exposed healthy adult mice to 100% oxygen for 20 or 60 min. We then examined the c-kit-positive stem/progenitor cells and colony-forming cells and measured the cytokine/chemokine levels in peripheral blood. We also induced cardiac I/R injury in mice at 3 h after 60 min of oxygen exposure and examined the recruitment of inflammatory cells and the fibrotic area in the heart. The proportion of c-kit-positive stem/progenitor cells significantly increased in peripheral blood at 3 and 24 h after oxygen exposure for either 20 or 60 min (p < .01 vs. control). However, the abundance of colony-forming cells in peripheral blood conversely decreased at 3 and 24 h after oxygen exposure for only 60 min (p < .05 vs. control). Oxygen exposure for either 20 or 60 min resulted in significantly decreased plasma vascular endothelial growth factor levels at 3 h, whereas oxygen exposure for only 60 min reduced plasma insulin-like growth factor 1 levels at 24 h (p < .05 vs. control). Protein array indicated the increase in the levels of some cytokines/chemokines, such as CXCL6 (GCP-2) at 24 h after 60 min of oxygen exposure. Moreover, oxygen exposure for 60 min enhanced the recruitment of Ly6g- and CD11c-positive inflammatory cells at 3 days (p < .05 vs. control) and increased the fibrotic area at 14 days in the heart after I/R injury (p < .05 vs. control). Prolonged oxygen exposure induced the mobilization and functional impairment of stem/progenitor cells and likely enhanced inflammatory responses to exacerbate cardiac I/R injury in healthy mice.

Nicaraven inhibits TNFα-induced endothelial activation and inflammation through suppression of NF-κB signaling pathway.

Inflammation-induced activation and dysfunction of endothelial cells play an important role in the pathology of multiple vascular diseases. Nicaraven, a potent hydroxyl radical scavenger, has recently been found to have anti-inflammatory roles; however, the mechanism of its action is not fully understood. Here we investigated the effects of Nicaraven on tumor necrosis factor α (TNFα) - induced inflammatory response in human umbilical vein endothelial cells and we explore the underlying mechanisms related to the nuclear factor-κB (NF-κB) signaling pathway. Our results showed that Nicaraven significantly reduced the reactive oxygen species production after TNFα stimulation. Nicaraven suppressed TNFα-induced mRNA expression of multiple adhesion molecules and pro-inflammatory cytokines, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, MCP-1, TNFα, interleukin-1ß (IL-1ß), IL-6, and IL-8. In addition, Nicaraven inhibited monocyte adhesion and reduced the protein levels of VCAM-1 and ICAM-1. Mechanistically, Nicaraven prevented TNFα-induced activation of NF-κB signaling pathway by suppressing the phosphorylation of NF-κB p65, IκBα, and IκB kinase (IKK)α/ß, stabilizing IκBα, and inhibiting the translocation of p65 from cytosol to nucleus. Finally, we showed that Nicaraven improved the functions of endothelial cells, seen as the upregulation of endothelial nitric oxide synthase and increased nitric oxide levels. Our findings indicated that Nicaraven effectively inhibits TNFα-induced endothelial activation and inflammatory response at least partly through inhibiting NF-κB signaling pathway.

Nicaraven Attenuates Postoperative Systemic Inflammatory Responses-Induced Tumor Metastasis.

BACKGROUND: Inflammation has been demonstrated to promote cancer metastasis. Due to the well-known systemic inflammatory responses (SIR) after major surgery, it is critical to investigate and attenuate SIR-induced tumor metastasis of cancer patients suffering surgical procedures. METHODS: C57BL/6 mice were intravenously injected with Lewis lung cancer cells at 6, 24, and 72 h after the induction of intestinal ischemia/reperfusion (I/R) injury. We found that the number of tumor nodules significantly increased in lungs of mice injected with cancer cells at 6 h but not at 24 and 72 h after I/R injury. The administration of nicaraven 30 min before and 24 h after I/R injury effectively attenuated the enhanced tumor metastasis to lungs. Protein array showed the increase of various cytokines in plasma of mice at 6 h after I/R injury, but many of them were attenuated by the administration of nicaraven. Immunostaining indicated the increase of Ly6g-, CD206-, and CD11c-positive inflammatory cells in the lungs, but it was also attenuated by nicaraven administration. CONCLUSIONS: Postoperative SIR-induced tumor metastasis have been clearly evidenced in our experimental model, and the administration of nicaraven may ameliorate the SIR-induced tumor metastasis by suppressing inflammatory responses.

Bmi-1 high-expressing cells enrich cardiac stem/progenitor cells and respond to heart injury.

Bmi-1 gene is well recognized as an oncogene, but has been recently demonstrated to play a role in the self-renewal of tissue-specific stem cells. By using Bmi-1GFP/+ mice, we investigated the role of Bmi-1 in cardiac stem/progenitor cells and myocardial repair. RT-PCR and flow cytometry analysis indicated that the expression of Bmi-1 was significantly higher in cardiac side population than the main population from CD45- Ter119- CD31- heart cells. More Sca-1+ cardiac stem/progenitor cells were found in Bmi-1 GFPhi subpopulation, and these Bmi-1 GFPhi heart cells showed the potential of differentiation into SMM+ smooth muscle-like cells and TnT+ cardiomyocyte-like cells in vitro. The silencing of Bmi-1 significantly inhibited the proliferation and differentiation of heart cells. Otherwise, myocardial infarction induced a significantly increase (2.7-folds) of Bmi-1 GFPhi population, mainly within the infarction and border zones. These preliminary data suggest that Bmi-1hi heart cells are enriched in cardiac stem/progenitor cells and may play a role in myocardial repair.

Fabrication of Synthetic Mesenchymal Stem Cells for the Treatment of Acute Myocardial Infarction in Mice.

RATIONALE: Stem cell therapy faces several challenges. It is difficult to grow, preserve, and transport stem cells before they are administered to the patient. Synthetic analogs for stem cells represent a new approach to overcome these hurdles and hold the potential to revolutionize regenerative medicine. OBJECTIVE: We aim to fabricate synthetic analogs of stem cells and test their therapeutic potential for treatment of acute myocardial infarction in mice. METHODS AND RESULTS: We packaged secreted factors from human bone marrow-derived mesenchymal stem cells (MSC) into poly(lactic-co-glycolic acid) microparticles and then coated them with MSC membranes. We named these therapeutic particles synthetic MSC (or synMSC). synMSC exhibited a factor release profile and surface antigens similar to those of genuine MSC. synMSC promoted cardiomyocyte functions and displayed cryopreservation and lyophilization stability in vitro and in vivo. In a mouse model of acute myocardial infarction, direct injection of synMSC promoted angiogenesis and mitigated left ventricle remodeling. CONCLUSIONS: We successfully fabricated a synMSC therapeutic particle and demonstrated its regenerative potential in mice with acute myocardial infarction. The synMSC strategy may provide novel insight into tissue engineering for treating multiple diseases.

Immunomodulatory effect of mesenchymal stem cells: Cell origin and cell quality variations.

The immunomodulatory property of mesenchymal stem cells (MSCs) has been previously reported. Still it is unclear if this property can be affected by the cell origin and cell quality. Using primary MSCs expanded from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) of mice, we investigated whether the immunomodulatory property of MSCs varied with cell origin and cell quality (early- vs. late-passaged BM-MSCs). BM-MSCs (p1) and AD-MSCs (p1) had a typical spindle shape, but morphological changes were observed in late-passaged BM-MSCs (p6). A pathway-focused array showed that the expression of chemokine/cytokine genes varied with different cell origins and qualities. By co-culturing with spleen mononuclear cells (MNC) for 3 days, the expression of CD4 was suppressed by all types of MSCs. By contrast, the expression of CD8 was suppressed by BM-MSCs and increased by AD-MSCs. The expression ratio of CD206 to CD86 was at a comparable level after co-culture with AD-MSCs and BM-MSCs, but was lower with late-passaged BM-MSCs. AD-MSCs highly induced the release of IL6, IL-10 and TGF-ß in culture medium. Compared with early-passaged BM-MSCs (p1), late-passaged BM-MSCs (p6) released less TGF-ß. Our data suggests that the immunomodulatory properties of MSCs vary with cell origin and cell quality and that BM-MSCs of good quality are likely the optimal source of immunomodulation.

µ-Crystallin controls muscle function through thyroid hormone action.

µ-Crystallin (Crym), a thyroid hormone-binding protein, is abnormally up-regulated in the muscles of patients with facioscapulohumeral muscular dystrophy, a dominantly inherited progressive myopathy. However, the physiologic function of Crym in skeletal muscle remains to be elucidated. In this study, Crym was preferentially expressed in skeletal muscle throughout the body. Crym-knockout mice exhibited a significant hypertrophy of fast-twitch glycolytic type IIb fibers, causing an increase in grip strength and high intensity running ability in Crym-null mice. Genetic inactivation of Crym or blockade of Crym by siRNA-mediated knockdown up-regulated the gene expression of fast-glycolytic contractile fibers in satellite cell-derived myotubes in vitro These alterations in Crym-inactivated muscle were rescued by inhibition of thyroid hormone, even though Crym is a positive regulator of thyroid hormone action in nonmuscle cells. The results demonstrated that Crym is a crucial regulator of muscle plasticity, controlling metabolic and contractile properties of myofibers, and thus the selective inactivation of Crym may be a potential therapeutic target for muscle-wasting diseases, such as muscular dystrophies and age-related sarcopenia.-Seko, D., Ogawa, S., Li, T.-S., Taimura, A., Ono, Y. µ-Crystallin controls muscle function through thyroid hormone action.

Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.

XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency.

BACKGROUND: Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair mechanism in human cells. The final rejoining step requires DNA ligase IV (LIG4) together with the partner proteins X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor. Patients with mutations in genes encoding LIG4, XRCC4-like factor, or the other NHEJ proteins DNA-dependent protein kinase catalytic subunit and Artemis are DSB repair defective and immunodeficient because of the requirement for NHEJ during V(D)J recombination. OBJECTIVE: We found a patient displaying microcephaly and progressive ataxia but a normal immune response. We sought to determine pathogenic mutations and to describe the molecular pathogenesis of the patient. METHODS: We performed next-generation exome sequencing. We evaluated the DSB repair activities and V(D)J recombination capacity of the patient's cells, as well as performing a standard blood immunologic characterization. RESULTS: We identified causal mutations in the XRCC4 gene. The patient's cells are radiosensitive and display the most severe DSB repair defect we have encountered using patient-derived cell lines. In marked contrast, a V(D)J recombination plasmid assay revealed that the patient's cells did not display the junction abnormalities that are characteristic of other NHEJ-defective cell lines. The mutant protein can interact efficiently with LIG4 and functions normally in in vitro assays and when transiently expressed in vivo. However, the mutation makes the protein unstable, and it undergoes proteasome-mediated degradation. CONCLUSION: Our findings reveal a novel separation of impact phenotype: there is a pronounced DSB repair defect and marked clinical neurological manifestation but no clinical immunodeficiency.

The c-MYC-ABCB5 axis plays a pivotal role in 5-fluorouracil resistance in human colon cancer cells.

c-MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c-MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c-MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5-fluorouracil (5-FU)-based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c-MYC increased the survival rate following 5-FU treatment in human colon cancer cells, and knockdown of endogenous c-MYC decreased it. Furthermore, c-MYC knockdown decreased the expression level of ABCB5, which is involved in 5-FU resistance. Using a chromatin immunoprecipitation assay, we found that c-MYC bound to the ABCB5 promoter region. c-MYC inhibitor (10058-F4) treatment inhibited c-MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5-FU treatment as expected, and the ABCB5 expression level was increased in 5-FU-resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5-FU and 10058-F4 treatment significantly decreased tumorigenicity in nude mice compared with 5-FU or 10058-F4 treatment alone. 10058-F4 treatment decreased the ABCB5 expression level in the presence or absence of 5-FU. In contrast, 5-FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c-MYC confers resistance to 5-FU through regulating ABCB5 expression in human colon cancer cells.