Netrin-1 induces the anti-apoptotic and pro-survival effects of B-ALL cells through the Unc5b-MAPK axis.

BACKGROUND: B-cell acute lymphoblastic leukemia (B-ALL) comprises over 85% of all acute lymphoblastic leukemia (ALL) cases and is the most common childhood malignancy. Although the 5 year overall survival of patients with B-ALL exceeds 90%, patients with relapsed or refractory B-ALL may suffer from poor prognosis and adverse events. The axon guidance factor netrin-1 has been reported to be involved in the tumorigenesis of many types of cancers. However, the impact of netrin-1 on B-ALL remains unknown. METHODS: The expression level of netrin-1 in peripheral blood samples of children with B-ALL and children without neoplasia was measured by enzyme-linked immunosorbent assay (ELISA) kits. Then, CCK-8 cell proliferation assays and flow cytometric analysis were performed to detect the viability and apoptosis of B-ALL cells (Reh and Sup B15) treated with exogenous recombinant netrin-1 at concentrations of 0, 25, 50, and 100 ng/ml. Furthermore, co-immunoprecipitation(co-IP) was performed to detect the receptor of netrin-1. UNC5B expression interference was induced in B-ALL cells with recombinant lentivirus, and then CCK-8 assays, flow cytometry assays and western blotting assays were performed to verify that netrin-1 might act on B-ALL cells via the receptor Unc5b. Finally, western blotting and kinase inhibitor treatment were applied to detect the downstream signaling pathway. RESULTS: Netrin-1 expression was increased in B-ALL, and netrin-1 expression was upregulated in patients with high- and intermediate-risk stratification group of patients. Then, we found that netrin-1 induced an anti-apoptotic effect in B-ALL cells, implying that netrin-1 plays an oncogenic role in B-ALL. co-IP results showed that netrin-1 interacted with the receptor Unc5b in B-ALL cells. Interference with UNC5B was performed in B-ALL cells and abolished the antiapoptotic effects of netrin-1. Further western blotting was applied to detect the phosphorylation levels of key molecules in common signaling transduction pathways in B-ALL cells treated with recombinant netrin-1, and the FAK-MAPK signaling pathway was found to be activated. The anti-apoptotic effect of netrin-1 and FAK-MAPK phosphorylation was abrogated by UNC5B interference. FAK inhibitor treatment and ERK inhibitor treatment were applied and verified that the FAK-MAPK pathway may be downstream of Unc5b. CONCLUSION: Taken together, our findings suggested that netrin-1 induced the anti-apoptotic effect of B-ALL cells through activation of the FAK-MAPK signaling pathway by binding to the receptor Unc5b. Video Abstract.

Kinetic modeling of Chinese hamster ovary cell culture: factors and principles.

As a host for therapeutic protein expression, Chinese hamster ovary (CHO) cells are widely utilized in the mainstream biopharmaceutical industry. Cell culture process development plays an important role in transitioning laboratory research to manufacturing. Among different mathematic tools, kinetic modeling is commonly achieved through analyzing cell culture data to design process parameters, optimize media, and scale up bioreactors. In this review, we examine key factors for upstream process development, and summarize currently used kinetic modeling strategies. In addition, two original examples of kinetic modeling application optimizing cell culture performance are presented. A comprehensive understanding is provided for the kinetic modeling and its applications in cell culture process development.

[Expression of ß-integrin family members in children with T-cell acute lymphoblastic leukemia].

OBJECTIVE: To study the expression of ß-integrin family members in children with T-cell acute lymphoblastic leukemia (T-ALL) and their significance. METHODS: Quantitative real-time PCR analyses were performed to assess the expression levels of ß-integrin family members in bone marrow samples from 22 children with newly-diagnosed T-ALL and 21 controls (16 children with non-malignant hematologic disease and 5 healthy donors with bone marrow transplantation). Jurkat cells were treated with integrin inhibitor arginine-glycine-aspartate (Arg-Gly-Asp, RGD) peptide. The cell viability and apoptosis rate were determined by CCK8 assay and flow cytometry respectively. RESULTS: The mRNA levels of integrins ß2, ß3, and ß5 were significantly lower in children with T-ALL than in controls (P<0.05). In T-ALL patients, high integrin ß3 expression was associated with lower white blood cell counts (<100×109/L), minimal residual disease (MRD) positivity, and day 33 bone marrow negative remission (P<0.05). In T-ALL patients, higher integrin ß5 expression was associated with relapse of T-ALL (P<0.05). Based on survival curve analysis, higher integrin ß3 expression was related to lower event-free survival and overall survival rates. RGD peptide treatment inhibited the proliferation of Jurkat cells and increased their apoptosis rate (P<0.05). CONCLUSIONS: ß-Integrin may play a role in the occurrence and development of T-ALL by affecting cell proliferation and apoptosis. The expression of integrin ß5 is closely related to the risk of relapse of T-ALL. The expression of integrin ß3 is closely related the treatment response and prognosis of T-ALL.

Netrin-1 inducing antiapoptotic effect of acute myeloid leukemia cells in a concentration-dependent manner through the Unc-5 netrin receptor B-focal adhesion kinase axis.

Acute myeloid leukemia (AML) is a hematological malignancy that commonly occurs in children. The prognosis of pediatric AML is relatively poor, thus threatening the patient's survival. The aberrant expression of the axon guidance factor, netrin-1, is observed in various types of malignancies, and it participates in the proliferation and apoptosis of tumor cells. Herein, we aimed to explore the role of netrin-1 in AML cells. Netrin-1 is highly expressed in AML patients. Proliferation and anti-apoptosis were observed in AML cells treated with netrin-1. The interaction between netrin-1 and Unc-5 netrin receptor B (UNC5B) was detected through coimmunoprecipitation, and UNC5B ribonucleic acid interference restrained the influence of netrin-1 on the AML cells. The phosphorylation of focal adhesion kinase-protein kinase B (FAK-Akt) was upregulated in AML cells treated with netrin-1. Both FAK and Akt inhibitors abrogated the effects of netrin-1 on the proliferation and apoptosis of AML cells. In conclusion, netrin-1 could promote the growth and reduce the apoptosis of AML cells in a concentration-dependent manner, and that these effects were mediated by activating the FAK-Akt signaling pathway via the UNC5B.

Inhibition of Integrin αvß3-FAK-MAPK signaling constrains the invasion of T-ALL cells.

The role of adhesion receptor integrin αvß3 in T-ALL was unclear. Firstly, we performed quantitative real-time PCR to assess medullary expression of integrin ß3(ITGB3) in T-ALL patients and high ITGB3 expression was relevant with the central nervous system leukemia(CNSL) incidence. Decreasing of cell invasion was observed in Jurkat and Molt4 treated with integrin αvß3 specific antibody and inhibitor as well as cells with ITGB3 interference. Further, phosphorylation of FAK, cRAF, MEK and ERK decreased in cells with integrin αvß3 inhibition or interference. Invasion decreased in T-ALL cells treated with FAK and ERK inhibitors. In conclusion, inhibition of integrin αvß3 signals significantly limits the cell invasion of T-ALL cells.

[Effect of Netrin-1 on VEGFA Expression in T-ALL Cells and Its Related Mechanism].

AbstractObjective: To investigate the effect of the axon guidance factor Netrin-1 on the expression of VEGFA in T cell acute lymphoblastic leukemia(T-ALL) and its related mechanism. METHODS: ELISA assays were applied to detect the levels of Netrin-1 and VEGFA in the bone marrow (BM) samples from children in the T-ALL and control group. The level of Netrin-1 and VEGFA were compared between control children and patients, and the liner correlation between Netrin-1 and VEGFA was analyzed. The T-ALL cells Jurkat and Molt-4 were culture in vitro, and the cells were treated with different concentration of Netrin-1 (0, 25, 50, 100 ng/ml) for 24 h, quantitative RT-PCR (qRT-PCR) and Western blot were used to detect the VEGFA expression in Jurkat, Molt-4 cells. The expression of Netrin-1 receptors in T-ALL cells was detected by qRT-PCR and the interaction between Netrin-1 and receptor in each cells was detected by co-IP. Furthermore, Western blot was used to detect the phosphorylation level of key prateins of AKT signal transduction pathway including Akt and mTOR in T-ALL cells treated with Netrin-1 (100 ng/ml). The expression of VEGFA and phosphorylation of AKT pathway transducers were detected by Western blot, after T-ALL cells treated with Netrin-1 (100 ng/ml) combined with inhibitors specific to Akt or mTOR. RESULTS: The expression level of Netrin-1 and VEGFA in T-ALL patients BM samples were both signi-ficantly higher than that of control group. And the expression level of Netrin-1 was positively correlated with that of VEGFA（r2=0974). With the increase of Netrin-1 concentration, the expression level of VEGFA also increased(P<0.05）. Netrin-1 interacted with its receptor, integrin-ß4 at the Netrin-1 concentration of 100 ng/ml. Further, the treatment of Netrin-1 could increase the phosphorylation of Akt and mTOR, which were the key transducers of AKT pathway. After treatment of T-ALL cells with Netrin-1 (100 ng/mL) and Akt inhibitor, the expression of VEGFA and phosphorylation of Akt or mTOR decreased. When the cells were treated with Netrin-1(100 ng/ml) and mTOR inbititor, the phosphorylation level of mTOR and the expression of VEGFA decreased, the phosphorylation level of Akt increased. CONCLUSION: The expression of Netrin-1 and VEGFA in bone marrow of childred with T-ALL were abnormal, and there was a linear relationship between them. Netrin-1 can interact with its receptor, integrin-ß4 and activate AKT transduction pathway to elevate the expression of VEGFA in T-ALL cells.

[Expression of Netrin-1 in Patients with Acute Lymphoblastic Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the correlation of the Netrin-1 expression level with the clinical characteristics in children with acute lymphoblastic leukemia (ALL) and to explore its possible regulatory mechanism. METHODS: ELISA was used to detect the expression level of Netrin-1 in peripheral blood serum from 48 child ALL patients (newly diagnosed, recurrent), and its relevance with clinical indicators was statistically analyzed. The blood serum samples from 27 children with non malignant hematological diseases were choosen as controls. Leukemia cell lines of Jurkat,Molt-4,SUP-B15 and Raji were cultivated in vitro, after treated with different concentrations of recombinant human Netrin-1 protein， the invasive ability of the cells was detected by Transwell method； the effect of Netrin-1 to the proli feration of cells was detected by CCK-8 method； The expression and phosphorylation level of key molecules, such as FAK,Erk1/2,PI3K and Akt signaling pathway were detected by Western blot. RESULTS: The expression of Netrin-1 in child patients was significantly higher than that of the control group (P<0.05). With the increasing of Netrin-1 level, the level of Plt (r=0.483, P<0.05) increased, while the level of WBC (r=-0.290, P<0.05) decreased, and there were no significant correlation with age, Hb level and the proportion of immature cells in bone marrow. When the concentration of Netrin-1 was 25-50 ng/ml, the level of Netrin-1 positively correlated with WBC (r=0.886, P<0.05) ; the level of Netrin-1 significantly decreased when the patient's WBC was ＞50×109/L and Plt ＞20×109/L(P=0.042，P=0.001); The expression level of Netrin-1 was significantly different in the risk group(P=0.017), and level of Netrin-1 in high-risk group was significantly higher than that in low risk group and middle risk group, but there was no significant difference of Netrin-1 expression in sex, hepatosplenomegaly, MRD, recurrence and chromosome abnormality. Netrin-1 could promote the invasiveness of the four kinds of cells (P<0.05). With the increase of Netrin-1 concentration, the number of cells increased at first and then decreased, and the number of cells in the invading chamber was the highest when the concentration of Netrin-1 was 100 ng/ml; the survival rate of the four kinds of cells significantly increased when the concentration of Netrin-1 was 25 ng/ml(P<0.05), and SUP-B15 cells showed the highest cell survival rate at a concentration of 100 ng/ml; The survival rate of the four kinds of cells showed a tendency : survival of cells increased at low concentration of Netrin-1 and survival of cells decreased at high concentration of Netrin-1. The results of Western blot showed that Netrin-1 activated the phosphorylation level of key molecules such as FAK,Erk1/2,PI3K,Akt signaling pathway (P<0.05). CONCLUSION: There is abnormal expression of Netrin-1 in serum of children with ALL. Netrin-1 may affect the occurrence and development of ALL by increasing the proliferation and invasiveness of leukemia cells, and may become a risk factor of ALL or a potential target in biotherapy.

[E-cadherin Expression in Children with Acute Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the correlation of E-cadherin expression level with the clinical characterastics in children with acute leukemia (AL), and to explore the possible regulatory mechanism. METHODS: Real-time quantitative RT-PCR was applied to detect the expression level of E-cadherin in bone marrow samples from 135 child patients diagnosed as AL, and its relevance with clinical indicators was statistically analyzed. The expression levels of E-cadherin, ß-catenin, and Akt/p-Akt were detected by using Western blot. The bone marrow samples from 22 children with non-malignant hematological diseases were used as controls. RESULTS: The expression level of E-cadherin significantly decreased in newly diagnosed patients with all 3 types of AL as compared with bone marrow samples from control group (P<0.01). In B-ALL group, compared with standard risk group, E-cadherin expression level significantly decreased in intermediate risk group (P<0.05). Moreover,the expression level of E-cadherin mRNA was also reduced in splenomegaly group (P<0.01). However, the correlation of E-cadherin level with clinical characteristics was not found in T-ALL and AML (P>0.05). The expression level of E-cadherin in the patients from Common-B-ALL group was higher than B-ALL patients with other immunophenotypes (P<0.01), while no significant difference was found among patients grouped by FAB classification. By the correlation analysis of measured data, lower E-cadherin expression level was found to be related with high WBC count and serum lactic dehydrogenase level (LDH) (r=-0.419, r=-0.269), but with low blood platelet count in B-ALL (r=0.335). In T-ALL, expression of E-cadherin was found to be negatively correlated with LDH and percentage of immature cells in the bone marrow (r=-0.567, r=-0.557). In addition, the lower expression of E-cadherin was also found to be related with WBC count and percentage of immature cells in the bone marrow in newly diagnosed AML patients (r=-0.368, r=-0.391). Compared with control group, the expression of E-cadherin was down-regulated significantly (P<0.01), while ß-catenin, Akt significantly was up-regulated in 3 types of AL patients (P<0.01). The expression of p-Akt and p-Akt/Akt was up-regulated significantly in T-ALL (P<0.01). CONCLUSION: Lower expression of E-cadherin is related factor of unfavourable prognosis in children with acute leukemia. The expression deficiency or down-regulation of E-cadherin may activate Wnt/ß-catenin and PI3K/ Akt signaling pathways to promote the genesis and progress of haematological malignancies, thus resulting in a series of malignant biological behaviors in cells. E-cadherin may be a new prognostic indicator for pediatric acute leukemia, thus to guide individualized hemotherapy.

HA117 endows HL60 cells with a stem-like signature by inhibiting the degradation of DNMT1 via its ability to down-regulate expression of the GGL domain of RGS6.

All-trans retinoic acid (ATRA) induces complete remission in almost all patients with acute promyelocytic leukemia (APL) via its ability to induce the in vivo differentiation of APL blasts. However, prolonged ATRA treatment can result in drug resistance. In previous studies, we generated a multi-drug-resistant HL60/ATRA cell line and found it to contain a new drug resistance-related gene segment, HA117. In this study, we demonstrate that ATRA induces multi-drug-resistant subpopulations of HL60 cells with a putative stem-like signature by up-regulating the expression of the new gene segment HA117. Western blot analysis and quantitative real-time PCR demonstrated that HA117 causes alternative splicing of regulator of G-protein signaling 6 (RGS6) and down-regulation of the expression of the GGL domain of RGS6, which plays an important role in DNA methyltransferase 1 (DNMT1) degradation. Moreover, DNMT1 expression was increased in multi-drug resistance HL60/ATRA cells. Knockdown of HA117 restored expression of the GGL domain and blocked DNMT1 expression. Moreover, resistant cells displayed a putative stem-like signature with increased expression of cancer steam cell markers CD133 and CD123. The stem cell marker, Nanog, was significantly up-regulated. In conclusion, our study shows that HA117 potentially promotes the stem-like signature of the HL60/ATRA cell line by inhibiting by the ubiquitination and degradation of DNMT1 and by down-regulating the expression of the GGL domain of RGS6. These results throw light on the cellular events associated with the ATRA-induced multi-drug resistance phenotype in acute leukemia.

[PRPS1 Expression in Children with Acute Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the correlation between the expression level of PRPS1 and the clinical characteristics in children with acute leukemia(AL). METHODS: Real-time quantitative RT-PCR and Western blot were used to detect the level of PRPS1 mRNA and protein expression in bone marrow samples from 176 patients diagnosed as AL (126 cases were newly diagnosed and 50 cases in complete remission), and its relevance with clinical indicators was statistically analyzed. The bone marrow samples from 21 children with non-malignant hematological disease were used as controls. RESULTS: (1)In B-ALL group, the level of PRPS1 mRNA in newly diagnosed patients were significantly higher than that in control and than that in complete remission patients (both P<0.0001). In T-ALL and AML group, differences was only observed between newly diagnosed patients and complete remission patients(both P<0.0001); (2)In B-ALL group, the expression level of PRPS1 increase with along risk enhancement (P<0.01), while no significant difference was observed in T-ALL (P>0.05). In AML patients, expression difference was shown between low risk group and high risk group(P<0.05); (3)High PRPS1 mRNA expression level were associated with high WBC counts and MRD positive in B-ALL patients (P=0.020, P=0.026, respectively); (4)Expression of NT5C2, an essential gene for relapse and drug resistance, was found to be positively correlated with PRPS1 expression in AL samples(P<0.05). CONCLUSION: High expression of PRPS1 is relevant factor of unfavourable prognosis in B-ALL children, which suggest PRPS1 may be a new indicator for prognosis of pediatric B-ALL and an index to guide individualized chemotherapy.

miRNA Profiling Reveals Dysregulation of RET and RET-Regulating Pathways in Hirschsprung's Disease.

Hirschsprung's disease (HSCR), the most common congenital malformation of the gut, is regulated by multiple signal transduction pathways. Several components of these pathways are important targets for microRNAs (miRNAs). Multiple miRNAs have been associated with the pathophysiology of HSCR, and serum miRNAs profiles of HSCR patients have been reported, but miRNA expression in HSCR colon tissue is almost completely unexplored. Using microarray technology, we screened colon tissue to detect miRNAs whose expression profiles were altered in HSCR and identify targets of differentially expressed miRNAs. Following filtering of low-intensity signals, data normalization, and volcano plot filtering, we identified 168 differentially expressed miRNAs (104 up-regulated and 64 down-regulated). Fifty of these mRNAs represent major targets of dysegulated miRNAs and may thus important roles in the pathophysiology of HSCR. Pathway analysis revealed that 7 of the miRNA targets encode proteins involved in regulation of cell proliferation and migration via RET and related signaling pathways (MAPK and PI3K/AKT). Our results identify miRNAs that play key roles in the pathophysiology of the complex multi-factorial disease HSCR.

[NT5C2 expression in children with acute leukemia and its clinical significance].

OBJECTIVE: To investigate the expression level and analyze the clinical significance of NT5C2, which is an nucleoside analogues metabolism related gene, in children with acute leukemia (AL). METHODS: Real-time PCR and immunohistochemistry were presented to detect the level of NT5C2 mRNA and its protein product cN- Ⅱ in bone marrow samples of 63 patients initially diagnosed with AL, 15 patients who achieved complete remission, 7 patients who relapsed and 16 non- hematologic malignancie controls. The expression of NT5C2 mRNA in different groups of AL and its relevance with clinical indicators were analyzed. RESULTS: ①The expression of NT5C2 mRNA in newly diagnosed B-ALL, TALL, AML and controls were 1.16 (0.89-2.25, 0.96 (0.74-1.25, 1.66 (0.84-3.15) and 0.88 (0.61-1.21), respectively. NT5C2 mRNA expression in AML (P<0.01) and B-ALL (P<0.05) cases were higher than that in controls; NT5C2 mRNA expression in T- ALL and in controls showed no significant difference (P>0.05). Changes of NT5C2 mRNA level were observed between preliminary diagnosis and complete remission in 15 patients. NT5C2 mRNA levels were significantly decreased in complete remission stage than that in newly diagnosis AL (P<0.01). NT5C2 mRNA levels of relapsed-refractory group were higher than that of complete remission group and controls (P<0.01). ② Immunohistochemical staining results revealed that NT5C2 protein levels were consistent with the trend of mRNA levels. ③NT5C2 mRNA levels in AML (r=0.434) and T-ALL (r=0.389) were positively correlated with risk classification (P<0.05). ④ During chemotherapy of patients with AML, the NR rate of bone marrow in NT5C2 high expression group was higher than that of low expression group after 9 days induction chemotherapy (35.2% vs 0) and before consolidation therapy (25.0% vs 0); The positive rate of minimal-residual disease (36.4% vs 14.3%) and relapse rate of AL (38.5% vs 28.6%) were increased in NT5C2 high expressed patients than that in low expressed patients, but all the differences were insignificant (P>0.05). CONCLUSION: High expression of NT5C2 was found to be a related risk factor of AL children with unfavourable prognosis. NT5C2 promises a new target for guiding individualized chemotherapy and evaluating the prognosis of childhood acute leukemia and monitoring recurrence.

[Establishment of HLH-like mouse model with CPG-ODN and IFN-γ].

OBJECTIVE: To establish a hemophagocytic lymphohistiocytosis (HLH)-like mouse model induced by CpG oligodeoxynucleotide (CpG-ODN1826) and interferon (IFN)-γ for further study on therapy. METHODS: Wild type adult C57BL/6 mice were administered with PBS or CpG-ODN1826 (50 µg) by intraperitoneal injection every two day and IFN-γ subcutaneous injection every day. Parameters of HLH were evaluated on day 10. RESULTS: As compared to control, HLH-like symptoms in CpG group were characterized with pancytopenia accompanied by increased ratios of monocytes, alanine aminotransferase [(198.7±54.2)IU/L], triglyceride level [(12.1±0.6)g/L], and serum ferritin [(708.4±11.8)pmol/L]; decreased albumin [(217.7±4.3)g/L], fibrinogen [(17.1±1.9)g/L] (all P<0.05). Hepatosplenomegaly was obvious in CpG group. The liver in CpG group had multifocal hepatocytes necrosis and perivascular inflammations. Spleen had expanding red pulp and hyperplastic nucleated cells. Furthermore, macrophages in the liver and spleen were largely activated. Hemophagocytosis were observed in liver, spleen and bone marrow smear. The CpG group was alive during experiment, other than significant decreased activity after the first injection of CpG-ODN. CONCLUSION: These data demonstrate that repeated administration of CpG-ODN1826 and IFN-γ could induce HLH-like symptoms without fatal condition in wild type C57B/L mice. This protocol could establish a mild HLH-like mouse model, which could be useful for further study on HLH.

Structural analysis uncovers lipid-binding properties of Notch ligands.

The Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system.