DEPTOR expression negatively correlates with mTORC1 activity and tumor progression in colorectal cancer.

The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers, including colorectal cancer (CRC). DEPTOR is an mTOR inhibitor whose expression is negatively regulated by mTOR. However, the role of DEPTOR in the development of CRC is not known. The aim of this study was to investigate the expression of DEPTOR and mTORC1 activity (P-S6) in a subset of CRC patients and determine their relation to tumor differentiation, invasion, nodal metastasis and disease-free survival. Here, Immunohistochemical expression of P-S6 (S235/236) and DEPTOR were evaluated in 1.5 mm tumor cores from 90 CRC patients and in 90 samples of adjacent normal mucosa by tissue microarray. The expression of P-S6 (S235/236) was upregulated in CRC, with the positive rate of P-S6 (S235/236) in CRC (63.3%) significantly higher than that in control tissues (36.7%, 30%) (p<0.05). P-S6 (S235/236) also correlated with high tumor histologic grade (p=0.002), and positive nodal metastasis (p=0.002). In contrast, the expression level of DEPTOR was correlated with low tumor histological grade (p=0.006), and negative nodal metastasis (p=0.001). Interestingly, P-S6 (S235/236) expression showed a significant negative association with the expression of DEPTOR in CRC (p=0.011, R= -0.279). However, upregulation of P-S6 (S235/236) (p=0.693) and downregulation of DEPTOR (p=0.331) in CRC were not significantly associated with overall survival. Thus, we conclude that expression of DEPTOR negatively correlates with mTORC1 activity and tumor progression in CRC. DEPTOR is a potential marker for prognostic evaluation and a target for the treatment of CRC.

[A clinical study of drug-related toxicities of CCLG-ALL 08 protocol for childhood acute lymphoblastic leukemia].

OBJECTIVE: The Chinese Children's Leukemia Group (CCLG)-acute lymphoblastic leukemia (ALL) 08 protocol for childhood ALL was established in 2008. This study aims to evaluate the drug-related toxicities of CCLG-ALL 08 protocol in the treatment of childhood ALL. METHODS: A total of 114 children with newly diagnosed ALL were treated with the CCLG-ALL 08 protocol. The protocol was divided into five phases: remission induction (VDLD), early reinforcement (CAM), consolidation therapy, delayed reinforcement (DIa & DIb) and maintenance treatment. Drug-related toxicities in each phase were evaluated according to the Common Terminology Criteria for Adverse Events version 4.0. RESULTS: Toxicities were more frequent in phase VDLD than other treatment phases, including hepatotoxicity (87.7%), dental ulcer (20.2%), hyperglycemia (20.2%), prolonged activated partial thromboplastin time (21.1%) and decreased fibrinogen (34.2%), with the incidence rates of severe adverse events at 7%, 0, 1.3%, 0.8% and 2.7% respectively. The incidence of allergic reaction to L-ASP was significantly higher in phase DIa than in phase VDLD (28.0% vs 7.9%; P<0.01), and there were no longer any allergic reactions in 15 patients who received continuing treatment with pegaspargase instead. There was no severe arrhythmia, myocardial ischemia, decreased left ventricular function, osteonecrosis, myopathy, organ failure or treatment-related mortality. CONCLUSIONS: The drug-related toxicities of CCLG-ALL 08 protocol are common in phase VDLD, but they are mild and reversible. There is no treatment-related mortality. The CCLG-ALL 08 protocol for childhood ALL is safe.

[Clinical features and outcome analysis of 83 childhood Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with HLH-2004 protocol].

OBJECTIVE: To investigate the clinical features of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH), to analysis the outcome of HLH-2004 protocol, and to explore the prognostic factors in EBV-HLH patients. METHODS: The clinical features at onset and outcome of HLH-2004 protocol from 83 pediatric patients with EBV-HLH enrolled from January 2006 to December 2009 in our hospital were analyzed retrospectively. Univariate and multivariate COX regression analysis were used to identify statistically significant prognostic factors. RESULTS: (1) Among the 83 patients, 45 were males and 38 were females. The age of onset ranged from 6 months to 14 years 4 months. 44 patients were treated with HLH-2004, and 3-year overall survival (OS) was (55.8 ± 7.9)%. (2) The most common clinical features of EBV-HLH included high fever, cytopenia, hepatosplenomegaly, and coagulopathy; The respiratory symptoms, angina phlogistic, skin rashes, neurologic abnormality were rare. 97.3% of patients showed an elevation of serum ferritin, liver dysfunction and lipid metabolism disorders was found in most of EBV-HLH patients. 89.0% of patient had hemophagocytosis in bone marrow at diagnosis of EBV-HLH. (3) COX regression analysis revealed that anemia degree, serum albumin < 30 g/L, CD4:CD8 abnormity, NK cell < 3%, treatment protocol were related with the prognosis significantly (P < 0.05). CONCLUSION: EBV-HLH in pediatric patients has severe clinical feature and poor prognosis. HLH-2004 protocol is an effective treatment for patients with EBV-HLH. Symptomatic treatment can't rescue the patients of EBV-HLH.

[Analysis of thirteen cases with secondary coagulation disorder caused by raticide exposure].

OBJECTIVE: To summarize the clinical characteristics of secondary coagulation disorders caused by exposure to poison (raticide) in children and to investigate the diagnosis and corresponding treatment. METHOD: The process of diagnosis, clinical characteristics, response to treatment and the prognosis were analyzed. RESULTS: The main clinical manifestation was mucosal bleeding (66.6%), including epistaxis, gingival bleeding, hematomas and so on. All these children were previously well and had no history of bleeding. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged, factor II was undetectable and the levels of factors VII, IX, and X were lower. The fibrinogen was normal. A raticide was detected in blood and urine of 13 children although 12 of the patients had no definite history of raticide ingestion. Prothrombin complex, fresh frozen plasma and vitamin K(1) were effective in these cases. However, 2 - 3 weeks later, 6 patients presented with recurrent bleeding. CONCLUSION: For children with secondary coagulation disorders of unknown cause, intoxication of raticide should be considered. The administration of blood coagulation factors and vitamin K(1) are effective in early treatment, and the treatment period should be more than 2 months. The PT and APTT should be followed up. Vitamin K(1) should be stopped when PT and APTT are normal.

[Successful treatment of a patient with Wiskott-Aldrich syndrome using hematopoietic stem cell transplantation--case report and literature review].

OBJECTIVE: Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency diseases. The patients with classical WAS have poor prognosis. The hematopoietic stem cell transplantation is the most effective method to cure WAS at present. In this report, a patient with WAS was cured with HLA identical sibling bone marrow transplantation (BMT). METHODS: Wiskott-Aldrich syndrome protein (WASP) was detected using flow cytometry and WASP were analyzed for the diagnosis. The bone marrow was collected from the elder sister who was the HLA identical sibling donor. A total of 4.38x10(8)/kg mononuclear cell (MNC) and 3.78x10(6)/kg CD34+ cells were collected and transfused into the patient after the conditioning regimen with busulfan/cyclophosphamide. Cyclosporine only was used for graft-versus-host disease prophylaxis. WASP and short tandem repeats (STR) were detected as the evidence of engraftment. RESULTS: The diagnosis was WAS: WASP (-IVS9+2T>C, WASP-negative). The patient received busulfan/cyclophosphamide 9 days before the transplantation. WBC decreased to 0.1x10(9)/L in d+4; The absolute number of neutrophils (ANC) was 0.8x10(9)/L in d+13, and exceeded 1.0x10(9)/L later on. From d(-9)-d+14 the patient was dependent on platelet transfusion. From d+15 the patient's PLT>50x10(9)/L and returned to normal after d+30. In d+9-d+10 mild GVHD (I degree) occurred but subsided after the steroid treatment. From d+50, WASP was detected positive and STR showed full donor DNA chimera. Follow-up for 510 d post-transplant, the patient suffered only from mild cold twice, no eczema, no bleeding occurred. The PLT is normal and no chronic GVHD occurred. The levels of IgG, IgM and IgA of the patient were approximately normal. CONCLUSION: The HLA-identical sibling's BMT seems to be the periorit treatment of choice for the WAS patient.

[Clinical study on childhood acute lymphoblastic leukemia diagnosed and treated with 04 Protocol in Chongqing, China].

OBJECTIVE: To analyze the clinical and laboratory data from acute lymphoblastic leukemia (ALL) patients and the results of treatment using 04 Protocol (suggested by the Pediatric Hematology Group of Chinese Medical Association in 2004). METHODS: This study included 88 children with ALL below the age of 18 years during the period from October 1, 2004 to June 30, 2007. Minimal inhibitory concentration (MIC) and clinical risk classification were done and the new chemotherapy regimen was used according to the protocol. Patients were stratified into low-risk (LR), medium-risk (MR), and high-risk (HR) groups. Life table method was used to estimate survival rate and statistical analysis was done by using software SPSS for Windows. RESULTS: From October 2004 to June 2007, 88 childhood ALL patients were treated with the 04 Protocol. Sixty-three (91.30%) patients attained complete remission (CR) and 17 patients lost to follow up. The overall 4-year-event-free survival (EFS) rate (+/- SE) was (59.73 +/- 7.22)%. EFS was (75.60 +/- 9.71)% in the LR (n = 30), (65.50 +/- 11.69)% in the MR (n = 20) and (44.03 +/- 12.36)% in the HR. Relapse occurred in 18.18% of patients. Seven (7.95%) of 88 patients with ALL died during he induction therapy. Infection was the most common cause of death. CONCLUSION: The outcome of patients treated with the 04 Protocol was favorable. Clinical risk classification and the leukemia cells of D19 are independent predictors of prognosis of ALL. High dose methotrexate played an important role in prevention and treatment of central nervous system leukemia. The mortality rate of this chemotherapeutic protocol during induction therapy was high.

[Hematopoiesis support of mesenchymal stem cells in children with aplastic anemia].

OBJECTIVE: The abnormality of hemopoietic inductive microenvironment (HIM) is involved in the pathophysiology of aplastic anemia (AA). Mesenchymal stem cells (MSC) are main source of bone marrow stromal cells which constitute the bone marrow HIM. Thus, the bone marrow failure in AA may be related to the function of MSC. The aim of the study was to investigate the hematopoiesis support function of MSC in children with AA in vitro. METHODS: Bone marrow samples were collected from 24 children with AA at diagnosis and 19 children with idiopathic thrombocytopenic purpura (ITP), infectious mononucleosis or lymphadenitis (controls). MSCs from bone marrow samples were isolated, cultured and expanded. Morphology, proliferation activity and colony forming unit-fibroblast (CFU-F) were measured. The ability of bone marrow MSC to adhere hemopoietic cells was assayed by MTT. The concentration of stem cell factor (SCF) released from MSC was tested using ELISA. Mononuclear cells (MNC) of bone marrow were plated onto a feeder layer formed by MSC. Cells count and BFU-E, CFU-GM, CFU-GMME productions were measured. RESULTS: The first and third passage time of MSC in children with AA was longer than that in the controls. The number of CFU-F in children with AA (15.70+/-5.78) was less than that in the controls (21.73+/-5.74) (P<0.05). The concentration of SCF in MSC supernatants in children with AA (30.69+/-16.82 pg/mL) was significantly lower than the controls (50.74+/-14.83 pg/mL) (P<0.01). The total MNC count and the number of BFU-E, CFU-GM and CFU-GMME colonies in the support of MSC in children with AA were significantly lower than those in the controls (P<0.01). CONCLUSIONS: The hematopoiesis support function of MSC was significantly reduced in children with AA in vitro. The decreased hematopoiesis support function of MSC may be related its decreased proliferation capacity and SCF release activity.

[Vitamin E inhibits homocysteine-mediated smooth muscle cell proliferation].

OBJECTIVE: To investigate the role of reactive oxygen species (ROS) and the effect of vitamin E on proliferation of vascular smooth muscle cells (VSMCs) induced by homocysteine. METHODS: DNA synthesis in the VSMCs cells was measured using [3H]-thymidine incorporation assay, and the cell number determined by trypan blue method. The level of ROS in the cells was determined using DCF-DA as the fluorescence probe. RESULTS: Homocysteine promoted VSMC DNA synthesis, proliferation, and ROS production. Cysteine resulted in increased ROS production in VSMCs, but had no significant effect on DNA synthesis and cell proliferation. Catalase significantly inhibited ROS production induced by homocysteine, but did not significantly inhibited homocysteine-mediated proliferation of VSMCs. While alpha-tocopherol and beta-tocopherol both suppressed increased ROS production induced by homocysteine in VSMCs, only alpha-tocopherol significantly inhibited homocysteine-mediated VSMC proliferation. CONCLUSION: ROS is not associated with VSMC proliferation, and vitamin E-induced suppression of VSMC proliferation is probably related to protein kinase C inhibition.

Mathematical model of phosphatidylinositol-4,5-bisphosphate hydrolysis mediated by epidermal growth factor receptor generating diacylglycerol.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is hydrolyzed in response to the tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and plays an important role in regulating cell proliferation and differentiation through the generation of second messengers diacylglycerol (DAG) and trisphosphate inositol (IP3) which lead to the activation of protein kinase C (PKC) and increased levels of intracellular calcium, respectively. In the paper, a mathematical model was established to simulate the accumulation of DAG due to PIP2 hydrolysis mediated by EGFR. Molecular mechanisms between DAG, PIP2, EGFR and phosphatidylinositol transfer protein (PITP) were explained successfully, and positive cooperativity which existed between phospholipase C-gamma1 (PLC-gamma1) and PIP2 was also explained. In the model the effects of parameters on simulation of PIP2 hydrolysis were analyzed and the efficacies of some molecular intervention strategies were predicted. To test the coherence between the model and the biological response to epidermal growth factor (EGF) in cells, the levels of DAG and the tyrosine phosphorylation-EGFRs in NIH3T3 mouse embryonic fibroblast (MEF) were determined by biochemical experiments which showed that the accumulation of DAG was a sigmoidal function of phosphorylation-EGFR concentration, and the consistency between the mathematical model and experimental results was confirmed. In brief, this mathematical model provided a new idea for the further study of the dynamic change of biological characteristics in inositol phospholipid hydrolysis, predicting the efficacy of molecular intervention and the relationship between the metabolisms of inositol phospholipid and other signal transduction pathways.

[Analysis and realization of the method for predicting physical pathways of allosteric communication in a protein family].

A fundamental goal in signal transduction study is to understand allosteric communication. The authors present hereby a statistical coupling analysis method (developed by Steve W. Lockless etc.) for quantitative mapping of the global network of amino acid positions in a protein and predicting a set of energetically coupled positions, which may constitute the physical pathways of allosteric communication in a protein family. Based on MATLAB, the authors realized this method and created histograms of amino acid distributions for all 63 395 entries (as of April 2004) in the Swiss-Prot database of eukaryotic proteins and calculated the mean values. The result was similar to that calculated by Steve W. Lockless in October 1998.

Hydrogen peroxide in the Burkitt's lymphoma cell line Raji provides protection against arsenic trioxide-induced apoptosis via the phosphoinositide-3 kinase signalling pathway.

Many anticarcinogenic drugs kill tumour cells by inducing apoptosis. We examined the effects of hydrogen peroxide (H(2)O(2)) on arsenic trioxide (As(2)O(3))-induced cell killing. Low concentrations of H(2)O(2) (200 micromol/l) inhibited the ability of As(2)O(3) to induce apoptosis in the Burkitt's lymphoma cell line Raji. H(2)O(2) altered the form of cell death from apoptosis to pyknosis/necrosis and also lowered the degree of cell killing by As(2)O(3). H(2)O(2) was capable of preventing caspase-3 activation induced by As(2)O(3) in Raji cells. Incubation of cells with a phosphoinositide-3 kinase (PI-3K) inhibitor, wortmannin (100 nmol/l), blocked the effects of H(2)O(2) on As(2)O(3)-induced caspase-3 activation. In addition, the PI-3K inhibitor partially blocked the effects of H(2)O(2) on up-regulation of Bcl-2 and Bcl-X(L) protein expression, down-regulation of Bax protein expression, and phosphorylation of Bcl-2 and IkappaBalpha. This investigation demonstrated for the first time that low concentrations of H(2)O(2) provide protection against the in vivo of As(2)O(3)-induced apoptosis. PI-3K plays a crucial role in enhancing cell survival during H(2)O(2), inhibiting As(2)O(3)-induced apoptosis in the Burkitt's lymphoma cells. As(2)O(3)-induced cancer cell apoptosis may be enhanced by certain antioxidants in the treatment protocol.

[Mathematical model of epidermal growth factor receptor-mediated lipid phosphatidylinositol(4,5)-bisphosphate hydrolyzation by phospholipase C-gamma1 activity].

OBJECTIVE: To investigate the dynamic characteristics of lipid phosphatidylinositol (4,5)-bisphosphate (PIP(2)) in plasma membrane hydrolyzed by phospholipase C-gamma1 in epidermal growth factor receptor(EGFR)-mediated signal pathway. METHODS: A mathematical model based on the law of mass action was established with differential equations to simulate metabolizable pathway of PIP(2). RESULTS: Differential equations of the key product concentration during hydrolysis of PIP(2) were formulated, and the effects of the parameters on these hydrolyzed products analyzed. CONCLUSION: This mathematical model provides foundation for further investigation of the dynamic changes of biological characteristics and the relations between the key product concentrations in PIP2 hydrolysis.