[Detection of O6-methylguanine-DNA methyltransferase promoter methylation in chemotherapy for glioma].

BACKGROUND AND OBJECTIVE: Epigenetic silencing of the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), is associated with the therapeutic response to methylating agents. This study was to assess the value of detecting the promoter methylation of MGMT gene in chemotherapy for glioma. METHODS: Methylation-specific PCR (MSP) was employed to detect MGMT promoter CpG island methylation in 39 samples of glioma taken from surgery. Western blot and immunohistochemistry were used to detect protein expression. MTT were employed to detect the sensitivity of two glioma cell lines to alkylating agents, ACNU and TMZ. The Kaplan-Meier curve was adopted to estimate the overall survival according to the methylation status of the MGMT promoter. RESULTS: Methylation of MGMT promoter CpG island was detectable in 46.2% of glioma tissues, but not in any normal tissues. The expression rate of MGMT protein was 61.5%. The status of MGMT methylation status was association with the protein level of MGMT (P<0.05). The MGMT gene was demethylated in glioma cell line SHG-44 following 5-Aza-CdR treatment; the expression of MGMT protein was restored and the resistance of SHG44 cells to alkylating agents was reversed. The overall survival was higher in patients with methylated MGMT promoter than in those with unmethylated MGMT promoter (P<0.05). CONCLUSIONS: The status of MGMT promoter CpG island methylation is closely correlated to MGMT protein expression and sensitivity of cells to alkylating agents in glioma. Detection of the methylated sequences of MGMT may be used as a predictive factor for the treatment of glioma.

[Correlation between DNA methylation of the ABO gene promoter CpG island and leukemia].

Recent studies have found that ABO blood group antigen is also closely related to the onset and development of many diseases. More and more attention is being paid to the decrease of A/B blood group antigen caused by some tumors. This study was purpose to investigate the correlation between DNA methylation of the ABO gene promoter CpG island and leukemia. The relative contents of ABH antigen on the surface of RBC from kinds of blood disease patients and healthy individuals were detected by using flow cytometry and confocal laser scanning microscopy. The DNA sequences and CpG methylation of ABO gene promoter in patients with hematopathy and healthy individuals, as well as the -102 site methylation of ABO gene promoter in patients with hematopathy and healthy individuals were detected by PCR and MSP-PCR respectively. The results showed that RBC from leukemia patients displayed different degree of A/B antigen decrease. The sequences of ABO gene promotor of patients with hematopathy were not different from healthy individuals indicating high conservation of promoter sequences. Comparison of sequences between patients with hematopathy and healthy individual indicated that CpG islands on ABO gene promoter either from blood disease patients or from healthy individual had no methylated site in AA patients, but C residues at position -102, -101, -100, -99 and -97 on the promoter of ABO gene in AML, CML, ALL and some MDS patients were methylated. It is concluded that methylation of CpG islands in promoter of ABO gene may result in AB antigen decrease in patients with leukemia. The methylation sites -102, -101, -100, -99 and -97 may be specific for leukemia. The methylation of site -102 can be used as a molecular marker in differential diagnosis for leukemias.

[Enhancing transfection efficiency of polyethylenimine by a hydrophobic peptide from bee venom].

The study was aimed to investigate the possibility of enhancing transfection efficiency of branched polyethylenimine (BPEI) in HeLa cells by hydrophobic tail of bee venom peptide (melittin). Hydrophobic tail of melittin was synthesized and its membrane permeable activity was evaluated by hemolysis test. The peptide was mixed with BPEI and the transfection efficiency was determined in HeLa cells by using green fluorescent protein gene (GFP) as a reporter gene. The cytotoxicity of the mixture was analyzed by MTT assay at 24 hours after transfection. The results indicated that the synthesized peptide had permeable activity leading to hemolysis in both neutral and acidic solution. At optimal condition, the peptide could significantly improve the transfection efficiency of BPEI and the cytotoxicity of the mixture was lower than BPEI itself. It is concluded that hydrophobic tail of melittin may be a potential enhancer to improve transfection efficiency mediated by cationic polymers in difficult to transfect cells.

[A pilot study on establishment of human/pig hematopoietic chimera model in fetal and neonatal pigs].

This study was aimed to explore the feasibility of transplanting human cord blood stem cells (HSC) into pre-immune fetal and neonatal pigs, and to investigate the self-renewal of HSC in the recipient pigs. The fetus and neonate were manipulated in sterile separated room and human donor cells were injected into fetus via fetus muscle or umbilical vein (dissectted womb) or into neonate via umbilical vein before cutting it. Human CD45(+) cells s were detected by labeling with human anti-CD45 antibody and analyzed by fluorescence activated cell sorting (FACS). The results showed that tested pigs developed as well as control and a definite proportion of human cells existed in peripheral blood of chimeric pig on day 60 after transplantation. In conclusion, the fetus and neonate pigs can tolerate a definite proportion of human antigens, and to establish the human/pig model of hematopoietic chimerism is possible.

[Effect of human plasma on preservation of red blood cells].

In order to study whether plasma can affect the structure and function of red blood cells during their storage period, the differences of pH value, concentration of K(+), Na(+), osmotic fragility, plasma hemoglobin, AchE, ATP, 2.3-DPG, P50 in suspended RBC, washed RBC, and RBC with various plasma volume at different storage times were compared. The results showed that plasma helped the blood to keep the RBC at high pH value, low K(+), high Na(+) and maintain RBC-ATP, oxygen carry capacity and deformability, but no effect on maintenance of osmotic fragility, and levels of plasma hemoglobin, AchE, ATP and 2.3-DPG was found in preservated blood. In conclusion, human plasma may be in favour of the preservation of red blood cells.

[Study on MGMT assay and tumor individual predictable chemotherapy].

How to predict and surmount the cell resistance in tumor chemotherapy is a prompt problem. We have observed that there were close correlation among O6-Methylguanine-DNA methyltransferase (MGMT) enzyme activity, protein expression and cell resistance to alkylating agents especially to nitrosourea anti-tumor compounds by a series of experiments including cell survival, xenografts in nude mice, tumor patient biopsy and molecule biology assay. We found that those tumors with high MGMT activity and abundance of MGMT protein were resistant to alkylating agents killing effect, while those with low MGMT activity and little MGMT protein were sensitive to alkylating anti-tumor drugs. We proposed a new tactics for tumor predictable chemotherapy treated with alkylating agents based on MGMT protein detection. By means of preparing MGMT monoclone antibody, we have succeeded in developing MGMT immunohistochemistry diagnostic kit.

[Preliminary in vitro study on modification of RBC with mPEG-BTC for resolving the problem of clinical difficulty in blood matching].

The aim of this study was to find an effective solution for difficulty of blood matching. Twenty nine cases with clinical difficult in blood matching were collected, classified by their etiological factors, and analyzed with all the antibodies in serum. RBC from health donor were incubated with mPEG-BTC at 25 degrees C for 1 hour. The coagulation of patient serum and donor RBC before and after mPEG-BTC camouflage was detected and compared by polybrene and antihuman globulin reagents. The result showed that 29 cases with difficult blood matching mainly suffered form blood diseases and tumors. The main antibody were Rh and autoantibody. Donor RBC modified by mPEG showed no coagulation with the blood serum in the patients with problems of blood matching. In conclusion, the modification of RBC with mPEG-BTC provides a useful strategy for resolving problem of clinical difficulty in blood matching.

[Selective myelo-protection by MDR1 and MnSOD genes regulated by a specific promoter].

OBJECTIVE: To study the specific protection of myeloid cells from chemotherapeutic agents and radiation. METHODS: The recombinant retroviral vectors containing MDR1 gene and MnSOD gene regulated by APN myeloid promoter were constructed and introduced into myeloblastic cell line KG1a and hepatoma cell line BEL7402. The resistance of the cells to antitumor drugs and radiation were analyzed by cell survival assay. In vivo, the murine bone marrow cells were isolated and infected by the retroviral particles, which were transplanted into recipient mouse treated with paclitaxel or X-ray. The murine white blood cell (WBC) was counted in order to assay the effects of MDR1 or MnSOD gene on hematopoiesis in the course of chemotherapy and radiotherapy. RESULTS: The resistance to chemotherapeutic agents such as cochicine, Vp-16, vincristine, doxorubcin and paclitaxel were elevated markedly by 10.6, 10.4, 11.2, 4.2 and 14.2 folds in KG1a cell line transduced with MDR1 gene. The resistance to radiation increased 3.7 folds at the dose of 10 Gy compared with parental cells in KGla cell line transduced with MnSOD gene derived by APN promoter. In contrast, the chemosensitivity and the radiosensitivity showed no significant change in BEL 7402 cell line transduced with MDR1 gene and MnSOD gene. In vivo, the WBC counts in the mouse introduced with MDR1 gene or MnSOD gene were higher than those in the control mouse (P < 0.01). CONCLUSION: The expression of MDR1 gene and MnSOD gene regulated by APN myeloid promoter is effective on myelo-specific protection without enhancing the resistance of tumor cells in vitro. The hematopoiesis can be reconstituted in vivo during anticancer drug or radiation treatment. This study may provide experimental evidence and new clues for myeloprotection of cancer patients being treated with chemotherapy and/or radiotherapy.

[Camouflage of HLA-I antigen in lymphocyte surface].

The objective of this study was to investigate the method and effect of blocking the specific reaction between lymphocyte HLA-I antigen and its antibody. The lymphocytes were disposed with 12 mmol/L methoxypolyethelene glycol benzotriazol carbonate (mPEG-BTC) in concentration gradient in PBS (pH 7.4) at 22 degrees C. The effect of the modified lymphocytes was detected by microlymphocytotoxicity assay. The results showed that lymphocytes modified by mPEG-BTC did not react with related HLA-I antibodies in microcytotoxicity test. It is suggested that the specific reaction between HLA-I antigen of lymphocyte and HLA-I antibodies can be completely camouflaged by mPEG-BTC in PBS (pH 7.4) under 22 degrees C room temperature.