Progress of study on sideroblastic anemia and its possible gene therapy--review / 中国实验血液学杂志

It was thought that delta-aminolevulinate synthase (ALAS) is the rate-limiting enzyme in the heme biosynthetic pathway. Actually there are two isozymes of ALAS and ALAS2 (erythroid delta-aminolevulinate synthase), they play the leading role in the hemoglobin biosynthetic pathway. Mutations in ALAS2 gene causes X-linked sideroblastic anemia (XLSA). About 25 different mutations in ALAS2 gene have been identified in XLSA patients and two of them were reported by our laboratory. It is possible to cure the patients with XLSA by gene therapy because it is a single gene disorder.

Construction of recombinant vector expressing ALAS2 gene in X-linked sideroblastic anemia / 中国实验血液学杂志

X-linked sideroblastic anemia (XLSA) is caused by mutations of erythroid-specific 5-aminolevulinate synthetase (ALAS2) gene. In this study a eukaryotic expression vector of ALAS2 was constructed and transfected into eukaryotic cells to observe the expression of ALAS2 gene. The full length cDNA of ALAS2 gene was inserted into plasmid pDs-red2-N1, named pDs-red2-N1/ALAS2. Then, the vector was transfected into K562 cells via electroporation. At 48 hours after transfection, total RNA from K562 cells was extracted, expressions of ALAS2 gene and protein with red fluorescence in the K562 cells were detected by RT-PCR and flow cytometry, respectively. The vector was also transfected into COS 7 cells via liposome. Both mRNA and protein expression in COS7 cells were detected by RT-PCR and fluorescence microscopy. The result showed that after the pDs-red2-N1/ALAS2 eukaryotic expression vector was digested by KpnI and BamHI, two fragments of 4 700 bp and 1 764 bp were displayed by electrophoresis on agarose gel. Sequence method confirmed that the sequence was correct. RT-PCR amplified the total RNA extracted from the transfected K562 and COS7 cells, and could find mRNA of ALAS2 gene that can't be found in K562 and COS7 cells usually. The expressions of both fluorescein and ALAS2 were significantly increased. The percentage of positive cells reached about 19.2% and 10.7%, respectively. ALAS2 expression lasted for 10 days in COS7 cells and the peak was at the third day. It is concluded that the eukaryotic expression vector of ALAS2 gene is successfully constructed; K562 and COS7 cells transfected with the vector via electroporation and liposome can express ALAS2 protein. So, the vector has the potential in gene replacement and can be used for patients with XLSA in future.

Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to eradicate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells.</p><p><b>METHODS</b>CIK cells were obtained from peripheral blood and induced by IFN-gamma, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method.</p><p><b>RESULTS</b>mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21% - 37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3 - 45.8 times, and that to colchicines to 6.7 - 11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered.</p><p><b>CONCLUSIONS</b>CIK cells were successfully transfected with mdr1 cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.</p>

CIK cells acquired multidrug resistance and maintained cytotoxic activity to tumor cells after mdr1 gene transfection / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate whether the cytokine-induced killer (CIK) cells could acquire multidrug resistance and maintain the original cytotoxic activity after multidrug resistance (mdr1) genes transfection.</p><p><b>METHODS</b>CIK cells were generated from peripheral blood cultured with IFN-gamma, CD(3) monoclonal antibody, IL-2, IL-1 and transfected with a plasmid (pHamdr) containing human mdr1 gene via electroporation. RT-PCR method was used to assay mRNA expression of mdr1 gene in transfected CIK cells, flow cytometry with anti-P-gp monoclonal antibody to detect P-glycoprotein (P-gp) expression on CIK cells membrane, and MTT assay to compare both the multidrug resistance to doxorubicin and colchicines and cytotoxic activity to human mammary cancer cell line MCF7 between transfected and non-transfected CIK cells.</p><p><b>RESULTS</b>mdr1 expression was detected in the transfected CIK cells. There was a strong expression of P-gp on the transfected CIK cells and the percentages of P-gp positive cells were 21% - 37% (average 27%). The IC(50) of transfected CIK cells to doxorubicin was 22.3 - 45.8 times and 6.7 - 11.35 times to colchicines of those of non-transfected CIK cells. The cytotoxic activity to MCF7 remained unchanged (P > 0.05).</p><p><b>CONCLUSION</b>It demonstrated that CIK cells transfected with mdr1 gene via electroporation could express multidrug resistance successfully without changes of cytotoxic activity.</p>