Improvement of combination chemotherapy tolerance by introduction of polycistronic retroviral vector drug resistance genes MGMT and MDR1 into human umbilical cord blood CD34+ cells.
We obtained a full-length cDNA fragment encoding human O(6)-methylguanine-DNA-methyltransferase (MGMT) from the liver tissue of a patient with cholelithiasis by RT-PCR and confirmed by DNA sequencing. The polycistronic retrovirus vector G1Na-MGMT-Neo(r)-IRES-MDR1 was constructed and verified by restriction endonuclease analysis and DNA sequencing. The vector was transfected into packaging cells GP+E86 and PA317 by the LipofectAMINE method. Cord blood CD34+ cells were transfected with the supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hematopoietic growth factors. PCR, RT-PCR, Southern Blot, Western Blot, FACS and MTT analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 5.8-6.3-fold stronger resistance to P-glycoprotein effluxed drugs and 5-fold to BCNU than untransduced cells. The polycistronic retrovirus vector mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.
Wang
Jishi
J
Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou 215006, People's Republic of China. jswang_yg@yahoo.com
Chen
Zixing
Z
Xia
Xueming
X
Lu
Daru
D
Xue
Jingluen
J
Ruan
Changgeng
C
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D000970:Antineoplastic Agents / Q000633:toxicity*; D002330:Carmustine / Q000633:toxicity; D018076:DNA, Complementary; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D005796:Genes; D019450:Genes, MDR; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D020014:K562 Cells; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014750:Vincristine / Q000633:toxicity
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD; D018952:Antigens, CD34; D000970:Antineoplastic Agents; D018076:DNA, Complementary; D016298:Hematopoietic Cell Growth Factors; D014750:Vincristine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine