Improvement of combination chemotherapy tolerance by introduction of polycistronic retroviral vector drug resistance genes MGMT and MDR1 into human umbilical cord blood CD34⁺ cells

Abstract 

We obtained a full-length cDNA fragment encoding human O⁶-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.

Keywords: MGMT gene, MDR1 gene, Retrovirus vector, Gene therapy, Gene co-expression, Hematopoietic stem cell, Cord blood

Abbreviations: MGMT, O⁶-methylguanine-DNA-methyltransferase, MDR1, multidrug resistancegene-1, IRES, internal ribosome entry site, RT-PCR, reverse transcript polymerase chain reaction, MTT, 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazoliumbromide, UCB, umbilical cord blood, BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea, AGT, O⁶-alkylguanine-DNA-alkyltransferase protein