title: [Improvement of combination chemotherapy tolerance of human umbilical cord blood CD(34)(+) cells transducted with double drug resistance genes by a bicistronic retroviral vector].
abstract: OBJECTIVE
  To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human aldehyde dehydrogenase class 3 (ALDH3) and multidrug resistance gene (MDR1) could increase resistance to 4-hydroxycyclophosphamide (4-HC) and P-glycoprotein effluxed drugs.
  
  
  METHODS
  A bicistronic retroviral vector G1Na-ALDH3-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD(34)(+) cells were enriched with a high-gradient magnetic cell sorting system (MACS), and then repeatedly transfected with supernatant of retrovirus containing human ALDH3 and MDR1 cDNA under stimulation of hematopoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT assay were used to evaluate the transfection and expression of the double genes.
  
  
  RESULTS
  The purity of cord blood CD(34)(+) cells was approximately 91% and the recovery rate was 72%. The highest titer of recombinant amphotropic retrovirus in the supernatant was up to 6.5 x 10(5) CFU/ml. The efficiency of gene transduction was 18%, 20% and 16.7% tested by colony formation, PCR and FACS, respectively. Rhodamine 123 efflux showed 16% transduced cells with P-gp function. No helper virus was found by both nested PCR and rescue assay. The MTT analysis showed a 3.5 to 6.8-fold increase of resistance of transducted cells to cyclophosphamide and P-glycoprotein effluxes drug as compared with the nontransduced cells.
  
  
  CONCLUSIONS
  The efficiency and co-expression of this dual genes transfer system provided a foundation for ameliorating combination chemotherapy toxicity in clinical trial.
authors.affiliation: Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou Medical College, Suzhou 215006, China.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: X
authors.initials: X
authors.lastname: Xia
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: D
authors.initials: D
authors.lastname: Lu
authors.forename: J
authors.initials: J
authors.lastname: Xue
authors.forename: C
authors.initials: C
authors.lastname: Ruan
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D045744:Cell Line, Tumor; D003520:Cyclophosphamide / Q000494:pharmacology; D004358:Drug Therapy; D004361:Drug Tolerance; D005312:Fetal Blood / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000821:virology; D006801:Humans; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018952:Antigens, CD34; C030863:corneal protein 54, bovine; D003520:Cyclophosphamide; D000444:Aldehyde Dehydrogenase