A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34(+) cells to improve combination chemotherapy tolerance

Citation
Js. Wang et al., A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34(+) cells to improve combination chemotherapy tolerance, CHIN MED J, 114(1), 2001, pp. 25-29
Citations number
11
Language
INGLESE
art.tipo
Article
Categorie Soggetti
General & Internal Medicine
Journal title
CHINESE MEDICAL JOURNAL
ISSN journal
0366-6999 → ACNP
Volume
114
Issue
1
Year of publication
2001
Pages
25 - 29
Database
ISI
SICI code
0366-6999(200101)114:1<25:ABRVTI>2.0.ZU;2-E
Abstract
Objective To study whether human umbilical cord blood CD34(+) cells transdu ced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resist ance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamid e (4-HC) and P-glycoprotein effluxed drugs. Methods A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructe d and used to transfect the packaging cell lines GP + E86 and PA317 by Lipo fectAMINE method, using the medium containing VCR and 4-HC agents for cloni ng selection and ping-ponging supernatant infection between the ecotropic p roducer clone and the amphotropic producer clone, we obtained high titer am photropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retr ovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopo ietic growth factors. Results Bicistronic retroviral vector construction was verified by restrict ion endonuclease analysis. Polymerase chain reaction (PCR), reverse transcr iption (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell s orting (FACS) method and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol ium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed ef ficiently. The transgenes recipient cells confered 4-fold stronger resistan ce to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduc ed cells. Conclusion The bicistronic retroviral vector-mediated transfer of two diffe rent types of drug resistance genes into human cord blood CD34(+) cells and co-expression provided an experimental foundation for improving combinatio n chemotherapy tolerance in tumor clinical trial.