Genetic modification of hematopoietic progenitor cells for combined resistance to 4-hydroperoxycyclophosphamide, vincristine, and daunorubicin.
OBJECTIVE
To investigate whether human peripheral blood hematopoietic progenitor cells (PBPC) modified with human aldehyde dehydrogenase class-3 gene (ALDH-3) and multidrug resistance gene 1 (MDR1) would increase chemotherapy resistance to 4-hydroperoxycyclophosphamide (4-HC) and -glycoprotein effluxed drugs.


METHODS
A bicistronic retroviral vector G1Na-ALDH3-IRES-MDR1 cDNA was constructed and used to transfect the packaging cell lines PA317 by electroporation. CD34+ PBPC were isolated with a high-gradient magnetic cell sorting system (MACS), and then were transfected with supernatant of retrovirus containing human ALDH-3 and MDR1 cDNA. PCR, RT-PCR, Southern blot, Northern blot, FACS, and MTT assay were used to evaluate the transfection and expression of the transgene in target cells.


RESULTS
The bicistronic retroviral vector construction was verified by PCR and restriction endonuclease analysis. Dual drug resistance genes were integrated into the genomic DNA of CD34+ PBPC and expressed efficiently. The efficiency of gene transfection in CD34+ PBPC was tested to be 18 % on colonies. Nested PCR and Neor rescue assay indicated that no helper virus was present in this system. Compared with the untransduced cells, transgene recipient cells conferred 4.5-fold resistance to 4-HC, 6.6-fold and 7.8-fold resistance to P-glycoprotein effluxed drug, vincristine and daunorubicin, respectively.


CONCLUSIONS
Efficient transduction of two different types of drug resistance genes into human peripheral blood hematopoietic progenitor cells and the co-expression may decrease cumulative myelosuppression of combination chemotherapy.
Wang
J S
JS
Department of Hematology, the Affiliated Hospital of Guiyang Medical College, Guiyang 550004,China. jswang@shmu.edu.cn
Fang
Q
Q
Sun
D J
DJ
Chen
J
J
Zhou
X L
XL
Lin
G W
GW
Lu
H Z
HZ
Fei
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D000444:Aldehyde Dehydrogenase; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003630:Daunorubicin / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D018274:Electroporation; D019450:Genes, MDR; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D019076:Transgenes; D014750:Vincristine / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014750:Vincristine; D003520:Cyclophosphamide; D000444:Aldehyde Dehydrogenase; C011272:perfosfamide; D003630:Daunorubicin