[Expression of D-animo acid oxidase gene in K562 cells and the cytotoxicity of D-alanine to the cells].
OBJECTIVE
To explore the feasibility of expression of R. gracilis D-amino acid oxidase(DAAO) gene in leukemia cell line K562 and the cytotoxicity of D-Alanine to the cells.


METHODS
DAAO cDNA was cloned into retroviral vector pLSN and pLDAAOSN was generated. The vector was then packaged with phiNXA and the virus titer was measured with NIH3T3 cells. Leukemia cell line K562 was infected with the viral supernatant. The positive clones were obtained by G418 selection and named KDAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in KDAAO. KDAAO was treated with different concentrations of D-Alanine.


RESULTS
pLDAAOSN was confirmed containing the full-length of DAAO cDNA. Infectious titer generated by the packaging cells was 5.2 x 10(6) cfu/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in KDAAO and expression of DAAO mRNA. Preliminary observation suggested that D-Ala could effectively kill KDAAO.


CONCLUSIONS
DAAO/D-Ala suicide gene system might be useful in cancer gene therapy.
Zhai
Y
Y
Department of Hematology, Changhai Hospital of the Second Military Medical University, Shanghai 200433, China.
Wang
J
J
Zhou
H
H
D000409:Alanine / Q000494:pharmacology*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D003605:D-Amino-Acid Oxidase / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D051379:Mice; D041681:NIH 3T3 Cells; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D003605:D-Amino-Acid Oxidase; D000409:Alanine