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[Study on in vitro killing activity of DAAO/D-Ala system to K562e cells]
Jianmin Wang1, Yongping Zhai, Yusheng Zhang
1Department of Hematology, Changhai Hospital of the Second Military Medical University, Shanghai 200433, China.
Objective:
To investigate the in vitro killing efficiency of D-amino acid oxidase (DAAO)/D-alanine (D-Ala) system on K562e cells.
Methods:
K(DfGC) cell line stably expressing DAAO was obtained by retrovirus transfection technique. The integration and expression of DAAO gene were identified by PCR and in situ hybridization. The killing activities of D-Ala to DAAO(+) cells alone or the mixtures of DAAO(+) and DAAO(-) cells in different ratios were observed. H(2)O(2) production by K(DfGC) cell was measured by phenol red oxidation assay.
Results:
PCR and in situ hybridization analysis confirmed the integration of DAAO gene in positive clone and its mRNA expression. There was no significant difference in cell proliferation between the two kinds of K562 cells. Ninety percent of K(DfGC) cells was killed by 12.5 mmol/L D-Ala after 24 hour-treatment and the H(2)O(2) levels were in accord with the killing activities of D-Ala. When K(DfGC) was mixed with K562e at different ratio, no significant bystander effect could be found after treating with 15.0 mmol/L D-Ala for 24 hours.
Conclusion:
The leukemia cell line K562e was sensitive to DAAO/D-Ala system and there was no significant bystander effects observed within this cells.