[In vitro purging effect of CD3AK/iNOS on leukemia cells].
OBJECTIVE
LAK cells have been applied to purge minimal residual leukemia cells in allogeneic hematopoietic stem cell transplantation(AHSCT) in clinic practice. CD3AK cells belong to T lymphocytes activated by anti-CD3McAb. This study was to construct nitric oxide donor CD3AK/iNOS through transfecting inducible nitric oxide synthase (iNOS) gene into human CD3AK cells by retroviral vector, and investigate the cytotoxic activity of CD3AK/iNOS to leukemia cell lines K562 and K562/ADM.


METHODS
The amphotropic packaging cell line PA317 transfected with iNOS gene was cultivated to obtain viral supernatant. Human peripheral blood mononuclear cells (PBMNCs) were isolated and activated by anti-CD3McAb and low dose of interleukin-2 (IL-2). CD3AK cells were incubated with viral supernatant. The amount of nitric oxide (NO) and the activity of iNOS in the cultured supernatant of CD3AK/iNOS were evaluated. The cytotoxic activities of CD3AK/iNOS and CD3AK cells to K562 and K562/ADM cells were evaluated by MTT assay.


RESULTS
The contents of NO excreted by CD3AK/iNOS and CD3AK cells were (378.60+/-41.57) micromol/L and (98.07+/-22.31) micromol/L, respectively (P<0.001); the activities of iNOS synthesized by CD3AK/iNOS and CD3AK cells were (20.77+/-2.49) U/ml and (9.81+/-1.96) U/ml, respectively (P<0.001). The cytotoxic activities of CD3AK/iNOS cells to K562 and K562/ADM cells were significantly stronger than those of CD3AK [(64.85+/-18.13)% vs. (45.66+/-17.46)%, P<0.05; (63.80+/-9.93)% vs. (47.85+/-12.01)%, P<0.05].


CONCLUSIONS
The content of NO and activity of iNOS synthesized and excreted by CD3AK/iNOS cells are largely increased compared with those of CD3AK cells. CD3AK/iNOS cells have more significant cytotoxic activity to K562 and K562/ADM cells than CD3AK cells, but its cytotoxic activities to K562 and K562/ADM cells are similar.
Shao
Qian-Wen
QW
Department of Hematology, Affiliated Zhongda Hospital, Southeast University, Nanjing, Jiangsu 210009, P. R. China.
Chen
Bao-An
BA
Zhu
Liang-Jun
LJ
Du
Juan
J
Shu
Yong-Qian
YQ
Shao
Ze-Ye
ZY
D000818:Animals; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D015979:Killer Cells, Lymphokine-Activated / Q000201:enzymology / Q000276:immunology*; D008213:Lymphocyte Activation; D051379:Mice; D041681:NIH 3T3 Cells; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D017252:CD3 Complex; D011994:Recombinant Proteins; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II