[Purging effects of CD3AK/iNOS in vitro on primary leukemic cells from chronic myeloid leukemia patients].
To investigate the purging effect of CD3AK/iNOS on primary leukemic cells from chronic myeloid leukemia patients in vitro, amphotropic packaging cell line PA317 transfected with the whole length of iNOS gene was cultivated, amplified and screened by G418. The viral titer was determined by the NIH3T3 cells. Human peripheral blood mononuclear cells were isolated and activated by anti-CD3 monoclonal antibody in vitro. CD3AK cells were incubated with viral supernatant and selected by G418. Resistant clones were assayed for iNOS gene expression by RT-RCR. The content of nitric oxide and the activity of iNOS in the culture supernatant of CD3AK/iNOS were evaluated by the method of Griess. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene was detected by serial dilution semi-quantitative net RT-PCR assay. The results showed that anti-G418 positive packaging cell line PA317 transfected with the whole length of iNOS gene clones could stably synthesize and excrete recombinant retroviral vectors. The titer of recombinant retroviral vectors was 1.0 x 10(5) CFU/ml. After being transfected by recombinant retroviral supernatant, the iNOS cDNA was expressed in CD3AK/iNOS. The content of NO and activity of iNOS that synthesized and excreted by CD3AK/iNOS were notably increased, compared with those of CD3AK. There were statistically significant differences in NO content and iNOS activity between two groups. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene in all of them was down-regulated by serial dilution semi-quantitative RT-PCR assay. It is concluded that construction of CD3AK/iNOS can markedly increase the content of NO and the activity of iNOS, which can be more efficient in in vitro purging leukemia cells for autologous hematopoietic stem cell transplantation.
Chen
Qing
Q
Department of Hematology, The Zhongda Hospital Affiliated to Southeast University, Nanjing 21009, China.
Chen
Bao-An
BA
Zhu
Min-Sheng
MS
Zhu
Liang-Jun
LJ
Du
Juan
J
Wang
Jun
J
Cheng
Jian
J
Zhao
Gang
G
Ding
Jia-Hua
JH
Sun
Yun-Yu
YY
Shao
Ze-Ye
ZY
D000328:Adult; D000368:Aged; D000818:Animals; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000276:immunology / Q000473:pathology*; D008297:Male; D051379:Mice; D008875:Middle Aged; D041681:NIH 3T3 Cells; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D017252:CD3 Complex; D012333:RNA, Messenger; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D016044:Fusion Proteins, bcr-abl