[Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism].
OBJECTIVE
Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.


METHODS
2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.


RESULTS
After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.


CONCLUSIONS
2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.
Chen
Ying-hu
YH
Division of Hematology-Oncology, Children's Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Tang
Yong-min
YM
Shen
Hong-qiang
HQ
Song
Hua
H
Yang
Shi-long
SL
Shi
Shu-wen
SW
Qian
Bai-qin
BQ
Xu
Wei-qun
WQ
Ning
Bo-tao
BT
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D018941:Antigens, CD19; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D019833:Genistein / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D000922:Immunotoxins / Q000276:immunology / Q000494:pharmacology*; D015448:Leukemia, B-Cell / Q000276:immunology
D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D000922:Immunotoxins; D019833:Genistein