Effects Of Inhibiting Bioactivity Of SDF-1 On Proliferation, Apoptosis And Chemosensitivity Of Human Acute Leukemia Cell Line HL-60

Objective: Leukemic marrow minimal residual disease is an important reason for the recurrence of acute leukemia, in which, bone marrow stroma microenvironment plays a important role, but the mechanisms are still not completely clarified. It was found recently that stromal cell derived factor-1 (SDF-1), excreted by bone marrow stromal cells, and CXCR4, the only receptor of SDF-1 in vivo, expressed on membrame of hemopoietic stem cells and various kinds of leukemia cells at different levels, plays a extexmely important role in survival and apoptosis of leukemia cells as well as mobilization and homing of hemopoietic stem cells. .Whether the intervention on this chemokine/receptor axis (SDF-l/CXCIU) affects the survival, proliferation, apoptosis and the chemotheraputic sensitivity of leukemia cells in bone marrow and plays an active role in eradicating of minimal residual disease? These problems were not investigated profoundly at present. So investigating the relationships between SDF-1/CXCR4 axis and leukemia cells were of importance in comprehending the mechanism of MRD formation, and in providing theoretical support to a new strategy for MRD reversion and the therapy of acute leukemia by means of modifing the leukemic hemopoietic microenvironment.Methods: Bone marrow stromal cells were grown from bone marrow mononuclear cells obtained from 13 cases of normal and 14 cases of leukemia individuals and were cultured by Dexter long-time culture. When confluent stromal cell layer formed, it was co-cultured with acute myelocytic leukemia cell line HL-60. In this co-cultured system, SDF-1 bioactivities were blocked with 10 jag/ml anti-CXCR4 monoclonal antibody 12G5. According to existence of 12G5 or not, all samples were divided into experiment group and control group, and then at diffirent time the correlated index was assayed as follows: (1) The expression of CXCR4 on HL-60 cells membrane was detected with flow cytometry. (2) The content of SDF-1 in supernatant from marrow stromal cell cultivation was investigated by enzyme-linkedFund National Scientific Fundatko of China (No. 30170396) 2immunoadsordent assay (ELIS A) . (3) The adhesion status of HL-60 cells on marrow stromal cell layer were observed with invert microscope and the adhesion rates were calculated . (4) Survival rates of HL-60 cells were assayed by means of trypan blue exclusion and cell growth curve was plotted meanwhile. (5) Cell cycle and apoptosis rates of HL-60 cells were investigated by flow cytometry. (6) The expression of PCNA and apotosis related protein such as BCL-2 and Fas were assayed by immunohistochemical technique. (7) 20 |ag/ml or 40 ug/ml Ara-C was added in co-culture as the same time as 12G5 incubation, and then the chemosensitivities of HL-60 cells were assayed by MTT and the morphology results were compared.Results: Main results were as follows:(1) There was middle degree expression of CXCRj on HL-60 membrame. As the time of 12G5 incubation went by, at 0, 2 and 6h, the expression of CXCRt decreased gradually(P<0.05).(2) The mean content of SDF-1 in supernatant from leukemic stromal cell culture was 2941.483 + 374.671 pg/ml, significantly higher than that from normal stromal culture, mean 1534.713?95.198 pg/ml (PO.05).(3) In normal stromal cell co-culture, by 2 h after 12G5 incubation, there was no leukemia cell adherent to marrow stromal cell layer, on the contrary, some HL-60 cells scattered on stroma in control group. In leukemic stromal cell co-culture, adherent cells in experiment group reduced significantly. When assess the adhesion rates assayed before and by 24 h after 12G5 incubation, correlated results showed that adhesion rates in experiment group were 19.1 + 8.6 % and 39.4?.9 % in normal and leukemia stromal cell co-culture, but those in control group were 48.7 + 5.3% and 51.4+5.9%, respectively, significantly higher than those in experiment group(P<0.05).(4) At 0, 24, 48, 72, 96 h after incubation with 12G5 in normal stromal cell co-culture, survival rates of HL-60 cell wer...