Experimental Study Of The Damaging Effect Of NO-enhanced Radiochemotherapy On Leukemia Cells

Objective: To construct 3T3 cells overexpressing iNOS gene, to observe whether NO could enhance the damaging effect of cyclophosphamide (CAP) and radiation on L1210 cells in separated culture condition, and to find some useful clues for leukemia therapy.Methods: 1. Experimental materials: L1210 cells, 3T3 cells, DH5α, pcDNA3.0-iNOS plasmid, pcDNA3.0 plasmid, home-made insert-wells. 2. Methods: ⑴Plasmid was identified by routine methods. 3T3 cells transfected by dioleoyltrimethylamino propane (DOTAP) were identified by RT-PCR, immunohistochemistry. NO production in culture medium was detected by Griess reaction. ⑵ Cell culture groups: L1210 cells co-cultured with different transfected 3T3 cells were divided into three groups: pcDNA3.0-iNOS plasmid transfected (iPT) group, pcDNA3.0 plasmid transfected (PT) group, and iPT plus DEVD-CHO (inhibitor of Caspase-3) group. ⑶ Methods for detection: The direct effect of NO and NO-induced apoptosis of L1210 cells in different groups at 12, 24, 48, and 72 h were detected by trypan blue rejecting dyeing (TRD) and TUNNEL, respectively. The cell cycles at 4, 8, 12, 24, 48, and 72 h were detected by flow cytometry.Results: 1. Identification of transfected 3T3 cells: ⑴ Identification of plasmid: After digestion of the plasmid with HindⅢ and XbaⅠ, two bands of the target DNA were harvested by agarose gel electrophoresis. Results of the plasmid sequenced at T7 and BGH, respectively, were coincident with the Genebank. ⑵ Identification of transfected 3T3 cells by RT-PCR: Band of the target DNA according to the specially designed primers was harvested in iPT group by agarose gel electrophoresis, but none was harvested in PT group and control group. ⑶ Identification of transfected 3T3 cells by immunohistochemistry: Green-yellow fluorescence protein was observed in 3T3 cells in iPT group but none was observed in 3T3 cells in PT group and control group. ⑷ NO production in culture medium: NO production in iPT group medium was significantly higher than that in PT group and control group at every time point (P < 0.01), but there was no significant difference in NO production between PT group and control group at every time point (P > 0.05). 2. The effects of NO on L1210 cells: ⑴ The direct effect of NO on L1210 cells: The rate of TRD in iPT group during 24 - 72 h was significantly lower than that in PT group (P < 0.05). However, there was no significant difference between iPT group and iPT plus DEVD-CHO group except at 48 h time point. ⑵ The effects of NO on apoptosis of L1210 cells: The apoptosis of L1210 cells in iPT group at 12 h (P < 0.05) and during 24 - 72 h (P < 0.01) was significantly higher than that in PT group and iPT plus DEVD-CHO group during 24 –72 h (P < 0.05). 3. NO-enhanced damaging effect of 12 Gy radiation on L1210 cells: ⑴ The direct effect of NO on L1210 cells after radiation: The rate of TRD of L1210 cells during 12 – 72 h was significantly lower in iPT group than that in PT group (P < 0.05). There was also significant difference between iPT group and iPT plus DEVD-CHO group during 24 – 72 h (P < 0.05). ⑵ The effects of NO on apoptosis of L1210 cells after radiation: The apoptosis of L1210 cells during 24 – 72 h after radiation in iPT group was significantly higher than that in PT group (P < 0.05). There was also significant difference between iPT group and iPT plus DEVD-CHO group during 24 – 72 h after radiation (P <0.05). ⑶ The effects of NO on cell cycles of L1210 cells after radiation: L1210 cells at G1 phase at 4 h after radiation increased more quickly in iPT group but declined more slowly during 24 – 72 h after radiation than that in PT group (P < 0.05). L1210 cells at S phase declined more quickly at 4 h after radiation in iPT group but increased more quickly during 24 - 72 h after radiation than that in PT group (P < 0.05). In iPT plus DEVD-CHO group , L1210 cells at G1 phase increased more slowly at 4 h than that in iPT group but declined more quickly during 24 – 72 h after radiation (P<0.05). L1210 cells at S phase declined more...