Studies Of Hydrogen Peroxide On HL-60 Cell Immortalization And Relationship Between Dosage And Effects

Inunortalization is a specific property of tumor cells, which can be affected by many factors. Some authors reported that free radical or reactive oxygen species (ROS) could modulate cell immortalization. The production and scavenging of free radicals keep balance in normal living body. Many kinds of active free radicals in environment are easy to react with some substances to affect growth, differentiation and death of cell by cooperation with endogenous free radicals in body. Recent years, many studies demonstrated that reactive oxygen species could change the action of cells enzyme, concentration of cAMP, cGMP to affect cells differentiation; ROS could affect DNA directly; ROS could affect ionic channel and signal transduction; those effects of free radicals depend on their dosage. In this experiment, H202 was used as exogenous ROS or free radical to study their effects on cell immortalization. The cultivated HL-60 cells were affected by different concentration of hydrogen peroxide. Then the number of live cells was recorded. The vitality of HL-60 cells was measured by MTT methods. Cell cycle was analyzed by distribution of DNA that measured by flow cytometry after I-IL-60 cells were synchronized and cultivated. The concentration of Ca2~ was detected by Interactive Luster Cytometer (ILC). The activity of telomerase was measured by TRAP-Hyb Kit and the length of telomere was measured by North2South Direct HRP Labeling and Detection Kit. The experiment includes: the effect of hydrogen peroxide on cell living state of HL-60 cell; the effect of hydrogen peroxide on the concentration of inside Ca2~ of cell and the effect of hydrogen peroxide on the activity of telomerase and length of telomere. The results showed: (I) There were different actions of H202 in distinct concentration on cell living state; low concentration of H202 irritated the living cell number, the effect of the Group of 2 ii mol U?was especially significance; middling concentration of H202(lOO I-knol 11) induced apoptosis; high concentration of H202(5mniol U? stimulated cell necrosis in certain range of dosage. (2) The inside Ca2~ of cell was related to dosage and time, those were affected by certain concentration of H202. Low concentration of hydrogen peroxide made concentration of inside Ca2~ of HL-60 cells decrease and soon increased to normal level. When concentration of H2O2(1O~mol U? was used, concentration of Ca2~ decreased and lasted for a long time, then increased to certain level, which was kept this state in high level compared with normal group. When high concentration of H202(O.5mmol U? was used, concentration of Ca2~ decreased quickly. (3) After the cultivated HL-60 cells were affected by different concentration of hydrogen peroxide, the activity of telomerase was increased than that in the control group. Some dose-effect relationship was existed in this experiment. If concentration of H202 exceed to some limit, the activity of telomerase could decrease. After HL-60 cells were affected by different concentration of hydrogen peroxide and cultured for forty days, the length of telomere was shorter than that in the control group. The results suggested that free radicals could affect condition of HL-60 cell, and showed some dose-effect relation. Low concentration of hydrogen peroxide could irritate increase of the living cell number, and the reason likely was due to shorten the cell cycle. Middling concentration of H202 could induce apoptosis, and high concentration of H202 could lead...