关键词: 放线菌素D, 条件培养液, p53, 细胞周期

Abstract:

OBJECTIVE:  To observe the effect of conditioned medium(CM) obtained from actinomycin D(ACTD)-exposed cells on the mRNA and protein levels of p53 and cell cycle distribution in bystander V79 cells and to explore the probable mechanism underlying the bystander effect induced by ACTD. METHODS：V79 cells were treated with 4.0 mg/L ACTD for 1 h. Then conditioned medium was collected at 4，8，12 and 24 h to culture bystander cells for 24 h to observe the bystander effect. RT-PCR method was used to detect the mRNA levels of p53 in bystander cells. Immunohistochemistry staining was adopted to determine the protein expression of p53. Flow cytometry was used to observe the cell cycle distribution of bystander cells. RESULTS：p53 mRNA levels in CM-treated bystander cells were higher than the control. With time p53 mRNA level gradually decreased from 4-12 h while increased in 24 h CM group. The protein expression of p53 indicated as mean gray values and integrated optical density increased in CM-treated groups except for 12 h CM group (P＜0.05). p53 expression reduced with time (4-12 h) then increased in 24 h CM group which was associated with p53 mRNA. The cell number in G0/G1 phase all increased in CM-treated bystander cells amongst which 4 h CM was the highest and decreased gradually with time till 12 h then reduced in 24 h CM. The cell number in S phase decreased in CM-treated bystander cells but there was no difference between various CM groups (P>0.05). The cell number in G2/M phase decreased significantly in 4 h CM heaviest then increased gradually close to control level at 12 h and reduced again in 24 h CM (P＜0.05). CONCLUSION：ACTD could induce bystander effect in V79 cells by from ACTD-exposed cells influencing p53 gene expression and interrupting cell cycle progression.

Key words: actinomycin D, conditioned medium, p53, cell cycle