The effect of p21 antisense oligodeoxynucleotides on the radiosensitivity of nasopharyngeal carcinoma cells with normal p53 function.
OBJECTIVE
p21(WAF1/CIP1) is transcriptionally activated by p53 and is required for G1 to S phase progression. p21 plays a critical role in DNA repair after DNA damage. Thus, cells with defective p21 may result in an enhancement of radiation induced apoptosis and improved radiosensitivity. We tested the hypothesis that p21 antisense oligodeoxynucleotides (p21 AS ODNs) can be used to reduce p21 expression level and increase radiosensitivity in CNE-1-wtp53 nasopharyngeal carcinoma cell line with normal p53 function.


METHODS
The p21 antisense oligodeoxynucleotides (p21 AS ODNs) and the random control oligodeoxynucleotides (p21 RD ODNs) were synthesized. p21 AS ODNs sequence: 5'-TGTCATGCTGGTCTGCCGCC-3'; p21 RD ODNs sequence: 5'-CCGGTGAACGAGCGAGCACA-3'. p21 AS ODNs and p21 RD ODNs were transfected into CNE-1-wtp53 nasopharyngeal carcinoma cell line. The protein expression levels of P21 were evaluated using Western blotting analysis. Cell cycle progression and apoptotic cells were assessed by flow cytometric analysis. The clonogenic survival assay was performed to determine the survival fraction. The parameters D0, Dq, and N for the single-hit multitarget model and the parameters alpha, beta, alpha/beta, and SF2 for the linear-quadratic model were calculated. BALB/c nude mice were used to investigate the effect of p21 AS ODNs on the radiosensitivity of nasopharyngeal xenografts in vivo.


RESULTS
p21 AS ODNs were detected mainly in plasma with fluorescence microscopy investigation. P21 protein level dramatically decreased and the amount of apoptotic cells increased in p21 AS ODNs transfected cells than in p21 RD ODNs transfected cells after irradiation. The percentage of G1 arrest decreased in p21 AS ODNs transfected cells 24 h after radiation, then G2 arrest decreased 48 h after radiation. The values of D0, Dq, SF2 decreased and alpha value increased in p21 AS ODNs transfected cells than in control cells. The inhibition rate in tumor xenografts exposed to X ray of 10 Gy alone was 39.1%, while it was 51.4% in xenografts injected with p21 AS ODNs before exposure to radiation. Unfortunately, there was no significant difference between these two groups (P < 0.05).


CONCLUSIONS
p21 Antisense oligodeoxynucleotides led to inhibition of P21 protein expression, loss of G1 arrest, increase of apoptosis in CNE-1-wtp53 nasopharyngeal carcinoma cell line in vitro and inhibited tumor growth in vivo. Antisense oligodeoxynucleotides may become a promising strategy to enhance radiosensitivity in nasopharyngeal carcinoma cells with normal p53 function.
Liu
Xiu-Fang
XF
State Key Laboratory of Oncology in Southern China, China.
Xia
Yun-Fei
YF
Li
Man-Zhi
MZ
Wang
Hong-Mei
HM
He
Yu-Xiang
YX
Zheng
Mei-Lian
ML
Yang
Hui-Ling
HL
Huang
Wen-Lin
WL
D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D003114:Colony-Forming Units Assay; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009303:Nasopharyngeal Neoplasms / Q000473:pathology* / Q000532:radiotherapy*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism*; D011836:Radiation Tolerance / Q000528:radiation effects*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016376:Oligonucleotides, Antisense; D016159:Tumor Suppressor Protein p53