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 AND MATERIALS: 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). CONCLUSION: 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.

[Establishment of three-dimensional culture models related to different stages of nasopharyngeal carcinogenesis].

BACKGROUND & OBJECTIVE: Since three-dimensional culture simulates in vivo microenvironment better than planar culture, the biological character of cells in three-dimensional culture model are more similar with that of living tissues. This study was to establish three-dimensional culture models that represent different stages of nasopharyngeal caicinogenesis, and provide information to elucidate the related molecular events. METHODS: Non-tumor nasopharyngeal biopsy specimens were used to culture for normal nasopharyngeal epithelial cells. Early and late passages of normal nasopharyngeal epithelial cell line NPNE2 from nasopharyngeal biopsy specimens, immortalized nasopharyngeal epithelial cell line NP69SV40T, nasopharyngeal carcinoma (NPC) cell line SUNE-1 and its high metastatic subclone 5-8F were cultured in Matrigel to establish three-dimensional models. RESULTS: The cells grew out of non-tumor nasopharyngeal biopsy specimens displayed the morphologic characteristics as epithelia, and could proliferate in vitro for 8-10 passages before senescence. Immunocytochemical staining of cytokeratin revealed that the cells were of epithelial origin. All of the cells, except late passages of NPNE2 cells, could proliferate in the three-dimensional culture system. NPNE2 and NP69SV40T cells mainly developed reticular structures in morphology, and formed few clones with clear and smooth edges as well as tight intercellular junctions. SUNE-1 and 5-8F cells formed clones with irregular morphology, unclear edge, and loose intercellular junctions. In addition, the clones formed by 5-8F cells also developed a lot of pseudopodia, but developed no reticular structure. Late passages of NPNE2 cells formed no clone and reticular structure in the three-dimensional culture. CONCLUSIONS: Normal nasopharyngeal epithelial cells can be successfully cultured in vitro from naspharyngeal biopsy specimens. The three-dimensional culture models, established with normal nasopharyngeal epithelial cells, immortalized nasopharyngeal epithelial cells, and NPC cells, may represent the different stages of nasopharyngeal carcinogenesis.

[Effect of EBV encoded BARF1 gene on malignant transformation of human epithelial cell line HBE].

BACKGROUND & OBJECTIVE: The BARF(1) (BamHI A right frame1)gene,encoded by Epstein-Barr virus(EBV),may have oncogenic properties,which leaded to immortalization of a monkey kidney epithelial cell line and malignant transformation of human B lymphocytes and murine fibroblasts. This study was designed to evaluate effect of BARF(1) on cell transformation and tumorgenesis on human epithelia and its mechanisms. METHODS: BARF(1) was amplified from B95-8 cell line by polymerase chain reaction (PCR). Eukaryotic recombinant vector pcDNA(3)-BARF(1) was constructed,and transfected into human bronchial epithelial cell line HBE,and expression of BARF(1) in the transfected cells was detected by Western blot. Growth status,proliferation rate,ability of colony formation in soft agar,and tumorigenicity of HBE cells transfected with BARF(1) were studied. RESULTS: BARF(1) gene promoted proliferation and transformation of HBE cells. After injected with 1 x 10(7) HBE cells transfected with BARF(1),tumor nodes were observed in 2 of 6 mice at the 10th day. CONCLUSION: BARF(1),as a virus oncogene,may play an important role in malignant transformation of HBE cells.