Rao Zhi-guo, Zhang Ji-ren, Zheng Yan-fang, Zhou Hui, Qu Liang-hu
Cancer Center, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, P. R. China.
Ai Zheng. 2002 Feb;21(2):149-52.
BACKGROUND & OBJECTIVE: Human papillomavirus (HPV) are the most important etiologic factor for cervical carcinoma. E6 virus gene is one of the most important oncogene and expression levels of E6 transforming oncoproteins of high risk HPV genotypes, such as HPV16, appear to be necessary for maintaining the malignant phenotype. Radiation treatment represents a standardized and effective modality for contemporary cervical carcinoma therapy. The goal of this study was to investigate the radiation sensitizing effect of anti-HPV16 E6-ribozyme on cervical carcinoma cell line.
With the method of lipofectin transfection, the anti-HPV16 E6 ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell line, which named as CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in the three kinds of cells were detected by Northern blot. The growth rates of the CaSKi and transfected cells were examined by cell count and their sensitivity to radiotherapy were examined by colony formation test. The apoptosis rates of each cell was determined by PI/Annexin V stained methods. Expressions of p53, bcl-2, and bax were determined by Flow cytometry analysis.
Anti-HPV16 E6-ribozyme can be expressed stably in transfected CaSKi-R cells. Northern blot showed that E6 mRNA was less in CaSKi-R than in CaSKi and CaSKi-P. The growth rate of CaSKi-R was much slower than that of CaSKi and CaSKi-P. The sensitivity of CaSKi-R cells to radiotherapy increased more than that of CaSKi and CaSKi-P cells. The ability of colony formation decreased (P < 0.05), while the apoptosis rates of CaSKi-R cells increased more than that of CaSKi and CaSKi-P cells(P < 0.01). Anti-HPV16 E6-ribozyme did significantly upregulate expression of p53, bax protein, and downregulate the expression of bcl-2 protein before and after radiotherapy (P < 0.01).
CaSKi-R cells transfected with Anti-HPVE 6-rivozyme showed growth inhibition and increased sensitivity to radiotherapy.
人乳头瘤病毒（HPV）是宫颈癌最重要的致病因素。E6病毒基因是最重要的致癌基因之一，高危型HPV基因型（如HPV16）的E6转化癌蛋白的表达水平似乎是维持恶性表型所必需的。放射治疗是当代宫颈癌治疗的一种标准化有效方式。本研究的目的是探讨抗HPV16 E6核酶对宫颈癌细胞系的放射增敏作用。
采用脂质体转染法，将抗HPV16 E6核酶和空真核表达质粒分别转染入CaSKi细胞系，分别命名为CaSKi-R和CaSKi-P。通过RNA斑点杂交观察转染细胞中核酶的表达。用Northern印迹法检测三种细胞中E6 mRNA的含量。通过细胞计数检测CaSKi细胞和转染细胞的生长速率，通过集落形成试验检测它们对放疗的敏感性。采用PI/Annexin V染色法测定各细胞的凋亡率。通过流式细胞术分析测定p53、bcl-2和bax的表达。
抗HPV16 E6核酶能在转染的CaSKi-R细胞中稳定表达。Northern印迹显示，CaSKi-R细胞中的E6 mRNA比CaSKi和CaSKi-P细胞中的少。CaSKi-R细胞的生长速率比CaSKi和CaSKi-P细胞慢得多。CaSKi-R细胞对放疗的敏感性比CaSKi和CaSKi-P细胞增加得更多。集落形成能力下降（P<0.05），而CaSKi-R细胞的凋亡率比CaSKi和CaSKi-P细胞增加得更多（P<0.01）。放疗前后，抗HPV16 E6核酶显著上调p53、bax蛋白的表达，下调bcl-2蛋白的表达（P<0.01）。
转染抗HPV E6核酶的CaSKi-R细胞表现出生长抑制和对放疗敏感性增加。
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