宫颈癌INK4a基因过表达及其与HPV感染的关系研究
|
摘要
背景与目的抑癌基因INK4a在多种肿瘤中阴性表达,但在几乎100%宫颈癌中过表达,究其发生原因,目前尚无定论。人乳头瘤病毒(human papilloma virus, HPV)感染是宫颈癌发生的必要条件,其中50%以上是HPV16型。HPV的E6和E7基因整合到宫颈上皮细胞并持续表达是引发宫颈癌的主要原因,同时也是维持宫颈癌恶性表型的必要条件。宫颈癌INK4a过表达是否与HPV感染有关,目前尚无统一认识。本研究拟通过RNA干扰技术,以抑制HPV16E7表达,进而检测对SiHa和CaSki细胞INK4a基因表达的影响;同时,比较不同HPV感染状态的宫颈癌细胞CaSki(HPV16感染),HeLa(HPV18感染),C-33A(无HPV感染)中INK4a表达水平,综合分析宫颈癌INK4a过表达与HPV感染的相关关系,初步阐明INK4a基因在宫颈癌中异常表达的可能机制,为宫颈癌的预防和早期诊断提供理论和实验依据。
方法
1.应用免疫组织化学技术检测126例HPV感染及未感染宫颈癌(cervical cancer,CC)、宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)及慢性宫颈炎中INK4a表达规律及其与HPV感染之间的相互关系
2.利用RNA干扰技术,针对HPV16E7的siRNA干涉SiHa和CaSki细胞,使HPV16E7 mRNA沉默
3.应用RT-PCR技术,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞HPV16E7及INK4a mRNA
4.应用Western blot技术,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞HPV16E7及INK4a蛋白表达
5.应用流式细胞仪,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞周期分布情况
6.联合应用RT-PCR和Western blot技术,比较不同HPV感染状态的宫颈癌细胞CaSki(HPV16感染),HeLa(HPV18感染),C-33A(无HPV感染)中INK4a表达水平
结果
1. 126例宫颈病变组织中INK4a表达规律及其与HPV感染之间的关系如下:
(1) 126例宫颈病变组织中,CC,CIN,慢性宫颈炎中p16~(INK4a)阳性表达率分别为100.00(50/50)、71.93(41/57)、42.11(8/19),三者间差异有显著性意义(p<0.05)。(2) 89例hr-HPV(+)宫颈病变组织中,CC,CIN,慢性宫颈炎中p16~(INK4a)阳性表达率分别为100.00%(42/42)、76.32%(29/38)、44.44%(4/9),三者间差异有显著性意义(p<0.05)。(3) 37例hr-HPV(-)宫颈病变组织中,CC,CIN,慢性宫颈炎中p16~(INK4a)阳性表达率分别为100.00%(8/8)、63.16%(12/19)、40.00%(4/10),三者间差异有显著性意义(p<0.05)。(4) 57例CIN中,CIN1,CIN2,CIN3 p16~(INK4a)阳性表达率分别为36.84%(7/19)、81.82%(18/22)、100.00%(16/16),且三者间差异有显著性意义(p<0.05)。(5) 38例hr-HPV(+)CIN中,CIN1,CIN2,CIN3 p16~(INK4a)阳性表达率分别为37.50%(3/8)、73.33%(11/15)、100.00%(15/15),CIN3与CIN2和CIN1之间差异均有显著性意义(p<0.05),但CIN2和CIN1之间差异无显著性意义(p>0.05)。(6) 107例CC和CIN组织中p16~(INK4a)表达和hr-HPV DNA检测同时阳性71例,占66.36%(71/107);p16~(INK4a)阳性表达而hr-HPV(-)21例,占19.63%(21/107);p16~(INK4a)阴性表达而hr-HPV(+)8例,占7.57%(8/107);p16~(INK4a)表达或hr-HPV DNA检测单一阳性加上共阳性占93.46%(100/107)。Pearson相关分析CC和CIN中p16~(INK4a)表达与宫颈hr-HPV感染相互间无相关性(p>0.05)。(7) 47例CC和16例CIN3中p16~(INK4a)全部阳性表达,无论HPV感染状态如何;38例hr-HPV(+)CIN组织中p16~(INK4a)表达29例,占76.32%(29/38);19例hr-HPV(-)CIN组织中p16~(INK4a)表达12例,占63.16%(12/19),两者间差异没有显著性意义(p>0.05);15例hr-HPV(+)CIN2组织中p16~(INK4a)表达11例,占73.33%(11/15);7例hr-HPV(-)CIN2组织中p16~(INK4a)表达7例,占100.00%(7/7),两者间差异没有显著性意义(p>0.05);8例hr-HPV(+)CIN1组织中p16~(INK4a)表达3例,占37.50%(3/8);11例hr-HPV(-)CIN1组织中p16~(INK4a)表达4例,占36.36%(4/11);两者间差异没有显著性意义(p>0.05);9例hr-HPV(+)慢性宫颈炎中p16~(INK4a)表达4例,占44.44%(4/9);10例hr-HPV(-)慢性宫颈炎中p16~(INK4a)表达4例,占40.00%(4/10);两者间差异没有显著性意义(p>0.05)。比较分析发现,p16~(INK4a)蛋白在hr-HPV(+)和hr-HPV(-)宫颈病变组织中阳性表达率差异无显著性意义(p>0.05)。
2. siHPV16E7 001,siHPV16E7 002,siHPV16E7 003干扰CaSki细胞后,HPV16E7 mRNA的含量下降,其中siHPV16E7 001干扰后,HPV16E7 mRNA的含量下降最明显,约80%;不同浓度siHPV16E7 001干扰CaSki细胞后,60nM组干扰效率最强,与未干扰组比较差异有显著性意义(p<0.01),且细胞毒性轻微。
3.和SiHa-Nontransfection组(0.6033±0.0186)及SiHa-siNControl组(0.5500±0.0173)相比,在SiHa-siHPV16E7组(0.3067±0.0120)中,RT-PCR方法证明其HPV16E7 mRNA表达显著降低(p<0.01),Western blot方法亦证明SiHa-siHPV16E7组的HPV16E7蛋白表达量显著低于SiHa-Nontransfection组和SiHa-siNControl组(p<0.01)。提示通过RNA干扰方法能有效地抑制SiHa细胞的HPV16E7表达。
4.和CaSki-Nontransfection组(1.1733±0.0667)及CaSki-siNControl组(1.3933±0.1667)相比,在CaSki-siHPV16E7组(0.4033±0.0328)中,RT-PCR方法证明其HPV16E7 mRNA表达显著降低(p<0.01),Western blot方法亦证明CaSki-siHPV16E7组的HPV16E7蛋白表达量显著低于CaSki-Nontransfection组和CaSki-siNControl组(p<0.01)。提示通过RNA干扰方法能有效地抑制CaSki细胞的HPV16E7表达。
5.和SiHa-Nontransfection组(1.0033±0.0233)及SiHa-siNControl组(0.9267±0.0371)相比,在SiHa-siHPV16E7组(0.5833±0.0133)中,RT-PCR方法证明其INK4a mRNA表达显著降低(p<0.01),Western blot方法亦证明SiHa-siHPV16E7组的INK4a蛋白表达量显著低于SiHa-Nontransfection组和SiHa-siNControl组(p<0.01)。提示通过siHPV16E7干扰能有效地抑制SiHa细胞的INK4a表达。
6.和CaSki-Nontransfection组(0.7833±0.0088)及CaSki-siNControl组(0.8533±0.0353)相比,在CaSki-siHPV16E7组(0.4767±0.0145)中,RT-PCR方法证明其INK4a mRNA表达显著降低(p<0.01),Western blot方法亦证明CaSki-siHPV16E7组的INK4a蛋白表达量显著低于CaSki-Nontransfection组和CaSki-siNControl组(p<0.01)。提示通过siHPV16E7干扰能有效地抑制CaSki细胞的INK4a表达。
7.细胞周期分析结果表明:HPV16E7干扰SiHa细胞24h,48h,72h后,G0-G1期细胞百分数较SiHa-Nontransfection组依次增加了1.1%,0.7%,31.7%,进入S期的细胞百分数较SiHa-Nontransfection组依次减少了0.2%,2.5%,6.4%;HPV16E7干扰CaSki细胞24h,48h,72h后,G0-G1期细胞百分数较CaSki-Nontransfection组依次增加了8.7%,12.7%,15.6%,进入S期的细胞百分数较CaSki-Nontransfection组减少了1.3%,4.3%,14.9%。提示通过siHPV16E7干扰能将更多的细胞阻滞在G0-G1期。
8. RT-PCR检测不同HPV感染状态的宫颈癌细胞CaSki(0.3967±0.0491) ,HeLa(0.5567±0.1037),C-33A(0.5633±0.0722)中INK4a mRNA表达无明显差异(p>0.05);Western blot方法亦证明CaSki(0.4152±0.0255) , HeLa(0.5094±0.0361) , C-33A (0.5895±0.0409)中INK4a蛋白表达无显著性差异(p>0.05)。提示不同HPV感染状态的宫颈癌细胞中INK4a表达规律趋于一致。
结论
1.无论是否合并HPV感染,宫颈癌及CIN3组织中INK4a基因都过表达。
2. siHPV16E7可序列特异性下调宫颈癌SiHa,CaSki细胞HPV16E7基因水平,显著降低HPV16E7蛋白表达。
3.通过RNA干扰抑制HPV16E7表达后,能显著抑制INK4a基因表达,同时将更多的细胞阻滞在G0-G1期。
4.不同HPV感染状态的宫颈癌细胞中INK4a表达规律趋于一致。
综上提示,在合并HPV感染的宫颈癌中INK4a基因过表达与HPV感染有关;未合并HPV感染的宫颈癌中INK4a基因同样过表达,应另有其他机制存在。检测INK4a基因表达,对于宫颈癌的早期诊断和筛查具有非常有价值的临床意义。
Background and Objective Tumor suppressor gene INK4a encodes p16~(INK4a) protein and aberrant expression of p16~(INK4a) protein has been reported in various malignancies, whereas p16~(INK4a) overexpression has been detected in almost all precancerous and cancerous lesions of the cervix and it’s reason has been indefinite. Human papilloma virus(HPV) infection was essential for genesis of cervical carcinomas. About 50% of cervical cancers was related with HPV type 16(HPV16).After infection, E6 and E7 gene of HPV were integrated to genome of cervical epithelium. Continued expression of transforming oncoprotein E6 and E7 not only drives the neoplastic progression in cervical epithelium but also plays important role in maintaining malignant phenotype of cervical cancer cells. Correlations between p16~(INK4a) overexpression and HPV infection has no accepted argument. This study was carried out to assess the correlations between p16~(INK4a) expression and the HPV infection by HPV16E7 gene silencing using small interfering RNA(siRNA) in human cervical carcinomas cell lines SiHa and CaSki and by comparing p16~(INK4a) expression in CaSki(HPV16 infection), HeLa(HPV18 infection), C-33A(no HPV infection); to ascertain the role of p16~(INK4a) immunostaining as an early biomarker of uterine cervix carcinomas.
Methods
1. The expression of p16~(INK4a) protein was detected in 126 cervical lesions samples by immunohistochemical staining, including cervical cancer(CC),cervical intraepithelial neoplasia(CIN) and cervicitis with high-risk Human papillomavirus(hr-HPV) infected or no, and to investigate the relationships of p16~(INK4a) protein expression with hr-HPV state.
2. RNA interference(RNAi) originated by chemically synthetic small interference RNA (siRNA) could induce HPV16E7 gene silencing in cervical carcinomas cell lines SiHa and CaSki.
3. Expressions of HPV16E7 and INK4a mRNA were examined by semiquantitative reverse transcription polymerase chain(RT-PCR) in SiHa-Nontransfection, SiHa -siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7,CaSki-siNControl cells respectively.
4. Expressions of HPV16E7 and INK4a protein were examined by Western blot in SiHa-Nontransfection, SiHa-siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7, CaSki-siNControl cells respectively.
5. Cell cycle analysis were assessed by flowcytometry(FCM)in SiHa-Nontransfection, SiHa-siHPV16E7 and CaSki-Nontransfection, CaSki-siHPV16E7, cells respectively.
6. Expressions of INK4a mRNA and protein were examined by RT-PCR and Western blot in CaSki, HeLa and C-33A cells with or without HPV16E7 infection,respectively.
Results
1. Immunostaining for p16~(INK4a) and relationship between p16~(INK4a) immunoreactivity and HPV in 126 cervical lesions samples: (1) The positive expression rates of p16~(INK4a) in CC,CIN and cervicitis samples were 100.00(50/50), 71.93%(41/57) and 42.11%(8/19) respectively, and there was obvious difference between the three groups(p<0.05). (2)The positive expression rates of p16~(INK4a) in CC,CIN and cervicitis samples(n=89)with HPV infection were 100.00%(42/42), 76.32%(29/38) and 44.44%(4/9) respectively, and there was obvious difference between the three groups(p<0.05). (3) The positive expression rates of p16~(INK4a) in CC,CIN and cervicitis samples(n=37) without HPV infection were 100.00% (8/8), 63.16%(12/19) and 40.00%(4/10) respectively, and there was obvious difference between the three groups(p<0.05). (4) The positive expression rates of p16~(INK4a) in CIN1,CIN2 and CIN3 samples(n=57) were 36.84%(7/19), 81.82%(18/22) and 100.00%(16/16), respectively, and there was obvious difference between the three groups(p<0.05). (5) The positive expression rates of p16~(INK4a) in CIN1,CIN2 and CIN3 samples(n=57) with HPV infection were 37.50%(3/8), 73.33%(11/15) and 100.00%(15/15) respectively, and there was obvious difference between CIN3 and CIN2 or CIN1, but there was no obvious difference between CIN2 and CIN1(p>0.05). (6) In 107 CC and CIN, 71(66.36%) cases showed simultaneous positive expression of p16~(INK4a) proteins and hr-HPV DNA, 21(19.63%) cases showed positive expression of p16~(INK4a) and negative expression of hr-HPV DNA, 8(7.57%) cases showed negative expression of p16~(INK4a) and positive expression of hr-HPV DNA, the positive rates of p16~(INK4a) and/or hr-HPV DNA expressions was 93.46%, whereas there was no significant relationship between p16~(INK4a) expression and hr-HPV DNA infection(p>0.05). (7) p16~(INK4a) protein was positive in all of CC(n=47) and CIN3(n=16) cases with or without HPV infection. The positive expression rates of p16~(INK4a) in hr-HPV(+) and hr-HPV(-) CIN samples were 76.32%(29/38) and 63.16%(12/19) respectively, and there was no obvious difference between the two groups(p<0.05), as well as in hr-HPV(+) and hr-HPV(-) cervicitis tissues, the positive rates of p16~(INK4a) expression were 44.44%(4/9) and 40.00%(4/10) respectively. This expression increases from cervicitis, CIN to CC, and the significant difference was observed between the groups of CC, CIN and cervicitis with hr-HPV infected or no(p<0.05).
2. When CaSki cells were interfered by siHPV16E7 001,siHPV16E7 002 and siHPV16E7 003, the levels of mRNA encoding HPV16E7 in cells was reduced, especially siHPV16E7 001 can make HPV16E7 mRNA reduce by 80%. Moreover, the most suitable multiplicity of infection of siHPV16E7 001 for CaSki cells was 60nM.
3. Compared with SiHa-Nontransfection(0.6033±0.0186) and SiHa-siNControl (0.5500±0.0173), HPV16E7 mRNA expression(0.3067±0.0120) surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group(p<0.01), and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in SiHa cells could be inhibited efficiently by RNAi.
4. Compared with CaSki-Nontransfection(1.1733±0.0667) and CaSki-siNControl (1.3933±0.1667), HPV16E7 mRNA expression(0.4033±0.0328) surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group(p<0.01), and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in CaSki cells could be inhibited efficiently by RNAi.
5. Compared with SiHa-Nontransfection(1.0033±0.0233) and SiHa-siNControl (0.9267±0.0371), INK4a mRNA expression(0.5833±0.0133) surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group(p<0.01), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in SiHa cells could be inhibited efficiently by siHPV16E7 interference.
6. Compared with CaSki-Nontransfection(0.7833±0.0088) and CaSki-siNControl (0.8533±0.0353), INK4a mRNA expression(0.4767±0.0145) surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group(p<0.01), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in CaSki cells could be inhibited efficiently by siHPV16E7 interference.
7. Cell cycle analysis showed that siHPV16E7 induced accumulation of SiHa and CaSki cells in G0-G1 phase by 31.7% and 15.6% with a decrease in the percentage of cells in S-phase by 6.4% and 14.9% relative to control. These sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.
8. Compared with CaSki(0.3967±0.0491) and HeLa(0.5567±0.1037), INK4a mRNA expression(0.5633±0.0722) surveyed by RT-PCR had no obvious difference in C-33A cells (p>0.05), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression was at equal pace in CC cells with or without HPV infection.
Conclusion
1. Overexpression of INK4A gene in precancerous and cancerous lesions of the cervix with or without HPV infection.
2. These sequence specific siRNAs showed a blockbuster effect in downregulation of HPV16E7 gene expression in CC cells SiHa and CaSki.
3. INK4a expression in SiHa and CaSki cells could be inhibited efficiently by siHPV16E7 interference,moreover, these sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.
4. INK4a expression was with one accord in CC cells with or without HPV infection. Therefore/ In a word, the above mentioned conclusion suggested that INK4a expression was at equal pace in CC cells with or without HPV infection. Overexpression of p16 INK4a protein is associated with HPV in cervical cell carcinoma and dysplasia with HPV infection,whereas there are else mechanisms on overexpression of p16 INK4a in CC and dysplasia without HPV infection. Overexpression of INK4a gene help to define the screening and early diagnosis of cervical cancer cases.
方法
1.应用免疫组织化学技术检测126例HPV感染及未感染宫颈癌(cervical cancer,CC)、宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)及慢性宫颈炎中INK4a表达规律及其与HPV感染之间的相互关系
2.利用RNA干扰技术,针对HPV16E7的siRNA干涉SiHa和CaSki细胞,使HPV16E7 mRNA沉默
3.应用RT-PCR技术,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞HPV16E7及INK4a mRNA
4.应用Western blot技术,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞HPV16E7及INK4a蛋白表达
5.应用流式细胞仪,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞周期分布情况
6.联合应用RT-PCR和Western blot技术,比较不同HPV感染状态的宫颈癌细胞CaSki(HPV16感染),HeLa(HPV18感染),C-33A(无HPV感染)中INK4a表达水平
结果
1. 126例宫颈病变组织中INK4a表达规律及其与HPV感染之间的关系如下:
(1) 126例宫颈病变组织中,CC,CIN,慢性宫颈炎中p16~(INK4a)阳性表达率分别为100.00(50/50)、71.93(41/57)、42.11(8/19),三者间差异有显著性意义(p<0.05)。(2) 89例hr-HPV(+)宫颈病变组织中,CC,CIN,慢性宫颈炎中p16~(INK4a)阳性表达率分别为100.00%(42/42)、76.32%(29/38)、44.44%(4/9),三者间差异有显著性意义(p<0.05)。(3) 37例hr-HPV(-)宫颈病变组织中,CC,CIN,慢性宫颈炎中p16~(INK4a)阳性表达率分别为100.00%(8/8)、63.16%(12/19)、40.00%(4/10),三者间差异有显著性意义(p<0.05)。(4) 57例CIN中,CIN1,CIN2,CIN3 p16~(INK4a)阳性表达率分别为36.84%(7/19)、81.82%(18/22)、100.00%(16/16),且三者间差异有显著性意义(p<0.05)。(5) 38例hr-HPV(+)CIN中,CIN1,CIN2,CIN3 p16~(INK4a)阳性表达率分别为37.50%(3/8)、73.33%(11/15)、100.00%(15/15),CIN3与CIN2和CIN1之间差异均有显著性意义(p<0.05),但CIN2和CIN1之间差异无显著性意义(p>0.05)。(6) 107例CC和CIN组织中p16~(INK4a)表达和hr-HPV DNA检测同时阳性71例,占66.36%(71/107);p16~(INK4a)阳性表达而hr-HPV(-)21例,占19.63%(21/107);p16~(INK4a)阴性表达而hr-HPV(+)8例,占7.57%(8/107);p16~(INK4a)表达或hr-HPV DNA检测单一阳性加上共阳性占93.46%(100/107)。Pearson相关分析CC和CIN中p16~(INK4a)表达与宫颈hr-HPV感染相互间无相关性(p>0.05)。(7) 47例CC和16例CIN3中p16~(INK4a)全部阳性表达,无论HPV感染状态如何;38例hr-HPV(+)CIN组织中p16~(INK4a)表达29例,占76.32%(29/38);19例hr-HPV(-)CIN组织中p16~(INK4a)表达12例,占63.16%(12/19),两者间差异没有显著性意义(p>0.05);15例hr-HPV(+)CIN2组织中p16~(INK4a)表达11例,占73.33%(11/15);7例hr-HPV(-)CIN2组织中p16~(INK4a)表达7例,占100.00%(7/7),两者间差异没有显著性意义(p>0.05);8例hr-HPV(+)CIN1组织中p16~(INK4a)表达3例,占37.50%(3/8);11例hr-HPV(-)CIN1组织中p16~(INK4a)表达4例,占36.36%(4/11);两者间差异没有显著性意义(p>0.05);9例hr-HPV(+)慢性宫颈炎中p16~(INK4a)表达4例,占44.44%(4/9);10例hr-HPV(-)慢性宫颈炎中p16~(INK4a)表达4例,占40.00%(4/10);两者间差异没有显著性意义(p>0.05)。比较分析发现,p16~(INK4a)蛋白在hr-HPV(+)和hr-HPV(-)宫颈病变组织中阳性表达率差异无显著性意义(p>0.05)。
2. siHPV16E7 001,siHPV16E7 002,siHPV16E7 003干扰CaSki细胞后,HPV16E7 mRNA的含量下降,其中siHPV16E7 001干扰后,HPV16E7 mRNA的含量下降最明显,约80%;不同浓度siHPV16E7 001干扰CaSki细胞后,60nM组干扰效率最强,与未干扰组比较差异有显著性意义(p<0.01),且细胞毒性轻微。
3.和SiHa-Nontransfection组(0.6033±0.0186)及SiHa-siNControl组(0.5500±0.0173)相比,在SiHa-siHPV16E7组(0.3067±0.0120)中,RT-PCR方法证明其HPV16E7 mRNA表达显著降低(p<0.01),Western blot方法亦证明SiHa-siHPV16E7组的HPV16E7蛋白表达量显著低于SiHa-Nontransfection组和SiHa-siNControl组(p<0.01)。提示通过RNA干扰方法能有效地抑制SiHa细胞的HPV16E7表达。
4.和CaSki-Nontransfection组(1.1733±0.0667)及CaSki-siNControl组(1.3933±0.1667)相比,在CaSki-siHPV16E7组(0.4033±0.0328)中,RT-PCR方法证明其HPV16E7 mRNA表达显著降低(p<0.01),Western blot方法亦证明CaSki-siHPV16E7组的HPV16E7蛋白表达量显著低于CaSki-Nontransfection组和CaSki-siNControl组(p<0.01)。提示通过RNA干扰方法能有效地抑制CaSki细胞的HPV16E7表达。
5.和SiHa-Nontransfection组(1.0033±0.0233)及SiHa-siNControl组(0.9267±0.0371)相比,在SiHa-siHPV16E7组(0.5833±0.0133)中,RT-PCR方法证明其INK4a mRNA表达显著降低(p<0.01),Western blot方法亦证明SiHa-siHPV16E7组的INK4a蛋白表达量显著低于SiHa-Nontransfection组和SiHa-siNControl组(p<0.01)。提示通过siHPV16E7干扰能有效地抑制SiHa细胞的INK4a表达。
6.和CaSki-Nontransfection组(0.7833±0.0088)及CaSki-siNControl组(0.8533±0.0353)相比,在CaSki-siHPV16E7组(0.4767±0.0145)中,RT-PCR方法证明其INK4a mRNA表达显著降低(p<0.01),Western blot方法亦证明CaSki-siHPV16E7组的INK4a蛋白表达量显著低于CaSki-Nontransfection组和CaSki-siNControl组(p<0.01)。提示通过siHPV16E7干扰能有效地抑制CaSki细胞的INK4a表达。
7.细胞周期分析结果表明:HPV16E7干扰SiHa细胞24h,48h,72h后,G0-G1期细胞百分数较SiHa-Nontransfection组依次增加了1.1%,0.7%,31.7%,进入S期的细胞百分数较SiHa-Nontransfection组依次减少了0.2%,2.5%,6.4%;HPV16E7干扰CaSki细胞24h,48h,72h后,G0-G1期细胞百分数较CaSki-Nontransfection组依次增加了8.7%,12.7%,15.6%,进入S期的细胞百分数较CaSki-Nontransfection组减少了1.3%,4.3%,14.9%。提示通过siHPV16E7干扰能将更多的细胞阻滞在G0-G1期。
8. RT-PCR检测不同HPV感染状态的宫颈癌细胞CaSki(0.3967±0.0491) ,HeLa(0.5567±0.1037),C-33A(0.5633±0.0722)中INK4a mRNA表达无明显差异(p>0.05);Western blot方法亦证明CaSki(0.4152±0.0255) , HeLa(0.5094±0.0361) , C-33A (0.5895±0.0409)中INK4a蛋白表达无显著性差异(p>0.05)。提示不同HPV感染状态的宫颈癌细胞中INK4a表达规律趋于一致。
结论
1.无论是否合并HPV感染,宫颈癌及CIN3组织中INK4a基因都过表达。
2. siHPV16E7可序列特异性下调宫颈癌SiHa,CaSki细胞HPV16E7基因水平,显著降低HPV16E7蛋白表达。
3.通过RNA干扰抑制HPV16E7表达后,能显著抑制INK4a基因表达,同时将更多的细胞阻滞在G0-G1期。
4.不同HPV感染状态的宫颈癌细胞中INK4a表达规律趋于一致。
综上提示,在合并HPV感染的宫颈癌中INK4a基因过表达与HPV感染有关;未合并HPV感染的宫颈癌中INK4a基因同样过表达,应另有其他机制存在。检测INK4a基因表达,对于宫颈癌的早期诊断和筛查具有非常有价值的临床意义。
Background and Objective Tumor suppressor gene INK4a encodes p16~(INK4a) protein and aberrant expression of p16~(INK4a) protein has been reported in various malignancies, whereas p16~(INK4a) overexpression has been detected in almost all precancerous and cancerous lesions of the cervix and it’s reason has been indefinite. Human papilloma virus(HPV) infection was essential for genesis of cervical carcinomas. About 50% of cervical cancers was related with HPV type 16(HPV16).After infection, E6 and E7 gene of HPV were integrated to genome of cervical epithelium. Continued expression of transforming oncoprotein E6 and E7 not only drives the neoplastic progression in cervical epithelium but also plays important role in maintaining malignant phenotype of cervical cancer cells. Correlations between p16~(INK4a) overexpression and HPV infection has no accepted argument. This study was carried out to assess the correlations between p16~(INK4a) expression and the HPV infection by HPV16E7 gene silencing using small interfering RNA(siRNA) in human cervical carcinomas cell lines SiHa and CaSki and by comparing p16~(INK4a) expression in CaSki(HPV16 infection), HeLa(HPV18 infection), C-33A(no HPV infection); to ascertain the role of p16~(INK4a) immunostaining as an early biomarker of uterine cervix carcinomas.
Methods
1. The expression of p16~(INK4a) protein was detected in 126 cervical lesions samples by immunohistochemical staining, including cervical cancer(CC),cervical intraepithelial neoplasia(CIN) and cervicitis with high-risk Human papillomavirus(hr-HPV) infected or no, and to investigate the relationships of p16~(INK4a) protein expression with hr-HPV state.
2. RNA interference(RNAi) originated by chemically synthetic small interference RNA (siRNA) could induce HPV16E7 gene silencing in cervical carcinomas cell lines SiHa and CaSki.
3. Expressions of HPV16E7 and INK4a mRNA were examined by semiquantitative reverse transcription polymerase chain(RT-PCR) in SiHa-Nontransfection, SiHa -siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7,CaSki-siNControl cells respectively.
4. Expressions of HPV16E7 and INK4a protein were examined by Western blot in SiHa-Nontransfection, SiHa-siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7, CaSki-siNControl cells respectively.
5. Cell cycle analysis were assessed by flowcytometry(FCM)in SiHa-Nontransfection, SiHa-siHPV16E7 and CaSki-Nontransfection, CaSki-siHPV16E7, cells respectively.
6. Expressions of INK4a mRNA and protein were examined by RT-PCR and Western blot in CaSki, HeLa and C-33A cells with or without HPV16E7 infection,respectively.
Results
1. Immunostaining for p16~(INK4a) and relationship between p16~(INK4a) immunoreactivity and HPV in 126 cervical lesions samples: (1) The positive expression rates of p16~(INK4a) in CC,CIN and cervicitis samples were 100.00(50/50), 71.93%(41/57) and 42.11%(8/19) respectively, and there was obvious difference between the three groups(p<0.05). (2)The positive expression rates of p16~(INK4a) in CC,CIN and cervicitis samples(n=89)with HPV infection were 100.00%(42/42), 76.32%(29/38) and 44.44%(4/9) respectively, and there was obvious difference between the three groups(p<0.05). (3) The positive expression rates of p16~(INK4a) in CC,CIN and cervicitis samples(n=37) without HPV infection were 100.00% (8/8), 63.16%(12/19) and 40.00%(4/10) respectively, and there was obvious difference between the three groups(p<0.05). (4) The positive expression rates of p16~(INK4a) in CIN1,CIN2 and CIN3 samples(n=57) were 36.84%(7/19), 81.82%(18/22) and 100.00%(16/16), respectively, and there was obvious difference between the three groups(p<0.05). (5) The positive expression rates of p16~(INK4a) in CIN1,CIN2 and CIN3 samples(n=57) with HPV infection were 37.50%(3/8), 73.33%(11/15) and 100.00%(15/15) respectively, and there was obvious difference between CIN3 and CIN2 or CIN1, but there was no obvious difference between CIN2 and CIN1(p>0.05). (6) In 107 CC and CIN, 71(66.36%) cases showed simultaneous positive expression of p16~(INK4a) proteins and hr-HPV DNA, 21(19.63%) cases showed positive expression of p16~(INK4a) and negative expression of hr-HPV DNA, 8(7.57%) cases showed negative expression of p16~(INK4a) and positive expression of hr-HPV DNA, the positive rates of p16~(INK4a) and/or hr-HPV DNA expressions was 93.46%, whereas there was no significant relationship between p16~(INK4a) expression and hr-HPV DNA infection(p>0.05). (7) p16~(INK4a) protein was positive in all of CC(n=47) and CIN3(n=16) cases with or without HPV infection. The positive expression rates of p16~(INK4a) in hr-HPV(+) and hr-HPV(-) CIN samples were 76.32%(29/38) and 63.16%(12/19) respectively, and there was no obvious difference between the two groups(p<0.05), as well as in hr-HPV(+) and hr-HPV(-) cervicitis tissues, the positive rates of p16~(INK4a) expression were 44.44%(4/9) and 40.00%(4/10) respectively. This expression increases from cervicitis, CIN to CC, and the significant difference was observed between the groups of CC, CIN and cervicitis with hr-HPV infected or no(p<0.05).
2. When CaSki cells were interfered by siHPV16E7 001,siHPV16E7 002 and siHPV16E7 003, the levels of mRNA encoding HPV16E7 in cells was reduced, especially siHPV16E7 001 can make HPV16E7 mRNA reduce by 80%. Moreover, the most suitable multiplicity of infection of siHPV16E7 001 for CaSki cells was 60nM.
3. Compared with SiHa-Nontransfection(0.6033±0.0186) and SiHa-siNControl (0.5500±0.0173), HPV16E7 mRNA expression(0.3067±0.0120) surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group(p<0.01), and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in SiHa cells could be inhibited efficiently by RNAi.
4. Compared with CaSki-Nontransfection(1.1733±0.0667) and CaSki-siNControl (1.3933±0.1667), HPV16E7 mRNA expression(0.4033±0.0328) surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group(p<0.01), and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in CaSki cells could be inhibited efficiently by RNAi.
5. Compared with SiHa-Nontransfection(1.0033±0.0233) and SiHa-siNControl (0.9267±0.0371), INK4a mRNA expression(0.5833±0.0133) surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group(p<0.01), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in SiHa cells could be inhibited efficiently by siHPV16E7 interference.
6. Compared with CaSki-Nontransfection(0.7833±0.0088) and CaSki-siNControl (0.8533±0.0353), INK4a mRNA expression(0.4767±0.0145) surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group(p<0.01), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in CaSki cells could be inhibited efficiently by siHPV16E7 interference.
7. Cell cycle analysis showed that siHPV16E7 induced accumulation of SiHa and CaSki cells in G0-G1 phase by 31.7% and 15.6% with a decrease in the percentage of cells in S-phase by 6.4% and 14.9% relative to control. These sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.
8. Compared with CaSki(0.3967±0.0491) and HeLa(0.5567±0.1037), INK4a mRNA expression(0.5633±0.0722) surveyed by RT-PCR had no obvious difference in C-33A cells (p>0.05), and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression was at equal pace in CC cells with or without HPV infection.
Conclusion
1. Overexpression of INK4A gene in precancerous and cancerous lesions of the cervix with or without HPV infection.
2. These sequence specific siRNAs showed a blockbuster effect in downregulation of HPV16E7 gene expression in CC cells SiHa and CaSki.
3. INK4a expression in SiHa and CaSki cells could be inhibited efficiently by siHPV16E7 interference,moreover, these sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.
4. INK4a expression was with one accord in CC cells with or without HPV infection. Therefore/ In a word, the above mentioned conclusion suggested that INK4a expression was at equal pace in CC cells with or without HPV infection. Overexpression of p16 INK4a protein is associated with HPV in cervical cell carcinoma and dysplasia with HPV infection,whereas there are else mechanisms on overexpression of p16 INK4a in CC and dysplasia without HPV infection. Overexpression of INK4a gene help to define the screening and early diagnosis of cervical cancer cases.
引文
1. Dehn D,Torkko KC,Shroyer KR.Human papillomavirus testing and molecular markers of cervical dysplasia and carcinoma.Cancer.2007;111(1):1-14.
2. Nam EJ, Kim JW, Kim SW, et al. The expressions of the Rb pathway in cervical intraepithelial neoplasia; predictive and prognostic significance. Gynecologic Oncology. 2007; 104(1):207-211.
3. Hinkula M, Pukkala E, Kyyronen P, et al. A population-based study on the risk of cervical cancer and cervical intraepithelial neoplasia among grand multiparous worsen in Finland. Br J Cancer. 2004; 90 (5):1025-1029.
4. Gibson SL, Dai CY, Lee HW, et al. Inhibition of colon tumor progression and angiogenesis by the Ink4a/Arf locus. Cancer-Res. 2003; 63(4):742-746.
5. Kuo ML, Duncavage EJ, Mathew R, et al. Arf induces p53-dependent and -independent antiproliferative genes. Cancer-Res. 2003; 63(5):1046-1053.
6. Silva J, Silva JM, Dominguez G, et al. Concomitant expression of p16INK4a and p14ARF in primary breast cancer and analysis of inactivation mechanisms. J-Pathol. 2003; 199(3):289-297.
7. Kamijo T, Zindy F, Roussel MF, et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell. 1997; 91(5): 649-659.
8. Gessner C, Liebers U, KuhnH, et al. BAX and p16INK4A are independent positive prognostic markers for advanced tumour stage of nonsmall cell lung cancer. Eur Respir J. 2002; 19(1):134-140.
9. Nicholson SA, Okby NT, Khan MA, et al. Alterations of p14ARF, p53, and p73 genes involved in the E2F-1-mediated apoptotic pathways in non-small cell lung carcinoma. Cancer Res. 2001; 61(14):5636-5643.
10. Tannapfel A, Busse C, Weinans L, et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas. Oncogene. 2001; 20(48):7104-7109.
11. Caca K, Feisthammel J, Klee K, et al. Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines. Int J Cancer. 2002; 97(4):481-488.
12. Sharpless NE. INK4a/ARF: a multifunctional tumor suppressor locus. Mutat-Res. 2005;576(1-2):22-38.
13. Kanao H. Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer. Cancer Lett. 2004; 213(1):31-37.
14. Feng W, Xiao J, Zhang Z, et al. Senescence and apoptosis in carcinogenesis of cervical squamous carcinoma. Mod Pathol. 2007;20(9):961-966.
15. Lau WM, Ho TH, Hui KM. p16INK4A-silencing augments DNA damage-induced apoptosis in cervical cancer cells. Oncogene. 2007; 26(41):6050-6060.
16. Kamijo T, Weber JD, Zambetti G, et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc-Natl-Acad-Sci-U-S-A. 1998; 95(14): 8292-8297.
17. Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell. 1998; 92(6): 725-734.
18. Zochbauer-Muller S, Fong KM, Virmani AK, et al. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer-Res. 2001; 61(1):249-255.
19. Redman R, Rufforny I, Liu C, et al. The Utility of p16(Ink4a) in Discriminating Between Cervical Intraepithelial Neoplasia 1 and Nonneoplastic Equivocal Lesions of the Cervix. Arch Pathol Lab Med. 2008;132(5):795-799.
20. Samama B, Schaeffer C, Boehm N. P16 expression in relation to human papillomavirus in liquid-based cervical smears. Gynecol Oncol. 2008;109(2):285-290.
21. McKay JA, Douglas JJ, Ross VG, et al. Analysis of key cell-cycle checkpoint proteins in colorectal tumours. J-Pathol. 2002; 196(4): 386-393.
22. van der Avoort IA, Shirango H, Hoevenaars BM, et al. Vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways. Int J Gynecol Pathol. 2006; 25(1):22-29.
23. Benevolo M, Vocaturo A, Mottolese M, et al. Clinical role of p16INK4a expression in liquid-based cervical cytology: correlation with HPV testing and histologic diagnosis. Am J Clin Pathol. 2008;129(4):606-612.
24. Ishikawa M, Fujii T, Saito M, et al. Overexpression of p16INK4a as an indicator for human papillomavirus oncogenic activity in cervical squamous neoplasia. Int J Gynecol Cancer. 2006;16(1):347-353.
25. Kanao H, Enomoto T, Ueda Y, et al. Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer. Cancer Lett. 2004; 213(1):31-37.
26. Khoo CM, Carrasco DR, Bosenberg MW, et al. Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse. Proc Natl Acad Sci U S A. 2007;104(10):3931-3936.
27. Blokx WA, Lesterhuis JJ, Andriessen MP, et al. CDKN2A(INK4A-ARF)mutation analysis to distinguish cutaneous melanoma metastasis from a second primary melanoma. Am J Surg Pathol. 2007; 31(4):637-641.
28. O'Neill CJ, McCluggage WG. p16 expression in the female genital tract and its value in diagnosis. Adv Anat Pathol. 2006; 13(1):8-15.
29. Liang J, Mittal KR, Wei JJ, et al. Utility of p16INK4a, CEA, Ki67, P53 and ER/PR in the differential diagnosis of benign, premalignant, and malignant glandular lesions of the uterine cervix and their relationship with Silverberg scoring system for endocervical glandular lesions. Int J Gynecol Pathol. 2007; 26(1):71-75.
30. Eleuterio J Jr, Giraldo PC, Goncalves AK, et al. Prognostic markers of high-grade squamous intraepithelial lesions: the role of p16INK4a and high-risk human papillomavirus. Acta Obstet Gynecol Scand. 2007; 86(1):94-98.
31. Norman I, Brismar S, Zhu J, et al. p16(INK4a) immunocytochemistry in liquid-based cervical cytology: is it feasible for clinical use? Int J Oncol. 2007;31(6):1339-1343.
32. Redman R, Rufforny I, Liu C, et al. The Utility of p16(Ink4a) in Discriminating Between Cervical Intraepithelial Neoplasia 1 and Nonneoplastic Equivocal Lesions of the Cervix. Arch Pathol Lab Med. 2008;132(5):795-799.
33. Hashi A, Xu JY, Kondo T, et al. p16INK4a overexpression independent of human papillomavirus infection in lobular endocervical glandular hyperplasia. Int J Gynecol Pathol. 2006; 25(2):187-194.
34. Galmiche L, Coste-Burel M, Lopes P, et al. The expression of p16 is not correlated with HPV status in CINI. Histopathology. 2006; 48(6):1365-2559.
35. Denny L. The prevention of cervical cancer in developing countries. BJOG. 2005; 112(9):1204-1212.
36. Santin AD. Gene expression profiles of primary HPV16- and HPV18-infected early stage cervical cancers and normal cervical epithelium: identification of novel candidatemolecular markers for cervical cancer diagnosis and therapy. Virology. 2005; 331(2): 269-291.
37. Unger ER. Human papillomavirus and cervical cancer. Emerg-Infect-Dis. 2004; 10(11):2031-2032.
38. Dallenbach-Hellweg G. Traditional and new molecular methods for early detection of cervical cancer. Arkh-Patol. 2004; 66(5):35-39.
39. Schellekens MC. Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. Gynecol-Oncol. 2004; 93(1):49-53.
40. Horn LC, Reichert A, Oster A, et al. Immunostaining for p16INK4a Used as a Conjunctive Tool Improves Interobserver Agreement of the Histologic Diagnosis of Cervical Intraepithelial Neoplasia. Am J Surg Pathol. 2008 Jan 24 [Epub ahead of print]
41. Zamore PD, Tuschl T, Sharp PA, et al. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23nt intervals. Cell. 2000; 101:25-33.
42. Filipowicz W, Jaskiewicz L, Kolb FA, et al. Post-transcriptional gene silencing by siRNAs and miRNAs. Curr Opin Struct Biol. 2005; 15(3):331-341.
43. Cervantes J, Lema C, Valentina-Hurtado L, et al. HLA-DRB1*1602 allele is positively associated with HPV cervical infection in Bolivian Andean women. Hum Immunol. 2003; 64(9): 890-895.
44. Gostout BS, Strome SE, Clayton AC, et al. Two cases of coincident carcinomas of the head and neck and the uterine cervix. Gynecol Oncol. 2002; 85(2):376-380.
45. Ghaderi M, Nikitina-Zake L, Wallin K, et al. Tumor necrosis factor A and MHC class I chain related gene A (MIC-A) polymorphisms in Swedish patients with cervical cancer. Hum Immunol. 2001; 62(10):1153-1158.
46. Veress G, Murvai M, Szarka K, et al. Transcriptional activity of human papillomavirus type 16 variants having deletions in the long control region. Eur J Cancer. 2001; 37(15):1946-1952.
47. Lee SJ, Cho YS, Cho MC, et al. Both E6 and E7 oncoproteins of human papillomavirus
16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells. J Immunol. 2001; 167(1): 497-504.
48. Pham JW, Sontheimer EJ. Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway. J Biol Chem. 2005;280(47):39278-39283.
49. Gilmore IR, Fox SP, Hollins AJ, et al. Delivery Strategies for siRNA-mediated Gene Silencing. Curr Drug Deliv. 2006; 3(2):147-155.
50. Jacks T,Weinberg RA.CeII-cycle control and its watchman.Nature. 1996; 381: 643-644.
51. Morgan DO.Prociples of CDK regulation. Nature. 1995;374:131-134.
52. Jacks T,Weinberg RA.The expanding role of cell cycle regulators.Science.1998; 280:1035-1036.
53. Hartwell LH, Kastan KB.Cell cycle control and cancer.Science.1994; 266:1821-1828.
54. Steef M.Cyclins and cancer:wheels within wheels.Lancet. 1994; 343:931-932.
55. Clurman BE,Roberts JM.Cell cycle and cancer.JNCI. 1995;87:1499-1501.
56. Billich A. HPV vaccine Medimmune/GlaxoSmithKline. Curr Opin Investig Drugs. 2003;
4(2):210-213.
57. Moniz M, Ling M, Hung CF, et al. HPV DNA vaccines. Front Biosci.2003; 8:D55-68.
58. Plummer M, Franceschi S. Strategies for HPV prevention. Virus Res.2002;89 (2):285-293.
59. Ghim SJ, Basu PS, Jenson A. Cervical Cancer:Etiology, Pathogenesis, Treatment., and Future Vaccines. Asian Pac J Cancer Prev. 2002; 3 (3):207-214.
60.汤钊酞.主编.现代肿瘤学第二版.上海医科大学出版社.2000年9月.P899-904.
61. Svrjanen S, Shabalova I, Petrovichev N, et al. Sexual habits and human papillomavirus infection among females in three New Independent Spates of the former Soviet Union. Sex Transm Dis. 2003; 30 (9): 680-684.
62. Schel lekens MC, Di jkman A, Aziz MF, et al. Gynecol Oncol.Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. 2004; 93 (1):49-53.
63. Giuliano AR, Papenfuss M, De Galaz EM, et al.Risk factors for squamous intraepithelial lesions (SIL) of the cervix among women residing at the US-Mexico border. Int J Cancer. 2004;109 (1):112-118.
64. Sierra-Tomes CH, yring SK, Au WW. Int J Gynecol Cancer. Risk contribution of sexual behavior and cigarette smoking to cervical neoplasia. 2003;13 (5):617-625.
65.李连第,鲁风珠,张思维等.中国恶性肿瘤死亡率20年变化趋势和近期预测分析.中华肿瘤杂志. 1997; 19: 3-9.
66.李洁,刘宝印, zur Hauson H等.中国妇女宫颈癌组织中人乳头瘤病毒感染及其地理分布的调查.中华实验临床病毒学杂志.1996; 10 (1): 50-55.
67. Lee SA, Kang D, Seo SS, et al. Multiple HPV infection in cervical cancer screened by HPV DNA Chip. Cancer Lett. 2003;198(2):187-192.
68. Schiffman M, Castle PE. human papillomavirus:epidemiology and public health. Arch Pathol Lab Med. 2003;127(8):930-934.
69. Franco EL, Schlecht NF, Saslow D. The epidemiology of cervical cancer. Cancer J. 2003; 9 (5):348-359.
70. Munoz N, Bosch FX, de San jose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer.N Engl J Med.2003;348(6):518-527.
71. Clifford GM, Smith JS, Plummer M, et al. Human papillomavirus types in invasive cervical cancer worldwide:a meta-analysis. Br J Cancer. 2003;88(1):63-73.
72. Speich N, Schmitt C, Bollmann R, et al. Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing:genotype spectrum of patients from the west German area. J Med Microbiol. 2004; 53 (Pt 2):125-128.
73. Webster K, Taylor A, Gaston K. Estrogen and progesterone increase the levels of apoptosis induced by the human papillomavirus type 16 E2 and E7 proteins. J Gen Virol. 2001; 82 (pt1):201-213.
74. DeFi l ippi s RA, Goodwin EC, Wu L, et al. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. J Virol. 2003; 77 (2):1551-1563.
75. Stevenson M, Lucy C, Tulie E, et al. Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia. Journal of General Virology. 2000;
81:1825-1832.
76. DeFilippis RA, Goodwin EC, Wu L, et al. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in Hel.a cervical carcinoma cells. J Virol.2003; 77 (2):1551-1563.
77. Castellsague X,Bosch X,Virus Res, et al. Environmental co-factors in HPV carclnogenesis. 2002; 89 (2):191-199.
78. Ressler S, Scheiden R, Dreier K, et al. High-risk human papillomavirus E7 oncoprotein detection in cervical squamous cell carcinoma. Clin Cancer Res.2007; 13(23):7067-7072.
79. Mansour M, Touka M, Hasan U, et al. E7 properties of mucosal human papillomavirustypes 26, 53 and 66 correlate with their intermediate risk for cervical cancer development. Virology. 2007; 367(1):1-9.
80. Tarnm I, Schurnacher A, Karawajew L, et al. Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apopotosis and tumor regression on vivo. Cancer Gene Ther. 2002; 9(8):641-650.
81. Serr CJ. Parsing Ink4a /Arf: "pure" p16-null mice. Cell. 2001; 106:531-534.
82. Hu L. Human papillomavirus genotyping and p16INK4a expression in cervical intraepithelial neoplasia of adolescents. Mod-Pathol. 2005; 18(2):267-273.
83. Ivanova TA, Golovina DA, Zavalishina LE, et al. Up-regulation of expression and lack of 5'CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas. BMC-Cancer. 2007; 7: 47.
84. Chiesa-Vottero AG, Malpica A, Deavers MT, et al. Immunohistochemical overexpression of p16 and p53 in uterine serous carcinoma and ovarian high-grade serous carcinoma. Int J Gynecol Pathol. 2007; 26(3):328-333.
85. Gurney AM. enter E.The use of small interfering RNA to elucidate the activity and function of ion channel genes in an intact tissue.. J Pharmacol Toxicol Methods. 2005; 51(3 ):253-62.
86. Lee KY,D'Acquisto F,Hayden MS, et al. PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation.Science. 2005; 308(5718):114-118.
87. Brummelkamp TR, Bernads R, Agami R. System for stable expression of short interfering RNAs in Mammalian cells.Science. 2002; 296:550-553.
88. Walboomers JM, Jacobs MV,Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[J]. J-Pathol. 1999; 189(1): 12-19
89. Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, et al. Relation of human papillomavirus status to cervical lesions and consequences for cervical-cancer screening: a prospective study. Lancet. 1999; 354(9172): 20-25
90. Bosch FX, Manos MM, Munoz N, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group[J]. J-Natl-Cancer-Inst. 1995; 87(11): 796-802
91. Liu MC, Marshall JL, Pestell RG. Novel strategies in cancer therapeutics:targeting enzymes involved in cell cycle regulation and cellular proliferation.Curr Cancer DrugTargets. 2004; 4(5):403-424.
92. Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells.Nature. 2001; 414:105-111.
93. Hunter T.Oncoprotein networks.Cell. 1997; 88:333-346.
94. Weinberg RA.How Cancer arises.Sci Am.1996;275:62-70.
95. Goldfarb M,Shimizu K,Perucho M.Isolation and preliminary characterization of a human Transforming gene from T24 bladder carcinoma cells.Nature. 1982;296:404-409.
96. Shih C,Weinberg RA. Isolation of a transforming sequence from a human bladder carcinoma cell line.Cell. 1982;29:161-169.
97. Friend SH,Bernards R,Rogel S.A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986;323:643-646.
98. Baker SJ,Markowitz S,Fearon ER.Suppression of human colorectal carcinoma cell growth by wild-type p53.Science. 1990;249:912-915.
99. Pawson T.Protein modules and signalling networks.Nature. 1995; 373:573-580.
100. Khoo CM, Carrasco DR, Bosenberg MW, et al. Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse. Proc Natl Acad Sci U S A. 2007; 104(10):3931-3936.
101. Franco EL, Schlecht NF, Saslow D. The epidemiology of cervical cancer. Cancer J. 2003;9(5):318-359.
102. Ghim SJ, Basu PS, Jenson A. Cervical Cancer:Etiology, Pathogenesis, Treatment, and Future Vaccines. Asian Pac J Cancer Prev. 2002; 3 (3): 207-214.
103. Bosch FX, Manos MM, Munoj N, et al. Prevalence of human papillomavirus in cervical cancer. a worldwide perspective. J Natl Cancer Inst. 1995; 87(11):796-802.
104. Dell G, Gaston K. Contributions in the domain of cancer research: human papillomaviruses and their role in cervical cancer. Cell. Mol. Life Sci. 2001; 58: 1923-1942.
105. Syrjanen S, Shabalova I, Petrovichev N, et al. Sexual habits and human papillomavirus infection among females in three New Independent States of the former Soviet Union. Sex Transm Dis. 2003;30 (9):680-684.
106. Lee SA, Kang D, Seo SS, et al. Multiple HPV infection in cervical cancer screened by HPV DNA Chip. Cancer Lett. 2003; 198(2):187-192.
107. Schiffman M, Castle PE. Human papillomavirus:epidemiology and public health. Arch Pathol Lab Med. 2003;127 (8):930-934.
108. Munoz N, Bosch FX, de Sanjose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003; 348(6): 518-527.
109. Speich N, Schmitt C, BoIImann R, et al. Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing: genotype spectrum of patients from the west German area. J Med Microbiol. 2004; 53(2):125-128.
110. Yoshida T, Sano T, Kanuma T, et al. Immunochemical analysis of HPV L1 capsid protein and p16 protein in liquid-based cytology samples from uterine cervical lesions. Cancer. 2008;114(2):83-88.
111. Koutsky LA, Holmes KK, Critchlow J, et al. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papi1lomavirus infection. N Engl J Med. 1992; 327:1272-1278.
112. Cuzick J, Szareqsky A, Terry G, et al. Human papillomavirus testing in promary cervical screening. Cancer. 1995; 347:1533-1536.
113. Schiffrnan MH, Bauer HM, Hoover RN, et al. Epiderniologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst. 1993; 85:958-964.
114. Sherman ME, Kurman RJ. Intraepithelian carcinoma of the cervix: Reflections on half a century of progress. Cancer. 1998; 83:22-43.
115. Bratti MC, Rodriguez AC, Schiffman M, et al. Description of a seven-year prospective study of human papillomavirus infection and cervical neoplasia among 10000 women in Guanacaste, Costa Rica.Rev Panam Salud Publica. 2004;15(2):75-89.
116. Lee SA,Kang D,screened Seo SS, et al. Multiple HPV infection in cervical cancer screened by HPV DNA Chip. Cancer Lett. 2003; 198(2):187-192.
117. Sierra-Tomes CH, Tyring SK, Au WW. Int J Gynecol Cancer. contribution of sexual behavior and cigarette smoking to cervical neoplasia. 2003;13(5):617-625.
118. Fernandez Vl. Pathogenesis of mantle-cell lymphoma: all oncogenic roads lead to dysregulation of cell cycle and DNA damage response pathways. J-Clin-Oncol. 2005; 23(26):6364-6369.
119. Park IK. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003; 423(6937):302-305.
120. Itahana K. Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1. Mol-Cell-Biol. 2003; 23(1):389-401.
121. Liu WH. Inhibition effect of antisense Bmi-1 on Jurkat cells. Zhonghua Xue Ye Xue Za Zhi. 2005; 26(9):554-556.
122. Tamm I, Schumacher A, Karawajew L, et al. Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo. Cancer Gene Ther. 2002; 9(8):641-650.
1. Macaluso M, Montanari M, Cinti C, et al. Modulation of cell cycle components by epigenetic and genetic events. Semin Oncol, 2005, 32(5):452-457
2. Gibson SL, Dai CY, Lee HW, et al. Inhibition of colon tumor progression and angiogenesis by the Ink4a/ Arf locus. Cancer Res, 2003,63(4):742-746
3. Sharpless NE.INK4a/ARF: a multifunctional tumor suppressor locus.Mutat Res, 2005,576(1-2):22-38
4. Maeda T, Hobbs RM, Merghoub T, et al. Role of the proto-oncogene Pokemon in cellular transformation and ARF repression. Nature, 2005,433(7023): 278-285
5. Tammela J, Odunsi K. Gene expression and prognostic significance in ovarian cancer. Minerva Ginecol, 2004, 56(6):495-502
6. Ibanez de Caceres I, Battagli C, Esteller M, et al. Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients. Cancer, Res. 2004,64(18):6476-6481
7. Wiley A, Katsaros D, Chen H, et al. Aberrant promoter methylation of multiple genes in malignant ovarian tumors and in ovarian tumors with low malignant potential. Cancer, 2006 Jun 13; [Epub ahead of print]
8. Katsaros D, Cho W, Singal R, et al. Methylation of tumor suppressor gene p16 and prognosis of epithelial ovarian cancer. Gynecol Oncol, 2004, 94(3):685-692
9. Salvesen HB, Kumar R, Stefansson I, et al. Low frequency of BRAF and CDKN2A mutations in endometrial cancer. Int-J-Cancer, 2005,115(6): 930-934
10. Semczuk A, Boltze C, Marzec B, et al. p16INK4A alterations are accompanied by aberrant protein immunostaining in endometrial carcinomas. J Cancer Res Clin Oncol, 2003,129(10):589-596
11. Watanabe J, Nishizaki R, Jobo T, et al. Expression of tumor suppressor gene product p14ARF in endometrioid adenocarcinoma of the uterine corpus. Int J Gynecol Pathol, 2004,23(3):234-240
12. Denny L. The prevention of cervical cancer in developing countries. BJOG, 2005,112(9):1204-1212
13. Ishikawa M, Fujii T, Saito M, et al. Overexpression of p16 INK4a as an indicator for human papillomavirus oncogenic activity in cervical squamous neoplasia. Int J Gynecol Cancer, 2006,16(1):347-353
14. Murphy N, Ring M, Killalea AG, et al. p16IN K4A as a marker for cervical dyskaryosis :CIN and cGIN in cervical biopsies and ThinPrep smears, J Clin Pathol. 2003,56 (1):56-63
15. Kanao H, Enomoto T, Ueda Y, et al. Correlation between p14(ARF) / p16 ( IN K4A) expression and HPV infection in uterine cervical cancer. Cancer Lett, 2004 ,213 (1) :31-37
16. van der Avoort IA, Shirango H, Hoevenaars BM, et al. Vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways. Int J GynecolPathol, 2006,25(1):22-29
17. Knopp S, Bjorge T, Nesland JM, et al. p16INK4a and p21Waf1/Cip1 expression correlates with clinical outcome in vulvar carcinomas. Gynecol Oncol, 2004,95(1):37-45
18. Wang M, Wei J, Zhang J. Replacement of t he p16 gene in human ovarian cancer cells. Chin Med J ( Eng1), 2001,114(8) :857-859
19. Allagher S, Kefford RF, Rizos H. Enforced expression of p14ARF Induces p53 - dependent cycle arrest but not apoptosis. Cell Cycle, 2005,4 (3):465 - 472
20. Guillaume N, PhilippH, Berlinda V, et al. p14ARF induces G2 cell cycle arrest in p53 - and p21 - deficient cells by down - regulating p34 -cdc- kinase activity. J Biol Chem, 2005,280(8):7118– 7130
1. Xue Q, Sano T, Kashiwabara K, et al. Aberrant expression of pRb, p16, p14ARF, MDM2, p21 and p53 in stage I adenocarcinomas of the lung[J]. Pathol Int, 2002, 52(2): 103-109.
2. Gibson SL, Dai CY, Lee HW, et al. Inhibition of colon tumor progression and angiogenesis by the Ink4a/Arf locus[J]. Cancer-Res, 2003, 63(4): 742-746.
3. Kuo ML, Duncavage EJ, Mathew R, et al. Arf induces p53-dependent and -independent antiproliferative genes[J]. Cancer-Res, 2003, 63(5): 1046-1053.
4.胡义德,高楠,曹世龙.肺癌INK4a/ARF基因缺失与突变研究进展[J].国外医学·呼吸系统分册, 2000, 20(2): 102-104.
5. Silva J, Silva JM, Dominguez G, et al. Concomitant expression of p16INK4a and p14ARF in primary breast cancer and analysis of inactivation mechanisms[J]. J-Pathol,2003, 199(3): 289-297.
6. Kamijo T, Zindy F, Roussel MF, et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF[J]. Cell, 1997, 91(5): 649-659.
7. Gessner C, Liebers U, KuhnH, et al. BAX and p16INK4A are independent positive prognostic markers for advanced tumour stage of nonsmall cell lung cancer[J]. Eur Respir J, 2002, 19(1): 134-140.
8. Nicholson SA, Okby NT, Khan MA, et al. Alterations of p14ARF, p53, and p73 genes involved in the E2F-1-mediated apoptotic pathways in non-small cell lung carcinoma[J]. Cancer Res, 2001, 61(14): 5636-5643.
9. Tannapfel A, Busse C, Weinans L, et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas[J]. Oncogene, 2001, 20(48): 7104-7109.
10. Caca K, Feisthammel J, Klee K, et al. Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines[J]. Int J Cancer, 2002, 97(4): 481-488.
11. Edmunds SC, Kelsell DP, Hungerford JL, et al. Mutational analysis of selected genes in the TGFbeta, Wnt, pRb, and p53 pathways in primary uveal melanoma[J]. Invest Ophthalmol Vis Sci, 2002, 43(9): 2845-2851.
12. Ko JY, Lee TC, Hsiao CF, et al. Definition of three minimal deleted regions by comprehensive allelotyping and mutational screening of FHIT,p16(INK4A), and p19(ARF) genes in nasopharyngeal carcinoma[J]. Cancer, 2002, 94(7): 1987-1996.
13. Esteller M, Tortola S, Toyota M, et al. Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status[J].Cancer Res, 2000, 60(1): 129-133.
14. Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells[J]. Nature, 2002, 416(6880): 552-556.
15. Zochbauer-Muller S, Fong KM, Virmani AK, et al. Aberrant promoter methylation of multiple genes in non-small cell lung cancers[J]. Cancer Res, 2001, 61(1): 249-255.
16. Lamy A, Sesboue R, Bourguignon J, et al. Aberrant methylation of the CDKN2a/p16INK4a gene promoter region in preinvasive bronchial lesions: a prospective study in high-risk patients without invasive cancer[J]. Int J Cancer, 2002, 100(2):189-193.
17. Hirao T, Bueno R, Chen CJ, et al. Alterations of the p16(INK4) locus in human malignant mesothelial tumors[J]. Carcinogenesis, 2002, 23(7): 1127-1130.
18. Wang M, Wei J, Zhang J. Replacement of the p16 gene in human ovarian cancer cells[J]. Chin Med J (Engl), 2001, 114(8): 857-859.
19. Katsuda K, Kataoka M, Uno F, et al. Activation of caspase-3 and cleavage of Rb are associated with p16-mediated apoptosis in human non-small cell lung cancer cells[J]. Oncogene, 2002, 21(13): 2108-2113.
20. Rui HB, Su JZ. Co-transfection of p16(INK4a) and p53 genes into the K562 cell line inhibits cell proliferation[J]. Haematologica, 2002, 87(2): 136-142.
21. Adachi Y, Chandrasekar N, Kin Y, et al. Suppression of glioma invasion and growth by adenovirus-mediated delivery of a bicistronic construct containing antisense uPAR and sense p16 gene sequences[J]. Oncogene, 2002, 21(1): 87-95.
22. Gao N, Hu YD, CaoXY, et al. The exogenous wild-type p14ARF gene induces growth arrest and promotes radiosensitivity in human lung cancer cell lines[J]. J Cancer Res Clin Oncol, 2001, 127(6): 359-367.
2. Nam EJ, Kim JW, Kim SW, et al. The expressions of the Rb pathway in cervical intraepithelial neoplasia; predictive and prognostic significance. Gynecologic Oncology. 2007; 104(1):207-211.
3. Hinkula M, Pukkala E, Kyyronen P, et al. A population-based study on the risk of cervical cancer and cervical intraepithelial neoplasia among grand multiparous worsen in Finland. Br J Cancer. 2004; 90 (5):1025-1029.
4. Gibson SL, Dai CY, Lee HW, et al. Inhibition of colon tumor progression and angiogenesis by the Ink4a/Arf locus. Cancer-Res. 2003; 63(4):742-746.
5. Kuo ML, Duncavage EJ, Mathew R, et al. Arf induces p53-dependent and -independent antiproliferative genes. Cancer-Res. 2003; 63(5):1046-1053.
6. Silva J, Silva JM, Dominguez G, et al. Concomitant expression of p16INK4a and p14ARF in primary breast cancer and analysis of inactivation mechanisms. J-Pathol. 2003; 199(3):289-297.
7. Kamijo T, Zindy F, Roussel MF, et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell. 1997; 91(5): 649-659.
8. Gessner C, Liebers U, KuhnH, et al. BAX and p16INK4A are independent positive prognostic markers for advanced tumour stage of nonsmall cell lung cancer. Eur Respir J. 2002; 19(1):134-140.
9. Nicholson SA, Okby NT, Khan MA, et al. Alterations of p14ARF, p53, and p73 genes involved in the E2F-1-mediated apoptotic pathways in non-small cell lung carcinoma. Cancer Res. 2001; 61(14):5636-5643.
10. Tannapfel A, Busse C, Weinans L, et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas. Oncogene. 2001; 20(48):7104-7109.
11. Caca K, Feisthammel J, Klee K, et al. Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines. Int J Cancer. 2002; 97(4):481-488.
12. Sharpless NE. INK4a/ARF: a multifunctional tumor suppressor locus. Mutat-Res. 2005;576(1-2):22-38.
13. Kanao H. Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer. Cancer Lett. 2004; 213(1):31-37.
14. Feng W, Xiao J, Zhang Z, et al. Senescence and apoptosis in carcinogenesis of cervical squamous carcinoma. Mod Pathol. 2007;20(9):961-966.
15. Lau WM, Ho TH, Hui KM. p16INK4A-silencing augments DNA damage-induced apoptosis in cervical cancer cells. Oncogene. 2007; 26(41):6050-6060.
16. Kamijo T, Weber JD, Zambetti G, et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc-Natl-Acad-Sci-U-S-A. 1998; 95(14): 8292-8297.
17. Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell. 1998; 92(6): 725-734.
18. Zochbauer-Muller S, Fong KM, Virmani AK, et al. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer-Res. 2001; 61(1):249-255.
19. Redman R, Rufforny I, Liu C, et al. The Utility of p16(Ink4a) in Discriminating Between Cervical Intraepithelial Neoplasia 1 and Nonneoplastic Equivocal Lesions of the Cervix. Arch Pathol Lab Med. 2008;132(5):795-799.
20. Samama B, Schaeffer C, Boehm N. P16 expression in relation to human papillomavirus in liquid-based cervical smears. Gynecol Oncol. 2008;109(2):285-290.
21. McKay JA, Douglas JJ, Ross VG, et al. Analysis of key cell-cycle checkpoint proteins in colorectal tumours. J-Pathol. 2002; 196(4): 386-393.
22. van der Avoort IA, Shirango H, Hoevenaars BM, et al. Vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways. Int J Gynecol Pathol. 2006; 25(1):22-29.
23. Benevolo M, Vocaturo A, Mottolese M, et al. Clinical role of p16INK4a expression in liquid-based cervical cytology: correlation with HPV testing and histologic diagnosis. Am J Clin Pathol. 2008;129(4):606-612.
24. Ishikawa M, Fujii T, Saito M, et al. Overexpression of p16INK4a as an indicator for human papillomavirus oncogenic activity in cervical squamous neoplasia. Int J Gynecol Cancer. 2006;16(1):347-353.
25. Kanao H, Enomoto T, Ueda Y, et al. Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer. Cancer Lett. 2004; 213(1):31-37.
26. Khoo CM, Carrasco DR, Bosenberg MW, et al. Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse. Proc Natl Acad Sci U S A. 2007;104(10):3931-3936.
27. Blokx WA, Lesterhuis JJ, Andriessen MP, et al. CDKN2A(INK4A-ARF)mutation analysis to distinguish cutaneous melanoma metastasis from a second primary melanoma. Am J Surg Pathol. 2007; 31(4):637-641.
28. O'Neill CJ, McCluggage WG. p16 expression in the female genital tract and its value in diagnosis. Adv Anat Pathol. 2006; 13(1):8-15.
29. Liang J, Mittal KR, Wei JJ, et al. Utility of p16INK4a, CEA, Ki67, P53 and ER/PR in the differential diagnosis of benign, premalignant, and malignant glandular lesions of the uterine cervix and their relationship with Silverberg scoring system for endocervical glandular lesions. Int J Gynecol Pathol. 2007; 26(1):71-75.
30. Eleuterio J Jr, Giraldo PC, Goncalves AK, et al. Prognostic markers of high-grade squamous intraepithelial lesions: the role of p16INK4a and high-risk human papillomavirus. Acta Obstet Gynecol Scand. 2007; 86(1):94-98.
31. Norman I, Brismar S, Zhu J, et al. p16(INK4a) immunocytochemistry in liquid-based cervical cytology: is it feasible for clinical use? Int J Oncol. 2007;31(6):1339-1343.
32. Redman R, Rufforny I, Liu C, et al. The Utility of p16(Ink4a) in Discriminating Between Cervical Intraepithelial Neoplasia 1 and Nonneoplastic Equivocal Lesions of the Cervix. Arch Pathol Lab Med. 2008;132(5):795-799.
33. Hashi A, Xu JY, Kondo T, et al. p16INK4a overexpression independent of human papillomavirus infection in lobular endocervical glandular hyperplasia. Int J Gynecol Pathol. 2006; 25(2):187-194.
34. Galmiche L, Coste-Burel M, Lopes P, et al. The expression of p16 is not correlated with HPV status in CINI. Histopathology. 2006; 48(6):1365-2559.
35. Denny L. The prevention of cervical cancer in developing countries. BJOG. 2005; 112(9):1204-1212.
36. Santin AD. Gene expression profiles of primary HPV16- and HPV18-infected early stage cervical cancers and normal cervical epithelium: identification of novel candidatemolecular markers for cervical cancer diagnosis and therapy. Virology. 2005; 331(2): 269-291.
37. Unger ER. Human papillomavirus and cervical cancer. Emerg-Infect-Dis. 2004; 10(11):2031-2032.
38. Dallenbach-Hellweg G. Traditional and new molecular methods for early detection of cervical cancer. Arkh-Patol. 2004; 66(5):35-39.
39. Schellekens MC. Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. Gynecol-Oncol. 2004; 93(1):49-53.
40. Horn LC, Reichert A, Oster A, et al. Immunostaining for p16INK4a Used as a Conjunctive Tool Improves Interobserver Agreement of the Histologic Diagnosis of Cervical Intraepithelial Neoplasia. Am J Surg Pathol. 2008 Jan 24 [Epub ahead of print]
41. Zamore PD, Tuschl T, Sharp PA, et al. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23nt intervals. Cell. 2000; 101:25-33.
42. Filipowicz W, Jaskiewicz L, Kolb FA, et al. Post-transcriptional gene silencing by siRNAs and miRNAs. Curr Opin Struct Biol. 2005; 15(3):331-341.
43. Cervantes J, Lema C, Valentina-Hurtado L, et al. HLA-DRB1*1602 allele is positively associated with HPV cervical infection in Bolivian Andean women. Hum Immunol. 2003; 64(9): 890-895.
44. Gostout BS, Strome SE, Clayton AC, et al. Two cases of coincident carcinomas of the head and neck and the uterine cervix. Gynecol Oncol. 2002; 85(2):376-380.
45. Ghaderi M, Nikitina-Zake L, Wallin K, et al. Tumor necrosis factor A and MHC class I chain related gene A (MIC-A) polymorphisms in Swedish patients with cervical cancer. Hum Immunol. 2001; 62(10):1153-1158.
46. Veress G, Murvai M, Szarka K, et al. Transcriptional activity of human papillomavirus type 16 variants having deletions in the long control region. Eur J Cancer. 2001; 37(15):1946-1952.
47. Lee SJ, Cho YS, Cho MC, et al. Both E6 and E7 oncoproteins of human papillomavirus
16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells. J Immunol. 2001; 167(1): 497-504.
48. Pham JW, Sontheimer EJ. Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway. J Biol Chem. 2005;280(47):39278-39283.
49. Gilmore IR, Fox SP, Hollins AJ, et al. Delivery Strategies for siRNA-mediated Gene Silencing. Curr Drug Deliv. 2006; 3(2):147-155.
50. Jacks T,Weinberg RA.CeII-cycle control and its watchman.Nature. 1996; 381: 643-644.
51. Morgan DO.Prociples of CDK regulation. Nature. 1995;374:131-134.
52. Jacks T,Weinberg RA.The expanding role of cell cycle regulators.Science.1998; 280:1035-1036.
53. Hartwell LH, Kastan KB.Cell cycle control and cancer.Science.1994; 266:1821-1828.
54. Steef M.Cyclins and cancer:wheels within wheels.Lancet. 1994; 343:931-932.
55. Clurman BE,Roberts JM.Cell cycle and cancer.JNCI. 1995;87:1499-1501.
56. Billich A. HPV vaccine Medimmune/GlaxoSmithKline. Curr Opin Investig Drugs. 2003;
4(2):210-213.
57. Moniz M, Ling M, Hung CF, et al. HPV DNA vaccines. Front Biosci.2003; 8:D55-68.
58. Plummer M, Franceschi S. Strategies for HPV prevention. Virus Res.2002;89 (2):285-293.
59. Ghim SJ, Basu PS, Jenson A. Cervical Cancer:Etiology, Pathogenesis, Treatment., and Future Vaccines. Asian Pac J Cancer Prev. 2002; 3 (3):207-214.
60.汤钊酞.主编.现代肿瘤学第二版.上海医科大学出版社.2000年9月.P899-904.
61. Svrjanen S, Shabalova I, Petrovichev N, et al. Sexual habits and human papillomavirus infection among females in three New Independent Spates of the former Soviet Union. Sex Transm Dis. 2003; 30 (9): 680-684.
62. Schel lekens MC, Di jkman A, Aziz MF, et al. Gynecol Oncol.Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. 2004; 93 (1):49-53.
63. Giuliano AR, Papenfuss M, De Galaz EM, et al.Risk factors for squamous intraepithelial lesions (SIL) of the cervix among women residing at the US-Mexico border. Int J Cancer. 2004;109 (1):112-118.
64. Sierra-Tomes CH, yring SK, Au WW. Int J Gynecol Cancer. Risk contribution of sexual behavior and cigarette smoking to cervical neoplasia. 2003;13 (5):617-625.
65.李连第,鲁风珠,张思维等.中国恶性肿瘤死亡率20年变化趋势和近期预测分析.中华肿瘤杂志. 1997; 19: 3-9.
66.李洁,刘宝印, zur Hauson H等.中国妇女宫颈癌组织中人乳头瘤病毒感染及其地理分布的调查.中华实验临床病毒学杂志.1996; 10 (1): 50-55.
67. Lee SA, Kang D, Seo SS, et al. Multiple HPV infection in cervical cancer screened by HPV DNA Chip. Cancer Lett. 2003;198(2):187-192.
68. Schiffman M, Castle PE. human papillomavirus:epidemiology and public health. Arch Pathol Lab Med. 2003;127(8):930-934.
69. Franco EL, Schlecht NF, Saslow D. The epidemiology of cervical cancer. Cancer J. 2003; 9 (5):348-359.
70. Munoz N, Bosch FX, de San jose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer.N Engl J Med.2003;348(6):518-527.
71. Clifford GM, Smith JS, Plummer M, et al. Human papillomavirus types in invasive cervical cancer worldwide:a meta-analysis. Br J Cancer. 2003;88(1):63-73.
72. Speich N, Schmitt C, Bollmann R, et al. Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing:genotype spectrum of patients from the west German area. J Med Microbiol. 2004; 53 (Pt 2):125-128.
73. Webster K, Taylor A, Gaston K. Estrogen and progesterone increase the levels of apoptosis induced by the human papillomavirus type 16 E2 and E7 proteins. J Gen Virol. 2001; 82 (pt1):201-213.
74. DeFi l ippi s RA, Goodwin EC, Wu L, et al. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. J Virol. 2003; 77 (2):1551-1563.
75. Stevenson M, Lucy C, Tulie E, et al. Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia. Journal of General Virology. 2000;
81:1825-1832.
76. DeFilippis RA, Goodwin EC, Wu L, et al. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in Hel.a cervical carcinoma cells. J Virol.2003; 77 (2):1551-1563.
77. Castellsague X,Bosch X,Virus Res, et al. Environmental co-factors in HPV carclnogenesis. 2002; 89 (2):191-199.
78. Ressler S, Scheiden R, Dreier K, et al. High-risk human papillomavirus E7 oncoprotein detection in cervical squamous cell carcinoma. Clin Cancer Res.2007; 13(23):7067-7072.
79. Mansour M, Touka M, Hasan U, et al. E7 properties of mucosal human papillomavirustypes 26, 53 and 66 correlate with their intermediate risk for cervical cancer development. Virology. 2007; 367(1):1-9.
80. Tarnm I, Schurnacher A, Karawajew L, et al. Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apopotosis and tumor regression on vivo. Cancer Gene Ther. 2002; 9(8):641-650.
81. Serr CJ. Parsing Ink4a /Arf: "pure" p16-null mice. Cell. 2001; 106:531-534.
82. Hu L. Human papillomavirus genotyping and p16INK4a expression in cervical intraepithelial neoplasia of adolescents. Mod-Pathol. 2005; 18(2):267-273.
83. Ivanova TA, Golovina DA, Zavalishina LE, et al. Up-regulation of expression and lack of 5'CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas. BMC-Cancer. 2007; 7: 47.
84. Chiesa-Vottero AG, Malpica A, Deavers MT, et al. Immunohistochemical overexpression of p16 and p53 in uterine serous carcinoma and ovarian high-grade serous carcinoma. Int J Gynecol Pathol. 2007; 26(3):328-333.
85. Gurney AM. enter E.The use of small interfering RNA to elucidate the activity and function of ion channel genes in an intact tissue.. J Pharmacol Toxicol Methods. 2005; 51(3 ):253-62.
86. Lee KY,D'Acquisto F,Hayden MS, et al. PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation.Science. 2005; 308(5718):114-118.
87. Brummelkamp TR, Bernads R, Agami R. System for stable expression of short interfering RNAs in Mammalian cells.Science. 2002; 296:550-553.
88. Walboomers JM, Jacobs MV,Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[J]. J-Pathol. 1999; 189(1): 12-19
89. Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, et al. Relation of human papillomavirus status to cervical lesions and consequences for cervical-cancer screening: a prospective study. Lancet. 1999; 354(9172): 20-25
90. Bosch FX, Manos MM, Munoz N, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group[J]. J-Natl-Cancer-Inst. 1995; 87(11): 796-802
91. Liu MC, Marshall JL, Pestell RG. Novel strategies in cancer therapeutics:targeting enzymes involved in cell cycle regulation and cellular proliferation.Curr Cancer DrugTargets. 2004; 4(5):403-424.
92. Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells.Nature. 2001; 414:105-111.
93. Hunter T.Oncoprotein networks.Cell. 1997; 88:333-346.
94. Weinberg RA.How Cancer arises.Sci Am.1996;275:62-70.
95. Goldfarb M,Shimizu K,Perucho M.Isolation and preliminary characterization of a human Transforming gene from T24 bladder carcinoma cells.Nature. 1982;296:404-409.
96. Shih C,Weinberg RA. Isolation of a transforming sequence from a human bladder carcinoma cell line.Cell. 1982;29:161-169.
97. Friend SH,Bernards R,Rogel S.A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986;323:643-646.
98. Baker SJ,Markowitz S,Fearon ER.Suppression of human colorectal carcinoma cell growth by wild-type p53.Science. 1990;249:912-915.
99. Pawson T.Protein modules and signalling networks.Nature. 1995; 373:573-580.
100. Khoo CM, Carrasco DR, Bosenberg MW, et al. Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse. Proc Natl Acad Sci U S A. 2007; 104(10):3931-3936.
101. Franco EL, Schlecht NF, Saslow D. The epidemiology of cervical cancer. Cancer J. 2003;9(5):318-359.
102. Ghim SJ, Basu PS, Jenson A. Cervical Cancer:Etiology, Pathogenesis, Treatment, and Future Vaccines. Asian Pac J Cancer Prev. 2002; 3 (3): 207-214.
103. Bosch FX, Manos MM, Munoj N, et al. Prevalence of human papillomavirus in cervical cancer. a worldwide perspective. J Natl Cancer Inst. 1995; 87(11):796-802.
104. Dell G, Gaston K. Contributions in the domain of cancer research: human papillomaviruses and their role in cervical cancer. Cell. Mol. Life Sci. 2001; 58: 1923-1942.
105. Syrjanen S, Shabalova I, Petrovichev N, et al. Sexual habits and human papillomavirus infection among females in three New Independent States of the former Soviet Union. Sex Transm Dis. 2003;30 (9):680-684.
106. Lee SA, Kang D, Seo SS, et al. Multiple HPV infection in cervical cancer screened by HPV DNA Chip. Cancer Lett. 2003; 198(2):187-192.
107. Schiffman M, Castle PE. Human papillomavirus:epidemiology and public health. Arch Pathol Lab Med. 2003;127 (8):930-934.
108. Munoz N, Bosch FX, de Sanjose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003; 348(6): 518-527.
109. Speich N, Schmitt C, BoIImann R, et al. Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing: genotype spectrum of patients from the west German area. J Med Microbiol. 2004; 53(2):125-128.
110. Yoshida T, Sano T, Kanuma T, et al. Immunochemical analysis of HPV L1 capsid protein and p16 protein in liquid-based cytology samples from uterine cervical lesions. Cancer. 2008;114(2):83-88.
111. Koutsky LA, Holmes KK, Critchlow J, et al. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papi1lomavirus infection. N Engl J Med. 1992; 327:1272-1278.
112. Cuzick J, Szareqsky A, Terry G, et al. Human papillomavirus testing in promary cervical screening. Cancer. 1995; 347:1533-1536.
113. Schiffrnan MH, Bauer HM, Hoover RN, et al. Epiderniologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst. 1993; 85:958-964.
114. Sherman ME, Kurman RJ. Intraepithelian carcinoma of the cervix: Reflections on half a century of progress. Cancer. 1998; 83:22-43.
115. Bratti MC, Rodriguez AC, Schiffman M, et al. Description of a seven-year prospective study of human papillomavirus infection and cervical neoplasia among 10000 women in Guanacaste, Costa Rica.Rev Panam Salud Publica. 2004;15(2):75-89.
116. Lee SA,Kang D,screened Seo SS, et al. Multiple HPV infection in cervical cancer screened by HPV DNA Chip. Cancer Lett. 2003; 198(2):187-192.
117. Sierra-Tomes CH, Tyring SK, Au WW. Int J Gynecol Cancer. contribution of sexual behavior and cigarette smoking to cervical neoplasia. 2003;13(5):617-625.
118. Fernandez Vl. Pathogenesis of mantle-cell lymphoma: all oncogenic roads lead to dysregulation of cell cycle and DNA damage response pathways. J-Clin-Oncol. 2005; 23(26):6364-6369.
119. Park IK. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003; 423(6937):302-305.
120. Itahana K. Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1. Mol-Cell-Biol. 2003; 23(1):389-401.
121. Liu WH. Inhibition effect of antisense Bmi-1 on Jurkat cells. Zhonghua Xue Ye Xue Za Zhi. 2005; 26(9):554-556.
122. Tamm I, Schumacher A, Karawajew L, et al. Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo. Cancer Gene Ther. 2002; 9(8):641-650.
1. Macaluso M, Montanari M, Cinti C, et al. Modulation of cell cycle components by epigenetic and genetic events. Semin Oncol, 2005, 32(5):452-457
2. Gibson SL, Dai CY, Lee HW, et al. Inhibition of colon tumor progression and angiogenesis by the Ink4a/ Arf locus. Cancer Res, 2003,63(4):742-746
3. Sharpless NE.INK4a/ARF: a multifunctional tumor suppressor locus.Mutat Res, 2005,576(1-2):22-38
4. Maeda T, Hobbs RM, Merghoub T, et al. Role of the proto-oncogene Pokemon in cellular transformation and ARF repression. Nature, 2005,433(7023): 278-285
5. Tammela J, Odunsi K. Gene expression and prognostic significance in ovarian cancer. Minerva Ginecol, 2004, 56(6):495-502
6. Ibanez de Caceres I, Battagli C, Esteller M, et al. Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients. Cancer, Res. 2004,64(18):6476-6481
7. Wiley A, Katsaros D, Chen H, et al. Aberrant promoter methylation of multiple genes in malignant ovarian tumors and in ovarian tumors with low malignant potential. Cancer, 2006 Jun 13; [Epub ahead of print]
8. Katsaros D, Cho W, Singal R, et al. Methylation of tumor suppressor gene p16 and prognosis of epithelial ovarian cancer. Gynecol Oncol, 2004, 94(3):685-692
9. Salvesen HB, Kumar R, Stefansson I, et al. Low frequency of BRAF and CDKN2A mutations in endometrial cancer. Int-J-Cancer, 2005,115(6): 930-934
10. Semczuk A, Boltze C, Marzec B, et al. p16INK4A alterations are accompanied by aberrant protein immunostaining in endometrial carcinomas. J Cancer Res Clin Oncol, 2003,129(10):589-596
11. Watanabe J, Nishizaki R, Jobo T, et al. Expression of tumor suppressor gene product p14ARF in endometrioid adenocarcinoma of the uterine corpus. Int J Gynecol Pathol, 2004,23(3):234-240
12. Denny L. The prevention of cervical cancer in developing countries. BJOG, 2005,112(9):1204-1212
13. Ishikawa M, Fujii T, Saito M, et al. Overexpression of p16 INK4a as an indicator for human papillomavirus oncogenic activity in cervical squamous neoplasia. Int J Gynecol Cancer, 2006,16(1):347-353
14. Murphy N, Ring M, Killalea AG, et al. p16IN K4A as a marker for cervical dyskaryosis :CIN and cGIN in cervical biopsies and ThinPrep smears, J Clin Pathol. 2003,56 (1):56-63
15. Kanao H, Enomoto T, Ueda Y, et al. Correlation between p14(ARF) / p16 ( IN K4A) expression and HPV infection in uterine cervical cancer. Cancer Lett, 2004 ,213 (1) :31-37
16. van der Avoort IA, Shirango H, Hoevenaars BM, et al. Vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways. Int J GynecolPathol, 2006,25(1):22-29
17. Knopp S, Bjorge T, Nesland JM, et al. p16INK4a and p21Waf1/Cip1 expression correlates with clinical outcome in vulvar carcinomas. Gynecol Oncol, 2004,95(1):37-45
18. Wang M, Wei J, Zhang J. Replacement of t he p16 gene in human ovarian cancer cells. Chin Med J ( Eng1), 2001,114(8) :857-859
19. Allagher S, Kefford RF, Rizos H. Enforced expression of p14ARF Induces p53 - dependent cycle arrest but not apoptosis. Cell Cycle, 2005,4 (3):465 - 472
20. Guillaume N, PhilippH, Berlinda V, et al. p14ARF induces G2 cell cycle arrest in p53 - and p21 - deficient cells by down - regulating p34 -cdc- kinase activity. J Biol Chem, 2005,280(8):7118– 7130
1. Xue Q, Sano T, Kashiwabara K, et al. Aberrant expression of pRb, p16, p14ARF, MDM2, p21 and p53 in stage I adenocarcinomas of the lung[J]. Pathol Int, 2002, 52(2): 103-109.
2. Gibson SL, Dai CY, Lee HW, et al. Inhibition of colon tumor progression and angiogenesis by the Ink4a/Arf locus[J]. Cancer-Res, 2003, 63(4): 742-746.
3. Kuo ML, Duncavage EJ, Mathew R, et al. Arf induces p53-dependent and -independent antiproliferative genes[J]. Cancer-Res, 2003, 63(5): 1046-1053.
4.胡义德,高楠,曹世龙.肺癌INK4a/ARF基因缺失与突变研究进展[J].国外医学·呼吸系统分册, 2000, 20(2): 102-104.
5. Silva J, Silva JM, Dominguez G, et al. Concomitant expression of p16INK4a and p14ARF in primary breast cancer and analysis of inactivation mechanisms[J]. J-Pathol,2003, 199(3): 289-297.
6. Kamijo T, Zindy F, Roussel MF, et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF[J]. Cell, 1997, 91(5): 649-659.
7. Gessner C, Liebers U, KuhnH, et al. BAX and p16INK4A are independent positive prognostic markers for advanced tumour stage of nonsmall cell lung cancer[J]. Eur Respir J, 2002, 19(1): 134-140.
8. Nicholson SA, Okby NT, Khan MA, et al. Alterations of p14ARF, p53, and p73 genes involved in the E2F-1-mediated apoptotic pathways in non-small cell lung carcinoma[J]. Cancer Res, 2001, 61(14): 5636-5643.
9. Tannapfel A, Busse C, Weinans L, et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas[J]. Oncogene, 2001, 20(48): 7104-7109.
10. Caca K, Feisthammel J, Klee K, et al. Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines[J]. Int J Cancer, 2002, 97(4): 481-488.
11. Edmunds SC, Kelsell DP, Hungerford JL, et al. Mutational analysis of selected genes in the TGFbeta, Wnt, pRb, and p53 pathways in primary uveal melanoma[J]. Invest Ophthalmol Vis Sci, 2002, 43(9): 2845-2851.
12. Ko JY, Lee TC, Hsiao CF, et al. Definition of three minimal deleted regions by comprehensive allelotyping and mutational screening of FHIT,p16(INK4A), and p19(ARF) genes in nasopharyngeal carcinoma[J]. Cancer, 2002, 94(7): 1987-1996.
13. Esteller M, Tortola S, Toyota M, et al. Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status[J].Cancer Res, 2000, 60(1): 129-133.
14. Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells[J]. Nature, 2002, 416(6880): 552-556.
15. Zochbauer-Muller S, Fong KM, Virmani AK, et al. Aberrant promoter methylation of multiple genes in non-small cell lung cancers[J]. Cancer Res, 2001, 61(1): 249-255.
16. Lamy A, Sesboue R, Bourguignon J, et al. Aberrant methylation of the CDKN2a/p16INK4a gene promoter region in preinvasive bronchial lesions: a prospective study in high-risk patients without invasive cancer[J]. Int J Cancer, 2002, 100(2):189-193.
17. Hirao T, Bueno R, Chen CJ, et al. Alterations of the p16(INK4) locus in human malignant mesothelial tumors[J]. Carcinogenesis, 2002, 23(7): 1127-1130.
18. Wang M, Wei J, Zhang J. Replacement of the p16 gene in human ovarian cancer cells[J]. Chin Med J (Engl), 2001, 114(8): 857-859.
19. Katsuda K, Kataoka M, Uno F, et al. Activation of caspase-3 and cleavage of Rb are associated with p16-mediated apoptosis in human non-small cell lung cancer cells[J]. Oncogene, 2002, 21(13): 2108-2113.
20. Rui HB, Su JZ. Co-transfection of p16(INK4a) and p53 genes into the K562 cell line inhibits cell proliferation[J]. Haematologica, 2002, 87(2): 136-142.
21. Adachi Y, Chandrasekar N, Kin Y, et al. Suppression of glioma invasion and growth by adenovirus-mediated delivery of a bicistronic construct containing antisense uPAR and sense p16 gene sequences[J]. Oncogene, 2002, 21(1): 87-95.
22. Gao N, Hu YD, CaoXY, et al. The exogenous wild-type p14ARF gene induces growth arrest and promotes radiosensitivity in human lung cancer cell lines[J]. J Cancer Res Clin Oncol, 2001, 127(6): 359-367.