Genomic instability and cancer: lessons learned from human papillomaviruses

High-risk HPV E6 and E7 oncoproteins cooperate to subvert critical host cell cycle checkpoint control mechanisms in order to promote viral genome replication. This results not only in aberrant proliferation but also in host cellular changes that can promote genomic instability. The HPV-16 E7 oncoprotein was found to induce centrosome abnormalities thereby disrupting mitotic fidelity and increasing the risk for chromosome missegregation and aneuploidy. In addition, expression of the high-risk HPV E7 oncoprotein stimulates DNA replication stress as a potential source of DNA breakage and structural chromosomal instability. Proliferation of genomically unstable cells is sustained by several mechanisms including the accelerated degradation of claspin by HPV-16 E7 and the degradation of p53 by the high-risk HPV E6 oncoprotein. These results highlight the oncogenic potential of aberrant proliferation and opens new avenues for prevention of malignant progression, not only in HPV-associated cervical cancer but also in non-virally associated malignancies with disrupted cell cycle checkpoint control mechanisms.     

Centrosome clustering and chromosomal (in)stability: a matter of life and death

Centrosome abnormalities occur commonly in cancer, and contribute to chromosomal instability and tumorigenesis. New evidence on a phylogenetically conserved mechanism termed 'centrosomal clustering' provides exciting insights into how cells with supernumerary centrosomes adapt to avoid lethal multipolar divisions. Here, we highlight the emerging molecular basis of centrosome clustering, and its impact on asymmetric divisions of stem cells, chromosomal (in)stability and malignant transformation. Finally, pharmacological inhibition of centrosome clustering promises to selectively target tumor cells.     

Aurora-A在宫颈癌及癌前病变中的表达以及与HPV感染的相关性研究

目的 分析正常宫颈、不同级别宫颈上皮内瘤变(Cervical intraepithelial neoplasia,CIN)和宫颈鳞状细胞癌(Cervical squamous cell carcinoma,SCC)组织中Aurora-A的表达差异,观察其异常表达对宫颈癌变过程的影响及与人乳头瘤病毒(Human papilloma virus,HPV)感染的相关性,了解Aroura-A在CIN与宫颈癌发病机制中的作用。方法 选取已进行宫颈管分泌物高危型HPV检测的宫颈活检或手术标本存档蜡块100例,其中正常宫颈组织20例,CIN1级组织20例、CIN 2级组织20例、CIN 3级组织20例,宫颈癌全部选取的是宫颈鳞状细胞癌组织20例。采用免疫组化方法检测Aurora-A蛋白的表达水平,并分析Aurora-A表达与HR-HPV感染的相关性。结果 Aurora-A在CIN以及宫颈癌中存在高表达(P＜0.05),且其阳性表达率随着宫颈病变程度的加深而增强,两者呈正相关性(r=0.475,P＜0.001);在高级别宫颈上皮内瘤变(CIN2、3)组织中,Aurora-A表达与HPV感染间存在正相关(V=0.591,P＜0.05)。结论 Aurora-A的异常表达可能与宫颈癌的发生发展密切有关,Aurora-A可能作为早期诊断CIN或宫颈癌的重要分子生物学指标,同时也可能是宫颈癌的潜在治疗靶点。     

Radiation-induced mitotic cell death and glioblastoma radioresistance: A new regulating pathway controlled by integrin-linked kinase,hypoxia-inducible factor 1alpha and survivin in U87 cells

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity.In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.     

Centrosome amplification as a putative prognostic biomarker for the classification of urothelial carcinomas

Recent studies have reported that centrosome amplification is closely related to chromosomal instability and patient prognosis in human malignancies. The purpose of this study was to elucidate the relationship between centrosome amplification and genomic alterations in urothelial carcinomas. Centrosomes were evaluated by immunohistochemistry using anti–γ-tubulin antibody. Array-based comparative genomic hybridization technology using DNA chips spotted with 4030 bacterial artificial chromosome clones was applied to 70 urothelial carcinomas to examine DNA copy number aberrations. Studying aberrations in the number of chromosomes 7, 9, and 17 using fluorescence in situ hybridization allowed the estimation of the degree of chromosomal instability. DNA copy number gains at 20p12.2, 5p15.2, 5p15.31, and 17q25.3 and losses at 17p12, 8p22, 2q37.3, 5q31.1, and 2q37.3 were more frequent in tumors with centrosome amplification than in those without it. The total numbers of DNA copy number aberrations and frequency of chromosomal instability were also larger in tumors with centrosome amplification than in those without it (P = .0263 and P < .0001, respectively). These parameters were more closely associated with centrosome amplification than with the subjectively assigned tumor grade (P = .0405 and P = .0020, respectively). Thus, these data suggest that centrosome amplification may have great potential as a biomarker for improved objective classification of urothelial carcinoma and estimation of prognosis.     

The human papillomavirus E7 oncoprotein

The human papillomavirus (HPV) E7 oncoprotein shares functional similarities with such proteins as adenovirus E1A and SV40 large tumor antigen. As one of only two viral proteins always expressed in HPV-associated cancers, E7 plays a central role in both the viral life cycle and carcinogenic transformation. In the HPV viral life cycle, E7 disrupts the intimate association between cellular differentiation and proliferation in normal epithelium, allowing for viral replication in cells that would no longer be in the dividing population. This function is directly reflected in the transforming activities of E7, including tumor initiation and induction of genomic instability.     

癌基因B-RafV600E导致黑色素瘤Sbcl2和SK-MEL31细胞染色体不稳定性的分子机制研究

目的 研究癌基因B-RafV600E导致肿瘤细胞染色体不稳定的分子机制.方法 采用RNA干扰技术敲除稳定表达B-RafV600E基因的黑色素瘤Sbcl2和SK-MEL31细胞中内源性单核纺锤体蛋白激酶(Mps1)基因表达,免疫荧光染色技术检测中心体及纺锤体结构.HU-arrest分析法观察Mps1基因缺失对癌基因B-RafV600E致肿瘤细胞中心体过度复制及多极纺锤体形成的影响.结果 未敲除内源性Mps1基因的表达B-RafV600E基因的Sbcl2和SK-MEL31细胞中36％出现中心体过度复制及多极纺锤体,Mps1基因被敲除后上述异常细胞比例降低至6％.结论 B-RafV600E可能通过Mps1调控中心体过度复制及多极纺锤体结构的形成,影响肿瘤细胞染色体不稳定性及非整倍体细胞的出现.     

原发性肝癌中p15、p16基因缺失和STK15基因过表达的研究

目的 探讨肿瘤组织p15、p16基因缺失和STK15基因过表达与原发性肝癌的关系。 方法 术中取原发性肝癌组织和相应癌旁正常组织标本30例,术前均未经化学疗法及放射疗法治疗。提取DNA并应用聚合酶链反应(PCR)技术检测p15第二外显子(p15E2)和p16第二外显子(p16E2)纯台缺失。提取RNA,逆转录合成cDNA,应用PCR技术检测STK15基因的表达。以β-肌动蛋白基因作为内对照。测定癌组织和癌旁正常组织中STK15基因和β-肌动蛋白基因的平均密度值(ADV)。 结果 在癌组织中p15E2缺失率为13.3％(4／30),p16E2缺失率为16.7％(5／30),p15E2和p16E2共缺失率为6.7％(2／30)。在30例癌组织中有19例(63.3％)STK15基因表达高于邻近正常组织。STK15基因ADV／β-肌动蛋白基因ADV比值,癌组织为1.53±0.31,癌旁正常组织为0.91±0.25,t=2.86,P<0.01。 结论 p15E2、p16E2纯合缺失和STK15过表达可能在原发性肝癌的发生发展中发挥一定作用。     

中心体异常扩增与宫颈鳞癌发生的相关性研究

目的　探讨中心体异常扩增在宫颈鳞状细胞癌发生发展过程中的作用及意义。方法　选取２００７年６月至２００９年６月暨南大学附属第一医院病理科的宫颈病变组织石蜡标本５６例,包括正常宫颈上皮１０例、宫颈上皮内瘤变２６例及宫颈鳞癌２０例。组织切片标本采用间接免疫荧光染色,通过荧光显微镜观察组织细胞内中心体的状态。结果　正常宫颈上皮、宫颈ＣＩＮⅠ均未发现中心体异常扩增,ＣＩＮⅡ及ＣＩＮⅢ发现中心体异常扩增,异常率分别为（３.０５±２.０１）％和（４.８３±３.４７）％;宫颈鳞癌Ⅰ、Ⅱ、Ⅲ期均存在中心体异常扩增,异常率分别为（６.０８±２.０３）％、（６.４１±３.５３）％和（６.９８±３.７０）％;宫颈鳞癌组中心体异常扩增率为（６.３３±２.７２）％,高于ＣＩＮ组的（２.５０±３.０６）％（Ｐ＜０.０１）;宫颈低分化鳞癌组中心体异常扩增率高于中分化组,分别为（７.７１±３.１３）％和（５.５９±２.２８）％（Ｐ＞０.０５）;中心体异常扩增率随ＣＩＮ病变程度及宫颈鳞癌分期增加而递增,随宫颈鳞癌细胞分化程度的降低而增加。结论　中心体异常扩增是宫颈上皮内瘤变及宫颈鳞癌中普遍存在的细胞学形态的异常现象,并可能与宫颈鳞癌的发展进程相关。因此,中心体异常扩增作为一个客观的细胞形态学异常指标可以辅助宫颈上皮内瘤变及宫颈鳞癌的诊断。     

Involvement of centrosomes in nuclear irregularity of thyroid carcinoma cells

Nuclear irregularities including nuclear pseudoinclusions and nuclear grooves are characteristic of papillary thyroid carcinoma cells and are regarded as important diagnostic clues in histopathology. We observed nuclear features of thyroid carcinoma cell lines (KTC-1 and TPC-1) in various culture conditions and performed immunocytochemical examinations for cytoskeleton molecules to clarify the morphogenesis of thyroid carcinoma nuclei. We found that nuclear irregularities presenting as bean-like nuclei (BLNs) and donut-like nuclei (DLNs) appeared in cells from confluent cultures, but not in cells from sparse cultures. On immunocytofluorescence analyses, clusters of γ-tubulin, representing a centrosome, frequently localized at the indentation of BLNs or in the hole of DLNs of thyroid carcinoma cells. In conclusion, we suggest that cell-to-cell contact may affect nuclear changes such as BLNs and DLNs in cancer cell lines and that centrosomes may be involved in the morphogenetic process of these nuclear changes.