Physical state of HPV16 and chromosomal mapping of the integrated form in cervical carcinomas
Physical state of HPV16 and chromosomal mapping of the integrated form in cervical carcinomas
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DOI：
10.1097/00019606-200103000-00008
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发表时间：
2001-03-01
影响因子：
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通讯作者：
Johansson, B
中科院分区：
文献类型：
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作者：
Kalantari, M;Blennow, E;Johansson, B
使用基于限制性酶切、自身连接和反向聚合酶链反应（rliPCR）的程序，作者调查了18例宫颈上皮内瘤变III（CIN III）病例和37例浸润性鳞状细胞癌中人乳头瘤病毒16型（HPV 16）的整合。发现所有18例CIN III病例（严重异型增生或高度鳞状上皮内病变）均携带游离型HPV，但其中1例样本含有混合游离型和整合型HPV。37例浸润性宫颈癌中有17例先前通过使用覆盖整个E1/E2基因的PCR鉴定为含有完全整合的HPV 16基因组，并且在16例病例中通过rliPCR证实了这一点。然而，一个案例中，表现出低水平的附加型脱氧核糖核酸除了占主导地位的整合形式。在先前分析中显示附加型形式的剩余20个癌样品中，使用rliPCR发现14个含有整合形式，并且4个含有多聚体附加型形式。因此，总共37个癌中的31个（84%）显示整合的HPV 16基因组。将来自五个癌病例的rliPCR产物克隆到质粒载体中，并用作“引物步移”脱氧核糖核酸测序的模板，以推断整合的HPV基因组侧翼的人类序列。在此基础上，细菌人工染色体（BAC）和π衍生的人工染色体（PAC）克隆，并作为探针在人类中期染色体的荧光原位杂交实验。荧光原位杂交实验的结果表明，HPV 16整合在染色体区域1 q25，3q 28，6p 25，11 p13和18 q22的证据。16个癌样本，含有附加型HPV 16，在长对照区进行测序。E2结合或沉默YY 1序列的变化的证据中发现，只有两个样品。
Using a procedure based on restriction enzyme cleavage, self-ligation, and inverse polymerase chain reaction (rliPCR), the authors investigated 18 cervical intraepithelial neoplasia III (CIN III) cases and 37 invasive squamous carcinomas for integration of human papillomavirus type 16 (HPV16). All eighteen CIN III cases (severe dysplasia or high-grade squamous intraepithelial lesion) were found to harbor episomal HPV, but one of the samples contained mixed episomal and integrated forms. Seventeen of 37 invasive cervical carcinoma samples were identified previously as containing the completely integrated HPV 16 genome by using PCR covering the entire E1/E2 gene, and this was confirmed by rliPCR in 16 cases. One case, however, showed a low level of episomal deoxyribonucleic acid in addition to the predominant integrated form. Of the remaining 20 carcinoma samples showing episomal forms in the previous analysis, 14 were found to contain integrated forms using rliPCR, and four contained multimeric episomal forms. Thus, in total, 31 of 37 of the carcinomas (84%) showed the integrated HPV16 genome. The rliPCR product from five carcinoma cases was cloned into a plasmid vector and used as a template for "primer walking" deoxyribonucleic acid sequencing to deduce human sequences flanking the integrated HPV genome. Based on this information, bacterial artificial chromosome (BAC) and pi-derived artificial chromosome (PAC) clones were obtained and used as probes in fluorescent in situ hybridization experiments on human metaphase chromosomes. The results of the fluorescent in situ hybridization experiments showed evidence for HPV16 integration in chromosome regions 1q25, 3q28, 6p25, 11p13, and 18q22. Sixteen carcinoma samples, containing episomal HPV16, were sequenced in the long control region. Evidence for changes in E2 binding or silencer YY1 sequences was found in only two samples.