Chakraborty Chandraditya, Roychowdhury Anirban, Samadder Sudip, Roy Anup, Mandal Ranajit Kumar, Basu Partha, Roychoudhury Susanta, Panda Chinmay Kumar
Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, West Bengal, India.
North Bengal Medical College and Hospital, Siliguri, West Bengal, India.
Biochem J. 2016 Oct 1;473(19):3221-36. doi: 10.1042/BCJ20160323. Epub 2016 Jul 25.
To understand the molecular mechanism of RB1 phosphorylation in basal-parabasal layers of normal cervix and during cervical cancer (CACX) development, we analyzed the alterations (expression/methylation/deletion/mutation) of RB1/phosphorylated RB1 (p-RB1) (ser807/811 and ser567) and two RB1 phosphorylation inhibitors, P16 and RBSP3, in disease-free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 28), CACX (n = 102) samples and two CACX cell lines. Immunohistochemical analysis revealed high/medium expression of RB1/p-RB1 (ser807/811 and ser567) and low expression of P16 and RBSP3 in proliferating basal-parabasal layers of majority of normal cervical epitheliums, irrespective of HPV16 infection. Interestingly, 35-52% samples showed high/medium expression of P16 in basal-parabasal layers of normal and had significant association with deleterious non-synonimous SNPs of P16. Methylation of P16 and RBSP3 in basal-parabasal layers of normal cervix (32 and 62%, respectively) showed concordance with their respective expressions in basal-parabasal layers. The methylation frequency of P16 and RBSP3 in basal-parabasal layers of normal did not change significantly in CIN and CACX. The deletion frequency of P16 and RB1 increased significantly with CACX progression. While, deletion of RBSP3 was high in CIN and comparable during CACX progression. P16 showed scattered and infrequent mutation in CACX. The alteration of P16 and RBSP3 was synergistic and showed association with overexpression of p-RB1 in tumors and associated with poor prognosis of patients. Thus, our data suggest that overexpression of p-RB1 in basal-parabasal layers of normal cervical epithelium was due to methylation/low functional-linked non-synonimous SNPs of P16 and RBSP3. This pattern was maintained during cervical carcinogenesis by additional deletion/mutation.
为了解正常宫颈基底层-副基底层以及宫颈癌（CACX）发生发展过程中RB1磷酸化的分子机制，我们分析了RB1/磷酸化RB1（p-RB1）（ser807/811和ser567）以及两种RB1磷酸化抑制剂P16和RBSP3在无病正常宫颈上皮（n = 9）、肿瘤相邻正常宫颈上皮（n = 70）、宫颈上皮内瘤变（CIN；n = 28）、CACX（n = 102）样本以及两种CACX细胞系中的改变（表达/甲基化/缺失/突变）。免疫组化分析显示，在大多数正常宫颈上皮的增殖性基底层-副基底层中，RB1/p-RB1（ser807/811和ser567）呈高/中度表达，而P16和RBSP3呈低表达，无论是否感染HPV16。有趣的是，35%-52%的样本在正常宫颈的基底层-副基底层中P16呈高/中度表达，且与P16的有害非同义单核苷酸多态性显著相关。正常宫颈基底层-副基底层中P16和RBSP3的甲基化（分别为32%和62%）与其在基底层-副基底层中的各自表达一致。正常宫颈基底层-副基底层中P16和RBSP3的甲基化频率在CIN和CACX中无显著变化。P16和RB1的缺失频率随CACX进展而显著增加。而RBSP3的缺失在CIN中较高，在CACX进展过程中相当。P16在CACX中表现为散在且罕见的突变。P16和RBSP3的改变具有协同性，且与肿瘤中p-RB1的过表达相关，并与患者的不良预后相关。因此，我们的数据表明，正常宫颈上皮基底层-副基底层中p-RB1的过表达是由于P16和RBSP3的甲基化/低功能相关非同义单核苷酸多态性。这种模式在宫颈癌发生过程中通过额外的缺失/突变得以维持。
