RB1 deletion in RB-pathway disrupted cells results in DNA damage and cancer progression. RB1 deletion in RB-pathway disrupted cells results in DNA damage and cancer progression

Proliferative control in cancer cells is frequently disrupted by mutations in the RB-pathway. Paradoxically, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB-pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage in CDKN2A deleted cancer cells when RB1 mutations are induced. We created isogenic RB1 wild type, heterozygous, and null genotypes. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immune compromised mice RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the pre-existing proliferative control deficit. Overall design: γH2AX localization was studied in three different genotypes of U2OS osteosarcoma cells: RB1+/+, RB1+/- and RB1-/-. As a control for possible histone density differences, histone 4 (H4) ChIP-Seq was also performed.

癌细胞的增殖调控常因RB通路（RB-pathway）突变而失调。矛盾的是，在RB通路已因另一突变而受损的癌细胞中，RB1突变可在肿瘤发生后期出现。本研究证实，当诱导RB1突变时，CDKN2A缺失的癌细胞中DNA损伤水平显著升高。我们构建了RB1野生型、杂合型及纯合缺失型的同基因（isogenic）细胞系。即使仅携带一个RB1突变拷贝的细胞，其基础DNA损伤水平及有丝分裂错误率均有所升高。此类DNA损伤的根源在于活性氧（reactive oxygen species）水平升高以及同源重组修复（homologous recombination repair）功能受损。将RB1突变细胞异种移植至免疫缺陷（immune compromised）小鼠体内后，其在受体小鼠肺部定植形成新肿瘤的倾向显著升高。本研究证实，肿瘤发生后期出现的RB1突变，可在已存在的增殖调控缺陷之外，进一步促进基因组不稳定性与癌症进展。整体实验设计：针对三种不同基因型的U2OS骨肉瘤细胞（RB1+/+、RB1+/-及RB1-/-），我们对γH2AX的定位情况进行了研究。为控制组蛋白密度差异带来的影响，本研究同时开展了组蛋白H4（H4）的染色质免疫沉淀测序（ChIP-Seq）实验。