Suzuki Satoshi, Iwaizumi Moriya, Tseng-Rogenski Stephanie, Hamaya Yasushi, Miyajima Hiroaki, Kanaoka Shigeru, Sugimoto Ken, Carethers John M
a First Department of Medicine , Hamamatsu University School of Medicine , Hamamatsu , Shizuoka , Japan.
b Division of Gastroenterology, Department of Internal Medicine and Department of Human Genetics, University of Michigan , Ann Arbor , MA , USA.
Cancer Biol Ther. 2016 Jul 2;17(7):760-8. doi: 10.1080/15384047.2016.1178430. Epub 2016 Apr 26.
Methyl-CpG binding domain protein 4 (MBD4) is a DNA glycosylase that can remove 5-fluorodeoxyuracil from DNA as well as repair T:G or U:G mismatches. MBD4 is a target for frameshift mutation with DNA mismatch repair (MMR) deficiency, creating a truncated MBD4 protein (TruMBD4) that lacks its glycosylase domain. Here we show that TruMBD4 plays an important role for enhancing 5-fluorouracil (5FU) sensitivity in MMR-deficient colorectal cancer cells. We found biochemically that TruMBD4 binds to 5FU incorporated into DNA with higher affinity than MBD4. TruMBD4 reduced the 5FU affinity of the MMR recognition complexes that determined 5FU sensitivity by previous reports, suggesting other mechanisms might be operative to trigger cytotoxicity. To analyze overall 5FU sensitivity with TruMBD4, we established TruMBD4 overexpression in hMLH1-proficient or -deficient colorectal cancer cells followed by treatment with 5FU. 5FU-treated TruMBD4 cells demonstrated diminished growth characteristics compared to controls, independently of hMLH1 status. Flow cytometry revealed more 5FU-treated TruMBD4 cells in S phase than controls. We conclude that patients with MMR-deficient cancers, which show characteristic resistance to 5FU therapy, may be increased for 5FU sensitivity via secondary frameshift mutation of the base excision repair gene MBD4.
甲基化CpG结合域蛋白4（MBD4）是一种DNA糖基化酶，可从DNA中去除5-氟脱氧尿嘧啶，并修复T:G或U:G错配。MBD4是DNA错配修复（MMR）缺陷导致移码突变的靶点，会产生一种缺乏糖基化酶结构域的截短型MBD4蛋白（TruMBD4）。在此我们表明，TruMBD4在增强MMR缺陷型结直肠癌细胞对5-氟尿嘧啶（5FU）的敏感性方面发挥重要作用。我们通过生化方法发现，TruMBD4与掺入DNA的5FU结合的亲和力高于MBD4。此前的报道表明，MMR识别复合物决定了5FU敏感性，而TruMBD4降低了该复合物对5FU的亲和力，这表明可能存在其他机制引发细胞毒性。为了分析TruMBD4对5FU的整体敏感性，我们在hMLH1功能正常或缺陷的结直肠癌细胞中过表达TruMBD4，随后用5FU处理。与对照组相比，经5FU处理的TruMBD4细胞生长特性减弱，且与hMLH1状态无关。流式细胞术显示，经5FU处理的TruMBD4细胞处于S期的比对照组更多。我们得出结论，对5FU治疗具有特征性耐药性的MMR缺陷型癌症患者，可能通过碱基切除修复基因MBD4的继发性移码突变而提高对5FU的敏感性。
