Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis

Abstract

Cisplatin is one of the most widely used anticancer agents, displaying activity against a wide variety of tumors. However, development of drug resistance presents a challenging barrier to successful cancer treatment by cisplatin. To understand the mechanism of cisplatin resistance, we investigated the role of damaged DNA binding protein complex subunit 2 (DDB2) in cisplatin-induced cytotoxicity and apoptosis. We show that DDB2 is not required for the repair of cisplatin-induced DNA damage, but can be induced by cisplatin treatment. DDB2-deficient noncancer cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin than cells with fully restored DDB2 function. Moreover, DDB2 expression in cisplatin-resistant ovarian cancer cell line CP70 and MCP2 was lower than their cisplatin-sensitive parental A2780 cells. Overexpression of DDB2 sensitized CP70 cells to cisplatin-induced cytotoxicity and apoptosis via activation of the caspase pathway and downregulation of antiapoptotic Bcl-2 protein. Further analysis indicates that the overexpression of DDB2 in CP70 cells downregulates Bcl-2 expression through decreasing Bcl-2 mRNA level. These results suggest that ovarian cancer cells containing high level of DDB2 become susceptible to cisplatin by undergoing enhanced apoptosis.

DDB2 association with PCNA is required for its degradation after UV-induced DNA damage

“…DDB2 co-localizes with p21 and PCNA at DNA damage sites In this work, a pcDNA3.1-DDB2 wild-type construct 6 was expressed in HeLa cells, and nuclear localization and expression levels were confirmed by immunofluorescence microscopy and western blotting, respectively (data not shown). To study the recruitment of the exogenous DDB2 to DNA damage sites, HeLa cells were transfected, then exposed to local UV-C (100 J/m 2 ) irradiation through filters with 3-μm pores.…”

“…26 Expression construct coding for DDB2 was kindly provided by Dr Q Wang. 6 To obtain DDB2 mutated in the PIPbox sequence (DDB2 mut ), the DDB2 For 3M: CCATCTGTCG CCCAGGGGGC CCAGCAGTCC GCCTTGCAC and DDB2 Rev 3M: GTGCAAGGCG GACTGCTGGG CCCCCTGGGC GACAGATGG primers were used. The mutated gene was cloned in the same expression vector of the wt form and the sequence was verified.…”

“…These results support the idea that DDB2 degradation is crucial for XPC binding to DNA damage sites during the initial steps of NER. 6,13,14 DDB2 regulates the cellular levels of the cell cycle inhibitor p21 CDKN1A (p21), an important player in the DNA damage response that was suggested to participate in various DNA repair processes thanks to its interaction with PCNA. 14 In particular, in NER p21 was found to disrupt the association of p300 with PCNA, 15 thereby removing the inhibitory effect on the acetylation of histones, 16 but also of NER factors, such as XPG.…”

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

“…Normal human fibroblasts OSU-2 cells were established and maintained in culture in our laboratory, DDB2-deficient Li-Fraumeni Syndrome fibroblast strain, designated 041 fibroblasts, were kindly provided by Dr. Michael Tainsky (MD Anderson Cancer center, Houston, TX), DDB2-expressing 041 cell line (041-N22) was established in our laboratory by stably transfecting pcDNA3.1-His-DDB2 into 041 cells. 32 These cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics. XP-A (GM05509), XP-C (GM02096), XP-D (GM03615), XP-E (GM01389), and XP-F (GM04313) fibroblasts were purchased from NIGMS Human Genetic Cell Repository (Camden, NJ), and were grown in MEM supplemented with 10% FCS and antibiotics.…”

“…11 However, DDB2 is required for the removal of UVinduced CPD but is not required for the repair of cisplatininduced intra-strand crosslinks. 28,[30][31][32] Thus, it appears that the requirement of DDB2 in UV-induced, but not cisplatininduced, XPG degradation is attributable to the ability of DDB2 to recognize DNA damage.…”