Metformin Induces Persistent DNMT1-Associated DNA–Protein Cross-Links under Glucose Deprivation

Metformin, a widely prescribed biguanide for the treatment of type 2 diabetes, is known to exhibit selective cytotoxicity under glucose-deprived conditions that mimic the metabolic environment of poorly vascularized solid tumors. We previously demonstrated, using chicken DT40 cells, that metformin induces DNA–protein cross-links (DPCs) under glucose deprivation. However, the molecular identity of these DPCs and their biological significance in human cells have remained unclear. In the present study, we investigated the nature of metformin-induced DNA damage under metabolic stress conditions using human lymphoblastoid TK6 cells. Metformin induced marked cytotoxicity and chromosome fragmentation under glucose-deprived conditions. Genetic analyses revealed marked hypersensitivity of SPRTN-deficient cells, whereas cells deficient in TDP1, TDP2, or both TDP1 and TDP2 exhibited only modest increases in sensitivity. FITC-based quantification demonstrated that metformin increased the overall DPC burden under both glucose-supplemented and glucose-deprived conditions. In contrast, DNA methyltransferase 1 (DNMT1)-associated DPCs were preferentially detected under glucose deprivation, whereas Topoisomerase I- or Topoisomerase II-associated DPCs were not substantially induced. Furthermore, DNA-associated DNMT1 showed increased levels and prolonged persistence following metformin treatment in sprtn/tdp1/tdp2 triple mutant cells. These findings suggest that the biological consequences of metformin-induced DPCs cannot be explained by their overall abundance alone and may also be related to differences in their molecular composition under glucose-deprived conditions.