Mitochondrial Targeting of HumanO6-Methylguanine DNA Methyltransferase Protects against Cell Killing by Chemotherapeutic Alkylating Agents

Abstract

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas

“…Consistent with this assertion, it is known that mtDNA mutations result in premature aging (49) or sensitize cells to mitochondrial-dependent apoptosis (50). Meanwhile, increased ROS can result in mutations of oncogenes and/or tumor suppressor genes, leading to the deregulation of cell-cycle progression and proliferation, the escape of some cells from apoptosis, and, finally, formation of cancer cells.…”

DNA Base Excision Repair: Evolving Biomarkers for Personalized Therapies in Cancer

“…A detailed discussion of individual pathways is beyond the scope of this chapter as several recent excellent reviews on DNA repair are available [1][2][3][4][5][6]. Briefly, direct repair is involved in the repair of alkylated bases (such as O 6 methyl guanine) by MGMT (O 6 methyl guanine DNA methyl transferases [7][8][9][10]. DNA mismatch repair (MMR) corrects base-base mismatches and insertion-deletion loops (IDLs) erroneously generated during DNA replication and by exogenous DNA damage [11][12][13].…”

Mitochondrial DNA repair in aging and disease

“…Transfection with nuclear-or mitochondrialtargeted MGMT generated resistance in these cells against the cytotoxic effects of chemotherapeutic drugs such as 2-chloroethylnitrosourea (BCNU) and temozolomide (TMZ). This effect was more dependent on mitochondrial than on nuclear MGMT, emphasizing the contribution of mtDNA repair mechanisms in cellular resistance to alkylation chemotherapy (135).…”

“…Evidence indicates that protection of mtDNA after alkylation is equally as important as protection of nuclear DNA. Cai et al carried out experiments with hematopoietic cell lines characterized by low repair activity for alkylation DNA damage (135). Transfection with nuclear-or mitochondrialtargeted MGMT generated resistance in these cells against the cytotoxic effects of chemotherapeutic drugs such as 2-chloroethylnitrosourea (BCNU) and temozolomide (TMZ).…”