Induction of DNA–protein cross-links by ionizing radiation and their elimination from the genome

Ionizing radiation produces various types of DNA lesions, such as base damage, single-strand breaks, double-strand breaks (DSBs), and DNA–protein cross-links (DPCs). Of these, DSBs are the most critical lesions underlying the lethal effects of ionizing radiation. With DPCs, proteins covalently trapped in DNA constitute strong roadblocks to replication and transcription machineries, and hence can be lethal to cells. The formation of DPCs by ionizing radiation is promoted in the absence of oxygen, whereas that of DSBs is retarded. Accordingly, the contribution of DPCs to the lethal events in irradiated cells may not be negligible for hypoxic cells, such as those present in tumors. However, the role of DPCs in the lethal effects of ionizing radiation remains largely equivocal. In the present study, normoxic and hypoxic mouse tumors were irradiated with X-rays [low linear energy transfer (LET) radiation] and carbon (C)-ion beams (high LET radiation), and the resulting induction of DPCs and DSBs and their removal from the genome were analyzed. X-rays and C-ion beams produced more DPCs in hypoxic tumors than in normoxic tumors. Interestingly, the yield of DPCs was slightly but statistically significantly greater (1.3- to 1.5-fold) for C-ion beams than for X-rays. Both X-rays and C-ion beams generated two types of DPC that differed according to their rate of removal from the genome. This was also the case for DSBs. The half-lives of the rapidly removed components of DPCs and DSBs were similar (<1 h), but those of the slowly removed components of DPCs and DSBs were markedly different (3.9–5 h for DSBs versus 63–70 h for DPCs). The long half-life and abundance of the slowly removed DPCs render them persistent in DNA, which may impede DNAtransactions and confer deleterious effects on cells in conjunction with DSBs

Participation of TDP1 in the repair of formaldehyde-induced DNA-protein cross- links in chicken DT40 cells

Proteins are covalently trapped on DNA to form DNA-protein cross-links (DPCs) when cells are exposed to DNA-damaging agents. Aldehyde compounds produce common types of DPCs that contain proteins in an undisrupted DNA strand. Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs topoisomerase 1 (TOPO1) that is trapped at the 3’-end of DNA. In the pres- ent study, we examined the contribution of TDP1 to the repair of formaldehyde-induced DPCs using a reverse genetic strategy with chicken DT40 cells. The results obtained showed that cells deficient in TDP1 were sensitive to formaldehyde. The removal of formal- dehyde-induced DPCs was slower in tdp1-deficient cells than in wild type cells. We also found that formaldehyde did not produce trapped TOPO1, indicating that trapped TOPO1 was not a primary cytotoxic DNA lesion that was generated by formaldehyde and repaired by TDP1. The formaldehyde treatment resulted in the accumulation of chromosomal break- ages that were more prominent in tdp1-deficient cells than in wild type cells. Therefore, TDP1 plays a critical role in the repair of formaldehyde-induced DPCs that are distinct from trapped TOPO1

Targeting cis-regulatory elements of FOXO family is a novel therapeutic strategy for induction of leukemia cell differentiation

Abstract Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation