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

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.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

Case Report: Recent Case Reports of Levant Blunt-Nosed Viper Macrovipera lebetina obtusa Snakebites in Iran.

Envenomation and death resulting from snakebites represent a significant public health problem worldwide, particularly in tropical and subtropical regions. The WHO has defined snakebite as a neglected tropical health concern. Bites from Macrovipera lebetina obtusa usually cause life-threatening systemic hemodynamic disturbances, reduced functionality of the kidneys, and other serious symptoms, including hypotension shock, edema, and tissue necrosis, at the bite site. Herein, we highlight five cases of M. l. obtusa envenomation that presented with wide-ranging manifestations. Many recovered cases were left with long-term musculoskeletal disabilities. In a particular case, a 15-year-old male patient was envenomed in his palm by an 80-cm M. l. obtusa. Within 12 hours, swelling extended to near the shoulder. Fasciotomy was performed on the forearm and part of the upper arm of this patient. Symptoms of severe localized pain and swelling, dizziness, weakness, low blood pressure, and itching around the bite area were documented. The patient remained in the hospital for 13 days.

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 present 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 formaldehyde-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 breakages 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.

Comparative study of the in vivo toxicity and pathophysiology of envenomation by three medically important Egyptian snake venoms.

Snakebite envenomation is a serious medical problem in many developing tropical and subtropical countries. Envenomation is registered by the World Health Organization as a neglected tropical disease due to critical shortages in the production of antivenom. Envenomation causes more than 100,000 deaths annually. Snakebites result in several effects to include edema, blistering, hemorrhage, necrosis and respiratory paralysis. Antivenom is the preferred treatment for the systemic effects of snakebite envenomation, though these are often ineffective in neutralizing venom toxin-induced local tissue damage. To effectively treat snakebites, it is important to determine the lethal potency and pathophysiological effects induced by specific snake venoms. In the current study, we compared the lethality, and the hemorrhagic and dermonecrotic activities of venoms from three snakes in Egypt that are the primary causes of local tissue necrosis. Our data show that the intraperitoneal median lethal doses (LD50) for Cerastes cerastes, Echis carinatus and Naja nigricollis venoms are 0.946, 1.744 and 0.341 mg/kg mouse body weight, respectively. These results indicated that N. nigricollis venom is the most toxic and significantly accelerated the time of death compared to the other two venoms. However, no hematoma or associated edema appeared upon sub-plantar injection of N. nigricollis venom into the mice hind paw. Two hours following intradermal injection of C. cerastes and E. carinatus venoms, macroscopic analysis of the inner surface of mouse skin showed severe hemorrhagic lesions, whereas only insignificant hemorrhagic lesion appeared in mice injected with the highest dose of N. nigricollis venom. Furthermore, the minimum necrotic doses (MND) for the same venoms were 43.15, and 70.87 µg/mouse, or not observed in the case of N. nigricollis venom, respectively. These LD50 values and pathophysiological results can be used to guide development of antivenom against bites by these dangerous Egyptian snakes.

DNA-protein cross-links: Formidable challenges to maintaining genome integrity.

DNA is associated with proteins that are involved in its folding and transaction processes. When cells are exposed to chemical cross-linking agents or free radical-generating ionizing radiation, DNA-associated proteins are covalently trapped within the DNA to produce DNA-protein cross-links (DPCs). DPCs produced by these agents contain cross-linked proteins in an undisrupted DNA strand. Some DNA-metabolizing enzymes that form covalent reaction intermediates can also be irreversibly trapped in the presence of inhibitors or DNA damage to give rise to abortive DPCs. The abortive DPCs often contain cross-linked proteins attached to the 5' or 3' end of a DNA strand break. In vitro studies show that steric hindrance caused by cross-linked proteins impedes the progression of DNA helicases and polymerases and of RNA polymerases. The modes and consequences by which DPCs impede replication and transcription processes are considerably different from those with conventional DNA lesions. Thus, DPCs are formidable challenges to maintaining genome integrity and faithful gene expression. Current models of DPC repair involve direct and indirect removal of DPCs. The direct mechanism works for DPCs that contain topoisomerase 2 attached to the 5' end of DNA. The Mre11-Rad50-Nbs1 complex cleaves the site internal to the DPC and directly releases a DPC-containing oligonucleotide. The indirect mechanism involves degradation of cross-linked proteins by proteasomes or the recently identified DPC proteases Wss1 and Sprtn to relieve steric hindrance of DPCs. The resulting peptide-cross-links might be processed by translesion synthesis or other canonical repair mechanisms: however, the exact mechanism remains to be elucidated.

Radiation-induced DNA-protein cross-links: Mechanisms and biological significance.

Ionizing radiation produces various DNA lesions such as base damage, DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-protein cross-links (DPCs). Of these, the biological significance of DPCs remains elusive. In this article, we focus on radiation-induced DPCs and review the current understanding of their induction, properties, repair, and biological consequences. When cells are irradiated, the formation of base damage, SSBs, and DSBs are promoted in the presence of oxygen. Conversely, that of DPCs is promoted in the absence of oxygen, suggesting their importance in hypoxic cells, such as those present in tumors. DNA and protein radicals generated by hydroxyl radicals (i.e., indirect effect) are responsible for DPC formation. In addition, DPCs can also be formed from guanine radical cations generated by the direct effect. Actin, histones, and other proteins have been identified as cross-linked proteins. Also, covalent linkages between DNA and protein constituents such as thymine-lysine and guanine-lysine have been identified and their structures are proposed. In irradiated cells and tissues, DPCs are repaired in a biphasic manner, consisting of fast and slow components. The half-time for the fast component is 20min-2h and that for the slow component is 2-70h. Notably, radiation-induced DPCs are repaired more slowly than DSBs. Homologous recombination plays a pivotal role in the repair of radiation-induced DPCs as well as DSBs. Recently, a novel mechanism of DPC repair mediated by a DPC protease was reported, wherein the resulting DNA-peptide cross-links were bypassed by translesion synthesis. The replication and transcription of DPC-bearing reporter plasmids are inhibited in cells, suggesting that DPCs are potentially lethal lesions. However, whether DPCs are mutagenic and induce gross chromosomal alterations remains to be determined.

Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets.

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,ß-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,ß-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage.

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 DNA transactions and confer deleterious effects on cells in conjunction with DSBs.

Translocation and stability of replicative DNA helicases upon encountering DNA-protein cross-links.

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0-14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15-36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.

Detection of DNA-protein crosslinks (DPCs) by novel direct fluorescence labeling methods: distinct stabilities of aldehyde and radiation-induced DPCs.

Proteins are covalently trapped on DNA to form DNA-protein crosslinks (DPCs) when cells are exposed to DNA-damaging agents. DPCs interfere with many aspects of DNA transactions. The current DPC detection methods indirectly measure crosslinked proteins (CLPs) through DNA tethered to proteins. However, a major drawback of such methods is the non-linear relationship between the amounts of DNA and CLPs, which makes quantitative data interpretation difficult. Here we developed novel methods of DPC detection based on direct CLP measurement, whereby CLPs in DNA isolated from cells are labeled with fluorescein isothiocyanate (FITC) and quantified by fluorometry or western blotting using anti-FITC antibodies. Both formats successfully monitored the induction and elimination of DPCs in cultured cells exposed to aldehydes and mouse tumors exposed to ionizing radiation (carbon-ion beams). The fluorometric and western blotting formats require 30 and 0.3 µg of DNA, respectively. Analyses of the isolated genomic DPCs revealed that both aldehydes and ionizing radiation produce two types of DPC with distinct stabilities. The stable components of aldehyde-induced DPCs have half-lives of up to days. Interestingly, that of radiation-induced DPCs has an infinite half-life, suggesting that the stable DPC component exerts a profound effect on DNA transactions over many cell cycles.

Repair and biochemical effects of DNA-protein crosslinks.

Genomic DNA is associated with various structural, regulatory, and transaction proteins. The dynamic and reversible association between proteins and DNA ensures the accurate expression and propagation of genetic information. However, various endogenous, environmental, and chemotherapeutic agents induce DNA-protein crosslinks (DPCs), and hence covalently trap proteins on DNA. Since DPCs are extremely large compared to conventional DNA lesions, they probably impair many aspects of DNA transactions such as replication, transcription, and repair due to steric hindrance. Recent genetic and biochemical studies have shed light on the elaborate molecular mechanism by which cells repair or tolerate DPCs. This review summarizes the current knowledge regarding the repair and biochemical effects of the most ubiquitous form of DPCs, which are associated with no flanked DNA strand breaks. In bacteria small DPCs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed by RecBCD-dependent homologous recombination (HR). NER does not participate in the repair of DPCs in mammalian cells, since the upper size limit of DPCs amenable to mammalian NER is smaller than that of bacterial NER. Thus, DPCs are processed exclusively by HR. The reactivation of the stalled replication fork at DPCs by HR seems to involve fork breakage in mammalian cells but not in bacterial cells. In addition, recent proteomic studies have identified the numbers of proteins in DPCs induced by environmental and chemotherapeutic agents. However, it remains largely elusive how DPCs affect replication and transcription at the molecular level. Considering the extremely large nature of DPCs, it is possible that they impede the progression of replication and transcription machineries by mechanisms different from those for conventional DNA lesions. This might also be true for the DNA damage response and signaling mechanism.