A Novel Cell-Free Assay Identifies the Curcumin Analog EF24 as a Potent Inhibitor of the Fanconi Anemia Pathway

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2654-2654
Author(s):  
Igor Landais ◽  
Aiming Sun ◽  
Stacie N Stone ◽  
Alexandra Sobeck ◽  
James P Snyder ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Core Complex ◽  
Curcumin Analog ◽  
The Core ◽  
Curcumin Analogs ◽  
Damage Response ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Abstract Objective: The Fanconi anemia (FA) pathway is a DNA damage response network involved in the cellular resistance against DNA interstrand crosslinks (ICLs). A recent study showed that the FA pathway is synthetic lethal with several other DNA repair genes (Kennedy, 2007) such as ATM, NBS1, RAD54B and TP53BP1. Defects in those genes have been linked to a wide range of inherited and sporadic hematological malignancies including B-CLL, ALL, AML, CML, non-Hodgkin lymphoma, mantle cell lymphoma and multiple myeloma. FA pathway inhibitors may therefore selectively kill malignant cells bearing these defects. Curcumin, a natural product, was the first identified FA pathway inhibitor with activity in the micromolar range in cells (Chirnomas, 2006). However, the poor bioavailability of curcumin hinders its clinical efficacy. Identification of a curcumin analog with better activity, bioavailability and low toxicity could overcome this obstacle. We recently developed a cell-free assay for FA pathway function using Xenopus egg extracts to test the activity of curcumin analogs. As a pilot study we evaluated how well the assay identified inhibitors of the FA pathway in human cells. Methods: Fourteen curcumin analogs previously assayed in the NCI anticancer cell line screen (Adams, 2004) were tested for their activity on the FA pathway. Xenopus egg extracts were used to measure the relative inhibitory activity of the analogs on FANCD2 monoubiquitylation (FANCD2-L) and phosphorylation of other DNA damage response proteins. The underlying mechanism of inhibition was explored by testing the integrity of the core complex, the recruitment of the core complex to DNA and chromatin, and analyzing DNA replication and proteasome activity. Activity of several analogs was confirmed in HeLa cells by evaluation of the inhibition of hydroxyurea (HU)-induced FANCD2-L and FANCD2 foci. Results: EF24 (Adams, 2005) and three structurally similar analogs were 10 times more active than curcumin for FANCD2-L inhibition in Xenopus extracts. These analogs inhibited Mre11 phosphorylation at similar concentrations but had no effect on RPA32 and H2AX phosphorylation. In contrast to curcumin, EF24 did not display significant proteasome inhibition activity and did not affect integrity of the core complex or its recruitment to DNA and chromatin, ruling out these mechanisms to explain inhibition of the FA pathway. In HU-treated HeLa cells, EF24 strongly inhibited FANCD2-L and FANCD2 foci with an IC50 of 350 nM, confirming the results observed in Xenopus extracts. Conclusions: EF24 is a more potent FA pathway inhibitor than curcumin both in Xenopus extracts and in human cells, and as such may be effective as a single agent in targeted therapies against hematological malignancies deficient in ATM, NBS1, RAD54B or TP53BP1. In addition, this study demonstrates that Xenopus extracts are a powerful tool to identify and evaluate small molecules that modulate the FA and other DNA damage response pathways.

scholarly journals Cip29 is phosphorylated following activation of the DNA damage response in Xenopus egg extracts

PLoS ONE ◽  
2017 ◽  
Vol 12 (7) ◽  
pp. e0181131 ◽  
Author(s):  
Janet Holden ◽  
Elaine M. Taylor ◽  
Howard D. Lindsay
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Damage Response ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Identification and Partial Characterization of a Novel Partner Protein for Fanconi Anemia Protein FANCM

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3104-3104
Author(s):  
Stacie Stone ◽  
Alexandra Sobeck ◽  
Igor Landais ◽  
Weidong Wang ◽  
Maureen Hoatlin
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Protein Complexes ◽  
S Phase ◽  
Chromatin Binding ◽  
Core Complex ◽  
Dna Structures ◽  
Partner Protein ◽  
Damage Response ◽  
Egg Extracts

Abstract Fanconi anemia (FA) is an inherited hematological disorder characterized by bone marrow failure, birth defects, and cancer susceptibility, typically leading to squamous cell carcinomas and acute myelogenous leukemia. Twelve FA genes have been described, eight of which function together in a multiprotein, upstream “FA core complex” to mediate the S-phase and DNA damage-induced monoubiquitylation of two downstream proteins, FANCD2 and FANCI. Despite this knowledge the precise function of the FA proteins is not well understood because they function as part of a network of proteins that have not been completely defined. Recently we developed a new animal model for FA research using extracts from the eggs of Xenopus laevis. Xenopus extracts are cell cycle synchronized and contain nuclear proteins that are stockpiled for DNA replication. We showed that FA gene orthologs (xFA), like their human counterparts, form complexes that are required for the monoubiquitylation of xFANCD2 in response to DNA damage. Xenopus laevis extracts are thus a powerful system to analyze the endogenous state of xFA protein complexes and their components in an S phase, replication-competent context. The objective of this study was to isolate protein complexes containing the xFA core complex protein, xFANCM and xFANCM-interacting proteins. Using a co-immunoprecipitation approach followed by mass spectrometry, we identified a novel protein-binding partner of xFANCM (termed xMIP-1, for xFANCM Interacting Protein 1). The interaction was confirmed by reciprocal coimmunoprecipitation in both Xenopus extracts and human cells. Surprisingly, co-fractionation demonstrated that xFANCM was present in two protein complexes during S phase; one containing FA core complex members (<900 kDa) as expected, and another previously undescribed complex (>900 kDa) containing xMIP-1. Because xMIP-1 is a partner protein of xFANCM we wanted to determine if xMIP-1, like xFANCM, was required for the monoubiquitylation of xFANCD2. This was done using a DNA stimulation assay, where upon immunodepletion of xMIP-1 from egg extracts, we were able to observe the monoubiquitylation of xFANCD2 in response to DNA structures as a size shift via immunoblot. The absence of xMIP-1 had no detectable effect on the monoubiquitylation of xFANCD2 suggesting that xMIP-1, unlike xFANCM, was not required for xFANCD2 monoubiquitylation. To explore a functional link between xFANCM and xMIP-1 we used egg extracts to show that xMIP-1, like xFANCM, was recruited to replicating chromatin and exhibited a size shift during the replication process. Furthermore immunodepletion of xFANCM from egg extracts reduced recruitment of xMIP-1 to replicating chromatin, suggesting that xMIP-1 chromatin binding was dependent on xFANCM. In contrast, xMIP-1 recruitment to replicating chromatin was not affected by the immunodepletion of other FA core complex proteins tested, suggesting that xMIP-1 chromatin binding is independent of the FA core complex. To further characterize the observed DNA binding activity of xMIP-1 we used the DNA stimulation assay and several defined DNA structures. Surprisingly xMIP-1 showed a double-stranded DNA stimulated mobility shift similar to those reported previously for xFANCD2 (Sobeck et al., 2007) and xMRE11 (Costanzo et al., 2001) suggesting xMIP-1 may play a role in the DNA damage response. Our data suggests xFANCM is a member of an S phase complex that has not been previously described with a “non-FA” partner protein that may function with xFANCM during the DNA damage response to maintain genomic stability.

scholarly journals APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Md Akram Hossain ◽  
Yunfeng Lin ◽  
Garrett Driscoll ◽  
Jia Li ◽  
Anne McMahon ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pancreatic Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Dna Damage Response ◽  
Human Pancreatic Cancer ◽  
Genome Integrity ◽  
Pancreatic Cancer Cells ◽  
Damage Response ◽  
Egg Extracts

The maintenance of genome integrity and fidelity is vital for the proper function and survival of all organisms. Recent studies have revealed that APE2 is required to activate an ATR-Chk1 DNA damage response (DDR) pathway in response to oxidative stress and a defined DNA single-strand break (SSB) in Xenopus laevis egg extracts. However, it remains unclear whether APE2 is a general regulator of the DDR pathway in mammalian cells. Here, we provide evidence using human pancreatic cancer cells that APE2 is essential for ATR DDR pathway activation in response to different stressful conditions including oxidative stress, DNA replication stress, and DNA double-strand breaks. Fluorescence microscopy analysis shows that APE2-knockdown (KD) leads to enhanced γH2AX foci and increased micronuclei formation. In addition, we identified a small molecule compound Celastrol as an APE2 inhibitor that specifically compromises the binding of APE2 but not RPA to ssDNA and 3′-5′ exonuclease activity of APE2 but not APE1. The impairment of ATR-Chk1 DDR pathway by Celastrol in Xenopus egg extracts and human pancreatic cancer cells highlights the physiological significance of Celastrol in the regulation of APE2 functionalities in genome integrity. Notably, cell viability assays demonstrate that APE2-KD or Celastrol sensitizes pancreatic cancer cells to chemotherapy drugs. Overall, we propose APE2 as a general regulator for the DDR pathway in genome integrity maintenance.

Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 723-723
Author(s):  
Alexandra Sobeck ◽  
Stacie Stone ◽  
Bendert deGraaf ◽  
Vincenzo Costanzo ◽  
Johan deWinter ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
S Phase ◽  
Dependent Manner ◽  
Core Complex ◽  
Damage Response ◽  
Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

scholarly journals Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Songli Zhu ◽  
Mohammadjavad Paydar ◽  
Feifei Wang ◽  
Yanqiu Li ◽  
Ling Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

scholarly journals Distinct roles of XRCC1 in genome integrity in Xenopus egg extracts

2019 ◽  
Vol 476 (24) ◽  
pp. 3791-3804 ◽  
Author(s):  
Steven Cupello ◽  
Yunfeng Lin ◽  
Shan Yan
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Dna Repair ◽  
Oxidative Dna Damage ◽  
Dna Lesions ◽  
Genome Integrity ◽  
Polymerase Beta ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Oxidative DNA damage represents one of the most abundant DNA lesions. It remains unclear how DNA repair and DNA damage response (DDR) pathways are co-ordinated and regulated following oxidative stress. While XRCC1 has been implicated in DNA repair, it remains unknown how exactly oxidative DNA damage is repaired and sensed by XRCC1. In this communication, we have demonstrated evidence that XRCC1 is dispensable for ATR-Chk1 DDR pathway following oxidative stress in Xenopus egg extracts. Whereas APE2 is essential for SSB repair, XRCC1 is not required for the repair of defined SSB and gapped plasmids with a 5′-OH or 5′-P terminus, suggesting that XRCC1 and APE2 may contribute to SSB repair via different mechanisms. Neither Polymerase beta nor Polymerase alpha is important for the repair of defined SSB structure. Nonetheless, XRCC1 is important for the repair of DNA damage following oxidative stress. Our observations suggest distinct roles of XRCC1 for genome integrity in oxidative stress in Xenopus egg extracts.

scholarly journals DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

2002 ◽  
Vol 158 (5) ◽  
pp. 863-872 ◽  
Author(s):  
Matthew P. Stokes ◽  
Ruth Van Hatten ◽  
Howard D. Lindsay ◽  
W. Matthew Michael
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Alkylating Agents ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Damaged Dna

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

scholarly journals DNA damage-induced replication arrest in Xenopus egg extracts

2003 ◽  
Vol 163 (2) ◽  
pp. 245-255 ◽  
Author(s):  
Matthew P. Stokes ◽  
W. Matthew Michael
Keyword(s):  
Dna Damage ◽  
Proliferating Cell Nuclear Antigen ◽  
Replication Fork ◽  
Nuclear Antigen ◽  
Chromosomal Replication ◽  
Replication Arrest ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Proliferating Cell

Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase α, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase α and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA.

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