Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Proteomic analysis of mismatch repair-mediated alkylating agent-induced DNA damage response.

ABSTRACT:

Background

Mediating DNA damage-induced apoptosis is an important genome-maintenance function of the mismatch repair (MMR) system. Defects in MMR not only cause carcinogenesis, but also render cancer cells highly resistant to chemotherapeutics, including alkylating agents. To understand the mechanisms of MMR-mediated apoptosis and MMR-deficiency-caused drug resistance, we analyze a model alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine, MNNG)-induced changes in protein phosphorylation and abundance in two cell lines, the MMR-proficient TK6 and its derivative MMR-deficient MT1.

Results

Under an experimental condition that MNNG-induced apoptosis was only observed in MutSa-proficient (TK6), but not in MutSa-deficient (MT1) cells, quantitative analysis of the proteomic data revealed differential expression and phosphorylation of numerous individual proteins and clusters of protein kinase substrates, as well differential activation of response pathways/networks in MNNG-treated TK6 and MT1 cells. Many alterations in TK6 cells are in favor of turning on the apoptotic machinery, while many of those in MT1 cells are to promote cell proliferation and anti-apoptosis.

Conclusions

Our work provides novel molecular insights into the mechanism of MMR-mediated DNA damage-induced apoptosis.

SUBMITTER: Chen X

PROVIDER: S-EPMC3848634 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage.

Project description:The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G(2) arrest after only one round of replication. In these G(2)-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G(2). In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2-hMSH6 from hMLH1-hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.

| S-EPMC2819642 | biostudies-literature

JAK2 regulates mismatch repair protein-mediated epigenetic alterations in response to oxidative damage.

Project description:At sites of chronic inflammation epithelial cells undergo aberrant DNA methylation that contributes to tumorigenesis. Inflammation is associated with an increase in reactive oxygen species (ROS) that cause oxidative DNA damage, which has also been linked to epigenetic alterations. We previously demonstrated that in response to ROS, mismatch repair proteins MSH2 and MSH6 recruit epigenetic silencing proteins DNA methyltransferase 1 (DNMT1) and polycomb repressive complex 2 (PRC2) members to sites of DNA damage, resulting in transcriptional repression of tumor suppressor genes (TSGs). However, it was unclear what signal is unique to ROS that results in the chromatin binding of MSH2 and MSH6. Herein, we demonstrate that in response to hydrogen peroxide (H2 O2 ), JAK2 localizes to the nucleus and interacts with MSH2 and MSH6. Inhibition or knockdown of JAK2 reduces the H2 O2 -induced chromatin interaction of MSH2, MSH6, DNMT1, and PRC2 members, reduces H2 O2 -induced global increase in trimethylation of lysine 27 of histone H3 (H3K27me3), and abrogates oxidative damage-induced transcriptional repression of candidate TSGs. Moreover, JAK2 mRNA expression is associated with CpG island methylator phenotype (CIMP) status in human colorectal cancer. Our findings provide novel insight into the connection between kinase activation and epigenetic alterations during oxidative damage and inflammation. Environ. Mol. Mutagen. 60:308-319, 2019. © 2018 Wiley Periodicals, Inc.

| S-EPMC8504556 | biostudies-literature

DNA Damage Response and Mismatch Repair Gene Defects in Advanced and Metastatic Prostate Cancer.

Project description:Alterations in DNA damage response (DDR) and related genes are present in up to 25% of advanced prostate cancers (PCa). Most frequently altered genes are involved in the homologous recombination repair, the Fanconi anemia, and the mismatch repair pathways, and their deficiencies lead to a highly heterogeneous spectrum of DDR-deficient phenotypes. More than half of these alterations concern non- BRCA DDR genes. From a therapeutic perspective, poly-ADP-ribose polymerase inhibitors have demonstrated robust clinical efficacy in tumors with BRCA2 and BRCA1 alterations. Mismatch repair-deficient PCa, and a subset of CDK12-deficient PCa, are vulnerable to immune checkpoint inhibitors. Emerging data point to the efficacy of ATR inhibitors in PCa with ATM deficiencies. Still, therapeutic implications are insufficiently clarified for most of the non- BRCA DDR alterations, and no successful targeted treatment options have been established.

| S-EPMC10846598 | biostudies-literature

A novel DNA damage response mediated by DNA mismatch repair in Caenorhabditis elegans: induction of programmed autophagic cell death in non-dividing cells.

Project description:DNA mismatch repair (MMR) contributes to genome integrity by correcting errors of DNA polymerase and inducing cell death in response to DNA damage. Dysfunction of MMR results in increased mutation frequency and cancer risk. Clinical researches revealed that MMR abnormalities induce cancers of non-dividing tissues, such as kidney and liver. However, how MMR suppresses cancer in non-dividing tissues is not understood. To address that mechanism, we analyzed the roles of MMR in non-dividing cells using Caenorhabditis elegans (C. elegans), in which all somatic cells are non-dividing in the adult stage. In this study, we used stable MMR-mutant lines with a balancer chromosome. First, we confirmed that deficiency of MMR leads to resistance to various mutagens in C. elegans dividing cells. Next, we performed drug resistance assays, and found that MMR-deficient adult worms were resistant to SN1-type alkylating and oxidizing agents. In addition, dead cell staining and reporter assays of an autophagy-related gene demonstrated that the cell death was autophagic cell death. Interestingly, this autophagic cell death was not suppressed by caffeine, implying that MMR induces death of non-dividing cells in an atl-1-independent manner. Hence, we propose the hypothesis that MMR prevents cancers in non-dividing tissues by directly inducing cell death.

| S-EPMC4575921 | biostudies-literature

Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKK? inhibition.

Project description:Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKK? inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKK? inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKK? inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKK? inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKK? inhibitors with nitrosoureas can be potentially exploited for melanoma therapy.

| S-EPMC5247288 | biostudies-literature

A novel alkylating agent Melflufen induces irreversible DNA damage and cytotoxicity in multiple myeloma cells.

Project description:Our prior study utilized both in vitro and in vivo multiple myeloma (MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti-MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2AX/ATR/CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2AX. Melflufen induces γ-H2AX, ATR, and CHK1 as early as after 2 h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2AX in melphalan-sensitive cells at 6 h and 24 h; no γ-H2AX induction was observed in melphalan-resistant cells even after 24 h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2 h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. Our data therefore suggest that melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in MM cells.

| S-EPMC4961600 | biostudies-literature

PARP-1 modulation of mTOR signaling in response to a DNA alkylating agent.

Project description:Poly(ADP-ribose) polymerase-1 (PARP-1) is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N'-nitro-N'-nitrosoguanine (MNNG), we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose) (PAR) synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD? and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK) is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC) can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS) production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.

| S-EPMC3480502 | biostudies-literature

Mismatch repair protein coordinate with SETD2 to activate DNA oxidative damage response and repair in human cells

Project description:There is mounting evidence that mismatch repair (MMR) proteins are involved in oxidative DNA damage response. SETD2, the histone H3K36 tri-methyltransferase, is a newly found factor participate in MMR pathway in human cells. In this study, we show that SETD2 can directly interact with MMR protein MutSα, and they are enriched and colocalized in chromatin in response to oxidative damage, which maybe the reason for the amplification of H3K36me3. Moreover, MutSα and SETD2 are essential for ATM and CHK2 activation upon H2O2 treatment, loss of SETD2 and MutSα will display impaired DNA damage response and oxidative DNA repair, which will accumulate oxidative damage and lead to more cell apoptosis and cell death. Our finding provided a novel SETD2 dependent mechanism for the oxidative DNA damage response together with MMR protein.

2022-07-06 | GSE163940 | GEO

A Novel Chemotherapeutic Agent to Treat Tumors with DNA Mismatch Repair Deficiencies.

Project description:Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a MMR-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S-phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS-induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency. Cancer Res; 76(14); 4183-91. ©2016 AACR.

| S-EPMC5033673 | biostudies-literature

Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage.

Project description:At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations. Previously, we demonstrated that oxidative damage causes epigenetic silencing proteins to become part of a large complex that is localized to GC-rich regions of the genome, including promoter CpG islands that are epigenetically silenced in cancer. However, whether these proteins were recruited directly to damaged DNA or during the DNA repair process was unknown. Here we demonstrate that the mismatch repair protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin. Hydrogen peroxide treatment induces the interaction of MSH2-MSH6 with DNMT1, suggesting that the recruitment is through a protein-protein interaction. Importantly, the reduction in transcription for genes with CpG island-containing promoters caused by oxidative damage is abrogated by knockdown of MSH6 and/or DNMT1. Our findings provide evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription, potentially preventing transcription from interfering with the repair process. This study uniquely brings together several factors that are known to contribute to colon cancer, namely inflammation, mismatch repair proteins, and epigenetic changes.

| S-EPMC4937888 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data