scholarly journals Candida albicans RFX2 Encodes a DNA Binding Protein Involved in DNA Damage Responses, Morphogenesis, and Virulence

2009 ◽  
Vol 8 (4) ◽  
pp. 627-639 ◽  
Author(s):  
Binghua Hao ◽  
Cornelius J. Clancy ◽  
Shaoji Cheng ◽  
Suresh B. Raman ◽  
Kenneth A. Iczkowski ◽  
...  
Keyword(s):  
Dna Damage ◽  
Candida Albicans ◽  
Wild Type ◽  
Time To Death ◽  
Solid Media ◽  
Solid Agar ◽  
Buccal Epithelial Cells ◽  
Damage Response ◽  
Dna Damage Responses ◽  
Strain Sc5314

ABSTRACT We previously showed that Candida albicans orf19.4590, which we have renamed RFX2, expresses a protein that is reactive with antibodies in persons with candidiasis. In this study, we demonstrate that C. albicans RFX2 shares some functional redundancy with Saccharomyces cerevisiae RFX1. Complementation of an S. cerevisiae rfx1 mutant with C. albicans RFX2 partially restored UV susceptibility and the repression of DNA damage response genes. DNA damage- and UV-induced genes RAD6 and DDR48 were derepressed in a C. albicans rfx2 null mutant strain under basal conditions, and the mutant was significantly more resistant to UV irradiation, heat shock, and ethanol than wild-type strain SC5314. The rfx2 mutant was hyperfilamentous on solid media and constitutively expressed hypha-specific genes HWP1, ALS3, HYR1, ECE1, and CEK1. The mutant also demonstrated increased invasion of solid agar and significantly increased adherence to human buccal epithelial cells. During hematogenously disseminated candidiasis, mice infected with the mutant had a significantly delayed time to death compared to the wild type. During oropharyngeal candidiasis, mice infected with the mutant had significantly lower tissue burdens in the oral cavity and esophagus at 7 days and they were less likely to develop disseminated infections because of mucosal translocation. The data demonstrate that C. albicans Rfx2p regulates DNA damage responses, morphogenesis, and virulence.

Characterization of Pph3-mediated dephosphorylation of Rad53 during methyl methanesulfonate-induced DNA damage repair in Candida albicans

2017 ◽  
Vol 474 (7) ◽  
pp. 1293-1306 ◽  
Author(s):  
Guangyin Yao ◽  
Junhua Wan ◽  
Qizheng Liu ◽  
Chunhua Mu ◽  
Yue Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Candida Albicans ◽  
Pathogenic Fungus ◽  
Genotoxic Stress ◽  
Methyl Methanesulfonate ◽  
Wild Type ◽  
Cycle Arrest ◽  
Damage Repair ◽  
Damage Response

Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus Candida albicans. The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in C. albicans. We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in pph3Δ/Δ cells. Phosphomimetic mutation of the three residues (rad53-3D) impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the rad53-3D mutant also occurred in the pph3Δ/Δ mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in C. albicans.

scholarly journals Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jia Feng ◽  
Shuangyan Yao ◽  
Yansong Dong ◽  
Jing Hu ◽  
Malcolm Whiteway ◽  
...  
Keyword(s):  
Dna Damage ◽  
Candida Albicans ◽  
Nucleotide Excision Repair ◽  
Dna Damage Response ◽  
Excision Repair ◽  
Content Type ◽  
Virulence Regulation ◽  
Nucleotide Excision ◽  
Damage Response

ABSTRACT In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18. RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicans. IMPORTANCE Candida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans. We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

scholarly journals DNA damage responses in murine Pre-B cells with genetic deficiencies in damage response genes

Cell Cycle ◽  
2019 ◽  
Vol 19 (1) ◽  
pp. 67-83 ◽  
Author(s):  
Cynthia L. Innes ◽  
Jill E. Hesse ◽  
Abigail J. Morales ◽  
Beth A. Helmink ◽  
Shepherd H. Schurman ◽  
...  
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
Damage Response ◽  
Dna Damage Responses

scholarly journals Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells

2013 ◽  
Vol 288 (23) ◽  
pp. 16212-16224 ◽  
Author(s):  
Elvira Crescenzi ◽  
Zelinda Raia ◽  
Francesco Pacifico ◽  
Stefano Mellone ◽  
Fortunato Moscato ◽  
...  
Keyword(s):  
Dna Damage ◽  
Tumor Cells ◽  
Dna Damage Response ◽  
Critical Role ◽  
Wild Type ◽  
Down Regulation ◽  
Senescent Phenotype ◽  
Response To Chemotherapy ◽  
Damage Response ◽  
Wild Type P53

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G2 arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser15 and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser15 phosphorylation is associated with enhanced phosphorylation at Ser46, increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.

Involvement of Death-Associated Protein Kinases In DNA Damage Responses of B-ALL Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3369-3369
Author(s):  
Magali Humbert ◽  
Michaela Medova ◽  
Barbara Geering ◽  
Wieslawa Blank-Liss ◽  
Hans-Uwe Simon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Gamma Irradiation ◽  
Protein Kinases ◽  
Dna Damage Response ◽  
Lymphoblastic Leukemia ◽  
Damage Response ◽  
Dna Damage Responses

Abstract Abstract 3369 Intact DNA damage response pathways are important for genomic fidelity of cells in order to avoid tumor formation. On the other hand, inhibition of DNA repair provides an important mechanism to enhance the therapeutic efficacy of DNA damaging agents such as gamma-irradiation. Thus, it is important to identify novel players in DNA damage response that might represent novel targets for combination therapies. Death-associated protein kinases (DAPK) are serine/threonine kinases believed to be involved in cell death and autophagy mechanisms, whereby particularly the role of DAPK1 has previously been investigated. The DAPK family is composed of five members: DAPK1, DAPK2 (or DRP-1), DAPK3 (or ZIP kinase), DRAK1 and DRAK2. DAPK1 and DAPK2 share 80% homology in the catalytic domain. Generally, the role of DAPK in DNA damage responses is not well studied. To analyze the role of DAPK1 and DAPK2 in response to gamma-irradiation, we used p53 wild-type REH B-cell acute lymphoblastic leukemia (B-ALL) cells as a model. In response to irradiation, DAPK1 protein expression increased paralleled by an increased of total p53, phospho-Ser20-p53 and p21WAF1/CIP1. DAPK2 expression, however, did not increase. Since upregulation of p21WAF1/CIP1, a classical p53 target in response to DNA damage leads to cell cycle arrest, we asked whether knocking down DAPK1 or DAPK2 might affect the cell cycle. Interestingly, knocking down DAPK2 but not DAPK1 led to a significant increase of S-phase cells upon irradiation. Moreover, knocking down DAPK2 attenuated the induction of DAPK1 upon irradiation indicating a DAPK2-DAPK1 cascade in DNA damage responses. Next, given the significant role of p21WAF1/CIP1 and p53 in DNA damage responses, we tested if DAPK2 might directly participate in a novel signaling pathway by interacting with these proteins. Indeed, pull down assays revealed that p21WAF1/CIP1 and p53 are novel DAPK2 interacting proteins. Clearly, further experiments are needed to define the DAPK2-DAPK1-p53- p21WAF1/CIP1 network in DNA repair pathways. In conclusion, we identified a novel role for DAPK1 and DAPK2 in DNA damage responses of B-ALL cells and propose a novel DAPK2/DAPK1/p53/ p21WAF1/CIP1 DNA damage regulatory pathway. Disclosures: No relevant conflicts of interest to declare.

Effect of inhibition of receptor tyrosine kinase AXL by a selective small molecular inhibitor R428 (BGB321) on DNA damage repair response in ovarian cancer cells.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15640-e15640
Author(s):  
Ruby Yun-Ju Huang ◽  
Xun Hui Yeo ◽  
Wai Leong Tam
Keyword(s):  
Dna Damage ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Receptor Tyrosine Kinase ◽  
Dna Damage Repair ◽  
Damage Repair ◽  
Damage Response ◽  
Dna Damage Responses

e15640 Background: AXL is a receptor tyrosine kinase that is often overexpressed in many cancers. It contributes to tumor progression, metastasis and drug resistance through activating downstream signaling cascades, making it an emerging therapeutic target. The first-in-class AXL inhibitor R428 (BGB321) was approved by the FDA for the treatment of relapsed or refractory acute myeloid leukemia. R428 (BGB321) was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal (Mes) molecular subtype. Recently, a novel role of AXL in the regulation of DNA damage responses has been described. In this study, we explored further the role of AXL in mediating DNA damage responses by using OC as a disease model. Methods: OC cell lines were treated with R428. Accumulation of γH2AX positive foci was assessed for DNA damage response. Western blotting for γH2AX, ATM and ATR levels were performed. Dose response curves of ATR inhibitors were generated by treating OC cells with the fixed dose of R428 (IC20 concentration of each cell line). Results: AXL inhibition by using R428 resulted in the increase of DNA damage foci in Mes OC cells SKOV3 and HeyA8. This occurred concurrently with the up-regulation of classic DNA damage response signaling molecules such as γH2AX, ATM and ATR. The IC50 of the ATR inhibitor significantly decreased for 2-3 folds in all OC cell lines tested. AXL inhibitor R428 sensitized both BRCA-mutated and non-BRCA-mutated OC cells to a potent and highly selective ATR inhibitor. Conclusions: Our results showed that AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress, paving ways to the rationale for potential combinatory use of AXL and DNA damage repair inhibitors.

scholarly journals Role of Calcineurin in Stress Resistance, Morphogenesis, and Virulence of a Candida albicans Wild-Type Strain

2006 ◽  
Vol 74 (7) ◽  
pp. 4366-4369 ◽  
Author(s):  
Teresa Bader ◽  
Klaus Schröppel ◽  
Stefan Bentink ◽  
Nina Agabian ◽  
Gerwald Köhler ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Type Strain ◽  
Pulmonary Infection ◽  
Wild Type Strain ◽  
Selection Marker ◽  
Wild Type ◽  
Environmental Signals ◽  
Successful Infection ◽  
Strain Sc5314

ABSTRACT By generating a calcineurin mutant of the Candida albicans wild-type strain SC5314 with the help of a new recyclable dominant selection marker, we confirmed that calcineurin mediates tolerance to a variety of stress conditions but is not required for the ability of C. albicans to switch to filamentous growth in response to hypha-inducing environmental signals. While calcineurin was essential for virulence of C. albicans in a mouse model of disseminated candidiasis, deletion of CMP1 did not significantly affect virulence during vaginal or pulmonary infection, demonstrating that the requirement for calcineurin for a successful infection depends on the host niche.

scholarly journals DNA Damage-Response Pathway in Lymphoma Determines Interactions with Macrophages By Altered PD-L1 Expression and Exosome Formation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 275-275
Author(s):  
Daniela Vorholt ◽  
Elena Izquierdo-Alvarez ◽  
Benedict Sackey ◽  
Jan Schmitz ◽  
Nadine Nickel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Research Funding ◽  
Therapeutic Response ◽  
Wild Type ◽  
Lymphoma Cells ◽  
Damage Response ◽  
Dna Damage Response Pathway ◽  
Support Research

Abstract The tumor microenvironment is characterized by multiple interactions of transformed malignant cells with non-transformed stroma or immune cells. Particularly macrophages play a pivotal role in this network determining disease progression and therapeutic response. In previous work we could show that macrophages are an essential mediator of therapeutic response in the synergistic response to the administration of the chemoimmunotherapy. The combination treatment strongly increases tumor clearance by repolarization of tumor-associated macrophages from a suppressive to an activated phenotypic state. Here, se analyzed the functional implications of the DNA damage response pathway for the generation of the ASAP and synergy in chemoimmunotherapy. We attempted to disrupt DNA damage response pathway in lymphoma cells generated from the hMB humanized Double-Hit-Lymphoma model by knock-down of key elements like ATM, DNA-PK or p53. We could prevent the formation of the stimulatory cytokine release effect on macrophage phagocytic capacity. Here, p53 status displays a key regulatory role on macrophage mediated malignant cell depletion. TP53 activation via Nutlin-3A treatment of lymphoma cell enhances ADCP in in p53 wild-type cells, while not displaying enhancement in p53-deficient lymphoma cells. Addressing the treatment in vivo using the hMB model for modeling of Double-Hit Lymphoma bearing mice we could demonstrate diminished ASAP and ADCP for p53-deficient lymphoma treated with cyclophosphamide in vivo. Using primary human CLL patient cells comparing both wild-type and p53-deficient status, the p53-deficient CLL cells failed to induce the stimulatory, cytokine-mediated effect on macrophage phagocytosis in response to combination treatment as seen with the p53 proficient CLL cells. Using a CLL mouse model by treating Eµ-TCL1/p53wt/wt as well as Eµ-TCL1p53-/- mice we could show that low-dose cyclophosphamide treated Eµ-TCL1p53-/- mice failed to induce an antibody mediated stimulatory effect on macrophage phagocytosis capacity as seen with Eµ-TCL1/p53wt/wt mice. A similar effect was seen for primary multiple myeloma cells in response to daratumumab displaying significantly less ADCP of p53-deficient multiple myeloma cells. As for the mechanism of p53-defined interaction within the tumor microenvironment we subjected p53-wild-type and p53-deficient lymphoma cells for proteomic analysis. Here we could identify a significantly deregulated protein expression profile for exosome release in p53 deficient lymphoma cells. Verifying this finding by assessing size and frequency exosomes released by respective cell populations we reveal profound changes induced by p53 loss. Furthermore we could identify up-regulation of PD-L1 in p53-deficient cells. Blocking this checkpoint in the ADCP assay could significantly restore phagocytic capacity of macrophages and overall therapeutic response. In this work, we indicate that p53 functional status determines phagocytic function and therapeutic response to monoclonal antibodies. We can verify this finding in independent models in vitro and in vivo as in primary CLL and myeloma patient cells. We furthermore identify altered exosome profiles and checkpoint inhibitor expression in lymphoma cells as underlying mechanism of macrophage modulation. Finally our ongoing research offers possibility to reveal and tailor new combinatorial treatment approaches for chemo-refractory patients. Disclosures Wendtner: Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding. Hallek:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Pallasch:Gilead: Research Funding.

scholarly journals SIRT1 protects the heart against doxorubicin-induced cardiotoxicity by mediating the DNA damage response via deacetylation of histone H2AX

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
A Kuno ◽  
R Hosoda ◽  
Y Horio
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Caspase 3 ◽  
Tunel Staining ◽  
H9c2 Cells ◽  
Wild Type ◽  
Histone H2ax ◽  
Funding Sources ◽  
Damage Response ◽  
Damaged Dna

Abstract Background Doxorubicin induces DNA damage not only in tumor cells but also in the cardiomyocyte, and accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. We previously found that cardiomyocyte-specific deletion of SIRT1, a NAD+-dependent histone/protein deacetylase, worsens doxorubicin-induced cardiotoxicity in mice. However, its molecular mechanism remains unclear. Phosphorylation of histone H2AX at Ser139 catalyzed by ATM (mutated in ataxia-telangiectasia) at the sites of DNA damage is a critical mediator for DNA repair. Purpose Here, we tested the hypothesis that deacetylation of H2AX by SIRT1 mediates DNA damage response to counteract doxorubicin-induced cardiotoxicity. Methods and results Wild-type (WT) mice and tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (SIRT1-cKO) mice at 3 month of age received doxorubicin (4 IP injections of 5 mg/kg/week) or a vehicle. Immunoblotting of myocardial lysates from mice 1 week after final doxorubicin showed that doxorubicin increased phospho-Ser139-H2AX level by 1.6-fold in WT, but such a response was blunted in SIRT1-cKO. Ser1981-phosphorylations of ATM induced by doxorubicin were similar in WT and SIRT1-cKO. DNA fragmentation evaluated by TUNEL staining revealed that the increase in TUNEL-positive nuclei by doxorubicin was more in SIRT1-cKO (0.13% to 0.38%) than those in WT (0.07% to 0.19%), suggesting higher DNA damage in SIRT1-cKO. In H9c2 cardiomyocytes, knockdown of SIRT1 also abolished the doxorubicin-induced Ser139-phosphorylation of H2AX without changing phospho-ATM levels. Increases in DNA damage evaluated by comet assay and cleavage of caspase-3 by doxorubicin were also enhanced in SIRT1-knockdown cells. Immunostaining for acetyl-Lys5-H2AX in the heart sections revealed that acetyl-Lys5-H2AX levels were increased in SIRT1-cKO by 58% compared with those in WT. In H9c2 cells, acetyl-Lys5-H2AX level was also increased by SIRT1 knockdown and reduced by expression of wild-type SIRT1. To test the role of the increased acetyl-Lys5-H2AX level under SIRT1 inhibition, we generated a mutant in which Lys5 was substituted to glutamine (K5Q H2AX) as a mimic of acetylated Lys5. In COS7 cells expressing WT or K5Q H2AX, Ser139-phosphorylation induced by doxorubicin was suppressed in K5Q mutant. In addition, doxorubicin-induced cleavage of caspase-3 was enhanced in H9c2 cells expressing K5Q H2AX as well as S139A H2AX, that cannot be phosphorylated at Ser139, compared with cells expressing WT H2AX. Conclusions These findings suggest that the increased Lys5 acetylation of H2AX via SIRT1 inhibition interferes Ser139 phosphorylation, leading to accumulation of damaged DNA and promotion of the apoptotic response. Such regulation of the DNA damage response contributes to protection by SIRT1 against doxorubicin-induced cardiotoxicity. FUNDunding Acknowledgement Type of funding sources: None.

Sign in / Sign up

Export Citation Format

Share Document