scholarly journals Targeting DNA Damage Response as a Strategy to Treat HPV Infections

2019 ◽  
Vol 20 (21) ◽  
pp. 5455 ◽  
Author(s):  
N. Sanjib Banerjee ◽  
Dianne Moore ◽  
Cameron J. Parker ◽  
Thomas R. Broker ◽  
Louise T. Chow
Keyword(s):  
Cervical Cancer ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Dna Amplification ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Primary Human Keratinocytes ◽  
Viral Dna ◽  
Raft Culture ◽  
Damage Response

Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90–99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.

scholarly journals Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer

2020 ◽  
Vol 295 (50) ◽  
pp. 17169-17186
Author(s):  
Mysore S. Veena ◽  
Santanu Raychaudhuri ◽  
Saroj K. Basak ◽  
Natarajan Venkatesan ◽  
Parameet Kumar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Dna Damage ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Cell ◽  
Dna Damage Response ◽  
S Phase ◽  
Cervical Cancer Cell ◽  
Damage Response

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

scholarly journals BK Polyomavirus Activates the DNA Damage Response To Prolong S Phase

2019 ◽  
Vol 93 (14) ◽  
Author(s):  
Joshua L. Justice ◽  
Jason M. Needham ◽  
Sunnie R. Thompson
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
S Phase ◽  
Genome Replication ◽  
Viral Production ◽  
Viral Dna ◽  
Bk Polyomavirus ◽  
Damage Response ◽  
Infected Cells

ABSTRACT BK polyomavirus (PyV) is a major source of kidney failure in transplant recipients. The standard treatment for patients with lytic BKPyV infection is to reduce immunosuppressive therapy, which increases the risk of graft rejection. PyVs are DNA viruses that rely upon host replication proteins for viral genome replication. A hallmark of PyV infection is activation of the DNA damage response (DDR) to prevent severe host and viral DNA damage that impairs viral production by an unknown mechanism. Therefore, we sought to better understand why BKPyV activates the DDR through the ATR and ATM pathways and how this prevents DNA damage and leads to increased viral production. When ATR was inhibited in BKPyV-infected primary kidney cells, severe DNA damage occurred due to premature Cdk1 activation, which resulted in mitosis of cells that were actively replicating host DNA in S phase. Conversely, ATM was required for efficient entry into S phase and to prevent normal mitotic entry after G2 phase. The synergistic activation of these DDR kinases promoted and maintained BKPyV-mediated S phase to enhance viral production. In contrast to BKPyV infection, DDR inhibition did not disrupt cell cycle control in uninfected cells. This suggests that DDR inhibitors may be used to specifically target BKPyV-infected cells. IMPORTANCE BK polyomavirus (BKPyV) is an emerging pathogen that reactivates in immunosuppressed organ transplant patients. We wanted to understand why BKPyV-induced activation of the DNA damage response (DDR) enhances viral titers and prevents host DNA damage. Here, we show that the virus activates the DNA damage response in order to keep the infected cells in S phase to replicate the viral DNA. The source of DNA damage was due to actively replicating cells with uncondensed chromosomes entering directly into mitosis when the DDR was inhibited in BKPyV-infected cells. This study clarifies the previously enigmatic role of the DDR during BKPyV infection by demonstrating that the virus activates the DDR to maintain the cells in S phase in order to promote viral replication and that disruption of this cell cycle arrest can lead to catastrophic DNA damage for the host.

scholarly journals Cell cycle inertia underlies a bifurcation in cell fates after DNA damage

2021 ◽  
Vol 7 (3) ◽  
pp. eabe3882
Author(s):  
Jenny F. Nathans ◽  
James A. Cornwell ◽  
Marwa M. Afifi ◽  
Debasish Paul ◽  
Steven D. Cappell
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Tracking ◽  
Time Lapse ◽  
S Phase ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Cell Fates ◽  
Damage Response ◽  
Time Lapse Imaging

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

scholarly journals Expression of DNA damage response proteins and complete remission after radiotherapy of stage IB–IIA of cervical cancer

2006 ◽  
Vol 94 (11) ◽  
pp. 1683-1689 ◽  
Author(s):  
C Beskow ◽  
L Kanter ◽  
Å Holgersson ◽  
B Nilsson ◽  
B Frankendal ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dna Damage ◽  
Complete Remission ◽  
Dna Damage Response ◽  
Stage Ib ◽  
Damage Response

scholarly journals NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines

2011 ◽  
Vol 414 (1) ◽  
pp. 123-128 ◽  
Author(s):  
Burcu Erbaykent-Tepedelen ◽  
Besra Özmen ◽  
Lokman Varisli ◽  
Ceren Gonen-Korkmaz ◽  
Bilge Debelec-Butuner ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cell Lines ◽  
Cancer Cell ◽  
Dna Damage Response ◽  
Prostate Cancer Cell ◽  
S Phase ◽  
Prostate Cancer Cell Lines ◽  
Damage Response ◽  
Phase Entry

scholarly journals Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Dipon Das ◽  
Molly L. Bristol ◽  
Nathan W. Smith ◽  
Claire D. James ◽  
Xu Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Life Cycle ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Dna Damage Response ◽  
Human Papillomaviruses ◽  
Repair Protein ◽  
Damage Response ◽  
Dna Repair Protein

ABSTRACTHuman papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.IMPORTANCEHPV16 is the major viral human carcinogen responsible for between 3 and 4% of all cancers worldwide. Following infection, this virus activates the DNA damage response (DDR) to promote its life cycle and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here, we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall, the results suggest a mechanism by which SIRT1 deacetylation of WRN promotes its interaction with E1-E2-replicating DNA to control the levels and fidelity of that replication.

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.

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