Differential effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA damage, and cell cycle analysis

106 Background: Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer death in U.S. men. The retinoblastoma tumor suppressor protein, RB, plays a critical role in cell cycle regulation and loss of RB has been observed in 25-30% of prostate cancers. We have previously shown that RB loss results in a castrate resistant phenotype, however the consequences of RB status with regard to radiation response are unknown. We hypothesized that RB loss would downregulate the G1-S cell cycle checkpoint arrest normally induced by irradiation, inhibit DNA repair, and subsequently sensitize cells to ionizing radiation. Methods: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAP-C4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of RB using shRNA). Gamma H2AX assays were used to quantitate DNA damage and PARP cleavage and Caspase 3 were used to quantitate apoptosis. FACS analysis with BrdU was used to assess the cell cycle. Cell survival was measured using the clonogenic cell survival assay. In vivo work was performed in nude mice with tumor xenografts. Results: We observed that loss of RB increased radioresponsiveness in both transient and clonogenic cell survival assays in both hormone sensitive and castrate resistant cell lines (p<0.05). Cell death was not mediated through increased apoptosis nor was perturbations in cell cycle noted. However, loss of RB effected DNA repair as measured by gamma H2AX staining as well as cellular senescence. In vivo xenografts of the RB deficient tumors exhibited diminished tumor mass, lower PSA kinetics and decreased tumor growth after treatment with single fraction of ionizing radiation in comparison to RB intact tumors (p<0.05). Conclusions: Loss of RB results in a differential response to ionizing radiation. Isogenic cells with RB knockdown are more sensitive to DNA damage and result in reduced cell survival. The underlying mechanism appears to be related to DNA damage repair and cellular senescence.

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Differential effects of ambient diesel carcinogens on inflammation and DNA damage response in cells relevant for lung toxicity

Toxicology Letters ◽

10.1016/j.toxlet.2014.06.120 ◽

2014 ◽

Vol 229 ◽

pp. S25

Author(s):

Eleonora Longhin ◽

Johan Øvrevik ◽

Maurizio Gualtieri ◽

Annike Totlandsdal ◽

Steen Mollerup ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Lung Toxicity ◽

Differential Effects ◽

Damage Response ◽

In Cells

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Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma

Blood ◽

10.1182/blood-2015-07-658823 ◽

2015 ◽

Vol 126 (26) ◽

pp. 2821-2831 ◽

Cited By ~ 27

Author(s):

Lu Dai ◽

Jimena Trillo-Tinoco ◽

Yueyu Cao ◽

Karlie Bonstaff ◽

Lisa Doyle ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Tumor Progression ◽

Cell Survival ◽

Cell Cycle Arrest ◽

Primary Effusion Lymphoma ◽

Cycle Arrest ◽

Key Points ◽

Met Inhibitor

Key Points The HGF/c-MET pathway has a complex network to control KSHV+ PEL cell survival. The c-MET inhibitor induces PEL apoptosis and suppresses tumor progression in vivo.

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DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

10.1101/2021.05.28.445639 ◽

2021 ◽

Author(s):

Kartika Venugopal ◽

Pawel Nowialis ◽

Yang Feng ◽

Daniil E Shabashvili ◽

Cassandra M Berntsen ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Dna Damage ◽

Replication Fork ◽

Dna Methyltransferase ◽

De Novo ◽

Nucleoside Analogs ◽

Cellular Systems ◽

Double Labeling ◽

In Cells

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

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Relationship between the loss of the retinoblastoma tumor suppressor and radiosensitivity.

Journal of Clinical Oncology ◽

10.1200/jco.2011.29.7_suppl.34 ◽

2011 ◽

Vol 29 (7_suppl) ◽

pp. 34-34 ◽

Cited By ~ 1

Author(s):

R. B. Den ◽

S. Ciment ◽

A. Sharma ◽

H. Mellert ◽

S. Mc-Mahon ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Cycle ◽

Dna Damage ◽

Tumor Suppressor ◽

Ionizing Radiation ◽

Cell Survival ◽

Retinoblastoma Tumor Suppressor ◽

Clonogenic Cell ◽

Retinoblastoma Tumor

34 Background: Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer death in U.S. men. The retinoblastoma tumor suppressor protein, RB, plays a critical role in cell cycle regulation. Loss of RB has been observed in 25–30% of prostate cancers and is correlated with increasing tumor stage and grade. The clinical consequences of RB loss are unknown. We have previously shown that RB loss results in a castrate resistant phenotype. We hypothesized that RB loss would downregulate the G1-S cell cycle arrest normally induced by irradiation, inhibit DNA repair, and subsequently sensitize cells to mitotic catastrophe. Methods: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAP-C4 cells and hormone resistant C42 cells; stable knockdown of RB using shRNA). Gamma H2AX assays were used to quantitate DNA damage and PARP cleavage and Caspase 3 were used to quantitate apoptosis. FACS analysis with BrdU was used to assess the cell cycle. Cell survival was measured using the clonogenic cell survival assay. In vivo work was performed in nude mice with tumor xenografts. Results: We observed that loss of RB increased radioresponsiveness in both transient and clonogenic cell survival assays in all cell lines (p<0.05). Cell death was not mediated through increased apoptosis, however, there was increased cell cycling despite the presence of DNA damage in the RB knockdown cells. In vivo xenografts of the RB deficient tumors exhibited diminished tumor mass, lower PSA kinetics and decreased tumor growth after treatment with single fraction of ionizing radiation in comparison to RB intact tumors (p<0.05). Conclusions: Loss of RB results in a differential response to ionizing radiation. Isogenic cells with RB knockdown are more sensitive to DNA damage and result in reduced cell survival. RB status is integral to determining which therapeutic modality should be employed in the management of prostate cancer. No significant financial relationships to disclose.

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Specific Role of Chk1 Phosphorylations in Cell Survival and Checkpoint Activation

Molecular and Cellular Biology ◽

10.1128/mcb.01611-06 ◽

2007 ◽

Vol 27 (7) ◽

pp. 2572-2581 ◽

Cited By ~ 111

Author(s):

Hiroyuki Niida ◽

Yuko Katsuno ◽

Birendranath Banerjee ◽

M. Prakash Hande ◽

Makoto Nakanishi

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Protein Kinase ◽

Cell Survival ◽

Cell Cycle Checkpoints ◽

Mitotic Catastrophe ◽

Wild Type ◽

Checkpoint Activation ◽

Multifunctional Protein

ABSTRACT Chk1 is a multifunctional protein kinase that plays essential roles in cell survival and cell cycle checkpoints. Chk1 is phosphorylated at multiple sites by several protein kinases, but the precise effects of these phosphorylations are largely unknown. Using a knockout-knockin system, we examined the abilities of Chk1 mutants to reverse the defects of Chk1-null cells. Wild-type Chk1 could rescue all the defects of Chk1-null cells. Like endogenous Chk1, wild-type Chk1 localized in both the cytoplasm and the nucleus, and its centrosomal association was enhanced by DNA damage. The mutation at S345 resulted in mitotic catastrophe, impaired checkpoints, and loss of the ability to localize in the cytoplasm, but the mutant retained the ability to be released from chromatin upon encountering genotoxic stressors. In contrast, the mutation at S317 resulted in impaired checkpoints and loss of chromatin release upon encountering genotoxic stressors, but its mutant retained the abilities to prevent mitotic catastrophes and to localize in the cytoplasm, suggesting the distinct effects of these phosphorylations. The forced immobilization of S317A/S345A in centrosomes resulted in the prevention of apoptosis in the presence or absence of DNA damage. Thus, two-step phosphorylation of Chk1 at S317 and S345 appeared to be required for proper localization of Chk1 to centrosomes.

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Exploring NCX4040, an Aspirin Derivative, as a Potential Treatment for Benign Prostatic Hyperplasia

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1556 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A765-A765

Author(s):

Preksha Vaibhav Shahagadkar ◽

Gnanasekar Munirathinam

Keyword(s):

Cell Cycle ◽

Benign Prostatic Hyperplasia ◽

Cell Cycle Arrest ◽

Colony Formation ◽

Annexin V ◽

Prostatic Hyperplasia ◽

Cell Cycle Analysis ◽

Cycle Arrest ◽

Anti Inflammatory ◽

In Cells

Abstract Benign Prostatic hyperplasia (BPH) is a leading cause of lower urinary tract symptoms which affects men above 50 years of age. Chronic inflammation and abnormal proliferation of stromal and epithelial cells are implicated in BPH disease onset. The symptoms of BPH include back pain and difficulty in emptying bladder. Finasteride, sildenafil and tamsulosin are some of the drugs used to ease difficulty urinating and relax the muscles of the gland. Transurethral resection of the prostate or laser surgery can be performed to treat severe symptoms. However, these therapies have deleterious effects such as low blood pressure, ejaculatory dysfunction, and lump formation. Hence, there is an unmet need for potential drugs against BPH. Nonsteroidal anti-inflammatory drugs (NSAIDs) have proven to be effective in cancers but their applicability in BPH condition is yet to be fully explored. Aspirin, one of the NDSAIDs, has anti-tumor and anti-inflammatory properties at higher doses. NCX4040, a nitric oxide releasing derivative of aspirin, could prove to be effective against BPH, since it can inhibit abnormal cell proliferation and serve as a vasodilator. We hypothesize that NCX4040 would be an effective drug to treat BPH. BPH-1 epithelial and WPMY-1 stromal cells were used as in vitro models of BPH. MTT assay was performed to check the inhibitory effect of NCX4040 and blocking agents like catalase and N-acetyl-L-cysteine (NAC) were explored on cells after treatment. Clonogenic assay was done to explore the colony formation ability of cells. Spheroid assay was performed to analyze the anti-proliferative effect of NCX4040. Annexin V/PI and cell cycle analysis was performed to check for apoptosis and cell cycle arrest in the cells. Western blot was done to assess the signaling molecules altered by NCX4040 in BPH-1 cells. Confocal immunofluorescence was employed to analyze the dynamics of actin filaments after treatment in cells. Our studies revealed that NCX4040 inhibited the cell viability of BPH-1 and WPMY-1 in a dose dependent manner with IC50 predicted at 5µM and 2.5µM respectively. Of note, catalase and NAC blocked the effect of NCX4040 on prostate cells. Colony formation assay result implied a gradual decrease in the number of colonies of cells treated with NCX4040 with 2.5µM and 5µM doses. Spheroid assay in BPH-1 cells showed inhibitory effects after treatment. Cell cycle analysis by flowcytometry inferred that cell cycle arrest at G2/M phase and annexin V analysis indicated that activation of apoptosis in cells following treatment. Phalloidin staining showed decrease in the actin filament intensity in cells. At the molecular level, NCX4040 downregulated the expression of key markers such as RhoA, p65, COX-2, PCNA, Cyclin D3, and PDE-5 in BPH-1 cells. Taken together, NCX4040 could be used as a potential agent to manage BPH with minimal side effects, which needs further evaluation in animal models.

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Adaptation to DNA damage as a bet-hedging mechanism in a fluctuating environment

10.1101/2021.03.02.433602 ◽

2021 ◽

Author(s):

Pierre Roux ◽

Delphine Salort ◽

Zhou Xu

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Cell Survival ◽

Genome Instability ◽

Population Level ◽

Signalling Pathway ◽

Genome Integrity ◽

Dna Damage Checkpoint ◽

Bet Hedging

AbstractIn response to DNA damage, efficient repair is essential for cell survival and genome integrity. In eukaryotes, the DNA damage checkpoint is a signalling pathway that coordinates this response and arrests the cell cycle to provide time for repair. However, when repair fails or when the damage is not repairable, cells can eventually bypass the DNA damage checkpoint and undergo cell division despite persistent damage, a process called adaptation to DNA damage. Interestingly, adaptation occurs with a delayed timing compared to repair and shows a large variation in time, two properties that may provide a survival advantage at the population level without interfering with repair. Here, we explore this idea by mathematically modelling cell survival in response to DNA damage and focusing on adaptation parameters. We find that the delayed adaptation timing indeed maximizes survival, but its heterogeneity is beneficial only in a fluctuating damage-inducing environment. Finally, we show that adaptation does not only contribute to survival but also to genome instability and mutations, which might represent another criterion for its selection through-out evolution. Overall, we propose that adaptation can act as a bet-hedging mechanism for cell survival in response to DNA damage.

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Adaptation to DNA damage as a bet-hedging mechanism in a fluctuating environment

Royal Society Open Science ◽

10.1098/rsos.210460 ◽

2021 ◽

Vol 8 (8) ◽

pp. 210460

Author(s):

Pierre Roux ◽

Delphine Salort ◽

Zhou Xu

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Cell Survival ◽

Genome Instability ◽

Population Level ◽

Signalling Pathway ◽

Genome Integrity ◽

Dna Damage Checkpoint ◽

Bet Hedging

In response to DNA damage, efficient repair is essential for cell survival and genome integrity. In eukaryotes, the DNA damage checkpoint is a signalling pathway that coordinates this response and arrests the cell cycle to provide time for repair. However, when repair fails or when the damage is not repairable, cells can eventually bypass the DNA damage checkpoint and undergo cell division despite persistent damage, a process called adaptation to DNA damage. Interestingly, adaptation occurs with a delayed timing compared with repair and shows a large variation in time, two properties that may provide a survival advantage at the population level without interfering with repair. Here, we explore this idea by mathematically modelling cell survival in response to DNA damage and focusing on adaptation parameters. We find that the delayed adaptation timing indeed maximizes survival, but its heterogeneity is beneficial only in a fluctuating damage-inducing environment. Finally, we show that adaptation does not only contribute to survival but also to genome instability and mutations, which might represent another criterion for its selection throughout evolution. Overall, we propose that adaptation can act as a bet-hedging mechanism for cell survival in response to DNA damage.

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