Role of PARP1 on DNA damage induced by mineral silicate chrysotile in bronchial epithelial and pleural mesothelial cells
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Chrysotile
PARP1
Comet Assay
DNA damage and defective DNA repair are extensively linked to neurodegeneration in Parkinson’s disease (PD), but the underlying molecular mechanisms remain poorly understood. Here, we determined that the PD-associated protein DJ-1 plays an essential role in modulating DNA double-strand break (DSB) repair. Specifically, DJ-1 is a DNA damage response (DDR) protein that can be recruited to DNA damage sites, where it promotes DSB repair through both homologous recombination and nonhomologous end joining. Mechanistically, DJ-1 interacts directly with PARP1, a nuclear enzyme essential for genomic stability, and stimulates its enzymatic activity during DNA repair. Importantly, cells from PD patients with the DJ-1 mutation also have defective PARP1 activity and impaired repair of DSBs. In summary, our findings uncover a novel function of nuclear DJ-1 in DNA repair and genome stability maintenance, and suggest that defective DNA repair may contribute to the pathogenesis of PD linked to DJ-1 mutations.
PARP1
Non-homologous end joining
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Chrysotile
Brucite
Talc
Magnesite
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Comet Assay
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Abstract Temozolomide is the first-choice DNA alkylating agent and has been commonly used in oncology over the last several decades. The cytotoxicity that temozolomide elicits through the methylation of guanine and adenine residues often becomes the limiting factor in effective treatment. Despite growing evidence of dysregulated alkylating DNA damage repair as a driving force of genome instability leading to cancer, neurological diseases, and premature aging, little is currently known about the coordinated role of PARP1 and MGMT (O6-methylguanine methyltransferase) enzymes in the repair of temozolomide-induced methylated DNA lesions. A major PARP1 function in DNA damage is facilitation of repair of the methylated DNA lesions, N6-methyladenine and N7-methylguanine, via base excision repair (BER); while MGMT restores guanine in O6-methylguanine (O6meG), the most cytotoxic adduct, by a one-step catalysis. It is generally thought that BER and MGMT represent two distinct mechanisms for removing DNA damage induced by alkylating agents; however, using a number of advanced cell-free and cell-based approaches, we provided evidence for direct (DNA-independent) and indirect (through PARylation) interaction between PARP1 and MGMT and demonstrated a functional crosstalk between these repair pathways. Particularly, O6meG repair activity is increased once PARP1 PARylates MGMT. Further, longer (more clinically relevant) exposure to temozolomide induced stronger MGMT PARylation in cells, indicating that the PARP1-MGMT interaction is important for enhanced O6meG repair and cell survival. As PARP1-MGMT complex forms in a variety of cancer cell types, our findings have strong implications for the development of an effective cancer therapy for cells dependent on PARP1 and MGMT mediated DNA repair. Citation Format: Jodie D. Cropper, Dauren S. Alimbetov, Kevin T. Brown, Andrew Robles, Rostislav I. Likhotvorik, James T. Guerra, Yidong Chen, Youngho Kwon, Raushan T. Kurmasheva. O6-methylguanine as the junction of MGMT and PARP1-mediated repair pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6209.
PARP1
Temozolomide
O-6-methylguanine-DNA methyltransferase
Synthetic Lethality
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Rat pleural mesothelial cells (RPMC) were treated, in vitro , in a two-stage model of carcinogenesis. RPMC obtained from passage 12th were treated once with 1 μg/ml of benzo[a]pyrene (BP) and from passage 13th to 40th with 0.4 μg/cm 2 of chrysotile fibres (Chr). Transformation was determined by the observation of the colonies formed when the cells were plated at low density. Colonies were classified into four classes according to the level of lack in contact inhibition between cells (0 to III). BP as well as chrysotile fibres were potent in inducing a high proportion of type III colonies which were not observed in control series. In addition, there was no synergistic effect between BP and Chr. These results would indicate that chrysotile fibres do not act as a promoter on rat pleural mesothelial cells in culture, but induce morphologically transformed colonies.
Chrysotile
Microgram
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DNA repair is a tightly coordinated stress response to DNA damage, which is critical for preserving genome integrity. Accruing evidence suggests that metabolic pathways have been correlated with cellular response to DNA damage. Here, we show that fatty acid oxidation (FAO) is a crucial regulator of DNA double-strand break repair, particularly homologous recombination repair. Mechanistically, FAO contributes to DNA repair by activating poly(ADP-ribose) polymerase 1 (PARP1), an enzyme that detects DNA breaks and promotes DNA repair pathway. Upon DNA damage, FAO facilitates PARP1 acetylation by providing acetyl-CoA, which is required for proper PARP1 activity. Indeed, cells reconstituted with PARP1 acetylation mutants display impaired DNA repair and enhanced sensitivity to DNA damage. Consequently, FAO inhibition reduces PARP1 activity, leading to increased genomic instability and decreased cell viability upon DNA damage. Finally, our data indicate that FAO serves as an important participant of cellular response to DNA damage, supporting DNA repair and genome stability.
PARP1
Replication protein A
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The genome of each cell is under constant threat from various forms of DNA damage. In order to protect themselves from this danger, cells possess a number of pathways able to resolve DNA lesions. The addition of poly(ADPribose) is a post-translational modification produced by attaching successive ADP-ribose moieties to a protein acceptor, forming long chains. Enzymes called poly(ADP-ribose) polymerases (PARPs) catalyse the production of these modifications, and a number of different PARPs have been linked to the process of DNA repair, including PARP1, PARP2 and PARP3. How these enzymes might function together to facilitate the repair of different lesions is unclear. Furthermore, inhibitors that target these enzymes are in clinical use for their ability to kill homologous recombination deficient tumour cells, through a mechanism of synthetic lethality. Which subset of PARPs is necessary to inhibit to achieve maximum efficacy of these agents has not been assessed. I use genome editing to generate cells disrupted for these PARPs in different combinations. Whilst loss of PARP1 compromises cellular tolerance to homologous recombination deficiency, this is independent of the status of PARP2 and PARP3, indicating the development of PARP1-specific inhibitors may hold therapeutic potential. In contrast to these observations, I uncover strong redundancy between PARP1 and PARP2 in the repair of damaged DNA bases through the base excision repair (BER) pathway. I also identify BER independent roles of both PARP1 and PARP2 in resolving replication forks that have collided with BER-intermediates, through promoting the stability of Rad51 nucleofilaments via an Fbh1-dependent mechanism. Thus PARP1 and PARP2 perform two closely-linked functions in response to cellular base damage promoting resolution of these lesions directly through BER, and stabilising replication forks which have encountered BER intermediates.
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Synthetic Lethality
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Chrysotile
Mesothelium
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Long term inhalation studies and intraperitoneal injection studies in rats were undertaken with a series of chrysotile asbestos dusts. Three dust samples were generated from chrysotile modified by the wet dispersion process (WDC) and one was from unmodified chrysotile. Following a 1 year inhalation period, all the chrysotile samples proved extremely fibrogenic and carcinogenic and there were no significant differences between the WDC dusts and normal chrysotile. In all experimental groups approximately 25% of animals developed pulmonary carcinomas and in the oldest rats advanced interstitial fibrosis occupied on average 10% of all lung tissue. In the injection studies all the dust samples produced mesotheliomas in over 90% of animals. Very little chrysotile remained in the lungs of the animals that survived longest following dust inhalation and what there was was present as individual chrysotile fibrils. It is suggested that chrysotile is potentially the most harmful variety of asbestos as shown in these and other animal studies but that it is removed from lung tissue quite rapidly. In the long lived human species this may mean that except where exposure levels are very high and of long duration, chrysotile should be less hazardous than other asbestos types.
Chrysotile
Inhalation exposure
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Comet Assay
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