The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Brian Marples - One of the best experts on this subject based on the ideXlab platform.
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the effects of G2 Phase enrichment and checkpoint abrogation on low dose hyper radiosensitivity
International Journal of Radiation Oncology Biology Physics, 2010Co-Authors: S A Krueger, G D Wilson, Evano Piasentin, M C Joiner, Brian MarplesAbstract:Purpose An association between low-dose hyper-radiosensitivity (HRS) and the "early" G2/M checkpoint has been established. An improved molecular understanding of the temporal dynamics of this relationship is needed before clinical translation can be considered. This study was conducted to characterize the dose response of the early G2/M checkpoint and then determine whether low-dose radiation sensitivity could be increased by synchronization or chemical inhibition of the cell cycle. Methods and Materials Two related cell lines with disparate HRS status were used (MR4 and 3.7 cells). A double–thymidine block technique was developed to enrich the G2-Phase population. Clonogenic cell survival, radiation-induced G2-Phase cell cycle arrest, and deoxyribonucleic acid double-strand break repair were measured in the presence and absence of inhibitors to G2-Phase checkpoint proteins. Results For MR4 cells, the dose required to overcome the HRS response (approximately 0.2 Gy) corresponded with that needed for the activation of the early G2/M checkpoint. As hypothesized, enriching the number of G2-Phase cells in the population resulted in an enhanced HRS response, because a greater proportion of radiation-damaged cells evaded the early G2/M checkpoint and entered mitosis with unrepaired deoxyribonucleic acid double-strand breaks. Likewise, abrogation of the checkpoint by inhibition of Chk1 and Chk2 also increased low-dose radiosensitivity. These effects were not evident in 3.7 cells. Conclusions The data confirm that HRS is linked to the early G2/M checkpoint through the damage response of G2-Phase cells. Low-dose radiosensitivity could be increased by manipulating the transition of radiation-damaged G2-Phase cells into mitosis. This provides a rationale for combining low-dose radiation therapy with chemical synchronization techniques to improve increased radiosensitivity.
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Is low-dose hyper-radiosensitivity a measure of G2-Phase cell radiosensitivity?
Cancer and Metastasis Reviews, 2004Co-Authors: Brian MarplesAbstract:Low-dose hyper-radiosensitivity describes a phenomenon by which cells die from excessive sensitivity to small single doses of ionizing radiation below ∼20–30 cGy. This review describes experimental data that strongly imply that low-dose hyper-radiosensitivity is exclusively associated with the survival response of cells in the G2 Phase of the cycle. This G2-centric concept arose when the characteristic cell survival pattern that denotes low-dose hyper-radiosensitivity was not detected in the radiation survival response of cell populations enriched in G1 or S Phase cells. In contrast, an extended or exaggerated low-dose hyper-radiosensitivity response was evident from populations selected to contain only G2 Phase cells by flow cytometry cell sorting. The historical data that has defined the field of low-dose hyper-radiosensitivity will be considered with respect to the radiation sensitivity of, and cell cycle checkpoints specific to, G2 Phase cells. A working model of the putative mechanism of low-dose hyper-radiosensitivity will be presented within this context. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell-cycle checkpoint that is specific to cells irradiated in the G2 Phase. This early G2 Phase checkpoint has been demonstrated to exhibit a dose expression profile that is comparable to the cell-survival pattern that defines low-dose hyper-radiosensitivity and is therefore a likely key regulator of the phenomenon.
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Is low-dose hyper-radiosensitivity a measure of G2-Phase cell radiosensitivity?
Cancer and Metastasis Reviews, 2004Co-Authors: Brian MarplesAbstract:Low-dose hyper-radiosensitivity describes a phenomenon by which cells die from excessive sensitivity to small single doses of ionizing radiation below approximately 20-30 cGy. This review describes experimental data that strongly imply that low-dose hyper-radiosensitivity is exclusively associated with the survival response of cells in the G2 Phase of the cycle. This G2-centric concept arose when the characteristic cell survival pattern that denotes low-dose hyper-radiosensitivity was not detected in the radiation survival response of cell populations enriched in G1 or S Phase cells. In contrast, an extended or exaggerated low-dose hyper-radiosensitivity response was evident from populations selected to contain only G2 Phase cells by flow cytometry cell sorting. The historical data that has defined the field of low-dose hyper-radiosensitivity will be considered with respect to the radiation sensitivity of, and cell cycle checkpoints specific to, G2 Phase cells. A working model of the putative mechanism of low-dose hyper-radiosensitivity will be presented within this context. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell-cycle checkpoint that is specific to cells irradiated in the G2 Phase. This early G2 Phase checkpoint has been demonstrated to exhibit a dose expression profile that is comparable to the cell-survival pattern that defines low-dose hyper-radiosensitivity and is therefore a likely key regulator of the phenomenon.
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An association between the radiation-induced arrest of G2-Phase cells and low-dose hyper-radiosensitivity: a plausible underlying mechanism?
Radiation research, 2003Co-Authors: Brian Marples, Bradly G. Wouters, Michael C. JoinerAbstract:Abstract Marples, B., Wouters, B. G. and Joiner, M. C. An Association between the Radiation-Induced Arrest of G2-Phase Cells and Low-Dose Hyper-Radiosensitivity: A Plausible Underlying Mechanism? Radiat. Res. 160, 38–45 (2003). The survival of asynchronous and highly enriched G1-, S- and G2-Phase populations of Chinese hamster V79 cells was measured after irradiation with 60Co γ rays (0.1–10 Gy) using a precise flow cytometry-based clonogenic survival assay. The high-dose survival responses demonstrated a conventional relationship, with G2-Phase cells being the most radiosensitive and S-Phase cells the most radioresistant. Below 1 Gy, distinct low-dose hyper-radiosensitivity (HRS) responses were observed for the asynchronous and G2-Phase enriched cell populations, with no evidence of HRS in the G1- and S-Phase populations. Modeling supports the conclusion that HRS in asynchronous V79 populations is explained entirely by the HRS response of G2-Phase cells. An association was discovered between the occurren...
Brian Gabrielli - One of the best experts on this subject based on the ideXlab platform.
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JIP4 is a PLK1 binding protein that regulates p38MAPK activity in G2 Phase
Cellular signalling, 2015Co-Authors: Alex Pinder, Dorothy Loo, Brittney S. Harrington, Vanessa Oakes, Michelle M. Hill, Brian GabrielliAbstract:Cell cycle progression from G2 Phase into mitosis is regulated by a complex network of mechanisms, all of which finally control the timing of Cyclin B/CDK1 activation. PLK1 regulates a network of events that contribute to regulating G2/M Phase progression. Here we have used a proteomics approach to identify proteins that specifically bind to the Polobox domain of PLK1. This identified a panel of proteins that were either associated with PLK1 in G2 Phase and/or mitosis, the strongest interaction being with the MAPK scaffold protein JIP4. PLK1 binding to JIP4 was found in G2 Phase and mitosis, and PLK1 binding was self-primed by PLK1 phosphorylation of JIP4. PLK1 binding is required for JIP4-dependent p38MAPK activation in G2 Phase during normal cell cycle progression, but not in either G2 Phase or mitotic stress response. Finally, JIP4 is a target for caspase-dependent cleavage in mitotically arrested cells. The role for the PLK1–JIP4 regulated p38MAPK activation in G2 Phase is unclear, but it does not affect either progression into or through mitosis.
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Truncated MEK1 is required for transient activation of MAPK signalling in G2 Phase cells.
Cellular signalling, 2013Co-Authors: Tanya Pike, Nichole Giles, Charlotte Widberg, Andrew Goodall, Elizabeth Payne, John F. Hancock, Brian GabrielliAbstract:The primary endpoint of signalling through the canonical Raf-MEK-ERK MAP kinase cascade is ERK activation. Here we report a novel signalling outcome for this pathway. Activation of the MAP kinase pathway by growth factors or phorbol esters during G2 Phase results in only transient activations of ERK and p90RSK, then suppression to below control levels. A small peak of ERK and p90RSK activation in early G2 Phase cells was identified, and inhibition of this delayed entry into mitosis. The previously identified, proteolytically cleaved form of MEK1 termed tMEK (truncated MEK1), is also induced with G2 Phase MAPK pathway activation. We demonstrate that addition of recombinant mutants of MEK1 with an N-terminal truncation similar to that of tMEK also inhibited ERK and p90RSK activations and delayed progression into mitosis. Only catalytically inactive forms of tMEK were capable of these effects, but surprisingly, phosphorylation on the activating Ser218/222 sites was also required. A lack of MEK1 or ability to accumulate tMEK resulted in the absence of the feedback inhibition of ERK and p90RSK activations. tMEK is a novel output from the canonical MAP kinase signalling pathway, acting in a MAPK signalling-regulated dominant negative manner to inhibit ERK and p90RSK activations, acting as a dampening mechanism to reduce the magnitude or duration of MAPK pathway signalling in G2/M Phase.
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A UVR-induced G2-Phase checkpoint response to ssDNA gaps produced by replication fork bypass of unrepaired lesions is defective in melanoma
The Journal of investigative dermatology, 2012Co-Authors: Matthew Wigan, Sandra Pavey, Alex Pinder, Andrew Burgess, Nichole Giles, Shushyan Wong, Richard A. Sturm, Brian GabrielliAbstract:UVR is a major environmental risk factor for the development of melanoma. Here we describe a coupled DNA-damage tolerance (DDT) mechanism and G2-Phase cell cycle checkpoint induced in response to suberythemal doses of UVR that is commonly defective in melanomas. This coupled response is triggered by a small number of UVR-induced DNA lesions incurred during G1 Phase that are not repaired by nucleotide excision repair (NER). These lesions are detected during S Phase, but rather than stalling replication, they trigger the DDT-dependent formation of single-stranded DNA (ssDNA) gaps. The ssDNA attracts replication protein A (RPA), which initiates ATR–Chk1 (ataxia telangiectasia and Rad3-related/checkpoint kinase 1) G2-Phase checkpoint signaling, and colocalizes with components of the RAD18 and RAD51 postreplication repair pathways. We demonstrate that depletion of RAD18 delays both the resolution of RPA foci and exit from the G2-Phase arrest, indicating the involvement of RAD18-dependent postreplication repair in ssDNA gap repair during G2 Phase. Moreover, the presence of RAD51 and BRCA1 suggests that an error-free mechanism may also contribute to repair. Loss of the UVR-induced G2-Phase checkpoint results in increased UVR signature mutations after exposure to suberythemal UVR. We propose that defects in the UVR-induced G2-Phase checkpoint and repair mechanism are likely to contribute to melanoma development.
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G2 Phase cell cycle arrest in human skin following UV irradiation
Oncogene, 2001Co-Authors: Sandra Pavey, Terry Russell, Brian GabrielliAbstract:The contribution of the short wavelength ultraviolet (UV) component of sunlight to the aetiology of skin cancer has been widely acknowledged, although its direct contribution to tumour initiation or progression is still poorly understood. The loss of normal cell cycle controls, particularly checkpoint controls, are a common feature of cancer. UV radiation causes both G1 and G2 Phase checkpoint arrest in vitro cultured cells. In this study we have investigated the cell cycle responses to suberythemal doses of UV on skin. We have utilized short-term whole organ skin cultures, and multi parameter immunohistochemical and biochemical analysis to demonstrate that basal and suprabasal layer melanocytes and keratinocytes undergo a G2 Phase cell cycle arrest for up to 48 h following irradiation. The arrest is associated with increased p16 expression but no apparent p53 involvement. This type of organ culture provides a very useful model system, combining the ease of in vitro manipulation with the ability to perform detailed molecular analysis in a normal tissue environment.
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cdc25 dependent activation of cyclin a cdk2 is blocked in G2 Phase arrested cells independently of atm atr
Oncogene, 2001Co-Authors: Sandra Pavey, Sherilyn Goldstone, Alistair R R Forrest, J Sinnamon, Brian GabrielliAbstract:Cyclin A/cdk2 is active during S and G2 Phases of the cell cycle, but its regulation and function during G2 Phase is poorly understood. In this study we have examined the regulation of cyclin A/cdk2 activity during normal G2 Phase progression and in genotoxin-induced G2 arrest. We show that cyclin A/cdk2 is activated in early G2 Phase by a cdc25 activity. In the G2 Phase checkpoint arrest initiated in response to various forms of DNA damage, the cdc25-dependent activation of both cyclin A/cdk2 and cyclin B1/cdc2 is blocked. Ectopic expression of cdc25B, but not cdc25C, in G2 Phase arrested cells efficiently activated both cyclin A/cdk2 and cyclin B1/cdc2. Finally, we demonstrate that the block in cyclin A/cdk2 activation in the G2 checkpoint arrest is independent of ATM/ATR. We speculate that the ATM/ ATR-independent block in G2 Phase cyclin A/cdk2 activation may act as a further layer of checkpoint control, and that blocking G2 Phase cyclin A/cdk2 activation contributes to the G2 Phase checkpoint arrest.
James E. Ferrell - One of the best experts on this subject based on the ideXlab platform.
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The Apparent Requirement for Protein Synthesis during G2 Phase Is due to Checkpoint Activation
Cell Reports, 2020Co-Authors: Sarah Lockhead, Alisa Moskaleva, Julia Kamenz, Yuxin Chen, Minjung Kang, Anay Reddy, Silvia D.m. Santos, James E. FerrellAbstract:Summary Protein synthesis inhibitors (e.g., cycloheximide) block mitotic entry, suggesting that cell cycle progression requires protein synthesis until right before mitosis. However, cycloheximide is also known to activate p38 mitogen-activated protein kinase (MAPK), which can delay mitotic entry through a G2/M checkpoint. Here, we ask whether checkpoint activation or a requirement for protein synthesis is responsible for the cycloheximide effect. We find that p38 inhibitors prevent cycloheximide-treated cells from arresting in G2 Phase and that G2 duration is normal in approximately half of these cells. The Wee1 inhibitor MK-1775 and Wee1/Myt1 inhibitor PD0166285 also prevent cycloheximide from blocking mitotic entry, raising the possibility that Wee1 and/or Myt1 mediate the cycloheximide-induced G2 arrest. Thus, protein synthesis during G2 Phase is not required for mitotic entry, at least when the p38 checkpoint pathway is abrogated. However, M Phase progression is delayed in cycloheximide-plus-kinase-inhibitor-treated cells, emphasizing the different requirements of protein synthesis for timely entry and completion of mitosis.
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The apparent requirement for protein synthesis during G2 Phase is due to checkpoint activation
2019Co-Authors: Sarah Lockhead, Alisa Moskaleva, Julia Kamenz, Yuxin Chen, Minjung Kang, Anay Reddy, Silvia D.m. Santos, James E. FerrellAbstract:Abstract Protein synthesis inhibitors (e.g. cycloheximide) prevent cells from entering mitosis, suggesting that cell cycle progression requires protein synthesis until right before mitotic entry. However, cycloheximide is also known to activate p38 MAPK, which can delay mitotic entry through a G2/M checkpoint. Here we asked whether checkpoint activation or a requirement for protein synthesis is responsible for the cycloheximide effect. We found that p38 inhibitors prevent cycloheximide-treated cells from arresting in G2 Phase, and that G2 duration is normal in about half of these cells. The Wee1/Myt1 inhibitor PD0166285 also prevents cycloheximide from blocking mitotic entry, raising the possibility that Wee1 and/or Myt1 mediate the cycloheximide-induced G2 arrest. Thus, the ultimate trigger for mitotic entry appears not to be the continued synthesis of mitotic cyclins or other proteins. However, M-Phase progression was delayed in cycloheximide-plus-kinase-inhibitor-treated cells, emphasizing the different requirements of protein synthesis for timely entry and completion of mitosis. Impact statement Cycloheximide arrests cells in G2 Phase due to activation of p38 MAPK, not inhibition of protein synthesis, arguing that protein synthesis in G2 Phase is not required for mitotic entry.
Pietro Pichierri - One of the best experts on this subject based on the ideXlab platform.
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the mammalian mismatch repair protein msh2 is required for correct mre11 and rad51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 Phase
Oncogene, 2003Co-Authors: Annapaola Franchitto, Pietro Pichierri, Rita Piergentili, Marco Crescenzi, Margherita Bignami, Fabrizio PalittiAbstract:In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established. We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair. Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2+/+ and MSH2-/- mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells. Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-Phase cells. Finally, MSH2-/- cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis. Consistent with the premature release from the G2-Phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells. Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 Phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.
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Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 Phase of the cell cycle.
Mutation research, 2000Co-Authors: Pietro Pichierri, Annapaola Franchitto, Pasquale Mosesso, L. Proietti De Santis, Adayabalam S. Balajee, F. PalittiAbstract:Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 Phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 Phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 Phase processes involving the WRN protein.
Kay A. O. Ellem - One of the best experts on this subject based on the ideXlab platform.
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A cyclin D-Cdk4 activity required for G2 Phase cell cycle progression is inhibited in ultraviolet radiation-induced G2 Phase delay.
The Journal of biological chemistry, 1999Co-Authors: Brian Gabrielli, J Sinnamon, Boris Sarcevic, Graeme J. Walker, Marina Castellano, Xue-qing Wang, Kay A. O. EllemAbstract:Cyclin D-Cdk4 complexes have a demonstrated role in G1 Phase, regulating the function of the retinoblastoma susceptibility gene product (Rb). Previously, we have shown that following treatment with low doses of UV radiation, cell lines that express wild-type p16 and Cdk4 responded with a G2 Phase cell cycle delay. The UV-responsive lines contained elevated levels of p16 post-treatment, and the accumulation of p16 correlated with the G2 delay. Here we report that in UV-irradiated HeLa and A2058 cells, p16 bound Cdk4 and Cdk6 complexes with increased avidity and inhibited a cyclin D3-Cdk4 complex normally activated in late S/early G2 Phase. Activation of this complex was correlated with the caffeine-induced release from the UV-induced G2 delay and a decrease in the level of p16 bound to Cdk4. Finally, overexpression of a dominant-negative mutant of Cdk4 blocked cells in G2 Phase. These data indicate that the cyclin D3-Cdk4 activity is necessary for cell cycle progression through G2 Phase into mitosis and that the increased binding of p16 blocks this activity and G2 Phase progression after UV exposure.
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Ultraviolet light-induced G2 Phase cell cycle checkpoint blocks cdc25-dependent progression into mitosis
Oncogene, 1997Co-Authors: B G Gabrielli, Andrew K Mccormack, J.m. Clark, Kay A. O. EllemAbstract:In response to low doses of ultraviolet (U.V.) radiation, cells undergo a G2 delay. In this study we have shown that the G2 delay results in the accumulation of inactive forms of cyclin B1/cdc2 and both the G2 and mitotic complexes of cyclin A/cdk. This appears to be through a block in the cdc25-dependent activation of these complexes. The expression and localisation of cyclin A and cyclin B1/cdk complexes are similar in U.V.-induced G2 delay and normal early G2 Phase cells. Cdc25B and cdc25C also accumulate to normal G2 levels in U.V. irradiated cells, but the mitotic phosphorylation associated with increased activity of both cdc25B and cdc25C is absent. The cdc25B accumulates in the nucleus of U.V. irradiated cells and in normal G2 Phase cells. Thus the block in cyclin B/cdc2 activation is in part due to the physical separation of cyclin B/cdc2, localised in the cytoplasm, from the cdc25B and cdc25C phosphatases localised in the nucleus. The data positions the U.V.-induced G2 checkpoint at either the S/G2 transition or early G2 Phase, prior to the activation of cyclin A/cdk2.
