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1
Mantel, Charlie, and Hal E. Broxmeyer. "Embryonic Stem Cells Bypass Numerous Cell Cycle Checkpoints; Not Just G1." Blood 112, no. 11 (November 16, 2008): 1331. http://dx.doi.org/10.1182/blood.v112.11.1331.1331.
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Abstract It was recently demonstrated that human and mouse embryonic stem cells (ESC) have deficiencies in the mitotic spindle assembly checkpoint (SAC) and it’s uncoupling to apoptosis which leads to polyploidy (Mantel et.al. BLOOD10:4518; 2007), a source of genetic instability in ESC in-vitro. The G1 checkpoint is also absent in ESC, a fact already known. It was also shown that p53 phosphorylation is absent in SAC-bypassed murine ESC in contrast to somatic cells with intact checkpoints (Mantel, et.al. CELL CYCLE 7:484; 2008). This lack of p53 phosporylation likely contributes to apoptosis uncoupling and polyploidization in ESC after microtubule/spindle damage and SAC-bypass. Microtubule/spindle damage in somatic cells eventually causes M-phase slippage where cells enter a 4C-G1 state that has 4C DNA content, no cyclin B1, and highly phosphorylated Rb. 4C-G1 status has not been investigated in ESC. We have now begun studies to determine mechanisms of checkpoint-bypass and polyploidization in ESC using intracellular flow cytometric analysis and here we report on the phosphorylation status of Rb in polyploid ESC. Because histone acetylation has been linked to cell cycle checkpoint function and because chromatin structure is more “open” in ESC, we investigated the oscillatory acetylations of the four core nucleosomal histones during checkpoint-bypass in ESC. The effects of DNA strand breaks on cell cycle checkpoints in ESC were also investigated. Results demonstrated that Rb is highly phosphorylated at several sites when ESC are in a cell cycle phase consistent with that seen in somatic cells in 4C-G1 after microtubule damage. It is concluded that ESC polyploidization is accompanied by 4C-G1-exit without apoptosis, which contrasts to 4C-G1-exit in somatic cells that do initiate apoptosis. There were also pronounced differences in acetylation oscillations on histone H4 and histone H2B compared to histone H3 and histone H2A during checkpoint activation and bypass. Total histones increased linearly as DNA content increased, as expected. Bivalent histone acetylation/methylation site, histone H3K9, changed little during checkpoint-bypass. However, DNA strand breakage revealed that S, G2, and the following G1 DNA-damage checkpoints also appeared to be bypassed in ESC. Most unusual is the polyploidization after DNA strand breakage, which may be due to aborted G2/M phases, but not to SAC activation since DNA strand breakage is not known to activate the SAC. DNA damage caused polyploidy without accumulation of cells in 4C-G1, as noted by lack of Rb phosphorylation, lack of p53 phosphorylation (as previously determined), but with an increase in total p53 in all phases of the cell cycle including 8C/polyploid. We conclude that mouse ESC can bypass numerous cell cycle checkpoints and fail to couple them to apoptosis initiation. This could be related to differences in histone acetylation, Rb phosphorylation, and the absence of p53 phosphorylation when compared to results of similar studies of somatic cells. Bypass of numerous checkpoints is a likely source of genetic instability in ESC cultured in-vitro.
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Naiki, Takahiro, Toshiyasu Shimomura, Tae Kondo, Kunihiro Matsumoto, and Katsunori Sugimoto. "Rfc5, in Cooperation with Rad24, Controls DNA Damage Checkpoints throughout the Cell Cycle inSaccharomyces cerevisiae." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 5888–96. http://dx.doi.org/10.1128/mcb.20.16.5888-5896.2000.
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ABSTRACT RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Δ mutants are defective in all the G1-, S-, and G2/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine115 residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neitherrfc5-1 nor rad24-K115R single mutants are defective in the G1- and G2/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24K115R fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G1-, S- and G2/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.
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Mantel, Charlie, Stephen E. Braun, Suzanna Reid, Octavian Henegariu, Lisa Liu, Giao Hangoc, and Hal E. Broxmeyer. "p21cip-1/waf-1 Deficiency Causes Deformed Nuclear Architecture, Centriole Overduplication, Polyploidy, and Relaxed Microtubule Damage Checkpoints in Human Hematopoietic Cells." Blood 93, no. 4 (February 15, 1999): 1390–98. http://dx.doi.org/10.1182/blood.v93.4.1390.
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Abstract A recent hypothesis suggests that tumor-specific killing by radiation and chemotherapy agents is due to defects or loss of cell cycle checkpoints. An important component of some checkpoints is p53-dependent induction of p21cip-1/waf-1. Both p53 and p21 have been shown to be required for microtubule damage checkpoints in mitosis and in G1 phase of the cell cycle and they thus help to maintain genetic stability. We present here evidence that p21cip-1/waf-1 deficiency relaxes the G1 phase microtubule checkpoint that is activated by microtubule damage induced with nocodazole. Reduced p21cip-1/waf-1expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and thus helps to maintain genomic integrity. The inability to efficiently upregulate p21cip-1/waf-1 in p21cip-1/waf-1antisense-expressing cells in response to microtubule damage could uncouple the centrosome cycle from the DNA cycle and lead to nuclear abnormalicies and polyploidy. A centrosome duplication checkpoint could be a new target for novel chemotherapy strategies.
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Mantel, Charlie, Stephen E. Braun, Suzanna Reid, Octavian Henegariu, Lisa Liu, Giao Hangoc, and Hal E. Broxmeyer. "p21cip-1/waf-1 Deficiency Causes Deformed Nuclear Architecture, Centriole Overduplication, Polyploidy, and Relaxed Microtubule Damage Checkpoints in Human Hematopoietic Cells." Blood 93, no. 4 (February 15, 1999): 1390–98. http://dx.doi.org/10.1182/blood.v93.4.1390.404k25_1390_1398.
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A recent hypothesis suggests that tumor-specific killing by radiation and chemotherapy agents is due to defects or loss of cell cycle checkpoints. An important component of some checkpoints is p53-dependent induction of p21cip-1/waf-1. Both p53 and p21 have been shown to be required for microtubule damage checkpoints in mitosis and in G1 phase of the cell cycle and they thus help to maintain genetic stability. We present here evidence that p21cip-1/waf-1 deficiency relaxes the G1 phase microtubule checkpoint that is activated by microtubule damage induced with nocodazole. Reduced p21cip-1/waf-1expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and thus helps to maintain genomic integrity. The inability to efficiently upregulate p21cip-1/waf-1 in p21cip-1/waf-1antisense-expressing cells in response to microtubule damage could uncouple the centrosome cycle from the DNA cycle and lead to nuclear abnormalicies and polyploidy. A centrosome duplication checkpoint could be a new target for novel chemotherapy strategies.
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Xu, Bo, Seong-Tae Kim, Dae-Sik Lim, and Michael B. Kastan. "Two Molecularly Distinct G2/M Checkpoints Are Induced by Ionizing Irradiation." Molecular and Cellular Biology 22, no. 4 (February 15, 2002): 1049–59. http://dx.doi.org/10.1128/mcb.22.4.1049-1059.2002.
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ABSTRACT Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G1, S, and G2 phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G2 checkpoint and a prolonged G2 arrest after IR, suggesting the existence of different types of G2 arrest. Two molecularly distinct G2/M checkpoints were identified, and the critical importance of the choice of G2/M checkpoint assay was demonstrated. The first of these G2/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G2 at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G2 cells must be used to assess this arrest. In contrast, G2/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G2/M accumulation after IR is not affected by the early G2/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G2 arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G2 checkpoints appear to affect survival following irradiation. Thus, two different G2 arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.
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Xu, Bo, Seong-tae Kim, and Michael B. Kastan. "Involvement of Brca1 in S-Phase and G2-Phase Checkpoints after Ionizing Irradiation." Molecular and Cellular Biology 21, no. 10 (May 15, 2001): 3445–50. http://dx.doi.org/10.1128/mcb.21.10.3445-3450.2001.
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ABSTRACT Cell cycle arrests in the G1, S, and G2phases occur in mammalian cells after ionizing irradiation and appear to protect cells from permanent genetic damage and transformation. Though Brca1 clearly participates in cellular responses to ionizing radiation (IR), conflicting conclusions have been drawn about whether Brca1 plays a direct role in cell cycle checkpoints. Normal Nbs1 function is required for the IR-induced S-phase checkpoint, but whether Nbs1 has a definitive role in the G2/M checkpoint has not been established. Here we show that Atm and Brca1 are required for both the S-phase and G2 arrests induced by ionizing irradiation while Nbs1 is required only for the S-phase arrest. We also found that mutation of serine 1423 in Brca1, a target for phosphorylation by Atm, abolished the ability of Brca1 to mediate the G2/M checkpoint but did not affect its S-phase function. These results clarify the checkpoint roles for each of these three gene products, demonstrate that control of cell cycle arrests must now be included among the important functions of Brca1 in cellular responses to DNA damage, and suggest that Atm phosphorylation of Brca1 is required for the G2/M checkpoint.
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Hopkins, Michael, John J. Tyson, and Béla Novák. "Cell-cycle transitions: a common role for stoichiometric inhibitors." Molecular Biology of the Cell 28, no. 23 (November 7, 2017): 3437–46. http://dx.doi.org/10.1091/mbc.e17-06-0349.
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The cell division cycle is the process by which eukaryotic cells replicate their chromosomes and partition them to two daughter cells. To maintain the integrity of the genome, proliferating cells must be able to block progression through the division cycle at key transition points (called “checkpoints”) if there have been problems in the replication of the chromosomes or their biorientation on the mitotic spindle. These checkpoints are governed by protein-interaction networks, composed of phase-specific cell-cycle activators and inhibitors. Examples include Cdk1:Clb5 and its inhibitor Sic1 at the G1/S checkpoint in budding yeast, APC:Cdc20 and its inhibitor MCC at the mitotic checkpoint, and PP2A:B55 and its inhibitor, alpha-endosulfine, at the mitotic-exit checkpoint. Each of these inhibitors is a substrate as well as a stoichiometric inhibitor of the cell-cycle activator. Because the production of each inhibitor is promoted by a regulatory protein that is itself inhibited by the cell-cycle activator, their interaction network presents a regulatory motif characteristic of a “feedback-amplified domineering substrate” (FADS). We describe how the FADS motif responds to signals in the manner of a bistable toggle switch, and then we discuss how this toggle switch accounts for the abrupt and irreversible nature of three specific cell-cycle checkpoints.
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Fulka, Josef, Judy Bradshaw, and Robert Moor. "Meiotic cycle checkpoints in mammalian oocytes." Zygote 2, no. 4 (November 1994): 351–54. http://dx.doi.org/10.1017/s0967199400002197.
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Recent Spectacular achievements have enabled the identification of key molecules responsible for mitotic cell cycle progression through the stages of G1, the gap before DNA replication; S, the phase of DNA synthesis; G2, the gap before chromosome segregation; and M, mitosis itself. The last stage has been most intensively studied, where MPE, maturation promotion factor, has been found responsible for the nuclear events associated with chromosomal segregation and the prodcution of two identical daughter cells (see Murray & Hunt, 1993).
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Morgan, S. E., C. Lovly, T. K. Pandita, Y. Shiloh, and M. B. Kastan. "Fragments of ATM which have dominant-negative or complementing activity." Molecular and Cellular Biology 17, no. 4 (April 1997): 2020–29. http://dx.doi.org/10.1128/mcb.17.4.2020.
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The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogated the S-phase checkpoint after ionizing irradiation and enhanced radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complemented radiosensitivity and the S-phase checkpoint and reduced chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM.
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Koledova, Zuzana, Leona Kafkova, Sonja Hennemann, Alwin Kraemer, and Vladimir Divoky. "Cdk2 Kinase Activity Is Not Abrogated after DNA Damage in Mouse Embryonic Stem Cells." Blood 110, no. 11 (November 16, 2007): 3371. http://dx.doi.org/10.1182/blood.v110.11.3371.3371.
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Abstract Stem cells have gained special attention for their implication in medicine where stem-cell based therapies promise powerful approach to treat different disorders, and, on the other hand, cancer stem cells have been suggested as important novel targets for the treatment of cancer. To successfully develop new therapies, a deeper understanding of the biology of stem cells is necessary. Embryonic stem (ES) cells are naturally immortalized pluripotent cells derived from early mammalian embryos. ES cells are characterized by unique self-renewal and differentiation abilities, as well as by special features of cell cycle regulation and DNA damage response. In general, when DNA damage occurs, checkpoint pathways are activated, preventing replication of damaged DNA and/or division of cells with damaged DNA. Somatic cells employ checkpoints throughout the whole cell cycle; in ES cells functional checkpoints have been described in S and G2 phases only. In somatic cells the G1/S transition is governed by Cdk2-cyclin E complex. G1-checkpoint mechanisms lead to inhibition of Cdk2 activity via two parallel pathways: Chk1/Chk2-Cdc25A and p53-p21. It has been suggested proteins of these pathways are not functional in ES cells. We aimed to unravel the causes of G1 checkpoint non-functionality in mouse ES (mES) cells. To analyze the events after DNA damage in G1 phase, we synchronized mES cells (lines HM-1 and V6.5) in M phase by nocodazole treatment. After release from nocodazole arrest the cells were gamma-irradiated (γ) in early and late G1 phase. We observed activation of both Chk2-Cdc25A and p53-p21 pathways in mES cells after DNA damage by γ-irradiation. However, FACS cell cycle analysis revealed that after γ-induced DNA damage mES cells did not arrest in G1; instead, cell cycle arrest occurred only at the G2/M boundary. Measurements of Cdk2 kinase activity in γ-irradiated and mock-treated mES cells revealed that although Cdk2-activating phosphatase Cdc25A is degraded after γ-irradiation, Cdk2 activity is not diminished. Since it has been reported earlier that in mES cells Chk2 is mislocalized to centrosomes, we speculated that full function of other cell cycle regulatory proteins might be hampered by aberrant localization as well. Our immunolocalization studies showed that both Cdk2 and its phosphorylated, inactive form (P-Thr14/Tyr15-Cdk2) localize to centrosomes in mES cells. This could, at least partially, influence its accessibility by interacting factors such as Cdc25A and explain the lack of Cdk2 activity downregulation after DNA damage despite activated checkpoint pathways. In conclusion, DNA damage in mES cells (lines HM-1 and V6.5) elicits fast activation of both Chk2-Cdc25A and p53-p21 G1 checkpoint pathways. However, since Cdk2 activity is not reduced after DNA damage, mES cells do not arrest in G1 phase. Other factors than those identified in somatic cells, including aberrant localization of cell cycle regulatory proteins, could play important roles in the regulation of cell cycle progression in mES cells. These factors lead to sustained Cdk2 kinase activity even in the presence of DNA damage.
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Matson, Jacob Peter, Amy M. House, Gavin D. Grant, Huaitong Wu, Joanna Perez, and Jeanette Gowen Cook. "Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence." Journal of Cell Biology 218, no. 7 (June 11, 2019): 2169–84. http://dx.doi.org/10.1083/jcb.201902143.
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To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.
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Siede, W., A. S. Friedberg, I. Dianova, and E. C. Friedberg. "Characterization of G1 checkpoint control in the yeast Saccharomyces cerevisiae following exposure to DNA-damaging agents." Genetics 138, no. 2 (October 1, 1994): 271–81. http://dx.doi.org/10.1093/genetics/138.2.271.
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Abstract The delay of S-phase following treatment of yeast cells with DNA-damaging agents is an actively regulated response that requires functional RAD9 and RAD24 genes. An analysis of cell cycle arrest indicates the existence of (at least) two checkpoints for damaged DNA prior to S-phase; one at START (a G1 checkpoint characterized by pheromone sensitivity of arrested cells) and one between the CDC4- and CDC7-mediated steps (termed the G1/S checkpoint). When a dna1-1 mutant (that affects early events of replicon initiation) also carries a rad9 deletion mutation, it manifests a failure to arrest in G1/S following incubation at the restrictive temperature. This failure to execute regulated G1/S arrest is correlated with enhanced thermosensitivity of colony-forming ability. In an attempt to characterize the signal for RAD9 gene-dependent G1 and G1/S cell cycle arrest, we examined the influence of the continued presence of unexcised photoproducts. In mutants defective in nucleotide excision repair, cessation of S-phase was observed at much lower doses of UV radiation compared to excision-proficient cells. However, this response was not RAD9-dependent. We suggest that an intermediate of nucleotide excision repair, such as DNA strand breaks or single-stranded DNA tracts, is required to activate RAD9-dependent G1 and G1/S checkpoint controls.
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Paciotti, Vera, Michela Clerici, Maddalena Scotti, Giovanna Lucchini, and Maria Pia Longhese. "Characterization of mec1Kinase-Deficient Mutants and of New Hypomorphic mec1Alleles Impairing Subsets of the DNA Damage Response Pathway." Molecular and Cellular Biology 21, no. 12 (June 15, 2001): 3913–25. http://dx.doi.org/10.1128/mcb.21.12.3913-3925.2001.
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ABSTRACT DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage. The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects. We show thatmec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Δ cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response. Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G1 or during S phase. Conversely, an excess of Mec1kd inMEC1 cells does not abrogate the G2/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable. In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G1/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G2. The finding that these mutants, although indistinguishable frommec1Δ cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents.
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Mantel, Charlie R., Stephen E. Braun, Younghee Lee, Young-June Kim, and Hal E. Broxmeyer. "The interphase microtubule damage checkpoint defines an S-phase commitment point and does not requirep21waf-1." Blood 97, no. 5 (March 1, 2001): 1505–7. http://dx.doi.org/10.1182/blood.v97.5.1505.
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Cell cycle checkpoints ensure orderly progression of events during cell division. A microtubule damage (MTD)-induced checkpoint has been described in G1 phase of the cell cycle (G1MTC) for which little is known. The present study shows that the G1MTC is intact in activated T lymphocytes from mice with the p21waf-1 gene deleted. However, p21waf-1 gene deletion does affect the ratio of cells that arrest at the G1MTC and the spindle checkpoint after MTD. The G1MTC arrests T lymphocytes in G1 prior to cdc2 up-regulation and prior to G1arrest by p21waf-1. Once cells have progressed past the G1MTC, they are committed to chromosome replication and metaphase progression, even with extreme MTD. The G1MTC is also present in a human myeloid cell line deficient in p21waf-1gene expression. The p21-independent G1MTC may be important in cellular responses to MTD such as those induced by drugs used to treat cancer.
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Pomerening, Joseph R., Jeffrey A. Ubersax, and James E. Ferrell. "Rapid Cycling and Precocious Termination of G1 Phase in Cells Expressing CDK1AF." Molecular Biology of the Cell 19, no. 8 (August 2008): 3426–41. http://dx.doi.org/10.1091/mbc.e08-02-0172.
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In Xenopus embryos, the cell cycle is driven by an autonomous biochemical oscillator that controls the periodic activation and inactivation of cyclin B1-CDK1. The oscillator circuit includes a system of three interlinked positive and double-negative feedback loops (CDK1 -> Cdc25 -> CDK1; CDK1 ⊣ Wee1 ⊣ CDK1; and CDK1 ⊣ Myt1 ⊣ CDK1) that collectively function as a bistable trigger. Previous work established that this bistable trigger is essential for CDK1 oscillations in the early embryonic cell cycle. Here, we assess the importance of the trigger in the somatic cell cycle, where checkpoints and additional regulatory mechanisms could render it dispensable. Our approach was to express the phosphorylation site mutant CDK1AF, which short-circuits the feedback loops, in HeLa cells, and to monitor cell cycle progression by live cell fluorescence microscopy. We found that CDK1AF-expressing cells carry out a relatively normal first mitosis, but then undergo rapid cycles of cyclin B1 accumulation and destruction at intervals of 3–6 h. During these cycles, the cells enter and exit M phase-like states without carrying out cytokinesis or karyokinesis. Phenotypically similar rapid cycles were seen in Wee1 knockdown cells. These findings show that the interplay between CDK1, Wee1/Myt1, and Cdc25 is required for the establishment of G1 phase, for the normal ∼20-h cell cycle period, and for the switch-like oscillations in cyclin B1 abundance characteristic of the somatic cell cycle. We propose that the HeLa cell cycle is built upon an unreliable negative feedback oscillator and that the normal high reliability, slow pace and switch-like character of the cycle is imposed by a bistable CDK1/Wee1/Myt1/Cdc25 system.
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Deneka, Alexander Y., Margret B. Einarson, John Bennett, Anna S. Nikonova, Mohamed Elmekawy, Yan Zhou, Jong Woo Lee, Barbara A. Burtness, and Erica A. Golemis. "Synthetic Lethal Targeting of Mitotic Checkpoints in HPV-Negative Head and Neck Cancer." Cancers 12, no. 2 (January 28, 2020): 306. http://dx.doi.org/10.3390/cancers12020306.
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Head and neck squamous cell carcinomas (HNSCC) affect more than 800,000 people annually worldwide, causing over 15,000 deaths in the US. Among HNSCC cancers, human papillomavirus (HPV)-negative HNSCC has the worst outcome, motivating efforts to improve therapy for this disease. The most common mutational events in HPV-negative HNSCC are inactivation of the tumor suppressors TP53 (>85%) and CDKN2A (>57%), which significantly impairs G1/S checkpoints, causing reliance on other cell cycle checkpoints to repair ongoing replication damage. We evaluated a panel of cell cycle-targeting clinical agents in a group of HNSCC cell lines to identify a subset of drugs with single-agent activity in reducing cell viability. Subsequent analyses demonstrated potent combination activity between the CHK1/2 inhibitor LY2606268 (prexasertib), which eliminates a G2 checkpoint, and the WEE1 inhibitor AZD1775 (adavosertib), which promotes M-phase entry, in induction of DNA damage, mitotic catastrophe, and apoptosis, and reduction of anchorage independent growth and clonogenic capacity. These phenotypes were accompanied by more significantly reduced activation of CHK1 and its paralog CHK2, and enhanced CDK1 activation, eliminating breaks on the mitotic entry of cells with DNA damage. These data suggest the potential value of dual inhibition of CHK1 and WEE1 in tumors with compromised G1/S checkpoints.
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Sheen, Joon-Ho, and Robert B. Dickson. "Overexpression of c-Myc Alters G1/S Arrest following Ionizing Radiation." Molecular and Cellular Biology 22, no. 6 (March 15, 2002): 1819–33. http://dx.doi.org/10.1128/mcb.22.6.1819-1833.2002.
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ABSTRACT Study of the mechanism(s) of genomic instability induced by the c-myc proto-oncogene has the potential to shed new light on its well-known oncogenic activity. However, an underlying mechanism(s) for this phenotype is largely unknown. In the present study, we investigated the effects of c-Myc overexpression on the DNA damage-induced G1/S checkpoint, in order to obtain mechanistic insights into how deregulated c-Myc destabilizes the cellular genome. The DNA damage-induced checkpoints are among the primary safeguard mechanisms for genomic stability, and alterations of cell cycle checkpoints are known to be crucial for certain types of genomic instability, such as gene amplification. The effects of c-Myc overexpression were studied in human mammary epithelial cells (HMEC) as one approach to understanding the c-Myc-induced genomic instability in the context of mammary tumorigenesis. Initially, flow-cytometric analyses were used with two c-Myc-overexpressing, nontransformed immortal lines (184A1N4 and MCF10A) to determine whether c-Myc overexpression leads to alteration of cell cycle arrest following ionizing radiation (IR). Inappropriate entry into S phase was then confirmed with a bromodeoxyuridine incorporation assay measuring de novo DNA synthesis following IR. Direct involvement of c-Myc overexpression in alteration of the G1/S checkpoint was then confirmed by utilizing the MycER construct, a regulatable c-Myc. A transient excess of c-Myc activity, provided by the activated MycER, was similarly able to induce the inappropriate de novo DNA synthesis following IR. Significantly, the transient expression of full-length c-Myc in normal mortal HMECs also facilitated entry into S phase and the inappropriate de novo DNA synthesis following IR. Furthermore, irradiated, c-Myc-infected, normal HMECs developed a sub-G1 population and a >4N population of cells. The c-Myc-induced alteration of the G1/S checkpoint was also compared to the effects of expression of MycS (N-terminally truncated c-Myc) and p53DD (a dominant negative p53) in the HMECs. We observed inappropriate hyperphosphorylation of retinoblastoma protein and then the reappearance of cyclin A, following IR, selectively in full-length c-Myc- and p53DD-overexpressing MCF10A cells. Based on these results, we propose that c-Myc attenuates a safeguard mechanism for genomic stability; this property may contribute to c-Myc-induced genomic instability and to the potent oncogenic activity of c-Myc.
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Rancourt, Raymond C., Peter C. Keng, Christopher E. Helt, and Michael A. O'Reilly. "The role of p21CIP1/WAF1 in growth of epithelial cells exposed to hyperoxia." American Journal of Physiology-Lung Cellular and Molecular Physiology 280, no. 4 (April 1, 2001): L617—L626. http://dx.doi.org/10.1152/ajplung.2001.280.4.l617.
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Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21CIP1/WAF1, which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2′-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.
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Ouadid-Ahidouch, Halima, Morad Roudbaraki, Philippe Delcourt, Ahmed Ahidouch, Nathalie Joury, and Natalia Prevarskaya. "Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression." American Journal of Physiology-Cell Physiology 287, no. 1 (July 2004): C125—C134. http://dx.doi.org/10.1152/ajpcell.00488.2003.
Full textAbstract:
We have previously reported that the hEAG K+ channels are responsible for the potential membrane hyperpolarization that induces human breast cancer cell progression into the G1 phase of the cell cycle. In the present study, we evaluate the role and functional expression of the intermediate-conductance Ca2+-activated K+ channel, hIK1-like, in controlling cell cycle progression. Our results demonstrate that hIK1 current density increased in cells synchronized at the end of the G1 or S phase compared with those in the early G1 phase. This increased current density paralleled the enhancement in hIK1 mRNA levels and the highly negative membrane potential. Furthermore, in cells synchronized at the end of G1 or S phases, basal cytosolic Ca2+ concentration ([Ca2+]i) was also higher than in cells arrested in early G1. Blocking hIK1 channels with a specific blocker, clotrimazole, induced both membrane potential depolarization and a decrease in the [Ca2+]i in cells arrested at the end of G1 and S phases but not in cells arrested early in the G1 phase. Blocking hIK1 with clotrimazole also induced cell proliferation inhibition but to a lesser degree than blocking hEAG with astemizole. The two drugs were essentially additive, inhibiting MCF-7 cell proliferation by 82% and arresting >90% of cells in the G1 phase. Thus, although the progression of MCF-7 cells through the early G1 phase is dependent on the activation of hEAG K+ channels, when it comes to G1 and checkpoint G1/S transition, the membrane potential appears to be primarily dependent on the hIK1-activity level.
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Burt, Janis M., Tasha K. Nelson, Alexander M. Simon, and Jennifer S. Fang. "Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells." American Journal of Physiology-Cell Physiology 295, no. 5 (November 2008): C1103—C1112. http://dx.doi.org/10.1152/ajpcell.299.2008.
Full textAbstract:
In addition to providing a pathway for intercellular communication, the gap junction-forming proteins, connexins, can serve a growth-suppressive function that is both connexin and cell-type specific. To assess its potential growth-suppressive function, we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed: cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression, and progression through the G1/S checkpoint was delayed, resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G1. Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37, with decreases in G1 and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G1/S checkpoint.
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Graeber, T. G., J. F. Peterson, M. Tsai, K. Monica, A. J. Fornace, and A. J. Giaccia. "Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status." Molecular and Cellular Biology 14, no. 9 (September 1994): 6264–77. http://dx.doi.org/10.1128/mcb.14.9.6264-6277.1994.
Full textAbstract:
It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.
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Clerici, Michela, Veronica Baldo, Davide Mantiero, Francisca Lottersberger, Giovanna Lucchini, and Maria Pia Longhese. "A Tel1/MRX-Dependent Checkpoint Inhibits the Metaphase-to-Anaphase Transition after UV Irradiation in the Absence of Mec1." Molecular and Cellular Biology 24, no. 23 (December 1, 2004): 10126–44. http://dx.doi.org/10.1128/mcb.24.23.10126-10144.2004.
Full textAbstract:
ABSTRACT In Saccharomyces cerevisiae, Mec1/ATR plays a primary role in sensing and transducing checkpoint signals in response to different types of DNA lesions, while the role of the Tel1/ATM kinase in DNA damage checkpoints is not as well defined. We found that UV irradiation in G1 in the absence of Mec1 activates a Tel1/MRX-dependent checkpoint, which specifically inhibits the metaphase-to-anaphase transition. Activation of this checkpoint leads to phosphorylation of the downstream checkpoint kinases Rad53 and Chk1, which are required for Tel1-dependent cell cycle arrest, and their adaptor Rad9. The spindle assembly checkpoint protein Mad2 also partially contributes to the G2/M arrest of UV-irradiated mec1Δ cells independently of Rad53 phosphorylation and activation. The inability of UV-irradiated mec1Δ cells to undergo anaphase can be relieved by eliminating the anaphase inhibitor Pds1, whose phosphorylation and stabilization in these cells depend on Tel1, suggesting that Pds1 persistence may be responsible for the inability to undergo anaphase. Moreover, while UV irradiation can trigger Mec1-dependent Rad53 phosphorylation and activation in G1- and G2-arrested cells, Tel1-dependent checkpoint activation requires entry into S phase independently of the cell cycle phase at which cells are UV irradiated, and it is decreased when single-stranded DNA signaling is affected by the rfa1-t11 allele. This indicates that UV-damaged DNA molecules need to undergo structural changes in order to activate the Tel1-dependent checkpoint. Active Clb-cyclin-dependent kinase 1 (CDK1) complexes also participate in triggering this checkpoint and are required to maintain both Mec1- and Tel1-dependent Rad53 phosphorylation, suggesting that they may provide critical phosphorylation events in the DNA damage checkpoint cascade.
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Orren, D. K., L. N. Petersen, and V. A. Bohr. "A UV-responsive G2 checkpoint in rodent cells." Molecular and Cellular Biology 15, no. 7 (July 1995): 3722–30. http://dx.doi.org/10.1128/mcb.15.7.3722.
Full textAbstract:
We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed.
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Mohanty, Suchismita, Atish Mohanty, Natalie Sandoval, Victoria Bedell, Joyce Murata-Collins, Jun Wu, Anna Scuto, Dennis D. Weisenburger, and Vu N. Ngo. "Cyclin D1 Maintains Mantle Cell Lymphoma Through CDK4-Independent Regulation Of DNA Replicative Checkpoints." Blood 122, no. 21 (November 15, 2013): 2512. http://dx.doi.org/10.1182/blood.v122.21.2512.2512.
Full textAbstract:
Abstract Mantle cell lymphoma (MCL) is rarely curable and therapy resistance often leaves few viable treatment options for patients. Previous studies have identified the importance of cyclin D1 (CCND1) translocation and overexpression in MCL pathogenesis, which leads to increased cyclin-dependent kinase 4 (CDK4) activity and accelerated cell cycle progression. However, targeting this abnormal cell cycle control, mainly through CDK4 inhibition causes only G1-phase growth arrest without significant cell death (Marzec et al. 2006). In contrast, prolonged inhibition of CCND1 with RNA interference induces apoptosis in MCL cell lines (Weinstein et al. 2012), suggesting an essential function of CCND1 independent of CDK4 activity. The mechanism of this non-catalytic role of CCND1 in maintaining MCL cell survival is largely unknown. To clarify the cell cycle role of CCND1 in addition to its CDK4-dependent function, we compared the effects of CCND1 and CDK4 silencing on MCL cell survival. MCL cell lines co-expressing GFP and doxycycline-inducible shRNA targeting CCND1 or CDK4 were generated. Cells with similar GFP expression levels were FACS sorted to normalize for shRNA expression. Both CCND1 and CDK4 silencing resulted in G1-phase arrest, but only CCND1-silenced cells demonstrated a marked increase in apoptosis. Investigation of the potential cause of apoptosis revealed significant accumulation of DNA double-strand breaks following CCND1 ablation, as measured by nuclear gamma-H2AX focus formation. Interestingly, CCND1-silenced cells exhibited a significant increase in 53BP1+ nuclear bodies in G1-phase, reminiscent of 53BP1 foci observed by Lukas and colleagues in cells undergoing aphidicolin-induced replication stress (Lukas et al. 2011). Analysis of replication fork movement in CCND1-depleted cells showed substantially reduced fork speed and increased frequency of origin firing, both of which are indicative of replication stress. In contrast, knockdown of CDK4 did not result in slower forks or increase in the frequency of origin firing. Genomic instability associated with replication stress was also apparent in CCND1-silenced cells, including increased micronucleus formation and recurrent chromatid gaps or breaks detected by cytokinesis-block assay and karyotyping, respectively. Analysis of DNA replicative and damage checkpoints revealed that both ATR-CHEK1 and ATM-CHEK2 pathways were activated by phosphorylation following CCND1 silencing in MCL cell lines, a xenograft animal model, and primary tumor samples, but not in non-MCL tumors. Interestingly, this activation (with the exception of ATM phosphorylation) was unsustainable over time and did not cause down-regulation of the downstream targets CDC25 and CDK1/2 but, instead, we observed an increase in CDC25A/B protein levels and CDK1/2 activity, indicating defective cell cycle checkpoints. Exposing CCND1-silenced cells to replication stress-inducing or DNA-damaging agents such hydroxyurea, aphidicolin, etoposide or ionizing radiation further amplified the checkpoint defects seen in unperturbed cells. We did not observe any significant difference in this checkpoint signaling in control and CDK4 knockdown cells under these conditions. Furthermore, CCND1-deficient cells were more sensitive to pharmacological inhibition of ATR and CHEK1 but not ATM, confirming a constitutive role of CCND1 in the ATR-CHEK1 pathway. In conclusion, these studies revealed an unexpected CDK4-independent role of CCND1 in maintaining DNA replicative checkpoints to prevent replication stress and genome instability in MCL cells. As most cancer treatments rely on agents that create DNA replication stress, targeting this function of CCND1 could provide a rational approach to overcome resistance to conventional therapies in MCL. Disclosures: No relevant conflicts of interest to declare.
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Crider, David G., Luis J. García-Rodríguez, Pallavi Srivastava, Leonardo Peraza-Reyes, Krishna Upadhyaya, Istvan R. Boldogh, and Liza A. Pon. "Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression." Journal of Cell Biology 198, no. 5 (August 27, 2012): 793–98. http://dx.doi.org/10.1083/jcb.201205193.
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The Chk2-mediated deoxyribonucleic acid (DNA) damage checkpoint pathway is important for mitochondrial DNA (mtDNA) maintenance. We show in this paper that mtDNA itself affects cell cycle progression. Saccharomyces cerevisiae rho0 cells, which lack mtDNA, were defective in G1- to S-phase progression. Deletion of subunit Va of cytochrome c oxidase, inhibition of F1F0 adenosine triphosphatase, or replacement of all mtDNA-encoded genes with noncoding DNA did not affect G1- to S-phase progression. Thus, the cell cycle progression defect in rho0 cells is caused by loss of DNA within mitochondria and not loss of respiratory activity or mtDNA-encoded genes. Rad53p, the yeast Chk2 homologue, was required for inhibition of G1- to S-phase progression in rho0 cells. Pif1p, a DNA helicase and Rad53p target, underwent Rad53p-dependent phosphorylation in rho0 cells. Thus, loss of mtDNA activated an established checkpoint kinase that inhibited G1- to S-phase progression. These findings support the existence of a Rad53p-regulated checkpoint that regulates G1- to S-phase progression in response to loss of mtDNA.
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Navadgi-Patil, Vasundhara M., and Peter M. Burgers. "Cell-cycle-specific activators of the Mec1/ATR checkpoint kinase." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 600–605. http://dx.doi.org/10.1042/bst0390600.
Full textAbstract:
Mec1 [ATR (ataxia telangiectasia mutated- and Rad3-related) in humans] is the principle kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. The heterotrimeric checkpoint clamp, 9-1-1 (checkpoint clamp of Rad9, Rad1 and Hus1 in humans and Ddc1, Rad17 and Mec3 in S. cerevisiae; Ddc1-Mec3-Rad17) and the DNA replication initiation factor Dpb11 (human TopBP1) are the two known activators of Mec1. The 9-1-1 clamp functions in checkpoint activation in G1- and G2-phase, but its employment differs between these two phases of the cell cycle. The Ddc1 (human Rad9) subunit of the clamp directly activates Mec1 in G1-phase, an activity identified only in S. cerevisiae so far. However, in G2-phase, the 9-1-1 clamp activates the checkpoint by two mechanisms. One mechanism includes direct activation of Mec1 by the unstructured C-terminal tail of Ddc1. The second mech-anism involves the recruitment of Dpb11 by the phosphorylated C-terminal tail of Ddc1. The latter mechanism is highly conserved and also functions in response to replication stress in higher eukaryotes. In S. cerevisiae, however, both the 9-1-1 clamp and the Dpb11 are partially redundant for checkpoint activation in response to replication stress, suggesting the existence of additional activators of Mec1.
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Graeber, T. G., J. F. Peterson, M. Tsai, K. Monica, A. J. Fornace, and A. J. Giaccia. "Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status." Molecular and Cellular Biology 14, no. 9 (September 1994): 6264–77. http://dx.doi.org/10.1128/mcb.14.9.6264.
Full textAbstract:
It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.
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Bouchard, Michael, Stavros Giannakopoulos, Edith H. Wang, Naoko Tanese, and Robert J. Schneider. "Hepatitis B Virus HBx Protein Activation of Cyclin A–Cyclin-Dependent Kinase 2 Complexes and G1 Transit via a Src Kinase Pathway." Journal of Virology 75, no. 9 (May 1, 2001): 4247–57. http://dx.doi.org/10.1128/jvi.75.9.4247-4257.2001.
Full textAbstract:
ABSTRACT Numerous studies have demonstrated that the hepatitis B virus HBx protein stimulates signal transduction pathways and may bind to certain transcription factors, particularly the cyclic AMP response element binding protein, CREB. HBx has also been shown to promote early cell cycle progression, possibly by functionally replacing the TATA-binding protein-associated factor 250 (TAFII250), a transcriptional coactivator, and/or by stimulating cytoplasmic signal transduction pathways. To understand the basis for early cell cycle progression mediated by HBx, we characterized the molecular mechanism by which HBx promotes deregulation of the G0 and G1 cell cycle checkpoints in growth-arrested cells. We demonstrate that TAFII250 is absolutely required for HBx activation of the cyclin A promoter and for promotion of early cell cycle transit from G0 through G1. Thus, HBx does not functionally replace TAFII250 for transcriptional activity or for cell cycle progression, in contrast to a previous report. Instead, HBx is shown to activate the cyclin A promoter, induce cyclin A–cyclin-dependent kinase 2 complexes, and promote cycling of growth-arrested cells into G1 through a pathway involving activation of Src tyrosine kinases. HBx stimulation of Src kinases and cyclin gene expression was found to force growth-arrested cells to transit through G1 but to stall at the junction with S phase, which may be important for viral replication.
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McArthur, G. A., J. Raleigh, A. Blasina, C. Cullinane, D. Dorow, N. Conus, R. J. Hicks, et al. "Imaging with FLT-PET demonstrates that PF-477736, an inhibitor of CHK1 kinase, overcomes a cell cycle checkpoint induced by gemcitabine in PC-3 xenografts." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 3045. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.3045.
Full textAbstract:
3045 Background: The development of strategies to monitor the molecular and cellular response to novel agents that target the cell cycle is vital to provide proof of mechanism and biological activity of these compounds. The protein kinase CHK1 is activated following DNA damage in the S and G2-phases of the cell cycle and mediates cell cycle arrest. In vitro studies demonstrate that inhibition of CHK1 can overcome cell cycle arrest induced by DNA damage and enhance cytotoxic activity of DNA damaging agents. In vivo studies show that combining DNA damaging agents with a CHK1 inhibitor potentiates antitumor activity. We hypothesize that functional imaging with 18F-fluorine-L-thymidine (FLT), a PET-tracer where tumor uptake is maximal in the S and G2 phases of the cell cycle can be used to non-invasively monitor the induction and therapeutic inhibition of a cell cycle checkpoint in vivo. Methods: Nude mice harbouring PC-3 xenografts were treated with vehicle controls, gemcitabine, the CHK1-inhibitor PF-477736 or gemcitabine + PF-477736. FLT-PET scans were performed and tumors harvested for ex-vivo biomarkers to assess S-phase, M-phase and DNA-repair. Results: Gemcitabine induced a 8.3 ±0.8 fold increase in tumoral uptake of FLT at 21 hours that correlated with a 3.3 ±0.2-fold increase in thymidine kinase activity and S-phase arrest as demonstrated by BrdU incorporation and elevated expression of cyclin-A. Treatment with PF-477736 at 17 hours after gemcitabine abrogated the early FLT-flare at 21 hours by 82% (p<0.001). This was associated with both an increased fraction of cells in mitosis and G1-phase of the cell cycle as determined by phos-histone H3 and flow cytometry. Furthermore, the combination of gemcitabine and PF-477736 enhanced DNA damage as measured by phos-gamma-H2AX and significantly delayed tumor growth when compared to tumors treated with gemcitabine alone. Conclusion: These data clearly indicate that the CHK1-inhibitor PF-477736 can overcome the cell cycle checkpoint induced by gemcitabine and increase associated DNA damage in tumors in-vivo. The PET studies indicate that functional imaging with FLT-PET is a promising strategy to monitor responses to therapeutic agents that target cell cycle checkpoints. [Table: see text]
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Schwartz, Gary K. "Development of Cell Cycle Active Drugs for the Treatment of Gastrointestinal Cancers: A New Approach to Cancer Therapy." Journal of Clinical Oncology 23, no. 20 (July 10, 2005): 4499–508. http://dx.doi.org/10.1200/jco.2005.18.341.
Full textAbstract:
The cell cycle represents a series of tightly integrated events that allow the cell to grow and proliferate. An essential part of the cell cycle machinery is the cyclin-dependent kinases (CDKs). When activated, the CDKs provide a means for the cell to move from one phase of the cell cycle to the next (G1 to S or G2 to M). The cell cycle serves to protect the cell from genotoxic stress. In the setting of DNA damage, the CDKs are inhibited and the cell undergoes cell-cycle arrest. This provides the cell the opportunity to repair its own damaged DNA before it resumes cell proliferation. If a cell continues to cycle with its damaged DNA intact, the apoptotic machinery is triggered and the cell will undergo apoptosis. In essence, cell cycle arrest at these critical checkpoints represents a survival mechanism, which provides the tumor cell the opportunity to escape the effects of lethal DNA damage induced by chemotherapy. Over the past several years, a series of new targeted agents has been developed that promote apoptosis of DNA damaged tumor cells either during cell cycle arrest or following premature cell cycle checkpoint exit, such that tumor cells re-enter the cell cycle before DNA repair is complete. An understanding of the cell cycle and its relationship to p53 are critical for the successful clinical development of these agents for the treatment of patients with gastrointestinal cancers.
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Kumaraswamy, Easwari, and Ramin Shiekhattar. "Activation of BRCA1/BRCA2-Associated Helicase BACH1 Is Required for Timely Progression through S Phase." Molecular and Cellular Biology 27, no. 19 (July 30, 2007): 6733–41. http://dx.doi.org/10.1128/mcb.00961-07.
Full textAbstract:
ABSTRACT BACH1 (also known as FANCJ and BRIP1) is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1. Previous biochemical and functional analyses have suggested a role for the BACH1 homolog in Caenorhabditis elegans during DNA replication. Here, we report the association of BACH1 with a distinct BRCA1/BRCA2-containing complex during the S phase of the cell cycle. Depletion of BACH1 or BRCA1 using small interfering RNAs results in delayed entry into the S phase of the cell cycle. Such timely progression through S phase requires the helicase activity of BACH1. Importantly, cells expressing a dominant negative mutation in BACH1 that results in a defective helicase displayed increased activation of DNA damage checkpoints and genomic instability. BACH1 helicase is silenced during the G1 phase of the cell cycle and is activated through a dephosphorylation event as cells enter S phase. These results point to a critical role for BACH1 helicase activity not only in the timely progression through the S phase but also in maintaining genomic stability.
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Morita, Eiji, Akitoshi Nakashima, Hironobu Asao, Hiroyuki Sato, and Kazuo Sugamura. "Human Parvovirus B19 Nonstructural Protein (NS1) Induces Cell Cycle Arrest at G1 Phase." Journal of Virology 77, no. 5 (March 1, 2003): 2915–21. http://dx.doi.org/10.1128/jvi.77.5.2915-2921.2003.
Full textAbstract:
ABSTRACT Human parvovirus B19 infects predominantly erythroid precursor cells, leading to inhibition of erythropoiesis. This erythroid cell damage is mediated by the viral nonstructural protein 1 (NS1) through an apoptotic mechanism. We previously demonstrated that B19 virus infection induces G2 arrest in erythroid UT7/Epo-S1 cells; however, the role of NS1 in regulating cell cycle arrest is unknown. In this report, by using paclitaxel, a mitotic inhibitor, we show that B19 virus infection induces not only G2 arrest but also G1 arrest. Interestingly, UV-irradiated B19 virus, which has inactivated the expression of NS1, still harbors the ability to induce G2 arrest but not G1 arrest. Furthermore, treatment with caffeine, a G2 checkpoint inhibitor, abrogated the B19 virus-induced G2 arrest despite expression of NS1. These results suggest that the B19 virus-induced G2 arrest is not mediated by NS1 expression. We also found that NS1-transfected UT7/Epo-S1 and 293T cells induced cell cycle arrest at the G1 phase. These results indicate that NS1 expression plays a critical role in G1 arrest induced by B19 virus. Furthermore, NS1 expression significantly increased p21/WAF1 expression, a cyclin-dependent kinase inhibitor that induces G1 arrest. Thus, G1 arrest mediated by NS1 may be a prerequisite for the apoptotic damage of erythroid progenitor cells upon B19 virus infection.
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Poli, Alessandro, Sara Mongiorgi, Lucio Cocco, and Matilde Y. Follo. "Protein kinase C involvement in cell cycle modulation." Biochemical Society Transactions 42, no. 5 (September 18, 2014): 1471–76. http://dx.doi.org/10.1042/bst20140128.
Full textAbstract:
Protein kinases C (PKCs) are a family of serine/threonine kinases which act as key regulators in cell cycle progression and differentiation. Studies of the involvement of PKCs in cell proliferation showed that their role is dependent on cell models, cell cycle phases, timing of activation and localization. Indeed, PKCs can positively and negatively act on it, regulating entry, progression and exit from the cell cycle. In particular, the targets of PKCs resulted to be some of the key proteins involved in the cell cycle including cyclins, cyclin-dependent kinases (Cdks), Cip/Kip inhibitors and lamins. Several findings described roles for PKCs in the regulation of G1/S and G2/M checkpoints. As a matter of fact, data from independent laboratories demonstrated PKC-related modulations of cyclins D, leading to effects on the G1/S transition and differentiation of different cell lines. Moreover, interesting data were published on PKC-mediated phosphorylation of lamins. In addition, PKC isoenzymes can accumulate in the nuclei, attracted by different stimuli including diacylglycerol (DAG) fluctuations during cell cycle progression, and target lamins, leading to their disassembly at mitosis. In the present paper, we briefly review how PKCs could regulate cell proliferation and differentiation affecting different molecules related to cell cycle progression.
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Havens, Courtney G., Alan Ho, Naohisa Yoshioka, and Steven F. Dowdy. "Regulation of Late G1/S Phase Transition and APCCdh1 by Reactive Oxygen Species." Molecular and Cellular Biology 26, no. 12 (June 15, 2006): 4701–11. http://dx.doi.org/10.1128/mcb.00303-06.
Full textAbstract:
ABSTRACT Proliferating cells have a higher metabolic rate than quiescent cells. To investigate the role of metabolism in cell cycle progression, we examined cell size, mitochondrial mass, and reactive oxygen species (ROS) levels in highly synchronized cell populations progressing from early G1 to S phase. We found that ROS steadily increased, compared to cell size and mitochondrial mass, through the cell cycle. Since ROS has been shown to influence cell proliferation and transformation, we hypothesized that ROS could contribute to cell cycle progression. Antioxidant treatment of cells induced a late-G1-phase cell cycle arrest characterized by continued cellular growth, active cyclin D-Cdk4/6 and active cyclin E-Cdk2 kinases, and inactive hyperphosphorylated pRb. However, antioxidant-treated cells failed to accumulate cyclin A protein, a requisite step for initiation of DNA synthesis. Further examination revealed that cyclin A continued to be ubiquitinated by the anaphase promoting complex (APC) and to be degraded by the proteasome. This antioxidant arrest could be rescued by overexpression of Emi1, an APC inhibitor. These observations reveal an intrinsic late-G1-phase checkpoint, after transition across the growth factor-dependent G1 restriction point, that links increased steady-state levels of endogenous ROS and cell cycle progression through continued activity of APC in association with Cdh1.
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Johnston, J. B., G. Wang, J. W. Barrett, S. H. Nazarian, K. Colwill, M. Moran, and G. McFadden. "Myxoma Virus M-T5 Protects Infected Cells from the Stress of Cell Cycle Arrest through Its Interaction with Host Cell Cullin-1." Journal of Virology 79, no. 16 (August 15, 2005): 10750–63. http://dx.doi.org/10.1128/jvi.79.16.10750-10763.2005.
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ABSTRACT The myxoma virus (MV) M-T5 gene encodes an ankyrin repeat protein that is important for virus replication in cells from several species. Insight was gained into the molecular mechanisms underlying the role of M-T5 as a host range determinant when the cell cycle regulatory protein cullin-1 (cul-1) was identified as a cellular binding partner of M-T5 and found to colocalize with the protein in both nuclear and cytosolic compartments. Consistent with this interaction, infection with wild-type MV (vMyxlac) or a deletion mutant lacking M-T5 (vMyxT5KO) differentially altered cell cycle progression in a panel of permissive and nonpermissive cells. Cells infected with vMyxlac transitioned rapidly out of the G0/G1 phase and preferentially accumulated at the G2/M checkpoint, whereas infection with vMyxT5KO impeded progression through the cell cycle, resulting in a greater percentage of cells retained at G0/G1. Levels of the cul-1 substrate, p27/Kip-1, were selectively increased in cells infected with vMyxT5KO compared to vMyxlac, concurrent with decreased phosphorylation of p27/Kip-1 at Thr187 and decreased ubiquitination. Compared to cells infected with vMyxlac, cell death was increased in vMyxT5KO-infected cells following treatment with diverse stimuli known to induce cell cycle arrest, including infection itself, serum deprivation, and exposure to proteasome inhibitors or double-stranded RNA. Moreover, infection with vMyxlac, but not vMyxT5KO, was sufficient to overcome the G0/G1 arrest induced by these stimuli. These findings suggest that M-T5 regulates cell cycle progression at the G0/G1 checkpoint, thereby protecting infected cells from diverse innate host antiviral responses normally triggered by G0/G1 cell cycle arrest.
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Dhar, Sonu, Jeremy A. Squire, M. Prakash Hande, Raymund J. Wellinger, and Tej K. Pandita. "Inactivation of 14-3-3ς Influences Telomere Behavior and Ionizing Radiation-Induced Chromosomal Instability." Molecular and Cellular Biology 20, no. 20 (October 15, 2000): 7764–72. http://dx.doi.org/10.1128/mcb.20.20.7764-7772.2000.
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ABSTRACT Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G2 and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3ς gene has been reported to be a checkpoint control gene, since it promotes G2 arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3ς−/− cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G1 checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G2 after irradiation with gamma rays, corroborating the role of the 14-3-3ς protein in G2/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G2 checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3ς−/− cells are defective in maintaining G2 arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G2/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.
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Karlsson, Christina, Stephanie Katich, Anja Hagting, Ingrid Hoffmann, and Jonathon Pines. "Cdc25b and Cdc25c Differ Markedly in Their Properties as Initiators of Mitosis." Journal of Cell Biology 146, no. 3 (August 9, 1999): 573–84. http://dx.doi.org/10.1083/jcb.146.3.573.
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We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.
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Gharote, Mukul Arvind. "Chronomodulation of cyclin-dependent kinases 4/6 inhibitor may reduce hematological toxicities? A review of literature." International Journal of Molecular and Immuno Oncology 3, no. 3 (October 26, 2018): 78. http://dx.doi.org/10.18203/issn.2456-3994.intjmolimmunooncol20184342.
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<p class="s3">Nucleotide excision repair, DNA damage checkpoints, and apoptosis are under the influence of the circadian rhythm1. Circadian rhythm is defined as oscillations in the behavior and biochemical changes in an individual that repeats itself after the span of 24 h approximately. Cyclin-dependent kinase (CDK) inhibition causes cell cycle arrest and subsequent circadian stage-dependent gating of cells at G2-M interface of the cell cycle. Few anecdotes have suggested that chronomodulation reduces hematological toxicity in cell cycle-specific chemotherapy, especially S1-specific chemotherapy. In a study conducted by Boucher et al., 2016, circadian rhythm plays a role in the regulation of human mesenchymal stem cells (hMSCs) differentiation and division and likely represents key factor in maintaining hMSCs properties. If we apply the knowledge of circadian clock, then we know the fact that bone marrow stem cells (BMSCs) are under the control of circadian rhythm and G1-S phase of cell division cycle occurs at the early morning period of solar day. If CDK4/6 plasma peak level coincides with G1-S phase of BMSCs, then theoretically cytopenia may occur, which again is the sign of CDK4/6 action but is also the reason of its toxicity. Chronomodulation studies of CDK4/6 inhibitor may reduce hematological toxicity of CDK4/6 inhibitor.</p>
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Chen, Zihao, and Chunhe Li. "Quantifying the Landscape and Transition Paths for Proliferation–Quiescence Fate Decisions." Journal of Clinical Medicine 9, no. 8 (August 10, 2020): 2582. http://dx.doi.org/10.3390/jcm9082582.
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The cell cycle, essential for biological functions, experiences delicate spatiotemporal regulation. The transition between G1 and S phase, which is called the proliferation–quiescence decision, is critical to the cell cycle. However, the stability and underlying stochastic dynamical mechanisms of the proliferation–quiescence decision have not been fully understood. To quantify the process of the proliferation–quiescence decision, we constructed its underlying landscape based on the relevant gene regulatory network. We identified three attractors on the landscape corresponding to the G0, G1, and S phases, individually, which are supported by single-cell data. By calculating the transition path, which quantifies the potential barrier, we built expression profiles in temporal order for key regulators in different transitions. We propose that the two saddle points on the landscape characterize restriction point (RP) and G1/S checkpoint, respectively, which provides quantitative and physical explanations for the mechanisms of Rb governing the RP while p21 controlling the G1/S checkpoint. We found that Emi1 inhibits the transition from G0 to G1, while Emi1 in a suitable range facilitates the transition from G1 to S. These results are partially consistent with previous studies, which also suggested new roles of Emi1 in the cell cycle. By global sensitivity analysis, we identified some critical regulatory factors influencing the proliferation–quiescence decision. Our work provides a global view of the stochasticity and dynamics in the proliferation–quiescence decision of the cell cycle.
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Hochwagen, Andreas, Gunnar Wrobel, Marie Cartron, Philippe Demougin, Christa Niederhauser-Wiederkehr, Monica G. Boselli, Michael Primig, and Angelika Amon. "Novel Response to Microtubule Perturbation in Meiosis." Molecular and Cellular Biology 25, no. 11 (June 1, 2005): 4767–81. http://dx.doi.org/10.1128/mcb.25.11.4767-4781.2005.
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ABSTRACT During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G1 or G2, instead of metaphase. Cells arrest in G1 if microtubule perturbation occurs as they enter the meiotic cell cycle and in G2 if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.
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Chakraverty, Ronjon K., Jonathan M. Kearsey, Thomas J. Oakley, Muriel Grenon, Maria-Angeles de la Torre Ruiz, Noel F. Lowndes, and Ian D. Hickson. "Topoisomerase III Acts Upstream of Rad53p in the S-Phase DNA Damage Checkpoint." Molecular and Cellular Biology 21, no. 21 (November 1, 2001): 7150–62. http://dx.doi.org/10.1128/mcb.21.21.7150-7162.2001.
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ABSTRACT Deletion of the Saccharomyces cerevisiae TOP3gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391–8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Δ strains. We show that top3Δ mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion,top3Δ strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Δ mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.
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Rodriguez, Antonio, Eun Joo Jung, and Erik K. Flemington. "Cell Cycle Analysis of Epstein-Barr Virus-Infected Cells following Treatment with Lytic Cycle-Inducing Agents." Journal of Virology 75, no. 10 (May 15, 2001): 4482–89. http://dx.doi.org/10.1128/jvi.75.10.4482-4489.2001.
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ABSTRACT While Epstein-Barr virus (EBV) latency-associated gene expression is associated with cell cycle progression, the relationship between the EBV lytic program and the cell cycle is less clear. Using four different EBV lytic induction systems, we address the relationship between lytic cycle activation and the cell cycle. In three of these systems, G0 or G1 cell growth arrest signaling is observed prior to detection of the EBV immediate-early gene product Zta. In tetradecanoyl phorbol acetate-treated P3HR1 cultures and in 5-iodo-2′-deoxyuridine-treated NPC-KT cultures, cell cycle analysis of Zta-expressing cell populations showed a significant G1bias during the early stages of lytic cycle progression. In contrast, treatment of the cell line Akata with anti-immunoglobulin (Ig) results in rapid induction of immediate-early gene expression, and accordingly, activation of the immediate-early gene product Zta precedes significant anti-Ig-induced cell cycle effects. Nevertheless, cell cycle analysis of the Zta-expressing population following anti-Ig treatment shows a bias for cells in G1, indicating that anti-Ig-mediated induction of Zta occurs more efficiently in cells traversing G1. Last, although 5-azacytidine treatment of Rael cells results in a G1 arrest in the total cell population which precedes the induction of Zta, cell cycle analysis of the Zta-expressing population shows a significant bias for cells with an apparent G2/M DNA content. This bias may result, in part, from activation of Zta expression following demethylation of the Zta promoter during S-phase. Together, these studies indicate that induction of Zta occurs through several distinct mechanisms, some of which may involve checkpoint signaling.
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RUSSO, Gian Luigi, Christian VAN DEN BOS, Ann SUTTON, Paola COCCETTI, Maurizio D. BARONI, Lilia ALBERGHINA, and Daniel R. MARSHAK. "Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae." Biochemical Journal 351, no. 1 (September 26, 2000): 143–50. http://dx.doi.org/10.1042/bj3510143.
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The CDK (cyclin-dependent kinase) family of enzymes is required for the G1-to-S-phase and G2-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G1 phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317–20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G1 stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G1 progression.
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Kumar, Subodh, Srikanth Talluri, Mariateresa Fulciniti, Masood A. Shammas, and Nikhil C. Munshi. "Elevated APEX1 Disrupts G2/M Checkpoint, Contributing to Evolution and Survival of Myeloma Cells." Blood 126, no. 23 (December 3, 2015): 2997. http://dx.doi.org/10.1182/blood.v126.23.2997.2997.
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Abstract Cell cycle checkpoints provide the cell with time to repair chromosomal DNA damage before its replication (G1) and also prior to its segregation (G2), thus ensuring integrity, maintenance and protection of genome. Although proper functioning of both checkpoints is essential, G2/M has a special significance as a potentially lethal double-strand break in DNA escape repair and persist from G2 into mitosis, it may recombine in G1 to produce gene rearrangements. Moreover, G2 is the phase where homologous recombination (HR) can utilize a sister chromatid as a template to provide error-free repair. There is ample evidence that supports the role of defective G2/M checkpoint and dysregulated HR in genomic rearrangements and evolution in cancer. Previously, we have shown that elevated APEX1 contributes to dysregulated HR and genome stability in multiple myeloma (MM), and its upregulation leads to genomic instability and tumorigenesis in animal model. To further understand the role of APEX1 in myeloma, we investigated the impact of elevated APEX1 on cell cycle checkpoint/s and in the cellular response to genotoxic exposure. Our investigation using antibody array and subsequent confirmation with immunoprecipitation experiments demonstrated that APEX1 interacts with cyclin B and PLK in myeloma cells. A key step in progression from G2 to mitosis is the activation of cyclin B-CDK1 complex, which subsequently activates PLK to ensure G2/M progression. Based on observed interaction of APEX1 with cyclin B/PLK, we hypothesized that elevated APEX1 disrupts G2 checkpoint by mediating progression into mitosis. To test this, we inhibited APEX1 in myeloma cells by a small molecule as well as by shRNA targeting this gene, and investigated the impact on cell cycle checkpoints using a unique phospho-antibody array which allows investigation of 238 relevant proteins and their phosphorylation status. APEX1 inhibition by small molecule led to downregulation (> 2-fold) of many proteins/phosphorylations involved in the activation of cyclin B-CDK1 complex and other mediators of G2/M progression (including CDC25A, CDC25A, CDK1, ABL1), and upregulation of proteins/phosphorylations involved in G2/M arrest, including CDK1-phospho-Tyr15, 14-3-3 zeta-phospho-Ser58, p53-phospho-Ser15, and MYT and WEE which are involved in negative regulation of cyclin B-CDK1 complex. To further investigate the role of APEX1 in G2/M progression, myeloma cell lines (ARP, RPMI 8226, MM1S, LR5, H929) were treated with APEX1 inhibitor and subjected to cell cycle analysis using flow cytometery. Compared to control cells, all five APEX1-inhibitor treated cell lines showed a strong G2/M block, ranging from 5- to 10-fold increase in the fraction of cells in G2 phase in a dose dependent manner. The G2/M cell cycle arrest of APEX1-treated myeloma cells was further supported by reduced cell viability of treated myeloma cells (RPMI, H929, MM1S, ARP and U266); IC50 of inhibitor in myeloma cell lines ranged from 1.2 to 4 µM. Co-treatment with APEX1 inhbitor also sensitized myeloma cells to Melphalan. Consistent with these data, shRNA-mediated knockdown (KD) of APEX1 in RPMI cells was associated with 4-fold increase in the fraction of cells in G2, relative to control cells. APEX1-KD was also associated with reduction in cell viability (by 40%) and sensitization to melphalan. Our results therefore suggest that elevated APEX1 disrupts G2 checkpoint and sets a stage for genomic rearrangements by allowing persistance of DNA damage from G2 into mitosis. Dysfunctional G2 checkpoint, combined with APEX1-mediated dysregulation of HR, could be attributed to APEX1 associated genomic instability and oncogenic transformation. Therefore, inhibitors of APEX1, alone or in combination with other agents, have potential to make myeloma cells static. Disclosures No relevant conflicts of interest to declare.
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Hitomi, Masahiro, Ke Yang, Andrew W. Stacey, and Dennis W. Stacey. "Phosphorylation of Cyclin D1 Regulated by ATM or ATR Controls Cell Cycle Progression." Molecular and Cellular Biology 28, no. 17 (July 7, 2008): 5478–93. http://dx.doi.org/10.1128/mcb.02047-07.
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ABSTRACT Cyclin D1 is required at high levels for passage through G1 phase but must be reduced to low levels during S phase to avoid the inhibition of DNA synthesis. This suppression requires the phosphorylation of Thr286, which is induced directly by DNA synthesis. Because the checkpoint kinase ATR is activated by normal replication as well as by DNA damage, its potential role in regulating cyclin D1 phosphorylation was tested. We found that ATR, activated by either UV irradiation or the topoisomerase IIβ binding protein 1 activator, promoted cyclin D1 phosphorylation. Small interfering RNA against ATR inhibited UV-induced Thr286 phosphorylation, together with that seen in normally cycling cells, indicating that ATR regulates cyclin D1 phosphorylation in normal as well as stressed cells. Following double-stranded DNA (dsDNA) breakage, the related checkpoint kinase ATM was also able to promote the phosphorylation of cyclin D1 Thr286. The relationship between these checkpoint kinases and cyclin D1 was extended when we found that normal cell cycle blockage in G1 phase observed following dsDNA damage was efficiently overcome when exogenous cyclin D1 was expressed within the cells. These results indicate that checkpoint kinases play a critical role in regulating cell cycle progression in normal and stressed cells by directing the phosphorylation of cyclin D1.
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Toyn, J. H., A. L. Johnson, and L. H. Johnston. "Segregation of unreplicated chromosomes in Saccharomyces cerevisiae reveals a novel G1/M-phase checkpoint." Molecular and Cellular Biology 15, no. 10 (October 1995): 5312–21. http://dx.doi.org/10.1128/mcb.15.10.5312.
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Saccharomyces cerevisiae dbf4 and cdc7 cell cycle mutants block initiation of DNA synthesis (i.e., are iDS mutants) at 37 degrees C and arrest the cell cycle with a 1C DNA content. Surprisingly, certain dbf4 and cdc7 strains divide their chromatin at 37 degrees C. We found that the activation of the Cdc28 mitotic protein kinase and the Dbf2 kinase occurred with the correct relative timing with respect to each other and the observed division of the unreplicated chromatin. Furthermore, the division of unreplicated chromatin depended on a functional spindle. Therefore, the observed nuclear division resembled a normal mitosis, suggesting that S. cerevisiae commits to M phase in late G1 independently of S phase. Genetic analysis of dbf4 and cdc7 strains showed that the ability to restrain mitosis during a late G1 block depended on the genetic background of the strain concerned, since the dbf4 and cdc7 alleles examined showed the expected mitotic restraint in other backgrounds. This restraint was genetically dominant to lack of restraint, indicating that an active arrest mechanism, or checkpoint, was involved. However, none of the previously described mitotic checkpoint pathways were defective in the iDS strains that carry out mitosis without replicated DNA, therefore indicating that the checkpoint pathway that arrests mitosis in iDS mutants is novel. Thus, spontaneous strain differences have revealed that S. cerevisiae commits itself to mitosis in late G1 independently of entry into S phase and that a novel checkpoint mechanism can restrain mitosis if cells are blocked in late G1. We refer to this as the G1/M-phase checkpoint since it acts in G1 to restrain mitosis.
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Jiang, Guochun, and Aziz Sancar. "Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences." Molecular and Cellular Biology 26, no. 1 (January 1, 2006): 39–49. http://dx.doi.org/10.1128/mcb.26.1.39-49.2006.
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ABSTRACT We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.
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Shivakumar, Latha, John Minna, Toshiyuki Sakamaki, Richard Pestell, and Michael A. White. "The RASSF1A Tumor Suppressor Blocks Cell Cycle Progression and Inhibits Cyclin D1 Accumulation." Molecular and Cellular Biology 22, no. 12 (June 15, 2002): 4309–18. http://dx.doi.org/10.1128/mcb.22.12.4309-4318.2002.
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ABSTRACT The RASSF1A locus at 3p21.3 is epigenetically inactivated at high frequency in a variety of solid tumors. Expression of RASSF1A is sufficient to revert the tumorigenicity of human cancer cell lines. We show here that RASSF1A can induce cell cycle arrest by engaging the Rb family cell cycle checkpoint. RASSF1A inhibits accumulation of native cyclin D1, and the RASSF1A-induced cell cycle arrest can be relieved by ectopic expression of cyclin D1 or of other downstream activators of the G1/S-phase transition (cyclin A and E7). Regulation of cyclin D1 is responsive to native RASSF1A activity, because RNA interference-mediated downregulation of endogenous RASSF1A expression in human epithelial cells results in abnormal accumulation of cyclin D1 protein. Inhibition of cyclin D1 by RASSF1A occurs posttranscriptionally and is likely at the level of translational control. Rare alleles of RASSF1A, isolated from tumor cell lines, encode proteins that fail to block cyclin D1 accumulation and cell cycle progression. These results strongly suggest that RASSF1A is an important human tumor suppressor protein acting at the level of G1/S-phase cell cycle progression.
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Cann, Kendra L., and Geoffrey G. Hicks. "Regulation of the cellular DNA double-strand break response." Biochemistry and Cell Biology 85, no. 6 (December 2007): 663–74. http://dx.doi.org/10.1139/o07-135.
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DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1–S, intra-S phase, and G2–M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.
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Boothman, David A., Eric Odegaard, Chin-Rang Yang, Kelly Hosley, and Marc S. Mendonca. "Molecular analyses of adaptive survival responses (ASRs): role of ASRs in radiotherapy." Human & Experimental Toxicology 17, no. 8 (August 1998): 448–53. http://dx.doi.org/10.1177/096032719801700809.
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Adaptive survival responses (ASRs), whereby cells demonstrate a survival advantage when exposed to very low doses of ionizing radiation (IR) 4-24 h prior to a high dose challenge, were first reported over 15 years ago. These responses were linked to hormesis, which implied that exposure to low levels of IR may be beneficial to the cell. We postulate that increased survival does not necessarily mean that the treatment is beneficial.Studies at the molecular level indicate that ASRs are the result of misregulated cell cycle checkpoint responses, occurring in the G1 phase of the cell cycle after IR. Specific gene products (i.e., PCNA, cyclin D1, cyclin A, XIP8, xip5 and xip13) appear to control these cell cycle checkpoint responses. Certain neoplastic cells show potent ASRs because they bypass checkpoints which would otherwise lead to apoptosis or other forms of cell death (possibly necrosis), and/or these cancer cells lack genetic factors, such as specific caspases (cysteine aspartate-specific proteases), that control apoptosis. Alterations in these cell cycle checkpoints or apoptotic responses may also occur during IR-induced stress responses in normal cells, at critical times (10-18 days posttreatment) following IR. One IR-induced protein, XIP8, may be a critical controlling factor at this point where delayed-onset apoptosis occurs. Additionally, we have shown that the presence or absence (i.e., SCID cells) of nonhomologous DNA double strand break repair did not seem to influence ASRs, suggesting that ASRs may be caused by signal transduction stress responses.ASRs may be beneficial to survival, however, the consequence(s) of that survival may be dire. For example, many neoplastic cells exhibited far greater ASRs than normal cells. Additionally, ASRs were induced by as little as 1 cGy and and were enhanced by repeated exposures of low level radiation. The implications for radiotherapy are that when a patient arrives for port film imaging during the course of therapy, the dose-rate, overall level of exposure, and time between port film exposure and high dose IR treatment become potentially important factors for improved efficacy of treatment of certain cancers. Further research is warranted to determine what molecular factors are most important for ASRs, and current work is focusing on XIP8.