Journal articles: 'G2/M block'

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Bork, Ulrich, Wing-Kee Lee, Anna Kuchler, Thomas Dittmar, and Frank Thévenod. "Cadmium-induced DNA damage triggers G2/M arrest via chk1/2 and cdc2 in p53-deficient kidney proximal tubule cells." American Journal of Physiology-Renal Physiology 298, no. 2 (February 2010): F255—F265. http://dx.doi.org/10.1152/ajprenal.00273.2009.

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Carcinogenesis is a multistep process that is frequently associated with p53 inactivation. The class 1 carcinogen cadmium (Cd2+) causes renal cancer and is known to inactivate p53. G2/mitosis (M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in Cd2+-exposed p53-deficient renal cells are unknown. In p53-inactivated kidney proximal tubule (PT) cells, comet assay experiments showed that Cd2+ (50–100 μM) induced DNA damage within 1–6 h. This was associated with peak formation of reactive oxygen species (ROS) at 1–3 h, measured with dihydrorhodamine 123, and G2/M cell cycle arrest at 6 h, which were abolished by the antioxidant α-tocopherol (100 μM). Cd2+-induced G2/M arrest was enhanced approximately twofold on release from cell synchronization (double thymidine block or nocodazole) and resulted in approximately twofold increase of apoptosis, indicating that G2/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 μM), which relieves G2/M transition block, abolished Cd2+-induced G2 arrest and increased apoptosis. This was accompanied by prevention of Cd2+-induced cyclin-dependent kinase cdc2 phosphorylation at tyrosine 15, as shown by immunofluorescence microscopy and immunoblotting. The data indicate that in p53-inactivated PT cells Cd2+-induced ROS formation and DNA damage trigger signaling of checkpoint activating kinases ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) to cause G2/M arrest. This may promote survival of premalignant PT cells and Cd2+ carcinogenesis.

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Paris, J., P. Leplatois, and P. Nurse. "Study of the higher eukaryotic gene function CDK2 using fission yeast." Journal of Cell Science 107, no. 3 (March 1, 1994): 615–23. http://dx.doi.org/10.1242/jcs.107.3.615.

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In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis. In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2. Temperature-sensitive mutations in S. pombe cdc2 that arrest the cell cycle in both G1 and G2 phases are not complemented by CDK2. We have used S. pombe to investigate what functions CDK2 can perform. We found that overexpression of the human homologue (HsCDK2) caused cell cycle arrest in G2/M showing that HsCDK2 interfered with mitotic events. Xenopus CDK2 (XlCDK2) overexpression did not cause cell cycle arrest and could rescue the G1 block but not the G2 block of a cdc2-M26 ts strain. A mutant XlCDK2-R33, which is inactive as a kinase, failed to rescue the G1 block, suggesting that the protein kinase activity of CDK2 is required to enter the cell cycle in these circumstances. We designed screens to select mutants that would require XlCDK2 expression for viability, hoping to isolate new gene functions interacting with, or that could be replaced by, XlCDK2 in G1, or new cdc2 mutants altered solely in their G1 role. From these screens several cell cycle mutants were selected that were XlCDK2-dependent. These were all cdc2 mutants altered only in their G2/M function. Therefore XlCDK2 can influence both the G1/S and G2/M transition points of cdc2 in S. pombe.

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Choudhuri, Tathagata, Subhash C. Verma, Ke Lan, Masanao Murakami, and Erle S. Robertson. "The ATM/ATR Signaling Effector Chk2 Is Targeted by Epstein-Barr Virus Nuclear Antigen 3C To Release the G2/M Cell Cycle Block." Journal of Virology 81, no. 12 (April 4, 2007): 6718–30. http://dx.doi.org/10.1128/jvi.00053-07.

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ABSTRACT Epstein-Barr virus (EBV) infects most of the human population and persists in B lymphocytes for the lifetime of the host. The establishment of latent infection by EBV requires the expression of a unique repertoire of genes. The product of one of these viral genes, the EBV nuclear antigen 3C (EBNA3C), is essential for the growth transformation of primary B lymphocytes in vitro and can regulate the transcription of a number of viral and cellular genes important for the immortalization process. This study demonstrates an associated function of EBNA3C which involves the disruption of the G2/M cell cycle checkpoint. We show that EBNA3C-expressing lymphoblastoid cell lines treated with the drug nocodazole, which is known to block cells at the G2/M transition, did not show a G2/M-specific checkpoint arrest. Analyses of the cell cycles of cells expressing EBNA3C demonstrated that the expression of this essential EBV nuclear antigen is capable of releasing the G2/M checkpoint arrest induced by nocodazole. This G2/M arrest in response to nocodazole was also abolished by caffeine, suggesting an involvement of the ATM/ATR signaling pathway in the regulation of this cell cycle checkpoint. Importantly, we show that the direct interaction of EBNA3C with Chk2, the ATM/ATR signaling effector, is responsible for the release of this nocodazole-induced G2/M arrest and that this interaction leads to the serine 216 phosphorylation of Cdc25c, which is sequestered in the cytoplasm by 14-3-3. Overall, our data suggest that EBNA3C can directly regulate the G2/M component of the host cell cycle machinery, allowing for the release of the checkpoint block.

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Liu, Chang, Jing Nie, Rensheng Wang, and Weidong Mao. "The Cell Cycle G2/M Block Is an Indicator of Cellular Radiosensitivity." Dose-Response 17, no. 4 (October 1, 2019): 155932581989100. http://dx.doi.org/10.1177/1559325819891008.

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Background: Determination of the radiosensitivity of a specific tumor is essential to its precision tumor radiotherapy, but the measurement of cellular radiosensitivity with a routine colony forming assay is both labor- and time-consuming. An alternative option allowing rapid and precise prediction of radiosensitivity is necessary. Methods: In this study, we exposed 4 in vitro cultured cell lines to various doses of X-rays or carbon ions and then measured their radiosensitivities with a routine colony-forming assay, and monitored the kinetics of cell cycle distribution with routine propidium iodine staining and flow cytometry. Results: Based on the results, we correlated cellular radiosensitivity with a dynamic assay of cell cycle distribution, specifically, the negative correlation of cellular radiosensitivity with the accumulated G2/M arrested cells at 48 hours after exposure. The higher the proportion of accumulated G2/M arrested cells at 48 hours after exposure, the lower the radiosensitivity of the cell line, that is, the higher radioresistance of the cell line. Conclusion: These findings provide an optional application of regular cell cycle analysis for the prediction of tumor radiosensitivity.

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Kiang, Lee, Christian Heichinger, Stephen Watt, Jürg Bähler, and Paul Nurse. "Cyclin-Dependent Kinase Inhibits Reinitiation of a Normal S-Phase Program during G2 in Fission Yeast." Molecular and Cellular Biology 29, no. 15 (June 1, 2009): 4025–32. http://dx.doi.org/10.1128/mcb.00185-09.

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ABSTRACT To achieve faithful replication of the genome once in each cell cycle, reinitiation of S phase is prevented in G2 and origins are restricted from refiring within S phase. We have investigated the block to rereplication during G2 in fission yeast. The DNA synthesis that occurs when G2/M cyclin-dependent kinase (CDK) activity is depleted has been assumed to be repeated rounds of S phase without mitosis, but this has not been demonstrated to be the case. We show here that on G2/M CDK depletion in G2, repeated S phases are induced, which are correlated with normal G1/S transcription and attainment of doublings in cell size. Mostly normal mitotic S-phase origins are utilized, although at different efficiencies, and replication is essentially equal across the genome. We conclude that CDK inhibits reinitiation of S phase during G2, and if G2/M CDK is depleted, replication results from induction of a largely normal S-phase program with only small differences in origin usage and efficiency.

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Bolton, Diane L., and Michael J. Lenardo. "Vpr Cytopathicity Independent of G2/M Cell Cycle Arrest in Human Immunodeficiency Virus Type 1-Infected CD4+ T Cells." Journal of Virology 81, no. 17 (June 6, 2007): 8878–90. http://dx.doi.org/10.1128/jvi.00122-07.

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ABSTRACT The mechanism of CD4+ T-cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected individuals remains unknown, although mounting evidence suggests that direct viral cytopathicity contributes to this loss. The HIV-1 Vpr accessory protein causes cell death and arrests cells in the G2/M phase; however, the molecular mechanism underlying these properties is not clear. Mutation of hydrophobic residues on the surface of its third alpha-helix disrupted Vpr toxicity, G2/M arrest induction, nuclear localization, and self-association, implicating this region in multiple Vpr functions. Cytopathicity by virion-delivered mutant Vpr protein correlated with G2/M arrest induction but not nuclear localization or self-association. However, infection with whole virus encoding these Vpr mutants did not abrogate HIV-1-induced cell killing. Rather, mutant Vpr proteins that are impaired for G2/M block still prevented infected cell proliferation, and this property correlated with the death of infected cells. Chemical agents that inhibit infected cells from entering G2/M also did not reduce HIV-1 cytopathicity. Combined, these data implicate Vpr in HIV-1 killing through a mechanism involving inhibiting cell division but not necessarily in G2/M. Thus, the hydrophobic region of the third alpha-helix of Vpr is crucial for mediating G2/M arrest, nuclear localization, and self-association but dispensable for HIV-1 cytopathicity due to residual cell proliferation blockade mediated by a separate region of the protein.

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Bielaszewska, Martina, Bhanu Sinha, Thorsten Kuczius, and Helge Karch. "Cytolethal Distending Toxin from Shiga Toxin-Producing Escherichia coli O157 Causes Irreversible G2/M Arrest, Inhibition of Proliferation, and Death of Human Endothelial Cells." Infection and Immunity 73, no. 1 (January 2005): 552–62. http://dx.doi.org/10.1128/iai.73.1.552-562.2005.

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ABSTRACT Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing Escherichia coli (STEC) O157 and particular non-O157 serotypes. Here we investigated the biological effects of CDT-V from STEC O157:H− (strain 493/89) on human endothelial cells, which are believed to be major pathogenetic targets in severe STEC-mediated diseases. CDT-V caused dose-dependent G2/M cell cycle arrest leading to distension, inhibition of proliferation, and death in primary human umbilical vein endothelial cells (HUVEC) and two endothelial cell lines, EA.hy 926 cells (HUVEC derived) and human brain microvascular endothelial cells (HBMEC). The cell cycle effects of CDT-V were cell type specific. In HUVEC and EA.hy 926 cells, CDT-V caused a slowly developing but persistent G2/M block which resulted in delayed nonapoptotic cell death. In contrast, in HBMEC, CDT-V induced a rapidly evolving but transient G2/M block which was followed by progressive, mostly apoptotic cell death. In both HBMEC and EA.hy 926 cells, G2/M arrest was preceded by the early accumulation of a phosphorylated inactive form of cdc2 kinase. Significant G2/M arrest and inhibition of proliferation in both HUVEC and each of the endothelial cell lines were induced by 2 to 15 min of exposure to CDT-V, indicating that the effects of the toxin are irreversible. CDT-V-treated HBMEC and EA.hy 926 cells displayed fragmented nuclei and expressed phosphorylated histone protein H2AX, indicative of DNA damage followed by a DNA repair response. Our data demonstrate that CDT-V causes irreversible damage to human endothelial cells and thus may contribute to the pathogenesis of STEC-mediated diseases.

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Trielli, M. O., P. R. Andreassen, F. B. Lacroix, and R. L. Margolis. "Differential Taxol-dependent arrest of transformed and nontransformed cells in the G1 phase of the cell cycle, and specific-related mortality of transformed cells." Journal of Cell Biology 135, no. 3 (November 1, 1996): 689–700. http://dx.doi.org/10.1083/jcb.135.3.689.

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Taxol (paclitaxel) induces a microtubule hyperassembled state, and effectively blocks cells in mitosis. Here we report that Taxol also induces a stable late-G1 block in nontransformed REF-52 and WI-38 mammalian fibroblast cells, but not in T antigen-transformed cells of the same parental lineage. G1 arrest is characterized by partially dephosphorylated pRb, and inactive cdk2 kinase. Nontransformed cells recover normally from Taxol arrest. In contrast, T antigen transformed cells continue inappropriately past both G1 and G2-M in the presence of Taxol, and undergo a rapid death upon release. These results demonstrate a microtubule sensitive step in G1 regulation of nontransformed fibroblast cells. Also, Taxol selectively induces death of transformed cells, possibly because they slip the Taxol-dependent G1 arrest, as well as G2/M arrest, which are both specific to nontransformed cells.

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Whitehouse, Chris A., Paul B. Balbo, Everett C. Pesci, Daniel L. Cottle, Peter M. Mirabito, and Carol L. Pickett. "Campylobacter jejuni Cytolethal Distending Toxin Causes a G2-Phase Cell Cycle Block." Infection and Immunity 66, no. 5 (May 1, 1998): 1934–40. http://dx.doi.org/10.1128/iai.66.5.1934-1940.1998.

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ABSTRACT Cytolethal distending toxin (CDT) from the diarrheagenic bacteriumCampylobacter jejuni was shown to cause a rapid and specific cell cycle arrest in HeLa and Caco-2 cells. Within 24 h of treatment, CDT caused HeLa cells to arrest with a 4N DNA content, indicative of cells in G2 or early M phase. Immunofluorescence studies indicated that the arrested cells had not entered M phase, since no evidence of tubulin reorganization or chromatin condensation was visible. CDT treatment was also shown to cause HeLa cells to accumulate the inactive, tyrosine-phosphorylated form of CDC2. These results indicated that CDT treatment results in a failure to activate CDC2, which leads to cell cycle arrest in G2. This mechanism of action is novel for a bacterial toxin and provides a model for the generation of diarrheal disease byC. jejuni and other diarrheagenic bacteria that produce CDT.

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Muratori, M., I. Nicoletti, G. B. Vannelli, M. Luconi, E. Macorsini, M. Serio, G. Forti, and M. Maggi. "Genistein induces a G2/M block and apoptosis in human uterine adenocarcinoma cell lines." Endocrine Related Cancer 4, no. 2 (June 1, 1997): 203–18. http://dx.doi.org/10.1677/erc.0.0040203.

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Bujor, L., M. Quaia, and M. G. Trovò. "168 P Taxol as radiosensitizer in vitro: is the G2/M block a prerequisite?" European Journal of Cancer 32 (January 1996): S34. http://dx.doi.org/10.1016/0959-8049(96)84925-2.

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Allard, David, Michael Stoker, and Ermanno Gherardi. "A G2/M Cell Cycle Block in Transformed Cells by Contact with Normal Neighbours." Cell Cycle 2, no. 5 (September 12, 2003): 482–85. http://dx.doi.org/10.4161/cc.2.5.494.

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Jella, Ester Ruchama, Agus Suryanto, and Lilik Setyobudi. "DAMPAK APLIKASI MULSA DAN GENERASI UMBI BIBIT (G2, G3, LOKAL) PADA TANAMAN KENTANG (Solanum tuberosum LINN)." BUANA SAINS 17, no. 2 (January 4, 2018): 153. http://dx.doi.org/10.33366/bs.v17i2.815.

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Sub-optimal environment condition and the use of low-quality seed tubers is a constraint to increase the yield of potato tubers. The purpose of this study was to determine the impact of the various applications of mulch and seed tuber G2, G3, local on growth and yield of potato tubers, as well as to temperature and soil moisture. The research was conducted in June-December, 2013 in the highlands (2232.66 m asl) in Village Ranupani, District Senduro, Lumajang, East Java Province. A randomized block design was used with a combination of treatments: without mulch (tm), black plastic mulch silver (mphp), blue plastic mulch (mpb), Chromolaena odorata mulch (mCo) and the mother bulb G2, G3, local. There are 12 combinations of treatment was repeated 3 times. The combination of these treatments consist of: tm+G2, mphp+G2, mpb+G2, mCo+G2, tm+G3, mphp+G3, mpb+G3, mCo+G3, tm+local, mphp+local, mpb+local, mCo+local. Data were analyzed using the F test with a level of 5%. If a significantly different among treatment followed by LSD test 5%. The results showed the use mphp on local seed tubers are able to provide growth and potato tubers yield optimum is 641.76 g plant-1 ( 21.39 t ha-1) compared to other treatments, but the tuber yield did not differ significantly with the use mpb in seed tubers G2.

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Tien, Amy L., Sucharita Senbanerjee, Atul Kulkarni, Raksha Mudbhary, Bernadette Goudreau, Shridar Ganesan, Kirsten C. Sadler, and Chinweike Ukomadu. "UHRF1 depletion causes a G2/M arrest, activation of DNA damage response and apoptosis." Biochemical Journal 435, no. 1 (March 15, 2011): 175–85. http://dx.doi.org/10.1042/bj20100840.

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UHRF1 [ubiquitin-like protein, containing PHD (plant homeodomain) and RING finger domains 1] is required for cell cycle progression and epigenetic regulation. In the present study, we show that depleting cancer cells of UHRF1 causes activation of the DNA damage response pathway, cell cycle arrest in G2/M-phase and apoptosis dependent on caspase 8. The DNA damage response in cells depleted of UHRF1 is illustrated by: phosphorylation of histone H2AX on Ser139, phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr15. Moreover, we find that UHRF1 accumulates at sites of DNA damage suggesting that the cell cycle block in UHRF1-depleted cells is due to an important role in damage repair. The consequence of UHRF1 depletion is apoptosis; cells undergo activation of caspases 8 and 3, and depletion of caspase 8 prevents cell death induced by UHRF1 knockdown. Interestingly, the cell cycle block and apoptosis occurs in p53-containing and -deficient cells. From the present study we conclude that UHRF1 links epigenetic regulation with DNA replication.

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Ydenberg, Casey A., and Mark D. Rose. "Antagonistic regulation of Fus2p nuclear localization by pheromone signaling and the cell cycle." Journal of Cell Biology 184, no. 3 (February 2, 2009): 409–22. http://dx.doi.org/10.1083/jcb.200809066.

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When yeast cells sense mating pheromone, they undergo a characteristic response involving changes in transcription, cell cycle arrest in early G1, and polarization along the pheromone gradient. Cells in G2/M respond to pheromone at the transcriptional level but do not polarize or mate until G1. Fus2p, a key regulator of cell fusion, localizes to the tip of the mating projection during pheromone-induced G1 arrest. Although Fus2p was expressed in G2/M cells after pheromone induction, it accumulated in the nucleus until after cell division. As cells arrested in G1, Fus2p was exported from the nucleus and localized to the nascent tip. Phosphorylation of Fus2p by Fus3p was required for Fus2p export; cyclin/Cdc28p-dependent inhibition of Fus3p during late G1 through S phase was sufficient to block exit. However, during G2/M, when Fus3p was activated by pheromone signaling, Cdc28p activity again blocked Fus2p export. Our results indicate a novel mechanism by which pheromone-induced proteins are regulated during the transition from mitosis to conjugation.

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Maqoud, Fatima, Angela Curci, Rosa Scala, Alessandra Pannunzio, Federica Campanella, Mauro Coluccia, Giuseppe Passantino, Nicola Zizzo, and Domenico Tricarico. "Cell Cycle Regulation by Ca2+-Activated K+ (BK) Channels Modulators in SH-SY5Y Neuroblastoma Cells." International Journal of Molecular Sciences 19, no. 8 (August 18, 2018): 2442. http://dx.doi.org/10.3390/ijms19082442.

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The effects of Ca2+-activated K+ (BK) channel modulation by Paxilline (PAX) (10−7–10−4 M), Iberiotoxin (IbTX) (0.1–1 × 10−6 M) and Resveratrol (RESV) (1–2 × 10−4 M) on cell cycle and proliferation, AKT1pSer473 phosphorylation, cell diameter, and BK currents were investigated in SH-SY5Y cells using Operetta-high-content-Imaging-System, ELISA-assay, impedentiometric counting method and patch-clamp technique, respectively. IbTX (4 × 10−7 M), PAX (5 × 10−5 M) and RESV (10−4 M) caused a maximal decrease of the outward K+ current at +30 mV (Vm) of −38.3 ± 10%, −31.9 ± 9% and −43 ± 8%, respectively, which was not reversible following washout and cell depolarization. After 6h of incubation, the drugs concentration dependently reduced proliferation. A maximal reduction of cell proliferation, respectively of −60 ± 8% for RESV (2 × 10−4 M) (IC50 = 1.50 × 10−4 M), −65 ± 6% for IbTX (10−6 M) (IC50 = 5 × 10−7 M), −97 ± 6% for PAX (1 × 10−4 M) (IC50 = 1.06 × 10−5 M) and AKT1pser473 dephosphorylation was observed. PAX induced a G1/G2 accumulation and contraction of the S-phase, reducing the nuclear area and cell diameter. IbTX induced G1 contraction and G2 accumulation reducing diameter. RESV induced G2 accumulation and S contraction reducing diameter. These drugs share common actions leading to a block of the surface membrane BK channels with cell depolarization and calcium influx, AKT1pser473 dephosphorylation by calcium-dependent phosphatase, accumulation in the G2 phase, and a reduction of diameter and proliferation. In addition, the PAX action against nuclear membrane BK channels potentiates its antiproliferative effects with early apoptosis.

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Goh, Phuay-Yee, and Uttam Surana. "Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae." Molecular and Cellular Biology 19, no. 8 (August 1, 1999): 5512–22. http://dx.doi.org/10.1128/mcb.19.8.5512.

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ABSTRACT Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G1/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4(cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1cells. However, the previously characterized mutationcdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G2/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G2/M transition when released from hydroxyurea-induced S-phase arrest. A second function forCDC4 in late S or G2 phase was further confirmed by the observation that cells lacking the CDC4gene are arrested both at G1/S and at G2/M. We subsequently isolated additional temperature-sensitive mutations in theCDC4 gene (such as cdc4-12) that render the mutant defective in both G1/S and G2/M transitions at the restrictive temperature. While the G1/S block in both cdc4-12 and cdc4Δ mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G2/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.

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Saldivar, Joshua C., Stephan Hamperl, Michael J. Bocek, Mingyu Chung, Thomas E. Bass, Fernanda Cisneros-Soberanis, Kumiko Samejima, et al. "An intrinsic S/G2 checkpoint enforced by ATR." Science 361, no. 6404 (August 23, 2018): 806–10. http://dx.doi.org/10.1126/science.aap9346.

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The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)–directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.

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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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Knight, Gillian L., Andrew S. Turnell, and Sally Roberts. "Role for Wee1 in Inhibition of G2-to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins." Journal of Virology 80, no. 15 (August 1, 2006): 7416–26. http://dx.doi.org/10.1128/jvi.00196-06.

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ABSTRACT The infectious cycle of human papillomavirus type 1 (HPV1) is accompanied by abundant expression of the full-length E1^E4 protein (17-kDa) and smaller E4 polypeptides (16-, 11-, and 10-kDa) that arise by sequential loss of N-terminal E1^E4 sequences. HPV1 E4 inhibits G2-to-M transition of the cell cycle. Here, we show that HPV1 E4 proteins mediate inhibition of cell division by more than one mechanism. Cells arrested by coexpression of E1^E4 (E4-17K) and a truncated protein equivalent to the 16-kDa species (E4-16K) contain inactive cyclin B1-cdk1 complexes. Inactivation of cdk1 is through inhibitory Tyr15 phosphorylation, with cells containing elevated levels of Wee1, the kinase responsible for inhibitory cdk1 phosphorylation. Consistent with these findings, overexpression of Wee1 enhanced the extent to which E4-17K/16K-expressing cells arrest in G2, indicating that maintenance of Wee1 activity is necessary for inhibition of cell division induced by coexpression of the two E4 proteins. Moreover, we have determined that depletion of Wee1 by small interfering RNA (siRNA) alleviates the G2 block imposed by E4-17K/16K. In contrast however, maintenance of Wee1 activity is not necessary for G2-to-M inhibition mediated by E4-16K alone, as overexpression or depletion of Wee1 does not influence the G2 arrest function of E4-16K. Cells arrested by E4-16K expression contain low levels of active cyclin B1-cdk1 complexes. We hypothesize that differential expression of HPV1 E4 proteins during the viral life cycle determines the host cell cycle status. Different mechanisms of inhibition of G2-to-M transition reinforce the supposition that distinct E4 functions are important for HPV replication.

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Wade, Mark, and Martin J. Allday. "Epstein-Barr Virus Suppresses a G2/M Checkpoint Activated by Genotoxins." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1344–60. http://dx.doi.org/10.1128/mcb.20.4.1344-1360.2000.

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ABSTRACT Several Epstein-Barr virus (EBV)-negative Burkitt lymphoma-derived cell lines (for example, BL41 and Ramos) are extremely sensitive to genotoxic drugs despite being functionally null for the tumor suppressor p53. They rapidly undergo apoptosis, largely from G2/M of the cell cycle. 5-Bromo-2′-deoxyuridine labeling experiments showed that although the treated cells can pass through S phase, they are unable to complete cell division, suggesting that a G2/M checkpoint is activated. Surprisingly, latent infection of these genotoxin-sensitive cells with EBV protects them from both apoptosis and cell cycle arrest, allowing them to complete the division cycle. However, a comparison with EBV-immortalized B-lymphoblastoid cell lines (which have functional p53) showed that EBV does not block apoptosis per se but rather abrogates the activation of, or signalling from, the checkpoint in G2/M. Furthermore, analyses of BL41 and Ramos cells latently infected with P3HR1 mutant virus, which expresses only a subset of the latent viral genes, showed that LMP-1, the main antiapoptotic latent protein encoded by EBV, is not involved in the protection afforded here by viral infection. This conclusion was confirmed by analysis of clones of BL41 stably expressing LMP-1 from a transfected plasmid, which respond like the parental cell line. Although steady-state levels of Bcl-2 and related proteins varied between BL41 lines and clones, they did not change significantly during apoptosis, nor was the level of any of these anti- or proapoptotic proteins predictive of the outcome of treatment. We have demonstrated that a subset of EBV latent gene products can inactivate a cell cycle checkpoint for monitoring the fidelity and timing of cell division and therefore genomic integrity. This is likely to be important in EBV-associated growth transformation of B cells and perhaps tumorigenesis. Furthermore, this study suggests that EBV will be a unique tool for investigating the intimate relationship between cell cycle regulation and apoptosis.

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Verderio, Paolo, Paolo Bonetti, Miriam Colombo, Laura Pandolfi, and Davide Prosperi. "Intracellular Drug Release from Curcumin-Loaded PLGA Nanoparticles Induces G2/M Block in Breast Cancer Cells." Biomacromolecules 14, no. 3 (February 15, 2013): 672–82. http://dx.doi.org/10.1021/bm3017324.

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Falzano, Loredana, Perla Filippini, Sara Travaglione, Alessandro Giamboi Miraglia, Alessia Fabbri, and Carla Fiorentini. "Escherichia coli Cytotoxic Necrotizing Factor 1 Blocks Cell Cycle G2/M Transition in Uroepithelial Cells." Infection and Immunity 74, no. 7 (July 2006): 3765–72. http://dx.doi.org/10.1128/iai.01413-05.

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ABSTRACT Evidence is accumulating that a growing number of bacterial toxins act by modulating the eukaryotic cell cycle machinery. In this context, we provide evidence that a protein toxin named cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli is able to block cell cycle G2/M transition in the uroepithelial cell line T24. CNF1 permanently activates the small GTP-binding proteins of the Rho family that, beside controlling the actin cytoskeleton organization, also play a pivotal role in a large number of other cellular processes, including cell cycle regulation. The results reported here show that CNF1 is able to induce the accumulation of cells in the G2/M phase by sequestering cyclin B1 in the cytoplasm and down-regulating its expression. The possible role played by the Rho GTPases in the toxin-induced cell cycle deregulation has been investigated and discussed. The activity of CNF1 on cell cycle progression can offer a novel view of E. coli pathogenicity.

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Nasr-Esfahani, M. M., and M. H. Johnson. "The origin of reactive oxygen species in mouse embryos cultured in vitro." Development 113, no. 2 (October 1, 1991): 551–60. http://dx.doi.org/10.1242/dev.113.2.551.

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The increase in production of reactive oxygen species such as H2O2 at the G2/M phase of the second cell cycle may be related to the in vitro block to development of mouse 2-cell embryos. The occurrence of the H2O2 rise is independent of the activation of the embryonic genome and of passage through the S, G2 and M phases of the first cell cycle and G1 and M phases of the second cell cycle, but does require the activation of the unfertilized oocyte. The H2O2 is produced via dismutation of superoxide by the enzyme superoxide dismutase. Production of superoxide via mitochondrial, NADPH-oxidase and xanthine/xanthine oxidase systems has been investigated. The evidence suggests that superoxide, and thereby H2O2, is produced by the xanthine/xanthine oxidase system, but an involvement of the other superoxide generating systems has not been excluded. The relation between H2O2 and development in vitro is discussed.

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Altznauer, Frank, Sibylla Martinelli, Shida Yousefi, Christine Thürig, Inès Schmid, Edward M. Conway, Martin H. Schöni, et al. "Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils." Journal of Experimental Medicine 199, no. 10 (May 17, 2004): 1343–54. http://dx.doi.org/10.1084/jem.20032033.

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Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.

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Dove, Brian, Gavin Brooks, Katrina Bicknell, Torsten Wurm, and Julian A. Hiscox. "Cell Cycle Perturbations Induced by Infection with the Coronavirus Infectious Bronchitis Virus and Their Effect on Virus Replication." Journal of Virology 80, no. 8 (April 15, 2006): 4147–56. http://dx.doi.org/10.1128/jvi.80.8.4147-4156.2006.

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ABSTRACT In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.

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Misteli, T., and G. Warren. "Mitotic disassembly of the Golgi apparatus in vivo." Journal of Cell Science 108, no. 7 (July 1, 1995): 2715–27. http://dx.doi.org/10.1242/jcs.108.7.2715.

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Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative conventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50–70 nm in diameter, accumulated in the vicinity of the stacks. The disappearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disassembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.

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Hart, Bruce, and John Schlager. "Okadaic Acid and Calyculin A Reverse Sulfur Mustard-Induced G2/M Cell-cycle Block in Human Keratinocytes." International Journal of Toxicology 15 (1996): 36–42. http://dx.doi.org/10.3109/10915819609048336.

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Harrison, Lawrence E., Qing Mei Wang, and George P. Studzinski. "Butyrate-Induced G2/M Block in Caco-2 Colon Cancer Cells is Associated with Decreased p34cdc2 Activity." Proceedings of the Society for Experimental Biology and Medicine 222, no. 2 (October 1999): 150–56. http://dx.doi.org/10.1046/j.1525-1373.1999.d01-125.x.

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Orchard, Craig B., Ilario Siciliano, David A. Sorrell, Angela Marchbank, Hilary J. Rogers, Dennis Francis, Robert J. Herbert, et al. "Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle." Plant Journal 44, no. 2 (September 20, 2005): 290–99. http://dx.doi.org/10.1111/j.1365-313x.2005.02524.x.

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Lu, K. P., S. A. Osmani, A. H. Osmani, and A. R. Means. "Essential roles for calcium and calmodulin in G2/M progression in Aspergillus nidulans." Journal of Cell Biology 121, no. 3 (May 1, 1993): 621–30. http://dx.doi.org/10.1083/jcb.121.3.621.

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nimT encodes a protein in Aspergillus nidulans that is required for tyrosine dephosphorylation of p34cdc2 and has a strong homology to cdc25-type proteins. Conditional mutation of nimT (nimT23 mutation) arrests cells in G2 at the restrictive temperature. After release of the temperature-sensitive nimT23 block, p34cdc2 undergoes tyrosine dephosphorylation and we showed that as cells entered mitosis, a rapid increase in calmodulin was observed. The increase in calmodulin and progression into mitosis were prevented by reducing extracellular Ca2+ levels to 2 nM. The calmodulin gene of a nimT23-containing strain was replaced with a hybrid gene in which calmodulin transcription was regulated by the alcA promoter (AlcCaM/T23). This allowed experimental manipulation of the level of intracellular calmodulin by the carbon source in the medium. When either extracellular Ca2+ or intracellular calmodulin levels were reduced at the nimT23 G2 arrest point, p34cdc2 remained tyrosine phosphorylated but the mitotic NIMA kinase encoded by nimA was not activated. Release of the temperature sensitive nimT23 arrest when either extracellular Ca2+ or calmodulin concentrations were low blocked tyrosine dephosphorylation of p34cdc2, activation of NIMA and progression of cells into mitosis. However, reduced levels of either Ca2+ or calmodulin had no effect on the increase in histone H1 kinase activity associated with p13 beads or the degree of phosphorylation of the majority of MPM-2-reacting proteins following release of the nimT23 mutation. These results demonstrate that both Ca2+ and calmodulin are important for progression into mitosis from the nimT23 arrest point in a pathway involving activation of both NIMA and p34cdc2 protein kinases.

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Uhal, B. D., and D. E. Rannels. "DNA distribution analysis of type II pneumocytes by laser flow cytometry: technical considerations." American Journal of Physiology-Lung Cellular and Molecular Physiology 261, no. 4 (October 1, 1991): L296—L306. http://dx.doi.org/10.1152/ajplung.1991.261.4.l296.

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Optimal conditions were established for determination of cell cycle phase fractions of freshly isolated or cultured adult rat type II pneumocytes (T2P). Propidium iodide staining of ethanol-fixed cells treated with ribonuclease (RNase) consistently yielded histograms with low coefficients of variation. Contaminating cells and cell clumps were eliminated during data acquisition through electronic gating based on anti-vimentin immunofluorescence and peak red fluorescence, respectively. Failure to delete contaminants, clumps or RNA resulted in overestimation of S or G2/M phase fractions by as much as 20-fold. When T2P were cultured on plastic at an initial density of 2.5 x 10(5)/cm2, the S phase fraction did not change over a culture interval in which thymidine incorporation rates increased almost 10-fold. In contrast, a significant increase in the G2/M phase fraction by day 2 of culture occurred with no significant increase in cell number. These results support the hypothesis that adult rat T2P, when subjected to customary conditions of primary culture, undergo cell cycle block in G2/M phases. The data also indicate that under these in vitro conditions, net thymidine incorporation by T2P may vary independently of the S phase fraction. The methods described in this report address basic considerations crucial to future applications of flow cytokinetics to the study of T2P proliferation and differentiation.

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Boschelli, F. "Expression of p60v-src in Saccharomyces cerevisiae results in elevation of p34CDC28 kinase activity and release of the dependence of DNA replication on mitosis." Molecular and Cellular Biology 13, no. 8 (August 1993): 5112–21. http://dx.doi.org/10.1128/mcb.13.8.5112.

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Expression of the oncogenic protein tyrosine kinase p60v-src in the yeast Saccharomyces cerevisiae has been shown to result in rapid cell death (J. S. Brugge, G. Jarosik, J. Andersen, A. Queral-Lustig, M. Fedor-Chaiken, and J. R. Broach, Mol. Cell. Biol. 7:2180-2187, 1987). Work described here demonstrates that v-Src expression results in accumulation of large-budded cells and a nuclear division block without blocking cytokinesis. Flow-cytometric analysis indicates that the DNA content of these cells is elevated beyond the G2 DNA content, and genetic studies indicate that v-Src expression causes aneuploidy. The activity of Cdc28 kinase, which controls the G1/S and G2/M transitions in S. cerevisiae, increases during galactose induction in a Src+ strain but not in an isogenic Src- strain. These observations indicate that v-Src expression disrupts p34CDC28 kinase regulation, allowing DNA replication to proceed in the absence of a prior mitotic event.

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Boschelli, F. "Expression of p60v-src in Saccharomyces cerevisiae results in elevation of p34CDC28 kinase activity and release of the dependence of DNA replication on mitosis." Molecular and Cellular Biology 13, no. 8 (August 1993): 5112–21. http://dx.doi.org/10.1128/mcb.13.8.5112-5121.1993.

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Expression of the oncogenic protein tyrosine kinase p60v-src in the yeast Saccharomyces cerevisiae has been shown to result in rapid cell death (J. S. Brugge, G. Jarosik, J. Andersen, A. Queral-Lustig, M. Fedor-Chaiken, and J. R. Broach, Mol. Cell. Biol. 7:2180-2187, 1987). Work described here demonstrates that v-Src expression results in accumulation of large-budded cells and a nuclear division block without blocking cytokinesis. Flow-cytometric analysis indicates that the DNA content of these cells is elevated beyond the G2 DNA content, and genetic studies indicate that v-Src expression causes aneuploidy. The activity of Cdc28 kinase, which controls the G1/S and G2/M transitions in S. cerevisiae, increases during galactose induction in a Src+ strain but not in an isogenic Src- strain. These observations indicate that v-Src expression disrupts p34CDC28 kinase regulation, allowing DNA replication to proceed in the absence of a prior mitotic event.

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Mashimo, K. "FLOW CYTOMETRIC AND FLUORESCENCE MICROSCOPIC ANALYSIS OF ETHANOL-INDUCED G2+M BLOCK: ETHANOL DOSE-DEPENDENTLY DELAYS THE PROGRESSION OF THE M PHASE." Alcohol and Alcoholism 34, no. 3 (May 1, 1999): 300–310. http://dx.doi.org/10.1093/alcalc/34.3.300.

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36

Hoffman, Robert M., Chengyu Wu, Shuya Yano, and Lei Zhang. "Comparison of traditional Chinese medicine (TCM) herbal mixture LQ and paclitaxel on the phase of cancer cell cycle blockage and cancer cell invasion." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e22014-e22014. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22014.

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e22014 Background: Although Traditional Chinese Medicine (TCM) has been used to treat cancer for thousands of years, the mechanisms of action are either poorly understood or unknown. In the present report, we use state-of-the-art technology to investigate the anti-cancer mechanism of the TCM herbal mixture LQ. Methods: Fluorescence ubiquitination-based cell cycle indicator (FUCCI) was used to monitor cell cycle arrest HeLa cells after LQ treatment. FUCCI-HeLa cells were cultured in two dimensional (2D) monolayer, Matrigel and 3D Gelfoam. Changes of cell cycle status were observed using the Olympus FV1000 confocal imaging system whereby cycling cells fluoresce green and quiescent cells red. Paclitaxel (Taxol) was used as the positive control. Results: Paclitaxel induced a G2/M cell cycle block. In contrast, LQ blocked FUCCI-HeLa cells in the G0/G1 phase of the cell cycle in all 3 culture models. In monolayer culture, the paclitaxel positive control had approximately 45% of the cells in G2/M phase. In contrast, the LQ-treated cells were mostly in the G0/G1 phase (>90%). In Matrigel culture, HeLa cells formed spheres. The spheres in the paclitaxel control group had 40% of the cells in G2/M phase, but only 15% in LQ-treated cultures. In 3D Gelfoam culture, cells grew along the structures of the Gelform. The paclitaxel positive control culture had approximately 45% of cells in G2/M phase. In contrast, the cells in LQ-treated culture were mostly in G0/G1 phase (>80%). The cells in pacilitaxel control group invaded to 250~300 µm deep in the Gelfoam, but only 150~200 µm deep in LQ-treated culture. Conclusions: The anti-cancer mechanism of TCM herbal mixture LQ involves cancer cell cycle arrest at G0/G1 and inhibition of cancer cell invasion.

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LEE, Joanna Y., Lisa G. LEONHARDT, and Lina M. OBEID. "Cell-cycle-dependent changes in ceramide levels preceding retinoblastoma protein dephosphorylation in G2/M." Biochemical Journal 334, no. 2 (September 1, 1998): 457–61. http://dx.doi.org/10.1042/bj3340457.

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Ceramide functions as a growth-inhibitory lipid-signalling molecule and might have a role in mediating the effects of extracellular agents on cell growth, differentiation and senescence. Here we investigate the roles of ceramide in cell cycle progression. With the use of the model of serum withdrawal, we were able to synchronize Wi-38 human diploid fibroblasts at different stages of cell cycle. Serum stimulation resulted in G0 to G1/S progression as determined by flow cytometric analysis and [3H]thymidine incorporation. Analyses of endogenous ceramide levels demonstrated that ceramide levels remained relatively constant on serum stimulation, indicating that ceramide might not be critical during G1/S transition. Treating exponentially growing Wi-38 human diploid fibroblasts with nocodazole led to cell cycle arrest at the G2/M phase of the cell cycle; 2 h after the removal of nocodazole, retinoblastoma (Rb) protein became dephosphorylated and the cells exited from G2/M and moved to the G1 phase of the new cycle. When cells were released from G2/M block by nocodazole, and before Rb protein dephosphorylation, endogenous ceramide levels transiently increased up to 2-fold at 0.5 h after the removal of nocodazole. Fumonisin B1, an inhibitor of ceramide synthase, inhibited the elevation of ceramide levels. Desipramine and SR33557, both acid sphingomyelinase inhibitors, did not have an appreciable effect on the elevation of ceramide levels. Furthermore, fumonisin B1 inhibited Rb protein dephosphorylation induced by endogenous ceramide but not by exogenous ceramide. These results demonstrate for the first time changes in ceramide during cell cycle progression and suggest that ceramide synthesized de novo might function as an endogenous modulator of Rb protein and cell cycle progression.

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Muñoz, Purificación, Malgorzata Z. Zdzienicka, Jean-Marie Blanchard, and Jacques Piette. "Hypersensitivity of Ku-Deficient Cells toward the DNA Topoisomerase II Inhibitor ICRF-193 Suggests a Novel Role for Ku Antigen during the G2 and M Phases of the Cell Cycle." Molecular and Cellular Biology 18, no. 10 (October 1, 1998): 5797–808. http://dx.doi.org/10.1128/mcb.18.10.5797.

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ABSTRACT Ku antigen is a heterodimer, comprised of 86- and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G2 at drug doses which had no effect on wild-type cells. Moreover, bypass of this G2 block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase IIα or -β. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G2/M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku antigen in the G2 and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.

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Giuliano, Kenneth A., Yih-Tai Chen, and D. Lansing Taylor. "High-Content Screening with siRNA Optimizes a Cell Biological Approach to Drug Discovery: Defining the Role of P53 Activation in the Cellular Response to Anticancer Drugs." Journal of Biomolecular Screening 9, no. 7 (October 2004): 557–68. http://dx.doi.org/10.1177/1087057104265387.

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Deciphering the effects of compounds on molecular events within living cells is becoming an increasingly important component of drug discovery. In a model application of the industrial drug discovery process, the authors profiled a panel of 22 compounds using hierarchical cluster analysis of multiparameter high-content screening measurements from nearly 500,000 cells per microplate. RNAi protein knockdown methodology was used with high-content screening to dissect the effects of 2 anticancer drugs on multiple target activities. Camptothecin activated p53 in A549 lung carcinoma cells pretreated with scrambled siRNA, exhibited concentration-dependent cell cycle blocks, and induced moderate microtubule stabilization. Knockdown of camptothecin-induced p53 protein expression with p53 siRNA inhibited the G1/S blocking activity of the drug and diminished its microtubule-stabilizing activity. Paclitaxel activated p53 protein at low concentrations but exhibited G2/M cell cycle blocking activity at higher concentrations where microtubules were stabilized. In cells treated with p53 siRNA, paclitaxel failed to activate p53 protein, but the knockdown did not have a significant effect on the ability of paclitaxel to stabilize microtubules or induce a G2/M cell cycle block. Thus, this model application of the use of RNAi technology within the context of high-content screening shows the potential to provide massive amounts of combinatorial cell biological information on the temporal and spatial responses that cells mount to treatment by promising therapeutic candidates.

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Berger, Gregory, Madeleine Lawrence, Stephane Hué, and Stuart J. D. Neil. "G2/M Cell Cycle Arrest Correlates with Primate Lentiviral Vpr Interaction with the SLX4 Complex." Journal of Virology 89, no. 1 (October 15, 2014): 230–40. http://dx.doi.org/10.1128/jvi.02307-14.

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ABSTRACTThe accessory genevpr, common to all primate lentiviruses, induces potent G2/M arrest in cycling cells. A recent study showed that human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of simian immunodeficiency virus (SIV) Vpr proteins. We show that SIV Vpr proteins differ in their ability to promote cell cycle arrest in human cells. While this is dependent on the DCAF1/DDB1/CUL4 ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with human SLX4 (huSLX4) and could be abolished by small interfering RNA (siRNA) depletion of any member of the SLX4 complex. In contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in grivet cells. Correspondingly, here cell cycle arrest correlated with interaction with the grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins and that the ability to dysregulate the Fanconi anemia DNA repair pathway is an essential function of Vprin vivo.IMPORTANCEThe Vpr protein of HIV-1 and related viruses is essential for the virusin vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data have shown that HIV-1 Vpr targets the Fanconi anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA cross-links. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. The results of these studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins and that the ability to dysregulate members of the Fanconi anemia DNA repair pathway is essential for HIV/SIV replicationin vivo.

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Mack, D., B. Kluxen, and J. Kruppa. "Accessibility to proteases of the cytoplasmic G protein domain of vesicular stomatitis virus is increased during intracellular transport." Journal of Cell Biology 109, no. 5 (November 1, 1989): 2057–65. http://dx.doi.org/10.1083/jcb.109.5.2057.

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G1 and G2 are two forms of the membrane-integrated G protein of vesicular stomatitis virus that migrate differently in gel electrophoresis because G1 is modified by high-mannose and G2 by complex-type oligosaccharide side chains. The cytoplasmic domain in G1 is less exposed to cleavage by several proteases than in G2 molecules. Acylation by palmitic acid as well as inhibition of carbohydrate processing by swainsonine and deoxynojirimycin resulted in the same pattern of proteolytic sensitivity of both glycoproteins as in untreated cells. In contrast, accessibility of the cytoplasmic domain to proteases did not change when the intracellular transport of the G protein was blocked in carbonyl cyanide m-chlorophenylhydrazone- or monensin-treated BHK-21 cells, respectively. The results suggest that the increase in accessibility of the cytoplasmic tail of the G protein occurs after the monensin block in the trans-Golgi and might reflect a conformational change of functional significance--i.e., making the cytoplasmic domain of the viral spike protein competent for its interaction with the viral core, inducing thereby the formation of the budding virus particle.

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Wang, Suiquan, Youhong Zhang, Joseph R. Biggs, Dong-Er Zhang, and Andrew S. Kraft. "Phosphorylation of AML1 (RUNX1) Controls Transcription, Nuclear Localization, and Is Regulated during DNA Damage." Blood 106, no. 11 (November 16, 2005): 2712. http://dx.doi.org/10.1182/blood.v106.11.2712.2712.

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Abstract We demonstrate that AML1 (RUNX1) is phosphorylated on S276, S293, T300, S303 and S462 after phorbol ester treatment in K562 cells, and these phosphorylations control the transcriptional activity of this protein and regulate varied promoters including TCRb, GM-CSF, M-CSF receptor, and myeloperoxidase gene (JBC279: 53116–53125, 2004). In addition, we find that phosphorylation of the AML1 protein controls the binding of AML1 to the nuclear matrix, and regulates its degradation (MCR3: 391–401, 2005). We have now derived rabbit phosphopeptide antisera to S276, S303, and S462 in the AML1 protein. Using these antibodies, we find that in unsynchronized Jurkat T cells AML1 contain low levels of phosphorylation at all three sites. To examine whether this phosphoryaltion is cell cycle regulated, we blocked cells in G1/S with aphidicolin or in G2/M with nocodazole. In contrast to G1/S block that had no effect on phosphorylation, nocodazole treatment induces marked increases in phosphorylation of S276, S303, and S462. The nocodazole increase in phosphorylation was markedly inhibited by staurosporine, a PKC inhibitor, and to a lesser extent by the JNK inhibitor SP600125, and the p38 inhibitor, SB202190. Using elutriation to examine untreated Jurkat cells, we demonstrate increases in the level of AML1 protein as cells progress through the cell cycle to G2/M. However, the actual level of phosphorylated protein as judged by phospho303 and 462 antibodies decreases. Nocodazole treatment of Jurkat cells not only induces G2/M arrest, but also induces apoptosis. We are currently investigating the hypothesis that DNA damage causes phosphorylation of AML1 that regulates both its activity and location.

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43

Carrasco-Garcia, Estefania, Isabel Martinez-Lacaci, Leticia Mayor-López, Elena Tristante, Mar Carballo-Santana, Pilar García-Morales, Maria Ventero Martin, Maria Fuentes-Baile, Álvaro Rodriguez-Lescure, and Miguel Saceda. "PDGFR and IGF-1R Inhibitors Induce a G2/M Arrest and Subsequent Cell Death in Human Glioblastoma Cell Lines." Cells 7, no. 9 (September 6, 2018): 131. http://dx.doi.org/10.3390/cells7090131.

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Glioblastomas are highly resistant to radiation and chemotherapy. Currently, there are no effective therapies for this type of tumor. Signaling mechanisms initiated by PDGFR and IGF-1R are important in glioblastoma, and inhibition of the signal transduction pathways initiated by these receptors could be a useful alternative strategy for glioblastoma treatment. We have studied the effects of the PDGFR inhibitor JNJ-10198409 (JNJ) and the IGF-1R inhibitor picropodophyllin (PPP) in glioblastoma cell lines as well as in primary cultures derived from patients affected by this type of tumor. JNJ and PPP treatment blocked PDGFR and IGF-1R signaling respectively and reduced Akt and Erk 1/2 phosphorylation. Both inhibitors diminished cell proliferation, inducing a G2/M block of the cell cycle. Cell death induced by JNJ was caspase-dependent, Annexin-V positive and caused PARP cleavage, especially in T98 cells, suggesting an apoptotic mechanism. However, cell death induced by PPP was not completely inhibited by caspase inhibitors in all cell lines apart from LN-229 cells, indicating a caspase-independent mechanism. Several inhibitors targeted against different cell death pathways could not block this caspase-independent component, which may be a non-programmed necrotic mechanism. Apoptotic arrays performed in T98 and LN-229 cells upon JNJ and PPP treatment revealed that procaspase 3 levels were augmented by both drugs in T98 cells and only by JNJ in LN229-cells. Furthermore, XIAP and survivin levels were much higher in LN-229 cells than in T98 cells, revealing that LN-229 cells are more susceptible to undergo caspase-independent cell death mechanisms. JNJ and PPP combination was more effective than each treatment alone.

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44

Shinomiya, Nariyoshi, Toshiya Takemura, Kazutsugu Iwamoto, and Makoto Rokutanda. "Caffeine induces S-phase apoptosis in cis-diamminedichloroplatinum-treated cells, whereas cis-diamminedichloroplatinum induces a block in G2/M." Cytometry 27, no. 4 (April 1, 1997): 365–73. http://dx.doi.org/10.1002/(sici)1097-0320(19970401)27:4<365::aid-cyto8>3.0.co;2-b.

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45

García-Luis, Jonay, and Félix Machín. "Fanconi Anaemia-Like Mph1 Helicase Backs up Rad54 and Rad5 to Circumvent Replication Stress-Driven Chromosome Bridges." Genes 9, no. 11 (November 17, 2018): 558. http://dx.doi.org/10.3390/genes9110558.

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Homologous recombination (HR) is a preferred mechanism to deal with DNA replication impairments. However, HR synapsis gives rise to joint molecules (JMs) between the nascent sister chromatids, challenging chromosome segregation in anaphase. Joint molecules are resolved by the actions of several structure-selective endonucleases (SSEs), helicases and topoisomerases. Previously, we showed that yeast double mutants for the Mus81-Mms4 and Yen1 SSEs lead to anaphase bridges (ABs) after replication stress. Here, we have studied the role of the Mph1 helicase in preventing these anaphase aberrations. Mph1, the yeast ortholog of Fanconi anaemia protein M (FANCM), is involved in the removal of the D-loop, the first JM to arise in canonical HR. Surprisingly, the absence of Mph1 alone did not increase ABs; rather, it blocked cells in G2. Interestingly, in the search for genetic interactions with functionally related helicases and translocases, we found additive effects on the G2 block and post-G2 aberrations between mph1Δ and knockout mutants for Srs2, Rad54 and Rad5. Based on these interactions, we suggest that Mph1 acts coordinately with these helicases in the non-canonical HR-driven fork regression mechanism to bypass stalled replication forks.

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46

Sikorski, R. S., W. A. Michaud, and P. Hieter. "p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains." Molecular and Cellular Biology 13, no. 2 (February 1993): 1212–21. http://dx.doi.org/10.1128/mcb.13.2.1212.

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CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

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47

Sikorski, R. S., W. A. Michaud, and P. Hieter. "p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains." Molecular and Cellular Biology 13, no. 2 (February 1993): 1212–21. http://dx.doi.org/10.1128/mcb.13.2.1212-1221.1993.

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CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

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48

Patel, Rajnikant, Elizabeth M. Wright, and Michael Whitaker. "Caffeine overrides the S-phase cell cycle block in sea urchin embryos." Zygote 5, no. 2 (May 1997): 127–38. http://dx.doi.org/10.1017/s0967199400003804.

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SummaryDuring the early mitotic cell cycles of the sea urchin embryo, the cell oscillates between S-phase and M-phase. In the presence of aphidicolin, a DNA synthesis inhibitor, a checkpoint control blocks the activation of the p34cdc2 protein kinase, by keeping it in the inactive, tyrosine phosphorylated form, and the embryos do not enter mitosis. Caffeine has been shown to bypass the G2/M-phase checkpoint in mammalian cells and in cycling Xenopus extracts and to induce mitosis despite the presence of damaged or unreplicated DNA. In this study we show that caffeine also induces mitosis and cell division in sea urchin embryos, in the presence of unreplicated DNA, by stimulating the tyrosine dephosphorylation of p34cdc2 and switching on its protein kinase activity. We also show that the caffeine-induced activation of the p34cdc2 protein kinase is not mediated by either of the two second messengers, calcium and cAMP, or by inhibition of the p34cdc2 tyrosine kinase. Thus, none of the mechanisms proposed for caffeine's action can explain how it overrides the S-phase checkpoint in the early cell cycles of the sea urchin embryo.

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Binder, Anke B., Antonio M. Serafin, and Lothar J. F. Bohm. "Abrogation of G2/M-Phase Block Enhances the Cytotoxicity of Daunorubicin, Melphalan and Cisplatin in TP53 Mutant Human Tumor Cells." Radiation Research 154, no. 6 (December 2000): 640–49. http://dx.doi.org/10.1667/0033-7587(2000)154[0640:aogmpb]2.0.co;2.

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50

Deplanque, Ga�l, Jocelyn C�raline, Marcia Chia-Miao Mah-Becherel, Jean-Pierre Cazenave, Jean-Pierre Bergerat, and Claudine Klein-Soyer. "Caffeine and the G2/M block override: A concept resulting from a misleading cell kinetic delay, independent of functional p53." International Journal of Cancer 94, no. 3 (2001): 363–69. http://dx.doi.org/10.1002/ijc.1478.

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Journal articles on the topic 'G2/M block'

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