Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.
ADVANCED SEARCH
Log in
Top
Breast Cancer Research
Published in: Breast Cancer Research 2/2012

Open Access 01-04-2012 | Research article

RAC1 GTPase plays an important role in γ-irradiation induced G2/M checkpoint activation

Authors: Ying Yan, Patrick M Greer, Phu T Cao, Ryan H Kolb, Kenneth H Cowan

Published in: Breast Cancer Research | Issue 2/2012

Login to get access

Abstract

Introduction

In response to gamma-irradiation (IR)-induced double-strand DNA breaks, cells undergo cell-cycle arrest, allowing time for DNA repair before reentering the cell cycle. G2/M checkpoint activation involves activation of ataxia telangiectasia mutated (ATM)/ATM- and rad3-related (ATR) kinases and inhibition of Cdc25 phosphatases, resulting in inhibition of Cdc2 kinase and subsequent G2/M cell-cycle arrest. Previous studies from our laboratory showed that the G2/M checkpoint activation after IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. In the present studies, we investigated the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) guanosine triphosphatase (GTPase) in IR-induced G2/M checkpoint response and ERK1/2 activation, as well as in cell survival after IR.

Methods

With Rac1-specific inhibitor, dominant negative mutant Rac1 (N17Rac1) and specific small interfering RNA, the effect of Rac1 on IR-induced G2/M checkpoint response and ERK1/2 activation was examined in human breast cancer cells. In addition, the effect of Rac1 on cell survival after irradiation was assessed by using Rac1-specific inhibitor.

Results

IR exposure of MCF-7 breast cancer cells was associated with a marked activation of Rac1 GTPase. Furthermore, inhibition of Rac1 by using specific inhibitor, dominant-negative Rac1 mutant, or specific siRNA resulted in attenuation of IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1, and Chk2 kinases, as well as phosphorylation of Cdc2-Tyr15. Moreover, Rac1 inhibition or decreased Rac1 expression also abrogated IR-induced phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) and ERK1/2. Ultimately, inhibition of Rac1 markedly increased cellular sensitivity to IR exposure, which involves induction of apoptosis.

Conclusion

Studies in this report suggest that Rac1 GTPase plays an essential role in the activation of IR-induced ERK1/2 signaling and subsequent G2/M checkpoint response. Furthermore, results also support a role for Rac1 in promoting cell survival after irradiation treatment.
Appendix
Available only for authorised users
Literature
1.
Motoyama N, Naka K: DNA damage tumor suppressor genes and genomic instability. Curr Opin Genet Dev. 2004, 14: 11-10.1016/j.gde.2003.12.003.CrossRefPubMed
2.
Iliakis G, Wang Y, Guan J, Wang H: DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene. 2003, 22: 5834-5847. 10.1038/sj.onc.1206682.CrossRefPubMed
3.
4.
Rhind N, Furnari B, Russell P: Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast. Genes Dev. 1997, 11: 504-511. 10.1101/gad.11.4.504.CrossRefPubMed
5.
Kharbanda S, Saleem A, Datta R, Yuan ZM, Weichselbaum R, Kufe D: Ionizing radiation induces rapid tyrosine phosphorylation of p34cdc2. Cancer Res. 1994, 54: 1412-1414.PubMed
6.
O'Connell MJ, Raleigh JM, Verkade HM, Nurse P: Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J. 1997, 16: 545-554. 10.1093/emboj/16.3.545.CrossRefPubMedPubMedCentral
7.
Lundgren K, Walworth N, Booher R, Dembski M, Kirschner M, Beach D: mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell. 1991, 64: 1111-1122. 10.1016/0092-8674(91)90266-2.CrossRefPubMed
8.
Parker LL, Atherton-Fessler S, Piwnica-Worms H: p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15. Proc Natl Acad Sci USA. 1992, 89: 2917-2921. 10.1073/pnas.89.7.2917.CrossRefPubMedPubMedCentral
9.
Bulavin DV, Higashimoto Y, Demidenko ZN, Meek S, Graves P, Phillips C, Zhao H, Moody SA, Appella E, Piwnica-Worms H, Fornace AJ: Dual phosphorylation controls Cdc25 phosphatases and mitotic entry. Nat Cell Biol. 2003, 5: 545-551. 10.1038/ncb994.CrossRefPubMed
10.
Kastan MB, Bartek J: Cell-cycle checkpoints and cancer. Nature. 2004, 432: 316-323. 10.1038/nature03097.CrossRefPubMed
11.
Prigent C, Dimitrov S: Phosphorylation of serine 10 in histone H3, what for?. J Cell Sci. 2003, 116: 3677-3685. 10.1242/jcs.00735.CrossRefPubMed
12.
Xu B, Kastan MB: Analyzing cell cycle checkpoints after ionizing radiation. Methods Mol Biol. 2004, 281: 283-292.PubMed
13.
Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD: Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma. 1997, 106: 348-360. 10.1007/s004120050256.CrossRefPubMed
14.
Sauve DM, Anderson HJ, Ray JM, James WM, Roberge M: Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation. J Cell Biol. 1999, 145: 225-235. 10.1083/jcb.145.2.225.CrossRefPubMedPubMedCentral
15.
Xu B, Kim ST, Lim DS, Kastan MB: Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol. 2002, 22: 1049-1059. 10.1128/MCB.22.4.1049-1059.2002.CrossRefPubMedPubMedCentral
16.
Yan Y, Black CP, Cowan KH: Irradiation-induced G2/M checkpoint response requires ERK1/2 activation. Oncogene. 2007, 26: 4689-4698. 10.1038/sj.onc.1210268.CrossRefPubMed
17.
Dent P, Yacoub A, Fisher PB, Hagan MP, Grant S: MAPK pathways in radiation responses. Oncogene. 2003, 22: 5885-5896. 10.1038/sj.onc.1206701.CrossRefPubMed
18.
Cui W, Yazlovitskaya EM, Mayo MS, Pelling JC, Persons DL: Cisplatin-induced response of c-jun N-terminal kinase 1 and extracellular signal-regulated protein kinases 1 and 2 in a series of cisplatin-resistant ovarian carcinoma cell lines. Mol Carcinog. 2000, 29: 219-228. 10.1002/1098-2744(200012)29:4<219::AID-MC1004>3.0.CO;2-D.CrossRefPubMed
19.
Wang X, McGowan CH, Zhao M, He L, Downey JS, Fearns C, Wang Y, Huang S, Han J: Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest. Mol Cell Biol. 2000, 20: 4543-4552. 10.1128/MCB.20.13.4543-4552.2000.CrossRefPubMedPubMedCentral
20.
Abbott DW, Holt JT: Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation. J Biol Chem. 1999, 274: 2732-2742. 10.1074/jbc.274.5.2732.CrossRefPubMed
21.
Tang D, Wu D, Hirao A, Lahti JM, Liu L, Mazza B, Kidd VJ, Mak TW, Ingram AJ: ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. J Biol Chem. 2002, 277: 12710-12717. 10.1074/jbc.M111598200.CrossRefPubMed
22.
Bosco EE, Mulloy JC, Zheng Y: Rac1 GTPase: a "Rac" of all trades. Cell Mol Life Sci. 2009, 66: 370-374. 10.1007/s00018-008-8552-x.CrossRefPubMed
23.
Schnelzer A, Prechtel D, Knaus U, Dehne K, Gerhard M, Graeff H, Harbeck N, Schmitt M, Lengyel E: Rac1 in human breast cancer: overexpression, mutation analysis, and characterization of a new isoform, Rac1b. Oncogene. 2000, 19: 3013-3020. 10.1038/sj.onc.1203621.CrossRefPubMed
24.
Fritz G, Just I, Kaina B: Rho GTPases are over-expressed in human tumors. Int J Cancer. 1999, 81: 682-687. 10.1002/(SICI)1097-0215(19990531)81:5<682::AID-IJC2>3.0.CO;2-B.CrossRefPubMed
25.
Jordan P, Brazao R, Boavida MG, Gespach C, Chastre E: Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors. Oncogene. 1999, 18: 6835-6839. 10.1038/sj.onc.1203233.CrossRefPubMed
26.
Singh A, Karnoub AE, Palmby TR, Lengyel E, Sondek J, Der CJ: Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation. Oncogene. 2004, 23: 9369-9380. 10.1038/sj.onc.1208182.CrossRefPubMed
27.
Brown JH, Del Re DP, Sussman MA: The Rac and Rho Hall of Fame: a decade of hypertrophic signaling hits. Circ Res. 2006, 98: 730-742. 10.1161/01.RES.0000216039.75913.9e.CrossRefPubMed
28.
Eblen ST, Slack JK, Weber MJ, Catling AD: Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes. Mol Cell Biol. 2002, 22: 6023-6033. 10.1128/MCB.22.17.6023-6033.2002.CrossRefPubMedPubMedCentral
29.
King AJ, Sun H, Diaz B, Barnard D, Miao W, Bagrodia S, Marshall MS: The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature. 1998, 396: 180-183. 10.1038/24184.CrossRefPubMed
30.
Slack-Davis JK, Eblen ST, Zecevic M, Boerner SA, Tarcsafalvi A, Diaz HB, Marshall MS, Weber MJ, Parsons JT, Catling AD: PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation. J Cell Biol. 2003, 162: 281-291. 10.1083/jcb.200212141.CrossRefPubMedPubMedCentral
31.
Nishida M, Tanabe S, Maruyama Y, Mangmool S, Urayama K, Nagamatsu Y, Takagahara S, Turner JH, Kozasa T, Kobayashi H, Sato Y, Kawanishi T, Inoue R, Nagao T, Kurose H: G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J Biol Chem. 2005, 280: 18434-18441.CrossRefPubMed
32.
Moore KA, Sethi R, Doanes AM, Johnson TM, Pracyk JB, Kirby M, Irani K, Goldschmidt-Clermont PJ, Finkel T: Rac1 is required for cell proliferation and G2/M progression. Biochem J. 1997, 326: 17-20.CrossRefPubMedPubMedCentral
33.
Michaelson D, Abidi W, Guardavaccaro D, Zhou M, Ahearn I, Pagano M, Philips MR: Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division. J Cell Biol. 2008, 181: 485-496. 10.1083/jcb.200801047.CrossRefPubMedPubMedCentral
34.
Soule HD, Maloney TM, Wolman SR, Peterson WD, Brenz R, McGrath CM, Russo J, Pauley RJ, Jones RF, Brooks SC: Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 1990, 50: 6075-6086.PubMed
35.
Zhao X, Malhotra GK, Lele SM, Lele MS, West WW, Eudy JD, Band H, Band V: Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate. Proc Natl Acad Sci USA. 2010, 107: 14146-14151. 10.1073/pnas.1009030107.CrossRefPubMedPubMedCentral
36.
Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y: Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA. 2004, 101: 7618-7623. 10.1073/pnas.0307512101.CrossRefPubMedPubMedCentral
37.
Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM, Abraham RT: Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 1999, 59: 4375-4382.PubMed
38.
Hall-Jackson CA, Cross DA, Morrice N, Smythe C: ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK. Oncogene. 1999, 18: 6707-6713. 10.1038/sj.onc.1203077.CrossRefPubMed
39.
Wei Q, Adelstein RS: Pitx2a expression alters actin-myosin cytoskeleton and migration of HeLa cells through Rho GTPase signaling. Mol Biol Cell. 2002, 13: 683-697. 10.1091/mbc.01-07-0358.CrossRefPubMedPubMedCentral
40.
Cook JA, Albacker L, August A, Henderson AJ: CD28-dependent HIV-1 transcription is associated with Vav, Rac, and NF-kappa B activation. J Biol Chem. 2003, 278: 35812-35818. 10.1074/jbc.M302878200.CrossRefPubMed
41.
Deng K, Gao Y, Cao Z, Graziani EI, Wood A, Doherty P, Walsh FS: Overcoming amino-nogo-induced inhibition of cell spreading and neurite outgrowth by 12-O-tetradecanoylphorbol-13-acetate-type tumor promoters. J Biol Chem. 2010, 285: 6425-6433. 10.1074/jbc.M109.071548.CrossRefPubMed
42.
Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A: The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992, 70: 401-410. 10.1016/0092-8674(92)90164-8.CrossRefPubMed
43.
Yan Y, Haas JP, Kim M, Sgagias MK, Cowan KH: BRCA1-induced apoptosis involves inactivation of ERK1/2 activities. J Biol Chem. 2002, 277: 33422-33430. 10.1074/jbc.M201147200.CrossRefPubMed
44.
Wang X, Gorospe M, Huang Y, Holbrook NJ: p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene. 1997, 15: 2991-2997. 10.1038/sj.onc.1201450.CrossRefPubMed
45.
Twigger K, Vidal L, White CL, De Bono JS, Bhide S, Coffey M, Thompson B, Vile RG, Heinemann L, Pandha HS, Errington F, Melcher AA, Harrington KJ: Enhanced in vitro and in vivo cytotoxicity of combined reovirus and radiotherapy. Clin Cancer Res. 2008, 14: 912-923. 10.1158/1078-0432.CCR-07-1400.CrossRefPubMed
46.
Kuo P-L, Hsu Y-L, Cho C-Y: Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther. 2006, 5: 3209-3221. 10.1158/1535-7163.MCT-06-0478.CrossRefPubMed
47.
Cai Z, Chattopadhyay N, Liu WJ, Chan C, Pignol J-P, Reilly RM: Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: comparison with manual counting. Int J Radiat Biol. 2011, 87: 1135-1146. 10.3109/09553002.2011.622033.CrossRefPubMed
48.
Xu B, Kim S, Kastan MB: Involvement of Brca1 in S-phase and G(2)-phase checkpoints after ionizing irradiation. Mol Cell Biol. 2001, 21: 3445-3450. 10.1128/MCB.21.10.3445-3450.2001.CrossRefPubMedPubMedCentral
49.
Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994, 371: 346-347. 10.1038/371346a0.CrossRefPubMed
50.
Wang Y, Ji P, Liu J, Broaddus RR, Xue F, Zhang W: Centrosome-associated regulators of the G(2)/M checkpoint as targets for cancer therapy. Mol Cancer. 2009, 8: 8-10.1186/1476-4598-8-8.CrossRefPubMedPubMedCentral
51.
Kuntz K, O'Connell MJ: The G(2) DNA damage checkpoint: could this ancient regulator be the Achilles heel of cancer?. Cancer Biol Ther. 2009, 8: 1433-1439.CrossRefPubMed
52.
Wang WH, Hullinger RL, Andrisani OM: Hepatitis B virus X protein via the p38MAPK pathway induces E2F1 release and ATR kinase activation mediating p53 apoptosis. J Biol Chem. 2008, 283: 25455-25467. 10.1074/jbc.M801934200.CrossRefPubMedPubMedCentral
53.
Zhou J, Lim CU, Li JJ, Cai L, Zhang Y: The role of NBS1 in the modulation of PIKK family proteins ATM and ATR in the cellular response to DNA damage. Cancer Lett. 2006, 243: 9-15. 10.1016/j.canlet.2006.01.026.CrossRefPubMedPubMedCentral
54.
Yoshida T, Zhang Y, Rivera Rosado LA, Chen J, Khan T, Moon SY, Zhang B: Blockade of Rac1 activity induces G1 cell cycle arrest or apoptosis in breast cancer cells through downregulation of cyclin D1, survivin, and X-linked inhibitor of apoptosis protein. Mol Cancer Ther. 2010, 9: 1657-1668. 10.1158/1535-7163.MCT-09-0906.CrossRefPubMed
55.
Taylor WR, Stark GR: Regulation of the G2/M transition by p53. Oncogene. 2001, 20: 1803-1815. 10.1038/sj.onc.1204252.CrossRefPubMed
56.
Lacroix M, Toillon R-A, Leclercq G: p53 and breast cancer, an update. Endocr Relat Cancer. 2006, 13: 293-325. 10.1677/erc.1.01172.CrossRefPubMed
57.
Watson NC, Di YM, Orr MS, Fornari FA, Randolph JK, Magnet KJ, Jain PT, Gewirtz DA: Influence of ionizing radiation on proliferation, c-myc expression and the induction of apoptotic cell death in two breast tumour cell lines differing in p53 status. Int J Radiat Biol. 1997, 72: 547-559. 10.1080/095530097143059.CrossRefPubMed
58.
Deckbar D, Jeggo PA, Lobrich M: Understanding the limitations of radiation-induced cell cycle checkpoints. Crit Rev Biochem Mol Biol. 2011, 46: 271-283. 10.3109/10409238.2011.575764.CrossRefPubMedPubMedCentral
59.
Kawabe T: G(2) checkpoint abrogators as anticancer drugs. Mol Cancer Ther. 2004, 3: 513-519.PubMed
60.
Gewirtz DA: Growth arrest and cell death in the breast tumor cell in response to ionizing radiation and chemotherapeutic agents which induce DNA damage. Breast Cancer Res Treat. 2000, 62: 223-235. 10.1023/A:1006414422919.CrossRefPubMed
Metadata
Title
RAC1 GTPase plays an important role in γ-irradiation induced G2/M checkpoint activation
Authors
Ying Yan
Patrick M Greer
Phu T Cao
Ryan H Kolb
Kenneth H Cowan
Publication date
01-04-2012
Publisher
BioMed Central
Published in
Breast Cancer Research / Issue 2/2012
Electronic ISSN: 1465-542X
DOI
https://doi.org/10.1186/bcr3164

Other articles of this Issue 2/2012

Breast Cancer Research 2/2012 Go to the issue

Research article

Insulin-like growth factor-1, insulin-like growth factor binding protein-3 and lobule type in the Nurses' Health Study II

Research article

ER and HER2 expression are positively correlated in HER2 non-overexpressing breast cancer

Research article

Occurrence of breast cancer subtypes in adolescent and young adult women

Research article

A genome-wide association study identifies a breast cancer risk variant in ERBB4 at 2q34: results from the Seoul Breast Cancer Study

Research article

Effects of BRCA2 cis-regulation in normal breast and cancer risk amongst BRCA2 mutation carriers

Research article

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits