DNA damage and homologous recombination signaling induced by thymidylate deprivation.
DNA damage is accepted as a consequence of thymidylate deprivation induced by chemotherapeutic inhibitors of thymidylate synthase (TS), but the types of damage and signaling responses remain incompletely understood. Thymidylate deprivation increases dUTP and uracil in DNA, which is removed by base excision repair (BER). Because BER requires a synthesis step, strand break intermediates presumably accumulate. Thymidylate deprivation also induces cell cycle arrest during replication. Homologous recombination (HR) is a means of repairing persistent BER intermediates and collapsed replication forks. There are also intimate links between HR and S-phase checkpoint pathways. In this study, the goals were to determine the involvement of HR-associated proteins and DNA damage signaling responses to thymidylate deprivation. When RAD51, which is a central component of HR, was depleted by siRNA cells were sensitized to raltitrexed (RTX), which specifically inhibits TS. To our knowledge, this is the first demonstration in mammalian cells that depletion of RAD51 causes sensitivity to thymidylate deprivation. Activation of DNA damage signaling responses was examined following treatment with RTX. Phosphorylation of replication protein A (RPA2 subunit) and formation of damage-induced foci were strikingly evident following IC(50) doses of RTX. Induction was much more striking following RTX treatment than with hydroxyurea, which is commonly used to inhibit replication. RTX treatment also induced foci of RAD51, gamma-H2AX, phospho-Chk1, and phospho-NBS1, although the extent of co-localization with RPA2 foci varied. Collectively, the results suggest that HR and S-phase checkpoint signaling processes are invoked by thymidylate deprivation and influence cellular resistance to thymidylate deprivation.
Yang
Zhengguan
Z
Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, 715 Sumter Street, Columbia, SC 29208, USA.
Waldman
Alan S
AS
Wyatt
Michael D
MD
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D003110:Colonic Neoplasms; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects; D005434:Flow Cytometry; D005492:Folic Acid / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D009711:Nucleotides / Q000494:pharmacology; D010766:Phosphorylation; D011799:Quinazolines / Q000494:pharmacology; D051135:Rad51 Recombinase / Q000187:drug effects / Q000235:genetics; D011995:Recombination, Genetic / Q000187:drug effects*; D051716:Replication Protein A / Q000187:drug effects / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology; D013938:Thymidine Monophosphate / Q000172:deficiency*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors
D004273:DNA, Neoplasm; D005493:Folic Acid Antagonists; D009711:Nucleotides; D011799:Quinazolines; D051716:Replication Protein A; D013876:Thiophenes; D013938:Thymidine Monophosphate; D005492:Folic Acid; D013940:Thymidylate Synthase; D051135:Rad51 Recombinase; C068874:raltitrexed
Oncogene. 2007 Dec 10;26(56):7720-30
18066084
Mol Cell Biol. 1986 Oct;6(10):3463-9
3796589
Mol Cancer Ther. 2004 Jun;3(6):747-53
15210861
DNA Repair (Amst). 2006 Mar 7;5(3):369-80
16412704
Anticancer Drugs. 1997 Jan;8(1):3-16
9147608
DNA Repair (Amst). 2007 May 1;6(5):561-77
17258515
DNA Repair (Amst). 2008 Oct 1;7(10):1624-35
18603020
Mol Cell Biol. 2005 May;25(9):3553-62
15831461
Oncogene. 2006 Aug 31;25(39):5359-69
16619043
EMBO Rep. 2002 Mar;3(3):248-54
11850399
Biochem Pharmacol. 2005 Nov 15;70(10):1458-68
16191427
Chem Res Toxicol. 2006 Dec;19(12):1580-94
17173371
Carcinogenesis. 2005 Mar;26(3):547-55
15564287
Science. 2007 May 25;316(5828):1160-6
17525332
DNA Repair (Amst). 2004 Aug-Sep;3(8-9):1015-24
15279788
Eur J Cancer. 2003 Feb;39(3):378-87
12565992
Cancer Res. 1979 Mar;39(3):797-9
427767
Cancer Chemother Pharmacol. 1998;42(5):357-62
9771948
J Cell Biochem. 2006 Dec 1;99(5):1452-62
16927366
Mol Cell Biol. 2007 Sep;27(17):6053-67
17591703
Mol Cell Biol. 2002 Jun;22(12):4189-201
12024032
Nature. 2004 Nov 18;432(7015):316-23
15549093
Mol Gen Genet. 1984;195(3):474-80
6381965
Can J Microbiol. 1988 Jul;34(7):905-7
2848620
EMBO J. 1998 Jan 15;17(2):598-608
9430650
Mol Cancer Ther. 2006 Aug;5(8):1935-43
16928813
DNA Repair (Amst). 2008 Feb 1;7(2):162-9
17942376
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6236-40
8692798
Genetics. 1986 Oct;114(2):375-92
3533714
Proc Natl Acad Sci U S A. 1980 Oct;77(10):6057-61
6449701
Cancer Res. 1996 Oct 15;56(20):4715-23
8840989
J Biol Chem. 2001 Dec 21;276(51):47759-62
11673449
EMBO Rep. 2006 Oct;7(10):1046-51
16964178
Cancer Res. 2006 Sep 1;66(17):8829-37
16951200
Biochem Pharmacol. 2008 Sep 15;76(6):697-706
18599024
DNA Repair (Amst). 2007 Jul 1;6(7):1018-31
17379580
J Mol Biol. 1994 Nov 4;243(4):611-20
7966286
Mutat Res. 2003 Nov 27;532(1-2):215-26
14643438
J Biol Chem. 2005 Feb 18;280(7):5516-26
15611052
Cancer Res. 2004 Sep 15;64(18):6588-94
15374972
DNA Repair (Amst). 2008 May 3;7(5):686-93
18243065
Cancer Res. 2007 Feb 1;67(3):940-5
17283124
Oncogene. 2002 Dec 16;21(58):8935-48
12483510
J Biol Chem. 2005 Apr 1;280(13):12927-34
15653682
Nat Cell Biol. 2005 Feb;7(2):195-201
15665856
Gastroenterology. 2007 Dec;133(6):1858-68
18054558
Mol Cell Biol. 1996 Dec;16(12):7133-43
8943369
Mutat Res. 1991 Jan;246(1):215-20
1824719
J Biol Chem. 2007 Aug 3;282(31):22939-52
17526493