CHK1 and CHK2 are differentially involved in mismatch repair-mediated 6-thioguanine-induced cell cycle checkpoint responses.
The DNA mismatch repair (MMR) system plays an important role in mediating a G2-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 micromol/L x 24 hours), activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G2 arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G1-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.
Yan
Tao
T
Department of Radiation Oncology, LTR 6068, University Hospitals of Cleveland/Ireland Cancer Center, 11100 Euclid Avenue, Cleveland, OH 44106-6068, USA.
Desai
Anand B
AB
Jacobberger
James W
JW
Sramkoski
R Michael
RM
Loh
Tamalette
T
Kinsella
Timothy J
TJ
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D064007:Ataxia Telangiectasia Mutated Proteins; D020137:Base Pair Mismatch; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D015179:Colorectal Neoplasms / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013866:Thioguanine / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins
D000964:Antimetabolites, Antineoplastic; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D002110:Caffeine; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D013866:Thioguanine; D019311:Staurosporine