Chk1- and claspin-dependent but ATR/ATM- and Rad17-independent DNA replication checkpoint response in HeLa cells.
When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM-independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM-dependent and ATR/ATM-independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38 alpha,beta are not involved in the ATR/ATM-independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM-independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry.
Rodríguez-Bravo
Verónica
V
Departament de Biologia Cellular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.
Guaita-Esteruelas
Sandra
S
Florensa
Roger
R
Bachs
Oriol
O
Agell
Neus
N
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000235:genetics*; D000071877:Checkpoint Kinase 1; D003114:Colony-Forming Units Assay; D019926:Cyclin B / Q000502:physiology; D056744:Cyclin B1; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D029867:Xenopus Proteins
D048868:Adaptor Proteins, Signal Transducing; C534699:CCNB1 protein, human; C488158:CLSPN protein, human; D018797:Cell Cycle Proteins; D019926:Cyclin B; D056744:Cyclin B1; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C113575:Rad17 protein, human; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D002110:Caffeine; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D006918:Hydroxyurea