Replication-dependent DNA damage response triggered by roscovitine induces an uncoupling of DNA replication proteins.
The cyclin-dependent kinase (CDK) inhibitor roscovitine is under evaluation in clinical trials for its antiproliferative properties. Roscovitine arrests cell cycle progression in G(1) and in G(2) phase by inhibiting CDK2 and CDK1, and possibly CDK7 and CDK9. However, the effects of CDK2 inhibition in S-phase cells have been not fully investigated. Here, we show that a short-term treatment with roscovitine is sufficient to inhibit DNA synthesis, and to activate a DNA damage checkpoint response, as indicated by phosphorylation of p53-Ser15, replication protein A, and histone H2AX. Analysis of DNA replication proteins loaded onto DNA during S phase showed that the amount of proliferating cell nuclear antigen (PCNA), a cofactor of DNA replication enzymes, was significantly reduced by roscovitine. In contrast, chromatin-bound levels of DNA polymerase delta, DNA ligase I and CDK2, were stabilized. Checkpoint inhibition with caffeine could rescue PCNA disassembly only partially, pointing to additional effects due to CDK2 inhibition and the presence of replication stress. These results suggest that in S-phase cells, roscovitine induces checkpoint-dependent and -independent effects, leading to stabilization of replication forks and an uncoupling between PCNA and PCNA-interacting proteins.
Savio
Monica
M
Dipartimento di Medicina Sperimentale, Patologia Generale, Università di Pavia, Pavia, Italy.
Cerri
Michaela
M
Cazzalini
Ornella
O
Perucca
Paola
P
Stivala
Lucia A
LA
Pichierri
Pietro
P
Franchitto
AnnaPaola
A
Meijer
Laurent
L
Prosperi
Ennio
E
D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage / Q000187:drug effects* / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects* / Q000235:genetics; D018816:Genes, cdc / Q000187:drug effects* / Q000502:physiology; D006657:Histones / Q000378:metabolism; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology*; D051716:Replication Protein A / Q000378:metabolism; D000077546:Roscovitine; D016196:S Phase / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C468783:H2AX protein, human; D006657:Histones; D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D047428:Protein Kinase Inhibitors; D011687:Purines; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; D000077546:Roscovitine; D004247:DNA; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases