Generation of replication-dependent double-strand breaks by the novel N2-G-alkylator S23906-1.
S23906-1, a new DNA alkylating agent that reacts with the exocyclic 2-NH2 group of guanine residues yielding monofunctional adducts, is currently under clinical evaluation in phase I trials. To investigate the mechanism of action of S23906-1, we compared parental KB-3-1 cells and KB/S23-500 cells that are 15-fold resistant to S23906-1. Cell death induced by 1 micromol/L S23906-1 in KB-3-1 cells was associated with their irreversible arrest in the G2-M phases of the cell cycle followed by apoptosis, whereas a proportion of the resistant KB/S23-500 cells were able to exit from the G2 arrest and divide, leading to a significantly lower rate of apoptosis. The attenuated apoptotic response was associated with decreased Chk2 protein phosphorylation, indicating that the DNA damage signaling pathways are more potently activated in the sensitive cells. However, similar rates of adduct formation and repair were measured in both cell lines. Exposure to S23906-1 induced a higher formation of DNA breaks, measured by the comet assay, in sensitive cells. In agreement, a histone H2AX phosphorylation assay revealed that S23906-1 induced double-strand breaks (DSB) in a dose- and time-dependent manner and that these were more persistent in the parental cells. These DSBs were found mainly in S-phase cells and inhibited by aphidicolin, suggesting that they are DNA replication-mediated DSBs. These results suggest that secondary DNA lesions play an important role in the cytotoxicity of this compound and make histone H2AX phosphorylation an attractive marker for monitoring the efficacy of S23906-1.
Léonce
Stéphane
S
Institut de Recherches Servier, Cancer Drug Discovery, Croissy sur Seine, France. stephane.leonce@fr.netgrs.com
Kraus-Berthier
Laurence
L
Golsteyn
Roy M
RM
David-Cordonnier
Marie-Hélène
MH
Tardy
Christelle
C
Lansiaux
Amélie
A
Poindessous
Virginie
V
Larsen
Annette K
AK
Pierré
Alain
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000175:Acronine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000478:Alkylation; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007624:KB Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004273:DNA, Neoplasm; C468783:H2AX protein, human; D006657:Histones; C436732:S 23906-1; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D000175:Acronine