Open AccessSynchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage
Author(s) -
Kurose A.,
Tanaka T.,
Huang X.,
Traganos F.,
Darzynkiewicz Z.
Publication year - 2006
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/j.1365-2184.2006.00380.x
Subject(s) - dna replication , eukaryotic dna replication , biology , dna re replication , cell cycle , control of chromosome duplication , aphidicolin , dna damage , microbiology and biotechnology , histone , s phase , proliferating cell nuclear antigen , dna synthesis , dna , cell , genetics
. Several methods to synchronize cultured cells in the cell cycle are based on temporary inhibition of DNA replication. Previously it has been reported that cells synchronized this way exhibited significant growth imbalance and unscheduled expression of cyclins A and B1. We have now observed that HL‐60 cells exposed to inhibitors of DNA replication (thymidine, aphidicolin and hydroxyurea), at concentrations commonly used to synchronize cell populations, had histone H2AX phosphorylated on Ser ‐139. This modification of H2AX, a marker of DNA damage (induction of DNA double‐strand breaks; DSBs), was most pronounced in S‐phase cells, and led to their apoptosis. Thus, to a large extent, synchronization was caused by selective kill of DNA replicating cells through induction of replication stress. In fact, similar synchronization has been achieved by exposure of cells to the DNA topoisomerase I inhibitor camptothecin, a cytotoxic drug known to target S‐phase cells. A large proportion of the surviving cells ‘synchronized’ by DNA replication inhibitors at the G 1 /S boundary had phosphorylated histone H2AX. Inhibitors of DNA replication, thus, not only selectively kill DNA replicating cells, induce growth imbalance and alter the machinery regulating progression through the cycle, but they also cause DNA damage involving formation of DSBs in the surviving (‘synchronized’) cells. The above effects should be taken into account when interpreting data obtained with the use of cells synchronized by inhibitors of DNA replication.