Irofulven (6-hydroxymethylacylfulvene, HMAF,
MGI 114) is one of a new class of
anticancer agents that are semisynthetic derivatives of the mushroom toxin
illudin S. Preclinical studies and clinical trials have demonstrated that
irofulven is effective against several
tumor types. Mechanisms of action studies indicate that
irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in
ovarian cancer cells, CHK2
kinase is activated by
irofulven while
CHK1 kinase is not activated even when treated at higher concentrations of the
drug. By using GM00847 human fibroblast expressing
tetracycline-controlled, FLAG-tagged
kinase-dead ATR (ATR.kd), it was demonstrated that ATR
kinase does not play a major role in
irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by
irofulven is mediated by the upstream ATM
kinase. Phosphorylation of ATM on Ser(1981), which is critical for
kinase activation, was observed in
ovarian cancer cell lines treated with
irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing
siRNA for ATM. Finally, experiments done with human
colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing
kinase-dead CHK2 in an
ovarian cancer cell line demonstrated that CHK2 activation contributes to
irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on
serine 20 is dependent on CHK2 after
irofulven treatment. In summary, we found that the
anticancer agent,
irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by
irofulven.