ATM-dependent CHK2 activation induced by anticancer agent, irofulven.
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.
Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia 26506, USA.
Jian
J
Wang
Timothy
T
Wiltshire
Yutian
Y
Wang
Carmenza
C
Mikell
Julian
J
Burks
Cynthia
C
Cunningham
Emily S
ES
Van Laar
Stephen J
SJ
Waters
Eddie
E
Reed
Weixin
W
Wang
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004268:DNA-Binding Proteins; D005260:Female; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010051:Ovarian Neoplasms; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects; D012694:Serine / Q000378:metabolism; D012717:Sesquiterpenes / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D000098550:Structural Maintenance of Chromosome Protein 1
D018906:Antineoplastic Agents, Alkylating; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D012717:Sesquiterpenes; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D000098550:Structural Maintenance of Chromosome Protein 1; D012694:Serine; C102714:irofulven; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases