Open AccessInduction of DNA strand breaks is critical to predict the cytotoxicity of gemtuzumab ozogamicin against leukemic cells
Author(s) -
Yamauchi Takahiro,
Matsuda Yasufumi,
Tasaki Toshiki,
Negoro Eiju,
Ikegaya Satoshi,
Takagi Kazutaka,
Yoshida Akira,
Urasaki Yoshimasa,
Ueda Takanori
Publication year - 2012
Publication title -
cancer science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.035
H-Index - 141
eISSN - 1349-7006
pISSN - 1347-9032
DOI - 10.1111/j.1349-7006.2012.02343.x
Subject(s) - calicheamicin , gemtuzumab ozogamicin , microbiology and biotechnology , biology , dna , cell culture , dna damage , cytotoxicity , antibody , in vitro , cd33 , biochemistry , genetics , stem cell , cd34
Gemtuzumab ozogamicin ( GO ) consists of the CD 33 antibody linked to calicheamicin. The binding of GO to the CD 33 antigen on leukemic cells results in internalization followed by the release of calicheamicin, thereby inducing DNA strand breaks. We hypothesized that the induction of DNA strand breaks would be a surrogate marker of GO cytotoxcity. Here, two GO ‐resistant variants ( HL / GO ‐ CSA [225‐fold], HL / GO [200‐fold]) were established by serially incubating human leukemia HL ‐60 cells with GO with or without a P ‐glycoprotein ( P ‐gp) inhibitor, cyclosporine A , respectively. The CD 33 positivity was reduced in both variants. The HL / GO ‐ CSA cells showed an increased multidrug resistance protein‐1 ( MRP 1) transcript, and an MRP 1 inhibitor partially reversed GO resistance. The HL / GO cells had neither P ‐gp nor MRP 1 overexpression. Microarray analysis and W estern blotting indicated elevated levels of DNA repair‐associated proteins in both variants. Two other leukemic subclones, showing either P ‐gp or MRP 1 overexpression, were also GO ‐resistant. Using single cell gel electrophoresis analysis, it was determined that GO ‐induced DNA strand breaks increased dose‐dependently in HL ‐60 cells, whereas the number of breaks was reduced in the GO ‐resistant cell lines. The induction of DNA strand breaks was correlated with GO sensitivity among these cell lines. The CD 33 positivity and the expression levels of transporters were not proportional to drug sensitivity. Using primary leukemic cells, the induction of DNA strand breaks appeared to be associated with GO sensitivity. Thus, GO ‐induced DNA strand breaks as the final output of the mechanism of action would be critical to predict GO cytotoxicity.