Induction of DNA strand breaks is critical to predict the cytotoxicity of gemtuzumab ozogamicin against leukemic cells.
Gemtuzumab ozogamicin (GO) consists of the CD33 antibody linked to calicheamicin. The binding of GO to the CD33 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 CD33 positivity was reduced in both variants. The HL/GO-CSA cells showed an increased multidrug resistance protein-1 (MRP1) transcript, and an MRP1 inhibitor partially reversed GO resistance. The HL/GO cells had neither P-gp nor MRP1 overexpression. Microarray analysis and Western blotting indicated elevated levels of DNA repair-associated proteins in both variants. Two other leukemic subclones, showing either P-gp or MRP1 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 CD33 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.
Department of Hematology and Oncology, University of Fukui, Eiheiji, Japan. tyamauch@u-fukui.ac.jp
Takahiro
T
Yamauchi
Yasufumi
Y
Matsuda
Toshiki
T
Tasaki
Eiju
E
Negoro
Satoshi
S
Ikegaya
Kazutaka
K
Takagi
Akira
A
Yoshida
Yoshimasa
Y
Urasaki
Takanori
T
Ueda
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000617:Aminoglycosides / Q000627:therapeutic use / Q000633:toxicity*; D061067:Antibodies, Monoclonal, Humanized / Q000627:therapeutic use / Q000633:toxicity*; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000627:therapeutic use / Q000633:toxicity*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004260:DNA Repair; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D000079982:Gemtuzumab; D015870:Gene Expression; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D011379:Prognosis; D063268:Sialic Acid Binding Ig-like Lectin 3; D055815:Young Adult
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000617:Aminoglycosides; D061067:Antibodies, Monoclonal, Humanized; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000970:Antineoplastic Agents; D000079982:Gemtuzumab; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D063268:Sialic Acid Binding Ig-like Lectin 3; C571163:CD33 protein, human; C421109:multidrug resistance-associated protein 1