Fludarabine resistance mediated by aminoglycoside‐3'phosphotransferase‐IIa and the structurally related eukaryotic cAMP‐dependent protein kinase

Abstract

While working with G418-resistant stably transfected cells, we realized the neomycin resistance (NeoR) gene, which encodes the aminoglycoside-3'-phosphotransferase-IIa [APH(3')-IIa], also confers resistance to the nucleoside analog fludarabine. Fludarabine is a cytostatic drug widely used in the treatment of hematologic and solid tumors, as well as in the conditioning of patients before transplantation of hematopoietic progenitors. We present evidence that NeoR-transfected cells do not incorporate fludarabine, thus avoiding DNA damage caused by the drug, evidenced by a lack of FANCD2 monoubiquitination and impaired apoptosis. A screening of other nucleoside analogs revealed that APH(3')-IIa only protects against ATP purine analogs. Moreover, APH(3')-IIa ATPase activity is inhibited by fludarabine monophosphate, suggesting that APH(3')-IIa blocks fludarabine incorporation into DNA by dephosphorylating its active fludarabine triphosphate form. Furthermore, overexpression of the catalytic subunit of the eukaryotic kinase PKA, which is structurally related to APHs, also provides resistance to fludarabine, anticipating its putative utility as a response marker to the drug. Our results preclude the use of Neo marker plasmids in the study of purine analogs and unveils a new resistance mechanism against these chemotherapeuticals.-Sánchez-Carrera, D., Bravo-Navas, S., Cabezón, E., Arechaga, I., Cabezas, M., Yáñez, L., Pipaón, C. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

Nucleoside triphosphate cosubstrates control the substrate profile and efficiency of aminoglycoside 3′-O-phosphotransferase type IIa

“…A subject of the present study, APHIJ3′)-IIa, encoded by the neomycin resistance (neo R ) gene aphIJ3′)-IIa, has been used as an AG resistance marker in molecular biology for decades, providing robust selection in AG-containing media. [17][18][19] A key issue to considering AGs as therapies or genetic selection tools is their modification by APHs. Therefore, a systematic investigation of the substrate-cosubstrate profile of APH enzymes is necessary.…”