Functional DNA repair system analysis in haematopoietic progenitor cells using host cell reactivation

Abstract

in individual DNA repair systems are involved in both de novo and therapy‐related acute myeloid leukaemia (t‐AML), as indicated by genetic markers involving nucleotide excision repair (NER gene polymorphisms), double‐strand‐break (DSB) or mismatch repair (microsatellite instability (MSI)). We modified a host cell reactivation (HCR) assay for functional DNA repair system analysis of living primary haematopoietic cells; 2×10⁵ normal peripheral blood lymphocytes (PBLs) and cord blood CD34+ progenitor cells were cryopreserved, thawed and transfected with 75–250 ng luciferase reporter plasmid (pCMVLuc) using DEAE‐dextran (0.1 mg/mL) in a transfection volume of 250 µL. We obtained luciferase activities of ∼300‐fold above background in CD34+ progenitor cells and ∼2000‐fold in PBLs, thus rendering these cells applicable for DNA repair analysis. We then evaluated the NER (UV‐irradiated pCMVLuc) and DSB repair capacity (linearized pCMVLuc) of normal lymphocytes and several leukaemic cell lineages. Kasumi‐1 and HL‐60 AML cells exhibited a reduced NER capacity compared to normal GM03715 lymphocytes, PBLs and CD34+ progenitor cells (6.2±0.9%, 6.5±0.9% vs. 12.3±1.8%, 13.5±0.7% and 13.5±2.0%, respectively). Kasumi‐1 AML cells exhibited a reduced DSB repair capacity compared to AG10107 and GM03715 normal lymphocytes as well as CEM acute T‐cell lymphoblastic leukaemia cells (6.4±0.8% vs. 10.8±0.7%, 27.3±1.1% and 20.5±1.6%, respectively). The modified HCR assay can be used for functional DNA repair analysis in living cells of patients with pre‐ and post‐leukaemic conditions as well as in leukaemic blasts to elucidate the role of DNA repair in de novo and t‐AML leukaemogenesis and to determine the individual susceptibility to t‐AML prior to chemotherapy.

• DNA repair
• haematopoietic stem cells
• leukaemia
• luciferases
• transfection

Acknowledgements

S.E., K.‐M.T. and D.L. were supported by grants from the Deutsche Forschungsgemeinschaft DFG (EM 63/3‐1 and GRK 1034). K.‐M. Thoms and J. Baesecke contributed equally to the work.