title: Functional DNA repair system analysis in haematopoietic progenitor cells using host cell reactivation.
abstract: Deficiencies 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 x 10(5) 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 microL. We obtained luciferase activities of approximately 300-fold above background in CD34+ progenitor cells and approximately 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 tells 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.
authors.affiliation: Department of Dermatology and Venerology, Georg-August-University Goettingen, Germany.
authors.forename: K M
authors.initials: KM
authors.lastname: Thoms
authors.forename: J
authors.initials: J
authors.lastname: Baesecke
authors.forename: B
authors.initials: B
authors.lastname: Emmert
authors.forename: J
authors.initials: J
authors.lastname: Hermann
authors.forename: T
authors.initials: T
authors.lastname: Roedling
authors.forename: P
authors.initials: P
authors.lastname: Laspe
authors.forename: D
authors.initials: D
authors.lastname: Leibeling
authors.forename: L
authors.initials: L
authors.lastname: Truemper
authors.forename: S
authors.initials: S
authors.lastname: Emmert
mesh_terms: D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004789:Enzyme Activation; D005260:Female; D017930:Genes, Reporter / Q000235:genetics*; D005821:Genetic Techniques; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007948:Leukemia, Monocytic, Acute / Q000235:genetics; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics; D008156:Luciferases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D008214:Lymphocytes / Q000145:classification / Q000378:metabolism*; D053842:Microsatellite Instability / Q000528:radiation effects; D010957:Plasmids / Q000235:genetics; D011832:Radiation Injuries / Q000235:genetics; D014162:Transfection; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D004247:DNA; D008156:Luciferases