Fludarabine-mediated inhibition of nucleotide excision repair induces apoptosis in quiescent human lymphocytes.
Incorporation of fludarabine, 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A), into replicating DNA inhibits further chain elongation and is the critical event in F-ara-A-mediated cytotoxicity. We have used the normal cellular process of nucleotide excision repair to create an opportunity for F-ara-A incorporation into the DNA of noncycling cells. Irradiation of quiescent lymphocytes with UV light (254 nm, 0. 5-30 J/m2) in the presence of [3H]F-ara-A produced a dose-dependent increase in F-ara-A monophosphate incorporation into DNA that reflected a 60-70% inhibition of DNA repair at 2 h. Lymphocytes pretreated with 3 micrometer F-ara-A for 2 h before irradiation with 0.5 or 2.0 J/m2 were incubated for 24 h in the presence or absence of F-ara-A. Morphological features of apoptosis and DNA cleavage into high molecular weight fragments were increased in cells treated with UV plus F-ara-A compared to those treated with UV or F-ara-A alone. FACScan analysis confirmed the morphological and biochemical results. A 2-h pulse of F-ara-A produced intracellular F-ara-ATP levels of 40 micrometer, and removal of F-ara-A from the media resulted in a monophasic elimination (r2 = 0.88) in F-ara-ATP levels with a half-life of 5.6 h. Lymphocytes undergoing apoptosis demonstrated a G0 DNA content, indicating that entry into the cell cycle was not required. This study demonstrates that F-ara-AMP is incorporated into DNA during UV-induced repair in quiescent lymphocytes and that this is associated with the inhibition of ongoing DNA repair and an increased incidence of apoptosis. Combinational therapies involving fludarabine with agents and modalities that initiate DNA repair may have clinical relevance in the treatment of human malignancies.
Sandoval
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Departments of Clinical Investigation and Hematology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Consoli
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Plunkett
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D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D053938:DNA Fragmentation / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects*; D005434:Flow Cytometry; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000528:radiation effects; D014466:Ultraviolet Rays; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology
D000970:Antineoplastic Agents; D014740:Vidarabine; C024352:fludarabine