Lymphoblasts already in the DNA synthesis phase of the cell cycle can be reversibly arrested at the R/G transition.
Early and late S-phase of the cell cycle are separated by the R-band/G-band (R/G) transition. This corresponds to the time at which R-band synthesis has been completed while G-band synthesis has yet to begin. The aim of this work was to study cell cycle kinetics during S-phase using different blocking agents: mimosine, methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine and an excess of thymidine. The stage at which these blocking agents arrest the cell cycle and their efficiency at blocking Epstein-Barr virus transformed lymphoblasts at the R/G transition were evaluated using flow cytometric techniques. Mimosine blocked 90% of the cells near the G1/S-phase boundary. Methotrexate, 5-fluoro-2'-deoxyuridine and 5-fluorouracil, and particularly thymidine, let a significant proportion of cells enter S-phase. The cells were released from the arrest state and their progression through early S-phase was monitored by flow cytometry. Before the cells reached the R/G transition, a second agent was added to inhibit cell cycle progression. For example, the use of mimosine followed by thymidine allowed up to 60% of the cells to be blocked at the R/G transition. The arrest of DNA replication at the R/G transition was confirmed by a marked decrease of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation, revealed by using bivariate flow cytometric analysis. The blocking agent was then removed and the cell cohort was released in the presence of BrdUrd so that replication banding analysis could be performed on the harvested mitotic cells. This yielded a mitotic index of approximately 10% and chromosomes showing replication bands. Flow cytometric analysis combined with cytogenetic banding analysis suggested that the R/G transition is an arrest point within the S-phase of the cell cycle and allowed us to conclude that only cells that have already initiated S-phase are blocked at this point. It corresponds to a susceptible site where S-phase can be arrested easily. The R/G transition could also be a regulatory checkpoint within S-phase, a checkpoint that could respond to imbalance in deoxyribonucleotide pools.
Lavoie
J
J
Division of Pathology, Department of Medical Biology, Faculty of Medicine, Laval University, Canada.
Drouin
R
R
D001973:Bromodeoxyuridine / Q000032:analysis; D002453:Cell Cycle / Q000187:drug effects; D002461:Cell Line, Transformed; D002871:Chromosome Banding; D002877:Chromosomes, Human / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000096:biosynthesis*; D004261:DNA Replication / Q000187:drug effects; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007621:Karyotyping; D007700:Kinetics; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D008898:Mimosine / Q000494:pharmacology; D016196:S Phase / Q000187:drug effects*; D013936:Thymidine / Q000494:pharmacology
D008898:Mimosine; D004247:DNA; D001973:Bromodeoxyuridine; D005472:Fluorouracil; D013936:Thymidine; D008727:Methotrexate