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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Flow cytometric analysis by bromodeoxyuridine/DNA assay of cell cycle perturbation of methotrexate-treated mouse L1210 leukemia cells.

The in vitro effects of methotrexate (MTX) on cell cycle progression and DNA synthesis of L1210 leukemia cells were studied by the bromodeoxyuridine (BrdUrd)/DNA analysis technique. Low dose (10(-8) M) MTX, which slightly inhibits clonal replication of the cells, delays progress across the S phase, and treatment for 24 h results in a slight increase of the S-phase population. Much higher doses (10(-7) M and 10(-6) M) of MTX, which strongly reduce the clonogenicity, prevented the progression of cells at the G1-S boundary and across the S phase, but not in the other phases. The cells arrested at the G1-S boundary were able to incorporate BrdUrd in the medium for 6-12 h after the start of treatment and then lost the ability to incorporate BrdUrd. By determining the colony inhibitory activity of MTX, it could be shown that not only S-phase cells but non-S-phase cells are susceptible to cytotoxicity of MTX. MTX-induced S-phase arrest is closely associated with an alteration in the distribution of BrdUrd-labeled cells, and MTX apparently inhibits BrdUrd incorporation into L1210 cells as the dose and duration of treatment increase. These results suggest that MTX-induced cell cycle perturbation is related to inhibition of DNA synthesis.[1]


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