We applied DAPI (4',6-diamidino-2-phenylindole) staining to the determination of nuclear DNA content in single megakaryocytes in 12 normal subjects and 12 patients with myelodysplastic syndrome (MDS). After the megakaryocytes had been identified on Wright-Giemsa stained smear and classified according to modified Feinendegen's classification, they were photographed. Then Wright-Giemsa stain was removed by immersion in 50% ethanol at 37 degrees C for 1 h and 100% methanol at 37 degrees C for 1 h. The specimens were then stained with DAPI solution (DAPI 0.01 mg/ml, pH 7.4 Tris-EDTA-2Na buffer solution and 0.01 M 2-mercaptoethylamine hydrochloride mixed at the ratio of 0.5:98.5:1.0) for more than 30 min. The amount of nuclear DNA in the previously identified megakaryocytes was measured by microcytofluorometry. The maximum population of megakaryocytes ploidy was in 16N in normal subjects, 8N in 10/12 MDS patients, and 4N in the remaining two patients. These findings suggest impairment of the development of the megakaryocytes nucleus in the MDS patients.
A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4',6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4 degrees C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.
The effects of N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BH-AC) on the cell cycle of murine leukemic cells (L 1210 cells) were compared with those of 1-beta-D-arabinofuranosylcytosine (ara-C), known to be effective for acute leukemia. In a cytokinetic study, a combination of Feulgen microcytofluorometry and tritiated thymidine autoradiography (3H-TdR ARG) was used to measure DNA content and to determine DNA synthesis simultaneously in a single cell. Administration of 200 mg/kg of BH-AC significantly prolonged the survival time of mice bearing L 1210. In addition, the cells in the G1 + S1 phases increased with time, accounting for 94.4 per cent of all cells measured at 48 h after the administration, compared to 73.7 per cent before administration. On the other hand, following the administration of 86 mg/kg of ara-C (equivalent to 200 mg/kg of BH-AC), the percentage of cells in the S1 + S2 phases increased maximally to 75.9 per cent at 18 h. Cytokinetic studies further showed that BH-AC administration blocks DNA synthesis of the cells in the S-phase for a longer period than does ara-C. These results suggest that the prolonged inhibition by BH-AC on DNA synthesis allows cells to accumulate in the S-phase to a greater degree.
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