The Expression of Aphidicolin Induced Fragile Sites in Human Peripheral Blood Lymphocytes / 대한해부학회지

An aphidicolin is a chemical agent which selectively inhibits DNA polymerase alpha in S phase of cell cycle. The purpose of this study is toinvestigate of chromosomal abnormalities including fragile sites induced by 0.2 microgram/ml and 0.4 ng/ml aphidicolin in lymphocyte cultures of six healthy individuals. The results were follows. 1. A significant decreasing in mitotic indexes in respect to control culture was observed with both aphidicolin concentrations used. 2. The cells showing chromosome aberrations and the total number of cytogeneticic alterations were significantly increased both aphidicolin treated cultures than control cultures. 3. The total numbers of chromosomal aberrations were increased in the concentration of 0.4 microgram/ml aphidicolin compared to 0.2 microgram/ml treated groups. 4. The most frequent type of chromosomal aberration is a gap. 5. A site showing a gap or break was defined as common fragile sites (c-fra) if it appeared more than 1% of cells analyzed and in at least three of six individuals studied with the same culture treatment. Using these criteria, 3p14, 4q12, 5p13, 6q16, 9p13, and 16q23 were induced in different proportions by different concentration of aphidicolin and four of these c-fras, 4q12, 5p13, 6q16, 9p13 have not been reported so far. This results support that aphidicolin induced fragile sites differently according to cultured cell or cultured conditions, and also suggest the mechanism that common fragile sites caused be closely related with the defect of DNA synthesis in the S phase of cell cycle.

Fragile sites induced by aphidicolin in lymphocytes, HaCat cells and MRC-5 cells / 대한해부학회지

To investigate fragile sites induced by aphidicolin which is a specific inhibitor of eukaryotic DNA polymerase a which is primarily associated with chromosomal DNA replication in human lymphocytes, HaCat cells (human keratinocytes) and MRC-5 cells (human embryonic lung fibroblast), we cultured each cells in RPMI 1640 with 10% fetal calf serum and 2% PHA. Treatment of the cells with aphidicolin was generally carried out for the last 24 hours of culturing. The drug was dissolved in DMSO and used at final concentrations of 0.05~0.15 mg/ml, corresponding to a maximum DMSO concentration of 0.028%. Karyotypes of each cells were performed by routine method, and 50 metaphases were scored for each culture for analysis of breakage rate. Experimental cells treated with APC showed a dose dependent sensitivity and the amounts of chromosome breakage induced by APC are the highest in concentration of 0.15 mg/ml. The frequency of fragile sites on each cells appeared in MRC-5 cells, lymphocytes and HaCat cells in order. The common fragile sites on all experiments was 16q23, and the common fragile sites on embryonic cells was 1p31. It can be concluded that gene or nucleic acid which is located on 16q23 is the most important factor to induce chromosomal breakage with sensitivity to aphidicolin and 1p31 is important site to induce chromosomal breakage in embryonal cells.

Resolution of DNA polymerase-α-primase complex and primase free DNA polymerase α from embryonic chicken brain.

DNA polymerase-α from embryonic chicken brain was resolved on DEAEcellulose into 3 component activities that remained distinct upon rechromatography. Product formation by each activity required exogenously added template-primer DNA, all 4 deoxynucleoside triphosphates, and a divalent metal cation. Each form incorporated [3H]- dTTP or [3H]-dCTP into a high molecular weight product that was identified as DNA by its chromatographic behavior and its sensitivity to DNase. High ionic strength, Nethylmaleimide, and the polymerase-α-specific inhibitor aphidicolin inhibited each activity; the apparent Ki, value of aphidicolin was 3·0 μΜ in each case. Based on these results, the 3 activities were identified as multiple forms of DNA polymerase-α . Experiments using embryonic chicken brains of various ages indicated that polymerase-α1, and polymerase-α3 reached maximal activity in 9-day-old embryos, while polymerase-α2 activity was elevated at a slightly later developmental stage. Using poly (dC) as template, high primase activity was detected in polymerase-α1, fractions.