Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry

We developed a simple and rapid method to study chromosome aberrations invo lving specific chromosomes using unstimulated human peripheral blood lympho cytes (HPBL). Premature chromosome condensation (PCC) was induced by incuba ting unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase i nhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase ran essential component of mitosis-promoting factor (MPF)I, which eliminated th e need for fusion with mitotic cells. OA concentration and duration of incu bation for PCC induction was optimized using mitogen-stimulated HPBL; a fin al concentration of 0.75 mu M incubated for 3 h was optimum, resulting in a pproximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the ad dition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p 34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration- dependent increase in PCC yield (30% to 42%). We demonstrate that this tech nique of inducing PCC in unstimulated HPBL is suitable for studying radiati on-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chrom osome painting. Cells with aberrant chromosome number 1 are characterized w ith more than two chromosome spots. The frequency of cells with aberrant ch romosome 1 increased with Co-60 gamma-radiation doses in the region 0-7.5 G y. The observed dose-effect relationship for the percentage of cells with a berrant chromosome 1 (Y) was explained by using both a linear [Y = (2.77 +/ - 0.230)D + 0.90 +/- 0.431, r(2) = 0.966] and a nonlinear power [Y = (5.70 +/- 0.46)D(0.61 +/- 0.05), r(2) = 0.9901) model. This technique can be appl ied to biological dosimetry of radiation exposures involving uniform whole- body low linear energy transfer (LET) exposures. (C) 2000 Published by Else vier Science B.V. All rights reserved.