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 involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition 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 p34(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 technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant 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 applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.
Prasanna
P G
PG
Applied Cellular Radiobiology Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603, USA. prasanna@mx.afrri.usuhs.mil
Escalada
N D
ND
Blakely
W F
WF
D016203:CDC2 Protein Kinase / Q000494:pharmacology; D017489:CDC28 Protein Kinase, S cerevisiae / Q000494:pharmacology; D002869:Chromosome Aberrations; D020223:Chromosome Painting; D002877:Chromosomes, Human / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects; D018844:Cyclin-Dependent Kinases / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects; D008934:Mitogens / Q000494:pharmacology; D009152:Mutagenicity Tests / Q000379:methods; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011874:Radiometry / Q000379:methods; D013997:Time Factors
D008934:Mitogens; D019319:Okadaic Acid; D016203:CDC2 Protein Kinase; D017489:CDC28 Protein Kinase, S cerevisiae; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases