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

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Abstract

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 p34cdc2/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 μM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34cdc2/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34cdc2/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, r2=0.966] and a nonlinear power [Y=(5.70±0.46)D(0.61±0.05), r2=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.

Introduction

Accidental exposure of human beings to radiation is a major concern. Development of simple and rapid methods is required for radiation dose assessment, which will benefit the treatment of exposed individuals. Muller and Streffer [1] published a comprehensive review of biological indicators of radiation damage, explaining current techniques of biological dosimetry for radiation dose assessment. After exposure to high doses of radiation, sufficient numbers of mitotic cells are not available for dose assessment by the routine chromosome aberration analysis. The premature chromosome condensation (PCC) assay, performed on an exposed individual's blood lymphocytes, is viewed as a rapid biodosimetry method of clinical significance [2], [3], [4].

Conventionally, PCC is induced by a fusion of test cells, such as human peripheral blood lymphocytes (HPBL), with mitotic cells, enabling analysis of chromosome aberrations in interphase nuclei. In these protocols, polyethylene glycol (PEG) or Sendai virus is used to alter membrane permeability to accomplish cell fusion so that diffusion of mitosis-promoting factors (MPFs) can occur from mitotic cells into the test cells to induce PCC. However, these methods of cell fusion are technically difficult and laborious, and PCC yield is often too low or not consistent [5]. Therefore, the search for alternative simple and rapid protocols is necessary and is being conducted in several laboratories [6], [7], [8], [9]. Earlier, inhibitors of type 1 and 2A protein phosphatases were used to induce PCC in proliferating cells [6], [7], [8], [9]. These studies evaluated for biological dosimetry applications, chromosome aberration analysis in PCC spreads induced by okadaic acid (OA) [6], [7] or calyculin A [8] and obtained 48 h after mitogen-stimulation. Durante et al. [8] demonstrated that simultaneous measurement of chromosome aberrations in G1 and M phases is possible by using whole-chromosome probe-fluorescence in situ hybridization (FISH) technique following exposure to 200 kVp X-rays. It was also shown that incubation of tumor cell lines in a cell culture medium containing OA or calyculin A results in PCC induction [9]. Using whole-chromosome-specific probes, chemically induced PCC spreads containing radiation-induced chromosome aberrations are readily identified as cells with more than 2 chromosome spots. A difference in radiosensitivity was demonstrated between radiosensitive (SCC61) and radioresistant (A549) cell lines [9].

An understanding of the molecular mechanisms involved in PCC induction by phosphatase inhibitors will aid the further development of this novel technique for rapid biodosimetry applications. Mechanisms regulating the onset of M phase, which is considered common to all eukaryotic cells, were reviewed by Nurse [10]. In mitosis, chromosome condensation is associated with p34cdc2/cyclin B kinase activity, hyperphosphorylation of histone H1, and phosphorylation of histone H3. Activity of p34cdc2 kinase is, therefore, considered essential for chromosome condensation as one important event in mitosis promotion [10]. Treatment of cells with phosphatase inhibitors, such as OA and fostriecin, results in PCC due to phosphorylation of histones H2A and H3 even in the absence of p34cdc2 kinase activity and H1 hyperphosphorylation [11]. We tested the hypothesis that the presence of p34cdc2/cyclin B kinase along with adenosine triphosphate (ATP) in a cell culture medium containing OA induces PCC in unstimulated HPBL and further improves the quality of chromosome condensation. We demonstrate that damage to specific chromosomes in unstimulated HPBL can be studied by FISH with whole-chromosome-specific probes in chemically-induced PCC spreads. This technique may be suitable for rapid biological dosimetry applications.

Section snippets

Human peripheral blood lymphocytes

After obtaining informed consent, peripheral blood from healthy adult donors was collected by phlebotomy into vacutainers containing ethylenediaminetetraacetic acid (Becton-Dickinson, Rutherford, NJ, USA). The Uniformed Services University of the Health Sciences' Human Use Committee, Bethesda, MD, approved the informed consent form. Lymphocytes were isolated, using a density gradient (Histopaque 1077, Sigma, St. Louis, MO, USA), and washed twice in phosphate-buffered saline (pH 7.0). The cells

PCC induction in mitogen-stimulated HPBL

We designed a study, using a mitogen-stimulated HPBL model system, to choose a suitable concentration of OA and duration of incubation, up to 24 h, that together would provide a reasonably high yield of PCC, for use with p34cdc2/cyclin B kinase to induce PCC in unstimulated cells. Since incubation of unstimulated HPBL in a cell culture medium containing OA alone did not result in PCC induction, PHA was used to help activate cell cycle progression. The HPBL were treated with OA at concentrations

Discussion

It is important to develop simple and reliable techniques for biological dosimetry of radiation exposures than currently used techniques such as analysis of chromosome aberrations in metaphase or PCC spreads after mitotic-cell fusion. We developed a chemical method of inducing PCC in unstimulated HPBL for analyzing aberrations involving specific chromosomes. This method is simple and does not require the high degree of technical expertise associated with alternative PCC-inducing protocols [5],

Acknowledgements

The Armed Forces Radiobiology Research Institute supported this research under work unit AFRRI-00-3. The views expressed are those of the authors; no endorsement by the Armed Forces Radiobiology Research Institute has been given or inferred. We thank K.S. Kumar and reviewers for their valuable critique of this manuscript; W.E. Jackson for assistance in statistics; Ranvir Floura, Massihullah Hamidi, and Keife Duffy for expert technical help; and D.K. Solyan for the editorial assistance. The

References (27)

  • M Durante et al.

    A simple method for simultaneous interphase–metaphase chromosome analysis in biodosimetry

    Int. J. Radiat. Biol.

    (1998)
  • J.M Coco-Martin et al.

    Detection of radiation-induced chromosome aberrations using fluorescence in situ hybridization in drug-induced premature chromosome condensations of tumor cell lines with different radiosensitivities

    Int. J. Radiat. Biol.

    (1997)
  • P Nurse

    Universal control mechanism regulating onset of M-phase

    Nature

    (1990)
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