int. j. radiat. biol 1998, vol. 74 , no. 4 , 457 ± 462
Technical Report A simple method for simultaneous interphase–metaphase chromosome analysis in biodosimetry M. DURANTE†*, Y. FURUSAWA† and E. GOTOH‡ ( R eceived 1 A pril 1 9 9 8 ; accepted 2 9 M ay 1 9 9 8 )
A b stract. P urpose: To nd a simple protocol for measuring chromosome
damage both in G1 and in G2/M chromosomes, to overcome problems related to low mitotic index and cell-cycle alterations in biodosimetric tests. M aterials and m ethods: The protocol is based on the use of calyculin A to induce premature chromosome condensation in human peripheral blood lymphocytes in diå erent phases of the cell cycle. Chromosom e exchanges were measured by uorescence in situ hybridization (chromosomes 2 and 4) in lymphocytes from four diå erent donors. Cells were exposed to 4 Gy X-rays and the results were compared to aberrations in M phase (colcemid block) and G0 ( premature chromosome condensation induced by fusion to mitotic hamster cells). R esults: Treatment with calyculin A produced a high fraction of chromosome condensation in diå erent phases of the cell cycle. Cells in G1 and G2/M could be scored simultaneously for biodosimetry by chromosome painting. The condensation index was 5–20 times higher than the mitotic index (colcemid alone). The calyculin A treatment did not produce a signi cant increase in the background of chromosomal aberrations or modify the yield of chromosomal aberrations scored after exposure to X-rays. C onclusions: Induction of chromosome condensation by calyculin A is a powerful biodosimetric tool, which provides a high number of spreads for analysis and overcomes problems related to poor in vitro growth or cell-cycle alterations.
1. Intro d u ctio n
Biological dosimetry of ionizing radiation is generally performed by scoring dicentrics and/or translocations in chromosomes from peripheral blood lymphocytes (PBL) at the rst mitosis following in vitro growth stimulation (for recent reviews see Edwards 1997, Lucas 1997). In the protocol recommended by IAEA (1986), PBL are stimulated for 48 h by phytohaemagglutinin (PHA), and during the last *Author for correspondence. † Space and Particle Radiation Group, National Institute of Radiological Sciences, 9– 1 Anagawa-4-chome, Inage-ku, Chiba 263–8555, Japan. e-mail:
[email protected] (on leave of absence from the Department of Physics, University ‘Federico II’, Napoli, Italy). ‡ Division of Genetic Resources, National Institute of Infectious Diseases, 1– 23–1, Toyama, Shinjuku-ku, Tokyo 162– 0052, Japan.
3 h of incubation colcemid is added to the cultures for mitotic cell accumulation. However, it is well known that this procedure has several problems. First, the mitotic index is extremely low in some individuals. Low mitotic indexes are common in the elderly, after accidental exposure to high radiation doses, as a consequence of immunological diseases, and in microgravity conditions. If chromosome painting is used, many slides have to be hybridized, and the biodosimetric test will be expensive and time consuming. Secondly, because only those cells reaching metaphase are collected, the scored population is selected and might not be representative of the exposed population. The selection of cells harvested at metaphase may occur during radiation therapy when partial-body exposures at high doses are delivered and after exposure to densely ionizing radiation, which e æ ciently induces cellcycle delay. For these reasons, premature chromosome condensation (PCC) was proposed as a biodosimetric tool several years ago (Pantelias and Maillie 1984). PCC are induced in G0-PBL by fusion to mitotic cells; the in vitro stimulation step therefore is eliminated and no cell selection occurs. PCC can be used in biodosimetry by scoring excess fragments in Giemsa-stained cells (Prasanna et al. 1997), dicentrics by C-banding (Pantelias et al. 1993), or translocations after chromosome painting (Durante et al. 1996, 1997). In particular, uorescence in situ hybridization (FISH) with whole-chromosome probes allows easy and reliable scoring of chromosomal interchanges in PCC. Nonetheless, PCC analysis has severe drawbacks compared with metaphase analysis. The fusion method is technically di æ cult, and the PCC index is generally much lower than the mitotic index. Recently, it was shown that calyculin A, an inhibitor of type 1 and type 2A protein phosphatases, induces premature chromosome condensation in PBL at any time of the cell cycle (Gotoh et al. 1995, Asakawa and Gotoh 1997). This study describes a simple protocol for chromosome condensation induc-
0955 –3002/98 $12.00 1998 Taylor & Francis Ltd
458
M . D urante et al.
tion in PBL that can be applied in biological dosimetry by chromosome painting. PBL are stimulated by PHA, blocked at the rst mitosis by colcemid supplementation after 24 h, and chromosomes are prematurely condensed by calyculin A after 46 h. Very high chromosome condensation indexes are obtained, and slides can successfully be painted by FISH. As will be demonstrated, this procedure does not produce an increase in the frequency of chromosomal aberration compared with conventional metaphase and G0-PCC analysis, and does not interfere with the radiation response of PBL. 2. M aterials a nd m e thod s
Premature chromosome condensation by calyculin A was compared for biological dosimetry with conventional mitotic block and PCC induction by fusion. The three protocols are schematically represented in gure 1, and described in detail below.
2.1. L y m phocy te isolation Although the protocol worked with whole blood samples, the best results were obtained with isolated lymphocytes. Approximately 4 ml of blood were collected in a Vacutainer CPT (Beckton-Dickinson, Lincoln Park, USA) from four di å erent donors (table 1). The tube was centrifuged for 30 min at 1700 g (3000 rpm), and the white layer containing mononuclear cells and platelets was collected by a pipette and transferred to a conical centrifuge tube (Falcon, Franklin Lakes, NJ, USA). PBS was added up to 14 ml, and the tube was centrifuged for 15 min at 300 g . The pellet was washed once again in PBS and nally resuspended in 10 ml RPMI 1640 medium (Gibco-BRL, Grand Island, NY, USA), supplemented with 20 % foetal calf serum, 1 % PHA, 1 % l glutamine, 1 % penicillin/streptomycin and 0·1 % sodium heparin (stock 176·2 units/mg).
2.2. C hrom osom e co ndensation Cells in complete medium were incubated in tightly closed centrifuge tubes at 37 ß C. After 24 h, 40 m l of colcemid (Wako Chemicals, Japan; stock 10 m g/ml, nal concentration: 40 ng/ml) was added to the medium, and the tube was incubated again for 22 h. Finally, 5 m l of calyculin A (Wako Chemicals; stock 0·1 m m in ethanol, stored at Õ 20 ß C. Final concentration: 50 n m ) was added and the tube was incubated for 1 h at 37 ß C.
2.3. C olcem id accum ulation expe rim ents For comparing the results with the IAEA suggested protocol, isolated lymphocytes were resuspended in complete medium and incubated at 37 ß C. After 45 h, 0·4 ml of colcemid ( nal concentration: 400 ng/ml) was added and the tubes were incubated again for 3 h before harvesting.
2.4. F usion
Figure 1.
Flow-chart of the protocols compared in the present study.
For comparison with G0-PCC, the polyethylene glycol (PEG) fusion method originally developed by Pantelias and Maillie (1983) was adopted. Isolated lymphocytes were incubated in RPMI1640 (without PHA) for 24 h, and subsequently fused to mitotic Chinese hamster ovary (CHO) cells from a frozen stock. Following 1 h of incubation for chromosome condensation, the cells were xed as described below. Details of the PCC induction by the PEG method have been reported elsewhere (Durante et al. 1996).
P rotocol for interphase± m etaphase chrom osom e analy sis
2.5. C hrom osom e spreads Tubes were centrifuged for 5 min at 2000 rpm and the pellet was carefully resuspended in 8 ml of 75 m m KCl. Tubes were then incubated for 20 min at 37 ß C. Then 2 ml of freshly-prepared xative solution (methanol5acetic acid = 3 51) was slowly added to the solution, and the tube was centrifuged again. The pellet was resuspended in xative and kept for 20 min on ice. After centrifugation, the cells were resuspended in 14 ml of xative, centrifuged, and stored at Õ 20 ß C in xative. 2.6. S lide preparation and ageing After two further washes in methanol5acetic acid solution, the pellet was resuspended in 0·5 ml xative and three drops were spread on humid slides at room temperature. Slides were air-dried and treated in RNAase (0·1 mg/ml) for 1 h at 37 ß C. Slides were then dehydrated in 70 % , 85 % and 100 % ethanol for 2 min each. After air-drying, the slides were baked overnight at 45 ß C on a slide warmer before hybridization. 2.7. C hrom osom e painting The slides were hybridized with wholechromosome-paint probes speci c for human chromosomes 2 (spectrum green) and 4 (spectrum orange). The protocol suggested by the manufacturer (Vysis Inc, Downers Grove, IL, USA) was basically followed, with few modi cations (Durante et al. 1996). Slides were counterstained in DAPI and viewed with an Olympus uorescence microscope equipped with lters for DAPI, Texas red and FITC. Only spreads in G1, G2 and M phases were scored for aberrations. Two bicolour chromosomes were scored as a reciprocal exchange. 2.8. C ell- cy cle stage evaluation For evaluation of the cell-cycle phase and of the condensation index, as well as mitotic index and G0-PCC index, slides were stained in 4 % Giemsa and observed with a light microscope. Spreads displaying 46 univalent chromosomes were classi ed as G1. In S phase, the spreads had a typical pulverized form. G2 and M phases presented two chromatids in each chromosomes. Because a clear distinction between G2 and M was sometimes di æ cult, all bivalent chromosomes were classi ed in the G2/M category. For evaluation of the cell-cycle round at the time of harvesting, selected samples were incubated in medium as described in §2.1 and supplemented with 10 mm bromodeoxyuridine and 10 mm
459
deoxycytidine (both chemicals supplemented by Sigma, St. Louis, MD, USA). The tube was wrapped in aluminium paper and processed as described above. Slides were stained for 20 min in 0·5 m g/ml Hoechst 33258 (Calbiochem, La Jolla, CA, USA), rinsed in PBS and exposed for 30 min to UV light. Slides were then incubated for 15 min at 65 ß C in 2 Ö SSC, rinsed in water, and nally stained in 4 % Giemsa. 2.9. I rradiations Isolated lymphocytes in the G0 phase, resuspended in their own plasma, were exposed to X-rays (200 kVp, 19·0 mA), with 0·5 mm Cu and 0·5 mm Al shielding. The exposure rate was 116 R/min and the total exposure was 423 R, corresponding to 4 Gy. PBL were then centrifuged and resuspended in growth medium immediately after exposure. 3. R e sults a nd d iscussio n
When cells were treated in calyculin A, 30–60 % of the PBL showed clearly condensed chromosomes. Examples are shown in gure 2. The distribution of the cells in the di å erent phases of the cell cycle is reported in table 1. Cells in the S phase cannot be used for biodosimetry, which limits to G1 and G2/M spreads. Nonetheless, the number of useful spreads is much higher than those obtained with conventional colcemid block or G0-PCC by fusion. Samples from older people, in particular, had an extremely low mitotic index, but an adequate number of spreads was obtained by calyculin A. Because colcemid was added after 24 h of culture, all these cells are presumably in the rst post-stimulation replication round. This procedure was necessary to make sure that all the cells analysed were at the rst replication round, an important requisite for biodosimetry (Bender et al. 1988). Samples treated for 22 h in 40 ng/ml colcemid without calyculin A supplementation had mitotic indexes similar to those obtained with 3 h of colcemid treatment, but no premature chromosome condensation in phases other than M was induced. Tests with harlequin staining proved that no cells in second mitosis, G2 or S phase, are present in the samples treated for 22 h in colcemid, while some second mitosis was observed with the IAEA protocol (table 1). It is known that cells treated for a prolonged time in colcemid at a low concentration are blocked at the rst mitosis (Sasaki and Norman 1966, Chen and Zhang 1992), but some investigators reported modi cations in spontaneous (Scarpato and Migliore 1996) or radiation-induced (Kanda et al. 1994) yields of aberrations following prolonged colcemid treatment.
460
M . D urante et al.
461
P rotocol for interphase± m etaphase chrom osom e analy sis Table 1.
The percentage of cells condensed in diå erent phases of the cell cycle following treatment with calyculin A or colcemid alone. Calyculin A a
Donor A B C D
Age
Sex
32 34 65 65
M F M F
G1 ( %) 8Ô 4Ô 11 Ô 9Ô
S ( %) 3 1 2 3
27 Ô 22 Ô 12 Ô 19 Ô
Colcemid b G2/M ( %) 18 Ô 36 Ô 10 Ô 14 Ô
5 3 2 5
M ( %) 3Ô 8Ô 0·07 Ô 0·2 Ô
4 3 3 5
Fusion c G0 ( %)
Second mitosis ( % of mitosis) 4Ô 7Ô 0 1Ô
1 3 0·04 0·1
0·05 Ô 0·06 Ô
2 1
0·01 0·01
1
Mean values are pooled from three separate experiments and errors represent standard deviations from the three experiments. Percentages refer to the whole lymphocyte pool, including uncondensed PBL, except for second mitosis in colcemid-block experiments, where the percentage is relative to the total number of mitotic cells scored. a Lymphocytes stimulated in RPMI1640 plus PHA 1 % for 47 h; colcemid (40 ng/ml) added after 24 h in culture; calyculin A (50 n m ) added after 46 h in culture. b Lymphocytes stimulated in RPMI1640 plus PHA 1 % for 48 h; colcemid (400 ng/ml) added after 45 h in culture. c Lymphocytes incubated for 24 h in RPMI1640 medium, then fused to mitotic CHO cells for G0-PCC induction.
In addition, the clastogenicity of calyculin A or its interactions with radiation are not known. Therefore, the frequency of chromosomal aberrations in control and irradiated samples treated with calyculin A were studied and the results were compared with colcemidblock (IAEA protocol) and G0-PCC (fusion) methods. Table 2.
Reciprocal exchanges (dicentrics plus translocations) scored in chromosomes 2 and 4. Calyculin A a (G1 + G2 + M)
Dose (Gy)
No. of slides hybridized
No. of cells scored
A
0
2
3007
B
0
2
3763
C
0
3
2373
D
0
2
2766
A
4
1
1612
C
4
1
1023
D
4
1
1212
Donor
Reciprocal exchanges (dicentrics and translocations) were scored by FISH painting in chromosomes 2 and 4 and the results are reported in table 2. Results with calyculin A were pooled from G1, G2 and M phases. Because the slides were prepared in a highly reproducible way for the di å erent methods (see §2.6),
Colcemid b (M)
Reciprocal No. of No. of exchanges slides cells (frequency Ô SE) hybridized scored 8 0·9) Ö 10 Õ 6 (1·6 Ô 0·6) Ö 10 Õ 10 (4·2 Ô 1·3) Ö 10 Õ 10 (3·6 Ô 1·1) Ö 10 Õ 819 (0·508 Ô 0·018) 604 (0·590 Ô 0·024) 632 (0·521 Ô 0·021) (2·7 Ô
3
3
3
3
7
2848
4
2003
8
578
5
398
2
196
8
126
5
131
Fusion c (G0)
Reciprocal No. of No. of exchanges slides cells (frequency Ô SE) hybridized scored 6 0·9) Ö 10 Õ 3 (1·5 Ô 0·9) Ö 10 Õ 2 (3·4 Ô 2·4) Ö 10 Õ 1 (2·5 Ô 2·5) Ö 10 Õ 95 (0·48 Ô 0·05) 68 (0·53 Ô 0·06) 63 (0·48 Ô 0·06) (2·1 Ô
3
3
3
3
Reciprocal exchanges (frequency Ô SE)
6
423
6
456
–
–
1 2·3) Ö 1 2·2) Ö –
–
–
–
3
76
–
–
44 0·09) –
–
–
–
(2·3 Ô (2·2 Ô
(0·58 Ô
10 Õ
3
10 Õ
3
Lymphocytes stimulated in RPMI1640 plus PHA 1 % for 47 h; colcemid (40 ng/ml) added after 24 h in culture; calyculin A (50 nM) added after 46 h in culture. b Lymphocytes stimulated in RPMI1640 plus PHA 1 % for 48 h; colcemid (400 ng/ml) added after 45 h in culture. c Lymphocytes incubated for 24 h in RPMI1640 medium, then fused to mitotic CHO cells for G0-PCC induction. a
Figure 2. Examples of chromosome spreads obtained after treatment in calyculin A in PBL exposed to 4 Gy X-rays. (A) G1-PCC following diå erential replication staining. An S-phase cell is also shown ( pointed to by the black arrow). (B) M and G1 cells following FISH painting. The picture was taken with an FITC lter. Two fragments in chromosome 2 are present in the G1 cell (white arrows). (C) A reciprocal exchange (white arrows) and a fragment (red arrow) in chromosome 2 in a G1 PBL (FITC lter).
462
P rotocol for interphase± m etaphase chrom osom e analy sis
the actual number of hybridized slides is also reported to give a practical indication of the advantages of the new method. Data reported in table 2 indicate that no signi cant di å erences are observed in the frequency of spontaneous and radiation-induced reciprocal exchanges when samples are treated with calyculin A ( plus 22 h colcemid), colcemid only (3 h), or scored in G0 (after fusion to mitotic hamster cells). It has also been observed that far fewer slides need to be hybridized when samples are collected after calyculin A treatment, re ecting the high chromosome condensation indexes reported in table 1. In conclusion, chromosome condensation induced by calyculin A in cells blocked in the rst mitosis by prolonged colcemid treatment seem to be a powerful method for biological dosimetry. The fraction of lymphocytes that can be scored is much higher than obtained with conventional colcemid block (M only) or fusion (G0 only). In addition, cells in di å erent phases of the cell cycle can be obtained on the same slides, thus avoiding problems related to cell-cycle selection. G0-PCC biodosimetry will be faster because the PBL are not grown for 2 days. It was not possible to induce condensation by calyculin A in G0 lymphocytes, probably because the level of maturation promoting factor (MPF) protein is extremely low in G0. Calyculin A is an inhibitor of protein serine/threonine phosphatases, and these proteins block the MPF during interphase. Therefore, PCC by calyculin A can be induced only when the MPF level is su æ ciently high in the cells. Although treatment with calyculin A requires lymphocyte activation in vitro , the much higher condensation index, the possibility of scoring di å erent phases simultaneously, and the great simplicity of the present method makes it preferable for practical applications. A cknow led gem e nts
MD is supported by the Science and Technology Agency ( Japan) fellowship no. 196121. R e fe rences Asakawa, Y. and Gotoh, E., 1997, A method for detecting
sister chromatid exchanges using prematurely condensed
chromosomes and immunogold-silver M utagenesis , 12, 175– 177.
staining.
Bender, M. A., Awa, A., Brooks, L., Evans, H. J., Groer, P . G., Littlefield , L. G., P ereira , C., P reston, R. J. and Wachholz, B., 1988, Current status of cytogenetic pro-
cedures to detect and quantify previous exposures to radiation. M utation R esearch , 196, 103– 159. Chen, D. and Zhang, C., 1992, A simple and convenient method for gaining pure populations of lymphocytes at the rst mitotic division in vitro. M utation R esearch , 282, 227– 229. Durante, M., George, K. and Yang, T. C., 1996, Biological dosimetry by interphase chromosome painting. R adiatio n R esearch , 145, 53– 60. Durante, M., George, K. and Yang, T. C., 1997, Biodosimetry of ionizing radiation by selective painting of prematurely condensed chromosomes in human lymphocytes. R adiatio n R esearch , 148, S45– S50. Edwards, A. A., 1997, The use of chromosomal aberrations in human lymphocytes for biological dosimetry. R adiatio n R esearch , 148, S39– S44. Gotoh, E., Asakawa, Y. and Kosaka, H., 1995, Inhibition of protein serine/threonine phosphatases directly induces premature chromosome condensation in mammalian somatic cells. B iom edical R esearch , 16, 63– 68. Kanda, R., Jiang, T., Hayata, I. and Kobayashi, S., 1994, E å ects of colcemid concentration on chromosome aberrations analysis in human lymphocytes. J ournal of R adiati on R esearch , 35, 41– 47. IAEA, 1986, Biological dosimetry: chromosome aberration analysis for dose assessment. International Atomic Energy Agency Technical Report No. 260 (Vienna: IAEA). Lucas, J. N., 1997, Dose reconstruction for individuals exposed to ionizing radiation using chromosome painting. R adiatio n R esearch , 148, S33– S38. P antelias, G. E., Iliakis, G. E., Sambani, C. D. and P olitis, G., 1993, Biological dosimetry of absorbed radiation by C-banding of interphase chromosomes in peripheral blood lymphocytes. I nternational J ournal of R adiati on B iology , 63, 349– 354. P antelias, G. E. and Maillie, H. D., 1983, A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol. S om atic C ell G enetics , 9, 533– 547. P antelias, G. E. and Maillie, H. D., 1984, The use of peripheral blood mononuclear cell prematurely condensed chromosomes for biological dosimetry. R adiatio n R esearch , 99, 140– 150. P rasanna, P . G. S., Kolanko, C. J., Gerstenber g, H. M. and Balkeley, W. F., 1997, Premature chromosome condensation assay for biodosimetry: studies with ssionneutrons. H ealth P hy sics , 72, 594– 600. Sasaki, M. S. and Norman, A., 1966, Proliferation of human lymphocytes in culture. N ature , 210, 913– 914. Scarpato, R. and Migliore, L., 1996, Comparison of spontaneous structural chromosome aberration frequency in 48 h-cultured human lymphocytes mitotically arrested by diå erent colcemid treatment. M utation R esearch , 361, 35– 39.
