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Characterization of ionizing radiation-induced ring chromosomes by atomic force microscopy
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 334 (2004) 251–256 www.elsevier.com/locate/yabio
Characterization of ionizing radiation-induced ring chromosomes by atomic force microscopy Masahiro Murakami¤, Reiko Kanda, Masako Minamihisamatsu, Isamu Hayata Radiation Hazards Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555, Japan Received 26 May 2004 Available online 28 August 2004
Abstract We applied atomic force microscopy (AFM) to the structural analysis of radiation-induced ring chromosomes. Constrictions observed on the metaphase ring chromosome were found to correspond to the centromere regions of the ring chromosome in comparison with the AFM image of the centromere of rod chromosomes and with the Xuorescence in situ hybridization (FISH) technique. Section analysis by AFM revealed that some ring-like chromosome fragments and ring-like chromatid fragments were thicker than standard chromosomes or chromatids, suggesting that they were ring chromosomes viewed edge on. Topographic analysis by AFM makes it possible to distinguish a ring viewed edge on that is diYcult to recognize as a ring by light microscopy and to discriminate between a centric ring chromosome and an acentric ring chromosome using the same slides prepared for light microscopy. 2004 Elsevier Inc. All rights reserved. Keywords: Atomic force microscopy; Centromere; Chromosome aberration; Prematurely condensed chromosome; Ring chromosome
The atomic force microscope invented by Binnig and Quate [1] has been used to visualize biological macromolecules at a nanometer level of resolution. Structures of chromosomes visualized by atomic force microscopy (AFM)1 have been reported [2–5]. When cells were exposed to ionizing radiation, chromosome aberrations were observed as a result of DNA damage and its misrepair. DNA damage induced with neutrons [6] or gamma rays [7] was analyzed by AFM. Chromosome aberrations, such as chromatid gaps and chromatid breaks induced with heavy ion radiation, were imaged, and the Wne structures inside of the gap region were analyzed by AFM [8]. *
Corresponding author. Fax: +81 43 255 6802. E-mail address: [email protected] (M. Murakami). 1 Abbreviations used: AFM, atomic force microscopy; PCC ring, premature condensed ring chromosome; DFM, dynamic force mode; FISH, Xuorescence in situ hybridization; FITC, Xuorescein isothiocyanate. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.07.023
Experimental data on the number of centromeres on each ring chromosome and on the number of ring chromosomes in each chromosome spread should provide useful information about the dose of radiation to which cells are exposed and the damage to DNA that is suVered [9]. When a radiation-induced ring chromosome is analyzed by light microscopy of a metaphase spread after Giemsa staining, discriminating between centric and acentric ring chromosomes is often diYcult and sophisticated techniques regarding slide preparation and microscopic analysis are required. Recently, a druginduced premature condensed ring chromosome (PCC ring) was used for biodosimetry for high-dose radiation exposure [10]. Also in this case, there is diYculty in distinguishing ring chromosomes viewed edge on from fragments. AFM with high resolution and high performance is expected to allow accurate distinction of ring chromosomes viewed edge on and fragments as well as development of an advanced technique with higher
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M. Murakami et al. / Analytical Biochemistry 334 (2004) 251–256
throughput. In the current study, we applied AFM to the structural analysis of radiation-induced ring chromosomes.
Materials and methods Preparation of metaphase ring chromosomes Peripheral blood was taken with a heparinized syringe from a healthy woman and was irradiated with X rays (200 kV, 20 mA) at doses of 5–20 Gy. Lymphocytes were then separated using vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ, USA) and were cultured in RPMI 1640 medium containing 20% fetal bovine serum, kanamycin (60 g/ml), 2% phytohemagglutinin (PHA HA-15, Murex Biotech, Dartford, UK), and 0.05 g/ml colcemid at 37 °C in a 5% CO2 atmosphere for 51 h. The cultured cells were treated with 0.075 M KCl for 20 min at 37 °C and then Wxed with methanol-acetic acid (3:1). Air-dried slides were made in warm and humid conditions [11]. Slides were then treated with RNase A (0.1 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 40 min at 37 °C, washed with water, and stained by Giemsa. Large metaphase ring chromosomes were selected for AFM by light microscopy. Preparation of prematurely condensed ring chromosomes For the analysis of prematurely condensed chromosomes, X-irradiated peripheral blood was stored at 37 °C for 3 h. Lymphocytes were then separated using vacutainer CPT tubes and were cultured in the same medium as the metaphase preparation at 37 °C in a 5% CO2 atmosphere for 48 h. Okadaic acid (Wako Pure Chemicals, Osaka, Japan), a free acid form, was added at 500 mM to the culture medium during the Wnal 1 h [10]. The slides prepared by an air-drying method were stained with Giemsa and mounted with EUKITT (Kindler O., Freiburg, Germany). Prematurely condensed chromosomes, including PCC rings, were identiWed under a light microscope and photographed by digital camera. Cells that exhibit attached sister chromatids are called G2/M cells, and those that exhibit separated sister chromatids are called M/A cells [10]. The round-shaped small chromosomal materials-chromosome fragments in G2/M cells and chromatid fragments in M/A cells-without obvious holes, which are diYcult to recognize as PCC rings by light microscopy after Giemsa staining, are called PCC ring-like chromosome fragments and PCC ring-like chromatid fragments, respectively. The PCC ring-like chromosome fragments in the G2/M-PCC cells and the PCC ring-like chromatid fragments in the M/APCC cells were selected for AFM. After light microscopy, the coverslip and EUKITT were removed in xylene and the slides were air-dried.
Atomic force microscopy Giemsa-stained metaphase chromosomes, PCC rings, PCC ring-like chromosome fragments, and PCC ringlike chromatid fragments were visualized with AFM (model SPI 3800N, Seiko Instruments, Chiba, Japan) in the dynamic force mode (DFM) with a 20-m scanner at room temperature [8]. Cantilevers for DFM-AFM (DF40, Seiko Instruments) were used for this experiment. The scan frequency was typically 2.0 Hz, and all images contained 512 £ 512 data points. Fluorescence in situ hybridization of centromeres After observation by AFM, some slides were destained with methanol/acetic acid, treated with RNase A, and processed for hybridization according to a dual staining method developed for the successive application of Giemsa staining and Xuorescence in situ hybridization (FISH) painting in the same metaphase [12–14]. The centromeric regions of all human chromosomes were labeled with biotinylated human pan centromeric chromosome paint (STAR FISH, Cambio, Cambridge, UK) conjugated with avidin-Xuorescein isothiocyanate (FITC) (Roche Diagnostics, Tokyo, Japan) according to the manufacturer’s instructions. Hybridization was done at 37 °C for 23 h. The chromosomes were counterstained with propidium iodide and observed under a Xuorescent microscope.
Results Analysis of centromere regions of metaphase ring chromosomes by AFM In the metaphase chromosome spread, large metaphase ring chromosomes were visualized by AFM (Fig. 1). On the ring chromosome, a constriction was identiWed by AFM imaging. This type of structure was observed in the cetromere region of rod chromosomes in the same nucleus. These structures were not recognizable by light microscopic observation. To identify the centromere regions of ring chromosomes, the metaphase spread analyzed by AFM was subjected to FISH. Three constrictions of single-ring chromosomes recognized by AFM were labeled by pan cetromeric DNA probes. Centromere regions of rod chromosomes in the same metaphase spread were also labeled by the probes. On the other hand, rod chromosomes without constrictions visualized with AFM were not labeled by the probes. Notably, two centromeres located in an adjacent position on the same chromosome were diYcult to detect by light microscopic observation after Giemsa staining but were easily distinguished by AFM imaging.
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253
Fig. 1. Light, Xuorescence, and AFM imaging of metaphase ring chromosome induced with X-irradiation (20 Gy). A metaphase ring chromosome (arrow) was observed by light microscope after Giemsa staining (upper left). Centromere regions were detected by the FISH technique with pan centromeric DNA probes (upper middle, white arrow). No signals were detected on the fragment (*). The same metaphase ring chromosome was visualized by AFM (upper right). Constrictions (white arrow) corresponding to centromere regions were observed on the metaphase ring or rod chromosome by AFM imaging. No constriction was observed on the fragment (*). When two centromeres were located adjacent to each other on the same chromosome, it was diYcult to identify each by light microscopic observation after Giemsa staining (upper left, arrowhead), but the two were distinguishable by FISH and AFM (upper middle and right, respectively, white arrowhead). A higher magniWcation AFM image of a metaphase ring chromosome is shown (lower left, bar D 1 m). Three constrictions (white arrow) corresponding to centromere regions were observed on the metaphase ring chromosome. Both chromatids of the ring chromosome that were diYcult to recognize by light microscope were visualized by AFM (white arrowhead). Results of the section analysis of the metaphase ring chromosome are shown in the lower panel. The height data were obtained along the indicated axis of the chromatid (lower middle) and are shown in the lower right panel. Two chromatids in the lower part were detected (white arrowhead).
The lower part of the ring chromosome was observed by AFM imaging. Both chromatids of the ring chromosome that are diYcult to recognize by light microscope were visualized by AFM. The height of this region of both chromatids was obviously lower than that of the other side of the chromosome (Fig. 1). Analysis of PCC rings by AFM A typical G2/M-PCC ring selected by light microscopy after Giemsa staining was visualized by AFM (Fig. 2). The height data were obtained by AFM. A sectional analysis of the PCC ring revealed a hollow in the center. A G2/M-PCC ring-like chromosome fragment was visualized by AFM (Fig. 3). Analysis showed that there was no hollow. A typical M/A-PCC ring selected by light microscopy after Giemsa staining was imaged by AFM (Fig. 4). The height data were obtained by AFM. A sectional analysis of the PCC ring revealed a hollow in the center. An M/A-PCC ring-like chromatid fragment was visualized by AFM (Fig. 5). Analysis showed that there was no hollow. Although there was a lower point around the center of this PCC ring-like
object, it was not signiWcantly diVerent in surface roughness from the other chromatids (data not shown). As summarized in Table 1, PCC ring-like chromosome fragments and PCC ring-like chromatid fragments were analyzed by AFM and classiWed into two types: standard and thick. Fragments classiWed as thick had surface heights that were signiWcantly larger (t test, P < 0.05) than the average. In the G2/M cells, the average heights of morphologically normal chromosomes, the one thick chromosome fragment, and the overlapping chromosomes (all in the same nucleus) were 143 § 2.6, 164 § 5.2, and 155 § 3.3 nm, respectively. In the G2/M cells, 7 PCC ring-like chromosome fragments were analyzed by AFM. Of these, 6 were classiWed as standard chromosome fragments and the remaining fragment was classiWed as a thick chromosome fragment. For example, in the M/A cells, in one nucleus the heights of the chromatids, the thick chromatid fragment, and the overlapping chromatids were 125 § 2.7, 165 § 5.3, and 155 § 3.5 nm, respectively. In the M/A cells, 31 PCC ringlike chromatid fragments were analyzed by AFM. Of these, 26 were classiWed as standard chromatid fragments and 5 were classiWed as thick chromatid fragments.
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Fig. 2. Light microscopic and AFM imaging of a G2/M-PCC ring induced with X-irradiation (10 Gy). A G2/M-PCC ring (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring was visualized by AFM (upper right, bar D 1 m). Results of the sectional analysis of the G2/M-PCC ring are shown in the lower panel. The height data were obtained along the indicated axis of the ring (lower left) and are shown in the lower right panel.
Fig. 3. Light microscopic and AFM imaging of a G2/M-PCC ring-like chromosome fragment induced with X-irradiation (10 Gy). A G2/M-PCC ringlike chromosome fragment (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring-like chromosome fragment was visualized by AFM (upper right, bar D 1 m). Results of the sectional analysis of the G2/M-PCC ring-like chromosome fragment are shown in the lower panel. The height data were obtained along the indicated axis of the chromosome (lower left) and are shown in the lower right panel.
Discussion In the same nucleus where the ring chromosomes were observed, constriction was typically visualized in the centromere region of the rod chromosome by AFM. Considering this result, constriction of the metaphase ring chromosome might correspond to the centromere of this chromosome when this shape is compared with that of the centromere region of other rod chromosomes in the same nucleus. This hypothesis was conWrmed when the same regions imaged as constrictions by AFM were
recognized as centromere regions by the FISH technique with pan centromeric DNA probes. When two centromeres were located in an adjacent position on the same chromosome, it was usually diYcult to identify each centromere by light microscopic observation after Giemsa staining, whereas the centromeres were distinguishable by AFM. In the current study, AFM and FISH analysis also revealed three centromeres detected on a single ring, whereas these structures were not identiWed by light microscopic observation.
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Fig. 4. Light microscopic and AFM imaging of an M/A-PCC ring induced with X-irradiation (15 Gy). An M/A-PCC ring (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring was visualized by AFM (upper right, bar D 1 m). Results of the section analysis of the M/A-PCC ring are shown in the lower panel. The height data were obtained along the indicated axis of the ring (lower left) and are shown in the lower right panel.
Fig. 5. Light microscopic and AFM imaging of an M/A-PCC ring-like chromatid fragment induced with X-irradiation (15 Gy). An M/A-PCC ringlike chromatid fragment (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring-like chromatid fragment was visualized by AFM (upper right, bar D 1 m). Results of the sectional analysis of the M/A-PCC ring-like chromatid fragment are shown in the lower panel. The height data were obtained along the indicated axis of the chromatid (lower left) and are shown in the lower right panel.
As reported by Hayata [15], Giemsa-stainable cytoplasmic substances were removed from cytogenetic slides. The application of a mild enzymatic treatment (RNase A and pepsin) to remove the cell material in the case of scanning force microscopy has been reported [16]. In the current study, we used RNase A treatment for preparation of the metaphase chromosome spread for AFM observation. One advantage of AFM is that one can obtain height information that is not possible to obtain by light microscopy. In the current study, we examined PCC
ring-like chromosome fragments and PCC ring-like chromatid fragments by AFM. We call these fragments “ring-like” because under a light microscope they resemble PCC rings but without a visible hole. Some of the chromosome materials classiWed as thick had a signiWcantly greater surface height than did the other chromosomes and chromatids in the same metaphase and were at least as high as the overlapping chromosomes or chromatids in the same cell. The wet chromatid diameter of human mitotic chromosomes measured by phase contrast microscopy, as well as Nomarski
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Table 1 AFM analysis of PCC ring-like chromosome fragments and PCC ringlike chromatid fragments Standard chromosome material
Thick chromosome material
Total
G2/M
6 (85.7%)
7 (100%)
M/A
26 (83.9%)
1 (14.3%) (H, 1.15) a 5 (16.1%) (H, 1.17) b (H, 1.26) b (H, 1.32) a (H, 1.48) a (H, 1.28) a
31 (100%)
Note. The round-shaped small chromosome materials (chromosome fragments in G2/M cells and chromatid fragments in M/A cells) without an obvious hole, which are diYcult to recognize as PCC rings by light microscopy after Giemsa staining, are called PCC ring-like chromosome fragments and PCC ring-like chromatid fragments, respectively. H, height relative to that of the other chromosome or chromatid in the same nucleus. a Height was equivalent to or higher than that of the overlapping chromosome or chromatid in the same nucleus. b No data were obtained for the height of the overlapping chromatid in the same nucleus.
interference contrast microscopy, is reported to be approximately 1.4 m [17]. The surface heights that we measured fell into a range that was consistent with this measurement. Considering these results, it is possible that the thick chromosome material is the PCC ring viewed edge on. These types of chromosome rings in metaphase are identiWed as darkly stained chromosome materials by light microscopy after Giemsa staining. In the case of PCC rings, however, diVerences in staining between PCC rings viewed edge on and other chromosome materials are not obvious. Using AFM, one can tell them apart. In most of the PCC ring-like chromosome or chromatid fragments, a hole in the center was not observed by AFM. The diYculty in distinguishing minute or dot deletions and acentric rings with the limits of optical resolution has been described in the case of smaller deletions [18]. Even with AFM, it is diYcult to identify PCC rings without holes because the surface roughness of this structure makes it diYcult to distinguish the shallow hollow of the smaller PCC ring (data not shown). Further study is needed to identify this type of small PCC ring. The application of AFM to the structural analysis of a ring chromosome makes it possible to discriminate between an acentric ring chromosome and a centric ring chromosome, to distinguish a ring chromosome viewed edge on from a fragment within a short period of time after the light microscopic observation of Giemsa-stained chromosome slides, and to improve the accuracy and eYciency of biological dosimetry.
Acknowledgment This work was supported in part by the Nuclear Cross-Over Research Project of the Ministry of Culture, Education, Sports, Science and Technology in Japan.
References [1] G. Binnig, C.F. Quate, Atomic force microscope, Phys. Rev. Lett. 56 (1986) 930–933. [2] B.G. De Grooth, C.A.J. Putman, High-resolution imaging of chromosome-related structures by atomic force microscopy, J. Microsc. 168 (1992) 239–247. [3] P. Rasch, U. Wiedemann, J. Wienberg, W.M. Heckl, Analysis of banded human chromosomes and in situ hybridization patterns by scanning force microscopy, Proc. Natl. Acad. Sci. USA 90 (1993) 2509–2511. [4] A. Musio, T. Marani, C. Frdiani, I. Sbrana, C. Ascoli, Longitudinal patterns similar to G-banding in untreated human chromosomes: evidence from atomic force microscopy, Chromosoma 103 (1994) 225–229. [5] D.M. Jondle, L. Ambrosio, J. Vesenka, E. Henderson, Imaging and manipulating chromosomes with the atomic force microscope, Chrom. Res. 3 (1995) 239–244. [6] D. Pang, B.L. Berman, S. Chasovskikh, J.E. Rodgers, O.A. Dritschilo, Investigation of neutron-induced damage in DNA by atomic force microscopy: experimental evidence of clustered DNA lesions, Radiat. Res. 150 (1998) 612–618. [7] M. Murakami, H. Hirokawa, I. Hayata, Analysis of radiation damage of DNA by atomic force microscopy in the comparison with agarose gel electrophoresis studies, J. Biochem. Biophys. Methods 44 (2000) 31–40. [8] M. Murakami, M. Minamihisamatsu, K. Sato, I. Hayata, Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy, J. Biochem. Biophys. Methods 48 (2001) 293–301. [9] International Atomic Energy Agency, Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assessment (technical reports series 260), International Atomic Energy Agency, Vienna, 1986. [10] R. Kanda, I. Hayata, D.C. Lloyd, Easy biodosimetry for high-dose radiation exposures using drug-induced prematurely condensed chromosomes, Intl. J. Radiat. Biol. 75 (1999) 441–446. [11] I. Hayata, H. Tabuchi, A. Furukawa, N. Okabe, M. Yamamoto, K. Sato, Robot system for preparing lymphocyte chromosome, J. Radiat. Res. 33 (Suppl.) (1992) 231–241. [12] R. Kanda, I. Hayayta, Comparison of the yields of translocations and dicentrics measured using conventional Giemsa staining and chromosome painting, Int. J. Radiat. Biol. 69 (1996) 701–705. [13] I. Hayta, C. Wang, W. Zhang, D. Chen, M. Minamihisamatsu, H. Morishima, Y. Yuan, L. Wei, T. Sugahara, Chromosome translocation in residents of the high background radiation areas in southern China, J. Radiat. Res. 41 (Suppl.) (2000) 69–74. [14] R. Kanda, Y. Yamagishi, I. Hayata, Sister chromatid exchanges in ring chromosomes following X-irradiation of human lymphocytes, Int. J. Radiat. Biol. (in press). [15] I. Hayata, Removal of stainable cytoplasmic substances from cytogenetic slide preparations, Biotechnol. Histochem. 68 (1993) 150–152. [16] J. Tamayo, M. Miles, Human chromosome structure studied by scanning force microscope after an enzymatic digestion of the covering cell material, Ultramicroscopy 82 (2000) 245–251. [17] P. Bak, A.L. Bak, J. Zeuthen, Characterization of human chromosomal unit Wbers, Chromosoma 73 (1979) 301–315. [18] J.R.K. Savage, ClassiWcation and relationships of induced chromosomal structural changes, J. Med. Genet. 12 (1975) 103–122.
Characterization of ionizing radiation-induced ring chromosomes by atomic force microscopy Masahiro Murakami¤, Reiko Kanda, Masako Minamihisamatsu, Isamu Hayata Radiation Hazards Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555, Japan Received 26 May 2004 Available online 28 August 2004
Abstract We applied atomic force microscopy (AFM) to the structural analysis of radiation-induced ring chromosomes. Constrictions observed on the metaphase ring chromosome were found to correspond to the centromere regions of the ring chromosome in comparison with the AFM image of the centromere of rod chromosomes and with the Xuorescence in situ hybridization (FISH) technique. Section analysis by AFM revealed that some ring-like chromosome fragments and ring-like chromatid fragments were thicker than standard chromosomes or chromatids, suggesting that they were ring chromosomes viewed edge on. Topographic analysis by AFM makes it possible to distinguish a ring viewed edge on that is diYcult to recognize as a ring by light microscopy and to discriminate between a centric ring chromosome and an acentric ring chromosome using the same slides prepared for light microscopy. 2004 Elsevier Inc. All rights reserved. Keywords: Atomic force microscopy; Centromere; Chromosome aberration; Prematurely condensed chromosome; Ring chromosome
The atomic force microscope invented by Binnig and Quate [1] has been used to visualize biological macromolecules at a nanometer level of resolution. Structures of chromosomes visualized by atomic force microscopy (AFM)1 have been reported [2–5]. When cells were exposed to ionizing radiation, chromosome aberrations were observed as a result of DNA damage and its misrepair. DNA damage induced with neutrons [6] or gamma rays [7] was analyzed by AFM. Chromosome aberrations, such as chromatid gaps and chromatid breaks induced with heavy ion radiation, were imaged, and the Wne structures inside of the gap region were analyzed by AFM [8]. *
Corresponding author. Fax: +81 43 255 6802. E-mail address: [email protected] (M. Murakami). 1 Abbreviations used: AFM, atomic force microscopy; PCC ring, premature condensed ring chromosome; DFM, dynamic force mode; FISH, Xuorescence in situ hybridization; FITC, Xuorescein isothiocyanate. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.07.023
Experimental data on the number of centromeres on each ring chromosome and on the number of ring chromosomes in each chromosome spread should provide useful information about the dose of radiation to which cells are exposed and the damage to DNA that is suVered [9]. When a radiation-induced ring chromosome is analyzed by light microscopy of a metaphase spread after Giemsa staining, discriminating between centric and acentric ring chromosomes is often diYcult and sophisticated techniques regarding slide preparation and microscopic analysis are required. Recently, a druginduced premature condensed ring chromosome (PCC ring) was used for biodosimetry for high-dose radiation exposure [10]. Also in this case, there is diYculty in distinguishing ring chromosomes viewed edge on from fragments. AFM with high resolution and high performance is expected to allow accurate distinction of ring chromosomes viewed edge on and fragments as well as development of an advanced technique with higher
252
M. Murakami et al. / Analytical Biochemistry 334 (2004) 251–256
throughput. In the current study, we applied AFM to the structural analysis of radiation-induced ring chromosomes.
Materials and methods Preparation of metaphase ring chromosomes Peripheral blood was taken with a heparinized syringe from a healthy woman and was irradiated with X rays (200 kV, 20 mA) at doses of 5–20 Gy. Lymphocytes were then separated using vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ, USA) and were cultured in RPMI 1640 medium containing 20% fetal bovine serum, kanamycin (60 g/ml), 2% phytohemagglutinin (PHA HA-15, Murex Biotech, Dartford, UK), and 0.05 g/ml colcemid at 37 °C in a 5% CO2 atmosphere for 51 h. The cultured cells were treated with 0.075 M KCl for 20 min at 37 °C and then Wxed with methanol-acetic acid (3:1). Air-dried slides were made in warm and humid conditions [11]. Slides were then treated with RNase A (0.1 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 40 min at 37 °C, washed with water, and stained by Giemsa. Large metaphase ring chromosomes were selected for AFM by light microscopy. Preparation of prematurely condensed ring chromosomes For the analysis of prematurely condensed chromosomes, X-irradiated peripheral blood was stored at 37 °C for 3 h. Lymphocytes were then separated using vacutainer CPT tubes and were cultured in the same medium as the metaphase preparation at 37 °C in a 5% CO2 atmosphere for 48 h. Okadaic acid (Wako Pure Chemicals, Osaka, Japan), a free acid form, was added at 500 mM to the culture medium during the Wnal 1 h [10]. The slides prepared by an air-drying method were stained with Giemsa and mounted with EUKITT (Kindler O., Freiburg, Germany). Prematurely condensed chromosomes, including PCC rings, were identiWed under a light microscope and photographed by digital camera. Cells that exhibit attached sister chromatids are called G2/M cells, and those that exhibit separated sister chromatids are called M/A cells [10]. The round-shaped small chromosomal materials-chromosome fragments in G2/M cells and chromatid fragments in M/A cells-without obvious holes, which are diYcult to recognize as PCC rings by light microscopy after Giemsa staining, are called PCC ring-like chromosome fragments and PCC ring-like chromatid fragments, respectively. The PCC ring-like chromosome fragments in the G2/M-PCC cells and the PCC ring-like chromatid fragments in the M/APCC cells were selected for AFM. After light microscopy, the coverslip and EUKITT were removed in xylene and the slides were air-dried.
Atomic force microscopy Giemsa-stained metaphase chromosomes, PCC rings, PCC ring-like chromosome fragments, and PCC ringlike chromatid fragments were visualized with AFM (model SPI 3800N, Seiko Instruments, Chiba, Japan) in the dynamic force mode (DFM) with a 20-m scanner at room temperature [8]. Cantilevers for DFM-AFM (DF40, Seiko Instruments) were used for this experiment. The scan frequency was typically 2.0 Hz, and all images contained 512 £ 512 data points. Fluorescence in situ hybridization of centromeres After observation by AFM, some slides were destained with methanol/acetic acid, treated with RNase A, and processed for hybridization according to a dual staining method developed for the successive application of Giemsa staining and Xuorescence in situ hybridization (FISH) painting in the same metaphase [12–14]. The centromeric regions of all human chromosomes were labeled with biotinylated human pan centromeric chromosome paint (STAR FISH, Cambio, Cambridge, UK) conjugated with avidin-Xuorescein isothiocyanate (FITC) (Roche Diagnostics, Tokyo, Japan) according to the manufacturer’s instructions. Hybridization was done at 37 °C for 23 h. The chromosomes were counterstained with propidium iodide and observed under a Xuorescent microscope.
Results Analysis of centromere regions of metaphase ring chromosomes by AFM In the metaphase chromosome spread, large metaphase ring chromosomes were visualized by AFM (Fig. 1). On the ring chromosome, a constriction was identiWed by AFM imaging. This type of structure was observed in the cetromere region of rod chromosomes in the same nucleus. These structures were not recognizable by light microscopic observation. To identify the centromere regions of ring chromosomes, the metaphase spread analyzed by AFM was subjected to FISH. Three constrictions of single-ring chromosomes recognized by AFM were labeled by pan cetromeric DNA probes. Centromere regions of rod chromosomes in the same metaphase spread were also labeled by the probes. On the other hand, rod chromosomes without constrictions visualized with AFM were not labeled by the probes. Notably, two centromeres located in an adjacent position on the same chromosome were diYcult to detect by light microscopic observation after Giemsa staining but were easily distinguished by AFM imaging.
M. Murakami et al. / Analytical Biochemistry 334 (2004) 251–256
253
Fig. 1. Light, Xuorescence, and AFM imaging of metaphase ring chromosome induced with X-irradiation (20 Gy). A metaphase ring chromosome (arrow) was observed by light microscope after Giemsa staining (upper left). Centromere regions were detected by the FISH technique with pan centromeric DNA probes (upper middle, white arrow). No signals were detected on the fragment (*). The same metaphase ring chromosome was visualized by AFM (upper right). Constrictions (white arrow) corresponding to centromere regions were observed on the metaphase ring or rod chromosome by AFM imaging. No constriction was observed on the fragment (*). When two centromeres were located adjacent to each other on the same chromosome, it was diYcult to identify each by light microscopic observation after Giemsa staining (upper left, arrowhead), but the two were distinguishable by FISH and AFM (upper middle and right, respectively, white arrowhead). A higher magniWcation AFM image of a metaphase ring chromosome is shown (lower left, bar D 1 m). Three constrictions (white arrow) corresponding to centromere regions were observed on the metaphase ring chromosome. Both chromatids of the ring chromosome that were diYcult to recognize by light microscope were visualized by AFM (white arrowhead). Results of the section analysis of the metaphase ring chromosome are shown in the lower panel. The height data were obtained along the indicated axis of the chromatid (lower middle) and are shown in the lower right panel. Two chromatids in the lower part were detected (white arrowhead).
The lower part of the ring chromosome was observed by AFM imaging. Both chromatids of the ring chromosome that are diYcult to recognize by light microscope were visualized by AFM. The height of this region of both chromatids was obviously lower than that of the other side of the chromosome (Fig. 1). Analysis of PCC rings by AFM A typical G2/M-PCC ring selected by light microscopy after Giemsa staining was visualized by AFM (Fig. 2). The height data were obtained by AFM. A sectional analysis of the PCC ring revealed a hollow in the center. A G2/M-PCC ring-like chromosome fragment was visualized by AFM (Fig. 3). Analysis showed that there was no hollow. A typical M/A-PCC ring selected by light microscopy after Giemsa staining was imaged by AFM (Fig. 4). The height data were obtained by AFM. A sectional analysis of the PCC ring revealed a hollow in the center. An M/A-PCC ring-like chromatid fragment was visualized by AFM (Fig. 5). Analysis showed that there was no hollow. Although there was a lower point around the center of this PCC ring-like
object, it was not signiWcantly diVerent in surface roughness from the other chromatids (data not shown). As summarized in Table 1, PCC ring-like chromosome fragments and PCC ring-like chromatid fragments were analyzed by AFM and classiWed into two types: standard and thick. Fragments classiWed as thick had surface heights that were signiWcantly larger (t test, P < 0.05) than the average. In the G2/M cells, the average heights of morphologically normal chromosomes, the one thick chromosome fragment, and the overlapping chromosomes (all in the same nucleus) were 143 § 2.6, 164 § 5.2, and 155 § 3.3 nm, respectively. In the G2/M cells, 7 PCC ring-like chromosome fragments were analyzed by AFM. Of these, 6 were classiWed as standard chromosome fragments and the remaining fragment was classiWed as a thick chromosome fragment. For example, in the M/A cells, in one nucleus the heights of the chromatids, the thick chromatid fragment, and the overlapping chromatids were 125 § 2.7, 165 § 5.3, and 155 § 3.5 nm, respectively. In the M/A cells, 31 PCC ringlike chromatid fragments were analyzed by AFM. Of these, 26 were classiWed as standard chromatid fragments and 5 were classiWed as thick chromatid fragments.
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Fig. 2. Light microscopic and AFM imaging of a G2/M-PCC ring induced with X-irradiation (10 Gy). A G2/M-PCC ring (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring was visualized by AFM (upper right, bar D 1 m). Results of the sectional analysis of the G2/M-PCC ring are shown in the lower panel. The height data were obtained along the indicated axis of the ring (lower left) and are shown in the lower right panel.
Fig. 3. Light microscopic and AFM imaging of a G2/M-PCC ring-like chromosome fragment induced with X-irradiation (10 Gy). A G2/M-PCC ringlike chromosome fragment (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring-like chromosome fragment was visualized by AFM (upper right, bar D 1 m). Results of the sectional analysis of the G2/M-PCC ring-like chromosome fragment are shown in the lower panel. The height data were obtained along the indicated axis of the chromosome (lower left) and are shown in the lower right panel.
Discussion In the same nucleus where the ring chromosomes were observed, constriction was typically visualized in the centromere region of the rod chromosome by AFM. Considering this result, constriction of the metaphase ring chromosome might correspond to the centromere of this chromosome when this shape is compared with that of the centromere region of other rod chromosomes in the same nucleus. This hypothesis was conWrmed when the same regions imaged as constrictions by AFM were
recognized as centromere regions by the FISH technique with pan centromeric DNA probes. When two centromeres were located in an adjacent position on the same chromosome, it was usually diYcult to identify each centromere by light microscopic observation after Giemsa staining, whereas the centromeres were distinguishable by AFM. In the current study, AFM and FISH analysis also revealed three centromeres detected on a single ring, whereas these structures were not identiWed by light microscopic observation.
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Fig. 4. Light microscopic and AFM imaging of an M/A-PCC ring induced with X-irradiation (15 Gy). An M/A-PCC ring (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring was visualized by AFM (upper right, bar D 1 m). Results of the section analysis of the M/A-PCC ring are shown in the lower panel. The height data were obtained along the indicated axis of the ring (lower left) and are shown in the lower right panel.
Fig. 5. Light microscopic and AFM imaging of an M/A-PCC ring-like chromatid fragment induced with X-irradiation (15 Gy). An M/A-PCC ringlike chromatid fragment (arrow) was observed by light microscope after Giemsa staining (upper left). The same PCC ring-like chromatid fragment was visualized by AFM (upper right, bar D 1 m). Results of the sectional analysis of the M/A-PCC ring-like chromatid fragment are shown in the lower panel. The height data were obtained along the indicated axis of the chromatid (lower left) and are shown in the lower right panel.
As reported by Hayata [15], Giemsa-stainable cytoplasmic substances were removed from cytogenetic slides. The application of a mild enzymatic treatment (RNase A and pepsin) to remove the cell material in the case of scanning force microscopy has been reported [16]. In the current study, we used RNase A treatment for preparation of the metaphase chromosome spread for AFM observation. One advantage of AFM is that one can obtain height information that is not possible to obtain by light microscopy. In the current study, we examined PCC
ring-like chromosome fragments and PCC ring-like chromatid fragments by AFM. We call these fragments “ring-like” because under a light microscope they resemble PCC rings but without a visible hole. Some of the chromosome materials classiWed as thick had a signiWcantly greater surface height than did the other chromosomes and chromatids in the same metaphase and were at least as high as the overlapping chromosomes or chromatids in the same cell. The wet chromatid diameter of human mitotic chromosomes measured by phase contrast microscopy, as well as Nomarski
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Table 1 AFM analysis of PCC ring-like chromosome fragments and PCC ringlike chromatid fragments Standard chromosome material
Thick chromosome material
Total
G2/M
6 (85.7%)
7 (100%)
M/A
26 (83.9%)
1 (14.3%) (H, 1.15) a 5 (16.1%) (H, 1.17) b (H, 1.26) b (H, 1.32) a (H, 1.48) a (H, 1.28) a
31 (100%)
Note. The round-shaped small chromosome materials (chromosome fragments in G2/M cells and chromatid fragments in M/A cells) without an obvious hole, which are diYcult to recognize as PCC rings by light microscopy after Giemsa staining, are called PCC ring-like chromosome fragments and PCC ring-like chromatid fragments, respectively. H, height relative to that of the other chromosome or chromatid in the same nucleus. a Height was equivalent to or higher than that of the overlapping chromosome or chromatid in the same nucleus. b No data were obtained for the height of the overlapping chromatid in the same nucleus.
interference contrast microscopy, is reported to be approximately 1.4 m [17]. The surface heights that we measured fell into a range that was consistent with this measurement. Considering these results, it is possible that the thick chromosome material is the PCC ring viewed edge on. These types of chromosome rings in metaphase are identiWed as darkly stained chromosome materials by light microscopy after Giemsa staining. In the case of PCC rings, however, diVerences in staining between PCC rings viewed edge on and other chromosome materials are not obvious. Using AFM, one can tell them apart. In most of the PCC ring-like chromosome or chromatid fragments, a hole in the center was not observed by AFM. The diYculty in distinguishing minute or dot deletions and acentric rings with the limits of optical resolution has been described in the case of smaller deletions [18]. Even with AFM, it is diYcult to identify PCC rings without holes because the surface roughness of this structure makes it diYcult to distinguish the shallow hollow of the smaller PCC ring (data not shown). Further study is needed to identify this type of small PCC ring. The application of AFM to the structural analysis of a ring chromosome makes it possible to discriminate between an acentric ring chromosome and a centric ring chromosome, to distinguish a ring chromosome viewed edge on from a fragment within a short period of time after the light microscopic observation of Giemsa-stained chromosome slides, and to improve the accuracy and eYciency of biological dosimetry.
Acknowledgment This work was supported in part by the Nuclear Cross-Over Research Project of the Ministry of Culture, Education, Sports, Science and Technology in Japan.
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