Int. J. Radiat. Biol., Vol. 82, No. 1, January 2006, pp. 31 – 38
Cell cycle arrest and apoptosis in Caenorhabditis elegans germline cells following heavy-ion microbeam irradiation
TOMOKO SUGIMOTO1, KUMIKO DAZAI1, TETSUYA SAKASHITA2, TOMOO FUNAYAMA2, SEIICHI WADA2, NOBUYUKI HAMADA2,3, TAKEHIKO KAKIZAKI2, YASUHIKO KOBAYASHI2,3 & ATSUSHI HIGASHITANI1 1
Graduate School of Life Sciences, Tohoku University, Sendai, 2Microbeam Radiation Biology Group, Japan Atomic Energy Agency, Takasaki Gunma, and 3Graduate School of Medicine, Gunma University, Maebashi, Japan
(Received 22 September 2005; accepted 16 January 2006) Abstract Purpose: To investigate positional effects of radiation with an energetic heavy-ion microbeam on germline cells using an experimental model metazoan Caenorhabditis elegans. Materials and methods: The germline cells were irradiated with raster-scanned broad beam or collimated microbeam of 220 MeV 12C5þ particles delivered from the azimuthally varying field (AVF) cyclotron, and subsequently observed for cell cycle arrest and apoptosis. Results: Whole-body irradiation with the broad beam at the L4 larval stage arrested germ cell proliferation. When the tip region of the gonad arm was irradiated locally with the microbeam at the L4 stage, the same arrest was observed. When the microbeams were used to irradiate the pachytene region of the gonad arm, at a young gravid stage, radiation-induced apoptosis occurred in the gonad. In contrast, arrest and apoptosis were not induced in the non-irradiated neighboring region or the opposite gonad. Similar results were confirmed in the c-abl-1 (mammalian ortholog of cellular counterpart of Abelson murine leukemia virus) mutant that is hypersensitive to radiation-induced apoptosis. Conclusion: These results indicate that the microbeam irradiation is useful in characterizing tissue-specific, local biological response to radiation in organisms. DNA damage-induced cell cycle arrest and apoptosis were observed in locally irradiated regions, but there was little, if any, ‘bystander effect’ in the nematode.
Keywords: Bystander effect, germline cells, heavy-ion particles, microbeam, pachytene checkpoint apoptosis
Introduction Ionizing radiation and some chemical agents can cause double-strand breaks in DNA. All cells have systems to repair such breaks, as well as apoptotic systems to eliminate cells that have failed to repair DNA lesions. These activities fluctuate in tissuespecific and/or age-dependent manners. We have studied the effects of radiation on germline cells using the nematode Caenorhabditis elegans as an experimental model (Takanami et al. 2000, 2003). The C. elegans hermaphrodite is a convenient model system for the study of radiation effects, due to the linear process of the developmental phases in oogenesis. In adult hermaphrodites, germ cells in the tip region of the gonad arm divide mitotically,
entering meiotic prophase I, where they progress from the zygotene to the diakinesis stage, in the maturing oocyte. After irradiation, the mitotic cells undergo cell cycle arrest leading to a reduction of cell number but the remaining cells swell and increase in size (Stergiou & Hengartner 2004, Takanami et al. 2003, Gartner et al. 2000). The meiotic pachytene phase becomes radioresistant due to high expression of enzymes for meiotic homologous recombination (Takanami et al. 2000). Additionally, DNA damageinduced apoptosis can occur in the germ cells at the late pachytene stage of meiosis (Gartner et al. 2000). Most C. elegans genes involved in DNA damage response and apoptosis are highly conserved in mammals (Stergiou & Hengartner 2004). One of the genes C. elegans c-abl-1 (mammalian ortholog of
Correspondence: A. Higashitani, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. Tel: þ81 (0)22 217 5715. Fax: þ81 (0)22 217 5745. E-mail:
[email protected] The first two authors contributed equally to this work. ISSN 0955-3002 print/ISSN 1362-3095 online Ó 2006 Taylor & Francis DOI: 10.1080/09553000600577821
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cellular counterpart of Abelson murine leukemia virus) functions to antagonize p53-mediated germline apoptosis after ionizing radiation (Deng et al. 2004). The c-abl-1 defective mutants, therefore, are specifically hypersensitive to radiation-induced apoptosis in the germline. Recently, an energetic heavy-ion microbeam irradiation system was constructed for local irradiation of biological systems to investigate positional effects of radiation (Kobayashi et al. 1999, Funayama 2005). It is useful to block the function of cell(s) through DNA damage without disrupting the cells physically in other ways. Additionally, studies with microbeam irradiation on cultured mammalian cells, have demonstrated the existence of a ‘bystander effect’, in which cells that have received no irradiation show biological consequences from their neighboring irradiated cells (Hall & Hei 2003, Shao et al. 2003, Iyer & Lehnert 2000, Mothersill & Seymour 2001). As well as induction of micronuclei, apoptosis induced by ‘bystander effects’ was established in mammalian cells (Zu et al. 2005, Belyakov et al. 2005). Notably, Belyakov et al. (2005) found micronucleated and apoptotic cells in unirradiated human skin tissue induced by radiation damage up to 1 mm away. The consequences of the ‘bystander effect’ at the organ and whole body levels, however, are poorly understood. We studied local, tissuespecific responses to radiation, such as cell cycle arrest and apoptosis, in the germline cells of C. elegans. Using the heavy-ion microbeam irradiation apparatus, we also investigated whether or not a ‘bystander effect’ occurs in these germline cells.
Materials and methods C. elegans strains and culture conditions In this study, we used C. elegans N2 wild type hermaphrodites and the XR1 mutant strain, carrying an abl-1 (ok171) deletion allele (C. elegans Genetic Center). Hermaphrodites with the XR1 mutation are hypersensitive to radiation-induced apoptosis in germline cells (Deng et al. 2004). Standard methods were used to culture and manipulate C. elegans (Lewis & Fleming 1995), and all experiments were performed at 208C. Young gravid hermaphrodites were synchronized, and cultured on OP-50 nematode growth medium (NGM) plates for 24 h, after entering the L4 larval stage. Irradiation with
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C ion microbeam and broad beam
L4 larvae and young gravid hermaphrodites were irradiated with a raster-scanned broad beam or collimated microbeam of 220 MeV 12C5þ particles delivered from the azimuthally varying field (AVF)
cyclotron at the Takasaki Ion accelerators for Advanced Radiation Application (TIARA) of the Japan Atomic Energy Agency (JAEA). The incident energy of the carbon ions was 18.3 MeV amu71 and the linear energy transfer (LET) in the hermaphrodite exposed to the carbon ion-broad beam and the microbeam was 110 keV mm71 and 120 keV mm71, respectively. To convert particle fluence to dose in Gy the following relationship was used: Dose [Gy] ¼ 1.6610796LET [keV/mm] 6 Fluence [particle/cm2]. For local irradiation with the microbeam, L4 larvae and young gravid hermaphrodites were anesthetized with 5 – 10 mM sodium azide in M9 buffer for 15 – 30 min, and were restrained between a TaKaRa Slide Seal without removing the cover plastic seal unilaterally (10 mm610 mm: 25 ml size, TaKaRa Bio Inc., Shiga, Japan) and a glass coverslip (thickness No. 1: 0.12 – 0.17 mm, Matsunami Glass Co., Osaka Japan) with 25 ml of the sodium azide buffer. Hermaphrodites were irradiated locally with the microbeam on the inverted optical microscope that was installed below the vertical beam line (Figure 1a). The collimated heavy-ion beams were extracted into the air through a f20 mm diameter aperture (Kobayashi et al. 1999, Funayama 2005). The track penumbra dimension was estimated as d ¼ 12.30 mm (LET ¼ 120 keV mm71) with an equation described in Kiefer & Straaten (1986). Figure 1b showed an example of a field of etch pits on an ion track detector, CR39, (instead of the glass coverslip) irradiated with 1500 particles of 12C ions using the f20 mm aperture. Approximately 70% of the particles hit into f20 mm diameter, and the majority of the rest hit between f20 mm and f50 mm diameter. The average doses to f20 mm diameter circle, and to the 15-mm-wide annulus around the central f20 mm diameter circle were 70 Gy and 5.7 Gy, respectively. It only takes a few seconds to be bombarded with 1500 particles using an automatic MHz shutter. The actual number of particles that pass through every specimen retained between the Slide Seal and the glass coverslip was counted with a plastic scintillatorphotomultiplier tube assembly and a constant fraction discriminator, following automatic change of the lens for the optical microscope to the assembly. Figure 1c and d show the specimens of L4 larvae and young gravid hermaphrodite overlaid with a field of etch pits produced by 1500 particles. The L4 larvae and young gravid hermaphrodites were then washed with M9 buffer and allowed to recover on NGM agar. In the case of L4 larvae, the main endpoint was induction of cell cycle arrest in irradiated germline cells and unirradiated cells. Thirty-six hours after irradiation, germ cell proliferation was visualized using 40 ,6-diamidino-2-phenylindole (DAPI) nuclear staining, and the dimensions of the anterior
Effects of microbeam on germline in C. elegans
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Figure 1. Irradiation using a collimated microbeam of 220 MeV 12C5þ particles delivered from the AVF cyclotron at TIARA onto the nematode C. elegans. (a) Control monitor of the microbeam irradiation onto a young gravid hermaphrodite. The red circle indicates the irradiated region with f20 mm diameter aperture. (b) An example of a field of etch pits on a CR39 ion track detector irradiated with 1500 particles of 12C ions using the f20 mm aperture. (c) Targets of tip region, posterior curved region (pachytene phase), and tail region of L4 larvae, and (d) of middle region (pachytene phase) and of tail region of young gravid hermaphrodite are illustrated and are overlaid with the etch pit of figure 1b, respectively.
and posterior gonad were measured with the Image J system (NIH imaging software, National Institute of Standards and Technology, Gaithersburg, Maryland, USA). In the case of young gravid hermaphrodites, the endpoint was DNA damage-induced apoptosis in late pachytene cells. Five hours after irradiation, apoptosis in germ cells was monitored using the differential interference contrast (DIC) microscopy (Gartner et al. 2000, Deng et al. 2004).
Results Effect of 12C ion broad beam irradiation on germline cells Following whole-body exposure to 12C ion-broad beam, the survival of eggs from young gravid hermaphrodites of N2 wild type, and abl-1 mutants,
was measured at 0 – 4 h and 11 – 15 h (Figure 2). Eggs laid between 0 – 4 h were irradiated at a stage of embryogenesis after fertilization; however, eggs laid between 11 – 15 h were irradiated at the pachytene stage of prophase, in meiotic division I. The sensitivity of both wild type and abl-1 mutants was almost identical. The hatching (survival) rate for eggs laid at 0 – 4 h, following 20, 40, and 60 Gy of wholebody irradiation, was 65, 20 and 2%, respectively (Figure 2). Survival of eggs laid 11 – 15 h following irradiation did not decrease by more than 50% at 60 Gy (Figure 2). Additionally, we measured the number of germ cell corpses caused by radiation-induced germ cell apoptosis at 5 and 18 h following whole-body irradiation of young gravid hermaphrodites, for both wild type and abl-1. In the abl-1 mutant, radiationinduced germ cell apoptosis was raised 1.5 to 2 fold over that of the wild type (Figure 3).
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Figure 2. Hatching rate of eggs laid by wild type and abl-1 mutant C. elegans hermaphrodites following whole-body irradiation with broad beam of 12C ions. Nine young gravid hermaphrodites (N2 wild type, circles; and XR1 abl-1 (ok171), squares) were irradiated with 20, 40, 60, and 100 Gy of 12C ion particles. Following irradiation, eggs laid from 0 – 4 h (open marks) and from 11 – 15 h (closed marks) were collected, and their hatching rates scored. Standard deviation is indicated by a vertical bar.
following manner (Figure 4a, b). The dimensions of the anterior and posterior gonads decreased by 60% compared to those of non-irradiated controls (Figure 4b). In addition to disappearance of mitotic metaphase plates, swollen cells as a result of mitotic cell-cycle arrest leading to a reduction of cell number were revealed in both tip regions of anterior and posterior gonad 36 h after irradiation (Table I). Irregularly condensed apoptotic nuclei were also observed in the pachytene region of both gonads (Table I). When the tip region of the gonad arm was bombarded locally with 1500 particles of 12C ions using f20 mm microbeam (equivalent local dose in the area of f20 mm diameter circle to 70 Gy of broad beam irradiation), similar inhibition of germ cell proliferation and swollen cells were caused in the irradiated gonad (Figure 4a, b and Table I). In contrast, no significant inhibition was detected in the gonad on the non-irradiated, anterior side (Figure 4 and Table I). On the other hand, meiotic prophase cells were normally observed in either gonad and all eggs laid proceeded to hatch. When the middle region (pachytene nuclei) of the posterior gonad arm or the tail region was bombarded with 1500 particles, no inhibition, none of swollen cells, but normal mitotic metaphase plates were detected in either gonad (Figure 4 and Table I). In the case of pachytene nuclei bombarded with local irradiation, apoptotic condensed nuclei were frequently observed, but only in the irradiated gonad arm (Table I).
Pachytene checkpoint apoptosis following microbeam irradiation
Figure 3. Induction of germ cell apoptosis by whole-body irradiation with a broad beam of 12C ions. (a) Germ cell corpses induced by radiation were observed in the distal arm of one side of the gonad, using DIC microscopy. (b) Young gravid hermaphrodites from N2 wild type (n ¼ 12) and abl-1 mutants (n ¼ 12) were irradiated with 60 and 100 Gy of 12C ions, and germ cell corpses were counted after 5 h (open boxes) and 18 h (hatched boxes). Standard deviation is indicated by a vertical bar.
Inhibition of germ cell proliferation by microbeam irradiation At the L4 stage, whole-body irradiation with 460 Gy of 12C ions arrested germ cell proliferation in the
The number of germ cell corpses resulting from radiation-induced germ cell apoptosis was measured 5 h following 12C ion microbeam irradiation in the N2 wild type and the abl-1 mutant adult hermaphrodites. When the posterior middle region (pachytene nuclei) of the wild type gonad arm was bombarded with 1500 particles, the number of corpses in the irradiated posterior gonad increased from 0.6 + 0.7 to 3.4 + 1.5 (mean + SD) (Figure 5); however, in abl-1 deletion mutants, the number of corpses increased from 2 + 1.7 to 5.8 + 2.1 (mean + SD) (Figure 5). In contrast, no significant increase in the number of corpses was detected for the non-irradiated anterior gonad (Figure 5). Additionally, when the tail region was bombarded with 1500 particles, germ-cell apoptosis did not increase in either the posterior or anterior gonad (Figure 5). Germ-cell apoptosis occurred in both the posterior and anterior gonad, when irradiated with 100 Gy of a broad beam of 12C ions, and occurred in
Effects of microbeam on germline in C. elegans
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Figure 4. Arrest of germ cell proliferation caused by local irradiation with 12C ion microbeam. (a) Thirty six h following irradiation at the L4 stage, germ cell proliferation was visualized using DAPI, and typical results are indicated: A, non-irradiated control; B, whole-body irradiation (100 Gy) with broad beam of 12C particles; C, local irradiation of the tail region with 1500 particles, using f20 mm microbeam; D, local irradiation of the tip of the posterior gonad arm with 1500 particles, using f20 mm microbeam. Scale bar indicates 20 mm. (b) The dimensions of the anterior (open boxes) and posterior (shaded boxes) gonads were measured using the Image J system (NIH imaging software). Results of broad beam (60 and 100 Gy) and microbeam (1500 and 3000 particles/f20 mm) irradiation of the L4 stage: Whole worm (broad beam); tail (tail); pachytene region of the posterior gonad (pachyt); and the tip of the posterior gonad arm (tip). Twelve animals were used in each experiment. Standard deviation is indicated by a vertical bar.
the irradiated posterior gonad when irradiated by microbeam (Figure 5). Discussion Using the nematode C. elegans, we studied the sensitivity of the developmental phases of oogenesis to radiation (Takanami et al. 2000, 2003). We demonstrated that the meiotic pachytene oocytes were less sensitive to 220 MeV of 12C ions, than were post-fertilization, early embryos (Figure 2). This result is consistent with our previous investigations
(Takanami et al. 2000, 2003). High linear energy transfer (LET) radiation generates complex rearrangements of chromosomal structures, whereas low LET radiation only generates complex alterations at high doses (Sachs et al. 2000). High LET heavy-ion irradiation induces deletions that are generally greater than 1,000 base pairs; low LET induces base substitutions, and deletions of less than 100 base pairs (Masumura et al. 2002). The data in this study indicate that the meiotic homologous recombination system can effectively repair the more severe DNA damage caused by high LET radiation.
Control (non-irradiated) Whole-body irradiation (100 Gy) Local-irradiation of tail region (1500 particles) Pachytene region of posterior gonad (1500 particles) Tip region of posterior gonad (1500 particles)
Individuals showed more than 3 mitotic metaphase plates per gonad. bIndividuals showed more than 2 swollen cells per gonad. cIndividuals showed more than 3 apoptotic condensed nuclei per gonad.
7 (0b/12) þ (12b/12) 7 (0b/12) 7 (0b/12) 7 (0b/12) þ (11a/12) 7 (0a/12) þ (10a/12) þ (12a/12) þ (11a/12)
N2 wild type (n ¼ 12)
a
7 (0c/12) þ (7c/12) 7 (0c/12) þ (3c/12) 7 (0c/12) 7 (0b/12) þ (12b/12) 7 (0b/12) 7 (0b/12) þ (12b/12) þ (10a/12) 7 (0a/12) þ (10a/12) þ (11a/12) 7 (0a/12) 7 (0c/12) þ (4c/12) 7 (0c/12) 7 (0c/12) 7 (0c/12)
Pachytene region Apoptotic condensed nuclei Swollen cells with cell cycle arrest Swollen cells with cell cycle arrest Mitotic metaphase plates
Tip region
Anterior gonad
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Pachytene region Apoptotic condensed nuclei
C ions on germline cells of L4 larvae.
Tip region
Posterior gonad
Mitotic metaphase plates
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Table I. Effects of whole- and local-irradiation of
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Figure 5. Germ-cell apoptosis caused by 12C ion microbeam irradiation. The germ cell corpses in anterior (open boxes) and posterior (shaded boxes) gonads were observed 5 h following irradiation, using DIC microscopy. Results of broad beam (100 Gy) and microbeam (1500 particles) irradiation of the young gravid adult stage: Whole worm (broad); tail (tail); and pachytene region of the posterior gonad (pachyt). Twelve animals were used in each experiment. Standard deviation is indicated by a vertical bar.
Following local irradiation of the tip region of the gonad arm at the L4 stage with 12C ion microbeam, inhibition of germ cell proliferation and swollen cells were revealed in the targeted gonad arm (Figure 4 and Table I). On the other hand, neighboring meiotic prophase cells (pachytene nuclei) developed normally in the gonad irradiated on the tip region and all eggs laid were able to hatch. The inhibition and presence of swollen cells did not occur with microbeam irradiation at other sites, such as the middle region of the gonad arm, or the tail region (Figure 4 and Table I). The current study suggests that heavy-ion microbeam radiation could be used as a microsurgery tool, in a manner similar to that of laser ablation. Additionally, it is well known that ionizing radiation causes cell inactivation as a result of DNA damage, without causing significant damage to cytoplasm, cell membrane and so on. However, it is still necessary to develop a fine control system for target irradiation with charged particles. A similar observation was made in a study using laser ablation, in which the distal tip cells of the somatic gonad control germ cell proliferation, and germ cells in the gonad arm that have undergone laser ablation enter meiosis and differentiate into gametes (Kimble & White 1981). With the present system, we could not irradiate only the distal tip cells with microbeams. Approximately 15 – 20 germline cells including the distal tip cells, which expand in f50 mm diameter, were bombarded with 1500 particles of 12C ions using the f20 mm aperture (Figure 1). Microbeam irradiation induces pachytene checkpoint apoptosis in germ cells (Figure 5). Deng et al. (2004) reported that C. elegans ABL-1 antagonizes the germline apoptosis induced by g-radiation, but not by the DNA-alkylating agent ethylnitrosourea.
Effects of microbeam on germline in C. elegans The results in Figures 2, 3 and 5 indicate that the abl-1 mutant hermaphrodites are hypersensitive to apoptosis induced by heavy-ion particle irradiation, whereas their DNA repair activity (as evidenced by survival rate) was almost the same as wild-type. In both the hypersensitive mutant and the wild type hermaphrodites, germ cell corpses only occurred in the irradiated gonads, and were not found in the non-irradiated gonads on the opposite side, nor were they found following irradiation of the tail (Figure 5). These results indicate that DNA damage induced cell cycle arrest and apoptosis in the microbeam irradiated region of L4 larvae and young gravid hermaphrodites, respectively, but ‘bystander effects’ were not observed in non-irradiated neighboring regions. In contrast, in mammalian systems, many bystander effects have been observed and reported (Belyakov et al. 2005, Zu et al. 2005, Hall & Hei 2003, Shao et al. 2003, Iyer & Lehnert 2000, Mothersill & Seymour 2001). In particular, Belyakov et al (2005) recently reported that unirradiated cells up to 1 mm distant from irradiated cells showed a significant ‘bystander effect’ for induction of micronucleated and apoptotic cells in reconstituted human skin tissue. The 1 mm distance is just identical to the body size of C. elegans adult hermaphrodite. A possible reason for the absence or reduction of ‘bystander effect’ in C. elegans may be due to lack of communication between completely different organs (between gonad and body tail) and/or the defined part in the same organ (between meiotic pachytene nuclei and mitotic germline cells). Germline cells may be isolated and protected from radiationinduced ‘bystander’ responses. However, it is known that several materials, e.g., doubled stranded RNA (dsRNA) can be delivered from body cavity into germline cells of gonad. Another possibility is the absence of a nitric oxide (NO) signal transduction system, since no orthologs of a gene encoding an inducible NO synthase (iNOS) has been found in the genome. The NO signal transduction pathway, using iNOS, is one of the known radiation-induced bystander responses (Matsumoto et al. 2001, Shao 2002). Acknowledgements We thank the C. elegans Genetic Center for kindly supplying the mutant strain. We are also grateful to the staff of the TIARA of JAEA, for their assistance with the heavy-ion particle irradiation. This work was supported by the TIARA Cooperative Research Program (41043, 51044), by a Grant for Japan Space Forum and by a Grant-in-Aid for Scientific Research (16310033) from the Ministry of Education, Culture, Sports, Science, and Technology.
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