Sep 06, 2008

Kensuke Otsuka (Central Research Institute of Electric Power Industry)

Low-dose acute X-ray (0.5 Gy) for priming 2 weeks prior to a high-dose (6 Gy) challenge irradiation is known to improve survival rate of mice. In order to understand the mechanism of the whole-body adaptive response, we focused on recovery of hematopoietic cells after challenge irradiation. Bone marrow cells, erythrocytes and platelets in low-dose-preirradiated mice showed rapid recovery after the challenge irradiation than those in mice subjected only to the challenge irradiation. The rapid recovery of bone marrow cells after the challenge irradiation was consistent with the proliferation of primitive hematopoietic progenitor cells. A subpopulation of myeloid cells, which were descendants of primitive progenitor cells, rapidly recovered in the low-dose-preirradiated mice, whereas the number of B-lymphoid cells did not show a statistically significant increase. Furthermore, concentrations of several growth factors for myelopoiesis after the challenge irradiation were considerably increased by low-dose preirradiation. Thus the adaptive response induced by low-dose preirradiation in terms of the recovery kinetics of the number of hematopoietic cells may be due to the rapid recovery of the number of myeloid cells after high-dose irradiation.

References:
Otsuka et al. Recent insights into the biological action of heavy-ion radiation.Rapid myeloid recovery as a possible mechanism of whole-body radioadaptive response
Radiat Res 170: 307-315 (2008)

Sep 06, 2008

Mitsuaki Ojima (Oita University of Nursing and Health Sciences)

Phosphorylated ATM immunofluorescence staining was used to investigate the dose-response relationship for the number of DNA double-strand breaks (DSBs) induced in primary normal human fibroblasts irradiated with doses from 1.2 to 200 mGy.The induction of DSBs showed a supralinear dose-response relationship. Radiation-induced bystander effects may explain these findings. To test this hypothesis, the number of DSBs in cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; a supralinear dose-response relationship was not observed. Moreover, the number of DSBs obtained by subtracting the number of phosphorylated ATM foci in lindane-treated cells from the number of phosphorylated ATM foci in untreated cells was proportional to the dose at low doses (1.2-5 mGy) and was saturated at doses from 10-200 mGy. Thus the increase in the number of DSBs in the range of 1.2-5 mGy was largely due to radiation-induced bystander effects, while at doses >10 mGy, the DSBs may be induced mainly by dose-dependent direct radiation effects and partly by dose-independent radiation-induced bystander effects. The findings in our present study provide direct evidence of the dose-response relationship for radiation-induced bystander effects from broad-beam X rays.

References:
Ojima et al. DNA double strand breaks induced by very low X-ray doses are largely due to bystander effects
Radiat Res 170: 365-371 (2008)

Aug 31, 2008

Nobuyuki Hamada (Graduate School of Medicine, Gunma University)

During cancer therapy or long-term manned space travel, humans are exposed to energetic heavy ions that inactivate cells more effectively than photons like X-rays and gamma-rays. Growing evidence indicates that heavy ions overcome tumor radioresistance caused by p53 mutations, Bcl-2 overexpression and intra-tumor hypoxia, and have anti-angiogenic and anti-metastatic potential. Compared with heavy ions alone, the combination with chemical agents (a Bcl-2 inhibitor HA14-1, an anticancer drug docetaxel, and a halogenated pyrimidine analogue IUdR) or hyperthermia has been reported to further enhance tumor cell killing. Certain constituents of beer or melatonin ameliorate heavy ion-induced damage to normal cells. In addition to effects in cells directly targeted by heavy ions, evidence is increasing for non-targeted biological effects in cells that have not themselves been directly irradiated. Gap junctional intercellular communication, reactive oxygen species and nitric oxide have been proposed to underlie the bystander effect of heavy ions. This review focuses on recent findings regarding potential benefits of heavy ions for therapeutic use as well as on the bystander effect.

References:
Hamada et al. Recent insights into the biological action of heavy-ion radiation
J Radiat Res 50(1): 1-9 (2009)

Aug 21, 2008

Nobuyuki Hamada (Graduate School of Medicine, Gunma University)

The goal of radiation therapy for cancer is to eradicate tumors without harming healthy tissues. Despite considerable interest in combined modalities (e.g., chemoradiotherapy) or in molecularly targeted approaches to radiosensitize tumors for conventional photon therapy, such information is very limited for heavy-ion therapy. Our recent work has illustrated that the small-molecule Bcl-2 inhibitor HA14-1 renders human cervical cancer cells and their Bcl-2 overexpressing radioresistant counterparts, but not normal fibroblasts, more vulnerable to heavy ions in vitro. This suggests that Bcl-2 may be an attractive target for improving the efficacy of heavy-ion therapy. Further identification of the potential agents or combination regimens, which could enhance tumor control without aggravating or even with assuaging normal tissue complications, should maximize therapeutic outcome.

References:
Hamada et al. The small-molecule Bcl-2 inhibitor HA14-1 sensitizes cervical cancer cells, but not normal fibroblasts, to heavy-ion radiation
Radiother Oncol 89, 227-230 (2008)

Jul 15, 2008

Hiroshi Harada (Graduate School of Medicine, Kyoto University)

As the transcriptional activity of hypoxia-inducible factor 1 (HIF-1) is associated with resistance of tumor cells to current antitumor therapies, the spatiotemporal dynamics of HIF-1-active regions has been of great interest as a therapeutic target. In the present study, we established a unique cancer cell line, which changes color HIF-1-dependently, and monitored it during tumor progression. In imaging experiments, HIF-1-active cells appeared over the tumor xenograft, but dramatically decreased in number as blood vessels developed around the tumor. The remaining HIF-1-active cells at the center of the xenograft also disappeared after neovascularization. Thereafter, tumor growth was accelerated and HIF-1-active cells reappeared in different regions. The distance between HIF-1-active cells and the nearest vessels correlated to the diameter of the vessel (r = 0.801). These results provide a basic knowledge of how to estimate the spatiotemporal dynamics of HIF-1-active cells using information about the image-guided architecture of tumor blood vessels.

References:
Harada et al. Diameter of tumor blood vessels is a good parameter to estimate HIF-1-active regions in solid tumors
Biochem Biophys Res Commun 373(4):533-8 (2008)

Jun 25, 2008

Hiroshi Harada (Graduate School of Medicine, Kyoto University)

Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than the normal tissues. Hypoxia offers resistance to radiotherapy and anticancer chemotherapy, as well as predispose to increased tumor metastases. Furthermore, hypoxia induces hypoxia-inducible factor-1 (HIF-1), which in turn increases tumor angiogenesis. Thus, eradication of HIF-1-active/hypoxic tumor cells is very important for cancer therapy. We have previously reported that procaspase-3 fused with a von Hippel-Lindau (VHL)-mediated protein destruction motif of alpha subunit of HIF-1 (HIF-1alpha) containing Pro564, named TAT-ODD-procaspase-3 (TOP3), specifically induced cell death to hypoxic cells in vivo as well as in vitro. We now report that TOP3 also eradicates the radiation-induced HIF-1-active tumor cells. HIF-1 activity in the xenografts of human tumor cells, which express luciferase under the transcriptional control of HIF-1, were monitored and quantified daily with an in vivo bioluminescence photon-counting device. HIF-1 activity in tumors was more rapidly increased by ionizing radiation (IR) compared to untreated tumors. TOP3 efficiently decreased the HIF-1-activity in irradiated tumors as well as unirradiated ones, indicating TOP3 eradicated tumor cells with HIF-1-activity induced by IR as well as hypoxia. Eradication of HIF-1-active/hypoxic cells in the xenografts during irradiation exhibited significant suppression in angiogenesis and strong enhancement in a long-term growth suppression of tumor xenografts. These results further strengthen the argument that HIF-1-active/hypoxic cells play crucial roles in angiogenesis and radioresistance.

References:
Harada et al. Significance of HIF-1-active cells in angiogenesis and radioresistance
Oncogene 26, 7508-16 (2007)

Jun 25, 2008

Hiroshi Harada (Graduate School of Medicine, Kyoto University)

Hypoxia is closely associated with the radioresistance of tumours; therefore, targeting hypoxic areas is very important for cancer therapy. The aim of this study is to establish such a targeting strategy by applying a bacterial cytosine deaminase (BCD)/5-fluorocytosine (5-FC) gene therapy system and to examine whether the strategy enhances the efficacy of radiotherapy in a tumour xenograft. The hypoxia-responsive promoter 5HREp, in which five copies of the hypoxia-response element (HRE) enhance transcription from a cytomegalovirus minimal promoter, was employed to induce the expression of BCD under hypoxic conditions. The adenoviral vector Ad/5HREp-BCD, encoding the gene 5HREp-BCD, robustly induced BCD expression under hypoxic conditions and this led to significant cytotoxicity in combination with 5-FC in vitro. Intratumoral Ad/5HREp-BCD administration resulted in the expression of BCD at the border between normoxic and necrotic regions. The BCD/5-FC gene therapy enhanced the therapeutic effects of both single (12.5 Gy) and fractionated (3 Gy x 5 days) radiotherapy with few side effects and significantly increased tumour growth doubling time by up to 2.4-fold (P<0.01) and 2.5-fold (P<0.05), respectively. All of these results suggest that the present BCD/5-FC gene therapy has the ability to specifically target hypoxic tumour cells and significantly improves the control of tumour growth after radiotherapy.

References:
Liu et al. Adenovirus-mediated hypoxia-targeting cytosine deaminase gene therapy enhances radiotherapy in tumour xenografts
Br J Cancer 96, 1871-1878 (2007)

Jun 25, 2008

Hiroshi Harada (Graduate School of Medicine, Kyoto University)

The transcriptional activity of hypoxia-inducible factor-1 (HIF-1) is associated with tumor malignancies; therefore, it is important to comprehend its dynamism in solid tumors. However, a molecular imaging strategy to accurately access it remains to be developed. We constructed here a novel HIF-1-dependent reporter gene, 5HREp-ODD-luc, in which 5 copies of the hypoxia-response element (5HRE) enhance expression of the oxygen-dependent degradation (ODD) domain and luciferase (luc) fusion under hypoxia. Because the ODD domain caused the oxygen-dependent degradation of the ODD-Luc protein, the novel reporter gene showed little leak of luminescence under normoxia. Such a property caused an increase of the hypoxia-responsiveness up to about 4.7 x 10(4) -fold. Moreover, the ODD domain caused rapid degradation of the ODD-Luc protein under normoxia, the luminescence reflected the dynamism of HIF-1 activity in real-time. The superiority of the novel reporter gene will surely accelerate analysis of the intratumoral HIF-1 activity during tumor progression and cancer treatments.

References:
Harada et al. The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts
Biochem Biophys Res Commun 360, 791-796 (2007)

May 20, 2008

Tatsuhiko Imaoka (National Institute of Radiological Sciences)

The cancer risk from low-dose radiation is difficult to estimate because it should be far smaller than the background risk of spontaneous cancer development. Currently, such low-dose risks are estimated by extrapolating high-dose risks, though a large uncertainty accompanies this. Molecular signatures of radiation-related cancer could therefore improve the risk estimation at low doses. The present study attempted to distinguish between spontaneous and radiation-induced rat mammary cancers by comparing their gene expression profiles obtained with microarrays. Hierarchical clustering analysis successfully discriminated cancers that developed spontaneously (i.e., those arose in non-irradiated rats) from radiation-induced cancers (i.e., those developed in gamma-irradiated rats). Although most gene expressions were common, several tens of genes had different expression levels between spontaneous and radiogenic cancers. This was further confirmed by quantitative PCR on selected genes. Some of these genes had known important functions in the mammary gland, whereas others might have previously unidentified functions. Thus, gene expression analysis distinguished between spontaneous and radiogenic carcinomas, suggesting possible differences in their carcinogenic mechanisms.

References:
Imaoka et al. Gene expression profiling distinguishes between spontaneous and radiation-induced rat mammary carcinomas
J Radiat Res 49(4):349-60 (2008)

Apr 29, 2008

Nobuyuki Hamada (Graduate School of Medicine, Gunma University)

Ionizing radiation-induced bystander effects have been demonstrated in a variety of endpoints such as mutations, chromosome aberrations and cell death, which occur in nonirradiated bystander cells having received signals from directly irradiated cells [1,2]. However, little information exists on the bystander effect of heavy ions. Less irradiated cells should coexist with more nonirradiated counterparts in a population exposed to lower dose of higher-LET heavy ions [3], so that a clarification of the effects arising not merely in irradiated cells but in their bystander cells would be crucial to comprehend the mechanism of action of heavy ions. We have investigated heavy ion-induced bystander response in confluent human fibroblast cultures. First, microirradiation of 0.0003% of cells, and conventional broadfield irradiation were performed to see the effects in bystander and irradiated cells, respectively. Intriguingly, bystander cells manifested a more transient apoptotic response and delayed p53 phosphorylation, compared with irradiated cells [4]. Taken together, nearly three quarters of the genes whose expression changed in bystander cells were downregulated, and most of the genes upregulated in irradiated cells were downregulated in bystander cells [5]. These findings highlight the distinct response of irradiated and bystander cells. Furthermore, interleukin genes were upregulated in irradiated cells whereas its receptor gene was upregulated in bystander cells [5], suggestive of the signal transmission from irradiated to bystander cells. Second, chromosome aberrations were analyzed in cells treated with conditioned medium from X- or heavy ion-irradiated cells. We found the difference in the types of aberrations, but very little in the total aberration yields [6], indicating that bystander responses occur independently of radiation types but are induced through different mechanisms. Collectively, these induced bystander responses could be a defensive mechanism that would avert or minimize further expansion of aberrant cells.

References:
[1] Hamada et al. Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects
J Radiat Res 48, 87-95 (2007)
[2] Matsumoto, Hamada et al. Vanguards of paradigm shift in radiation biology: radiation-induced adaptive and bystander responses
J Radiat Res 48, 97-106 (2007)
[3] Funayama, Hamada et al. Heavy-ion microbeams-development and applications in biological studies
IEEE Trans Plasma Sci 36, 1432-40 (2008)
[4] Hamada et al. Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions
Mutat Res 639, 35-44 (2008)
[5] Iwakawa, Hamada et al. Expression profiles are different in carbon ion-irradiated normal human fibroblasts and their bystander cells
Mutat Res 642, 57-67 (2008)
[6] Kanasugi, Hamada et al. Role of DNA-PKcs in the bystander effect after low-or high-LET irradiation
Int J Radiat Biol 83, 73-80 (2007)

Apr 16, 2008

Yoshitaka Matsumoto (National Institute of Radiological Sciences)

The purpose of this study is to elucidate the molecular changes in response to carbon beams (C-ions) in melanoma. We examined expression profiles of 6 melanoma cell lines exposed to C-ions or X-rays with 2 Gy using single-color microarrays. Twenty-two genes, including NFKBIA, responded to C-ions in all six cell lines, based on analysis of variance (ANOVA) filtering (p < 0.001). We found 173 genes that responded in common to C-ions in four cell lines. We identified many down-regulated genes including the cell cycle - related genes that were more responsive to C-ions than X-rays. In contrast, most of the up-regulated genes including p53 target genes responded to both C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (p < 0.05). Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle - related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays.

References:
Matsumoto et al. Gene expression analysis in human malignant melanoma cell lines exposed to carbon beams
Int J Radiat Biol 84, 299-314 (2008)

Mar 25, 2008

Nobuyuki Hamada (Graduate School of Medicine, Gunma University)

Ionizing radiation-induced genomic instability has been demonstrated for a multitude of endpoints such as delayed reproductive death and chromosome instability, which arise in the descendants of survivors multiple generations after the initial insult. Our previous work has shown that delayed reductions in clonogenicity can increase LET-dependently and be most pronounced in normal human fibroblasts exposed to carbon ions (108 keV/µm) [1]. To gain insight into this potential cellular mechanism, morphological changes induced in colonies were further analyzed. The data have illustrated that carbon ions facilitate differentiation as a major response in the progeny of irradiated fibroblasts [2]. Accordingly, accelerated differentiation may potentially explain LET-dependent delayed loss of clonogenicity in normal human fibroblasts, and could be a defensive mechanism that would minimizes further expansion of aberrant cells. Moreover, LET-dependent delayed loss of clonogenicity was also found in human cervical cancer cells regardless of Bcl-2 overexpression [3].

References:
[1] Hamada et al. LET-dependent survival of irradiated normal human fibroblasts and their descendents
Radiat Res 166, 24-30 (2006)
[2] Hamada et al. Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts
Mutat Res 637, 190-196 (2008)
[3] Hamada et al. The survival of heavy ion-irradiated Bcl-2 overexpressing radioresistant tumor cells and their progeny
Cancer Lett 268, 76-81 (2008)

Mar 05, 2008

Motohiro Yamauchi (Graduate School of Biomedical Sciences, Nagasaki University)

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases cocomitantly with DNA double-strand break repair, some fraction of foci persists.To date, the physiological role of the persistent foci has been poorly understand. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1 Gy of X-rays. While the initial foci size was approximately 0.6 um, the one or two of persitent forcus (foci) grew, whose diameter reached 1.6 um or more in diameter at 24h after IR.All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1 Gy of X-rays and incubated for 24 h, the grown large phosphorylated ATM foci (>1.6 um) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6 um) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint. These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.

References:
Yamauchi et al. Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling
DNA Repair 7, 405-17 (2008)

Feb 29, 2008

Nobuyuki Hamada (Graduate School of Medicine, Gunma University)

The biological effectiveness is known to vary with the linear energy transfer (LET) of ionizing radiation. In contrast to conventional low-LET photons like X-rays and gamma-rays, energetic heavy ions offer potential therapeutic advantage of their excellent dose distribution and enhanced biological effectiveness. Hitherto, heavy-ion therapy has achived good cancer controllability while sparing critical normal organs. Overexpression of Bcl-2 occurs in nearly half of human cancers and has been associated with the resistance to conventional photons (referred hereinafter to as "radioresistance") and to various chemotherapeutic agents. Our recent work has shown that heavy ions can overcome in vitro tumor radioresistance caused by Bcl-2 overexpression, which may be potentially accounted for by the enhanced apoptotic response and prolonged G2/M arrest. Thus, heavy-ion therapy may be an important modality for Bcl-2 overexpressing radioresistant tumors. Together, other studies have documented that radioresistance of p53-mutated and hypoxic cellscan also be overcome with heavy ions. Altogether, heavy ions appear to effectively kill a wide variety of radioresistant tumors .

References:
Hamada et al. Energetic heavy ions overcome tumor radioresistance caused by overexpression of Bcl-2
Radiother Oncol 89, 231-236 (2008)

Feb 29, 2008

Tatsuhiko Imaoka (National Institute of Radiological Sciences)

Heavy-ion radiotherapy is expected to improve cancer treatment results by achieving accurate dose lacalization and a high relative biological effectiveness (RBE) due to the sharpe Bragg peak and high energy transfer (LET) of radiation. The carbon ion radiation of 290 MeV/u with 6-cm spread-out Bragg peak (SOBP) has been successfully used to treat several cancer types. However, the potential risk for developing secondary cancers is now becoming a matter of concern, and it is important to determine the RBE value of therapeutic heavy ions for inducting late occuring cancers. We aimed at providing two RBE values, one for risk estimation of theraputic and the other for consideration in radiation protection. We first used four rat strains to investigate the effect on mammary carcinogenicity of carbon ions of 290 MeV/u with 6-cm SOBP from HIMAC, a synchrotron facility at the National Institute of Radiological Sceiences. upon identifying the Sparague-Dawley rat as susceptible, we then studied the dose-effect relationship and calculated RBE. Carcinomas were then further analyzed for their molecular features such as estrogen recepter alpha status and mutations in the genes H-ras and Tp53. We found that carbon ions induced rat mammary carcinomas in a strain-dependent manner. An RBE value of 2 was appropriate for risk estimation of therapeutic doses, whereas a value of ~10 should be considered in protection against low doses. We found that most carcinomas were positive for estrogen recepter and that all tumors examined lacked H-ras and Tp53 mutations. These results have potential implications for ongoing thoracic heavy-ion radiotherapy, suggesting that genetically susceptible patients may be at higher risk for developing secondary breast cancers.

References:
Imaoka et al. High relative biologic effectiveness of carbon ion radiation on induction of rat mammary carcinoma and its lack of H-ras and Tp53 mutatuins
Int J Radiat Oncol Biol Phys 69(1):194-203 (2007)