Significance of manipulating tumour hypoxia and radiation dose rate in terms of local tumour response and lung metastatic potential,referring to the response of quiescent cell populations

The purpose of this study was to evaluate the influence of manipulating intratumour oxygenation status and radiation dose rate on local tumour response and lung metastases following radiotherapy, referring to the response of quiescent cell populations within irradiated tumours. B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation at high dose rate (HDR) or reduced dose rate (RDR) following treatment with the acute hypoxia-releasing agent nicotinamide or local hyperthermia at mild temperatures (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the quiescent (Q) and total (proliferating + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Following HDR irradiation, nicotinamide and MTH enhanced the sensitivity of the total and Q-cell populations, respectively. The decrease in sensitivity at RDR irradiation compared with HDR irradiation was slightly inhibited by MTH, especially in Q cells. Without γ-ray irradiation, nicotinamide treatment tended to reduce the number of lung metastases. With γ-rays, in combination with nicotinamide or MTH, especially the former, HDR irradiation decreased the number of metastases more remarkably than RDR irradiation. Manipulating both tumour hypoxia and irradiation dose rate have the potential to influence lung metastasis. The combination with the acute hypoxia-releasing agent nicotinamide may be more promising in HDR than RDR irradiation in terms of reducing the number of lung metastases.Many cells in solid tumours are quiescent in situ but still clonogenic [1]. Quiescent (Q) tumour cells are thought to be more resistant to low LET radiation because of their larger hypoxic fraction and greater capacity to recover from radiation-induced DNA damage than proliferating (P) tumour cells [1]. Actually, our original method for selectively detecting the response of intratumour Q cells [1] verified those characteristics of Q-cell population [1] and made it possible to evaluate the usefulness of various modalities for cancer therapy in terms of effectiveness against Q-cell populations within local tumours. Based on the characteristics of the response of intratumour Q cells to various DNA-damaging treatments obtained so far [1], more effective and useful treatment modalities for local tumour control can be developed.Metastasis is a leading cause of cancer deaths and involves a complex, multistep process by which tumour cells disseminate to distant sites to establish discontinuous secondary colonies [2, 3]. It was reported that acute and cyclic, but not chronic, hypoxia significantly increased the number of spontaneous lung metastases in mice by a factor of about 2, and that this effect was due to the influence of the acute hypoxia treatment on the primary tumour and not to other potential effects of the treatment such as damage to the lung epithelium [4, 5]. Based on this report, we recently reported the significance of injection of an acute hypoxia-releasing agent, nicotinamide, into tumour-bearing mice as a combined treatment with high dose rate (HDR) γ-ray irradiation in terms of repressing lung metastasis [6]. However, when combined with reduced dose rate (RDR) γ-ray irradiation, the significance of manipulating hypoxia within local solid tumours has not yet been clarified in terms of lung metastasis. Meanwhile, concerning local tumour control, it was already reported that manipulating hypoxia in solid tumours during RDR as well as HDR γ-ray irradiation influences the radiosensitivity of local tumours, especially with γ-rays [7].Thus, the aim of the current in vivo study is to elucidate the significance of the nicotinamide treatment as a combined treatment with RDR γ-ray irradiation in terms of lung metastases compared with the combination with mild temperature hyperthermia (MTH), which had already been shown to have the potential to manipulate intratumour hypoxia [8], and preferentially release diffusion-limited chronic hypoxia according to our previous reports [6, 9, 10]. In addition, concerning the local tumour response to γ-ray irradiation with or without nicotinamide or MTH, the effect not only on the total (P + Q) tumour cell population but also on the Q-cell population was also evaluated using an original method of detecting the response of Q cells in solid tumours [1].     

Reducing intratumour acute hypoxia through bevacizumab treatment, referring to the response of quiescent tumour cells and metastatic potential

Objectives

The aim was to evaluate the influence of bevacizumab on intratumour oxygenation status and lung metastasis following radiotherapy, with specific reference to the response of quiescent (Q) cell populations within irradiated tumours.

Methods

B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation following treatment with the acute hypoxia-releasing agent nicotinamide or local mild temperature hyperthermia (MTH) with or without the administration of bevacizumab under aerobic conditions or totally hypoxic conditions, achieved by clamping the proximal end of the tumours. Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In the other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.

Results

3 days after bevacizumab administration, acute hypoxia-rich total cell population in the tumour showed a remarkably enhanced radiosensitivity to γ-rays, and the hypoxic fraction (HF) was reduced, even after MTH treatment. However, the hypoxic fraction was not reduced after nicotinamide treatment. With or without γ-ray irradiation, bevacizumab administration showed some potential to reduce the number of lung metastases as well as nicotinamide treatment.

Conclusion

Bevacizumab has the potential to reduce perfusion-limited acute hypoxia and some potential to cause a decrease in the number of lung metastases as well as nicotinamide.It was believed that antiangiogenic therapy prevents tumour vascular growth and proliferation and deprives the tumour of oxygen and nutrients necessary for survival [1]. However, subsequent study has suggested that antiangiogenic therapy may also “normalise” the tumour vasculature for a short period of time, thereby providing a window of opportunity for improved drug delivery and enhanced sensitivity to radiation [1,2]. Tumour hypoxia results from either limited oxygen diffusion (chronic hypoxia) or limited perfusion (acute hypoxia) [3]. Furthermore, it has been reported that acute and cyclic, but not chronic, hypoxia significantly increases the number of spontaneous lung metastases, and that this effect is due to the influence of acute hypoxia treatment on the primary tumour [4,5].In this study, we attempted to analyse hypoxia in solid tumours after the administration of the vascular endothelial growth factor (VEGF) inhibitor, bevacizumab, using the acute hypoxia-releasing agent nicotinamide combined with γ-ray irradiation in terms of both local tumour response and lung metastasis compared with irradiation combined with mild temperature hyperthermia (MTH), which has already been shown to have the potential to release tumour cells from diffusion-limited chronic hypoxia [6,7]. In addition, for the local tumour response, the effect on the total (proliferating (P)+quiescent (Q)) tumour cell population and on the Q cell population was evaluated using our original method for detecting the response of Q cells in solid tumours [8].     

Radiosensitivity of pimonidazole-unlabelled intratumour quiescent cell population to γ-rays,accelerated carbon ion beams and boron neutron capture reaction

Objective

To detect the radiosensitivity of intratumour quiescent (Q) cells unlabelled with pimonidazole to accelerated carbon ion beams and the boron neutron capture reaction (BNCR).

Methods

EL4 tumour-bearing C57BL/J mice received 5-bromo-29-deoxyuridine (BrdU) continuously to label all intratumour proliferating (P) cells. After the administration of pimonidazole, tumours were irradiated with c-rays, accelerated carbon ion beams or reactor neutron beams with the prior administration of a ¹⁰B-carrier. Responses of intratumour Q and total (P+Q) cell populations were assessed based on frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of pimonidazole-unlabelled tumour cells was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole.

Results

Following c-ray irradiation, the pimonidazole-unlabelled tumour cell fraction showed significantly enhanced radiosensitivity compared with the whole tumour cell fraction, more remarkably in the Q than total cell populations. However, a significantly greater decrease in radiosensitivity in the pimonidazole-unlabelled cell fraction, evaluated using a delayed assay or a decrease in radiation dose rate, was more clearly observed among the Q than total cells. These changes in radiosensitivity were suppressed following carbon ion beam and neutron beam-only irradiaton. In the BNCR, the use of a ¹⁰B-carrier, especially L-para-boronophenylalanine-¹⁰B, enhanced the sensitivity of the pimonidazole-unlabelled cells more clearly in the Q than total cells.

Conclusion

The radiosensitivity of the pimonidazole-unlabelled cell fraction depends on the quality of radiation delivered and characteristics of the ¹⁰B-carrier used in the BNCR.

Advances in knowledge

The pimonidazole-unlabelled subfraction of Q tumour cells may be a critical target in tumour control.Human solid tumours are thought to contain moderately large fractions of quiescent (Q) tumour cells, which are not involved in the cell cycle and have stopped dividing, but which are as viable as established experimental animal tumour lines [1]. The presence of Q cells is probably due, at least in part, to hypoxia and the depletion of nutrition in the tumour core as a consequence of poor vascular supply [1]. As a result, with the exception of non-viable Q cells at the very edge of the necrotic rim where there is diffusion-limited hypoxia, Q cells are viable and clonogenic, but have ceased dividing.Using our method for selectively detecting the response of Q cells in solid tumours to treatment that damages DNA, the Q cell population in solid tumours has been shown to exhibit more resistance to conventional radiotherapy and chemotherapy [2]. The Q cell population has also been demonstrated to have greater capacity to recover from radiotherapeutic and chemotherapeutic agent-induced damage and to have a significantly larger hypoxic fraction (HF) irrespective of the p53 status of tumour cells [2]. However, the Q cell population in solid tumours has never been shown to be fully hypoxic [2]. Actually, the size of the HF of Q cell populations in SCC VII squamous cell carcinomas, implanted in the hind legs of C3H/He mice and with a diameter of 1 cm, was 55.1 ± 6.2% (mean ± standard error) [3]. Thus, this value was significantly less than 100%, indicating that the Q cell population undoubtedly includes oxygenated tumour cells.A few years ago, the universal detection of hypoxic cells in both tissues and cell cultures became possible using pimonidazole (a substituted 2-nitroimidazole) and a mouse immunoglobulin (Ig)G1 monoclonal antibody (MAb1) to stable covalent adducts formed through reductive activation of pimonidazole in hypoxic cells [4]. Here, we tried to selectively detect the response of the pimonidazole-unlabelled and probably oxygenated cell fraction of the Q cell population. To achieve this we combined our method for selectively detecting the response of Q cells in solid tumours with the method for detecting cell and tissue hypoxia using pimonidazole and MAb1 to pimonidazole.High-linear energy transfer (LET) radiation including neutrons is more effective [2] than low-LET X- or γ-radiation at inducing biological damage. High-LET radiation shows a higher relative biological effectiveness (RBE) value for cell killing, a reduced oxygen effect and a reduced dependence on the cell cycle [2,5], making it potentially superior to low-LET radiation in the treatment of malignant tumours. Reactor thermal and epithermal neutron beams available at our institute had been also shown to have a significantly higher RBE value than γ-rays in irradiated tumour cells in vivo [2]. Owing to a selective physical dose distribution and enhanced biological damage in target tumours, particle radiation therapy with protons or heavy ions has gained increasing interest worldwide, and many clinical centres are considering introducing radiation therapy with charged particles. However, almost all reports on the biological advantages of charged particle beams are based on effects only on total tumour cell populations as a whole using in vitro cell cultures or in vivo solid tumours [1,5].Intensity-modulated radiotherapy and stereotactic irradiation have become common as new radiotherapy modalities for the treatment of malignancies. These techniques often require precise positioning of patients and longer exposure times in a single treatment session [6,7]. Prolongation of irradiation time may induce adverse radiation effects and evokes major concern related to the dose-rate effect. Thus, there is a need to clarify the effect of a reduction in dose rate on the radiosensitivity of tumours in vivo.In the present study, the radiosensitivity of the pimonidazole-unlabelled cell fraction of the Q cell population, following cobalt-60 γ-ray or 290 MeV u⁻¹ accelerated carbon ion beam irradiation at both a high dose rate (HDR) and a reduced dose rate (RDR), was determined, compared with irradiation using reactor thermal neutron beams following the administration of a ¹⁰B-carrier at our institute. This is the first attempt to evaluate the sensitivity of oxygenated fractions of Q tumour cells in vivo in response to particle radiation.     

Evaluation of hypoxia-specific cytotoxic bioreductive agent-sodium borocaptate-10B conjugates as10B-carriers in boron neutron capture therapy

Purpose

To evaluate the usefulness of 5 new¹⁰B-compounds (TX-2091, TX-2095, TX-2097, TX-2100, and TX-2110) as¹⁰B-carriers in boron neutron capture therpy (BNCT). They were conjugates that had been synthesized from a hypoxia-specific cytotoxic bioreductive agent, quinoxaline oxide TX-402 and a clinically used¹⁰B-carrier, sodium borocaptate-¹⁰B (BSH).

Materials and Methods

The 5 new compounds were hybrid compounds that have both a hypoxic cytotoxin unit and a thermal neutron-sensitizing unit, BSH. These new compounds and BSH were administered intraperitoneally to SCC VII tumor-bearing mice. Then, the¹⁰B concentrations in the tumors and normal tissues were measured by γ-ray spectrometry. Subsequently, SCC VII tumor-bearing mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, then treated with TX-2100, which was chosen based on the results of the above-mentioned biodistribution analyses, or BSH in the same manner as in the biodistribution studies. Right after irradiation, during which intratumor¹⁰B concentrations were kept at levels similar to each other, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling [=quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in the total (P+Q) tumor cell population was determined from the tumors that were not pretreated with BrdU. Clonogenic cell survival was also determined in mice given no BrdU.

Results

¹⁰B biodistribution analyses in tumors, brain, skin, muscles, blood, and liver indicated that TX-2100 has the most favorable characteristics for concentrating a sufficient amount of¹⁰B in tumors and maintaining a high enough¹⁰B concentration during irradiation. In addition, TX-2100 had a significantly stronger radio-sensitizing effect with reactor thermal neutron beams than BSH on both total and Q cells in solid tumors. Further, TX-2100 clearly exhibited a radio-sensitizing effect with γ-rays not only on total cells but also on Q and hypoxic tumor cells, which was not achieved by BSH.

Conclusion

A¹⁰B-carrier that acts as a hypoxic cytotoxin on tumor cells as well as having the potential to keep¹⁰B in tumors and sensitize tumor cells more markedly than conventional¹⁰B-carriers, such as TX-2100, is a promising candidate for use in BNCT.     

Responses of total and quiescent cell populations in solid tumors to carbon ion beam irradiation (290 MeV/u) in vivo

PURPOSE: The aim of this study was to clarify the radiosensitivity of intratumor total cells and quiescent (Q) cells in vivo to accelerated carbon ion beams compared with gamma-ray irradiation. MATERIALS AND METHODS: Squamous cell carcinoma (SCC) VII tumor-bearing mice received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. They then were exposed to carbon ions (290 MeV/u) or gamma-rays. Immediately after and 12 h after irradiation, immunofluorescence staining for BrdU was used to assess the response of Q cells in terms of micronucleus frequency. The response of the total (P + Q) tumor cells was determined from the tumors not treated with BrdU. RESULTS: The apparent difference in radiosensitivity between total and Q cell populations under gamma-ray irradiation was markedly reduced with carbon ion beams, especially with a higher linear energy transfer (LET) value. Clearer recovery in Q cells than in total cells through delayed assay under gamma-ray irradiation was efficiently inhibited by carbon ion beams, especially those with a higher LET. CONCLUSION: In terms of the tumor cell-killing effect as a whole, including intratumor Q cells, carbon ion beams, especially with higher LET values, were extremely useful for suppressing the dependence on the heterogeneity within solid tumors as well as depositing the radiation dose precisely.     

Induction of radioresistance to accelerated carbon-ion beams in recipient cells by nitric oxide excreted from irradiated donor cells of human glioblastoma

Purpose : To investigate whether nitric oxide excreted from cells irradiated with accelerated carbon-ion beams modulates cellular radiosensitivity against irradiation in human glioblastoma A-172 and T98G cells. Materials and methods : Western-blot analysis of inducible nitric oxide synthase, hsp72 and p53, the concentration assay of nitrite in medium and cell survival assay after irradiation with accelerated carbon-ion beams were performed. Results : The accumulation of inducible nitric oxide synthase was caused by accelerated carbon-ion beam irradiation of T98G cells but not of A-172 cells. The accumulation of hsp72 and p53 was observed in A-172 cells after exposure to the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase to the medium. The radiosensitivity of A-172 cells was reduced in the conditioned medium of the T98G cells irradiated with accelerated carbonion beams compared with conventional fresh growth medium, and the reduction of radiosensitivity was abolished by the addition of an inducible nitric oxide synthase inhibitor to the conditioned medium. Conclusions : Nitric oxide excreted from the irradiated donor cells with accelerated carbon-ion beams could modulate the radiosensitivity of recipient cells. These findings indicate the importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to accelerated heavy ions.     

Effects of employing a ¹⁰B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy

Objectives

To evaluate the effects of employing a ¹⁰B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy (BNCT) by measuring the response of intratumour quiescent (Q) cells.

Methods

B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumours received reactor thermal neutron beam irradiation following the administration of a ¹⁰B-carrier [L-para-boronophenylalanine-¹⁰B (BPA) or sodium mercaptoundecahydrododecaborate-¹⁰B (BSH)] in combination with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P+Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.

Results

BPA-BNCT increased the sensitivity of the total tumour cell population more than BSH-BNCT. However, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. With or without a ¹⁰B–carrier, MTH enhanced the sensitivity of the Q cell population. Without irradiation, nicotinamide treatment decreased the number of lung metastases. With irradiation, BPA-BNCT, especially in combination with nicotinamide treatment, showed the potential to reduce the number of metastases more than BSH-BNCT.

Conclusion

BSH-BNCT in combination with MTH improves local tumour control, while BPA-BNCT in combination with nicotinamide may reduce the number of lung metastases.A neutron capture reaction in boron (¹⁰B(n,α)⁷Li) is, in principle, very effective at destroying tumours, provided a sufficient amount of ¹⁰B can be accumulated in the target tumour and a sufficient number of very low energy thermal neutrons can be delivered there. The two particles generated in this reaction have a high linear energy transfer (LET) and have a range of roughly the diameter of one or two tumour cells. It is theoretically possible to kill tumour cells without affecting adjacent healthy cells, if ¹⁰B atoms can be selectively accumulated in the interstitial space of tumour tissue and/or intracellular space of tumour cells. Thus, successful boron neutron capture therapy (BNCT) requires the selective delivery of large amounts of ¹⁰B to malignant cells [1].The two most common ¹⁰B-carriers used in clinical BNCT trials designed for the treatment of malignant gliomas, melanomas, inoperable head and neck tumours and oral cancer are sodium mercaptoundecahydrododecaborate-¹⁰B (sodium borocaptate-¹⁰B, BSH, Na₂¹⁰B₁₂H₁₁SH) and boronophenylalanine-¹⁰B (BPA, C₉H₁₂¹⁰BNO₄) [1]. In the case of BSH, the delivery of ¹⁰B relies on passive diffusion from blood to brain tumour through a disrupted blood–brain barrier [2]. Invariably, the use of BSH results in a high concentration of boron in blood and subsequent vascular damage during BNCT [3]. BPA is designed to be mostly taken up by active transport across the cancer cell membrane [4]. The transport mechanism is operative even in normal cells leading to the accumulation of BPA in normal brain, although at a lower rate. It has been suggested that tumour response can be improved by combining BSH and BPA [5].Many cancer cells in solid tumours are non-proliferating [quiescent (Q)], whose features are still unknown [6]. To improve cancer treatment, the response of Q cells to anticancer treatment should be determined because tumour cells that are quiescent in situ are often still clonogenic [6]. Meanwhile, within solid tumours, there are hypoxic areas resulting from either limited oxygen diffusion (chronic hypoxia) or limited perfusion (acute hypoxia) [7]. It has also been reported that acute and cyclic, but not chronic, hypoxia significantly increased the number of spontaneous lung metastases, and that this effect was due to the influence of acute hypoxia treatment on the primary tumour [8,9].Here, we tried to analyse the effects of ¹⁰B-carriers and of manipulating intratumour hypoxia on local tumour response and lung metastatic potential in BNCT using a readily metastasising murine melanoma cell line. The neutron capture reaction was performed with two ¹⁰B-carriers, BSH and BPA. Tumour hypoxia was manipulated by treatment with an acute hypoxia-releasing agent (nicotinamide) through its inhibiting action on temporary fluctuations in tumour blood flow [10,11] or mild temperature hyperthermia (MTH), which has been shown to have the potential to release tumour cells from diffusion-limited chronic hypoxia [10,11]. Concerning the local tumour response, the effect not only on the total [proliferating (P)+ Q] tumour cell population but also on the Q cell population was evaluated using our original method for selectively detecting the response of Q cells in solid tumours [12].     

Secondary radiation dose during high-energy total body irradiation

Aim

The goal of this work was to assess the additional dose from secondary neutrons and γ-rays generated during total body irradiation (TBI) using a medical linac X-ray beam.

Background

Nuclear reactions that occur in the accelerator construction during emission of high-energy beams in teleradiotherapy are the source of secondary radiation. Induced activity is dependent on the half-lives of the generated radionuclides, whereas neutron flux accompanies the treatment process only.

Materials and methods

The TBI procedure using a 18 MV beam (Clinac 2100) was considered. Lateral and anterior–posterior/posterior–anterior fractions were investigated during delivery of 2 Gy of therapeutic dose. Neutron and photon flux densities were measured using neutron activation analysis (NAA) and semiconductor spectrometry. The secondary dose was estimated applying the fluence-to-dose conversion coefficients.

Results

The main contribution to the secondary dose is associated with fast neutrons. The main sources of γ-radiation are the following: ⁵⁶Mn in the stainless steel and ¹⁸⁷W of the collimation system as well as positron emitters, activated via (n,γ) and (γ,n) processes, respectively. In addition to 12 Gy of therapeutic dose, the patient could receive 57.43 mSv in the studied conditions, including 4.63 μSv from activated radionuclides.

Conclusion

Neutron dose is mainly influenced by the time of beam emission. However, it is moderated by long source–surface distances (SSD) and application of plexiglass plates covering the patient body during treatment. Secondary radiation gives the whole body a dose, which should be taken into consideration especially when one fraction of irradiation does not cover the whole body at once.     

Comparison of cellular damage response to low-dose-rate 125I seed irradiation and high-dose-rate gamma irradiation in human lung cancer cells

PurposeTo investigate the difference of cellular response between low-dose-rate (LDR) ¹²⁵I seed irradiation and high-dose-rate (HDR) γ-irradiation in human lung cancer cells.Methods and MaterialsA549 and NCI-H446 cells with or without wortmannin (WM) treatment were exposed to ¹²⁵I seeds and γ-rays, respectively. Cell survival, micronuclei (MN) formation, and the expressions of Ku70/Ku80 proteins were measured.ResultsThere was a strong negative correlation between survival and MN formation for both irradiations, and the MN inductions of NCI-H446 were about twofolds of those of A549, and the survival of NCI-H446 was lower than that of A549, indicating the radiosensitivity of NCI-H446 cells was greater than that of A549 cells. Interestingly, at 4-Gy radiation, NCI-H446 cells were more sensitive to LDR irradiation than HDR irradiation. WM treatment enhanced the radiosensitivity of A549 cells evenly to ¹²⁵I seed and γ-irradiation, but this treatment led NCI-H446 cells to be more sensitive to LDR ¹²⁵I. Further results revealed that the expression of phosphorylated Ku80 protein was enhanced in irradiated A549, but in contrast, it was markedly decreased in NCI-H446 cells after 4-Gy LDR ¹²⁵I irradiation as that compared with γ-irradiated and nonirradiated cells.ConclusionNCI-H446 cells were more sensitive to LDR ¹²⁵I irradiation than HDR irradiation, and this sensitivity could be further enhanced by WM treatment. But no obvious differences of cellular response to both irradiations were observed in A549. Ku as molecular markers together with cell proliferation rate can be used to predict the radiosensitivity of tumor cells to LDR ¹²⁵I seed irradiation.     

Influence of dose-rate, post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes.

PURPOSE: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. MATERIALS AND METHODS: Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3 Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). RESULTS: A significant decrease in reproductive cell death was observed after 3 Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDRI analysed day 6, although not of statistical significance. Radiation-induced apoptosis was efficiently counteracted by growth factors up to 24-48 h after 3 Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR 1 cells plated day 6), a significant increase in reproductive cell death was found (3 Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR 1 plated day 1 and HDR6). CONCLUSIONS: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.     

Radioprotection of targeted and bystander cells by methylproamine

Introduction

Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells.

Methods

T98G glioma cells were treated with 15 μM methylproamine and exposed to ¹³⁷Cs γ-ray/X-ray irradiation and He²⁺ microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or γH2AX assay.

Results

Radioprotection of directly targeted T98G cells by methylproamine was observed for ¹³⁷Cs γ-rays and X-rays but not for He²⁺ charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He²⁺ ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed.

Discussion and conclusion

Methylproamine protected targeted cells from DNA damage caused by γ-ray or X-ray radiation but not He²⁺ ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received.

     

Influence of dose-rate,post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes

Purpose: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. Materials and methods : Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). Results: A significant decrease in reproductive cell death was observed after 3Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDR1 analysed day 6, although not of statistical significance. Radiation-induced apoptosis was e fficiently counteracted by growth factors up to 24-48h after 3Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR1 cells plated day 6), a significant increase in reproductive cell death was found (3Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR1 plated day 1 and HDR6). Conclusions: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.     

High-dose rate intracavitary irradiation for carcinoma of the uterine cervix

Aim

To investigate the adverse effect of treatment prolongation on the local control and survival of the cervical carcinoma of the uterus.

Patients and Method

Two hundred and sixteen patients with stages IIB and III cervical carcinoma treated with a combination of external radiation and high-dose rate (HDR) intracavitary irradiation between 1978 and 1989 were retrospectively studied. A multivariate analysis was used to determine the effect of treatment time on local control and survival.

Results

Overall treatment time was the most highly significant factors for local control in the multivariate analysis (p=0.0005). The 5-year cumulative relapse rates were significantly different with the treatment times 35 to 42 days: 9% versus 43 to 49 days: 19% versus 50 to 62 days: 42% (p=0.001). The second most significant parameter was stage classification (p=0.02). Concerning relapse-free survival, stage classification (p=0.0001), overall treatment time (p=0.0035) and hemoglobin level (p=0.0174) were the 3 most important prognostic factors, although there was no relationship between treatment time and late complications.

Conclusion

These results suggest that prolongation of treatment time is associated with decreased local control and survival in patients treated with external radiation and HDR intracavitary irradiation.     

The usefulness of a continuous administration of tirapazamine combined with reduced dose-rate irradiation using {gamma}-rays or reactor thermal neutrons

We clarified the usefulness of the continuous administration of tirapazamine (TPZ) in combination with reduced dose-rate irradiation (RDRI) using gamma-rays or reactor thermal neutrons. Squamous cell carcinoma (SCC) VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ in combination with conventional dose-rate irradiation (CDRI) or RDRI using gamma-rays or thermal neutrons. After irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cells was determined using tumours that were not pre-treated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Furthermore, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using gamma-rays or thermal neutrons. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.     

Assessment of the micronucleus assay as a biological dosimeter using cytokinesis-blocked lymphocytes from cancer patients receiving fractionated partial body-radiotherapy

Purpose

To assess the suitability of the cytokinesis block micronucleus assay as a biological dosimeter following in-vivo radiation using cancer patients undergoing radiotherapy.

Methods

Blood from 4 healthy donors was irradiated in vitro with γ-rays and the dose response of induced micronuclei in binucleate lymphocytes following cytokinesis block was determined. Micronucleus frequency was ascertained before and at intervals during radiotherapy treatment in 6 patients with various tumors in the pelvic region. Equivalent whole body doses (physical doses) at these times were calculated from radiation treatment plans and cumulative dose volume histograms.

Results

Linear dose response relationships were found for induced micronucleus frequency in lymphocytes resulting from both in-vitro and in-vivo irradiation. Doses resulting from in-vivo irradiation (biological doses) were estimated by substitution of micronucleus frequency observed in radiotherapy patients into the dose response curve from in-vitro irradiation of blood. The relationship between the biologically estimated dose (BD) and the calculated equivalent whole body dose (PD) was BD=0.868(±0.043)PD+0.117(±0.075).

Conclusion

The micronucleus assay appears to offer a reliable and consistent method for equivalent whole body radiation dose estimation, although our findings should be confirmed using lymphocytes from radiotherapy patients with tumors at anatomical sites other than the pelvis. Except at doses lower than about 0.4 Gy, the method yields dose estimates acceptably close to “true” physically determined doses. The assay can be performed relatively rapidly and can be used as a “first line” biological dosimeter in situations where accidental exposure to relatively high radiation doses has occurred.     

Survivin, a target to modulate the radiosensitivity of Ewing’s sarcoma

Background and purpose

Radiotherapy constitutes an essential element in the multimodal therapy of Ewing’s sarcoma. Compared to other sarcomas, Ewing tumors normally show a good response to radiotherapy. However, there are consistently tumors with a radioresistant phenotype, and the underlying mechanisms are not known in detail. Here we investigated the association between survivin protein expression and the radiosensitivity of Ewing’s sarcoma in vitro.

Material and methods

An siRNA-based knockdown approach was used to investigate the influence of survivin expression on cell proliferation, double-strand break (DSB) induction and repair, apoptosis and colony-forming ability in four Ewing’s sarcoma cell lines with and without irradiation.

Results

Survivin protein and mRNA were upregulated in all cell lines tested in a dose-dependent manner. As a result of survivin knockdown, STA-ET-1 cells showed reduced cell proliferation, an increased number of radiation-induced DSBs, and reduced repair. Apoptosis was increased by knockdown alone and increased further in combination with irradiation. Colony formation was significantly reduced by survivin knockdown in combination with irradiation.

Conclusion

Survivin is a radiation-inducible protein in Ewing’s sarcoma and its down-regulation sensitizes cells toward irradiation. Survivin knockdown in combination with radiation inhibits cell proliferation, repair, and colony formation significantly and increases apoptosis more than each single treatment alone. This might open new perspectives in the radiation treatment of Ewing’s sarcoma.     

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation

Purpose

Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines.

Methods

Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by ??H2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis.

Results

SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. ??H2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24?h before irradiation to the sarcoma cell cultures. In contrast, ??H2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type.

Conclusion

SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.     

Prognostic value of HIF-1α expression during fractionated irradiation

Background and purpose

Hypoxia and reoxygenation are important determinants of outcome after radiotherapy. HIF-1α is a key molecule involved in cellular response to hypoxia. HIF-1α expression levels have been shown to change after irradiation. The objective of the present study was to explore the prognostic value of HIF-1α expression during fractionated irradiation.

Materials and methods

Six human squamous cell carcinoma models xenografted in nude mice were analysed. Tumours were excised after 3, 5 and 10 fractions. HIF-1α expression was quantified by western blot. For comparative analysis, previously published data on local tumour control data and pimonidazole hypoxic fraction was used.

Results

HIF-1α expression in untreated tumours exhibited intertumoural heterogeneity and did not correlate with pimonidazole hypoxic fraction. During fractionated irradiation the majority of tumour models exhibited a decrease in HIF-1α expression, whereas in UT-SCC-5 no change was observed. Neither kinetics nor expression levels during fractionated irradiation correlated with local tumour control.

Conclusion

Our data do not support the use of HIF-1α determined during treatment as a biomarker to predict outcome after fractionated irradiation.