Consideration of hereditary effects in the radiological protection system: evolution and current status.

PURPOSE: The purpose of this paper is to provide an overview of the methodology used to estimate radiation genetic risks and quantify the risk of hereditary effects as outlined in the ICRP Publication 103. It aims to highlight the historical background and development of the doubling dose method for estimating radiation-related genetic risks and its continued use in radiological protection frameworks. RESULTS: This article emphasizes the complexity associated with quantifying the risk of hereditary effects caused by radiation exposure and highlights the need for further clarification and explanation of the calculation method. As scientific knowledge in radiation sciences and human genetics continues to advance in relation to a number of factors including stability of disease frequency, selection pressures, and epigenetic changes, the characterization and quantification of genetic effects still remains a major issue for the radiological protection system of the International Commission on Radiological Protection. CONCLUSION: Further research and advancements in this field are crucial for enhancing our understanding and addressing the complexities involved in assessing and managing the risks associated with hereditary effects of radiation.

Ethical and societal aspects of radiological protection for offspring and next generations.

PURPOSE: Over the last decade or so, ethical and societal aspects of radiological protection have received increasing attention. This is also reflected in the publications of the International Commission on Radiological Protection (ICRP). The current paper aims at identifying relevant ethical and societal topics which should receive attention in the context of radiological protection for offspring and next generations. MATERIALS AND METHODS: We present a non-comprehensive review of the subject, based on presentation made at an ICRP workshop in Budapest in 2022. We first discuss the ethical values promoted by ICRP, and the application of these values in cases of (potential) pre-conceptual and prenatal radiation exposures. We then consider experience gained after the Fukushima accident indicating particular societal concerns about the health effects of such exposures. RESULTS AND CONCLUSIONS: Beneficence/non-maleficence, prudence, justice and dignity, the "core values" of the system of radiological protection have special roles to play when heritable and/or in utero effects are to be considered. Prudence, in particular, must be taken account of in view of the fact that solid scientific data in humans are largely lacking in this area, and it is necessary to rely on insights from animal experiments as well as theoretical considerations. As regards societal considerations, the perception of risk among (potentially) affected populations needs to be taken seriously. Accountability, transparency, and inclusivity, the "procedural values" promoted by ICRP for the practical implementation of the system of radiological protection play a central role in overcoming skepticism and creating trust. Stakeholder involvement should emphasize cooperation and dialogue, which allows for the joint evaluation of an exposure situation by experts and affected people.

Ethical aspects in the use of radiation in medicine: update from ICRP Task Group 109.

Whereas scientific evidence is the basis for recommendations and guidance on radiological protection, professional ethics is critically important and should always guide professional behaviour. The International Commission on Radiological Protection (ICRP) established Task Group 109 to advise medical professionals, patients, families, carers, the public, and authorities about the ethical aspects of radiological protection of patients in the diagnostic and therapeutic use of radiation in medicine. Occupational exposures and research-related exposures are not within the scope of this task group. Task Group 109 will produce a report that will be available to the different interested parties for consultation before publication. Presently, the report is at the stage of a working document that has benefitted from an international workshop organised on the topic by the World Health Organization. It presents the history of ethics in medicine in ICRP, and explains why this subject is important, and the benefits it can bring to the standard biomedical ethics. As risk is an essential part in decision-making and communication, a summary is included on what is known about the dose-effect relationship, with emphasis on the associated uncertainties. Once this theoretical framework has been presented, the report becomes resolutely more practical. First, it proposes an evaluation method to analyse specific situations from an ethical point of view. This method allows stakeholders to review a set of six ethical values and provides hints on how they could be balanced. Next, various situations (e.g. pregnancy, elderly, paediatric, end of life) are considered in two steps: first within a realistic, ethically challenging scenario on which the evaluation method is applied; and second within a more general context. Scenarios are presented and discussed with attention to specific patient circumstances, and on how and which reflections on ethical values can be of help in the decision-making process. Finally, two important related aspects are considered: how should we communicate with patients, family, and other stakeholders; and how should we incorporate ethics into the education and training of medical professionals?

Ethics of Radiological Protection-recent developments.

The International Commission on Radiological Protection (ICRP) has recently reviewed the ethical foundations of its recommendations. The approach taken in its report is similar to principlism, i.e. the system of Beauchamp and Childress proposed in their 'Principles of Biomedical Ethics.' The commission identifies a number of 'core values' which have helped shape the evolution of the ICRP system of radiological protection, namely 'Beneficence and non-maleficence', 'Prudence', 'Justice' and 'Dignity'. In addition, 'procedural values' are cited that are important for the system's applications in practice, 'Accountability', 'Transparency' and 'Inclusiveness (Stakeholder Participation)'. It is emphasized that these values are common to or at least acceptable for people from different cultural backgrounds, which for an endeavour as global in nature as radiological protection seems to be quite important and appropriate. Thus, the ICRP document on 'Ethics of radiological protection' could set a standard for other areas.

ICRP Publication 138: Ethical Foundations of the System of Radiological Protection.

Abstract ­: Despite a longstanding recognition that radiological protection is not only a matter of science, but also ethics, ICRP publications have rarely addressed the ethical foundations of the system of radiological protection explicitly. The purpose of this publication is to describe how the Commission has relied on ethical values, either intentionally or indirectly, in developing the system of radiological protection with the objective of presenting a coherent view of how ethics is part of this system. In so doing, it helps to clarify the inherent value judgements made in achieving the aim of the radiological protection system as underlined by the Commission in Publication 103. Although primarily addressed to the radiological protection community, this publication is also intended to address authorities, operators, workers, medical professionals, patients, the public, and its representatives (e.g. NGOs) acting in the interest of the protection of people and the environment. This publication provides the key steps concerning the scientific, ethical, and practical evolutions of the system of radiological protection since the first ICRP publication in 1928. It then describes the four core ethical values underpinning the present system: beneficence/ non-maleficence, prudence, justice, and dignity. It also discusses how these core ethical values relate to the principles of radiological protection, namely justification, optimisation, and limitation. The publication finally addresses key procedural values that are required for the practical implementation of the system, focusing on accountability, transparency, and inclusiveness. The Commission sees this publication as a founding document to be elaborated further in different situations and circumstances.

Are the core values of the radiological protection system shared across cultures?

In spite of ongoing globalisation in many fields, the ethics of radiological protection have long been discussed almost exclusively in terms of 'Western' moral philosophy concepts such as utilitarianism or deontology. A cross-cultural discourse in this field is only just beginning. In 'Principles of Biomedical Ethics', Beauchamp and Childress suggested that there exists a 'common morality' which is 'not relative to cultures or individuals, because it transcends both'. They proposed four cross-culturally valid principles for decision making in medicine: respect for autonomy, non-maleficence, beneficence, and justice. A similar approach is being developed by the International Commission on Radiological Protection Task Group 94 on the ethics of radiological protection. Here, the core values are: human dignity, beneficence/non-maleficence, prudence, and justice. Other values could be added, such as consideration for the interests of society as a whole or the interests of future generations, or procedural values such as transparency and accountability; this paper will include a brief discussion on how they relate to the four basic principles. The main question to be addressed here, however, is whether the proposed core values are indeed part of a 'common morality'. This, as it will be argued, cannot be decided by a global opinion poll, but has to be based on an analysis of the written and oral traditions that have provided ethical orientation throughout history, and are still considered seminal by the majority of people. It turns out that there are indeed many commonalities across cultures, and that the concept of globally shared core values for the radiological protection system is not hopelessly idealistic.

Persistence of genetic damage in lymphocytes from former uranium miners.

Lymphocytes from former uranium miners who finished work underground one or more decades ago were analysed with respect to possibly persisting genetic damage induced by their radiation exposure. A modified micronucleus-centromere test was used which determined the frequency of micronucleus-containing binucleate cells after cytochalasin B treatment and the percentage of centromere-free micronuclei, assessed with the help of immunofluorescence labeling of centromere protein B. Whereas the overall frequency of micronucleus-containing cells was not significantly elevated above the level found in a control group, former miners showed a greater percentage of centromere-free micronuclei, i.e. micronuclei containing only acentric fragments. Our results are in excellent agreement with those of an earlier uranium miner study and lend support to the assumption that genetic damage from alpha radiation can persist for many years after exposure, possibly due to genomic instability. The frequency of micronucleus-containing cells, but not the percentage of centromere-free micronuclei, significantly increased with time since last exposure in the mines. This can be attributed, at least in part, to the fact that miners who have finished working underground longer ago tend to be older, and there is an increase of the frequency of micronucleus-containing cells with age.

Chromatin-bound PCNA as S-phase marker in mononuclear blood cells of patients with acute lymphoblastic leukaemia or multiple myeloma.

OBJECTIVES: Proliferating cell nuclear antigen (PCNA) has often been used as a marker to aid assessment of tumour growth fraction. This paper addresses the question of whether it can be used as an S-phase marker, when the non-chromatin-bound form of the protein is removed by pepsin treatment. MATERIALS AND METHODS: Cytofluorometric measurements were carried out after immunofluorescence staining of PCNA and counterstaining of DNA. S-phase fraction was determined with the help of windows on PCNA versus DNA scattergrams, or mathematically from DNA histograms. RESULTS: S-phase fractions obtained using the two methods correlated well, but did not always agree, exact discrepancies depending on the mathematical model used for histogram analysis. CONCLUSIONS: Determination of S-phase fractions with the help of PCNA immunofluorescence staining is possible, and probably more reliable than calculation of S-fractions from DNA histograms. It thus offers an alternative to assays involving BrdU labelling in vivo.

Relative biological effectiveness of 6 MeV neutrons with respect to cell inactivation and disturbances of the G1 phase.

The relative biological effectiveness (RBE) of neutrons and other types of densely ionizing radiation appears to be close to 1.0 for the induction of strand breaks, but considerably higher RBEs have been found for cellular end points such as colony-forming ability. This may be due to differences in the processing of strand breaks or to the involvement of other lesions whose yields are more dependent on radiation quality. Because cell cycle delays may be of great importance in the processing of DNA damage, we determined the RBE for disturbances of the G1 phase in four different cell types (Be11 melanoma, 4197 squamous cell carcinoma, EA14 glioma, GM6419 fibroblasts) and compared them with the RBE for cell inactivation. The method we used to determine the progress from G1 into S was as follows: Cells were serum-deprived for a number of days and then stimulated to grow with culture medium containing normal amounts of serum. Immediately before the change of medium, cells were exposed to graded doses of either 240 kV X rays or 6 MeV neutrons. At different times afterward, cells were labeled with BrdU and the numbers of active S-phase cells were assessed using two-parameter flow cytometry. For all four cell types, cells started to progress from G1 into S after a few hours. Radiation suppressed this process in all cases, but there were some interesting differences. For Be11 and 4197 cells, the most obvious effect was a delay in G1; the labeling index increased a few hours later in irradiated samples than in controls, and there was no significant effect on the maximum labeling index. For EA14 and GM6419 cells, although smaller doses were used because of greater radiosensitivity, a delay of the entry into S phase was again noticeable, but the most significant effect was a reduction in the maximum percentage of active S-phase cells after stimulation, indicating a permanent or long-term arrest in G1. The RBE for the G1 delay was the same for all four cell types, about 2.8, while the RBE for the G1 arrest varied between 3.2 for the most resistant Be11 cells and 1.7 for the most sensitive GM6419 cells. This trend was similar to that observed for the RBE for cell inactivation. If, as described above, the same number of strand breaks per dose is induced by neutrons and by X rays, the signal transduction cascade translates them into a greater G1 delay in the case of higher LET. This appears to be independent of repair capacity, because it is similar in all cell types we investigated. We therefore assume that a higher lesion density or the presence of other types of lesions is important for this relatively early effect. A G1 arrest, however, is more closely related to the later events leading to cell inactivation, where strand break repair does play a major role, influencing X-ray sensitivity more strongly than sensitivity to neutrons because of a lower repairability of lesions induced by higher-LET radiation.

Mutation induction in haploid yeast after split-dose radiation exposure. II. Combination of UV-irradiation and X-rays.

Split-dose protocols can be used to investigate the kinetics of recovery from radiation damage and to elucidate the mechanisms of cell inactivation and mutation induction. In this study, a haploid strain of the yeast, Saccharomyces cerevisiae, wild-type with regard to radiation sensitivity, was irradiated with 254-nm ultraviolet (UV) light and then exposed to X-rays after incubation for 0-6 hr. The cells were incubated either on nutrient medium or salt agar between the treatments. Loss of reproductive ability and mutation to canavanine resistance were measured. When the X-ray exposure immediately followed UV-irradiation, the X-ray survival curves had the same slope irrespective of the pretreatment, while the X-ray mutation induction curves were changed from linear to linear quadratic with increasing UV fluence. Incubations up to about 3 hr on nutrient medium between the treatments led to synergism with respect to cell inactivation and antagonism with respect to mutation, but after 4-6 hr the two treatments acted independently. Incubation on salt agar did not cause any change in the survival curves, but there was a strong suppression of X-ray-induced mutation with increasing UV fluence. On the basis of these results, we suggest that mutation after combined UV and X-ray exposure is affected not only by the induction and suppression of DNA repair processes, but also by radiation-induced modifications of cell-cycle progression and changes in the expression of the mutant phenotype.

Heterogeneity in 2-deoxy-D-glucose-induced modifications in energetics and radiation responses of human tumor cell lines.

PURPOSE: The glucose analog and glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), has been shown to differentially enhance the radiation damage in tumor cells by inhibiting the postirradiation repair processes. The present study was undertaken to examine the relationship between 2-DG-induced modification of energy metabolism and cellular radioresponses and to identify the most relevant parameter(s) for predicting the tumor response to the combined treatment of radiation + 2-DG. METHODS AND MATERIALS: Six human tumor cell lines (glioma: BMG-1 and U-87, squamous cell carcinoma: 4451 and 4197, and melanoma: MeWo and Be-11) were investigated. Cells were exposed to 2 Gy of Co-60 gamma-rays or 250 kVP X-rays and maintained under liquid-holding conditions 2-4 h to facilitate repair. 2-DG (5 mM, equimolar with glucose) that was added at the time of irradiation was present during the liquid holding. Glucose utilization, lactate production (enzymatic assays), and adenine nucleotides (high performance liquid chromatography and capillary isotachophoresis) were investigated as parameters of energy metabolism. Induction and repair of DNA damage (comet assay), cytogenetic damage (micronuclei formation), and cell death (macrocolony assay) were analyzed as parameters of radiation response. RESULTS: The glucose consumption and lactate production of glioma cell lines (BMG-1 and U-87) were nearly 2-fold higher than the squamous carcinoma cell lines (4197 and 4451). The ATP content varied from 3.0 to 6.5 femto moles/cell among these lines, whereas the energy charge (0.86-0.90) did not show much variation. Presence of 2-DG inhibited the rate of glucose usage and glycolysis by 30-40% in glioma cell lines and by 15-20% in squamous carcinoma lines, while ATP levels reduced by nearly 40% in all the four cell lines. ATP:ADP ratios decreased to a greater extent ( approximately 40%) in glioma cells than in squamous carcinoma 4451 and MeWo cells; in contrast, presence of 2-DG reduced ADP:AMP ratios by 3-fold in the squamous carcinoma 4451, whereas an increase was noted in the glioma cell line BMG-1. 2-DG significantly reduced the initial rates of DNA repair in all cells, resulting in an excess residual damage after 2 h of repair in BMG-1, U-87, and 4451 cell lines, whereas no significant differences could be observed in the other cell lines. Recovery from potentially lethal damage was also significantly inhibited in BMG-1 cells. 2-DG increased the radiation-induced micronuclei formation in the melanoma line (MeWo) by nearly 60%, while a moderate (25-40%) increase was observed in the glioma cell lines (BMG-1 and U-87). Presence of 2-DG during liquid holding (4 h) enhanced the radiation-induced cell death by nearly 40% in both the glioma cell lines, while significant effects were not observed in others. CONCLUSIONS: The modifications in energetics and radiation responses by 2-DG vary considerably among different human tumor cell lines, and the relationships between energy metabolism and various radiobiologic parameters are complex in nature. The 2-DG-induced modification of radiation response does not strictly correlate with changes in the levels of ATP. However, a significant enhancement of the radiation damage by 2-DG was observed in cells with high rates of glucose usage and glycolysis, which appear to be the two most important factors determining the tumor response to the combined treatment of 2-DG + radiation therapy.

G2-phase delays after irradiation and/or heat treatment as assessed by two-parameter flow cytometry.

Similar to what has been observed after irradiation, the fraction of G(2)-phase cells increases as a consequence of heat treatment. On the basis of cell cycle distributions alone, however, it is difficult to say whether the two results are related. In particular, comparison is complicated by the fact that the accompanying changes in the S-phase transition are different. These changes play a minor role after irradiation but constitute by far the most important cell cycle effect after heat treatment. Two-parameter flow cytometry was used here to study the proliferation of human melanoma cells in vitro. Cultures were pulse-labeled with BrdU after irradiation and/or heat treatment and were fixed either immediately or after a delay of up to 36 h. DNA-synthesizing cells were identified with the help of an FITC-conjugated antibody against BrdU; DNA was quantified after staining with propidium iodide. In this way, the cell cycle distribution could be determined and the progression through the cell cycle could be analyzed. From the movement of labeled cells through the cycle, in particular the appearance of labeled cells in the G(1) compartment (after they had gone through mitosis), the delay in G(2) phase could be determined. The duration of the G(2)/M phase in control cells was about 6 h. This was increased to 12, 13 and 16 h after irradiation (4 Gy X rays), heat treatment (1 h at 43 degrees C), and a combination of the two, respectively. In all these cases, the G(2)-phase block was completely overcome within 48 h after treatment, whereas changes in the S phase were still observable at this time. As expected, the radiation-induced G(2)-phase block was almost completely removed by incubating the cells with 5 or 10 mM caffeine. In the case of hyperthermia alone or in combination with radiation, however, caffeine was somewhat less effective. This does not mean, however, that the mechanisms involved are necessarily different. It can also be seen as a result of the differences in the time course of events. The long delay in S phase after heat treatment may lead to a loss of susceptibility to caffeine by the time the cells move into the G(2) phase.

p53 levels, cell cycle kinetics and radiosensitivity in two SV40 transformed Wi38VA13 fibroblast strains.

BACKGROUND: The tumor suppressor protein p53 which can mediate an ionizing radiation-induced G1 arrest in mammalian cells, forms complexes with SV40 large T antigen (L-T-Ag). We have analyzed the p53 levels, the capability to undergo a G1 arrest and the radiosensitivity of two SV40 transformed fibroblast strains differing in their large T antigen expression. MATERIAL AND METHODS: One of the two strains (VA13F) is the commercially available form of Wi38VA13, the other (VA13E) arose spontaneously from the original one in our laboratory. Their p53 levels were measured by means of flow cytometry (FCM) and Western blot (WB) with two p53 antibodies (Ab-3, clone PAb240; Ab-6, clone DO-1; both Oncogene Science). Cell cycle distributions were determined flow cytometrically after BrdU labeling at regular time intervals after exposure to 250 kV X-rays. Radiosensitivity was assessed in a clonogenicity assay. RESULTS: The p53 levels of the two strains corresponded to their large T antigen expression, presumably due to complex formation between the two proteins. The strain with a high p53 level did not show a G1 arrest and had a relatively high radiosensitivity, whereas the strain with a low p53 level showed a significant G1 arrest and a lower radiosensitivity. CONCLUSION: These results suggest that 1. complex formation between the large T antigen and p53 reduces the latter's functionality; 2. in these two strains the G1 arrest is one of the factors determining radiosensitivity.

A comparison of the potential therapeutic gain of p(66)/Be neutrons and d(14)/Be neutrons.

PURPOSE: To determine the relationship between photon sensitivity and neutron sensitivity and between neutron RBE and photon resistance for two neutron modalities (with mean energies of 6 and 29 MeV) using human tumor cell lines spanning a wide range of radiosensitivities, the principal objective being whether or not a neutron advantage can be demonstrated. METHODS AND MATERIALS: Eleven human tumor cell lines with mean photon inactivation doses of 1.65-4. 35 Gy were irradiated with 0-5.0 Gy of p(66)/Be neutrons (mean energy of 29 MeV) at Faure, S.A. and the same plating was irradiated on the same day with 0-10.0 Gy of Cobalt-gamma-rays. Twelve human tumor cell lines, many of which were identical with the above selection, and spanning mean photon inactivation doses of 1.75-4.08 Gy, were irradiated with 0-4 Gy of d(14)/Be neutrons (mean energy of 6 MeV) and with 0-10 Gy of 240 kVp X-rays at the Essen Klinikum. Cell survival was determined by the clonogenic assay, and data were fitted to the linear quadratic equation. RESULTS: 1. Using the mean inactivation dose, a significant correlation was found to exist between neutron sensitivity and photon sensitivity. However, this correlation was more pronounced in the Faure beam (r(2) = 0.89, p

Quiescence in S-phase and G1 arrest induced by irradiation and/or hyperthermia in six human tumour cell lines of different p53 status.

PURPOSE: Quiescent S-phase cells, i.e. cells with a DNA content intermediate between G1 and G2 that nevertheless do not synthesize DNA have been previously observed in human melanoma cells exposed to radiation and/or hyperthermia. This phenomenon has now been studied in more detail comparing six human tumour cell lines of different p53 status and thus different cell-cycle checkpoint control. MATERIALS AND METHODS: Two melanoma (Be11, MeWo), two squamous carcinoma (4197, 4451) and two glioma (EA14, U87) cell lines were used. Changes in the cell-cycle distribution after treatment were studied using two-parameter flow cytometry in order to measure DNA content and BrdU incorporation simultaneously. RESULTS: The fraction of unlabelled cells in the S-phase compartment was determined at daily intervals after treatment. Only background levels of such cells were seen in three of the cell lines (Be11, 4197, EA14). With the other three cell lines (MeWo, 4451, U87) we observed a time- and dose-dependent increase: a few days after treatment up to 20% of all cells did not incorporate BrdU. It is interesting to note that Bell, 4197 and EA14 are p53 wild-types and show a G1 block of several hours after irradiation and/or hyperthermia, while MeWo and 4451 are p53 mutants unable to exhibit such a delay, and U87 in spite of being a p53 wild-type has a reduced ability to do so. CONCLUSIONS: The MeWo, 4451 and U87 cell lines have less time available for the repair of DNA damage before entering into the S-phase, which leads to problems during replication and causes some kind of interphase death. Radiation-induced apoptosis does not seem to be involved here, as it is not unequivocally correlated with the induction of a G1 block or with p53 status.

Arginine deiminase inhibits proliferation of human leukemia cells more potently than asparaginase by inducing cell cycle arrest and apoptosis.

L-Asparaginase is used for the treatment of acute leukemias, but is sometimes ineffective or associated with severe side-effects. We report here that the enzyme arginine deiminase is approximately 100-fold more potent than L-asparaginase in inhibiting the proliferation of cultured human lymphatic leukemia cell lines while it appears to be less effective in leukemia cells of myeloid origin. The inhibition of cell proliferation involves cell growth arrest in the G1- and/or S-phase and eventually apoptotic cell death. Our results suggest the possibility of a future use of arginine deiminase for the therapy of leukemia.

Radiation and/or hyperthermia sensitivity of human melanoma cells after several days of incubation in media lacking serum or certain serum components.

PURPOSE: Complete serum and single growth factors have been reported to protect cells against the effects of hyperthermia. The phenomenon is not very well understood, especially with regard to the relative importance of the various serum components. There is also a need to clarify the possible involvement of changes in proliferation. The influence of serum and growth factors on radiation sensitivity has not been studied in detail. The present communication is an attempt to at least partly fill these gaps. Cells were exposed to X-rays and/or heat after incubation in sufficiently supplemented medium and media lacking certain serum components. Differences in clonogenic survival were recorded and related to the proliferative status under the various conditions. METHODS AND MATERIALS: Human melanoma cells (MeWo) were used throughout. Cells were incubated for 3 or 6 days (a) in Ham's F12 medium with 20% fetal calf serum; (b) in medium without serum, but supplemented with the following components: insulin, transferrin, Na-selenite, and a lipid-protein complex; (c) to (f) in media lacking one or the other of these components; (g) in unsupplemented medium. Colony formation ability served as a measure of cell survival after exposure to radiation (250 kV X-rays) and/or hyperthermia (heating at 43 degrees C). In parallel, cell proliferation was characterized by the bromodeoxyuridine (BrdU) labeling index and the Ki-67 growth fraction, both determined by two-parameter flow cytometry. RESULTS AND CONCLUSIONS: Sensitivity was hardly influenced by 3 days of incubation in medium containing no supplement at all, whereas after 6 days under the same conditions survival curves both of irradiated and hyperthermia-treated cells had a strongly reduced shoulder or were considerably steeper. Experiments with media lacking only one or the other serum component, showed that deprivation of insulin, transferrin, and sodium selenite led to no more than a modest increase in cell killing, whereas the effects in medium lacking lipid-protein complex were about the same as in medium containing no supplement at all. Sensitivity changes did not seem to be due to the lack of serum or certain of its components per se, but were very well correlated with changes in proliferation as characterized by BrdU labeling index or the Ki-67 growth fraction.

Quiescent S-phase cells as indicators of extreme physiological conditions in human tumor xenografts.

PURPOSE: During the last 20 years, evidence has been accumulating for the existence in animal and human tumors of quiescent S-phase cells, i.e. cells with an S-phase DNA content that do not actively synthesize DNA. In cell culture studies, quiescent S-phase cells have been observed under physiological conditions typical for poorly vascularized regions of tumors such as reduced pH, hypoxia, and glucose deprivation. Therefore, we studied the possible correlation between the frequency of quiescent S-phase cells and the oxygenation status as determined polarographically in a number of human tumor xenografts. METHODS AND MATERIALS: Five human tumor xenografts on nude mice were used. Oxygenation was measured polarographically with an Eppendorf pO2-Histograph in 24 to 30 individual tumors for each entity. Mice were injected intraperitoneally with 1 mg/30 g bodyweight bromodeoxyuridine (BrdU), tumors were excised 30 min later and prepared into a single-cell suspension. After immunofluorescence staining with an antibody against BrdU and staining of the DNA with propidium iodide, cells were measured in a FACScan flow cytometer and the frequency of cells in the S-phase compartment that did not incorporate BrdU was determined. RESULTS: In most cases, the frequency of measurements of an oxygen partial pressure <5 mm Hg in the tumor tissue increased with tumor volume. Likewise, the frequency of quiescent S-phase cells was generally higher in larger tumors. Taking all five tumor entities together, there was a highly significant correlation between tumor oxygenation and the occurrence of quiescent S-phase cells. CONCLUSIONS: Our data confirm earlier findings that inactive S-phase cells do exist in vivo. Because their frequency seems to be dependent (directly or indirectly) on the degree of oxygenation and has been shown to increase not only with hypoxia, but also with reduced pH and glucose deprivation in vitro, the frequency of inactive S-phase cells may be considered a summary indicator for extreme physiological conditions in tumors.

Radiation and/or hyperthermia sensitivity of human melanoma cells grown for several days in media with reduced pH.

BACKGROUND: There are a number of reports in the literature dealing with the influence of reduced extracellular pH on cellular radiation and/or heat sensitivity. The focus of these earlier studies has been on the effects of a short-term incubation under acidic conditions, whereas much less attention has been given to chronic acidosis which would seem to be more relevant with respect to the tumor micromilieu. As far as mechanisms are concerned, many authors have taken the view that the intracellular rather than the extracellular pH is decisive at least for heat sensitivity, but only rodent cells have been studied so far and again the effects of chronically low pH in the incubation medium have been largely neglected. We therefore studied human melanoma cells after incubation for up to 6 days in media with different pH between 6.5 and 7.3. MATERIAL AND METHODS: MeWo cells were used throughout. Cells were incubated for 0, 3 or 6 days in media with pH 6.5, 6.7, 6.9, 7.1 or 7.3. The sensitivity against 250 kV X-rays and hyperthermia at 43 degrees C were determined in the colony-forming assay. The intracellular pH was measured flow cytometrically using 5(and 6)-carboxyfluorescein. Calibration curves were established with cells incubated in different buffers containing nigericin to equilibrate intra- and extracellular pH. RESULTS: Cell growth was optimal with pH 7.3 and 7.1 in the medium, somewhat reduced at pH 6.9, and largely inhibited at pH 6.7 and 6.5. Radiation and/or hyperthermia sensitivities were noticeably increased after several days of incubation at reduced pH; the surviving fraction after 4 Gy and 1 h at 43 degrees C either alone or in combination being a factor of 2 to 4 lower at pH 6.5 than at 7.3. These changes in sensitivity could not be correlated with changes in the intracellular pH. Cells seemed to be capable of regulating this parameter very well; the flow cytometric measurements revealed that the intracellular pH was 7.2 +/- 0.2 irrespective of the extracellular pH in the range considered here. CONCLUSIONS: In contrast to the almost generally accepted hypothesis that intracellular pH is decisive for the heat sensitivity, the human melanoma cells studied here became sensitive after a few days of incubation under acidic conditions without changes in the intracellular pH. Other factors seem to be influencing the cellular response to radiation and/or heat under chronically low pH.

Arginine deiminase inhibits cell proliferation by arresting cell cycle and inducing apoptosis.

We have previously demonstrated that arginine deiminase inhibits the proliferation of vascular endothelial cells, but the mechanisms leading to growth inhibition have remained unclear. We report here that low concentrations of arginine deiminase purified from Mycoplasma arginini inhibit proliferation of various cultured cells by arresting the cell cycle in G(1) and/or S phase with higher arginine deiminase concentrations leading to subsequent apoptosis. Our results demonstrate that arginine deiminase inhibits cell proliferation not only by depletion of arginine, but also by mechanisms involving the cell cycle and death signals.