Immunohistochemical and genetic evidence of myeloperoxidase involvement in multiple sclerosis

The influence of sampling time on the frequencies of micronuclei, centromere-positive micronuclei and chromosome nondisjunction was investigated in binucleated lymphocytes following treatment with a known clastogen (mitomycin C) or an aneuploidy-inducing agent (vincristine sulfate). Cytochalasin B (6 micrograms/ml) was added 44 h after mitogen stimulation and cultures were harvested 12, 28, 36 and 48 h thereafter. Micronucleated cells and micronuclei were significantly induced by the two treatments at all sampling times. Furthermore, in situ hybridization with an 'all centromeres' probe showed that vincristine-induced micronuclei were prevalently centromere-positive whereas in mitomycin C-treated cultures only a minor fraction of induced micronuclei contained the hybridization signals. Chromosome nondisjunction rates, as measured by in situ hybridization with chromosome 7- and 11-specific alphoid probes, significantly increased following vincristine treatment. Chromosome nondisjunction and total micronucleus frequencies were found to increase with time both in controls and in mutagen-treated cultures, whereas the percentage of centromere-positive micronuclei in the different treatments was not influenced by the sampling time. Our data suggest that even in the presence of 6 micrograms/ml cytochalasin B, the abnormal segregation of binucleated cells may contribute to the baseline level of micronuclei and influence the results obtained. The introduction of a short cytochalasin B treatment (between 12 and 28 h) in the cytokinesis-blocked micronucleus assay may avoid the cytochalasin B effect on micronucleus frequencies.     

Evidence into practice, experimentation and quasi experimentation: are the methods up to the task?

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 gamma-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 ).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.     

The demonstration of heritable lethal mutations in the progeny of x-ray-irradiated CHO cells by micronucleus count in clone cells

PURPOSE: Low doses of ionizing radiation reduce the growth rates of clones following irradiation of the progenitor cells. Such reductions of clone growth have been proven by means of measurements of clone size distributions. The medians of such distributions can be used to quantify the radiation damage. Prolongations of generation times and cell death as result of heritable lethal mutations have been discussed as causes for the reduction of clone growth. MATERIAL AND METHODS: The cell number of a clone of hypotetraploid CHO-cells was compared to the frequency of micronucleated binucleated cells in the same clone using the cytokinesis-block-micronucleus method. The dose dependent reduction of clone sizes is measured by the difference of the medians (after log transformation) of the clone size distributions. RESULTS: At cytochalasin-B concentrations of 1 microgram/ml and after an incubation time of 16 h a yield of binucleated cells of about 50% was obtained. Median clone size differences as a measure of clonal radiation damage increased linearly with incubation times of 76, 100, 124, and 240 h following irradiation with 3, 5, 7, and 12 Gy. The frequency of binucleated clone cells with micronuclei strongly increased with decreasing clone size by a factor up to 20 following irradiation with 3, 5, and 7 Gy. The frequency of micronucleated binucleated clone cells was found to be independent of incubation time after irradiation. CONCLUSION: Radiation induced clone size reductions result from cell losses caused by intraclonal expression of micronuclei which have its origin in heritable lethal mutations. Measurements of clone size distributions can be done automatically. They can serve as predictive test for determination of median cell loss rates of surviving cell clones.     

A cross-sectional study on the relationship between depression and left ventricular hypertrophy

PURPOSE: The relationship between different forms of persistent radiation damage in irradiated cells was investigated in order to identify a common underlying mechanism. MATERIAL AND METHODS: V-79 Chinese hamster cells were irradiated with different doses of X-rays, neutrons and alpha-particles. In the progeny of surviving cells, up to 4 weeks after irradiation, delayed reproductive death, delayed micronuclei, delayed appearance of dicentric chromosomes and delayed apoptosis were investigated in parallel. RESULTS: A similar dose-response relationship was found for all endpoints, with a steep rise at low doses to a plateau at doses > 3 Gy. The target for inducing genomic instability by alpha-particles is larger than the nucleus. All chromosomes are equally involved in delayed breakage reunion events. CONCLUSION: The results indicate that non-lethal radiation damage to an extranuclear target leads to a persistent increase in clastogenic activity in the surviving irradiated cells.     

Partial volume rat lung irradiation: an evaluation of early DNA damage

PURPOSE: 1. To investigate early DNA damage induced in rat lung cells following single-dose, partial-volume irradiation (lung base and lung apex). 2. To determine the variation in DNA damage in different lung regions. 3. To investigate the possible mechanisms associated with early DNA damage after lung irradiation. METHODS AND MATERIALS: The whole lung or the upper or lower half of the entire lung of Sprague-Dawley rats was exposed to 10 Gy 60Co gamma rays. The animals were sacrificed at various times up to 42 h after irradiation. A trypsin-digested lung cell suspension was prepared and cells that attached to slides in the initial 24-h period were then grown in the presence of culture medium with cytochalasin-B for a further 72 h. Radiation-induced DNA damage was quantified in the cells (primarily fibroblasts) from both irradiated and unirradiated lung regions by using a well-characterized micronucleus assay. RESULTS: When the lungs were removed at 16-18 h after whole-lung irradiation, about 0.85 micronuclei (MN) per binucleate cell (BNC) were observed in the lung cells of the irradiated animals, compared to 0.02 MN/BNC in the lung cells of the controls. When only the lung base was irradiated, the frequency of micronuclei was 0.85 MN/BNC compared to 0.43 MN/BNC observed in cells from the irradiated lung apex. Of particular interest was the finding that the unirradiated lung apex also showed a large frequency of micronuclei (0.43 MN/BNC) after the irradiation of the lung base, whereas the unirradiated lung base showed only a marginal (approximately 2-fold) increase relative to the spontaneous frequency following irradiation of the lung apex. The changes in the frequency of micronuclei varied with the time at which the lungs were removed from the rats for early times, but had stabilized by 18 h after irradiation. Normal (unirradiated) cells grown in filtered or unfiltered conditioned media obtained from irradiated cell cultures showed an insignificant marginal increase in the number of micronuclei relative to the spontaneous frequency. Lung cells obtained from the lung base or the lung apex of healthy controls and irradiated separately in vitro showed no regional differences in the induction of micronuclei. Cells from the lungs of rats injected with superoxide dismutase, within 1 h prior to irradiation of the lung base, and processed 16-18 h after irradiation showed a reduction in the number of MN in the shielded lung apex, indicating the possible involvement of oxygen radicals. CONCLUSIONS: These data indicate that cells in the lung base sustain more DNA damage than those in the lung apex when either region is irradiated; however, when the whole lung, is irradiated, the lung damage observed is similar in the two regions. Also, out-of-field effects are observed for the lung apex but not the lung base. Possible mechanisms include a clastogenic (chromosome damaging) factor produced in the plasma following irradiation and/or the production of oxygen radicals by activated lymphocytes/monocytes. The partial blocking of the DNA damage, observed in the unirradiated lung apex following irradiation of the lung base, by superoxide dismutase, suggests that oxygen radicals are involved in this out-of-field effect. These radicals are likely produced as a result of the induction of inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) by the irradiation. The reason for the lack of an out-of-field effect in the lung base when the lung apex is irradiated is unknown, but may be due to the greater volume of lung irradiated in the lower lung field, because this is likely to affect the level of cytokines produced. Alternatively, it may reflect cytokines produced as a result of the partial liver irradiation that occurs with the lower lung field.     

Effects of short-term treatment with prednisolone and calcitriol on bone and mineral metabolism in normal men

PURPOSE: This study was conducted to clarify the relationship among the frequencies of micronuclei (MN) and apoptosis, and clonogenic cell survival after irradiation. MATERIALS AND METHODS: The frequencies of MN and apoptosis were compared in the surviving fraction in three human tumour cell lines and two rodent cell lines at various irradiation doses. RESULTS: The SHIN-3, DU-145 and CHO-K1 cells showed dose-dependent increases of MN per binucleate cell and an excellent correlation between the MN frequency and surviving fraction after irradiation. The F9 and COLO 320DM cells did not show this correlation. The number of apoptotic cells increased according to the increase in radiation dose in the F9 and COLO 320DM cells, but not in the SHIN-3, DU-145 or CHO-K1 cells. CONCLUSIONS: The detection of the MN frequency alone is insufficient to measure cellular intrinsic radiosensitivity. The simultaneous use of the MN assay and the detection of apoptotic cells would be more reliable as a method for predicting cell survival after radiation.     

Anti-CD38 prevents the development of the adaptive response induced by X-rays in human lymphocytes

Irradiation of human lymphocytes (1 cGy X-rays, 37 degrees C) evoked an approximately 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). The response was reflected in a lower micronucleus frequency but not in the DNA repair rate measured by the comet assay directly after the challenge dose. Treatment of lymphocytes with anti-CD38 antibody 1 h before irradiation with the adaptive dose prevented the development of the adaptive response measured as micronuclei frequency, but adaptation was not reflected in a lower rate of DNA repair, measured by the alkaline version of the 'comet' assay. In lymphocytes that were anti-CD38-treated and irradiated and or irradiated with the adaptive dose the rate of DNA repair was not changed. However, the mean DNA damage level in adapted anti-CD38-treated lymphocytes was significantly lower than that in the control lymphocytes at all time points. We conclude that ligation of CD38 by antibody initiates signalling that prevents the development of the adaptive response induced by X-rays. Lower chromosome damage revealed by the cytokinesis block-micronucleus test in the adapted lymphocytes is unrelated to DNA repair rate.     

Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH

In order to study the initial frequencies and define kinetics of the formation of chromosomal exchanges in X-irradiated human lymphocytes, the premature chromosome condensation (PCC) technique was employed in combination with fluorescence in situ hybridization (FISH) with a composite probe for human chromosome 8 and a pan-centromeric probe for the whole genome. Human lymphocytes were X-irradiated (0.5, 1, 2, 3, 4 and 6 Gy), fused with mitotic Chinese hamster ovary (CHO) cells immediately or 1, 3, 6, 12 and 18 h after irradiation. Immediately after irradiation chromosomal breaks, dicentrics and translocations showed a linear dose-response. Unrejoined chromosome breaks were the most frequent types of aberrations (about 85%) observed. About 15% of total aberrations were chromosome exchanges of 65% of these were translocations and 35% were dicentrics. The chromosomal exchanges initially observed were mostly incomplete, with no complex exchanges at doses of 1 and 2 Gy, at higher doses (3-6 Gy) complex exchanges were observed and their frequencies increased with increasing post incubation time. Following different recovery times, repair kinetics of breaks for different doses of irradiation was studied. The shapes of the curves obtained for breaks as well as chromosome exchanges were linear-quadratic. The linear yield component, alpha, is formed entirely in the fast process that can be manifested in the early plateau, while component beta developed slowly in the subsequent hours. The kinetics of breaks rejoining was exponential, almost 50% of breaks rejoined after 1 h and at 18 h about 20% of breaks remained. At low doses of 1 and 2 Gy most of the exchanges were formed immediately and at higher doses, the frequency of exchanges increased with kinetics similar to that observed for the rejoining of breaks. However, the kinetics was different for different doses of irradiation. The frequency of dicentrics increased at doses above 2 Gy following 3 h recovery time, but for the translocations effect was pronounced even at 1 h recovery time. The frequency of incomplete exchanges (i.e., terminal translocations) decreased with post irradiation time and at 18 h was 30-40% less than the frequency obtained immediately after irradiation. The increase in the total translocations as a function of time between irradiation and fusion was due to a rapid increase in complete exchanges (i.e., reciprocal translocations). The frequency of ring chromosomes immediately after irradiation, also increased linearly, however, it was 3-5 times lower than dicentrics and remained almost constant in number for different doses and at different post-irradiation times.     

Changes in RBE of 14-MeV (d + T) neutrons for V79 cells irradiated in air and in a phantom: is RBE enhanced near the surface?

PURPOSE: The relative biological effectiveness (RBE) for inactivation of V79 cells was determined as function of dose at the Heidelberg 14-MeV (d + T) neutron therapy facility after irradiation with single doses in air and at different depths in a therapy phantom. Furthermore, to assess the reproducibility of RBE determinations in different experiments we examined the relationship between the interexperimental variation in radiosensitivity towards neutrons with that towards low LET 60Co photons. METHODS: Clonogenic survival of V79 cells was determined using the colony formation assay. The cells were irradiated in suspension in small volumes (1.2 ml) free in air or at defined positions in the perspex phantom. Neutron doses were in the range, Dt = 0.5-4 Gy. 60Co photons were used as reference radiation. RESULTS: The radiosensitivity towards neutrons varied considerably less between individual experiments than that towards photons and also less than RBE. However, the mean sensitivity of different series was relatively constant. RBE increased with decreasing dose per fraction from RBE = 2.3 at 4 Gy to RBE = 3.1 at 0.5 Gy. No significant difference in RBE could be detected between irradiation at 1.6 cm and 9.4 cm depth in the phantom. However, an approximately 20% higher RBE was found for irradiation free in air compared with inside the phantom. Combining the two effects, irradiation with 0.5 Gy free in air yielded an approximately 40% higher RBE than a dose of 2 Gy inside the phantom. CONCLUSION: The measured values of RBE as function of dose per fraction within the phantom is consistent with the energy of the neutron beam. The increased RBE free in air, however, is greater than expected from microdosimetric parameters of the beam and may be due to slow recoil protons produced by interaction of multiply scattered neutrons or to an increased contribution of alpha particles from C(n, alpha) reactions near the surface. An enhanced RBE in subcutaneous layers of skin combined with an increase in RBE at low doses per fraction outside the target volume could potentially have significant consequences for normal tissue reactions in radiotherapy patients treated with fast neutrons.     

Age-dependent and lobe-specific spontaneous hyperplasia in the brown Norway rat prostate

PURPOSE: Response of quiescent (Q) and total tumor cells in solid tumors to neutron irradiation with three different cadmium (Cd) ratios was examined. The role of Q cells in tumor control was also discussed. METHODS AND MATERIALS: C3H/He mice bearing SCC VII tumors received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty minutes after intraperitoneal injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutrons, or those without 10B-compounds were irradiated with gamma rays. This neutron irradiation was performed using neutrons with three different cadmium (Cd) ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P + Q) tumor cells was determined from tumors that were not pretreated with BrdU. The sensitivity to neutrons was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). RESULTS: Without 10B-compounds, the MN frequency in Q cells was lower than that in the total cell population. The sensitivity difference between total and Q cells was reduced by neutron irradiation. Relative biological effectiveness (RBE) of neutrons compared with gamma rays was larger in Q cells than in total cells, and the RBE values for low-Cd-ratio neutrons tended to be larger than those for high-Cd-ratio neutrons. With 10B-compounds, MN frequency for each cell population was increased, especially for total cells. This increase in MN frequency was marked when high-Cd-ratio neutrons were used. BPA increased the MN frequency for total tumor cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that in BSH-treated Q cells. This tendency was clearly observed in high-Cd-ratio neutrons. CONCLUSION: From the viewpoint of enhancing the Q-cell sensitivity, tumors should be irradiated with high-Cd-ratio neutrons after BSH administration. However, normal tissue reaction remains to be examined because of its low tumor-to-normal tissue and tumor-to-blood biodistribution ratios.     

Reduction of radiation-induced chromosome aberration and apoptosis by dithiothreitol

We have examined in vitro and in vivo radioprotective effects of a well-known thiol-containing compound, dithiothreitol (DTT). The treatment of both 0.5 and 1 mM of DTT significantly increased clonogenic survival of gamma-ray irradiated Chinese hamster (V79-4) cells. In order to investigate the possible radioprotective mechanism of DTT, we measured gamma-ray induced chromosome aberration by micronucleus assay. In the presence of 0.5 mM or 1 mM DTT, the frequencies of micronuclei were greatly reduced in all dose range examined (1.5-8 Gy). Slightly higher reduction in micronucleus formation was observed in 1 mM DTT-treated cells than in 0.5 mM DTT-treated cells. In addition, incubation with both 0.5 and 1 mM of DTT prior to gamma-ray irradiation reduced nucleosomal DNA fragmentation at about same extent, this result suggests that treatment of DTT at concentrations of 0.5 and 1 mM reduced radiation-induced apoptosis. In vivo experiments, we also observed that DTT treatment reduced the incidence of apoptotic cells in mouse small intestine crypts. In irradiated control group 4.4 +/- 0.5 apoptotic cells per crypt were observed. In DTT-administered and irradiated mice, only 2.1 +/- 0.4 apoptotic cells per crypt was observed. In vitro and in vivo data obtained in this study showed that DTT reduced radiation-induced damages and it seems that the possible radioprotective mechanisms of action of DTT are prevention of chromosome aberration.     

Usefulness of micronucleus assay in radiosensitivity tests using human cancer cell lines

BACKGROUND: Recently, micronucleus assay is expected to be one of the radiosensitivity tests. The usefulness of micronucleus assay was compared with MTT assay and clonogenic assay using 5 human derived urological cancer cell lines, NBT-2, T24, PC3, OS-RC-2, and RERF-LC-AI in vitro. The correlation between the results in vitro assay and the radiation effects of nude mouse in vivo was investigated. METHODS: In vitro, the micronucleus frequency of 2 Gy radiation was scored in micronucleus assay. The survival fraction of 2 Gy radiation was obtained in MTT assay and clonogenic assay. The correlation between 3 assays was investigated. In vivo, cancer cells was inoculated to nude mouse and the tumor volume was measured at 3-7 days interval in control group and 10 Gy irradiated group. The tumor volume ratio in irradiated group to control group was calculated as a radiation effect in each cell lines, the correlation between this ratio in vivo and each value of 2 Gy radiation in vitro was studied. RESULTS: The correlation between micronucleus frequency and survival fraction in clonogenic assay was statistically significant (r = 0.941, p = 0.0169). But the correlation of the survival fraction between MTT assay and clonogenic assay is not statistically significant. The correlation between micronucleus frequency and the tumor volume ratio in vivo was statistically significant (r = 0.990, p = 0.0011). The correlation between survival fraction in clonogenic assay and the tumor volume ratio in vivo was also statistically significant (r = 0.914, p = 0.0298). However, the correlation between survival fraction in MTT assay and the tumor volume ratio in vivo was not statistically significant (r = 0.782, p = 0.118). CONCLUSION: In this 5 cell lines, micronucleus assay was most correlated to nude mouse radiation effect. This result suggested the possibility of micronucleus assay to be a better predictive method than clonogenic assay for radiosensitivity test.     

Enhancement of fractionated-dose irradiation by retinoic acid plus interferon

PURPOSE: To evaluate the relative cytotoxicity of fractionated-dose radiation in the presence and absence of 13-cis-retinoic acid (RA) plus alpha-2a-interferon (IFN), as a function of overall treatment time. METHODS AND MATERIALS: Studies were performed with the human squamous cell carcinoma line FaDu, in vitro. Attached exponential phase cells were treated with RA + IFN for 8-10 h and then exposed to single graded doses of radiation, or 1 to 6 doses of radiation at 2 Gy per dose, or to 5 doses of radiation at 2 Gy/dose with a time interval of 4-24 h between treatments. Following irradiation, the cells were incubated with drugs present throughout colony formation, and the fraction of survivors in the presence and absence of the combined drugs was calculated. RESULTS: For single graded-dose irradiation, the surviving fraction ratio at 2 Gy in the absence vs. presence of drugs was 1.27 +/- 0.19 in 3 repeat experiments. Following administration of 6 doses of radiation at 2 Gy/fraction with a 5-h time interval between treatments and, after correcting for cell proliferation between treatments, the surviving fractions differed by a factor of 3.25, again indicating an average difference in survival of 1.26 after each of the 6 2-Gy/fractions. Treatment with 5 2-Gy doses of irradiation with 24 vs. 4 h elapsing between doses, resulted in a 3-fold greater decrease in survival in the presence of drugs vs. no drug. The relatively greater cell kill due to 24 vs. 4 h between treatments was due to drug inhibition of cell proliferation between the more prolonged treatments. CONCLUSIONS: The results of this study indicate that retinoic acid plus interferon both sensitizes and inhibits cell proliferation during treatment. These results suggest that this combination of radiation and drugs, when used concurrently, may be effective for inhibiting tumor cell proliferation or accelerated repopulation during clinical fractionated radiotherapy.     

Abscess formation in Listeria monocytogenes-infected gamma delta T cell deficient mouse mutants involves alpha beta T cells

To evaluate the usefulness of the transgenic Muta mouse for investigating radiation-induced mutations in vivo, we have examined the effects of whole-body X irradiation and compared them to the effects of ultraviolet light. The spontaneous mutation frequencies in young adults were about 7 x 10(-5) in the spleen, liver and skin. The mutation frequencies 1 week after a lethal dose of X radiation (8 Gy) were 3.2, 2.6 and 2.7 times the spontaneous levels in the spleen, liver and skin, respectively. When the skin was irradiated with 10 kJ m-2 of UVB, the mutation frequency increased about 6 times. The mutation frequencies induced by an acute dose of 4 Gy or by a fractionated dose of 11.7 Gy (0.15 Gy x 78 times, 3 times/week) in the spleen and liver were less than 2-fold the spontaneous levels at 16 weeks after irradiation. A comparison of X and UV radiation was also conducted with cultured cells derived from the mouse embryo. UVC of 5 J m-2 raised the mutation frequency to 15 times that of unirradiated cells, while 10 Gy X rays raised it 2.6 times. The findings indicate that the Muta mouse is less sensitive to X-ray-induced mutation than UV-induced mutation.     

Proliferation and clonal survival of human lung cancer cells treated with fractionated irradiation in combination with paclitaxel

PURPOSE: This study was performed to determine the effects of a continuous exposure to paclitaxel (taxol) in combination with fractionated irradiation on cell proliferation and survival. METHODS AND MATERIALS: Human lung carcinoma cells (SW1573) were given a daily treatment with 3 Gy of x-rays during 5 days in the continuous presence of 5 nM taxol. The surviving fraction and the total number of cells were determined every 24 h before and immediately after irradiation. RESULTS: Irradiation with 5 x 3 Gy and 5 nM taxol cause approximately the same inhibition of cell proliferation. In combination these treatments have an additional effect and the cell population increases no further after the first 24 h. Whereas the cells become more resistant to taxol after the first 24 h with a minimum survival of 42%, taxol progressively reduces the population of surviving cells in combination with x-rays when the number of fractions increases, up to 25-fold relative to irradiation alone. The enhancement effect of 5 nM taxol is likely to be attributed to an inhibition of the repopulation during fractionated irradiation and not to an increased radiosensitivity. Only after treatment with 10 or 100 nM taxol for 24 h, which is attended with a high cytotoxicity, is moderate radiosensitization observed. CONCLUSION: Taxol, continuously present at a low concentration with little cytotoxicity, causes a progressive reduction of the surviving cell population in combination with fractionated irradiation, mainly by an inhibition of the repopulation of surviving cells between the dose fractions.     

Effect of ionizing radiation on cell-cycle progression and cyclin B1 expression in human melanoma cells

In the present study we investigated the effect of gamma-irradiation (2.5 and 10 Gy) on cell-cycle progression of a human melanoma cell line, M14, characterized by a moderate radiosensitivity (SF2 = O.5). Flow cytometric analysis showed a dose-dependent S-phase accumulation, which was detectable 8 hr after treatment with 2 and 5 Gy and was still persistent at 12 hr after 10 Gy exposure. Such a delay in S-phase was paralleled or followed by an accumulation of cells in G2M, which was transient at the lowest radiation doses and still persistent at 72 hr after 10 Gy. Such an accumulation was, at least in part, due to a block in G2-M transition, as demonstrated by mitotic index analysis. Bivariate flow cytometric analysis of DNA content and cyclin B1 expression showed that, following 2 and 5 Gy, the fraction of cyclin B1-expressing cells was superimposable upon that of G2M cells. Conversely, in cells treated with 10 Gy, the fraction of cyclin B1-expressing cells was half the G2M cell fraction. Northern-blot analysis indicated that the radiation-induced decrease in cyclin B1 protein expression was accompanied by a reduced cyclin B mRNA level. On the whole, our results indicate a direct inhibitory effect of 10 Gy irradiation on cyclin B1 expression as a possible cause for the persistent G2 block in irradiated M14 cells.     

Radiation response of porcine RPE cells in vitro

PURPOSE: To investigate the antiproliferative effect of ionizing radiation on retinal pigment epithelial (RPE) cells that are supposed to play a major role in the pathogenesis of proliferative vitreoretinopathy (PVR). METHODS: RPE cells from pig eyes were irradiated with doses ranging from 4 to 16 Gy (1 Gray = 1 Joule/kilogram). Cells were counted at 1, 2, 3, and 4 weeks (Experiment 1) or 1, 2, 4, and 6 weeks (Experiment 2) after treatment. In Experiment 3, cells were trypsinized 24 h after radiation and seeded again. Colonies were counted 10 days later, and the surviving fraction was determined. RESULTS: The numbers of cells and colonies were inversely correlated to the doses applied. In Experiment 2, cell numbers of radiated cultures remained stable during the time of follow-up, whereas, in Experiment 1, significant proliferation occurred in treated cultures as well as in controls. This may be due to the higher growing rate that was found in the cultures of Experiment 2, compared to those of Experiment 1, at the time of radiation. In Experiment 3, a D0 value of 0.72 Gy was found. CONCLUSIONS: Proliferation of RPE cells can be suppressed by irradiation in a dose-dependent manner. Therefore, radiotherapy may be useful in the treatment of PVR. Its effect probably depends on the stage or activity of PVR at the time of radiation.     

Variation in radiosensitivity due to cell age and split-dose recovery in polykaryons induced by cytochalasin

We have investigated the properties of an in vitro cell survival assay that uses as its endpoint the ability to form polyploid cells (polykaryons) in the presence of cytochalasin B (CB). The criterion for survival is that a polykaryon-forming unit (PFU) must reach the arbitrary DNA content of at least 16C. The age-dependence of PFU sensitivity to 137Cs irradiation was determined using V79-379A cells synchronized at mitosis. Cells assayed as PFUs demonstrated much less variation in radiosensitivity with age than did clonogens, but the changes in curve shape were qualitatively similar. In both assays mitotic cells yielded an exponential survival curve while that obtained at 5 h (mid-late S) had a marked quadratic component. Owing to the small overall variation in PFU survival with age, at doses greater than about 25 Gy the surviving fraction at 5 h was lower than in mitosis. In both V79-379A and HeLa S3 cells, PFUs demonstrated a capacity for split-dose recovery and yielded recovery ratios at 2.6 at 50 Gy in V79 and 1.5 at 20 Gy in HeLa. Since these ratios were much lower than in clonogens at the same dose, we suggest that this is consistent with an association that we have previously demonstrated between PFU response and the clonogenic initial slope. In an attempt to clarify the DNA lesions to which PFUs may be sensitive, we determined PFU response following exposure to 254-nm UV irradiation. In contrast with ionizing radiation, PFU response to UV was very similar to that of clonogens. This suggests that following UV exposure the absence of cytokinesis in polykaryons may confer less protection than in the case of ionizing radiation, possibly due to fundamental differences in the spectrum of DNA lesions produced.     

Apoptosis in the chicken bursa of fabricius induced by X-irradiation

Immature B lymphocytes in the chicken bursa of Fabricius have previously been reported to undergo apoptosis by low doses of ionizing radiation. In the present study, newly hatched chickens were subjected to whole-body X-irradiation, and the bursa of Fabricius was examined at various postirradiation times by light and electron microscopy to obtain information on the change of ultrastructure of irradiated bursal cells as well as on the time course and dose-response for the induction of apoptosis. Histological examination by light microscopy showed that pyknotic cells started to increase in the bursa within a few hours after irradiation and the frequency of occurrence reached a maximum at 6 hr. An evident increase of the pyknotic cells in number was observed at a dose of as little as 1 Gy, and the frequency increased with increases in the dose, reaching over 90% at 15 Gy. Electron microscopy of the irradiated bursa revealed typical apoptotic morphology such as chromatin condensation, nuclear fragmentation, formation of apoptotic bodies and phagocytosis of pyknotic cells. Induction of apoptosis was also confirmed by the appearance of a typical DNA ladder pattern on agarose gel. Thus, the present results demonstrate that the chicken bursal cells are hypersensitive to X-irradiation with regard to induction of apoptosis, and that the apoptotic bursal cells exhibit most of the ultrastructural features known to be typical of apoptosis.     

Radioprotective effects of diltiazem on cytogenetic damage and survival in gamma ray exposed mice

Diltiazem, a calcium ion channel blocker, already in use in cardiovascular therapeutics, has been observed to protect against bone marrow damage (cytogenetic damage, cell death) and mortality in whole body irradiated mice. The micronuclei fraction in bone marrow cells of whole body irradiated (60Co gamma rays, 2.0 Gy) mice was reduced from 2.24 +/- 0.23% to about 0.74 +/- 0.33% by preirradiation administration (-20 min) of 110 mg/kg body wt. diltiazem (ip). Endogenous colony forming unit counts in spleen of mice administered 110 mg/kg body wt. (-20 min) of diltiazem before 10 Gy whole body irradiation were 6 times more than untreated irradiated controls. Pretreatment with diltiazem accelerated the recovery of radiation induced weight loss also. Diltiazem (110 mg/kg body wt, -20 min) enhanced 30 day survival to about 95% and 85% after lethal whole body absorbed dose of 9 and 10 Gy respectively and also mitigated radiation induced life- span shortening. Post-irradiation (10 Gy) administration of diltiazem (+20 to 30 min) enhanced survival from about 2 to 15% only but was highly significant (P < 0.001). Possible modes of radioprotective action of diltiazem have been discussed.