Correlation between cell survival and micronuclei-induction in HeLa cells treated with adriamycin after exposure to various doses of gamma-radiation.

The effect of 10 microg/ml of adriamycin (doxorubicin) post-treatment was studied in HeLa cells exposed to 0, 0.5, 1, 2 and 3 Gy of gamma radiation. The survival of HeLa cells declined in a dose dependent manner in both irradiation+PBS and irradiation+ADR groups. Treatment of adriamycin immediately after irradiation resulted in a significant decline in the cell survival. The surviving fraction of HeLa cells reduced to 0.61 after exposure to 0. 5 Gy in the irradiation+ADR group, whereas a similar effect (i.e. surviving fraction of 0.61) was obtained for 3 Gy in the irradiation+PBS group. In contrast, the frequency of micronuclei increased in a dose dependent manner in both irradiation+PBS and irradiation+ADR groups. A significant elevation in the frequency of micronuclei was observed in the latter when compared with the former group. The dose response for both groups was linear quadratic. The cell proliferation indices also showed a dose dependent decline in both the groups. The decline in the cell proliferation was significantly higher in the irradiation+ADR group when compared with the irradiation+PBS group. A close correlation between the cell survival and micronuclei induction was observed in both groups, where the cell survival declined with the elevation in the micronuclei frequency. The relationship between cell survival and micronuclei induction was linear quadratic.

Increased radiation-induced chromosome breakage after progesterone addition at the G1/S-phase transition.

Pregnant females appear to have an increased chromosomal sensitivity to gamma-irradiation. This hypersensitivity was found to parallel the increase of gestation hormone amounts [M. Ricoul, L. Sabatier, B. Dutrillaux, Increased chromosome radiosensitivity during pregnancy, Mutat. Res. 374(1997) 73-78]. An in vitro experiment was developed to study the effect of progesterone. We performed irradiations of whole blood from normal human donors and chromosome were analysed in first generation metaphases. By comparison to untreated controls, all cultures in which progesterone was added around the 24th h of culture exhibited an increased frequency of chromosome rearrangements, principally dicentrics and rings, which confirms the role of progesterone in the results of in vivo studies. BrdU incorporation studies suggested that progesterone was particularly efficient just before the entry into S-phase, which corresponds to the G1/S transition period. Cultures with an increased frequency of chromosome breakage had a slightly higher mitotic index than controls. It is suggested that progesterone may stimulate DNA repair in cells which reached the end of G1-phase with unrepaired breaks. This would allow the cells to enter the S-phase and survive, although some illegitimate repair leads to chromosome rearrangements, visible at the following metaphase.

A method to score micronuclei in vivo using cytochalasin B-induced cytokinesis block.

The present paper describes an in vivo micronucleus assay using Cytochalasin B (CyB). Mice bearing three different tumours, fibrosarcoma (Swiss albino mice), B16 F1 melanoma (C57 BL) and Ehrlich ascites carcinoma (Swiss albino mice), were injected with repeated doses of CyB at different time intervals and binucleate cells were scored at 24, 36, 48, 60 and 72 h after CyB injection. It was found that three doses of 3+2+2 mg/kg CyB administered intraperitoneally (i.p.) at 12-h intervals effectively blocked cytokinesis. The maximum number of binucleated cells (BNC) was scored at 60 h after the last CyB dose. This dose schedule was also effective in scoring micronuclei in BNC after irradiation.

A histopathological assessment of the response of rectal adenocarcinoma to combination chemo-radiotherapy: relationship to apoptotic activity, p53 and bcl-2 expression.

AIMS: To investigate the use of pre-operative chemo-irradiation in downstaging advanced rectal cancer prior to surgical resection. METHODS: We examined the pathological effects of chemo-irradiation on 24 rectal tumours and correlated the efficacy of treatment with the level of apoptosis, mitosis, P53 and bcl-2 protein expression on pre-treatment biopsies. RESULTS: All tumours were resectable following chemo-irradiation. Six cancers showed complete regression with no viable tumour in the resection specimen. A significant correlation was found between spontaneous tumour apoptosis and tumour regression. CONCLUSIONS: Our results suggest that in rectal cancer the apoptotic rate in untreated tumour tissue may predict sensitivity to radiation and cytotoxic agents. No relationship was found between regression and mitotic rate, p53 or bcl-2 expression.

Proliferative activity and micronucleus frequency after radiation of lung cancer cells as assessed by the cytokinesis-block method and their relationship to clinical outcome.

We previously proposed a new assay using the cytokinesis-block micronucleus (MN) technique to estimate the fraction of cells undergoing mitosis in vitro [dividing fraction (DF)], potential doubling time (Tpot), and radiosensitivity (in terms of MN frequency) of human tumors. In the present study, we applied this technique to primary lung cancers to evaluate their biological characteristics, and the assay results for the proliferative activity were compared with the treatment outcome. Tumor tissues were disaggregated to single cells, which were cultured in the presence of cytochalasin B after (or without) radiation. At intervals, the proportion of multinucleate cells (its maximum value is the DF), the average number of nuclei/cell, and MNs in binucleate cells were scored. The Tpot was the extrapolated time for the nuclei:cell ratio to reach 2.0. Of the 71 tumor samples obtained, the DF and Tpot were evaluable in 61 (86%), and the MN frequency was evaluable in 52 (73%). The median DF and Tpot values were 23% and 7.7 days, respectively, for adenocarcinoma (n = 41), 26% and 8.9 days for squamous cell carcinoma (n = 13), 27% and 6.5 days for large cell carcinoma (n = 3), and 30% and 7.0 days for small cell carcinoma (n = 4). There was no significant difference in the mean DF or Tpot values according to the histological type or disease stage. The mean MN frequency after 2 Gy of radiation (minus the 0 Gy frequency) was 0.15 for adenocarcinoma, 0.17 for squamous cell carcinoma, 0.16 for large cell carcinoma, and 0.20 for small cell carcinoma. The MN frequency after radiation was positively correlated with both the DF and the baseline (at 0 Gy) MN frequency. In non-small cell lung cancer, a DF above the median was associated with an increased recurrence rate after operation, and the Tpot was correlated with the time until relapse in patients who developed recurrence. Although the clinical significance of the MN frequency needs to be clarified in future studies, the DF and Tpot determined by this assay appear to be good parameters of tumor proliferative activity.

Protection by black tea and green tea against UVB and UVA + B induced skin cancer in hairless mice.

The effects of green and black tea consumption on the early indices of UVB and UVA + B skin damage in hairless mice have been studied in the absence of any chemical tumour initiators or promoters. Black tea consumption was associated with a reduction in the number of sunburn cells in the epidermis of mice 24 h after UVA + B irradiation, although there was no effect of green tea. Other indices of early damage such as necrotic cells or mitotic figures were not affected. Neutrophil infiltration as a measure of skin redness was slightly lowered by tea consumption in the UVB group. Consumption of either green or black tea resulted in significantly fewer skin papillomas and tumours induced by UVA + B light, however black tea provided better protection against UVB-induced tumours than green tea. This study confirms earlier reports that tea consumption can reduce the incidence of skin cancer in hairless mice, and indicates that black tea may afford more protection against simulated solar irradiation than green tea.

Use of hematopoietic cytokines to accelerate the recovery of the immune system in irradiated mice.

In our previous studies aimed at designing appropriate strategies to accelerate recovery of the immune system after irradiation, we found that the hematopoietic cytokine recombinant murine (rmu) interleukin (IL)-3 was able to induce differentiation and growth of thymocytes and splenic T and B lymphocytes in mice exposed to x-rays (200-500 cGy). The recovery, however, was complete at 7 days only after a dose of 200 cGy, whereas 2, 3, and 4 weeks were necessary to achieve full recovery after 300, 400, and 500 cGy, respectively. These studies were extended to investigate the effects of another hematopoietic cytokine, recombinant human (rhu) IL-11, a bone marrow stromal-derived cytokine, administered together with IL-3 to irradiated mice. The synergistic effect of the two cytokines was evident when relatively small doses of rhu IL-11 were injected with an optimal dose of rmu IL-3.

Spatial and temporal patterns of expression of epidermal growth factor, transforming growth factor alpha and transforming growth factor beta 1-3 and their receptors in mouse jejunum after radiation treatment.

The goal of the present study was to assess changes in proliferation in the mouse jejunum after irradiation and the role of the growth factors EGF, TGF-alpha and TGF-beta 1-3 in the proliferative response. Our working hypothesis was that feedback signals from the villus to cells in the crypt regulate proliferation, and that the growth factors EGF and TGF-alpha with their common receptor EGF-R are involved in stimulation of proliferation, while the growth factors TGF-beta 1-3 with their receptors TGF-beta RI and TGF-beta RII are involved in inhibition of proliferation during this regulation. Immunohistochemical detection methods and automated image analysis were used for objective quantification of growth factor expression. The data indicate that, after 5 Gy irradiation, growth stimulation in the crypts takes place before major changes in the villi are observed. However, the combination of the reduction in the cell number, the number of cells expressing TGF-beta 1-3 and the reduction in the level of expression of TGF-beta 1-3 in the villi may cause the release of crypt cells from regulatory growth inhibition and initiate a proliferation-stimulating signal by an increase in the production of TGF-alpha and EGF. Regulation of proliferation after initiation of a proliferative response seems to be related more to the growth factors EGF, TGF-alpha and TGF-beta 3 in the crypts than to villus cellularity or growth factor expression, supporting the concept of stem cell autoregulation as a mechanism of cell regeneration in the intestinal crypt.

Biomodulative effects induced by 805 nm laser light irradiation of normal and tumor cells.

The influence of light emitted from a diode laser centred at lambda = 805 nm was investigated on murine skeletal myotubes (C2), normal urothelial cells (HCV29), human squamous carcinoma cells of the gingival mucosa (ZMK) and urothelial carcinoma cells (J82) in a computer-controlled irradiation chamber. Cells were treated with varying fluences between 0 and 20 J cm-2. The response was tested by analysis of the mitotic index using single cell counting after Orcein staining and proliferation index based on BrdU incorporation during DNA synthesis. While the mitotic index of C2, HCV29 and J82 cells increased at a fluence of 4 J cm-2, irradiation with fluences of 20 J cm-2 resulted in a slight decrease. ZMK tumor cells showed a decrease of the mitotic index with both fluences. No significant differences could be determined when using irradiances between 10 mW cm-2 and 150 mW cm-2. The BrdU test after irradiation showed no significant effects compared to the controls in each cell line.

Gamma-ray-induced cell killing and chromosome abnormalities in the bone marrow of p53-deficient mice.

Resistance to the lethal effects of ionizing radiation has been demonstrated in a wide variety of cell types with defects in the p53 gene (thymocytes, splenic B and T cells, in vitro hemopoietic colony-forming cells and intestinal cells of the mouse, embryo cells of the rat, and human Burkitt's lymphoma cells). In contrast, Slichenmeyer et al. (Cancer Res. 53, 4164-4167, 1993) found no evidence of resistance in fibroblasts derived from p53 null mice. The aim of our study was to compare the radiation response of hemopoietic colony-forming cells (in vitro CFC) and of fibroblastoid colony-forming cells or units (CFU-F) within the same tissue (marrow) in p53 null mice (-/-), heterozygotes (+/-) and wild-type animals (+/+). We have also tested the hypothesis that, in proliferating cells, radiation-induced cell killing is mediated through chromosome damage by examining the relationship between these end points in hemopoietic cells of the three mouse types. Both in vitro CFC and CFU-F of -/- mice were resistant to cell killing compared with +/+ and +/- mice whose cellular sensitivities were indistinguishable. The resistance was characterized by a broader "shoulder" on the cell survival curve, i.e. a higher extrapolation number but similar D0 values using the multitarget model or a lower alpha coefficient using the linear-quadratic model. The frequency of chromosomally abnormal marrow cells after irradiation was similar for the three genotypes. However, marrow cells with aberrations carried more aberrations in -/- mice than in +/+ or +/- mice such that the total number of aberrations per 100 cells was higher in -/- mice. Since there were no differences in the yields of aberrations between genotypes in spleen lymphocytes or in CFU-F (both noncycling at the time of irradiation) and less mitotic inhibition in -/- marrow cells than in +/+ or +/- cells, the chromosomal radiosensitivity of -/- marrow hemopoietic cells might be related to reduced cell cycle delay allowing insufficient time for repair, but other explanations have been considered. We postulate that the radiation resistance of both hemopoietic CFC and CFU-F in -/- mice is a consequence of the failure of DNA/chromosome damage to trigger apoptosis or permanent cell cycle arrest to the same extent as in the +/+ or +/- mice: hence the lack of correlation between chromosome damage and cell death in the three mouse types.

In vivo effect of gamma ray pretreatment on sister-chromatid exchange induction by mitomycin C in murine bone marrow cells.

The effect of gamma ray pretreatment on sister-chromatid exchange induction by mitomycin C (MMC) was determined in murine bone marrow cells in vivo. A 30% reduction in the expected SCE frequency was observed assuming an additive effect. These results support the prediction of the replicative model for SCE formation with regard to the interactions between mutagens, and confirm previous results focused on the adaptive response.

Oncogenic transformation of C3H 10T1/2 cells exposed to alpha particles: sensitivity through the cell cycle.

Oncogenic transformation of synchronized C3H 10T1/2 cells was determined after exposure to 4.3 MeV alpha particles (LET = 101 keV/microns). Two synchronization techniques were tested using basic and modified protocols: one based on the release of cells from contact inhibition and the second on the mitotic shake-off method. Progression of cells through the cycle was followed as a function of time by flow cytometric analysis, DNA labeling for passage through S phase, the growth curve for the cell number and mitotic index measurements. The conclusion is that, although the release of cells from confluence provides higher yields of synchronized cells, mitotic shake-off proved to be the best way of collecting a synchronized population of minimally perturbed cells. Cells synchronized by mitotic shake-off were irradiated with 0.30 Gy in the interval between 2 and 10 h corresponding to G1 and early S phases. For comparison asynchronous populations were irradiated in parallel. Oncogenic transformation frequency, corrected for background, in mid-G1 phase was (18 +/- 4) x 10(-5) (average values of frequencies at 4 and 6 h) compared with the value of (8 +/- 4) x 10(-5) for the asynchronous population. While these data are suggestive of a trend toward a slightly increased sensitivity in mid-G1 phase, it is not statistically significant. The surviving fraction is constant in G1 phase.

Effect of visible light on some cellular and immune parameters.

The biological effect of visible light of low energy density was investigated in this study. The effects of diffuse (DL) and linearly polarized (LPL) light were compared on models in vitro and in vivo. Experiments in vitro were performed on human lymphocytes to study their blast-transformation and rosette-formation abilities. Both DL and LPL increased the number of blast-transformed cells even in a lymphocyte culture without PHA, and reduced rosette-formation of T lymphocytes. LPL had a more pronounced effect. In vivo exposure to DL and LPL of the spleens of tumour-bearing mice caused the appearance of factor(s) in their serum, inhibiting the incorporation in vitro of [3H]-thymidine into the tumour cells obtained from non-exposed animals. In the other series of experiments serum samples were taken from tumorous animals after the exposure of their spleens to LPL. Following the daily administration of these sera to another group of non-exposed tumorous mice a decreasing tendency of the mitotic kinetics of ascites tumour was observed. The application of visible (preferably linearly polarized) light for the stimulation of human immune competent cells, and clinical trials with extracorporeal irradiation of blood for the promotion of natural defences of an immune-repressed organism are suggested.

Defective G2 checkpoint function in cells from individuals with familial cancer syndromes.

The early events in the G2 checkpoint response to ionizing radiation (IR) were analyzed in diploid normal human fibroblasts (NHFs) and fibroblasts from patients with two heritable cancer syndromes. Exposure to gamma-radiation of asynchronously growing NHFs resulted in a rapid reduction in the number of cells in mitosis (G2 delay) and was accompanied by a quantitatively similar reduction in the p34CDC2/cyclin B in vitro histone H1 kinase activity as compared with sham-treated controls. This G2 delay was strong by 1 h following exposure to IR, maximal by 2 h, and was accompanied by an accumulation of tyrosine-phosphorylated p34CDC2 molecules. In contrast, fibroblasts from individuals with ataxia telangiectasia displayed significantly less reduction of the mitotic index or histone H1 kinase activity after IR. Low passage fibroblasts from individuals with Li-Fraumeni syndrome having one wild-type and one mutated p53 allele were similar to NHFs in their immediate G2 checkpoint response to IR, as were NHFs expressing the human papilloma virus type 16 E6 gene product (functionally inactivating p53) and low passage cells from p53-deficient mouse embryos. However, the p53-deficient fibroblasts were genomically unstable and became defective in their early G2 checkpoint response to IR. Furthermore, immortal Li-Fraumeni syndrome fibroblasts lacking wild-type p53 displayed an attenuated G2 checkpoint response. These results link the early events in G2 checkpoint response to IR in NHFs with a rapid inhibition of p34CDC2/cyclin B protein kinase activity and demonstrate that while not required for this immediate G2 delay, lack of p53 can lead to subsequent genetic alterations that result in defective G2 checkpoint function.

Morphology and morphometry of mice's terminal ileum wall exposed to x-ray

Foram estudados os aspcectos morfológicos da parede do íleo de camundongos da linhagem C58BL expostos aos raios X, corpo inteiro, na dose única de 154,8 mC/Kg (600 R) e também a morfometria das figuras de mitose e das células caliciformes à microscopia de luz comum. Observamos alteraçöes morfológicas intensas no epitélio da mucosa ileal no período de 24 horas após irradiaçäo, tendência a recuperaçäo às 72 horas e normalizaçäo às 144 horas. A análise morfométrica demonstrou o índice mitótico significantemente baixo (quase nulo) no período significantemente baixo (quase nulo) no período de 24 hotas depois da irradiaçäo, rebote às 72 horas e normalizaçäo do mesmo às 144 horas. O índice de células caliciformes evidenciou diminuiçäo significante no período de 72 horas após irradiaçäo e recuperaçäo às 144 horas

Morfologia e morfometria da parede do íleo terminal de camundongos expostos aos raios X / Morphology and morphometry of the terminal ileum wall of mices exposed to X ray

Foram estudados os aspectos morfológicos da parede do ileo de camundongos da linhagem C57BL expostos aos raios X, corpo inteiro, na dose única de 154,8mC/Kg (600 R) e também a morfometria das figuras de mitose e das células calciformes à microscopia de luz comum. Observamos alteraçöes morfológicas intensas no epitélio da mucosa ileal no período de 24 horas após irradiaçäo, tendência à recuperaçäo às 72 horas e normalizaçäo às 144 horas. A análise morfométrica demonstrou o índice mitótico significantemente baixo (quase nulo) no período de 24 horas depois da irradiaçäo, rebote às 72 horas e normalizaçäo do mesmo às 144 horas. O índice de células caliciformes evidenciou diminuiçäo significante no período de 72 horas após irradiaçäo e recuperaçäo às 144 horas

The effect of ultraviolet-B irradiation on the cell shedding rate of the corneal epithelium.

In this study cell shedding rates of the corneal epithelium were determined in vitro following a single suprathreshold dose of ultraviolet-B irradiation. Rabbit corneas were excised and superfused in pairs. The epithelial and endothelial surfaces were bathed with solutions containing essential ions and adjusted to appropriate pH and osmolality. One cornea in each pair was irradiated, while the other cornea served as a control. Shed cells were periodically collected from the corneal surface and the shedding rate determined by counting the number of cells in a 50 min time period. Following a latent period of about 3 h, the epithelial shedding rate accelerated, and remained elevated for at least 8 h after irradiation. This result suggests that suprathreshold doses of ultraviolet-B irradiation disrupt the normal orderly cell shedding process and homeostatic equilibrium of the corneal epithelium. The elevated cell shedding rate exposes subsurface nerve endings and causes the characteristic pain of photokeratitis.

Efectos de la radiación ultravioleta A y B sobre linfocitos humanos / Effects of A and B ultraviolet irradiation over human lymphocutes

Over the last decade, agreter decrease in the ozone level has occurred in the Southern Hemisphere. For each 1 per cent decrease in this level, a 2 per cent increase in biologically effective radiation occurs. Aiming to evaluate the biological effect produced by UV radiation, 10 blood samples coming from patients consulting for reproductive problems, were irradiated with visible and ultraviolet radiation A (treatment A) and visible and ultraviolet radiation B (treatment B) during 1 to 5 minutes. This dosage is comparable to the radiation received in Santiago at 13:00 h in a summer day. After irradiation, lymphocytes were cultured during 72 h and the number of altered metaphases was quantified. There was a significant increase in chromosomal alterations with treatment A (2.61, 2.43, 4.53 and 3.53 at 1, 2, 3 and 5 min respectively) and treatment B (3.06, 3.81, 3.3, 5.51, at 1, 2, 3 and 5 min respectively) compared with non-irradiated controls (0.8 and 0.72). There was a reduction in mitotic indices in irradiated cells. It is concluded that both types of UV radiation (A and B) produce chromosomal alternations in vitro, even using lower doses than those received during summer in the central region of Chile

Biochemical parameters of epidermal aging in the hairless mouse and the relationship to UV-carcinogenesis.

Epidemiological studies suggest that the incidence of cancer increases with age in both human and animal populations and that declining physiologic condition associated with aging might be responsible. Experimentally, the reverse has been most often observed, that is, older animals appear less susceptible to the induction of UV-carcinogenesis. Thus, we examined several biochemical parameters of epidermal macromolecular synthesis in hairless mice in an effort to gain insight into the role these processes play in physiological aging and their relationship to carcinogenesis. SKh-Hr-1 hairless mice were randomized into two groups (UV-irradiated and non-irradiated controls) and were two months of age at the start of irradiation and biochemical analyses. The UV group received 0.028 sunburn units (SBUs) daily (5 days wk-1) for 16 months from 40 watt BZS-WLG lamps. Stratum corneum turnover rates (SCR), cell label index (CLI), protein, DNA and RNA synthesis, and ornithine decarboxylase (ODC) induction were determined at monthly intervals over a period of two years. There were no age-related tendencies observed in SCR. CLI increased with age. Chronic, low-dose UV had no effect upon either of these parameters. Epidermal capacity for DNA and protein synthesis increased with age from 2 months to 12-15 months at which time both parameters peaked and then began to decline. UV significantly reduced (P < 0.04) the magnitude of DNA synthetic capacity at peak periods of synthesis but had no effect upon protein synthesis. RNA synthetic rates declined with age, reaching their lowest levels at 24 months. Further, a significant reduction (P < 0.001) in ODC inducibility occurred with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiation-induced chromosomal aberrations in mouse 10T1/2 cells: dependence on the cell-cycle stage at the time of irradiation.

Cell-cycle stage radiosensitivity for the induction of chromosome aberrations has been investigated in C3H 10T1/2 cells. Exponentially growing cells were irradiated with 3 Gy X-rays (80 kVp) or 0.6 Gy alpha-particles (LET = 101 keV/micron). The two doses produce the same survival level (37%) in the asynchronous population. Cells were harvested at four different times following irradiation and cell-cycle phase at the time of irradiation was assessed by using the differential replication staining technique. The frequency of chromosome aberrations produced in a given stage of the cell cycle was not constant as a function of the sampling time, but this could not be simply related to the existence of subphases exhibiting different radiosensitivity, because of cell-cycle perturbation introduced by radiation. X-radiation induced more exchanges than deletions, whereas a predominance of isochromatid deletions was observed after alpha-irradiation. This can be interpreted on the basis of the different patterns of energy deposition of densely- and sparsely-ionizing radiation. Both X- and alpha-rays produced a significant increase in the frequency of Robertsonian translocations when cells were exposed in G1 or S phase, but not in G2 phase.