Combination of pentoxifylline and α-galactosylceramide with radiotherapy promotes necro-apoptosis and leukocyte infiltration and reduces the mitosis rate in murine melanoma.

Despite the success for the treatment of melanoma such as targeted molecular therapy, the use of such treatments are expensive For this reason, this study was carried out to explore the anti-cancer properties of available drugs that are able to modify the melanoma prognosis. The study was conducted in two phases: Evaluation of pharmacological effects of pentoxifylline (PTX) administered above (60 mg/kg) which is the therapeutic dose that is aimed at reducing the side-effect of radiotherapy, and of α- galactosylceramide (GalCer) administered at 100 µg/kg, as well as their combination using a murine model (BDF1 mice) of melanoma cell line (B16-F1, ATCC). For the radiotherapy phase, 9 Gy was applied in the tumor area, before (3 days), during (30 min) and after (3 days) the PTX + GalCer treatment. In both study phases, the mitosis rate, leukocyte infiltration and necro-apoptosis were assessed using histological and immunohistochemical approach and tumor volume evaluation as biomarkers. All treatments showed good prognosis results estimated as reduction of mitosis rate (PTX + GalCer after radiotherapy and GalCer), increased leukocyte infiltrate (PTX + GalCer after radiotherapy and GalCer) and necro-apoptosis augmentation (PTX + GalCer after radiotherapy and radiotherapy control). Nevertheless, a lower development of tumor volume was found in GalCer treatment. In this way, it is possible to suggest that the integrated treatment with immuno-stimulators such as GalCer, plus drug used for peripheral vascular disease (PTX) after radiotherapy is probably an alternative for controlling aggressive melanoma in murine model.

Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

Automatic Detection of Mitosis and Nuclei From Cytogenetic Images by CellProfiler Software for Mitotic Index Estimation.

Mitotic Index (MI) estimation expressed as percentage of mitosis plays an important role as quality control endpoint. To this end, MI is applied to check the lot of media and reagents to be used throughout the assay and also to check cellular viability after blood sample shipping, indicating satisfactory/unsatisfactory conditions for the progression of cell culture. The objective of this paper was to apply the CellProfiler open-source software for automatic detection of mitotic and nuclei figures from digitized images of cultured human lymphocytes for MI assessment, and to compare its performance to that performed through semi-automatic and visual detection. Lymphocytes were irradiated and cultured for mitosis detection. Sets of images from cultures were analyzed visually and findings were compared with those using CellProfiler software. The CellProfiler pipeline includes the detection of nuclei and mitosis with 80% sensitivity and more than 99% specificity. We conclude that CellProfiler is a reliable tool for counting mitosis and nuclei from cytogenetic images, saves considerable time compared to manual operation and reduces the variability derived from the scoring criteria of different scorers. The CellProfiler automated pipeline achieves good agreement with visual counting workflow, i.e. it allows fully automated mitotic and nuclei scoring in cytogenetic images yielding reliable information with minimal user intervention.

Current status of biodosimetry based on standard cytogenetic methods.

Knowledge about dose levels in radiation protection is an important step for risk assessment. However, in most cases of real or suspected accidental exposures to ionizing radiation (IR), physical dosimetry cannot be performed for retrospective estimates. In such situations, biological dosimetry has been proposed as an alternative for investigation. Briefly, biodosimetry can be defined as individual dose evaluation based on biological endpoints induced by IR (so-called biomarkers). The relationship between biological endpoints and absorbed dose is not always straightforward: nausea, vomiting and diarrhoea, for example, are the most well-known biological effects of individual irradiation, but a precise correlation between those symptoms and absorbed dose is hardly achieved. The scoring of unstable chromosomal-type aberrations (such as dicentrics and rings) and micronuclei in mitogen-stimulated peripheral blood, up till today, has been the most extensively biodosimetry assay employed for such purposes. Dicentric assay is the gold standard in biodosimetry, since its presence is generally considered to be specific to radiation exposure; scoring of micronuclei (a kind of by-product of chromosomal damages) is easier and faster than that of dicentrics for dose assessment. In this context, the aim of this work is to present an overview on biodosimetry based on standard cytogenetic methods, highlighting its advantages and limitations as tool in monitoring of radiation workers' doses or investigation into accidental exposures. Recent advances and perspectives are also briefly presented.

Induction of mitotic crossing-over in diploid strains of Aspergillus nidulans using low-dose X-rays.

As a contribution towards detecting the genetic effects of low doses of genotoxic physical agents, this paper deals with the consequences of low-dose X-rays in the Aspergillus nidulans genome. The irradiation doses studied were those commonly used in dental clinics (1-5 cGy). Even very low doses promoted increased mitotic crossing-over frequencies in diploid strains heterozygous for several genetic markers including the ones involved in DNA repair and recombination mechanisms. Genetic markers of several heterozygous strains were individually analyzed disclosing that some markers were especially sensitive to the treatments. These markers should be chosen as bio-indicators in the homozygotization index assay to better detect the recombinogenic/carcinogenic genomic effects of low-dose X-rays.

Induction of mitotic crossing-over in diploid strains of Aspergillus nidulansusing low-dose X-rays

As a contribution towards detecting the genetic effects of low doses of genotoxic physical agents, this paper deals with the consequences of low-dose X-rays in the Aspergillus nidulans genome. The irradiation doses studied were those commonly used in dental clinics (1-5 cGy). Even very low doses promoted increased mitotic crossing-over frequencies in diploid strains heterozygous for several genetic markers including the ones involved in DNA repair and recombination mechanisms. Genetic markers of several heterozygous strains were individually analyzed disclosing that some markers were especially sensitive to the treatments. These markers should be chosen as bio-indicators in the homozygotization index assay to better detect the recombinogenic/carcinogenic genomic effects of low-dose X-rays.

The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells.

Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability.

The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells

Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability.

Cell cycle time and rate of entry of cells into mitosis in the small intestine of young rats.

Cell cycle time (T(C)) and the rate of entry of cells into mitosis (r(M)) in the jejunum and duodenum of young rats were investigated using the stathmokinetic method. The cell cycle times in the jejunum were 24.3 and 28.3 h in light and dark periods, respectively. Cell cycle times in the duodenum were 17.1 and 21.5 h in light and dark periods, respectively. Rates of entry of cells into mitosis in the jejunum were 1.2 and 1.1 cells/cell/h in light and dark periods and rates of entry of cells into mitosis in the duodenum were 1.4 and 1.8 cells/cell/h in light and dark periods, respectively. Although these changes to cell cycle time values are not statistically significant, the variation between the two periods should be considered in relation to its possible biological effects.

Histological changes in rat duodenum mucosa after whole-body gamma irradiation.

In order to understand the mechanisms of intestinal injuries due to ionizing radiation, various groups of rats have been whole-body irradiated by gamma-rays at two dose rates (1 Gy/min and 1 Gy/hr), three doses (1, 2 and 4 Gy) and two post-irradiation times (24 and 48 hr). Duodenum samples of the animals were prepared for light microscopy, according to classical methods for histology and TUNEL reaction. A small number of morphological differences were observed within the mucosa between the two dose rates used. The extent and the number of lesions were more important at the slower dose rate (1 Gy/hr) and increased with the total dose. Clear cavities were seen inside the lamina propria which appeared like capillaries free of blood cells. The mitotic index calculated from crypt cells showed a regular decrease with the dose, which was exacerbated at 48 hr post-irradiation. On the other hand, the apoptotic index increased with the dose and the postirradiation time. Our results lead to hypothesize another mechanism of intestinal mucosa renewal allowing to explain mucosa denudations observed after radiotherapy. Thus we propose a new concept in which the duodenal mucosa renewal may occur by whole villi shedding into the duodenal lumen.

Photo-induced cytomorphologic changes in an advanced cancer phase I clinical trial.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate whether the application of an Infrared Pulsed Laser Device (IPLD) photo-induced significant cytomorphologic changes during the monitoring of advanced cancer patients participating in a phase I clinical trial. MATERIALS AND METHODS: Patients were irradiated with an IPLD (904 nm pulsed at 3 MHz) under a one-dose, one-schedule, and one-procedure design. Total daily dose consisted of a Radiant Exposure of 4.5x10(5) J/m(2). Thirty-one tissue samples from eleven patients with progressive solid neoplastic diseases (TNM IV, UICC) were obtained at three intervals: Time 0 (15-90 days pre-treatment, n=11); Time I (2-5 months post-treatment; n=11); Time II (6-12 months post-treatment, n=09). Three blinded pathologists evaluated samples; scores were determined by consensus. Data were evaluated by using the Wilcoxon matched-pairs signed-rank test and Spearman rank correlation coefficient. The level of statistical significance was alpha=0.05. RESULTS: Increased apoptosis (Time I, P<0.003; Time II, P<0.007), necrosis (Time I, NS; Time II, P<0.01), cytoplasmic vacuoles (Time I, P<0.03; Time II, P<0.02), and nuclear vacuoles (Time I, NS; Time II, P<0.01), reduced cell size (Time I, P<0.007; Time II, P<0.01) and intercellular adhesion (Time I, P<0.01; Time II, P<0.02) were present in neoplastic cells after IPLD treatment. No apparent changes were noted in non-neoplastic cells. The Spearman rank correlation coefficient between apoptosis, necrosis, nuclear vacuoles, cytoplasmatic vacuoles, intercellular adhesion, and cell size was positive and highly significant (P<0.006). CONCLUSIONS: Although further research is necessary, our preliminary results support the novel possibility that the IPLD photo-induces chaotic dynamics that modulate complex physiologically reparative bioeffects.

Protective effect of ethanol on X-ray-induced mitotic recombination in Drosophila melanogaster.

The effect of ethanol pretreatment on X-ray-induced mitotic recombination in D. melanogaster females was investigated by means of the white/white+ (w/w+) spot test. White females inseminated by yellow males were allowed to oviposit for 8 hr on medium containing 5%, 7.5% and 10% (v/v) ethanol and submitted to 10 Gy of X-rays 52 hr after the beginning of the egg laying period (chronic treatments). For acute treatments 56 +/- 4-hr-old larvae grown in regular medium were held in petri dishes containing filter paper soaked with 50% (v/v) ethanol for 30 min before being irradiated with 10 Gy. The emerging heterozygous w/w+ females were inspected for the presence of white spots (LS) in their eyes. Acute ethanol pretreatments lead to a significant reduction in the frequency of LS. This is suggested to be due to the scavenging by ethanol of free radicals originating during irradiation. If so, the contribution of the indirect action of radiation to mitotic recombination induced by X-rays must be significant. Chronic ethanol pretreatments also resulted in a decrease of LS, though impairment of larval development by ethanol may have partly contributed to the effect observed. At the concentrations tested, ethanol by itself did not modify the frequency of LS observed in the control.

[Effect of irradiation and heat on the agglutination and mitogenic capacity of phytohemagglutinins]. / Efeito Da irradiação e do calor sobre a capacidade Aglutinante E Mitogênica De Fito-Hemaglutininas

The authors studied the effect of ionizing radiation on hemaglutinating and mitogenic activity of phytohemaglutinis (PHA) in solution. 10 Krad (electron beam) are needed for the destruction of 50% of the aglutinating capacity. The mitogenic effect is more resistent to irradiation (70 Krad for 50% inactivation) maybe because both effects are due to different molecules. Changes were also followed by electrophoresis in polyacrylamida. The resistence to irradiation showed to be exponential function of the concentration of PHA in solution.