INDUCED POLYPLOIDY AND SORTING OF DAMAGED DNA BY MICRONUCLEATION IN RADIORESISTANT RAT LIVER EPITHELIAL STEM-LIKE CELLS EXPOSED TO X-RAYS. / INDUKOVANA POLIPLOÏDIIa TA SORTUVANNIa POShKODZhENOÏ DNK MIKRONUKLEATsIIeIu V RADIOREZYSTENTNYKh EPITELIAL'NYKh STOVBUROVYKh KLITYNAKh PEChINKY ShchURIV ZA VPLYVU RENTGENIVS'KYKh PROMENIV.

OBJECTIVE: Rat liver stem-like epithelial cells (WB-F344) that under certain conditions may differentiate into hepa- tocyte and biliary lineages were subjected to acute X-irradiation with the aim to examine cell cycle peculiarities dur- ing the course of survival. MATERIALS AND METHODS: Suspensions of WB-F344 cells that grew as a monolayer and reached sub-confluence were irradiated with 1, 5, and 10 Gy of X-rays (2 Gy/min). As an intact control, sham-irradiated cells were used. After irra- diation, cells were plated into 25-cm2 tissue culture flasks to culture them for over several days without reaching contact inhibition. On days 1, 2, 3, and 5 post-irradiation, cells were harvested and examined for nuclear morpholo- gy and DNA ploidy by stoichiometric toluidine blue reaction and image cytometry. On days 7 and 9 post-irradiation, only heavily irradiated (10 Gy) cells were examined. Also, 10 Gy-irradiated cells were chosen for immunofluorescence staining to monitor persistence of DNA lesions (γ-H2AX), cell proliferation (Ki-67), and self-renewal factors charac- teristic for stem cells (OCT4 and NANOG). RESULTS: Radioresistance of WB-F344 cells was evidenced by the findings that they do not undergo rapid and mas- sive cell death that in fact was weakly manifested as apoptotic even in heavily irradiated cells. Instead, there was cell cycle progression delay accompanied by polyploidization (via Ki-67-positive mitotic slippage or via impaired cytokinesis) and micronucleation in a dose-dependent manner, although micronucleation to some extent went ahead of polyploidization. Polyploid cells amenable for recovering from DNA damage can mitotically depolyploidize. Many micronuclei contained γ-H2AX clusters, suggesting isolation of severely damaged DNA fragments. Both factors, OCT4 and NANOG, were expressed in the intact control, but became enhanced after irradiation. CONCLUSIONS: Although the fact of micronucleation is indicative of genotoxic effect, WB-F344 cells can probably escape cell death via sorting of damaged DNA by micronuclei. Induction of polyploidy in these cells can be adaptive to promote cell survival and tissue regeneration with possible involvement of self-renewal mechanism.

Disentangling the aneuploidy and senescence paradoxes: a study of triploid breast cancers non-responsive to neoadjuvant therapy.

Aneuploid cells should have a reduced proliferation rate due to difficulty in proceeding through mitosis. However, contrary to this, high aneuploidy is associated with aggressive tumour growth and poor survival prognosis, in particular in triploid breast cancer. A further paradox revolves around the observation that, while cell senescence should inhibit proliferation, the senescence marker p16INK4a correlates with poor treatment outcome in patients with a very aggressive triple-negative breast carcinoma (TNBC). In this study, we aim to pour light on the possible relationship of these conundrums with polyploidy of tumour cells. We performed detailed analysis of DNA histogram profiles in diagnostic core biopsies of 30 cases of operable breast cancer and found that near triploidy in TNBC and other forms correlated with weak or no response to neoadjuvant chemotherapy (NAC) as scored by Miller-Payne index. Polyploid cells in operation samples from tumours that were non-responsive to NAC treatment were Ki67 and CD44 positive. In addition, polyploid cells were positive for markers of embryonic stemness (OCT4, SOX2, NANOG) and senescence (p16INK4a). The relationship patterns between p16INK4a and NANOG were heterogeneous, with predominantly mutually exclusive expression but also synergistic and intermediate variants in the same samples. We conclude that the aneuploidy and senescence paradoxes can be explained by the mutual platform of polyploidy, conferring genomic and epigenetic instability as a survival advantage. Such cells are able to bypass aneuploidy restrictions of conventional mitosis and overcome the barrier of senescence by a shift to self-renewal, resulting in progression of cancer.

Characterization of breast cancer DNA content profiles as a prognostic tool.

Worldwide, breast cancer in women remains to be the most common malignancy that in a considerable proportion shows the resistance to genotoxic treatments and poor outcome. Chromosomal instability manifested as aneuploidy represents an integral cha-racteristics of the malignant genotype not only because of the selection of mutated aneuploid sub-clones that stipulate the tumor progression, but also because of the reversible endopolyploidy of tumor cells that serves for the endless maintenance of therapy-resistant tumor stem cells. Therefore, cytometric determination of DNA content in tissue samples for detecting malignancy, monitoring responses to therapy, and prognosing disease outcome needs to be revived. Both flow and image cytometry are most frequently used for generation of DNA content profiles (histograms), interpretation of which, however, may have some caveats. This review presents the major characterization criteria and analysis tools for breast cancer DNA histograms.

Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation.

UNLABELLED: The aim of this study was to investigate the ability of environmental nitrogen oxides or natural nitric oxide (NO) donors to modify free radicals ba-lance and development of genomic instability alone or in combination with ionizing radiation. METHODS: Genotoxicity and cytogenetic abnormalities were assessed in vitro in peripheral blood lymphocytes (PBL) isolated from healthy humans or in vivo in rats PBL. Human PBL were treated with physiologically relevant NO donor - S-Nitrosoglutathione and X-ray irradiation. The inhalation treatment of animals with NO was carried out in chamber with purified gaseous NO mixed inside with air. Levels of S-Nitrosohemoglobin and methemoglobin in the blood were assessed with electron paramagnetic resonance. The total level of reactive oxygen and nitrogen species in PBL was determined fluorometrically, and serum levels of reactive oxygen species was determined by spectrophotometric assay. DNA damages were assessed by alkaline single-cell gel electrophoresis. The frequency of chromosomal aberrations in human PBL measured with the conventional cytogenetic assay in metaphase cells on short-term (52 h) and long-term (72 h) cultures. RESULTS: Environmental nitrogen oxides or release of NO from stable complexes with biomolecules (such as S-Nitrosothiols) intensified generation of free radicals, DNA damage and development of genomic instability alone or in combination with ionizing radiation. Treatment of PBL by S-Nitrosoglutathione caused prevalent induction of chromatid type but irradiation - chromosome aberrations. The dose dependence of chromatid-type aberrations observed in human PBL after combined influence of S-Nitrosoglutathione and ionizing radiation indicates a crucial role of NO in the formation of chromosomal instability. CONCLUSION: NO can deregulate free radicals balance resulted in genotoxic effect, posttranslational modification of repair enzymes and thus coordinated development of genomic instability and increase of cancer risk.

Fractionated low-dose radiation exposure potentiates proliferation of implanted tumor cells.

AIM: The purpose of this study is to test whether whole-body fractionated exposure of tumor-free animals to low doses of low-LET radiation (at the total delivered dose of 1.0 Gy of X-rays) is capable of potentiating growth of subsequently implanted tumor cells. MATERIALS AND METHODS: Adult male rats were fractionally exposed to low doses of X-rays (10 acute exposures with 0.1 Gy each and with a frequency of 1 exposure per 3 days). The next day after the last irradiation rats were implanted with Guerin carcinoma (GC) cells. On the 12th and 18th days after implantation of GC cells, animals were sacrificed, and the mass of tumors was measured by weighing them, although the kinetics of tumor growth was also examined by daily measurements of the dimensions of tumors. Cytotoxic effects in the bone marrow were assessed flow cytometrically in acridine orange-stained unfractionated bone marrow cells using the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE). RESULTS: In irradiated rats, tumors grew apparently faster than in unirradiated rats for up to 18 days after implantation of GC cells. On the 18th day after implantation of GC cells the average value of the mass of tumors in irradiated rats was 2.8-fold higher compared with the average value of the mass of tumors in unirradiated rats (p < 0.05). On this day post-implantation, the bone marrow in irradiated animals was 1.8-fold more suppressed (as evidenced by decreased PCE/NCE ratios) than that in animals that were irradiated, but were not implanted with GC cells (p > 0.05), and was 1.4-fold more suppressed than that in animals that were not irradiated, but were implanted with GC cells (p > 0.05). CONCLUSION: Fractionated irradiation of tumor-free animals with low doses of X-rays potentiates proliferation of subsequently implanted GC cells. This potentiation seems to be associated with radiation-induced impaired hematopoiesis.

The long-range cytotoxic effect in tumor-bearing animals.

AIM: The relationship between cancer and patient health is still of great interest for experimental and clinical oncology. The tumor can adversely affect surrounding and distant tissues as well. However, effects of the tumor on distant tissues are much less studied than its effects on surrounding tissues. This study was aimed to test whether the tumor could trigger cytotoxic and/or genotoxic signals with respect to the distant proliferative tissue such as bone marrow. MATERIALS AND METHODS: Rats were subcutaneously implanted with Guerin carcinoma cells, and on the 12(th) and 18(th) days after implantation both cytotoxic and genotoxic effects were assessed by flow cytometry in acridine orange stained unfractionated bone marrow cells isolated from femur. The cytotoxic effect was assessed using ratios of the following cell populations: total nucleated cells (TNC)/total enucleated erythrocytes (TE); polychromatic erythrocytes (PCE)/normochromatic erythrocytes (NCE). The genotoxic effect was assessed by quantification of micronucleated PCE (MNPCE) within the population of PCE. RESULTS: A significant cytotoxic effect was observed in tumor-bearing animals on the 12(th) and 18(th) days after implantation (≈ 2-fold decrease in both TNC/TE and PCE/NCE ratios compared with corresponding parameters in control animals). There was also a genotoxic effect in these animals (a slight increase in the number of MNPCE), however, this effect was insignificant. The PCE/NCE ratio reversely correlated with the tumor weight which is suggestive of the link between erythropoietic cytotoxicity and tumor progression. CONCLUSION: Cytotoxic insult to the bone marrow is likely to be associated with the mechanism(s) triggered by distantly located tumors whose growth may correlate with the cytotoxic effect.

At a crossroads of cancer risk and aging: the role of telomeres.

The risk of overall cancer inevitably increases with advancing age. The cancer incidence rate is not constant within the human life span (it exponentially increases with advancing age). Aging itself is a complex biological process with a poorly understood mechanism of its regulation. The aging process, as evidenced from the survey of the chances for death for the large cohort of people of various age groups, manifests probably due to a progressive accumulation of diverse adverse changes that increase the risk of death. While an increase of cancer risk due to aging cannot be fully explained, the length of telomeres (biomarker of aging) appears to be important to predict this risk. Cellular senescence, which is believed to be associated with dysfunctional (shortened) telomeres, may contribute to the aging of a whole organism. Here, based on recent literature data, we investigate the possible link between telomere dysfunction associated cellular senescence and tumorigenesis.

Challenges and progress in predicting biological responses to incorporated radioactivity.

Prediction of risks and therapeutic outcome in nuclear medicine largely rely on calculation of the absorbed dose. Absorbed dose specification is complex due to the wide variety of radiations emitted, non-uniform activity distribution, biokinetics, etc. Conventional organ absorbed dose estimates assumed that radioactivity is distributed uniformly throughout the organ. However, there have been dramatic improvements in dosimetry models that reflect the substructure of organs as well as tissue elements within them. These models rely on improved nuclear medicine imaging capabilities that facilitate determination of activity within voxels that represent tissue elements of approximately 0.2-1 cm(3). However, even these improved approaches assume that all cells within the tissue element receive the same dose. The tissue element may be comprised of a variety of cells having different radiosensitivities and different incorporated radioactivity. Furthermore, the extent to which non-uniform distributions of radioactivity within a small tissue element impact the absorbed dose distribution is strongly dependent on the number, type, and energy of the radiations emitted by the radionuclide. It is also necessary to know whether the dose to a given cell arises from radioactive decays within itself (self-dose) or decays in surrounding cells (cross-dose). Cellular response to self-dose can be considerably different than its response to cross-dose from the same radiopharmaceutical. Bystander effects can also play a role in the response. Evidence shows that even under conditions of 'uniform' distribution of radioactivity, a combination of organ dosimetry, voxel dosimetry and dosimetry at the cellular and multicellular levels can be required to predict response.

Philosophy of aging.

Aging is a complex biological process with a poorly understood mechanism of its regulation. Although numerous theoretical and experimental reports were dedicated to this interesting problem, it is doubtful that a single theory or mechanism can explain the nature of aging. In order to facilitate our current understanding of the aging process, we propose to consider it from a philosophical point of view based on a few postulates.

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements.

BACKGROUND: The ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle. METHODS: Asynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks. RESULTS: Cell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively. CONCLUSIONS: Growth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.

Model for regulation of non-muscle myosins.

Diverse morphological changes of non-muscle eukaryotic cell are usually accompanied by dynamic remodeling in its cytoskeleton. The conformation/functional state of myosins is presumed to be markedly altered during cytoskeletal reorganization. Although myosins play critical roles in various forms of cellular movement and shape changes, the molecular basis for regulation of their activity is complicated and far from understood. A numerous experimental data show the phosphorylatable light chain (20 kDa), known as a regulatory light chain (RLC), as one of the key modulators of regulatory signals in the structure of myosin-II molecule. However, the clue that coordinates its phosphorylation state remains to be enigmatic. In the present work, we propose the most rationale mechanism which might regulate the state of RLC phosphorylation, and therefore, might control organization and activity of non-muscle myosins.

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria.

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.

Myosin regulatory light chain as a critical substrate of cell death: a hypothesis.

As known, gamma-interferon-induced cell death in HeLa cells can be mediated via the recently identified protein kinase, death associated protein (DAP) kinase which is localized to the microfilament system of the cytoskeleton. However, the downstream or upstream effectors of DAP kinase remain uncertain. In the present work, we hypothesize that the most probable substrate for DAP kinase is regulatory light chain of myosin II, by phosphorylating which the kinase can transduct death signals.

Short note: heterogeneous response of red blood cells to colloidal silica as a criterion for study of their membrane alterations: flow cytometric approach.

Since the red blood cells are characterized by the different resistance to action of the colloidal silica particles, this fact allows us to suppose that heterogeneity of the cell response to silica can be used as a criterion for the study of changes in the membrane systems. Flow cytometry has been proposed as one of most effective techniques for detecting a dynamic changes of cell morphology during silica-cell interaction.

Erythrocyte as a potential model for study of lifelong somatic mutations.

The detection and measurement of somatic cell mutation in vivo is an important subject of research for the assessment of human cancer risk induced by various environmental genotoxic factors. The possible mechanisms which influence the persistence of mutant cells of the hematoimmune system in the peripheral blood are presented. The erythroid system is a system which accumulates mutational lesions and so stably generates red blood cells with various phenotypic changes.

Possible selective elimination of red blood cells under the influence of colloidal silica.

The physical interaction between the red blood cells and colloidal silica leads to hemolysis. We propose that the nature of the red-cell elimination as the result of this interaction is not random. As the negatively charged silica particles at the physiological conditions electrostatically react with the positive sites of the outer side of the cell membrane, it seems reasonable to suppose that the cells with a low density of negative charges on the membrane surface area are more probable to be lysed in the first place.

Adsorption of aerosil on erythrocyte surface by flow cytometry measurements.

Light scattering properties of the erythrocytes in less than 0.01% aerosil suspension were measured. It was shown that light scattering properties of intact cells and cells associated with silica (aerosil) are different. It was also established that the aerosil particles are characterized by more intensive autofluorescence in the channel of green fluorescence than the erythrocytes. We propose that this observation allows a facile means of studying the interaction of purified silica and cell membranes. Hemolysis, as a result of interaction between the aerosil particles and the erythrocytes, was confirmed by photometric measurement of hemoglobin in the supernatant.