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.

MOS, aneuploidy and the ploidy cycle of cancer cells.

After DNA or spindle damage, p53-defective tumor cells undergo a complex cycle of reversible polyploidy. How this process occurs and more importantly, why, has recently become the focus of several research groups, prompting this review in which we discuss two related phenomena that accompany the reversible polyploidy of tumor cells: the induction of meiosis genes such as MOS and the decrease in genomic instability observed during the reversion from polyploidy to para-diploidy. The reversible polyploidy likely provides the means through which the balance between increased chromosome instability (CIN), driving genetic variation and decreased CIN, necessary for perpetuating these malignant clones, is maintained. These concepts are integrated with recent findings that many meiotic and self-renewal genes become activated during reversible polyploidy and lead us to the hypothesis that tumor cell immortality may be achieved through germline-like transmission.

Evaluation of male fertility potential by Toluidine Blue test for sperm chromatin structure assessment.

BACKGROUND: We have previously suggested that the Toluidine Blue (TB) test can be used for sperm chromatin structure assessment. In this study, we wished to evaluate the clinical applicability of the TB test in assessing male fertility potential using well-defined groups of fertile and infertile men. METHODS: Sixty-three fertile and 79 infertile men were tested. Infertility thresholds for the proportion of sperm with abnormal [TB dark cells (TBDCs)] and normal [TB light cells (TBLCs)] chromatin structure were set by the ROC curve analysis. RESULTS: Thresholds of 45% TBDC and 20% TBLC were highly predictive for infertility (specificity of the test: 92 and 90%, respectively), but they were poor predictors of the fertility (sensitivity of the test: 42 and 32%, respectively). Odds ratio for infertility was 7.5 [95% confidence interval (CI): 2.7-20.8] when the 45% TBDC threshold was used and 4.4 (95% CI: 1.7-11.6) when the 20% TBLC threshold was used. CONCLUSIONS: The TB test can be suggested for clinical use as a complementary test for standard semen analysis to diagnose male infertility.

Sperm chromatin structure and male fertility: biological and clinical aspects.

Sperm chromatin/DNA integrity is essential for the accurate transmission of paternal genetic information, and normal sperm chromatin structure is important for sperm fertilizing ability. The routine examination of semen, which includes sperm concentration, motility and morphology, does not identify defects in sperm chromatin structure. The origin of sperm DNA damage and a variety of methods for its assessment are described. Evaluation of sperm DNA damage appears to be a useful tool for assessing male fertility potential both in vivo and in vitro. The possible impact of sperm DNA defects on the offspring is also discussed.

Comparative study of cytochemical tests for sperm chromatin integrity.

Tests were carried out on sperm from 40 fertile and infertile men to evaluate 2 DNA in situ denaturation methods using acridine orange (AO; the modified Rigler-Roschlau method and the Tejada method), alongside routine aniline blue (AB) and toluidine blue (TB) tests in our modification, and in order to estimate and compare the practical value of different in situ cytochemical tests for sperm chromatin structure. In addition, the methods were applied to rat and boar spermiogenesis models. The sperm heads with abnormal versus normal chromatin structure were specified as orange-red versus green by the AO method, blue versus uncolored by the AB method, and purple-violet versus light blue by the TB method. A good correlation for the proportion of sperm heads with abnormal chromatin structure was found among all the methods (r = .63-.70; P < .01), which characterized all 4 techniques as sensitive enough to estimate in situ sperm DNA integrity. In our study, the average value of abnormal cells was 17% +/- 3.8% and 30.2% +/- 6.8% for the fertile and infertile groups of men, respectively, setting a threshold of 95% probability at 23% as judged by the Rigler-Roschlau method. This compared with 23.9% +/- 7.5% and 52.1% +/- 20.8% (P < or = .05) for the fertile and infertile groups, respectively, setting a threshold at 31%, as judged by the Tejada method. The technical advantages and disadvantages of each method are briefly reported. Key words: Fertility, DNA normality, sperm maturation.

Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage.

The relationships between delayed apoptosis, polyploid 'giant' cells and reproductive survivors were studied in p53-mutated lymphoma cells after DNA damage. Following severe genotoxic insult with irradiation or chemotherapy, cells arrest at the G(2)-M cell cycle check-point for up to 5 days before undergoing a few rounds of aberrant mitoses. The cells then enter endoreduplication cycles resulting in the formation of polyploid giant cells. Subsequently the majority of the giant cells die, providing the main source of delayed apoptosis; however, a small proportion survives. Kinetic analyses show a reciprocal relationship between the polyploid cells and the diploid stem line, with the stem line suppressed during polyploid cell formation and restituted after giant cell disintegration. The restituted cell-line behaves with identical kinetics to the parent line, once re-irradiated. When giant cells are isolated and followed in labelling experiments, the clonogenic survivors appear to arise from these cells. These findings imply that an exchange exists between the endocyclic (polyploid) and mitotic (diploid or tetraploid) populations during the restitution period and that giant cells are not always reproductively dead as previously supposed. We propose that the formation of giant cells and their subsequent complex breakdown and subnuclear reorganization may represent an important response of p53-mutated tumours to DNA damaging agents and provide tumours with a mechanism of repair and resistance to such treatments.

Release of mitotic descendants by giant cells from irradiated Burkitt's lymphoma cell line..

Polyploid giant cells are produced as part of the response of p53 mutant Burkitt's lymphoma cell lines to high doses of irradiation. Polyploid giant cells arise by endo-reduplication in the first week after a single 10 Gray dose of irradiation. Within the giant cells a sub-nuclear structure is apparent and within this, sub-nuclear autonomy is evident, as displayed by independent nuclear structure and DNA replication in different parts of the nucleus. The majority of these cells soon die as apoptotic polykaryons. However, approximately 10-20% of giant cells remain viable into the second week after irradiation and begin vigorous extrusion of large degraded chromatin masses. During the second week, the giant cells begin to reconstruct their nuclei into polyploid 'bouquets', where chromosome double-loops are formed. Subsequently, the bouquets return to an interphase state and separate into several secondary nuclei. The individual sub-nuclei then resume DNA synthesis with mitotic divisions and sequester cytoplasmic territories around themselves, giving rise to the secondary cells, which continue mitotic propagation. This process of giant cell formation, reorganization and breakdown appears to provide an additional mechanism for repairing double-strand DNA breaks within tumour cells.

Arrest in metaphase and anatomy of mitotic catastrophe: mild heat shock in two human osteosarcoma cell lines.

The exits from metaphase arrest and anatomy of mitotic catastrophe were studied in two human osteosarcoma cell lines, nontumorigenic HOS TE85 and its chemically transformed strain MNNG-HOS, applying mild genotoxic damage by heat shock at 41.8 degrees C for 24 h. Under these conditions, both cell lines doubled or tripled their mitotic index entering arrest in metaphase. On return to 37 degrees C, the arrest was either released or ended in apoptosis. The transformed strain showed a greater capacity to arrest in metaphase as well as a greater probability of developing the third pathway: to restitute this arrest in polyploid interphase. This, in turn, either entered an 'endocycle' or, following a delay, apoptosis. Thus, arrest in metaphase was a cross-point of the mitotic cycle, apoptosis, and endocycle. Mitotic catastrophe can morphologically manifest combinations of elements of these three processes.

Aberrations of cell cycle and cell death in normal development of the chick embryo growth plate.

The epiphyses of femurs from 7.5-15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, 'dark chondrocytes' representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours. This connection is briefly analysed in view of some current concepts of carcinogenesis.

Aberrant death in dark chondrocytes of the avian growth plate.

Growth plate chondrocytes of embryonic chick femurs were examined by electron microscopy, cytophotometry and autoradiography. Apart from the well-described 'light' chondrocyte, a different 'dark' type of chondrocyte was present, comprising 10 - 35% of the cell population. They were found at all stages of chondrocyte differentiation and in all ages of the femurs studied. Well developed rough endoplasmatic reticulum and Golgi complex, many secretory vesicles, energetically active mitochondria and a lot of glycogen, indicating high activity of the cytoplasm, were combined with low RNA synthesis, gentle margination and scattered compaction of the chromatin. DNA cytometry revealed that most of dark cells were diploid, but 15 - 30% were tetraploid, with the absence of an S-phase. Substantial loss of DNA was found in about 10% of dark chondrocytes. The TUNEL reaction demonstrated a limited number of DNA strand breaks. Advanced dark cells possessed the nuclear features of both apoptosis and necrosis. Besides chromomeric-chromonemic compaction, a chromatin arrangement similar to that of prometaphase and metaphase, as well as amitotic nuclear segregation, all of them degenerative, were found. Our interpretation is that the dark chondrocytes undergo an aberrant type of cell death which may be combined with aberrant cell cycle. Cell death of dark chondrocytes is preceded by a pre-mortal burst of secretion.

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes.

Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause--the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells.

Epigenetic selection as a possible component of transdifferentiation. Further study of the commitment of hypertrophic chondrocytes to become osteocytes.

Transdifferentiation of hypertrophic chondrocytes into osteogenic cells was induced in 14 day chick embryo femurs by cutting through the region of hypertrophic cartilage. The process was studied in organ culture, using electron microscopy, staining for alkaline phosphatase, immunocytochemistry of collagen type I and proliferative cell nuclear antigen, and in situ localization of DNA strand-breaks. In addition, DNA and RNA synthesis were studied by 3[H]-T and 3[H]-U radioautography. Loss of ECM components from the cut edge occurred in culture. During the 12 day period necessary for transdifferentiation we observed phenotypic instability and bi-potentiality, the death of some cells and the gradual promotion of the osteoblastic phenotype in the survivors. Transition from chondrocytic to osteoblastic phenotype progressed stepwise, through variable mosaic intermediates, and involved a few cell cycles including asymmetric (differential) divisions. Proliferating and apoptotic cells were found in close proximity. As judged by the relative proportion of apoptotic cells and composition of the surrounding intralacunar matrix, negative selection of intermediate cell types displaying chondrocytic and altered mosaic phenotypes occurred. When the osteoblastic lineage was finally established, apoptotic cells were no longer present. Our hypothesis is that after disruption of cell-cell or cell-matrix interactions and lack of growth factors certain cells are selected and channelled through proliferation into the new stable phenotype. This process is targeted by the environment through a set of pre-determined steps.

The phenotypic switch from chondrocytes to bone-forming cells involves asymmetric cell division and apoptosis.

We have investigated the early cellular events that take place during the phenotypic switch from hypertrophic chondrocytes to bone-forming cells in a) chondrocytes located inside intact lacunae after embryonic chick femurs had been cut through the hypertrophic cartilage and cultured for 1-15 days; and b) at the cartilage/marrow interface of femurs after short-term culture. Ultrastructural studies were combined with in situ methods localizing proliferating and apoptotic cells, and 3D-reconstructions of confocal images of the cartilage/marrow edge. The crucial event in the phenotypic switch was an asymmetric cell division which resulted in one daughter cell which underwent apoptosis and another viable daughter cell which subsequently differentiated to an osteogenic cell, i.e to a smaller basophilic cell that was positive for alkaline phosphatase, type I collagen, osteonectin, osteopontin, bone sialoprotein and osteocalcin and that, after 12-15 days in culture, could synthesize a mineralized bone matrix within intact lacunae. The present results suggest a mechanism whereby differentiated cells can change their phenotype. At least one mitotic division seems to be required to fix the commitment to the new phenotype.

Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis.

We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize the changing pattern of differentiation. The first responses to the cutting, seen after 2 d, were upregulation of alkaline phosphatase activity, synthesis of type I collagen and single-stranded DNA breaks, probably indicating a metastable state. Associated with the change from chondrogenic to osteogenic commitment was an asymmetric cell division with diverging fates of the two daughter cells, where one daughter cell remained viable and the other one died. The available evidence suggests that the viable daughter cell then divided and generated osteogenic cells, while the other daughter cell died by apoptosis. The results suggest a new concept of how changes in lineage commitment of differentiated cells may occur. The concepts also reconcile previously opposing views of the fate of the hypertrophic chondrocyte.

Differentiation and programmed cell death in Sa-45 tumour treated with bone inducer.

The relationship between differentiation and concerted cell death was studied using ultrastructural, histochemical and immunochemical methods in solid rat fibrosarcoma Sa-45 grown in the presence of demineralized bone matrix. The control tumour consisted mostly of undifferentiated cells with few poorly or moderately differentiated cells. In the presence of the inducer, cells with a more differentiated pattern appeared in the surrounding area. The proliferative activity in the presence of the inducer was 3 to 5 times lower but the apoptotic index was higher than in the controls. However, complete differentiation was induced only in stromal cells, whereas the parenchymal cells showed signs of enhanced but incomplete differentiation. The ultrastructural signs of programmed cell death progressed in them faster than the corresponding features of maturation, thus leaving differentiation incomplete.

Interphase genome as the active space: chromatin dynamics during chick embryo chondrogenesis.

The rearrangement of the chromatin that takes place during cytodifferentiation was studied using TV image analysis in chick limb bud cartilage stained for DNA. The redistribution of the chromatin was compatible with the Rabl orientation: chromatin was extended radially from the centromeric ring to the telomere pole in young chondroblasts, and contracted back in ageing chondrocytes. The direction and gradient of this redistribution correlate with the changes in DNA content within the chromocentres formed by pericentromeric heterochromatin. In turn, intercalary heterochromatin regulates the condensation of the adjacent euchromatin depending upon the position in this radial-polar gradient.

The chromatin network: image analysis of differentiating chick embryo chondrocytes.

The chromatin network was revealed in DNA stained cell imprints of developing chick embryo chrondrocytes using the recently developed method of TV image processing. The network consists of two classes of alveoles. The large alveoles, which accompany large chromocentres, are concentrated at the centromeric nuclear pole representing the most stable part of the network. The small alveoles, connected with the small chromocentres and located around the chromosome arms and telomeric ends, are dynamic. These smaller alveoles appear in differentiating chondroblasts and disappear in ageing chondrocytes. Ectopic conjunctions involving constitutive and intercalary heterochromatin are responsible for the network formation.