Biochemical and cellular mechanisms of low-dose effects

Low-dose irradiation is usually considered to be rather ineffective in producing biologically relevant effects. Yet, individual radiation absorption events within cell nuclei or whole cells interact stochastically with subcellular structures due to the multiple ionizations along primary or secondary particle tracks, depending on ionization density. Whereas radiation effects are usually seen in the context of structure and function of DNA, other cellular effects, perhaps influencing DNA by secondary biochemical mechanisms, also warrant attention. Thus, previous work from this laboratory with bone marrow that was obtained from whole-body exposed mice, has shown that single or few instantaneous radiation absorption events per cell from gamma-rays produce an acute and temporary partial inhibition of the enzyme thymidine kinase; the effect appears within about 1 h after the event, reaches its maximum at approximately 4 h and disappears completely within another 6 h. This pattern of enzyme inhibition is fully concordant with the pattern of inhibition of uptake of tritiated thymidine or 125I-labelled deoxyuridine into the DNA; also concordant is a temporary increase in the concentration of free thymidine in the blood serum of the exposed mice. The particular response of thymidine kinase was considered to relate to some, thus far unknown, repair systems and/or to a defence mechanism of the hit cells. In order to further elucidate the role of the acute and temporary partial inhibition of thymidine kinase in cellular metabolism, experiments were carried out in which mice were acutely exposed to 0.01 or 0.1 Gy and again exposed to the same dose at different times up to 12 h after the first exposure. At regular time intervals after the second exposure bone marrow cells were obtained and thymidine kinase activity was studied by various assays. The results indicate that the first acute irradiation conditioned the cells in such a way that the second acute irradiation produced either an enhanced inhibition and recovery of thymidine kinase activity, or no effect at all was seen, when the second irradiation was given between about 3 and 8 h after the first irradiation. From 8 to 12 h after the first irradiation the cells apparently resumed their original state, so that the second irradiation produced effects quite similar to those seen after a single irradiation in unconditioned cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histopathogenesis of Galls Induced by Meloidogyne naasi in Wheat Roots

Histopathogenesis of galls induced by Meloidogyne naasi in wheat roots was studied. Large numbers of larvae penetrated wheat root tips within 24 hr; larvae migrated both inter- and intracellularly, causing cortical hypertrophy. Giant cells were formed in the stele around the head of each nematode within 4 to 5 days. Initial pathological alterations in giant cell formation consisted of hypertrophy of protophloem and protoxylem cells, their nuclei and nucleoli. Giant ceils contained 2 to 8 agglomerated multinucleolate nuclei. Synchronous mitotic divisions were first observed 9 days after inoculation. After 21 days, giant cells became highly vacuolate. Observations 40 days after inoculation revealed a complete degeneration of cell contents in many giant cells but their thick walls remained intact. Abnormal xylem completely surrounded the degenerated or partially degenerated giant cells.     

Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita

The total numbers of nuclei in giant cells induced by Meloidogyne incognita in pea, lettuce, tomato, and broad bean were determined. Mature giant cells from pea had the most nuclei per giant cell with a mean of 59 ± 23, lettuce had the fewest with 26 ± 16, and tomato and broad bean were intermediate. The rate of increase in numbers of nuclei for all plant species was greatest during the first 7 days after inoculation. No mitotic activity was observed in giant cells associated with adult nematodes. Number of nuclei per giant cell doubled each day during the period of greatest mitotic activity, but number of total chromosomes per giant cell increased 20-fold per day at the same time. The hypothesis is presented that factor(s) responsible for the polyploid, mulfinucleate condition characteristic of giant cells may be different from factor(s) responsible for aneuploid numbers of chromosome per nucleus or for nuclear aberrations such as the presence of linked nuclei.     

CELL CYCLE KINETICS OF ENDOREDUPLICATION IN GAMMA-IRRADIATED ROOT MERISTEMS OF PISUM SATIVUM

Label and mitotic indices and microspectrophotometry of unlabeled interphase cells were used to measure the proportion of root meristem cells of Pisum sativum in each cell cycle stage after exposure to protracted gamma irradiation. Three seedling types were investigated: 1) intact seedlings, 2) seedlings with cotyledons detached and treated with lanolin paste applied to the area of cotyledon excision, and 3) seedlings with detached cotyledons and treated with a G2 Factor applied to the area of cotyledon excision in lanolin paste. In intact seedling meristems, predominant cell arrest occurred with a 4C amount of DNA while 0.30 of the cells underwent endoreduplication to arrest with an 8C amount of DNA. Only 0.07 cells arrested with a 2C amount of DNA. Polyploid cells were produced several days after the start of irradiation and were derived from a diploid cell population. In seedlings exposed to lanolin only, without cotyledons, most cells arrested with a 2C amount of DNA with no polyploid cells. In seedlings exposed to a G2 Factor in lanolin after cotyledon excision, most cells arrested with a 4C amount of DNA but no cells underwent endoreduplication. These experimental results suggest that the G2 Factor derived from cotyledons of Pisum sativum was necessary for predominant cell arrest in G2 but alone was not sufficient for the polyploidization step.     

Developmental changes in the ploidy of mouse implanting trophoblast cells in vitro

Shortly after the onset of implantation, polar mouse trophoblast cells proliferate and give rise to the ectoplacental cone, constituted by two distinct cell populations: undifferentiated, diploid cells and giant cells. Giant cells characteristically exhibit exaggerated dimensions and polyploid nuclei. In this study, we employ ectoplacental cones as a dynamic source of trophoblast giant cells to analyze cell proliferation, cell death, and ploidy under in vitro conditions. Our results show that DNA synthesis and the increase in the cell number are relevant only during the first 24 h of culture. Subsequently, DNA synthesis still occurs, mainly in the giant cell compartment, while the number of cells gradually decreases. Cell death by injury and apoptosis was also observed in the non-giant cell compartment of the ectoplacental cone. These findings suggest that the first 24 h of culture are crucial to the mitotic activity of the ectoplacental cone cells that gradually ceases, favoring the endoreduplication process. The DNA synthesis index during the subsequent experimental intervals emphasizes accumulation of DNA for the polyploidization. There was clear correlation between DNA content and nuclear dimension. The ploidy values for the trophoblast giant cells varied from 2C up to 368C in the giant cells, but were not as expressive as those known from in vivo conditions, probably due to the absence of regulatory factors specific to the embryonic-maternal interface. In situ hybridization and histochemistry for the nucleolus-organizing region showed that trophoblast nuclei have only two marker signals, indicative of a typical polytenic process. This present study elucidates important aspects of trophoblast behavior and provides new information on trophoblast physiology in vivo and in vitro.     

Nuclear protein content and cell progression kinetics following X irradiation

After irradiation with 4 Gy of X rays the nuclear protein and DNA contents (to determine cell-cycle position) of HeLa cells were determined by isolating nuclei and staining them with the fluorescent dyes fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for DNA. Immediately following irradiation there was no change in the shape of the bivariate (FITC-PI) histogram. At 3 and 4 h after irradiation the region of the histogram which corresponds to mitotic cells had disappeared. At 6 h nuclei reappeared in this region. The maximum rearrangement of the histogram (i.e., maximum accumulation of cells in G2 with minimum cells in G1) occurred at 10.5 h after irradiation, which is later than the time required for mitotic recovery. No change in nuclear protein content of cells in G1 and S was observed. However, beginning at 4 h after irradiation and continuing throughout the period of observation, a small (10-20%) but significant increase in nuclear protein content was observed for nuclei isolated from cells in G2. The increase in nuclear protein content may be part of the mechanism of G2 arrest and/or may reflect unbalanced growth.     

Antipodal complex development in the embryo sac of wheat

Dynamics of an antipodal complex formation in wheat (Tritiñum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of “truncated” ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.     

Characterization of abnormal nuclei in renal proximal tubules after irradiation

The distribution and kinetics of proximal tubular cells with abnormally large nuclei, which were observed in irradiated mouse kidneys before any other obvious histological effects, were investigated. Six months after the administration of 13 or 15 Gy, little histopathological change was noted, in the kidneys of C3H mice; however, proliferation of proximal tubular cells was stimulated, and some of these cells had abnormally large nuclei. The relative DNA content of these large nuclei was measured with a quantitative image analysis system. Most of the large nuclear cells had more than diploid DNA content. The labeling index of the large nuclei was higher than that of unselected proximal tubular nuclei. These cells might be hyperploid cells that are dying after having gone through an abortive mitotic division. Examination and quantitation of these abnormal nuclei should be useful in elucidating the steps involved in cell loss in the proximal tubules after irradiation and as an assay for radiation damage to the kidney.     

CHRONOCRISIS: When Cell Cycle Asynchrony Generates DNA Damage in Polyploid Cells

Polyploid cells contain multiple copies of all chromosomes. Polyploidization can be developmentally programmed to sustain tissue barrier function or to increase metabolic potential and cell size. Programmed polyploidy is normally associated with terminal differentiation and poor proliferation capacity. Conversely, non-programmed polyploidy can give rise to cells that retain the ability to proliferate. This can fuel rapid genome rearrangements and lead to diseases like cancer. Here, the mechanisms that generate polyploidy are reviewed and the possible challenges upon polyploid cell division are discussed. The discussion is framed around a recent study showing that asynchronous cell cycle progression (an event that is named “chronocrisis”) of different nuclei from a polyploid cell can generate DNA damage at mitotic entry. The potential mechanisms explaining how mitosis in non-programmed polyploid cells can generate abnormal karyotypes and genetic instability are highlighted.     

Giant cell formation in ectopic mouse trophoblast

Segmenting mouse ova, grafted beneath the kidney capsule of syngenic adult recipients, result in a growth of trophoblast, which changes from small, actively-dividing cells into giant trophoblast cells which degenerate 15 days after grafting. Similar giant cells are found in normal mouse placentas. Radioautography with ³H-thymidine, uridine, and leucine revealed cessation of DNA synthesis after day 8, with decline in RNA synthesis from day 10, and continued protein synthesis through day 15. Treatment with Colcemid reduced the graft size but failed to suppress giant cell formation. Treatment on days 4–7 of grafting with 5-fluorodeoxyuridine (FUdR), cyclohexamide, or actinomycin D resulted in giant cell suppression with the maintenance of healthy-appearing small trophoblast cells. These results confirm the early withdrawal of trophoblast grafts from the mitotic pool and the non-mitotic increase of trophoblast DNA, and demonstrate the apparent need for RNA and protein synthesis to support the development of trophoblast giant cells.     

Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells

In mammalian cells, cyclin E-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. We have targeted the murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2. Whereas single knockout mice were viable, double knockout embryos died around midgestation. Strikingly, however, these embryos showed no overt defects in cell proliferation. Instead, we observed developmental phenotypes consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. We derived trophoblast stem cells from double knockout E3.5 blastocysts. These cells retained the ability to differentiate into giant cells in vitro, but were unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication was a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells.     

Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis

? Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide. ? The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita. ? Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression. ? The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.     

Mitotic remodeling of the replicon and chromosome structure

Animal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.     

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.     

Quantitative investigation of reproduction of gonosomal condensed chromatin during trophoblast cell polyploidization and endoreduplication in the east-european field vole <Emphasis Type="Italic">Microtus rossiaemeridionalis</Emphasis>

Simultaneous determinations of DNA content in cell nuclei and condensed chromatin bodies formed by heterochromatized regions of sex chromosomes (gonosomal chromatin bodies, GCB) have been performed in two trophoblast cell populations of the East-European field vole Microtus rossiaemeridionalis: in the proliferative population of trophoblast cells of the junctional zone of placenta and in the secondary giant trophoblast cells. One or two GCBs have been observed in trophoblast cell nuclei of all embryos studied (perhaps both male and female). In the proliferative trophoblast cell population characterized by low ploidy levels (2–16c) and in the highly polyploid population of secondary giant trophoblast cells (32–256c) the total DNA content in GCB increased proportionally to the ploidy level. In individual GCBs the DNA content also rose proportionally to the ploidy level in nuclei both with one and with two GCBs in both trophoblast cell populations. Some increase in percentage of nuclei with 2–3 GCBs was shown in nuclei of the placenta junctional zone; this may be accounted for by genome multiplication via uncompleted mitoses. In nuclei of the secondary giant trophoblast cells (16–256c) the number of GCBs did not exceed 2, and the fraction of nuclei with two GCBs did not increase, which suggests the polytene nature of sex chromosomes in these cells. In all classes of ploidy the DNA content in trophoblast cell nuclei with the single GCB was lower than in nuclei with two and more GCBs. This can indicate that the single GCB in many cases does not derive from fusion of two GCBs. The measurements in individual GCBs suggest that different heterochromatized regions of the X- and Y-chromosome may contribute in GCB formation.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

Tissue-specific schedule of selective replication in Drosophila nasutoides

Summary Dramatic changes in the DNA composition of post-mitotic versus mitotic and germ line nuclei occur during development in different organisms. Drosophila nasutoides possesses n=4 chromosomes which were quantified with a microphotometer in females. The diploid (2 C) DNA content was 0.79 pg or 7.7×10⁸ nucleotide pairs, calculated from brain metaphases and calibrated with hen erythrocyte nuclei. The individual elements comprised X=9%, 2=16%, 3=13%, and 4=62% of the total complement. In polytene nuclei of larval salivary glands which had undergone 11 endoreplication cycles, chromosome 4 contained only 1.55% of total Feulgen DNA. Thus, in contrast with other Drosophila genomes, where under-replicating material is dispersed to all elements, a huge quantity of non-endoreplicating DNA is restricted to a single chromosome. This permits accurate determination of the timing of under-replication in the single cell. The data presented here suggest that the schedule is tissue-specific. Larval hind gut and salivary duct nuclei begin under-replication during the first endocycle, whereas adult and larval salivary glands mainly begin during the second cycle. In Malpighian tubules the onset of selective DNA syntheses occurs during either the first or the second endocycle.     

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10⁶ cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [³H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.     

Sequential analysis of nuclei transformation in stem nodulation sites ofSesbania rostrata Bbem (Leguminosae) during infection by specificrhizobium

This paper is a sequential analysis of qualitative and quantitative nuclear evolution inSesbania rostrata (Leguminosae) stem nodules. Before infection, the nuclei of the root primordia (nodulation site) cells show a 2G level of DNA. Immediately upon infection, the cells cease their mitotic activity and the nuclei begin synthesizing DNA up to a 32C level after the onset of the infection. The increase in the diameter of the nuclei of the infected cells is concomitant with the rise in the level of DKA. In the final phase of the evolution of these nodules, the nuclei of the infected cells undergo degenerative changes.