Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro.

Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat's age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.

Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH.

In order to study the initial frequencies and define kinetics of the formation of chromosomal exchanges in X-irradiated human lymphocytes, the premature chromosome condensation (PCC) technique was employed in combination with fluorescence in situ hybridization (FISH) with a composite probe for human chromosome 8 and a pan-centromeric probe for the whole genome. Human lymphocytes were X-irradiated (0.5, 1, 2, 3, 4 and 6 Gy), fused with mitotic Chinese hamster ovary (CHO) cells immediately or 1, 3, 6, 12 and 18 h after irradiation. Immediately after irradiation chromosomal breaks, dicentrics and translocations showed a linear dose-response. Unrejoined chromosome breaks were the most frequent types of aberrations (about 85%) observed. About 15% of total aberrations were chromosome exchanges of 65% of these were translocations and 35% were dicentrics. The chromosomal exchanges initially observed were mostly incomplete, with no complex exchanges at doses of 1 and 2 Gy, at higher doses (3-6 Gy) complex exchanges were observed and their frequencies increased with increasing post incubation time. Following different recovery times, repair kinetics of breaks for different doses of irradiation was studied. The shapes of the curves obtained for breaks as well as chromosome exchanges were linear-quadratic. The linear yield component, alpha, is formed entirely in the fast process that can be manifested in the early plateau, while component beta developed slowly in the subsequent hours. The kinetics of breaks rejoining was exponential, almost 50% of breaks rejoined after 1 h and at 18 h about 20% of breaks remained. At low doses of 1 and 2 Gy most of the exchanges were formed immediately and at higher doses, the frequency of exchanges increased with kinetics similar to that observed for the rejoining of breaks. However, the kinetics was different for different doses of irradiation. The frequency of dicentrics increased at doses above 2 Gy following 3 h recovery time, but for the translocations effect was pronounced even at 1 h recovery time. The frequency of incomplete exchanges (i.e., terminal translocations) decreased with post irradiation time and at 18 h was 30-40% less than the frequency obtained immediately after irradiation. The increase in the total translocations as a function of time between irradiation and fusion was due to a rapid increase in complete exchanges (i.e., reciprocal translocations). The frequency of ring chromosomes immediately after irradiation, also increased linearly, however, it was 3-5 times lower than dicentrics and remained almost constant in number for different doses and at different post-irradiation times.

Detection of total- and partial-body irradiation in a monkey model: a comparative study of chromosomal aberration, micronucleus and premature chromosome condensation assays.

PURPOSE: To investigate the efficacy of three cytogenetic methods (dicentrics, micronuclei (MN) and premature chromosome condensation (PCC) analysis) for assessment of the unirradiated fraction and the persistence of damage after total-body (TB) and partial-body (PB) irradiation of rhesus monkeys (Macaca mulatta). MATERIALS AND METHODS: Animals were exposed to X-rays (5 Gy), either TB or PB, with about 6% of marrow cells shielded. Blood samples were collected at different times after exposure, i.e. 1, 3 and 7 days, and cultures were set up for the different cytogenetic endpoints. In addition, blood count analysis was performed before and after irradiation. RESULTS: Blood count analysis was not suitable for discriminating between TB and PB exposure. By using Poisson or overdispersion distribution as the basis, it was not possible to distinguish TB from PB irradiation when dicentric chromosomes and MN were analysed. PCC analysis, in contrast, showed a Poisson distribution after TB exposure and overdispersion after PB exposure. Using the PCC assay, reliable dose estimates could be obtained up to 7 days after irradiation. CONCLUSIONS: For dicentrics and MN, shielding of 6% of bone marrow cells was found to be too small to estimate the unirradiated fraction accurately. The PCC technique was useful for dose assessment and the inhomogeneous exposure of 6% was detected within a short period of time after exposure.

Cell replication rates and processes concerning antibody production in vitro are not influenced by 2.45-GHz microwaves at physiologically normal temperatures.

Several contradictory papers concerning the effects of microwaves on living organisms and on in vitro cell suspensions have been published through the years. These papers are difficult to interpret, because temperature measurement data are often lacking. Reliable temperature measurements are important, because they enable one to determine whether the observed microwave effects are thermal or nonthermal. Therefore, a method was developed to investigate microwave effects on cellular processes, in which the temperature was precisely monitored during microwave treatment using a fiberoptic thermometer. This method involved the processes required for in vitro production of monoclonal antibodies. Monoclonal antibodies are vital ingredients in (microwave-stimulated) immunostaining techniques and ELISAs, which have become important techniques in neuroscience. The effects of 2.45-GHz microwaves on mouse myeloma and (neural) hybridoma cell replication rates and on antibody production were investigated. In addition, the effects on the cell fusion abilities of spleen lymphocytes and myeloma cells and on in vitro immunization were studied. The results of this study show no effects of microwaves on either of the processes mentioned using exposure times up to 5 h a day at a physiologically normal temperature of 37 degrees C. It was concluded that the effects of 2.45-GHz microwaves detected at higher temperatures are thermal effects and that no indications for nonthermal 2.45-GHz microwave effects exist under the exposure conditions used in the present study.

Asymmetric fusion between protoplasts of tomato (Lycopersicon esculentum Mill.) and gamma-irradiated protoplasts of potato (Solanum tuberosum L.): the effects of gamma irradiation.

This paper describes the aggregation of nuclei in heterokaryons of tomato and unirradiated or irradiated potato protoplasts and the effects of gamma irradiation of potato and tomato protoplasts on single- and double-stranded DNA fragmentation, DNA repair and DNA synthesis as revealed by alkaline and pulsed field gel electrophoresis and an immunocytochemical technique. The prospects for obtaining highly asymmetric somatic hybrids of tomato and gamma-irradiated potato are discussed.

[The kinetics of polykaryon formation in a HeLa cell culture after irradiation at doses of 5 and 10 Gy]. / Kinetika obrazovaniia polikarionov v kul'ture kletok HeLa posle oblucheniia v dozakh 5 i 10 Gr.

Monolayer culture of HeLa tumor cells irradiated at 5 Gy and 10 Gy doses was followed up in kinetics for 14 days. It was shown that starting with day 3 the cell monolayer is modified in such a way that some cells occur in 2- to 8-cell accumulations. The rate of formation of such groups and the number of cells in them were dose-related. The labeling index in these formations was several times higher than that in the irradiated population on the whole. The analysis of the grade of DNA synthesis synchronization in these accumulations showed that complete cell fusion and polykaryon formation after irradiation at the dose of 10 Gy occurred on day 4 whereas after 5 Gy, only on day 7. In both cases the status of complete fusion was 3 days in duration.

[Can postradiation cell fusion be a factor in the protection of a cell population?]. / Mozhet li postradiatsionnoe sliianie kletok byt' faktorom zashchity kletochnoi populiatsii?

From the analysis of the literature it is inferred that there is one more type of nonspecific protection which is based on the polyploidy process and functions at the cell population and tissue levels. With high radiation loads, this process maintains the functioning rate of the given tissue and the survival rate of the given cell type. A hypothesis is proposed that cell fusion and formation of somatic hybrids, that provide clone survival, are particularly effective in tumor tissues. This mechanism is activated at high radiation doses: fusion of injured cells and formation of polykaryons and then hybrids can give rise to cells capable of reproduction. The cellularity is restored.

Radiation fusion hybrids for human chromosomes 3 and X generated at various irradiation doses.

We have used a gamma-irradiation (2.5-25 krads) cell fusion procedure to generate human-hamster somatic cell hybrids (IHB, irradiated human fragments in B14-150 cells), retaining small fragments derived from human chromosomes 3 and X. By using Alu-element mediated PCR amplification and dot-blot hybridization with human alphoid or total human DNA as probes, 86 positive hybrids were identified and selected for further analysis. Nonisotopic fluorescence in situ hybridization (FISH) with human DNA in a set of eight hybrids demonstrated the presence of from one to eight human fragments per cell independent of irradiation dose. In contrast, a significant dose-dependent variation of fragment sizes was shown in the analysis of the 86 hybrids with markers previously mapped to 3p (seven markers) and to Xq (21 markers). Using the Xq27-28 region as a model, 40% of the hybrids generated at 5 krads or less were found to have retained fragments in the range of 3-30 Mb, 10% retained the whole chromosome arm, and the remaining 50% retained fragments of less than 2-3 Mb. The proportion of fragments of 3 Mb or larger decreased rapidly at higher irradiation doses and was very low (less than 6%) in hybrids generated at 25 krads. Upon further characterization, the 86 hybrids analyzed here will provide a mapping panel for the entire chromosomes 3 and X with an estimated resolution in the range of 1-2 Mb on average, a size range amenable to PFGE and YAC contig mapping.

Dose-dependent effects of ionizing radiation on in vitro myoblast fusion.

We have recently demonstrated by dielectric relaxation studies in the radio-frequency range that there is a sharp decrease in the conductivity and permittivity of the membranes of chick embryo myoblasts in vitro at the time of fusion (60 h) (Bonincontro et al. 1987). This sharp fall in membrane electrical parameters was subsequently shown to be due to changes in ionic flux, particularly of the Na+/K+ equilibrium (Santini et al. 1988). Ionizing radiation induces a wide variety of effects on biological membranes, including variations in membrane ionic transport. We wished to investigate if sublethal doses of gamma-irradiation could affect membrane electrical parameters and thus myoblast membrane fusion. Consequently, chick embryo myoblast aggregate cultures were irradiated with 3.25, 5.15 or 6.35 Gy at 24 h of culture. We found that the lower dose delays membrane fusion by about 10 h while the two higher doses block fusion up to 120 h of culture. Aggregates showed a very high cell viability. The possible mechanisms by which ionizing radiation causes these variations in myoblast membrane electrical properties and fusion are discussed.

[The induction of hepatocyte polyploidization in rats of various ages by ionizing irradiation with different LETs]. / Induktsiia poliploidizatsii gepatotsitov krys raznogo vozrasta ioniziruiushchim izlucheniem s razlichnoi LPE.

A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of nonproliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation.

Generation of a panel of somatic cell hybrids containing unselected fragments of human chromosome 10 by X-ray irradiation and cell fusion: application to isolating the MEN2A region in hybrid cells.

We have used X-ray irradiation and cell fusion to generate somatic cell hybrids containing fragments of human chromosome 10. Our experiments were directed towards isolating the region of the MEN2A gene in hybrids and to use those as the source of DNA for cloning and mapping new markers from near the MEN2A locus. A number of hybrid clones containing human sequences that are tightly linked to the MEN2A gene were identified. Some 25% of our hybrids, however, proved to contain more than one human chromosome 10-derived fragment or showed evidence of deletions and/or rearrangements. A detailed analysis of the human content of X-ray irradiation hybrids is required to assess the integrity and number of human fragments retained. Despite retention of multiple human-derived fragments, these hybrids will prove useful as cloning and mapping resources.

Six complementation groups for ionising-radiation sensitivity in Chinese hamster cells.

The ionising radiation-sensitive mutants irs 1, irs 2, irs 3, xrs-1 (or xrs-7), EM7 and XR-1 were fused to wild-type cells or to each other in pairs to create hybrid cells. These hybrids were checked chromosomally and their X-ray sensitivity tested. Each mutant was found to be recessive to wild-type and to complement the X-ray sensitivity of the other mutants. Thus there appear to be at least 6 complementation groups for ionising radiation sensitivity in Chinese hamster cells.

[Polyploidization of rat hepatocytes due to cell fusion caused by radiations of different LET]. / Poliploidizatsiia gepatotsitov krys, obuslovlennaia sliianiem kletok pri deistvii izluchenii s raznoi LPE.

The method of flow cytometry was used to study polyploidization of hepatocytes following X-, gamma-, and neutron-irradiation. Ionizing radiation was shown to induce cell polyploidization by two different ways: (1) cells and nuclei fusion, and (2) restriction of mitosis after DNA replication. RBE of 14 MeV neutrons with respect to fusion was about 5.10(3). With neutron irradiation, the sensitivity of cells by fusion was not lower than that by chromosome mutations.

Binucleate cell formation correlates to loss of colony-forming ability in X-irradiated cultured mammalian cells.

The relationship between binucleate cell formation and the loss of colony-forming ability was examined in several cultured mammalian cell lines irradiated with X rays. The maximum fraction of binucleate cells after X irradiation increased dose-dependently within the range in which reproductive cell death might predominate over interphase cell death. When the logarithm of percentage survival was plotted against the percentage binucleate cells, a similar correlation was found for all cell lines tested, with the exception of mouse leukemia L5178Y cells, the most radiosensitive cells used. These observations suggest that the fraction of binucleate cells in the cell population can serve as a measure of cellular radiation damage.

The action of caffeine on X-irradiated HeLa cells. VII. Evidence that caffeine enhances expression of potentially lethal radiation damage.

HeLa cells irradiated with 2 Gy of 220-kV X rays suffer a 60-70% loss of colony-forming ability which is increased to 90% by postirradiation treatment with 10 mM caffeine for 6 hr. The detailed postirradiation patterns of cell death and sister-cell fusion in such cultures and in cultures in which the colony-forming ability was brought to about the same level by treatment with a larger (4 Gy) X-ray dose alone or by longer (48 hr) treatment with 10 mM caffeine alone were recorded by time-lapse cinemicrography. Because the patterns of cell death and fusion differ radically in irradiated and in caffeine-treated cultures, the response of the additional cells killed by the combined treatment can be identified as X-ray induced rather than caffeine induced. The appearance of cultures after several days of incubation confirms the similarity of the post-treatment patterns of proliferation in cultures suffering enhanced killing to those occurring in cultures treated with larger doses of X rays alone. It is concluded that X rays do not sensitize cells to caffeine, but rather that caffeine enhances the expression of potentially lethal radiation-induced damage.

[Fusion of germ and embryonic cells and the prospects of using the somatic hybridization method in embryology]. / Sliianie polovykh i émbrional'nykh kletok i perspektivy ispol'zovaniia metoda somaticheskoi gibridizatsii v émbriologii.

The role of investigations in which the method of induced cell fusion was used in studying the oocyte maturation, fertilization and early embryogenesis of animals has been analyzed on the basis of the published and the authors' data. The problems of developing the method of induced cell fusion, its combining with the other experimental methods (microsurgery, cell fragmentation, etc.) and the perspectives of its using in embryology are discussed.

UV Irradiation induces an activity which stimulates Simian virus 40 rescue upon cell fusion.

UV irradiation of African green monkey cells greatly stimulated efficiency of simian virus 40 induction from simian virus 40-transformed Syrian hamster cells after cell fusion. The maximum inducing activity was observed at 15 to 20 h after irradiation but remained only transiently. The addition of cycloheximide after UV irradiation eliminated the stimulation of the activity.