Kunio Shinohara

X-Ray-Induced Cell Death: Apoptosis and Necrosis

X-ray-induced cell death in MOLT-4N1, a subclone of MOLT-4 cells, and M10 cells was studied with respect to their modes of cell death, apoptosis and necrosis. MOLT-4N1 cells showed radiosensitivity similar to that of M10 cells, a radiosensitive mutant of L5178Y, as determined by the colony formation assay. Analysis of cell size demonstrated that MOLT-4N1 cells increased in size at an early stage after irradiation and then decreased to a size smaller than that of control cells, whereas the size of irradiated M10 cells increased continuously. Apoptosis detected by morphological changes and DNA ladder formation (the cleavage of DNA into oligonucleosomal fragments) occurred in X-irradiated MOLT-4N1 cells but not in M10 cells. Pulsed-field gel electrophoresis showed that the ladder formation involved an intermediate-sized DNA (about 20 kbp). Most of the DNA was detected at the origin in both methods of electrophoresis in the case of M10 cells, though a trace amount of ladder formation was observed. Heat treatment of M10 cells induced apoptosis within 30 min after treatment, in contrast to MOLT-4N1 cells. The results suggest that apoptosis and necrosis are induced by X rays in a manner which is dependent on the cell line irrespective of the capability of the cells to develop apoptosis. DNA fragmentation was the earliest change observed in the development of apoptosis.

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET.

Abstract Ito, A., Nakano, H., Kusano, Y., Hirayama, R., Furusawa, Y., Murayama, C., Mori, T., Katsumura, Y. and Shinohara, K. Contribution of Indirect Action to Radiation-Induced Mammalian Cell Inactivation: Dependence on Photon Energy and Heavy-Ion LET. Radiat. Res. 165, 703–712 (2006). The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/μm to 440 keV/μm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/μm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.

Interphase Death and Reproductive Death in X-Irradiated MOLT-4 Cells

The killing effects of ionizing radiation on MOLT-4 cells were studied using colony formation assays, dye exclusion tests, and cell growth analysis. When MOLT-4 cells were exposed to X rays, the fraction of cells stained with erythrosin B increased markedly during the incubation after irradiation at 37 degrees C and reached a maximum within 24 h. In contrast, no such increase was observed for L5178Y cells or their derivative, radiosensitive mutant M10 cells, at a dose resulting in the same level of cell survival as measured by the colony formation assay. Detailed analysis of cell survival as measured by the dye exclusion test in comparison with that measured by the colony formation assay in MOLT-4 cells indicated that MOLT-4 cells are subject to interphase death as well as reproductive death. Analysis of subclones showed a wide variety of differences in the level of stained cells using the dye exclusion test. The results confirmed the population heterogeneity of MOLT-4 cells. Moreover, none of the subclones showed exactly the same cell survival when this was measured with the two assays. The present results strongly suggest that both modes of cell death may occur in single MOLT-4 cells. Possibly apoptosis is the mechanism accounting for both modes of death in these cells.

Heat-induced apoptosis and p53 in cultured mammalian cells

Heat-induced apoptosis was studied in M10 and MOLT-4 cells by determining nuclear morphological changes, decrease in cell size, DNA degradation into fragments of about 30 kbp, and the appearance of internucleosomal DNA fragments (DNA ladders). Morphological changes in the nucleus were detected within 30 min after heat-treatment at 44 degrees C in M10 cells, but much later (> 5 h) in MOLT-4 cells. In M10 cells, 30 kbp-DNA fragments were observed even at the end of the heat-treatment and decreased 10 min later, while the DNA ladder increased at 10-30 min after heat treatment. DNA fragments of 30 kbp appeared in MOLT-4 cells at 1 h after the heat-treatment and apparently accumulated for up to 24 h. Heat-treatment increased p53 protein in MOLT-4 cells but not in M10 cells. Analysis of the DNA sequence of the p53 gene revealed that M10 cells have a heterozygous mutation in codon 173 of exon 5. These results suggest that apoptosis is induced by hyperthermia in a cell-line dependent manner, that the formation of 30 kbp-DNA fragments is a very early event in apoptosis, that DNA fragmentation into a DNA ladder occurs via the 30 kbp fragments, and that apoptosis in heat-treated M10 cells may be independent of p53.

Induction of DNA Double-Strand Breaks and Cellular Migration Through Bystander Effects in Cells Irradiated With the Slit-Type Microplanar Beam of the Spring-8 Synchrotron

PURPOSE To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects. METHODS AND MATERIALS Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity of phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells. RESULTS More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by scattered X-rays and DNA dsbs. In support of this, more 53BP1 foci were observed in nonirradiated, conditioned medium-treated cells than in control cells (i.e., cells not treated with irradiation or conditioned medium). These results suggest that DNA dsbs were induced in nonirradiated cells by soluble factors in the culture medium. In addition, we observed cellular migration into areas irradiated with peak doses, suggesting that irradiated cells send signals that cause nonirradiated cells to migrate toward damaged cells. CONCLUSIONS Bystander effects are produced by factors secreted as a result of slit-type microplanar X-ray beam irradiation.

Application of X‐ray microscopy in analysis of living hydrated cells

Because there is a limit for analysis of fine hydrophilic cell structures of living cells in medium by ordinary techniques, including electron microscopy, the development of a new technology to overcome such limitation is highly desirable. In this regard, soft X‐ray microscopy (high‐resolution X‐ray imaging of structures), which does not require any special procedures for sample preparation, has been developed and applied to analyze structures of biological specimens. In this article, application of two types of X‐ray microscopes, which use laser‐produced plasma X‐rays or synchrotron radiation to image the structure of macrophage cells, is introduced as an example of a novel approach to analysis of biological specimens. Both types of X‐ray microscopy show the network of fine fibrillar surface structures on macrophages in medium. Ordinary transmission and scanning electron microscopy and light microscopy also show the presence of such structures, but electron microscopy showed alterations due to sample processing and light microscopy did not show a clear image due to low resolution. Thus, X‐ray microscopy has the potential capability to analyze structures of live cells in a hydrated condition and may reveal a function‐related structural alignment of cells in their natural form. Anat Rec (New Anat) 269:217–223, 2002.

A narrow microbeam is more effective for tumor growth suppression than a wide microbeam: an in vivo study using implanted human glioma cells

A narrow microbeam is shown to be more effective than a wide microbeam for microbeam radiation therapy.

Spectromicroscopic film dosimetry for high-energy microbeam from synchrotron radiation.

A microscope with band-pass filters was used to measure the optical-density distribution of GafChromic films irradiated with multi-slit microbeam X-rays. The planar width was 25 microm, and the center-to-center distance was 200 microm. The peak and valley dose rates in air were found to be 120 and 0.7Gy/s, respectively. In a polymethylmethacrylate phantom, the peak-to-valley dose ratio decreased to 80 at a 1-mm depth. Doses calculated with the PENELOPE code agreed with those around the peak but became smaller in the valley.

Correlation between Unirradiated Cell TP53 Protein Levels and Radiosensitivity in MOLT-4 Cells

MOLT-4 cells undergo apoptosis after X irradiation. A radiosensitive variant, MOLT-4N1, and a radioresistant variant, MOLT-4N2, have been studied with respect to their radiosensitivity and its relationship to the levels of TP53 protein (formerly known as p53). X irradiation induces apoptosis in both cell lines with the following difference: The induction of apoptosis in MOLT-4N2 cells occurred later than in MOLT-4N1 cells as determined by the morphological changes and DNA fragmentation. The levels of cell death measured by the dye exclusion test coincided with the levels of apoptosis in both cell lines, suggesting that radiation-induced cell killing is determined by the induction of apoptosis. Unirradiated MOLT-4N1 cells contained a significantly higher intracellular level of TP53 protein and a much higher level of TP53 mRNA compared to MOLT-4N2 cells. X irradiation led to an accumulation of TP53 protein in both cell lines that was greater in MOLT-4N1 cells. This accumulation of TP53 protein preceded changes in DNA degradation and ladder formation and in nuclear morphology. These results strongly suggest that the radiosensitivity of MOLT-4 cells correlates well with the unirradiated control levels of TP53 mRNA and TP53 protein, and that the quantitative levels of TP53 protein must reach a threshold for the cells to undergo apoptosis.

Cell killing induced by decay of 125I during the cell cycle: comparison of 125I-antipyrine with 125I-bovine serum albumin.

The lethal effects induced by the decay of 125I in synchronized L5178Y cells were studied by suicide experiments. We used 125I-antipyrine, which is freely diffusible in cells, and 125I-bovine serum albumin (BSA), which remains outside the cells. Synchronized cells mixed with either 125I-antipyrine or 125I-BSA were frozen and stored at -196 degrees C for various periods to accumulate 125I decays. A clonogenic assay was used to measure the killing of these stored cells. Cells in G1-S and G2-M phases were more sensitive than those in late S phase for both 125I treatments. The ratio of survival in late S to that in G1-S (late S/G1-S) obtained by the decay of 125I-antipyrine, however, was much smaller than that obtained by the decay of 125I-BSA in the dose range examined. These results suggest that Auger electrons emitted from 125I-antipyrine cause a high-LET-type effect. The reason for the high-LET effect must be the intracellular localization of 125I-antipyrine and its closer contact with DNA.