Low-dose ionizing radiation exposure represses the cell cycle and protein synthesis pathways in in vitro human primary keratinocytes and U937 cell lines.

The effects of the high-dose ionizing radiation used in radiotherapy have been thoroughly demonstrated in vitro and in vivo. However, the effects of low-dose ionizing radiation (LDIR) such as computed tomography-guided biopsies and X-ray fluoroscopy on skin cells remain controversial. This study investigated the molecular effects of LDIR on the human primary keratinocytes (HPKs) and U937 cells, monocytes-like cell lines. These cells were exposed to 0.1 Gray (Gy) X-ray as LDIR. The modulation of transcription was assessed using a cDNA array, and the protein expression after LDIR exposure was investigated using isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis at 24 hours. These effects were confirmed by immunoblotting analysis. The direct effects of LDIR on the U937 cells and HPKs and the bystander effects of irradiated HPKs on U937 cells were also investigated. LDIR downregulated c-Myc in both U937 cells and HPKs, and upregulated the p21WAF1/CIP1 protein expression in U937 cells along with the activation of TGFß and protein phosphatase 2A (PP2A). In HPKs, LDIR downregulated the mTOR signaling with repression of S6 and 4EBP1 activation. Similar changes were observed as bystander effects of LDIR. Our findings suggest that LDIR inhibits protein synthesis and induces the cytokines activation associated with inflammation via direct and bystander effects, which might recapitulate the effects of LDIR in inflammated skin structures.

The crosstalk between α-irradiated Beas-2B cells and its bystander U937 cells through MAPK and NF-κB signaling pathways.

Although accumulated evidence suggests that α-particle irradiation induced bystander effect may relevant to lung injury and cancer risk assessment, the exact mechanisms are not yet elucidated. In the present study, a cell co-culture system was used to investigate the interaction between α-particle irradiated human bronchial epithelial cells (Beas-2B) and its bystander macrophage U937 cells. It was found that the cell co-culture amplified the detrimental effects of α-irradiation including cell viability decrease and apoptosis promotion on both irradiated cells and bystander cells in a feedback loop which was closely relevant to the activation of MAPK and NF-κB pathways in the bystander U937 cells. When these two pathways in U937 cells were disturbed by special pharmacological inhibitors before cell co-culture, it was found that a NF-κB inhibitor of BAY 11-7082 further enhanced the proliferation inhibition and apoptosis induction in bystander U937 cells, but MAPK inhibitors of SP600125 and SB203580 protected cells from viability loss and apoptosis and U0126 presented more beneficial effect on cell protection. For α-irradiated epithelial cells, the activation of NF-κB and MAPK pathways in U937 cells participated in detrimental cellular responses since the above inhibitors could largely attenuate cell viability loss and apoptosis of irradiated cells. Our results demonstrated that there are bilateral bystander responses between irradiated lung epithelial cells and macrophages through MAPK and NF-κB signaling pathways, which accounts for the enhancement of α-irradiation induced damage.

Radiation-induced thymidine phosphorylase upregulation in rectal cancer is mediated by tumor-associated macrophages by monocyte chemoattractant protein-1 from cancer cells.

PURPOSE: The mechanisms of thymidine phosphorylase (TP) regulation induced by radiation therapy (XRT) in various tumors are poorly understood. We investigated the effect and mechanisms of preoperative XRT on TP expression in rectal cancer tissues. METHODS AND MATERIALS: TP expression and CD68 and monocyte chemoattractant protein-1 (MCP-1) levels in rectal cancer tissues and cancer cell lines were evaluated before and after XRT in Western blotting, immunohistochemistry, enzyme-linked immunoassay, and reverse transcription-polymerase chain reaction studies. Isolated peripheral blood monocytes were used in the study of chemotaxis under the influence of MCP-1 released by irradiated colon cancer cells. RESULTS: Expression of TP was significantly elevated by 9 Gy of XRT in most rectal cancer tissues but not by higher doses of XRT. In keeping with the close correlation of the increase in both TP expression and the number of tumor-associated macrophages (TAMs), anti-TP immunoreactivity was found in the CD68-positive TAMs and not the neoplastic cells. Expression of MCP-1 was increased in most cases after XRT, and this increase was strongly correlated with TP expression. However, this increase in MCP-1 expression occurred in tumor cells and not stromal cells. The XRT upregulated MCP-1 mRNA and also triggered the release of MCP-1 protein from cultured colon cancer cells. The supernatant of irradiated colon cancer cells showed strong chemotactic activity for monocyte migration, but this activity was completely abolished by neutralizing antibody. CONCLUSIONS: Use of XRT induces MCP-1 expression in cancer cells, which causes circulating monocytes to be recruited into TAMs, which then upregulate TP expression in rectal cancer tissues.

Can radiation-induced apoptosis be modulated by inhibitors of energy metabolism?

PURPOSE: To determine the effect of the inhibitors of energy metabolism, 2-deoxyglucose (2DG) and sodium azide, on radiation-induced apoptosis. MATERIALS AND METHODS: Radiation-induced apoptosis was determined in U937 monocytic leukaemia cells exposed to energy inhibitors post-irradiation. Apoptosis was scored microscopically using morphological criteria. Glycolysis was determined by assessing glucose consumption and lactate production. Adenine nucleotide levels were measured using a luciferase assay after enzymatic conversion to ATP. Respiration was measured using a Clark-type oxygen electrode. RESULTS: In addition to their apoptosis-inducing properties, both 2DG and azide modified post-irradiation apoptosis. 2DG induced apoptotic radiosensitization after exposure to lower concentrations (5 mM, 10 mM) up to 20 h post-irradiation while a level of radioprotection was found after 5 h exposure to higher doses up to 100 mM. By contrast, all doses of azide examined (5-50 mM) induced apoptotic radioprotection at all times examined. Glycolytic flux and ATP levels fell rapidly with increasing 2DG dose but energy charge remained unchanged. Glycolysis was less influenced by azide, with ATP levels being initially maintained after exposure but decreasing in a dose-dependent manner at 3 h post-irradiation. However, energy charge was unaffected by azide at the concentrations examined. CONCLUSIONS: Both 2DG and azide can influence radiation-induced apoptosis possibly through their effects on glycolysis and ATP levels. We suggest that modulation of energy metabolism provides mechanistic insight into radiation-induced apoptotic pathways.

The CFSE distribution assay is a powerful technique for the analysis of radiation-induced cell death and survival on a single-cell level.

BACKGROUND AND PURPOSE: To analyze radiation sensitivity of cells and to monitor cellular responses to irradiation, sensitive test systems for cell death and proliferation on a single-cell level are required. Traditionally, cellular radiation survival is measured using the clonogenic assay as the gold standard. Here it is reported, that labeling of cells with 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester (CFDASE) can be used as a highly sensitive assay to determine cellular response toward irradiation on a single-cell level. MATERIAL AND METHODS: The human malignant cell lines U937 (myelomonocytic, nonadherent), SW48 and SW480 (colorectal, adherent) were labeled with CFDASE, irradiated with either UVB (0-540 mJ/cm(2)), or X-rays (0-16 Gy). Cell death and proliferation were monitored by cytofluorometry and compared to the clonogenic assay for adherent SW48 and SW480 cells. RESULTS: Dividing nonadherent U937 cells displayed a shift in carboxyfluorescein (CF) fluorescence in parallel with an increased cell count indicating cell proliferation. By comparison, UVB-irradiated U937 cells did not show a shift in CF fluorescence and an increase in cell count indicating cell-cycle arrest. In a mixed cell culture, only the nonirradiated cells divided and concomitantly reduced their fluorescence. Calculating the number of cell divisions it was observed that the nonirradiated cells underwent approximately six cell divisions within 7 days, whereas the irradiated cells divided only once on average. The adherent SW480 colorectal cells showed a more pronounced cell-cycle arrest after irradiation with 240 mJ/cm(2) UVB as compared to cells treated with X-ray up to 16 Gy. Furthermore, the CFSE assay also discriminated colorectal cell lines of different intrinsic radiosensitivities and yielded results comparable to the standard clonogenic assay. CONCLUSION: Analysis of CF distribution can be employed as a powerful add-on to the clonogenic assay to simultaneously monitor cellular responses toward irradiation on a single-cell level. It constitutes an add-on to the clonogenic assay, especially for nonadherent cells.

ELF fields and photooxidation yielding lethal effects on cancer cells.

The lethal effect on human cancer cells was studied under three types of treatment: A) an ELF pulsed sinusoidal of 50 Hz electromagnetic field (PEMF) with amplitudes between 10 and 55 mT; B) the field and a cytostatic agent (actinomycin-C); and C) the field, the cytostatic agent, which has a photodynamic effect, and exposure to a halogen lamp. The results show a decreasing vitality of human K-562 and U-937 cancer cells in suspension with each additional treatment. Combination with other parameters as hyperthermia and/or hyperacidity could yield high killing rates by this noninvasive method.

7-hydroxystaurosporine (UCN-01) and ionizing radiation combine to inhibit the growth of Bcl-2-overexpressing U937 leukemia cells through a non-apoptotic mechanism.

A clinically relevant dose (2.0 Gy) of ionizing radiation (IR) was employed to determine if subsequent exposure to the protein kinase C (PKC) and Chk 1 inhibitor UCN-01 for 24 h could abrogate IR-induced G2/M arrest and promote apoptosis in U937 leukemic cells ectopically expressing Bcl-2 (U937/Bcl-2). To this end, empty-vector control (U937/pCEP4) and U937/Bcl-2 cells were exposed to two UCN-01 concentrations following IR: i) a 50 nM concentration, which by itself was minimally toxic to both cell lines, and ii) a 150 nM concentration, which modestly induced apoptosis (e.g., ~19%) in control cells after 24 h. The effects of UCN-01 on IR responses were examined in relation to apoptosis induction, suspension culture growth inhibition, loss of clonogenic survival, and cell cycle perturbations. IR (2 Gy) alone minimally induced apoptosis in both U937 transfectant cell lines (e.g., <5% at 24 h in each case). Although UCN-01 failed to potentiate IR-mediated apoptosis at either early (e.g., 24 h) or late (e.g., 72 h) intervals, exposure to 50 or 150 nM UCN-01 resulted in a significant, albeit modest, reduction in proliferation and colony formation in irradiated U937/pCEP4 and U937/Bcl-2 cells. Despite failing to enhance apoptosis, UCN-01 treatment abrogated IR-induced G2/M arrest in both cell lines, an event associated with enhanced activation of cyclin-dependent kinase 1 (cdk1), promotion of G0/G1 arrest, and dephosphorylation of the retinoblastoma protein (pRb). Together, these findings indicate that exposure of U937 cells ectopically-expressing Bcl-2 to the combination of UCN-01 + IR leads to a further reduction in cell proliferation, and that this phenomenon appears to involve a non-apoptotic mechanism.

Enhancement of hyperthermia-induced apoptosis by non-thermal effects of ultrasound.

To determine the effect of ultrasound on hyperthermia-induced apoptosis, we exposed U937 cells (in air-saturated suspension) to continuous 1 MHz ultrasound at intensities 0.5 or 1.0 W/cm(2), considered non-thermal and sub-threshold for inertial cavitation, while at 44.0 degrees C for 10 min. We found that 0.5 W/cm(2), in combination with hyperthermia, synergistically induced apoptosis. On the other hand, 1.0 W/cm(2) in combination with hyperthermia showed an augmented instant cell lysis but no significant change in the ratio of apoptosis. This result might be useful when apoptosis induction is desired over instant cell killing in cancer therapy.

Activation of natural interferon-alpha producing cells by apoptotic U937 cells combined with lupus IgG and its regulation by cytokines.

We recently demonstrated that IgG from patients with systemic lupus erythematosus (SLE) in combination with U937 cells made apoptotic by UV-irradiation, can induce interferon-alpha (IFN-alpha) production in normal peripheral blood mononuclear cells (PBMC). In the present study we show by flow cytometry that the actual IFN-alpha producing cells (IPC) among PBMC had the same phenotype (HLA-DR+, CD4+, CD11b-, CD11c-, CD14-, CD19-, CD32-, CD36+, CD40+, CD45RA+, CD68+, CD83+, CD86-, IL-3R+ and IL-10R-) and low frequency (approximately 2/10(4)PBMC) as the IPC activated by Herpes simplex virus type I. Consequently, these cells correspond to the natural IPC, also described as type 2 precursor dendritic cells. We also demonstrated that cytokines of possible importance in the pathogenesis in SLE had effects on the IFN-alpha production. Specifically, the IFN-alpha production was strongly increased by the type I IFNs, IFN-alpha and -beta, but markedly inhibited by IL-10 and also to some extent by TFN-alpha. In contrast, the cytokines IFN-gamma, IL-6, TGF-beta and GM-CSF had no clear effects. No production of IL-10 was detected in PBMC stimulated by apoptotic U937 cells and SLE IgG. These results may explain the cause of the ongoing IFN-alpha production in SLE patients and its relation to the autoimmune process.

The role of intracellular Ca(2+) in apoptosis induced by hyperthermia and its enhancement by verapamil in U937 cells.

PURPOSE: The relationship between apoptosis induced by 42 degrees C and 44 degrees C hyperthermia alone or in combination with verapamil and changes in intracellular Ca(2+) concentration ([Ca(2+)]i) was investigated in U937 cells. METHODS: Apoptosis induced by hyperthermia was assessed according to DNA fragmentation, nuclear morphologic changes, and expression of phosphatidylserine on the outside plasma cell membrane. These changes were measured by flow cytometry. The [Ca(2+)]i of individual cells after hyperthermia was monitored by a digital image-analyzing technique using Fura-2. RESULTS: Hyperthermia-induced apoptosis reached a plateau after 6 h and was found to be both time and temperature-dependent. DNA fragmentation was maximum at 44 degrees C after 30 min. Verapamil enhanced the apoptosis induced by 42 degrees C and 44 degrees C hyperthermia in normal cells and by 44 degrees C hyperthermia in thermotolerant cells. The number of cells containing higher [Ca(2+)]i (more than 200 nM) was significantly increased by hyperthermia and further elevated by the addition of verapamil in both normal and thermotolerant cells. Apoptosis induced by hyperthermia was markedly decreased by an intracellular Ca(2+) chelator, BAPTA-AM, in a dose-dependent manner. CONCLUSION: These results indicate that [Ca(2+)]i increase plays a crucial role in apoptosis induced by hyperthermia and the combined treatment with verapamil in normal and thermotolerant U937 cells. Furthermore, hyperthermia-combined drug therapy has potential significance in cancer therapy.

Spatial distribution of selected genetic loci in nuclei of human leukemia cells after irradiation.

Fluorescence in situ hybridization (FISH) combined with high-resolution cytometry was used to determine the topographic characteristics of the centromeric heterochromatin (of the chromosomes 6, 8, 9, 17) and the tumor suppressor gene TP53 (which is located on chromosome 17) in cells of the human leukemia cell lines ML-1 and U937. Analysis was performed on cells that were either untreated or irradiated with gamma rays and incubated for different intervals after exposure. Compared to untreated cells, homologous centromeres and the TP53 genes were found closer to each other and also closer to the nuclear center 2 h after irradiation. The spatial relationship between genetic elements returned to that of the unirradiated controls during the next 2-3 h. Statistical evaluation of our experimental results shows that homologous centromeres and the homologous genes are positioned closer to each other 2 h after irradiation because they are localized closer to the center of the nucleus (probably due to more pronounced decondensation of the chromatin related to repair). This radial movement of genetic loci, however, is not connected with repair of DSBs by processes involving homologous recombination, because the angular distribution of homologous sequences remains random after irradiation.

25 Hz electromagnetic field exposure has no effect on cell cycle distribution and apoptosis in U-937 and HCA-2/1cch cells.

It is reported that exposure to 50 Hz extremely low-frequency electromagnetic field (ELF-EMF) can produce apoptosis and small variations in cell cycle distribution on different cell lines. In order to study the effect of ELF-EMF on tumoral cells in vitro, two cell lines (U-937, from a histiocytic lymphoma, and HCA-2/1cch, from a human colon adenocarcinoma) were exposed to 25 Hz, 1.5 mT, for 2 h and 45 min. Cell cycle distribution, apoptosis (spontaneous and dexamethasone-induced) and cell growth were evaluated. Neither significant alteration in cell cycle phases nor induction of apoptosis was observed. Nevertheless, the relative cell number was found to decrease to 55.84+/-7.35% (p <0.05, Student's t-test) for HCA-2/1cch cells after exposure to EMF in the presence of dexamethasone. The presence of dexamethasone during the EMF exposure could probably produce a decrease in the cell growth of this cell line.

Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells.

PURPOSE: Apoptosis is currently being evaluated for its importance as a pathway of radiation-induced cell death. However, the difference in the mechanisms between premitotic and postmitotic apoptosis following X-irradiation remains not well understood. We show here that the human monoblastoid cell line U937 can be induced to undergo these two different types of apoptosis. METHODS AND MATERIALS: U937 cells were irradiated at a dose of 5 or 20 Gy, and the DNA fragmentation rate was measured by both flow cytometric analysis and gel electrophoresis. Activation of caspase-3 was detected by Western blot analysis and fluorogenic assay using acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC). Detection of mitochondrial transmembrane potential (DeltaPsi) was performed by using Rho123. Chasing of S-phase fraction following X-irradiation was performed after labeling with 5-bromo-2'-deoxyuridine (BrdU). Thymidine was used for synchronization of the cells. Inhibition of caspase-3 activity was achieved by Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). RESULTS: Time courses of the apoptotic rates, caspase activation, and DeltaPsi indicated that two different types of cell death were induced by the different X-ray doses. High-dose X-ray (20 Gy) induced a rapid and strong apoptosis, whereas low-dose X-ray (5 Gy) induced a slow and mild apoptosis. Cell-cycle analyses revealed that there was cell death before cell division in the former apoptosis but the cells must be dying after cell division in the latter apoptosis. By means of cell-cycle synchronization, the S-phase cells proved to be the most sensitive fraction to premitotic apoptosis, but an obvious difference in the susceptibility to cell death among the cell-cycle phases was not observed in postmitotic apoptosis. Ac-DEVD-CHO treatment effectively blocked caspase activity and premitotic apoptosis, but it failed to block postmitotic apoptosis. CONCLUSIONS: Irradiation of U937 cells at different X-ray doses induced two different types of apoptotic cell death, premitotic apoptosis and postmitotic apoptosis, which are characterized by the time course and cell-cycle specificity. Decision concerning these two types of apoptotic cell death may be made by the difference in the magnitude of cell damage following X-irradiation.

Inhibition of the mitogen activated protein kinase pathway potentiates radiation-induced cell killing via cell cycle arrest at the G2/M transition and independently of increased signaling by the JNK/c-Jun pathway.

The ability of low dose ionizing radiation (2 Gy) to modulate the activities of the mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK1) cascades in human monocytic leukemia (U937/pREP4) cells and in cells over-expressing dominant negative c-Jun (TAM67) (U937/TAM67) was investigated. Radiation exposure caused prolonged ( approximately 1 h) MAPK activations in U937 cells. In contrast, low dose irradiation weakly modulated JNK1 activity in these cells. Inhibition of the MAPK pathway by use of the specific MEK1/2 inhibitor (10 microM PD98059) in both U937/pREP4 and U937/TAM67 cells prior to radiation exposure permitted strong prolonged radiation-induced activations of JNK1. Expression of TAM67 decreased the ability of radiation to cause apoptosis compared to control transfected cells. However, combined MEK1/2 inhibition and radiation exposure in both cell types caused a large decrease in suspension culture growth and a large increase in apoptosis, when compared to either treatment alone. Reduced proliferation after combined irradiation and PD98059 treatment in both cell types correlated with prolonged cell cycle arrest in G2/M phase. Prolonged growth arrest was abolished when MEK1/2 inhibitor was removed 6 h following irradiation, which was associated with a reduction in apoptosis. The ability of MEK1/2 inhibition to cause prolonged G2/M growth arrest was reduced in U937 cells stably transfected with a p21Cip-1/WAF1 antisense construct (U937/p21AS). This data correlated with an enhancement of radiation-induced apoptosis and a reduced ability of MEK1/2 inhibition to potentiate apoptosis. Collectively our data demonstrate that inhibition of MEK1/2 function increases the radiation sensitivity of U937 cells, independently of c-Jun function, and decreases the ability of these cells to recover from the radiation-induced G2/M cell cycle checkpoint arrest. In addition, our data also demonstrate that the ability of MEK1/2 inhibition to potentiate radiation-induced cell death in U937 cells in part requires an ability of cells to express low levels of p21Cip-1/WAF1.

Alkyl-lysophospholipids activate the SAPK/JNK pathway and enhance radiation-induced apoptosis.

Alkyl-lysophospholipids (ALPs) represent a new class of antitumor drugs that induce apoptotic cell death in a variety of tumor cell lines. Although their precise mechanism of action is unknown, ALPs primarily act on the cell membrane, where they inhibit signaling through the mitogen-activated protein kinase (MAPK) pathway. Because stimulation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway is essential for radiation-induced apoptosis in certain cell types, we tested the effect of ALPs in combination with ionizing radiation on MAPK/SAPK signaling and apoptosis induction. Here, we present data showing that three ALPs, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, hexadecylphosphocholine, and the novel compound octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate (D-21266) induce time- and dose-dependent apoptosis in the human leukemia cell lines U937 and Jurkat T but not in normal vascular endothelial cells. Moreover, in combination with radiation, ALPs strongly enhance the induction of apoptosis in both leukemic cell lines. All tested ALPs not only prevented MAPK activation, but, like radiation, stimulated the SAPK/JNK cascade within minutes. A dominant-negative mutant of c-Jun inhibited radiation- and ALP-induced apoptosis, indicating a requirement for the SAPK/JNK pathway. Our data support the view that ALPs and ionizing radiation cause an enhanced apoptotic effect by modulating the balance between the mitogenic, antiapoptotic MAPK, and the apoptotic SAPK/JNK pathways. This type of modulation of specific signal transduction pathways in tumor cells may lead to the development of new therapeutic strategies.