David Eriksson

Radiation-induced cell death mechanisms

The main goal when treating malignancies with radiation therapy is to deprive tumor cells of their reproductive potential. One approach to achieve this is by inducing tumor cell apoptosis. Accumulating evidences suggest that induction of apoptosis alone is insufficient to account for the therapeutic effect of radiotherapy. It has become obvious in the last few years that inhibition of the proliferative capacity of malignant cells following irradiation, especially with solid tumors, can occur via alternative cell death modalities or permanent cell cycle arrests, i.e., senescence. In this review, apoptosis and mitotic catastrophe, the two major cell deaths induced by radiation, are described and dissected in terms of activating mechanisms. Furthermore, treatment-induced senescence and its relevance for the outcome of radiotherapy of cancer will be discussed. The importance of p53 for the induction and execution of these different types of cell deaths is highlighted. The efficiency of radiotherapy and radioimmunotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation. Strategies to use specific inhibitors that will manipulate key molecules in these pathways in combination with radiation might potentiate therapy and enhance tumor cell kill.

Cell Cycle Disturbances and Mitotic Catastrophes in HeLa Hep2 Cells following 2.5 to 10 Gy of Ionizing Radiation

Purpose: Experimental radioimmunotherapy delivering absorbed doses of 2.5 to 10 Gy has been shown to cause growth retardation of tumors. The purpose of this study was to elucidate the sequential molecular and cellular events occurring in HeLa Hep2 cells exposed to such doses. Methods: Dose-response curves, activation of cell cycle checkpoints, and mitotic behavior were investigated in HeLa Hep2 cells following 2.5- to 10-Gy irradiation by carrying out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blots, fluorescence-activated cell sorting analysis, and immunofluorescence stainings. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining was used to detect apoptosis. Results: A G2-M arrest was shown by fluorescence-activated cell sorting analysis. p53 and p21 were found to be up-regulated but were not immediately related to the arrest. The G2-M arrest was transient and the cells reentered the cell cycle still containing unrepaired cellular damage. This premature entry caused an increase of anaphase bridges, lagging chromosomal material, and multipolar mitotic spindles as visualized by propidium iodide staining and immunofluorescence staining with α-tubulin and γ-tubulin antibodies. Furthermore, a dose-dependent significant increase in centrosome numbers from 12.6 ± 6.6% to 67 ± 5.3% was identified as well as a dose-dependent increase of polyploid cells from 2.8 ± 1.3% to 17.6 ± 2.1% with the highest absorbed dose of 10 Gy. These disturbances caused the cells to progress into mitotic catastrophe and a fraction of these dying cells showed apoptotic features as displayed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining 5 to 7 days after irradiation. Conclusion: An absorbed dose of 2.5 to 10 Gy was shown to force HeLa Hep2 cells into mitotic catastrophe and delayed apoptosis. These might be important cell death mechanisms involved in tumor growth retardation following radioimmunotherapy of solid tumors.

Apoptosis in HeLa Hep2 Cells is Induced by Low-Dose, Low-Dose-Rate Radiation

Abstract Mirzaie-Joniani, H., Eriksson, D., Johansson, A., Löfroth, P-O., Johansson, L., Riklund Åhlström, K and Stigbrand, T. Apoptosis in HeLa Hep2 Cells is Induced by Low-Dose, Low-Dose-Rate Radiation. Radiat. Res. 158, 634–640 (2002). Radioimmunotherapy with radiolabeled antibodies may cause inhibition of the growth of epithelial tumors, despite low total radiation doses and comparatively low radiosensitivity of epithelial tumor cells. The induction of apoptosis by low-dose radiation, such as delivered in radioimmunotherapy, was investigated in vitro in human HeLa Hep2 carcinoma cells. The cultured cells were exposed to defined radiation doses from a 60Co radiation therapy source. The radiation source delivered 0.80 ± 0.032 (mean ± SD) Gy/min and the cells were given total doses of 1, 2, 5, 10 and 15 Gy. Using fluorescein-labeled Annexin V, followed by flow cytometry and DNA ladder analysis, apoptotic cells were detected and quantified. Radiation doses below 2 Gy did not cause any significant increase in apoptosis. Compared to control cells, apoptosis was pronounced after 5–10 Gy irradiation and was correlated to the radiation dose, with up to 42 ± 3.5% of the cells examined displaying apoptosis. Clonogenic assays confirmed significantly decreased viability of the cells in the interval 2 to 10 Gy with low-dose-rate radiation, 60 ± 2% compared to 2 ± 2%. Lethal effects on the tumor cells were also evaluated by an assay of the cytotoxic effects of the release of 51Cr. Significant cytotoxicity, with up to 64 ± 6% dead cells, was observed at 5 Gy. Similar results were obtained when the dose rate was reduced to 0.072 ± 0.003 Gy/min (mean ± SD). In the case of the 137Cs source, the dose rate could be reduced to 0.045 Gy/h, a level comparable to radioimmunotherapy, which induced significant apoptosis, and was most pronounced at 72–168 h postirradiation. It can be concluded that in vitro low-dose and low-dose-rate radiation induces apoptosis in epithelial HeLa Hep2 cells and thus may explain a mechanism by which pronounced inhibition of growth of HeLa Hep2 tumors at doses used in radioimmunotherapy has been obtained previously.

Radiation Induced Cell Deaths

The previous classification of radiation induced cell deaths into either necrosis or apoptosis is today recognized as too simplistic. New possibilities to make use of other death mechanisms, when treating malignant diseases with targeted therapy, include rapid or delayed apoptosis, mitotic catastrophes, autophagy or senescence induction. Targeted radioimmunotherapy typically delivers low doses with low dose-rate irradiation to tumors, and is able to induce this extended panorama of different death mechanisms, which will be discussed in this chapter.

Genome wide expression analysis of radiation-induced DNA damage responses in isogenic HCT116 p53+/+ and HCT116 p53−/− colorectal carcinoma cell lines

Abstract Purpose: To study the kinetics of gene expression alterations following radiation exposure of isogenic HCT116 p53 +/+ and HCT116 p53−/− cell lines. Materials and methods: Cells were exposed to 5 Gy of irradiation (Cs-137) and genome-wide temporal expression analysis using Illumina bead chip arrays was performed. Signalling pathways were explored using Metacore (Genego). Biological responses including cell cycle checkpoint activation, centrosome amplification and senescence induction were analyzed. Results: Significant differences in the radiation response were observed between the p53+/+ and the p53−/− cell lines. In p53+/+ cells concurrent G1- and G2-arrests were activated followed by senescence induction. Increased expression of genes associated with senescence, senescence associated secretory phenotype (SASP) and repression of genes essential for G2-M transition were detected. P53−/− cells arrested mainly in G2 followed by centrosome amplification, mitotic slippage and a subsequent increase of polyploid cells. Furthermore, changes in expression correlated well with these signs of mitotic catastrophe. Conclusions: The presence or absence of p53 triggers different signalling cascades with different endpoints. Elucidating these differences is important as it enables improvement of radiation treatment and could be used to develop new combination treatments with specific inhibitors of key regulators of these cell death modalities.

Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α and death receptor stimulation in resistant variants of the human U937 cell line

Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

Overview of 3GPP Release 14 Enhanced NB-IoT

In 3GPP LTE Release 13, Narrowband Internet of Things (NB-IoT) was standardized for providing wide-area connectivity for massive machine-type communications for IoT. In LTE Release 14, NB-IoT was further developed to deliver enhanced user experience in selected areas through the addition of features such as increased positioning accuracy, increased peak data rates, the introduction of a lower device power class, improved non-anchor carrier operation, multicast, and authorization of coverage enhancements. In this article, we provide an overview of these features introduced for NB-IoT in LTE Release 14. An analysis is given on the applicability of these features and their benefits to enhance the NB-IoT radio access technology.

Therapeutically Used Targeted Antigens in Radioimmunotherapy

Many antigens have been tested as targets for radioimmunotherapy with intact antibodies. Some of the early used targets have been found to be of decreasing interest due to low expression, extensive shedding or other reasons. Others have been found more useful due to their accessibility, amount available in the tumours, or the biological properties of the target antigen. In this chapter some of the most used antigens and their characteristics are presented.

Targeting Tumours with Radiolabeled Antibodies

The introduction of radiolabelled antibodies targeting the lymphocyte antigen CD20 in certain hematologic malignancies received positive attention and is now accepted as a treatment modality. Treating solid tumours with radiolabelled antibodies has, so far, not been met with the same appreciation and such therapy for the large groups of malignancies like colorectal, breast, prostate, ovarian, lung cancer and brain tumours still require improvements in order to gain acceptance. In this chapter limitations, possibilities and future directions to improve therapy with radiolabelled antibodies are discussed.

Radiation Induced DNA Damage Checkpoints

Radiation induced damage to DNA can be limited to exchanges of single DNA bases or extensive double-strand breaks. Nuclear proteins can sense these alterations and are able to cause cell cycle arrests at the G1/S, intra-S or G2/M checkpoints in the cell cycle, until the lesions undergo repair. If the induction of these cell cycle arrests is defective, genomic instability and aberrations in the cell cycle kinetics appear, which may cause cell death. In this chapter radiation induced effects on the cell cycle will be presented.