Lack of inverse dose-rate effect and binding of the retinoblastoma gene product in the nucleus of human cancer T-47D cells arrested in G2 by ionizing radiation

PURPOSE: To investigate the radiosensitivity of human breast cancer cells, T-47D, irradiated with low dose-rates and to study activation of the retinoblastoma gene product in the G1 and G2 phases during irradiation. MATERIALS AND METHODS: Cells were irradiated with (60)Co gamma-rays with dose-rates of 0.37 and 0.94 Gy h(-1). Cell survival was measured as the ability of cells to form colonies. Cells were extracted, fixed and stained for simultaneous measurements of nuclear-bound pRB content and DNA content. Cell nuclei were stained with monoclonal antibody PMG3-245 and Hoechst 33258 was used for additional staining of DNA. Two-parametric flow cytometry measurements of pRB and DNA content were performed using a FACSTAR(PLUS) flow cytometer. RESULTS: It was observed that irradiated cells were arrested in G2. No increase in radiation sensitivity was observed when the cells accumulated in G2. Irradiation of cells at both 0.37 and 0.94 Gy h(-1) resulted in exponential dose-survival curves with nearly equal alpha values, i.e. the same radiosensitivity. However, the retinoblastoma gene product was bound in the nucleus, i.e. hypophosphorylated, in about 15% of the cells arrested in G2. CONCLUSIONS: T47-D cells accumulate in G2 during low dose irradiation, but no inverse dose-rate effect, i.e. a more efficient inactivation of cells at lower than at higher dose-rates, was observed. A population of arrested G2 cells has pRB protein bound in the nucleus, and pRB therefore could play a role in protecting cells against radiation-induced cell death in G2.     

Contributions of AMPK and p53 dependent signaling to radiation response in the presence of metformin

Background and purpose

Metformin is commonly prescribed to treat type 2 diabetes, and has additional potential as a cancer prophylactic and therapeutic. Metformin activates AMPK that in turn can launch a p53-dependent metabolic checkpoint. Possible interactions between metformin and radiation are poorly understood. Since radiation-induced signaling also involves AMPK and p53, we investigated their importance in mediating responses to metformin and radiation.

Materials and methods

A549 cells, HCT116 cells wildtype or knockout for p53 or MEFs wildtype or double knockout for AMPKα1 and α2 were irradiated in the presence or absence of metformin. The impact of metformin on oxygen consumption and proliferation rates was determined, as well as clonogenic radiation survival.

Results

Metformin resulted in moderate radiation protection in all cell lines, irrespective of AMPK and p53. Loss of AMPK sensitized cells to the anti-proliferative effects of metformin, while loss of p53 promoted both the growth inhibitory and toxic effects of metformin. Consequently, overall cell death after radiation was similar with and without metformin irrespective of AMPK or p53 genotype.

Conclusions

The anti-proliferative activity of metformin may confer benefit in combination with radiotherapy, and this benefit is intensified upon loss of AMPK or p53 signaling.     

Autophagy is required during cycling hypoxia to lower production of reactive oxygen species

Background and purpose

Human tumors are characterized by the presence of cells that experience periodic episodes of hypoxia followed by reoxygenation. These cells are exposed to reactive oxygen species (ROS) upon reoxygenation and require adaptation to this stress by lowering ROS production or enhancing ROS-clearance for their survival. We hypothesized that autophagy, a lysosomal degradation pathway, may be involved in reducing ROS during periodic hypoxia through removal of ROS producing species.

Materials and methods

Human tumor cells (MCF-7, HT29, U373) were exposed to cycles of hypoxia (O₂ < 0.02%) and reoxygenation in the absence or presence of the autophagy inhibitor chloroquine (CQ). Clonogenic survival, ROS production and mitochondrial-DNA content were assessed. In addition, A549 cells overexpressing wild-type or K63-mutated ubiquitin (K63R) were analyzed for ROS production.

Results

Our data indicate that CQ treatment sensitizes cells to cycling hypoxia, due to increased production of ROS, associated with an incapacity to reduce mitochondrial content. Addition of the ROS-scavenger N-acetyl-cysteine increased cell viability and neutralized CQ-effects. Additionally, genetic prevention of K63-linked ubiquitin chains that are required for the removal of toxic protein aggregates by autophagy, resulted in increased ROS production.

Conclusions

Inhibition of autophagy substantially increases cell death induced by cycling hypoxia through increased ROS production, providing an opportunity to decrease the hypoxic fraction within tumors and enhance tumor therapy.     

Proteomic analysis of gene expression following hypoxia and reoxygenation reveals proteins involved in the recovery from endoplasmic reticulum and oxidative stress.

BACKGROUND AND PURPOSE: Human tumors are characterized by large variations in oxygen concentration and hypoxic tumors are associated with poor prognosis. In addition, tumors are subjected to periodic changes in oxygenation characterized by hypoxia followed by reoxygenation. Cellular adaptation to hypoxia is well documented, nevertheless little is known about adaptive mechanisms to reoxygenation. Here, we investigate the changes in protein expression during reoxygenation using proteomics. MATERIALS AND METHODS: HeLa cervix carcinoma cells were exposed to 4h of hypoxia (<0.01% O(2)) followed by 1h of reoxygenation. The cellular proteome was examined using 2D gel electrophoresis coupled with mass spectrometry. Validation and investigation of the underlying basis for induced protein expression was investigated using Western blot analysis and quantitative RT-PCR. RESULTS: We identified proteins involved in several cellular processes that are responsible for regulating RNA metabolism, protein synthesis and degradation, including ribosomal protein P0, VCP/p97 and FUSE binding protein 2. CONCLUSIONS: Our results suggest that these newly identified proteins function in pathways that may assist in the recovery of ER stress and protein synthesis during reoxygenation. These proteins may thus be important determinants of the behaviour and survival of tumor cells to transient hypoxic exposures.     

Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction

Background and purpose

Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this study was to determine the effect of preventing mTOR-dependent translation inhibition on hypoxic cell survival and tumour sensitivity towards irradiation.

Material and methods

The effect of eIF4E-overexpression on cell proliferation, hypoxia-tolerance, and radiation sensitivity was assessed using isogenic, inducible U373 and HCT116 cells.

Results

We found that eIF4E-overexpression significantly enhanced proliferation of cells under normal conditions, but not during hypoxia, caused by increased cell death during hypoxia. Furthermore, eIF4E-overexpression stimulated overall rates of tumour growth, but resulted in selective loss of hypoxic cells in established tumours and increased levels of necrosis. This markedly increased overall tumour sensitivity to irradiation.

Conclusions

Our results demonstrate that hypoxia induced inhibition of translational control through regulation of eIF4E is an important mediator of hypoxia tolerance and radioresistance of tumours. These data also demonstrate that deregulation of metabolic pathways such as mTOR can influence the proliferation and survival of tumour cells experiencing metabolic stress in opposite ways of nutrient replete cells.