Low dose-rate irradiation with [3H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model.

BACKGROUND: Earlier in vitro studies show that irradiation with an ultra-low dose-rate of 15 mGy/h delivered with [3H]-valine leads to loss of clonogenicity in hypoxic T-47D cells. Here, the aim was to determine if [3H]-valine could be used to deliver low dose-rate irradiation in a colorectal cancer model. METHODS: Clonogenicity was measured in cultured cancer cell line HT29 irradiated with 15 mGy/h combined with intermittent hypoxia. Mice with HT29 xenografts were irradiated by repeated injections of [3H]-valine intravenously. The activity in the tumor tissue was measured by scintillation counting and tumor growth, hypoxic fraction and tritium distribution within tumors were assessed by pimonidazole staining and autoradiography. RESULTS: Ultra-low dose-rate irradiation decreased clonogenicity in hypoxic colorectal cancer cells. In vivo, the tumor growth, hypoxic fraction and weight of the mice were similar between the treated and untreated group. Autoradiography showed no [3H]-valine uptake in hypoxic tumor regions in contrast to aerobic tissue. CONCLUSION: Continuous low-dose-rate irradiation was well tolerated by aerobic tissue. This indicates a potential use of low dose-rate irradiation to target hypoxic tumor cells in combination with high dose-rate irradiation to eradicate the well oxygenated tumor regions. However, [3H]-valine is not the appropriate method to deliver ultra-low dose-rate irradiation in vivo.

Hypothesis: Hypoxia induces de novo synthesis of NeuGc gangliosides in humans through CMAH domain substitute.

Immunotherapy is a growing field in cancer research. A privileged tumor-associated antigen that has received much attention is N-glycolyl (NeuGc) GM3. This ganglioside is present in several types of cancer, but is almost undetectable in human healthy tissues. However, its non-hydroxylated variant, NeuAc GM3, is abundant in all mammals. Due to a deletion in the human gene encoding the key enzyme for synthesis of NeuGc, humans, in contrast to other mammals, cannot synthesize NeuGc GM3. Therefore the presence of this ganglioside in human cancer cells represents an enigma. It has been shown that hypoxic conditions trigger the expression of NeuGc gangliosides, which not only serve as attractive targets for cancer therapy, but also as diagnostic and prognostic tumor marker. Here, we confirm hypoxia-induced expression of the NeuGc GM3 ganglioside also in HeLa cells and reveal several candidate proteins, in particular GM3 synthase and subunit B of respiratory complex II (SDHB), that may be involved in the generation of NeuGc GM3 by SILAC-based proteome analysis. These findings have the potential to significantly advance our understanding of how this enigmatic tumor-associated antigen is produced in humans, and also suggest a possible mechanism of action of anti-tumor antibodies that recognize hypoxia markers, such as 14F7.

Separation of two sub-groups with different DNA content after treatment of T-47D breast cancer cells with low dose-rate irradiation and intermittent hypoxia.

Background Previous studies have shown that combined treatment with internal ultra-low dose-rate irradiation selectively inactivated hypoxic T-47D breast cancer cells after three to five weeks of treatment. However, 2-3% of the hypoxic cells were found to survive and restart proliferation upon re-oxygenation. Purpose To investigate the metastatic potential and characteristics of radiosensitivity of these surviving cells, named T - 47 DS. Material and Methods The T - 47 DS cells were grown in ambient air without irradiation. A cloning experiment identified two sub-groups with different DNA content ([Formula: see text] and [Formula: see text]). Furthermore, radiosensitivity and presence of hyper-radiosensitivity (HRS) was measured by Co-60 challenge irradiation and relative migration was determined by scratch assays. Results The two subpopulations of T - 47 DS had different DNA content; one had abnormally high DNA content ([Formula: see text]) and one had DNA content similar to wild-type T-47D cells ([Formula: see text]). HRS was surprisingly present in cells of the cloned population [Formula: see text], but was absent in cells of both [Formula: see text] and T - 47 DS. The radio response of T - 47 DS, [Formula: see text] at higher radiation doses were similar to that of T-47D cells, and neither subpopulation showed increased migration compared with wild-type T-47D. Conclusion No increase in the risk of metastasis was found and only slight changes in radiosensitivity in response to conventional clinical doses was observed. Thus, the data suggest that if ultra-low dose-rate irradiation is used for targeting the hypoxic tumor fraction, conventional high dose-rate irradiation can be used to eradicate eventual surviving cells as well as cells in the well oxygenated areas of the tumor.

Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells.

Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

Cell inactivation by combined low dose-rate irradiation and intermittent hypoxia.

PURPOSE: To investigate in detail the earlier observed combined effect of low dose-rate ß-irradiation delivered at a dose-rate of 15 mGy/h and continued intermittent hypoxia that leads to extensive cell death after approximately 3-6 weeks. MATERIAL AND METHODS: Continuous low dose-rate ß-irradiation at a dose rate of 15, 1.5 or 0.6 mGy/h was given by incorporation of [(3)H]-labelled valine into cellular protein. The cells were cultivated in an atmosphere with 4% O2 using an INVIVO2 hypoxia glove box. Clonogenic capacity, cell-cycle distribution and cellular respiration were monitored throughout the experiments. RESULTS: After 3-6 weeks most cells died in response to the combined treatment, giving a surviving fraction of only 1-2%. However, on continued cultivation a few cells survived and restarted proliferation as the cellular oxygen supply increased with the reduced cell number. Irradiating the T-47D cells grown in an atmosphere with 4% O2 at dose-rates 10 and 25 times lower than 15 mGy/h did not have a pronounced effect on the clonogenic capacity with surviving fractions of 60-80%. CONCLUSIONS: Treatment of T-47D cells with low dose-rate ß-irradiation leads to a specific effect on intermittent hypoxic cells, inactivating more than 98% of the cells in the population. Given improved oxygen conditions, the few surviving cells can restart their proliferation.

The roles of TGF-ß3 and peroxynitrite in removal of hyper-radiosensitivity by priming irradiation.

PURPOSE: To investigate the mechanisms inducing and maintaining the permanent elimination of low dose hyper-radiosensitivity (HRS) in cells given a dose of 0.3 Gy at low dose-rate (LDR) (0.3 Gy/h). MATERIALS AND METHODS: Two human HRS-positive cell lines (T-47D, T98G) were used. The effects of pretreatments with transforming growth factor beta (TGF-ß) neutralizers, TGF-ß3 or peroxynitrite scavenger on HRS were investigated using the colony assay. Cytoplasmic levels of TGF-ß3 were measured using post-embedding immunogold electron microscopic analysis. RESULTS: TGF-ß3 neutralizer inhibited the removal of HRS by LDR irradiation. Adding 0.001 ng/ml TGF-ß3 to cells removed HRS in T98G cells while 0.01 ng/ml additionally induced resistance to higher doses. Cytoplasmic levels of TGF-ß3 were higher in LDR-primed cells than in unirradiated cells. The presence of the peroxynitrite scavenger uric acid inhibited the effect of LDR irradiation. Furthermore, the permanent elimination of HRS in LDR-primed cells was reversed by treatment with uric acid. The removal of HRS by medium from hypoxic cells was inhibited by adding TGF-ß3 neutralizer to the medium before transfer or by adding hypoxia inducible factor 1 (HIF-1) inhibitor chetomin to the cell medium during hypoxia. CONCLUSIONS: TGF-ß3 is involved in the regulation of cellular responses to small doses of acute irradiation. TGF-ß3 activation seems to be induced by low dose-rate irradiation by a mechanism involving inducible nitric oxide (iNOS) and peroxynitrite, or during cycling hypoxia by a mechanism most likely involving HIF-1. The study suggests methods to turn resistance to doses in the HRS-range on (by TGF-ß3) or off (by TGF-ß3 neutralizer or by peroxynitrite inhibition).

The role of nitric oxide radicals in removal of hyper-radiosensitivity by priming irradiation.

In this study, a mechanism in which low-dose hyper-radiosensitivity (HRS) is permanently removed, induced by low-dose-rate (LDR) (0.2-0.3 Gy/h for 1 h) but not by high-dose-rate priming (0.3 Gy at 40 Gy/h) was investigated. One HRS-negative cell line (NHIK 3025) and two HRS-positive cell lines (T-47D, T98G) were used. The effects of different pretreatments on HRS were investigated using the colony assay. Cell-based ELISA was used to measure nitric oxide synthase (NOS) levels, and microarray analysis to compare gene expression in primed and unprimed cells. The data show how permanent removal of HRS, previously found to be induced by LDR priming irradiation, can also be induced by addition of nitric oxide (NO)-donor DEANO combined with either high-dose-rate priming or exposure to prolonged cycling hypoxia followed by reoxygenation, a treatment not involving radiation. The removal of HRS appears not to involve DNA damage induced during priming irradiation as it was also induced by LDR irradiation of cell-conditioned medium without cells present. The permanent removal of HRS in LDR-primed cells was reversed by treatment with inducible nitric oxide synthase (iNOS) inhibitor 1400W. Furthermore, 1400W could also induce HRS in an HRS-negative cell line. The data suggest that LDR irradiation for 1 h, but not 15 min, activates iNOS, and also that sustained iNOS activation is necessary for the permanent removal of HRS by LDR priming. The data indicate that nitric oxide production is involved in the regulatory processes determining cellular responses to low-dose-rate irradiation.

Low dose hyper-radiosensitivity is eliminated during exposure to cycling hypoxia but returns after reoxygenation.

PURPOSE: To investigate the effect of cycling hypoxia on low dose hyper-radiosensitivity (HRS). MATERIALS AND METHODS: Human breast tumor T-47D cells were grown in a hypoxia workstation operated at 4% O(2) for 3-6 weeks and the pericellular oxygen concentration was recorded every 20 minutes. The presence of HRS in response to subsequent challenge irradiation was measured by clonogenic survival. RESULTS: T-47D cells adapted to growing with 4% O(2) in the gas phase but showed no HRS. However, HRS was recovered after between 48 h and two weeks of reoxygenation at 20% O(2). Medium transferred from the hypoxic T-47D cells removed HRS in recipient cells grown in ambient air. Cells irradiated with X-rays showed a shallower HRS-'dip' and a lower d(c)-value (dose where the change from the hypersensitive to the induced repair response is 63% complete) compared to cells irradiated with (60)Co γ-rays. CONCLUSIONS: Cycling hypoxia transiently eliminates HRS in T-47D cells in vitro. This may partly explain the diverging results of in vivo studies of HRS. The effect of cycling hypoxia on HRS is comparable to our previous findings for T-47D cells receiving medium transfer from cells irradiated with 0.3 Gy at 0.3 Gy/h.

Mechanisms of the elimination of low dose hyper-radiosensitivity in T-47D cells by low dose-rate priming.

PURPOSE: To investigate the mechanisms of elimination of low-dose hyper-radiosensitivity (HRS) in T-47D cells induced by 0.3 Gy low dose-rate (LDR) priming. MATERIALS AND METHODS: The mitotic ratio was measured using mitotic marker histone H3 phosphorylation in LDR primed as well as untreated T-47D cells. The HRS response in unprimed cells receiving medium which was irradiated after being harvested from unprimed cells was measured with or without serum present during cell conditioning. 4,6-benzylidene-D-glucose (BG) was used to inhibit protein synthesis during LDR priming. RESULTS: LDR primed T-47D cells were HRS-deficient and showed a decrease in mitotic ratio with increasing dose while unprimed, i.e., HRS-competent T-47D cells, showed no decrease in mitotic ratio for doses in the HRS-range. HRS was eliminated in LDR primed cells, in cells receiving medium transfer from LDR primed cells, and in cells receiving LDR irradiated medium harvested from unprimed cells. The efficacy of the transferred medium depended on the presence of serum during cell conditioning. LDR priming eliminated HRS even in the presence of protein synthesis inhibitor BG. CONCLUSIONS: LDR priming of T-47D cells as well as LDR priming of medium conditioned on T-47D cells induce a factor in the medium which cause the early G(2)-checkpoint to be activated in recipient cells by doses normally in the HRS dose-range.

The elimination of low-dose hyper-radiosensitivity by transfer of irradiated-cell conditioned medium depends on dose rate.

Irradiation of T-47D cells with 0.3 Gy delivered by a (60)Co source at a low dose rate of 0.3 Gy/h abolished low-dose hyper-radiosensitivity (HRS) for at least 14 months (with continuous cell culturing), while the same dose administered acutely (40 Gy/h) eliminated HRS for less than 24 h. Medium transferred from the low-dose-rate primed cells (low-dose-rate ICCM) to unirradiated cells eliminated HRS in recipient cells even if the donor cells had been cultivated for 14 months after the priming dose. Thus low-dose-rate priming activates mechanisms that involve modification or induction of a factor in the medium. This factor affects unirradiated cells in such a way that HRS is eliminated in cells exposed to medium from the primed cells. However, only cells directly exposed to low-dose-rate radiation induce or modify the putative factor, since unirradiated cells that were exposed to low-dose-rate ICCM regained HRS within 2 weeks of cultivation in fresh medium. The ability of ICCM to eliminate HRS in recipient cells is dependent on dose rate. However, an increase in clonogenic survival was observed in cells receiving only medium transfer without subsequent irradiation that was independent of dose rate.