Response of chronic hypoxic cells to low dose-rate irradiation.

PURPOSE: Compare the sensitivity of human cells in vitro to low dose-rate irradiation in air and in moderate hypoxia (4% O2). MATERIALS AND METHODS: Continuous low dose-rate beta-irradiation at a dose rate of 0.015 or 0.062 Gy/h was given to human T-47D breast cancer cells by incorporation of [3H] -labelled valine into cellular protein. Acute irradiation at a dose rate of 0.4 Gy/min was performed using [137Cs]gamma-irradiation. Cells were cultivated in an atmosphere with 4% O2 using an INVIVO2 hypoxia cabinet. RESULTS: When grown in ambient air with continuous irradiation, T-47D cells were able to continue growth for at least 23 weeks at a dose-rate of 0.015 Gy/h with a surviving fraction stabilized at around 60%. When the dose rate was increased to 0.062 Gy/h the cell culture died out after about 23 days (corresponding to about 22 Gy). When grown in an atmosphere with 4% O2 we surprisingly found that the continuously irradiated T-47D cells (0.015 Gy/h) were severely inhibited in their growth, and cell death became extensive after about 3 weeks while un-irradiated cells continued growth seemingly unaffected by this low oxygenation. Peri cellular oxygenation varied between 4% and below 0.1% over an ordinary passage due to diffusion-limitations through the 2 mm deep medium. Online O2-recordings over a whole passage showed that oxygen was more depleted in the irradiated compared to the un-irradiated cultures indicating increased respiration during irradiation. While cells growing attached to the bottom were inhibited and inactivated during irradiation it was found that cells attached high up in the neck region, i.e., having only a shallow layer of medium above them, survived and formed colonies. When cells cultivated in 4% O2 for 7 weeks were irradiated with acute doses of 137Cs gamma-rays, the radiosensitivity was the same as for cells cultivated in ambient air. CONCLUSION: Continuous irradiation with 0.015 Gy/h for several weeks results in a stronger inhibition for T-47D cells grown in an atmosphere with 4% as compared to 20% O2. The data indicate that this may be due to increased oxygen consumption resulting in more severe hypoxia in [3H]-incorporating compared to control (un-irradiated) cells.

1,25-Dihydroxyvitamin D3 attenuates adenylyl cyclase activity in rat thyroid cells: reduction of thyrotropin receptor number and increase in guanine nucleotide-binding protein Gi-2 alpha.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the most potent of the naturally occurring vitamin D metabolites. In rat thyroid FRTL-5 cells, 1,25-(OH)2D3 attenuated the increase in TSH-stimulated adenylyl cyclase activity obtained by removing TSH from the culture medium. When cells were incubated with 1,25-(OH)2D3 (10 nmol/liter; 4 days), the binding capacity for specific [125I]TSH binding decreased from 20.1 +/- 1.8 to 8.8 +/- 1.6 fmol/10(6) cells (mean +/- SEM; n = 4; P < 0.01) compared to that in control cells. The Kd did not change (mean +/- SEM, 0.46 +/- 0.09 vs. 0.25 +/- 0.07 nmol/liter; n = 4; P = NS). Western blotting revealed no change in the membrane content of the adenylyl cyclase (AC) stimulatory guanine nucleotide-binding protein (G-protein) alpha-subunit (Gs alpha) during 1,25-(OH)2D3 treatment. Similarly, levels of the AC inhibitory G-protein Gi-3 alpha- and G-protein beta-subunits were not altered by 1,25-(OH)2D3. However, Western blotting with antibodies recognizing both Gi-1 alpha and Gi-2 alpha was augmented 4-fold, presumably representing an increase in Gi-2 alpha only, as Gi-1 alpha messenger RNA (mRNA) was not detected in FRTL-5 cells. 1,25-(OH)2D3 (10 nmol/liter; 4 days) reduced cholera toxin (10 nmol/liter)-stimulated AC activity to 85% of the control value (P < 0.05), whereas forskolin (100 mumol/liter)-stimulated direct activation of AC was inhibited by 39%. The TSH receptor mRNA level correlated to the beta-actin mRNA was 2-fold higher in control cells compared to that in 1,25-(OH)2D3-treated cells 12 h after TSH removal. Only minor alterations in the Gs alpha mRNA/beta-actin mRNA and Gi-3 alpha mRNA/beta-actin mRNA ratios were observed during 1,25-(OH)2D3 treatment, whereas Gi-2 alpha mRNA increased 3-fold compared to that in control cells. No change in the resting intracellular Ca2+ concentration could be detected after 4 days of 1,25-(OH)2D3 treatment. Our studies show that 1,25-(OH)2D3 attenuates AC activity by reducing the TSH receptor number and increasing the level of the AC inhibitory G-protein Gi-2 alpha in FRTL-5 cells.

Regulation of protein synthesis in human cells exposed to extreme hypoxia.

Protein synthesis was recorded in NHIK 3025 cells when cultured either during extremely hypoxic conditions or following reoxygenation. The rate of total protein synthesis in NHIK 3025 cells was found to be reduced to 30-40% of the rate in control cells within 1 h after the start of hypoxia. After reoxygenation, the rate of total protein synthesis was found to increase rapidly back to the control level. To reveal the stage of regulation of hypoxia associated proteins (HAP), mRNA was isolated from NHIK 3025 cells and translated in an in vitro translation kit. The results indicated that the relative amount of mRNA, of at least two HAP (80 and 45 kD), was increased under extremely hypoxic conditions ([O2] < 4ppm). This implies that some HAP are regulated pre-translationally.

Pericellular oxygen monitoring with integrated sensor chips for reproducible cell culture experiments.

OBJECTIVES: Here we present an application, in two tumour cell lines, based on the Sensing Cell Culture Flask system as a cell culture monitoring tool for pericellular oxygen sensing. MATERIALS AND METHODS: T-47D (human breast cancer) and T98G (human brain cancer) cells were cultured either in atmospheric air or in a glove-box set at 4% oxygen, in both cases with 5% CO2 in the gas phase. Pericellular oxygen tension was measured with the help of an integrated sensor chip comprising oxygen sensor arrays. RESULTS: Obtained results illustrate variation of pericellular oxygen tension in attached cells covered by stagnant medium. Independent of incubation conditions, low pericellular oxygen concentration levels, usually associated with hypoxia, were found in dense cell cultures. CONCLUSIONS: Respiration alone brought pericellular oxygen concentration down to levels which could activate hypoxia-sensing regulatory processes in cultures believed to be aerobic. Cells in culture believed to experience conditions of mild hypoxia may, in reality, experience severe hypoxia. This would lead to incorrect assumptions and suggests that pericellular oxygen concentration readings are of great importance to obtain reproducible results when dealing with hypoxic and normoxic (aerobic) incubation conditions. The Sensing Cell Culture Flask system allows continuous monitoring of pericellular oxygen concentration with outstanding long-term stability and no need for recalibration during cell culture experiments. The sensor is integrated into the flask bottom, thus in direct contact with attached cells. No additional equipment needs to be inserted into the flask during culturing. Transparency of the electrochemical sensor chip allows optical inspection of cells attached on top of the sensor.

Oxygen consumption in T-47D cells immobilized in alginate.

OBJECTIVES: Encapsulation or entrapment of cells is increasingly being used in a wide variety of scientific studies for tissue engineering and development of novel medical devices. The effect on cell metabolism of such systems is, in general, not well characterized. In this work, a simple system for monitoring respiration of cells embedded in 3-D alginate cultures was characterized. MATERIALS AND METHODS: T-47D cells were cultured in alginate gels. Oxygen concentration curves were recorded within cell-gel constructs using two different sensor systems, and cell viability and metabolic state were characterized using confocal microscopy and commercially available stains. RESULTS: At sufficient depth within constructs, recorded oxygen concentration curves were not significantly influenced by influx of oxygen through cell-gel layers and oxygen consumption rate could be calculated simply by dividing oxygen loss in the system per time, by the number of cells. This conclusion was supported by a 3-D numeric simulation. For the T-47D cells, the oxygen consumption rate was found to be 61 ± 6 fmol/cell/h, 3-4 times less than has previously been found for these cells, when grown exponentially in monolayer culture. CONCLUSIONS: The experimental set-up presented here may be varied in multiple ways by changing the cell-gel construct 3-D microenvironment, easily allowing investigation of a variety of factors on cell respiration.

The retinoblastoma gene product is reversibly dephosphorylated and bound in the nucleus in S and G2 phases during hypoxic stress.

We have studied the role of the retinoblastoma susceptibility gene product (pRB) in the regulation of cell-cycle progression under extremely hypoxic conditions (< 4 ppm O2). pRB is a nuclear matrix-associated phosphoprotein that normally exerts its growth-regulatory effects during early-G1 phase of the cell cycle, where all pRB present has been assumed to be in the under-phosphorylated form and bound in the nucleus. The effect of hypoxia on pRB nuclear binding and its state of phosphorylation was studied by two methods: (a) two-parametric flow cytometric measurement of pRB versus DNA and (b) Western blotting. Pulse-chase and pulse labeling with BrdUrd was used to record cell-cycle progression under versus after extremely hypoxic conditions. We demonstrate that pRB is dephosphorylated and rebound in the nucleus in more than 90% of cells located in S and G2 under extremely hypoxic conditions. While inhibition of DNA synthesis was instantaneous under hypoxic conditions, dephosphorylation and rebinding to nuclear structures of pRB takes more than 4 h. Within this time span, cells in G2 complete mitosis and divide. The slow dephosphorylation of pRB indicates that pRB is neither associated with the instantaneous inhibition of DNA synthesis nor is it the cause of the oxygen-dependent restriction point located in late-G1. The observed dephosphorylation of pRB is not dependent on functional p53, suggesting that at least one of the mechanisms responsible for the dephosphorylation is due to hypoxic activation of a pRB-specific phosphatase in the absence of p53-dependent inhibition of pRB kinase activity. However, it cannot be ruled out the participation of pRB kinase inhibitors independent of p53 activation. Cells arrested in G1 during prolonged hypoxia resumed cell-cycle progression within 2-->24 h after reoxygenation, while cells arrested in S were unable to reenter cell-cycle progression after reoxygenation. The hypoxia-induced dephosphorylation of pRB was only partly reversible by reoxygenation. Reentry into the cell cycle induced by reoxygenation occurred concomitant with unbinding (hyperphosphorylation) of pRB. Thus, rephosphorylation of pRB seem to be the rate-limiting step for reentry into the cell cycle after reoxygenation. Although pRB seems to play a major role in suppression of cell growth under and following hypoxic stress, other factors seem to be responsible for the immediate hypoxia-induced arrest in late-G1 and S phases.

Hypoxia-induced apoptosis in human cells with normal p53 status and function, without any alteration in the nuclear protein level.

We have studied hypoxia-induced inactivation of cells from three established human cell lines with different p53 status. Hypoxia was found to induce apoptosis in cells expressing wild-type p53 (MCF-7 cells), but not in cells where p53 is either mutated (T-47D cells), or abrogated by expression of the HPV18 E6 oncoprotein (NHIK 3025 cells). Apoptosis was demonstrated by DNA fragmentation, using agarose gel electrophoresis of DNA and DNA nick end labeling (TUNEL). We demonstrate that extremely hypoxic conditions (< 4 ppm O2) do not cause any change of expression in the p53 protein level in these three cell lines. In addition, the localization of p53 in MCF-7 cells was found exclusively in the nucleus in only some of the cells both under aerobic and hypoxic conditions. Furthermore, no correlation was found between the p53-expression level and whether or not a cell underwent apoptosis. Flow cytometric TUNEL analysis of MCF-7 cells revealed that initiation of apoptosis occurred in all phases of the cell cycle, although predominantly for cells in S phase. Apoptosis was observed only during a limited time window (i.e., approximately 10 to approximately 24 h) after the onset of extreme hypoxia. While 66% of the MCF-7 cells lost their ability to form visible colonies following 15 h exposure to extreme hypoxia, only approximately 28% were induced to apoptosis, suggesting that approximately 38% were inactivated by other death processes. Commitment to apoptotic cell death was observed in MCF-7 cells even for oxygen concentrations as high as 5000 ppm. Our present results indicate that the p53 status in these three tumor cell lines does not have any major influence on cell's survival following exposure to extremely hypoxic conditions, whereas following moderate hypoxia, cells expressing functional p53 enhanced their susceptibility to cell death. Taken together, although these results suggest that functional p53 might play a role in the induction of apoptosis during hypoxia, other factors seem to be equally important.

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions.

1,25-dihydroxyvitamin D3 alters the effect of cAMP in thyroid cells by increasing the regulatory subunit type II beta of the cAMP-dependent protein kinase.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) attenuates the stimulatory effects of cAMP on proliferation and iodide uptake in rat thyroid FRTL-5 cells. This study examines the effects of 1,25-(OH)2D3 on the cAMP-dependent protein kinase (PKA). Cytosol proteins separated by anion exchange chromatography showed increased [3H]cAMP binding activity as well as increased kinase activity in the fractions containing PKA type II in 1,25-(OH)2D3 (10 nM)-treated cells compared to the control cells. Western blot analysis of 1,25-(OH)2D3-treated cells revealed a 4-fold increase in the cytosolic amount of the PKA regulatory subunit RII beta, whereas no changes were detected in the regulatory subunits RI alpha and RII alpha or the catalytic (C) subunit. Northern blot analyses showed a similar increase in RII beta mRNA in cells treated for 12 h with 1,25-(OH)2D3 (10 nM), and RII beta mRNA increased further to 10-fold above control cell level after 96 h of incubation. Iodide uptake was synergistically stimulated with both PKAI- and PKAII-directed pairs of cAMP analogs. The PKAI synergism was, however, inhibited by 1,25-(OH)2D3 treatment of the cells, whereas the PKAII synergism was unaffected. In conclusion, 1,25-(OH)2D3 attenuates both PKAI formation and PKAI-stimulated iodide uptake in rat thyroid FRTL-5 cells by increasing the level of RII beta without altering the other PKA subunit levels.