РАДИАЦИОННАЯ БИОЛОГИЯ. РАДИОЭКОЛОГИЯ, 2007, том 47, № 3, с. 281-285
PROCEEDINGS OF THE 4TH INTERNATIONAL WORKSHOP ON SPACE RADIATION RESEARCH = AND 17TH ANNUAL NASA SPACE RADIATION HEALTH =
INVESTIGATORS' WORKSHOP (Moscow-St.-Petersburg, June 5-9, 2006)
УДК 576:539.1.04
SHARED OXIDATIVE PATHWAYS IN RESPONSE TO GRAVITY-DEPENDENT LOADING AND g-IRRADIATION OF BONE MARROW-DERIVED SKELETAL CELL PROGENITORS
© 2007 H. Kondo1, C. Limoli1, N. D. Searby2, E. A. C. Almeida2, D. J. Loftus2, W. Vercoutere2, E. Morey-Holton2, E. Giedzinski1, R. Mojarrab2, D. Hilton2, R. K. Globus2*
1 Department of Radiation Oncology, University of California, Irvine, CA USA
2 Life Sciences Division, NASA Ames Research Center, Moffett Field, CA USA
Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of os-teoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.
Reactive oxygen species, hypergravity, bone marrow stromal cells, osteoblast.
Space is a hazardous environment with the potential to challenge the long-term health of astronauts. Reduced weightbearing activity clearly causes bone loss [1-5], but the effects of radiation on skeletal degeneration are not certain. Studies of cancer patients indicate that osteoporosis and bone fragility may ensue following radiotherapy [6-10]; however, these studies are difficult to interpret because cancer effects, chemotherapy and nutritional status may all interact with radiation effects. In experimental animals, both radiation [11] and musculoskeletal disuse [12] independently lead to a decrement in bone mass (osteopenia); whether these responses are due to direct effects of the stimuli on bone cells and their progenitors, or are secondary to other systemic factors is not yet known. Because weightlessness may exacerbate radiation's effects on bone tissue in space, it is important to determine possible risks and relevant mechanisms.
Oxidative stress may regulate cell growth, survival and differentiation in response to both radiation expo-
* Corresponding address: NASA Ames Research Center, MS/236-7, Moffett Field, CA 94035, ph.: 650-604-5247; fax: 650-604-3159; e-mail: Ruth.K.Globus@nasa.gov.
sure [13] and mechanical loading [14]. In certain cell types, radiation increases reactive oxygen species (ROS), including osteosarcoma cells [15] and neural precursors [16, 17]. We propose that gravity loading and radiation increase oxidative stress, which influences the viability and growth of osteoblast progenitors residing in bone marrow as adherent stromal cells. To test this idea, we applied hypergravity by transient centrifugation as a mechanical stimulus. The application of hypergravity to osteoblasts in culture causes deformation of the cyto-skeleton [17], proliferation [18, 19], and the release of prostaglandin E2, which is characteristic of many different types of mechanical stimuli [17, 18]. Thus, centrifugation provides a useful means to test our hypothesis that gravity and radiation directly stimulate ROS generation and regulate the viability and growth of primary osteo-blast precursors derived from the bone marrow.
MATERIALS AND METHODS
Bone marrow stromal cell (BMS) culture. Cells were flushed from the marrow of both tibiae and femora of 6 wk-12 wk old male C57BL6/J mice, plated at
Relative ROS Level
1.50 г
1.25 -
1.00 -
0.75
0125 0125 0125 0125
12
24 Dose, Gy
48
Post-irradiation Time, h
Fig. 1. Both centrifugation and irradiation increased reactive oxygen species in BMS cells.
On day 4 in culture, cells were harvested for the assessment of ROS using the fluorogenic dye, CM-H2DCFDA. All samples were normalized to controls from the same experiment. Bars shown are the means ± S.E.M. and are representative of 3-4 experiments with 2 to 4 replicates per experiment. Significance between data sets obtained from FACS analyses was determined by the Kolmogorov-Smirnov test (K-S test) provided with the Cell QuestTM software.
A. Centrifugation increased ROS levels. BMS cells from mice were centrifuged at the indicated doses (50-g) for 180 min on day 2 of culture. Relative ROS levels were normalized to the 1-g control. Hypergravity significantly increased ROS levels compared to 1-g controls (*p < 0.05).
B. Irradiation increased ROS levels. BMS cells from mice were gamma irradiated (137Cs) at the indicated doses (1, 2, and 5 Gy), 6, 12, 24 and 48 h prior to FACS analysis. Relative ROS levels were normalized to the non-irradiated control. Irradiation significantly increased ROS levels compared to the 0 Gy controls (p < 0.01).
4 x 105 cells/cm2 and grown in a-MEM supplemented with 15% fetal bovine serum, 1000 units/ml penicillin G sodium, 100 |g/ml streptomycin sulfate, and 0.25 |g/ml amphotericin B. To induce differentiation of BMS into osteoblasts, medium was supplemented with ascorbic acid (50 |g/ml) and ^-glycerophosphate (10 mM). Medium was changed on day 2 to remove excess floating cells and every 4 days thereafter.
Transient centrifugation. Cells were centrifuged in environmentally controlled conditions (85% humidity, 5% CO2) on day 2 in culture. Cultures were washed twice with PBS to remove floating cells prior to centri-fugation. Centrifugation was used to alter mechanical loading (5-g-50-g hypergravity), after which cells were returned to a tissue culture incubator for further growth.
Irradiation. Cells were exposed to a single dose (15 Gy) of irradiation using a 137Cs irradiator (1.0 Gy/min). Cells were irradiated at various times after plating.
ROS measurement. Day 4 cultures were treated for 1 h at 37°C with 5 |mM of the ROS-sensitive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluoresce-in diacetate, CM-H2DCFDA (Molecular Probes) [16]. Cells were harvested and analyzed directly afterward by fluorescence-activated cell sorting (FACS) (FACS-Calibur, Becton Dickenson).
DNA content. A DNA binding dye was used to quantify cell numbers spectrofluorometrically using a kit (CyQUANT, Invitrogen) based on CyQUANT GR dye, which exhibits strong fluorescence enhancement when bound to cellular nucleic acids. Frozen cells were thawed and lysed by addition of a buffer containing the CyQUANT GR dye; fluorescence was then measured directly.
Alkaline phosphatase activity. Cells were plated into 12-well plates and cultured for 7 days before lysis and analysis of the supernatants for protein content and ALP activity [20].
Nodule formation assay. Osteoblastogenesis was estimated by measuring production of mineralized matrix [5]. BMS were plated into 12-well plates, and medium was changed every 4 days. After 3 weeks, the cells were fixed with 100% ethanol then stained with 1% Alizarin red.
Statistical analysis. Results shown are representative of at least 3 independent experiments. Data were presented as means, and statistical evaluation was performed using ANOVA (Fishers PLSD), with p < 0.05 considered significant for measurements of dNa, alkaline phosphatase and nodule formation. For FACS analyses, significance between data sets was determined using the Kolmogorov-Smirnov test provided with CELLQUEST software (BD Biosciences, San Jose, CA). Results are presented as means ± standard error of the mean (S.E.M.).
*
Centrifugation increased total amount of DNA and ALP activity in bone marrow stromal cell cultures
g-Level Duration Centrifugation Cells per Well (x103) DNA per Well (centrifuged/control) ALP Activity (|mol/min/mg)
1-g - 42.5 ± 4.5 1 0.34 ± 0.09
5-g 5 min 67.5 ± 19.5 1.5 ± 0.4* n.d.
180 min 78.0 ± 8.5 1.8 ± 0.2# n.d.
10-g 5 min 73.5 ± 16.5 1.7 ± 0.4* n.d.
180 min 64.0 ± 5.0 1.5 ± 0.1* n.d.
50-g 5 min 98.0 ± 8.0 2.2 ± 0.2#$ 0.92 ± 0.24#
180 min 81.5 ± 11.0 1.8 ± 0.2# n.d.
Note. Cells were centrifuged on day 2 in culture, samples were harvested on day 4, then DNA was measured and normalized to cell number. For ALP, the cells were centrifuged on day 4, and ALP activity was measured on day 7.
* Indicates significant difference between the 1-g control and centrifuged group (p < 0.05).
# Indicates difference (p < 0.01).
$ Indicates difference (p < 0.05) between the 5-g 5 min and 50-g 5 min groups. n.d. indicates not done.
RESULTS
Both centrifugation and irradiation increased ROS generation in BMS
Cells were centri
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.