help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Riou, C.
Right arrow Articles by Rousset, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Riou, C.
Right arrow Articles by Rousset, B.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CYCLOHEXIMIDE
*HYDROGEN PEROXIDE
Endocrinology Vol. 140, No. 5 1990-1997
Copyright © 1999 by The Endocrine Society

ARTICLES

Susceptibility of Differentiated Thyrocytes in Primary Culture to Undergo Apoptosis after Exposure to Hydrogen Peroxide: Relation with the Level of Expression of Apoptosis Regulatory Proteins, Bcl-2 and Bax1

Catherine Riou, Helene Tonoli, Françoise Bernier-Valentin, Rachida Rabilloud, Pierre Fonlupt and Bernard Rousset

INSERM, U-369, Faculté de Médecine Lyon-RTH Laennec, 69372 Lyon Cedex 08, France

Address all correspondence and requests for reprints to: Prof. Bernard Rousset, INSERM U-369, rue Guillaume Paradin, Faculté de Médecine Lyon-RTH Laennec, 69372 Lyon Cedex 08, France. E-mail: u369{at}laennec.univ-lyon1.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyrocytes, that generate and use hydrogen peroxide (H2O2) to synthesize thyroid hormones, undergo apoptosis, as do most cell types, when exposed in vitro to H2O2. We have studied 1) the kinetics and the amplitude of the apoptotic response to H2O2 and 2) the relationship between the extent of the apoptosis-inducing effect of H2O2, the H2O2 degradation activity, and the level of expression of apoptosis regulatory proteins, Bcl-2 and Bax, in pig thyrocytes in primary culture. Cells were seeded at high density to obtain confluent monolayers and were cultured in the presence of TSH to maintain the expression of differentiation. H2O2 (10–300 µM) induced the appearance of cells with fragmented DNA (terminal transferase deoxy-UTP-fluorescein isothiocyanate nick end labeling-positive cells) at a maximum of 3–4 h after H2O2 addition and then the detachment of apoptotic cells from the cell monolayer. The proportion of detached cells increased with H2O2 concentration and amounted to up to 30% of the initial cell number after 24 h. The transient effect of H2O2 was related to its rapid degradation by cells and culture medium components (rate constant, ~0.1 min-1). Iterative additions of H2O2 produced cumulative apoptotic waves. The amplitude of the apoptotic response of thyrocytes to H2O2 progressively increased with the time of culture, up to 4-fold from days 1–8. This was not related to a change in the capacity of thyrocytes to degrade H2O2. During the same period of culture, the Bcl-2 cell content progressively decreased, whereas that of Bax concomitantly increased; thus, the Bcl-2/Bax ratio varied from about 6 on day 1 to 0.5 on day 10. These data show that the susceptibility of thyrocytes to undergo apoptosis increases with the time of culture and that the pronounced changes in the apoptotic status of thyrocytes might be linked to coordinate modifications of the level of expression of pro- and antiapoptotic regulatory proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS or programmed cell death serves many physiological and homeostatic functions, such as the reduction of cell number and the removal of unwanted, damaged, or potentially dangerous cells (1). Inappropriate activation or inhibition of apoptosis may cause or contribute to a variety of diseases. Many different signals originating from either within or outside the cells have been shown to influence the decision between life and death by apoptosis (2); these include extracellular survival factors, cell interactions, genotoxic agents, and oxidative damage.

The thyroid gland has a particular status toward apoptosis. Thyroid epithelial cells or thyrocytes are constantly subjected to the actions of reactive oxygen species that are potent inducers of apoptosis (3). Indeed, thyrocytes produce high amounts of hydrogen peroxide (H2O2) (4, 5, 6), which acts as an electron acceptor in the course of the oxidative reactions leading to the iodination of tyrosyl residues and coupling of iodinated tyrosines within thyroglobulin, the thyroid hormone precursor protein (7). Tight control mechanisms of H2O2 generation, utilization, and degradation should exist in the thyroid gland to protect thyrocytes against apoptosis.

H2O2 has been reported to induce apoptosis in a large variety of cell types (8, 9, 10, 11, 12, 13), including thyrocytes (14). However, the mechanisms by which H2O2 triggers apoptosis are not clearly elucidated. H2O2, while relatively inactive by itself, might be converted into highly reactive hydroxyl radical (OH•) by metal ions through Fenton or Haber-Weiss reactions (15, 16). There is substantial evidence that reactive oxygen species can activate the cell death program (3); however, above a certain level, reactive oxygen species have been shown to exert toxic actions through DNA damage and protein and lipid peroxidation, leading to massive cell alterations and cell death by necrosis, also termed accidental cell death (9, 17, 18). Experimental conditions that have been used to disclose the apoptosis-inducing effect of H2O2 appear strikingly heterogeneous. H2O2 concentrations varying from 10 µM to up to 8 mM (17, 19, 20, 21, 22, 23) have been reported. The duration of cell treatment as well as the time of assessment of cells undergoing apoptosis after H2O2 addition are also highly variable. As H2O2 is potentially unstable and subjected to degradation by cellular enzymes, the time course and amplitude of its action might depend on its disappearance rate. To properly document the apoptotic response of thyrocytes to an exposure to H2O2, we have studied the kinetics and the amplitude of the apoptosis-inducing effect of exogenous H2O2 in relation to its rate of degradation. The data we obtained led us to examine the relationship between the extent of the apoptotic response of thyrocytes to H2O2 and the H2O2 degradation activity of thyrocytes, on the one hand, and the level of expression of apoptosis regulatory proteins, Bcl-2 and Bax, on the other hand. Experiments have been performed on pig thyrocytes in primary culture that were 1) seeded at high density to obtain confluent monolayers (and inhibit cell proliferation) and 2) cultured in the presence of TSH to maintain the expression of thyroid cell differentiation (24). We report that 1) H2O2 caused transient apoptotic waves, the length of which appeared to be related to H2O2 catabolism; and 2) the amplitude of the apoptotic response of thyrocytes to H2O2 varied with the time of culture and in relation to the level of expression of apoptosis regulatory proteins of the Bcl-2 family.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of pig thyrocytes
Thyroid glands from adult pigs were obtained from the local slaughterhouse and processed within 1 h of death. Dispersed thyrocytes were prepared by a discontinuous trypsinization as previously described (25). The dissociation procedure results in a cell suspension composed of isolated cells and groups of few cells. Cells were extensively washed in Ham’s F-12 medium and then resuspended in the same medium containing 5% calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and amphotericin B (0.25 µg/ml). Pig thyrocytes were seeded in petri dishes (Falcon, Oxnard, CA) at a density of 0.5 x 106 cells/cm2 and cultured under a 95% air-5% CO2 atmosphere for 1–10 days. Bovine TSH (Sigma Chemical Co.), at a concentration of 1 mU/ml, was added 24 h after seeding and was maintained throughout the period of culture. The culture medium was changed every other day.

In situ detection and quantification of apoptotic cells by the TUNEL [terminal transferase deoxy (d)-UTP-fluorescein isothiocyanate (FITC) nick end labeling] method (26)
Cells attached to petri dishes (monolayer cells) and cells collected from the culture supernatant (cells that detached from petri dishes) and spread on glass slides by cytocentrifugation were fixed in 4% (wt/vol) paraformaldehyde in PBS, pH 7.4, for 20 min and permeabilized in 0.25% (vol/vol) Triton X-100 in PBS for 20 min at room temperature. Cells with fragmented nuclear DNA were detected using terminal deoxynucleotidyl transferase (0.5 U/ µl) and FITC-labeled dUTP (0.5 nmol/ µl) from Boehringer Mannheim (Mannheim, Germany); incubations were performed according to the manufacturer’s instructions. Fixed cells were then incubated with Hoechst 33258 reagent (2 µg/ml; Molecular Probes, Inc., Eugene, OR) for 10 min at room temperature to identify all nuclei. FITC-dUTP and Hoechst fluorescence were detected using the following filter combinations (excitation-emission): BP 360–380/LP470 for Hoechst and BP 450–490/LP 520 for FITC, installed on an Axiophot fluorescence microscope from Zeiss (Carl Zeiss, Inc., Oberkochen, Germany). The proportion of apoptotic cells was determined by dividing the number of cells with a TUNEL-positive nucleus, measured on 10–20 randomly taken fields, by the total number of cells (Hoechst-labeled nuclei) in the corresponding fields.

In some experiments, an additional labeling step was carried out. Before paraformaldehyde fixation, living cells were incubated with propidium iodide (1 µg/ml) for 15 min. Propidium iodide that does not cross lipid bilayers was used to identify cells with altered plasma membrane permeability properties.

Measurement of H2O2 degradation
H2O2 was measured using the scopoletin fluorescence assay (27). Scopoletin gives a fluorescence emission at 460 nm when excited at 350–400 nm. In the presence of H2O2, scopoletin is oxidized by horseradish peroxidase and converted into a nonfluorescent compound. The decrease in scopoletin fluorescence is directly proportional to H2O2 concentration. After addition of H2O2 to the culture medium (in the presence or absence of thyrocytes), samples of 30–100 µl were sequentially collected over a period of up to 60 min and immediately added to test tubes containing 53 µM scopoletin and 3.6 µg/ml horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) in 50 mM phosphate, pH 7.2. After 1 min at room temperature, scopoletin fluorescence was measured, and results were converted into H2O2 concentration values using a standard curve generated with a H2O2 concentration ranging from 0.1–2 µM.

Western blot analysis
Cells were collected by scraping in PBS containing aprotinin, leupeptin, and pepstatin (each at a concentration of 1 µg/ml) and were lysed by sonication for 20 s at 25 watts using the vibra-cell apparatus from Bioblock Scientific (Illkirch, France). The protein concentration was assayed by the Lowry method after solubilization in 0.1% desoxycholate. Proteins from total cell extracts (10 µg) were fractionated on 12% polyacrylamide slab minigels and electrotransferred onto Immobilon-P membrane from Millipore Corp. (Bedford, MA). Membranes were preincubated in PBS containing 0.2% (vol/vol) Tween-20 and 5% (wt/vol) low fat milk powder for 30 min and then incubated with either a monoclonal mouse antihuman Bcl-2 antibody (1:1000; clone 124, DAKO Corp., Copenhagen, Denmark) or polyclonal rabbit antihuman Bax antibodies (1:1000; catalogue no. sc 493, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature. Detection of immune complexes was performed using horseradish peroxidase-conjugated goat antimouse Ig antibodies or horseradish peroxidase-conjugated goat antirabbit Ig antibodies from Bio-Rad Laboratories, Inc. (Richmond, CA). After 1-h incubation at room temperature, horseradish peroxidase activity was detected using an enhanced chemiluminescence detection procedure with the ECL kit from Covalab (Lyon, France) and exposure to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY). Quantification of the intensity of the labeled spots was performed by densitometry.

Assay of DNA
Cells were scraped from the dishes in 100 mM Tris, 10 mM EDTA, and 3 M NaCl, pH 7.4, and lysed by sonication as mentioned above. The DNA assay was performed using the fluorometric method described by Labarca and Paigen (28) with Hoechst 33258 reagent and salmon sperm DNA to generate the standard curve. Cell number was deduced from DNA measurements considering that 106 cells contain 10 µg DNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetic and amplitude of H2O2-induced apoptosis of thyrocytes in primary culture
Addition of H2O2 into the culture medium of pig thyrocytes induced within 1 h a significant rise in the number of cells exhibiting signs of apoptosis, i.e. TUNEL-positive but propidium iodide-negative cells. The number of apoptotic cells within the monolayer continued to increase to reach a maximum at 3–4 h (Fig. 1AGo); 3 h after the addition of 300 µM H2O2, the proportion of TUNEL-positive cells was 6- to 8-fold higher than that observed in control conditions. From that time on, the proportion of TUNEL-positive cells progressively declined and reached the basal or control value after 24 h. The basal value corresponds to the proportion of TUNEL-positive cells (between 1–4/1000 cells) that was constantly found in control culture conditions. Monolayer cells becoming TUNEL positive after exposure to H2O2 were always propidium iodide negative. Cells undergoing apoptosis within the monolayer subsequently detached from the culture dish. In control culture conditions (untreated thyrocytes), there was no statistically significant change in the number of adherent cells over a period of 24 h. In response to H2O2, there was a progressive reduction of the adherent cell number; exposure of thyrocytes (cultured for 3 days) to 300 µM H2O2 led to a 15% loss of adherent cells within the following 24 h (Fig. 1AGo). The apoptotic features of cells that detached from the culture substratum are shown in Fig. 2Go. TUNEL and Hoechst double labeling of cellular material collected (by cytocentrifugation) from the culture supernatant of thyrocytes exposed to H2O2 showed that 70–80% of Hoechst-labeled nuclei were TUNEL positive. Double labeled structures of small size were also detected; they could represent nuclear fragments within apoptotic bodies. About 20% of floating cells corresponded to apparently normal cells.



View larger version (8K):
[in this window]
[in a new window]
  		Figure 1. Characteristics of the apoptotic response of thyrocytes to H2O2. A and B, Time course of action of H2O2 (A) and cycloheximide, CHX (B) as inducers of apoptosis. Pig thyrocytes cultured for 3 days in Ham’s F-12 medium plus 5% serum plus TSH (1 mU/ml) were exposed to 300 µM H2O2 or 1 µg/ml CHX. At the indicated times (from 1–24 h) cells were fixed and double labeled with FITC-dUTP (TUNEL labeling procedure) and Hoechst 33258 reagent. The proportion of TUNEL-positive cells ({triangleup} and {blacktriangleup}), i.e. apoptotic cells in the monolayer, was determined by dividing the number of FITC-labeled cells by the total number of cells (i.e. Hoechst-labeled cells) counted on 10–20 randomly taken fields (each containing between 200–300 cells). Adherent cells from petri dishes, run in parallel, were collected and quantified by DNA assay ({circ} and •). Open symbols, Untreated cells; closed symbols, cells exposed to H2O2 or CHX. C and D, Effects of increasing concentrations of H2O2 on the proportion of TUNEL-positive cells among the adherent cell population 3 h after the addition of H2O2 (C) and on the proportion of detached cells 24 h after H2O2 addition (D). The proportion of detached cells (expressed as a percentage of the initial cell number) was calculated as follows: initial - final adherent cell number/initial cell number x 100. Other experimental conditions were the same as in A and B.

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Double fluorescence labeling of thyrocytes detached from the cell monolayer in response to H2O2. Thyrocytes cultured for 3 days were exposed to 300 µM H2O2. Three hours after H2O2 addition, the medium was changed and replaced by fresh medium. Cells that detached from the monolayer during the following 3 h were collected by centrifugation, resuspended in calf serum, spread on glass slides by cytocentrifugation, and subjected to FITC-dUTP (TUNEL method) and Hoechst labeling steps. A, FITC fluorescence; B, Hoechst fluorescence. By comparing A and B, it can be seen that most cells are double labeled and thus correspond to apoptotic cells. The Hoechst fluorescence of apoptotic nuclei appears more intense than that of the nucleus of apparently normal (TUNEL-negative) cells.

 
The apoptosis-inducing effects of increasing concentrations of H2O2 were analyzed using two different parameters: 1) the proportion of TUNEL-positive cells 3 h after H2O2 addition (Fig. 1CGo), and 2) the proportion of detached cells after 24 h (Fig. 1DGo). The proportion of detached cells was determined as the difference between the initial (0 h) and the final (24 h) number of adherent cells (quantified by DNA assay) expressed as a percentage of the initial number of adherent cells. The higher the concentration of H2O2 (from 10–300 µM), the higher the number of cells undergoing apoptosis. However, at a concentration exceeding 300 µM, H2O2 exerted toxic effects, leading to a rapid and massive detachment of cells from the substratum that was not related to apoptosis.

The time course of H2O2-induced apoptosis markedly differed from that of cycloheximide (CHX)-induced apoptosis (Fig. 1BGo). CHX (1 µg/ml) induced a progressive augmentation of the fraction of TUNEL-positive cells over a 24-h period. This gradual commitment of thyrocytes into apoptosis was accompanied by a progressive reduction of the adherent cell population, amounting to about 8% after 24 h. This observation led us to consider that the transient action of H2O2 could be linked to its degradation rate. Figure 3Go shows that H2O2, added to thyrocytes (cultured for 3 days) in the complete culture medium, was degraded with first order kinetics; the disappearance rate constant was 0.1 min-1. Similar values were obtained when the starting H2O2 concentration varied from 50–300 µM. In the absence of cells, i.e. in the complete culture medium only (Ham’s F-12 medium and serum), H2O2 depletion was only partially slowed down; the rate constant was about 0.075 min-1. It appeared that both basal medium components and serum contributed to H2O2 degradation; however, components of Ham’s F-12 medium were more potent than serum, as the H2O2 depletion rate constant in serum-free medium was only 30% lower than that observed in complete medium (Fig. 3Go). To have access to the capacity of thyrocytes to degrade H2O2, we looked for a medium in which H2O2 was stable; PBS was found to fulfill this requirement (Fig. 3Go). In PBS, H2O2 was degraded by thyrocytes with first order kinetics; the rate constant was about 0.05 min-1. These data indicate that under the conditions used to analyze H2O2-induced apoptosis, H2O2 was disappearing from the culture medium with a half-life of 6–7 min. Thus, when the starting H2O2 concentrations were 100 and 300 µM, the time of exposure of thyrocytes to a concentration of H2O2 higher than a threshold concentration of 10 µM (the lower H2O2 efficient concentration) did not exceed 25 and 30 min, respectively.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 3. Kinetics of H2O2 depletion from the culture medium in the absence or presence of thyrocytes. Empty petri dishes (6 cm in diameter) or petri dishes in which thyrocytes had been cultured for 3 days were filled either with 4 ml fresh culture medium (CM) supplemented or not with 5% calf serum (S) or with 4 ml PBS. Temperature was equilibrated at 4 or 37 C, and H2O2 was introduced at a final concentration of 100 µM (time zero). At different time intervals, 30- to 100-µl aliquots were taken for H2O2 assay by the scopoletin fluorescence method as described in Materials and Methods. H2O2 concentration values were determined using a standard curve. Experimental conditions corresponding to each decay curve are indicated in the figure. Symbols and vertical bars represent the mean and SEM of three to five determinations; when not apparent, error bars were included in the size of the symbol.

 
To further document the relationship between the kinetics of the apoptotic response to H2O2 and H2O2 catabolism, we analyzed the time course of apoptosis of thyrocytes after repeated additions of H2O2. Results are reported in Fig. 4Go. H2O2 (300 µM), added at 3-h intervals, exerted cumulative effects. The number of TUNEL-positive cells increased to about the same extent 3 h after each H2O2 addition. These data show that the time course of action of H2O2, as an apoptotic inducer, is linked to its degradation rate.



View larger version (8K):
[in this window]
[in a new window]
  		Figure 4. Apoptosis of thyrocytes in response to repeated additions of H2O2. Thyrocytes cultured for 3 days were exposed either once (at 0 h), twice (at 0 and 3 h), or three times (at 0, 3, and 6 h) to 300 µM H2O2 as indicated by arrows. The proportion of TUNEL-positive cells was determined as described in Fig. 1Go. Measurements were performed at 0 h (untreated cells), at 3, 6, and 9 h when cells were exposed once to H2O2, at 6 and 9 h when cells were exposed twice to H2O2 (0 and 3 h), and at 9 h when cells were exposed three times to H2O2 (0, 3, and 6 h). Symbols and vertical bars represent the mean and SEM of triplicate determinations.

 
Changes in the susceptibility of thyrocytes in primary culture to undergo apoptosis after exposure to H2O2
In the course of the above-reported studies that were performed on thyrocytes cultured for 3–4 days, the apoptosis-inducing effects of H2O2 were sometimes measured on thyrocytes maintained in culture for longer periods of time. As the apoptotic response to H2O2 was always significantly different, we decided to determine how the time of culture could influence the susceptibility of thyrocytes to undergo apoptosis. The results of representative experiments are reported in Fig. 5Go. H2O2 concentration-apoptotic response (appearance of TUNEL-positive cells) curves gradually shifted to the left (toward low H2O2 concentrations) when thyrocytes were cultured for increasing periods of time. The curves in Fig. 5AGo show a 5- to 10-fold increase in the sensitivity of thyrocytes to H2O2 between days 3 and 7. Furthermore, the toxicity threshold was lowered; 300 µM H2O2 rapidly altered the cell integrity of 7-day cultured thyrocytes, whereas the same toxic effect was only obtained at 1 mM H2O2 in 3-day cultured thyrocytes. The amplitude of the apoptotic response to H2O2, assessed by the reduction of the adherent cell population, also varied as a function of the time of culture (Fig. 5BGo). The apoptosis-dependent cell loss (detachment from culture dishes) 24 h after an exposure to 30 µM H2O2 varied from about 10% on day 2 to more than 30% on day 8. Concurrently, there was a time-dependent enhancement of spontaneous apoptosis. This was associated with a significant reduction in the number of monolayer cells over the 8-day culture period under regular conditions; the number of adherent cells on day 8 represented 75–80% of the initial number of adherent cells (measured on day 1).



View larger version (9K):
[in this window]
[in a new window]
  		Figure 5. Influence of the time of culture on the susceptibility of thyrocytes to undergo apoptosis in response to H2O2. A, Changes in the apoptotic response of thyrocytes cultured for 3 or 7 days to increasing concentrations of H2O2. The proportion of TUNEL-positive cells was measured 3 h after H2O2 addition. Symbols and vertical bars represent the mean and SEM of values from three dishes. The dotted lines indicate H2O2 toxicity leading to massive cell detachment and cell death. B, Changes in the amplitude of H2O2-induced apoptosis of thyrocytes cultured for increasing periods of time. Thyrocytes cultured for 1, 4, or 7 days were exposed, or not, to 300 µM H2O2. Twenty-four hours later (on days 2, 5, and 8, respectively), the proportion of detached cells was determined as indicated in Fig. 1Go. Symbols and vertical bars represent the mean and SEM of triplicate determinations in a representative experiment.

 
To test whether the increase in the susceptibility of thyrocytes to undergo apoptosis in response to H2O2 was related to a decrease in the antioxidant cell functions, we measured the capacity of thyrocytes cultured for up to 7 days to degrade H2O2. Measurements of H2O2 degradation were performed using the conditions described in Fig. 3Go, i.e. cells in PBS exposed to 100 µM H2O2. The H2O2 degradation activity of thyrocytes calculated from the values of H2O2 disappearance rates were 1.8 ± 0.2, 2.1 ± 0.1, and 2.3 ± 0.2 nmol/min·106 cells on days 2, 4, and 7, respectively (mean ± SEM; n = 3). Thus, the capacity of thyrocytes to degrade H2O2 remained constant or slightly increased with time.

The changes in the apoptotic status of differentiated thyrocytes maintained in primary culture were also evidenced using another apoptosis inducer, CHX. The proportion of detached cells, determined 24 h after addition of CHX (1 µg/ml), increased from about 10% on day 2 to 32% on day 5 and 47% on day 8 (average values obtained from three independent cell cultures).

Expression of anti- and proapoptosis regulatory proteins by thyrocytes in primary culture
A family of homologous proteins, the Bcl-2 family, endowed with anti- or proapoptotic functions, are known to control or determine the susceptibility to apoptosis in many cell types (29, 30). We tried to identify members of this protein family in pig thyrocytes. Experiments were performed by Western blot using specific antibodies directed against either the antiapoptotic proteins, Bcl-2 and Bcl-Xl, or the proapoptotic protein, Bax. A 26-kDa species corresponding to Bcl-2 was detected in the 100,000 x g membrane fraction of thyrocytes (Fig. 6Go). In contrast, Bax protein migrating as a 21-kDa band was only found in the cytosolic fraction. Antibodies to Bcl-X (large and small forms) barely labeled a 26- to 29-kDa component that probably corresponded to Bcl-Xl, but did not detect Bcl-Xs with an expected molecular mass of 18 kDa (data not shown). The search for potential variations in the expression of apoptosis regulatory proteins was restricted to Bcl-2 and Bax. In thyrocytes freshly isolated from pig thyroid glands, the intensity of the Bcl-2 band was always higher than that of Bax. The apparent Bcl-2 and Bax cell contents were unchanged after 1 day of culture, but were subjected to large variations over the following days of culture (Fig. 7AGo). Bcl-2 progressively decreased to reach a value representing 40% of the initial level after 10 days. On the contrary, Bax cell content gradually increased to up to 6-fold on day 10. Thus, the Bcl-2/Bax ratio shifted from a value of 6 to about 0.5 within a 10-day period of culture (Fig. 7BGo).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Identification of Bcl-2 and Bax in pig thyroid cell extracts. Freshly dispersed thyrocytes were resuspended in PBS containing aprotinin, leupeptin, and pepstatin (each at a concentration of 1 µg/ml) and lysed by sonication. Samples of 10 µg protein from total cell extracts (T) and from particulate (P) and soluble (S) fractions resulting from a 100,000 x g centrifugation were analyzed by Western blot using either a monoclonal anti-Bcl-2 antibody (left panel) or polyclonal anti-Bax antibodies (right panel). Immune complexes were visualized using goat antimouse Ig or goat antirabbit Ig antibodies conjugated to horseradish peroxidase. The enzyme activity was detected by chemiluminescence. It must be noted that the intensity of the Bcl-2 and Bax bands cannot be directly compared. Indeed, as the Bax signal was less intense, the time of exposure of the film to the photon emission was longer.

 


View larger version (9K):
[in this window]
[in a new window]
  		Figure 7. Expression of Bcl-2 and Bax by pig thyrocytes in primary culture. Thyrocytes cultured for 1–10 days were collected, and 10 µg protein from total cell extracts were subjected to Western blot using anti-Bcl-2 or anti-Bax antibodies and the chemiluminescence detection procedure described in Fig. 6Go. The intensity of the Western blot signals (presented in the top panel) was quantified by densitometry, and values were used to calculate the Bcl-2/Bax ratio (bottom panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of double or triple fluorescence labeling of nuclear DNA of individual cells together with DNA assay of cell populations have allowed a detailed examination of the susceptibility of pig thyrocytes in primary culture to undergo apoptosis. Studies were performed on nonproliferating differentiated thyrocytes. First, cells were seeded at high density to obtain confluent monolayers and to prevent cell multiplication and apoptosis due to aborted entry into the cell cycle. It was verified that the proportion of labeled cells, after a 12-h incubation in the presence of bromodeoxyuridine on day 4, was negligible (<1 of 105). Second, after cell attachment to the dish, TSH was added to the culture medium to maintain the expression of differentiation and thus to avoid decay of cellular metabolism that could lead to alterations of cell defenses. In the presence of TSH, thyrocytes keep the iodide uptake activity and continue to express thyroperoxidase and thyroglobulin (24). Under these defined conditions, the proportion of cells exhibiting signs of apoptosis at any moment throughout the time frame of the experiments (7–8 days) and without addition of any apoptosis inducer remained low (from 0.1–0.5%). However, the fraction of apoptotic cells tended to increase at the later time of culture. This basal apoptotic rate probably accounts for the 20–25% reduction of the cell population over the 7- to 8-day culture period.

Pig thyrocytes constitutively expressed the proteins required to execute apoptosis as inhibition of protein synthesis by cycloheximide was followed by a rapid (within 3 h) increase in the proportion of cells undergoing apoptosis. This phenomenon has already been found in many cell types (31), including dog thyrocytes (32); in this latter study, however, cycloheximide-induced effects were observed after a 5-day treatment. It is thus probable that under normal culture conditions, intracellular factors prevent activation of proteins involved in executing the death program (33). In addition, extracellular signals and/or serum factors such as insulin-like growth factor I or platelet-derived growth factor, known to inhibit or prevent apoptosis (34), might also act as survival factors.

The oxidative stress generated by H2O2 addition to the culture medium induced a rapid, but transient, apoptotic response of thyrocytes. We show that the time course of effect of H2O2 is related to its short life-time, H2O2 is degraded by both cells and culture medium components. H2O2 that diffuses through lipid bilayers is expected to freely enter the cells. Once in the cytoplasm, two general enzyme systems, glutathione peroxidase and catalase, rapidly catabolize H2O2. The former enzyme has a high affinity for H2O2, whereas the latter becomes increasingly effective in degrading H2O2 with increasing H2O2 concentrations. Degradation of H2O2 by the culture medium components is less understood. Many components present in the culture medium, such as metal ions, Mn2+, Mg2+, Zn2+, and vitamins (35) as well as phenol red, through their antioxidant activity probably participate in H2O2 disappearance. The contribution of serum to the decomposition of H2O2 could be due to serum proteins themselves serving as oxidation substrates or to specific proteins playing the role of antioxidant, such as ceruloplasmin and transferrin (35). The instability of H2O2 in culture media has rarely been taken into account in studies using H2O2 as an inducer of apoptosis. Indeed, it is commonly reported that cells are treated with H2O2 for 24 h or more. In light of the present data, one can assume that in many reports, the real time of exposure of cells to H2O2 was far less than that announced and that analyses of the apoptotic response to H2O2 were most likely performed at times distant from the maximum of action of H2O2. From the kinetic data in Fig. 3Go, we estimated that the actual exposure time of thyrocytes to the oxidative stress generated by H2O2 was on the order of few minutes to 30 min (depending on the initial H2O2 concentration). The first cells committed to die in response to the application of an oxidative stress of that length appeared within 1 h. The apoptotic response of thyrocytes to H2O2 peaked after 3–4 h, but lasted 18–24 h; the proportion of TUNEL-positive cells, 18 h after H2O2 addition, was still significantly higher than that in controls. This observation is in keeping with previous data showing that apoptosis in an asynchronous process (36). The duration of the condemned phase of apoptosis, i.e. the delay between apoptosis induction by the oxidative stress and the entry in the active or execution phase of apoptosis, is highly variable. This asynchronous nature of cell death has also been observed in cells that were synchronized following mitosis (36) and in cells subjected to other proapoptotic culture conditions, such as deprivation of a growth factor (37).

Only a fraction of the cell population underwent apoptosis in response to H2O2. Within 24 h after exposure to 0.3–1 mM H2O2 (the highest concentration that did not cause immediate cell injury), cells committed to die amounted to about 30% of the total cell population. This might indicate that thyrocytes are not equally responsive or sensitive to reactive oxygen species or that a large proportion of cells have the capacity to overcome the action of the oxidative stress and/or to block apoptosis in its initiation or induction phase. The fact that repetitive oxidative stresses (see Fig. 4Go) caused successive waves of apoptosis would be in favor of the first hypothesis. As the proportion of cells undergoing apoptosis increased with the H2O2 concentration, it is reasonable to believe that the level of damage could play a key role in the determination of the fraction of cells committed to die. It is difficult to appreciate whether a dysregulation of the production, utilization, or degradation of H2O2 by thyrocytes in vivo could lead to a H2O2 concentration susceptible to induce apoptosis. Indeed, the H2O2 degradation activity of thyrocytes, varying from 2–5 nmol/min·106 cells (present data and Refs. 4, 38), appears 10–20 times higher than the thyrocyte H2O2 production rate, which is on the order of 0.2 nmol/min·106 cells (4, 5). Only particular pathophysiological situations should lead to a reversal of this production/degradation imbalance and create an oxidative stress triggering apoptosis.

Thyrocytes entering the active phase of apoptosis presented the biochemical and morphological changes now recognized for adherent cultured cells (39, 40, 41, 42, 43, 44). TUNEL-positive and propidium iodide-negative thyrocytes (corresponding to cells with fragmented DNA and intact membrane permeability properties) detected within the monolayer subsequently detached from the culture dish. The large majority of cells appearing in the culture medium represented apoptotic cells with late apoptotic features, including nuclear fragmentation and formation of apoptotic bodies. The presence of apparently normal cells (TUNEL-negative cells) into the floating cell population might indicate that cells undergoing apoptosis take along adjacent cells in the course of detachment from the cell monolayer. Apoptotic cells probably do not accumulate in the culture medium because degradation processes are continuing up to complete cell destruction; this probably leads to an overrating of the actual proportion of normal cells within the floating cell population and the quantification of apoptosis by numbering floating cells. By contrast, precise measurements of the decrease in the adherent cell population by DNA assay give a reasonable estimate of the proportion of cells that have undergone apoptosis. Assessment of apoptosis by quantifying cell detachment has been used for many other epithelial cells, such as hepatocytes (44) and mammary cells (41), and for nonepithelial cells, fibroblasts (39), myoblasts (42), and endothelial cells (43).

Depending on the time of culture, thyrocytes differently responded to a H2O2-generated oxidative stress. The longer the time of culture, the greater the susceptibility of thyrocytes to undergo apoptosis in response to a given H2O2 concentration. Concordant data were obtained from 1) measurements (by the TUNEL method) of the proportion of adherent cells entering apoptosis 3 h after H2O2 addition and 2) measurements of the fraction of the cell population that underwent apoptosis during the 24-h period following oxidative stress. Contrary to what was reported in other cell systems (21, 45), the changes in the apoptotic response of thyrocytes to H2O2 do not correlate with alterations in the level of H2O2 degradation enzyme activity. The fact that thyrocytes cultured for increasing periods of time were also becoming more prone to undergo apoptosis in response to another inducer, cycloheximide, led us to think that the modifications of the apoptotic status of thyrocytes might be related to changes in the overall regulatory mechanisms of apoptosis.

Within the complex network of proteins regulating apoptosis, proteins of the Bcl-2 family are recognized to play a central role (29, 30). Members within this wide family have been classified into prosurvival or antiapoptotic proteins, Bcl-2 and the Bcl-2 cohort, and proapoptotic proteins, Bax and structurally related members (reviewed in Ref. 46). Bcl-2 and Bax are expressed by most epithelial cells, including thyroid cells (47, 48, 49). Bcl-2 was easily detected in freshly dispersed pig thyrocytes, whereas Bax was present in lower amounts. In agreement with a recent report (50), Bax was soluble, and Bcl-2 was in a membrane-bound form. Analyses of the level of expression of these two proteins during a 10-day period of primary culture revealed time-dependent changes that correlated with the changes in the susceptibility of thyrocytes to undergo apoptosis (either spontaneously or in response to H2O2 or cycloheximide). The highest levels of Bcl-2 were observed during the early times of culture when cells had a low propensity to undergo apoptosis; this is in accordance with the known role of Bcl-2 in protecting cells from apoptosis in the face of a wide variety of cytotoxic insults (46). The progressive down-regulation of Bcl-2 and the increased expression of Bax (leading to a 10-fold reduction of the Bcl-2/Bax ratio) during the 8- to 10-day period of culture could account for the variations in the apoptotic status of thyrocytes. Indeed, as Bax is known to heterodimerize with Bcl-2 and to inhibit its prosurvival function (29), both the decrease in Bcl-2 and the increase in Bax might contribute to the progressive sensitization of thyrocytes toward inducers of apoptosis. Our data further document the concept that the relative concentrations of anti- and proapoptotic proteins may act as a rheostat for the cell suicide program (29).

To our knowledge, such variations in the level of expression of proteins regulating apoptosis have not been found in cells in primary culture. Modifications of the expression of Bcl-2 and/or Bax have been, mostly if not exclusively, reported in tumor cell lines, and when observed, changes in the Bcl-2/Bax ratio are less pronounced and generally related to variations in the expression of either Bcl-2 or Bax. Studies are in progress to try to identify 1) a medium component(s) or serum factor(s) responsible for the changes in the apoptotic status of thyrocytes in primary culture and 2) a factor(s) or signal(s) that might alter the apoptotic status and/or the Bcl-2 and Bax expression pattern of thyrocytes. These studies could provide information on how the expression of cell survival proteins of the Bcl-2 family is regulated in glandular epithelium.


   Footnotes
 
1 This work was supported in part by Grant 97020641 from Région Rhône-Alpes (France). Back

Received November 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Granville DL, Carthy CM, Hunt DWC, McManus BM 1998 Apoptosis: molecular aspects of cell death and disease. Lab Invest 78:893–913[Medline]
  2. Raff M 1998 Cell suicide for beginners. Nature 369:119–122
  3. Jacobson MD 1996 Reactive oxygen species and programmed cell death. Trends Biochem Sci 21:83–86[CrossRef][Medline]
  4. Corvilain B, Van Sande J, Laurent E, Dumont JE 1991 The H2O2-generating system modulates protein iodination and the activity of the pentose phosphate pathway in dog thyroid. Endocrinology 128:779–785[Abstract]
  5. Björkman U, Ekholm R 1992 Hydrogen peroxide generation and its regulation in FRTL-5 and porcine thyroid cells. Endocrinology 130:393–399[Abstract]
  6. Raspé E, Dumont J 1995 Tonic modulation of dog thyrocyte H2O2 generation and I- uptake by thyrotropin through the cyclic adenosine 3',5'-monophosphate cascade. Endocrinology 135:965–973
  7. Nunez J, Pommier J 1982 Formation of thyroid hormones. Vitam Horm 39:175–229[Medline]
  8. McConkey DJ, Hartzell P, Nicotera P, Wyllie AH, Orrenius S 1988 Stimulation of exogenous endonuclease activity in hepatocytes exposed to oxidative stress. Toxicol Lett 42:123–130[CrossRef][Medline]
  9. Lennon SV, Martin SJ, Cotter TG 1991 Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif 24:203–204[Medline]
  10. Ueda N, Shah SV 1992 Endonuclease-induced DNA dammage and cell death in oxidant injury to renal tubular cells. J Clin Invest 90:2593–2597
  11. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ 1993 Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251[CrossRef][Medline]
  12. Whittemore ER, Loo DT, Cotman CW 1994 Exposure of hydrogen peroxide induces cell death via apoptosis in cultured rat cortical neurons. NeuroReport 5:1485–1488[Medline]
  13. deBono DP, Yang WD 1995 Exposure to low concentrations of hydrogen peroxide causes delayed endothelial cell death and inhibits proliferation of surviving cells. Atherosclerosis 114:235–245[CrossRef][Medline]
  14. Riou C, Remy C, Rabilloud R, Rousset B, Fonlupt P 1998 H2O2 induces apoptosis of pig thyrocytes in primary culture. J Endocrinol 156:315–322[Abstract]
  15. Breen AP, Murphy JA 1995 Reactions of oxyl radicals with DNA. Free Radical Biol Med 18:1033–1077[CrossRef][Medline]
  16. Rosen GM, Pou S, Ramos CL, Cohen MS, Britigan BE 1995 Free radicals and phagocytic cells. FASEB J 9:200–209[Abstract]
  17. Sakamoto T, Repasky WT, Uchida K, Hirata A, Hirata F 1996 Modulation of cell death pathways to apoptosis and necrosis of H2O2-treated rat thymocytes by lipocortin I. Biochem Biophys Res Commun 220:643–647[CrossRef][Medline]
  18. Hampton MB, Orrenius S 1997 Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett 414:552–556[CrossRef][Medline]
  19. Sakagami H, Kuribayashi N, Iida M, Hagiwara T, Takahashi H, Yoshida H, Shiato F, Ohata H, Momose K, Takeda M 1996 The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death. Life Sci 58:1131–1138[CrossRef][Medline]
  20. Bladier C, Wolvetang EJ, Hutchinson P, de Haan JB, Kola I 1997 Response of a primary human fibroblast cell line to H2O2: senescence-like growth arrest or apoptosis? Cell Growth Differ 8:589–598[Abstract]
  21. Um HD, Orenstein JM, Wahl SM 1996 Fas-mediated apoptosis in human monocytes by reactive oxygen intermediate dependent pathway. J Immunol 156:3469–3477[Abstract]
  22. Satoh T, Sakai N, Enokido Y, Uchiyama Y, Hatanaka H 1996 Free radical-independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide-triggered apoptosis. J Biochem 120:540–546[Abstract/Free Full Text]
  23. Jiang MC, Yang-Yen HF, Lin JK, Yen J 1996 Differential regulation of p53, c-Myc, Bcl-2 and Bax protein expression during apoptosis induced by widely divergent stimuli in human hepatoblastoma cells. Oncogene 13:609–616[Medline]
  24. Paire A, Bernier-Valentin F, Rabilloud R, Watrin C, Selmi-Ruby S, Rousset B 1998 Expression of {alpha}- and ß-subunits and activity of Na+K+ATPase in pig thyroid cells in primary culture: modulation by thyrotropin and thyroid hormones. Mol Cell Endocrinol 146:93–101[CrossRef][Medline]
  25. Rousset B, Poncet C, Mornex R 1976 Evidence for a secretion of thyroxine by isolated hog thyroid cells. Biochim Biophys Acta 437:543–561[Medline]
  26. Gavrieli Y, Sherman Y, Ben-Sasson SA 1992 Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501[Abstract/Free Full Text]
  27. Michot JL, Deme D, Virion A, Pommier J 1985 Relation between thyroid peroxidase, H2O2 generating system and NADPH-dependent reductase activities in thyroid particular fractions. Mol Cell Endocrinol 41:211–221[CrossRef][Medline]
  28. Labarca C, Paigen K 1980 A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344–352[CrossRef][Medline]
  29. Oltvai Z, Milliman CL, Korsmeyer SJ 1993 Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619[CrossRef][Medline]
  30. Korsmeyer SJ, Yin XM, Oltvai ZN, Veis-Novack DJ, Linette GP 1995 Reactive oxygen species and the regulation of cell death by the Bcl-2 gene family. Biochim Biophys Acta 1271:63–66[Medline]
  31. Martin SJ 1993 Apoptosis: suicide, execution or murder? Trends Cell Biol 3:141–144
  32. Dremier S, Golstein J, Mosselmans R, Dumont JE, Galand P, Robaye B 1994 Apoptosis in dog thyroid cells. Biochem Biophys Res Commun 200:52–58[CrossRef][Medline]
  33. Thornberry NA, Lazebnik Y 1998 Caspases: enemies within. Science 281:1312–1316[Abstract/Free Full Text]
  34. Harrington EA, Bennett MR, Fanidi A, Evan GI 1994 cMyc-induced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO J 13:3286–3295[Medline]
  35. Berlett BS, Stadtman ER 1997 Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316[Free Full Text]
  36. Messam CA, Pittman RN 1998 Asynchrony and commitment to die during apoptosis. Exp Cell Res 238:389–398[CrossRef][Medline]
  37. Mills JC, Kim LH, Pittman RN 1997 Differentiation to an NGF-dependent state and apoptosis following NGF removal both occur asynchronously in cultures of PC12 cells. Exp Cell Res 231:337–345[CrossRef][Medline]
  38. Ekholm R, Björkman 1997 Glutathione peroxidase degrades intracellular hydrogen peroxide and thereby inhibits intracellular protein iodination in the thyroid epithelium. Endocrinology 138:2871–2878[Abstract/Free Full Text]
  39. Kulkarni GV, McCulloch CAG 1994 Serum deprivation induces apoptotic cell death in a subset of BALB/c 3T3 fibroblasts. J Cell Sci 107:1169–1179[Abstract]
  40. Simm A, Bertsch G, Frank H, Zimmermann U, Hoppe J 1997 Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis. J Cell Sci 110:819–828[Abstract]
  41. Pullan S, Wilson J, Metcalfe A, Edwards GM, Goberdhan N, Tilly J, Hickman JA, Dive C, Streuli CH 1996 Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium. J Cell Sci 109:631–642[Abstract/Free Full Text]
  42. Stewart CEH, Rotwein P 1996 Insulin-like growth factor-II is an autocrine survival factor for differentiated myoblasts. J Biol Chem 271:11330–11338[Abstract/Free Full Text]
  43. Zoellner H, Höfler M, Beckmann R, Hufnagl P, Vanyek E, Bielek E, Wojta J, Fabry A, Lockie S, Binder RB 1996 Serum albumin is a specific inhibitor of apoptosis in human endothelial cells. J Cell Sci 109:2571–2580[Abstract]
  44. Bayly AC, Roberts RA, Dive C 1994 Suppression of liver cell apoptosis in vitro by the non-genotoxic hepatocarcinogen and peroxisome proliferator nafenopin. J Cell Biol 125:197–203[Abstract/Free Full Text]
  45. Kim YK, Ko SH, Woo JS, Lee SH, Jung JS 1998 Difference in H2O2 toxicity between intact renal tubules and cultured proximal tubular cells. Biochem Pharmacol 56:489–495[CrossRef][Medline]
  46. Adams JM, Cory S 1998 The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326[Abstract/Free Full Text]
  47. Hockenbery DM, Zutter M, Hickey W, Nahm M, Korsmeyer SJ 1991 Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 88:6961–6965[Abstract/Free Full Text]
  48. Krajewski S, Krajewska M, Shabaik A, Miyashita T, Wang HG, Reed JC 1994 Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. Am J Pathol 145:1323–1336[Abstract]
  49. Branet F, Brousset P, Krajewski S, Schlaifer D, Selves J, Reed JC, Caron P 1996 Expression of the cell death-inducing gene Bax in carcinomas developed from the follicular cells of the thyroid gland. J Clin Endocrinol Metab 81:2726–2730[Abstract]
  50. Hsu YT, Youle RJ 1998 Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformation. J Biol Chem 273:10777–10783[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
Y. Song, N. Driessens, M. Costa, X. De Deken, V. Detours, B. Corvilain, C. Maenhaut, F. Miot, J. Van Sande, M.-C. Many, et al.
Roles of Hydrogen Peroxide in Thyroid Physiology and Disease
J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3764 - 3773.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. A. Gilbert, A. G. Gianoukakis, S. Salehi, J. Moorhead, P. V. Rao, M. Z. Khan, A. M. McGregor, T. J. Smith, and J. P. Banga
Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways.
J. Immunol., April 15, 2006; 176(8): 5084 - 5092.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
B. Corvilain, L. Collyn, J. van Sande, and J. E. Dumont
Stimulation by iodide of H2O2 generation in thyroid slices from several species
Am J Physiol Endocrinol Metab, April 1, 2000; 278(4): E692 - E699.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. De Deken, D. Wang, M.-C. Many, S. Costagliola, F. Libert, G. Vassart, J. E. Dumont, and F. Miot
Cloning of Two Human Thyroid cDNAs Encoding New Members of the NADPH Oxidase Family
J. Biol. Chem., July 21, 2000; 275(30): 23227 - 23233.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Kim, T.-H. Lee, E. S. Park, J. M. Suh, S. J. Park, H. K. Chung, O-Y. Kwon, Y. K. Kim, H. K. Ro, and M. Shong
Role of Peroxiredoxins in Regulating Intracellular Hydrogen Peroxide and Hydrogen Peroxide-induced Apoptosis in Thyroid Cells
J. Biol. Chem., June 9, 2000; 275(24): 18266 - 18270.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Riou, C.
Right arrow Articles by Rousset, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Riou, C.
Right arrow