Induction of thioredoxin, thioredoxin reductase and glutaredoxin activity in mouse skin by TPA, a calcium ionophore and other tumor promoters

Sushil Kumar and Arne Holmgren1

Department of Medical Biochemistry and Biophysics, Medical Nobel Institute for Biochemistry, Karolinska Institutet, S-171 77 Stockholm, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have measured the levels of thioredoxin, thioredoxin reductase and glutaredoxin enzyme activity in mouse skin following topical application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator and tumor promoter. The specific activity of thioredoxin and thioredoxin reductase in extracts from normal epidermis increased by 40 and 50%, respectively, after single or multiple application of TPA. Multiple applications (twice per week for 2 weeks) of TPA increased glutaredoxin activity by >300%. Induction of the proteins lasted several days. Other PKC activators, like 12-O-retinoylphorbol 13-acetate, mezerein, 1-oleoyl-2-acetylglycerol and the calcium ionophore A23187, also induced all the enzyme activities. Phorbol and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, weak activators of PKC, selectively induced the thioredoxin system only and did not influence glutaredoxin activity. Multiple applications of TPA to tumor initiated (7,12-dimethyl[a]benzanthracene-treated) skin resulted in elevated levels of both the thioredoxin and glutaredoxin systems when examined 6 days after the last phorbol ester treatment. Induction of thioredoxin, thioredoxin reductase and glutaredoxin activities by TPA and calcium ionophores may play a general role in the epigenetic mechanism of tumor promotion via thiol redox control mechanisms.

Abbreviations: ADF, adult human T cell leukemia-derived factor; DMBA, 7,12-dimethyl[a]benzanthracene; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); FA, fluocinolone acetonide; GSH, reduced glutathione; GSSG, oxidized glutathione; OAG, 1-oleoyl-2-acetylglycerol; 4-O-MeTPA, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; RA, retinoic acid; ROS, reactive oxygen species; RPA, 12-O-retinoylphorbol-13-acetate; TPA, 12-O-tetradecanoylphorbol-13-acetate; TPCK, tosylphenylalanine chloromethylketone; TRE, TPA-responsive element.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The initiation–promotion protocol of mouse skin tumorgenesis is a model to study the mechanism of chemical carcinogenesis (1). In this system, tumor initiation is performed with a single topical application of a carcinogen, at a sub-threshold dose (2). Initiated cells that do not differentiate (3,4) are stimulated to grow by the repeated application of a non-carcinogenic tumor promoter such as 12-O-tetradecanoylphorbol-13-acetate (TPA) (2). The mechanism by which TPA provides a selective growth and mitotic advantage to a tumor cell over a non-tumor cell is related to the expression of activated p21 and protein kinase C (PKC) (5). TPA activates PKC and stimulates differentiation in normal keratinocytes (6,7). Neoplastic keratinocytes carry a mutated c-Ha-ras gene and ignore the differentiation stimulus of TPA (3,5). This characteristic of the initiated cell is due to the expression of activated p21 plus aberrant activation of the PKC signalling pathway (8). Induced tyrosine phosphorylation and inhibition of calcium-independent PKC{delta} activity impart to c-Ha-ras mutated keratinocytes enhanced proliferative capacity and reduced sensitivity to the differentiation stimulus of TPA (810). It is thus proposed that tumor promotion results from selective clonal expansion of the differentiation-resistant tumor (initiated) phenotype following stimulated hyperproliferation of cells in the epidermis.

The mechanism of action of TPA is epigenetic and includes its interaction with the cell membrane followed by an altered program of gene expression and stimulation of cell differentiation and growth. The effects of TPA are remarkably pleiotropic and are explained by activation of the mitogen-responsive phospholipase C pathway (11), direct activation of PKC and intracellular mobilization of calcium (6), increased expression of the fos and jun family of proteins and subsequent binding of their dimerized protein product AP-1 to a TPA-responsive element (TRE) (12). Thus, transcription of genes encoding cell cycle-related, mitogen-responsive, structural and regulatory proteins is induced.

Some of the important biochemical responses activated by TPA, e.g. deoxyribonucleotide biosynthesis (13), DNA and protein synthesis (14), activation of enzymes like PKC (1517), ornithine decarboxylase (18,19) and transglutaminase (20) and binding of AP-1 with TRE (21,22) are thiol-dependent and therefore suggested to be under the control of the thioredoxin and glutaredoxin systems. However, it remains unknown if such redox regulatory systems (23) in vivo are influenced by TPA.

The thioredoxin system consists of the small protein thioredoxin (Mr 12 000), with a redox-active disulfide (-Cys-Gly-Pro-Cys-) that is reduced to a dithiol by NADPH, and the flavoprotein thioredoxin reductase (Mr 116 000 in mammalian cells) (24). The glutaredoxin system is composed of the small protein glutaredoxin (Mr 12 000), which also has a redox-active disulfide (Cys-Pro-Tyr-Cys) that is reduced to a dithiol by reduced glutathione (GSH) and NADPH plus glutathione reductase (25).

Since the thioredoxin and glutaredoxin systems operate as obligatory electron donors in both DNA synthesis and in redox regulation, we wanted to study expression of the proteins as influenced by TPA and other related growth-affecting compounds in an animal model. We selected the mouse skin model of carcinogenesis for the purpose.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
TPA, 7,12-dimethyl[a]benzanthracene (DMBA), phorbol, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (4-O-MeTPA), mezerein, 12-O-retinoylphorbol-13-acetate (RPA), the calcium ionophore A23187, NADPH, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), GSH, retinoic acid (RA), tosylphenylalanine chloromethylketone (TPCK) and fluocinolone acetonide (FA) were purchased from Sigma Chemical Co. (St Louis, MO). 2-Hydroxyethyldisulfide and insulin were procured from Aldrich-Europe (Belgium) and Nordisk Insulin A/S (Gentofte, Denmark), respectively. Glutathione reductase (yeast) was obtained from Boehringer (Mannheim, Germany). Thioredoxin and thioredoxin reductase were purified to homogeneity from calf thymus and Escherichia coli by methods described previously (26,27). All chemicals were of reagent grade.

Animals and treatment
Female NMRI albino mice of 6–8 weeks age were purchased from Anticimex AB (Stockholm, Sweden) and were kept in plastic cages with steel hoppers in temperature, light (12 h rhythm) and humidity controlled rooms. Only the animals with their hair cycle in the resting phase were used in this study. The animals were kept on a synthetic pellet diet and water ad libitum. The mice were shaved on a 2 cm2 area in the interscapular region and the treatment (200 nmol DMBA and/or 8 nmol TPA in 100 µl acetone to experimental animals or 100 µl acetone to the vehicle control group) was applied to the shaved areas. The treated animals were killed, unless otherwise indicated, 18 h after the last treatment. The treated area of skin was excised and placed in chilled Petri dishes with the dermis side up. Adherent fat was removed and the dermis was scraped off gently with chilled and blunt-end forceps. The epidermis thus separated was weighed, minced and homogenized in 50 mM Tris–HCl, 0.1 mM EDTA buffer, pH 7.5, at 4°C using a Polytron homogenizer. The epidermis homogenates (10% w/v) were centrifuged at 10 500 g for 20 min at 4°C and the supernatant (S-10.5) was used as the enzyme source. Glutaredoxin activity was assayed in the supernatants on the same day, whereas thioredoxin and thioredoxin reductase activity was assayed in samples kept frozen at –20°C overnight.

Enzyme assays
The mouse skin thioredoxin activity was determined by the micromethod of insulin reduction (24,26). All assay tubes contained 0.26 M HEPES, pH 7.6, 10 mM EDTA, 2 mM NADPH, 1 mM insulin and 100 nM purified calf thymus thioredoxin reductase plus the tissue extract or buffer in a final volume of 100 µ1. An aliquot of mouse skin supernatant S-10.5, containing ~5–10 µg of protein in rate limiting amount, was added to the assay mixture to start the reaction. After incubation at 37°C for 20 min, the reaction was stopped by addition of 0.5 ml of 6 M guanidine–HCl, 0.2 M Tris–HCl, pH 8.0, 1 mM DTNB and the absorbance was measured at 412 nm against a reagent blank containing all the components except the tissue extracts. Separate blanks were also used for each sample to determine the background content of DTNB-reactable SH groups available in the S-10.5 aliquots. The thioredoxin activity of mouse epidermal extract was calculated from the net absorbance at 412 nm as nmol SH groups formed in insulin using the expression (A412x0.6)/13.6, where 13.6 is the millimolar extinction coefficient for the product of the reaction of an SH group with DTNB (26). Results are expressed as pmol thioredoxin/mg protein.

Thioredoxin reductase activity in mouse skin was assayed by the same micromethod of insulin reduction. The assay mixture was also the same as described above, except that the aliquots of mouse skin supernatant S-10.5 contained 10–20 µg of protein in rate limiting amount as the enzyme source and that 18 µM E.coli thioredoxin (26) was included in the assay mixture to catalyze insulin reduction. Reagent blanks were included to determine the background content of SH groups in samples or the SH groups being generated spontaneously in insulin by the action of endogenous thioredoxin. These values were subtracted. Results are expressed as pmol thioredoxin reductase/mg protein.

Glutaredoxin activity was determined by the coupled enzyme reaction (25,27). In this assay the NADPH-dependent reduction of 2-hydroxyethyl disulfide was followed at 340 nm in a spectrophotometer (Zeiss, PMQIII) in the presence of 100 nM glutathione reductase and mouse skin supernatant S-10.5 containing 50–100 µg protein as the source of glutaredoxin activity. The assay mixture contained 100 µg/ml of bovine serum albumin, 1 mM GSH, 6 µg/ml yeast glutathione reductase, 0.4 mM NADPH, 0.1 M Tris–HCl, 2 mM EDTA, pH 8.0, and 0.7 mM 2-hydroxyethyl disulfide in a total volume of 500 µl. Non-enzymatic oxidation of NADPH in the background was first determined and the reaction was started by addition of 2-hydroxyethyl disulfide. The enzyme activity was calculated from the linear net {Delta}A340 per min using the expression ({Delta}A340 per minx0.5)/6.2 and expressed as pmol glutaredoxin/mg protein.

Protein determination
The protein was assayed by the dye binding method of Bradford (28) using bovine serum albumin as the standard.

Statistical analysis
Student's t-test was applied to statistically evaluate the results and P values were determined to show significant differences.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results summarized in Table IGo demonstrate the presence of thioredoxin, thioredoxin reductase and glutaredoxin activity in mouse epidermis.


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Table I. Effect of a single application of TPA on mouse skin thioredoxin and glutaredoxin activities
 
A single topical application of 8 nmol TPA to the skin increased the specific activity of epidermal thioredoxin, thioredoxin reductase and glutaredoxin. Relative to acetone-treated controls, increases of 40, 180 and 130% in the respective enzyme activities were observed (Tables I and IIGoGo). An increase in the dose of TPA (15 nmol in 100 µl acetone) resulted in similar effects (data not shown).


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Table II. Effect of a single application of TPA on mouse skin thioredoxin reductase activity
 
Since repeated applications of the phorbol ester are required to promote the growth of skin tumors, the effect of multiple applications of TPA (8 nmol in 100 µl acetone, twice per week for 2 weeks) relative to multiple applications of the vehicle control acetone was studied. Multiple treatments of TPA resulted in similar effects on thioredoxin and thioredoxin reductase activity as observed after single application of TPA (data not shown). However, the activity of glutaredoxin showed a large increase of >300% (Table IIIGo).


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Table III. Effect of multiple applications of TPA on mouse skin glutaredoxin activity
 
To investigate whether induction of the enzyme activities by the phorbol ester was stable, thioredoxin, thioredoxin reductase and glutaredoxin levels were studied in mouse skin up to a period of 10 days after TPA treatment. The results, summarized in Figure 1Go, show that the thioredoxin and thioredoxin reductase enzyme activities were initially down-regulated 6 h after treatment. However, once increased, 12 h after treatment, these remained elevated in the tissue for 6 days. An increase in glutaredoxin activity was registered only at 12 h after treatment. The maximum induction of glutaredoxin and thioredoxin reductase activity was 24 h after TPA treatment. A persistent increase in the steady-state levels of thioredoxin reductase and glutaredoxin activities in TPA-treated skin was seen during the 10 day study period. The steady-state levels of thioredoxin in TPA-treated skin, however, stablized at a higher level 6 days after treatment. Interestingly, the acetone treatment also influenced all the enzyme activities in mouse skin. However, the effect was less than for TPA but higher than day 0 status during the course of this study.





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Fig. 1. Mouse skin (a) thioredoxin, (b) thioredoxin reductase and (c) glutaredoxin activities after a single application of acetone (•) or TPA ({circ}). The conditions for enzyme assay and treatment of animals are described in Materials and methods. The values are means ± SEM from four observations in each group.

 
To investigate whether enzyme induction by TPA could be blocked by modifiers of mouse skin tumor promotion, the effects of a single application of FA, TPCK or RA (administered topically at a dose of 1, 10 or 10 µg, respectively, 1 h prior to TPA) on the induction of mouse skin thioredoxin, thioredoxin reductase and glutaredoxin activity was studied. The results revealed that these inhibitors failed to block the TPA-induced biochemical change (data not shown).

To investigate the specificity of TPA in regulating the activity of the thioredoxin and glutaredoxin systems, we studied the effects of phorbol, 4-O-MeTPA, RPA, mezerein, the non-phorbol but intercalating and hyperplasiogenic compound 1-oleoyl-2-acetylglycerol (OAG) (29) and the calcium ionophore A23187 on thioredoxin, thioredoxin reductase and glutaredoxin activities (Figure 2Go). The results showed that all the phorbol compounds, the calcium ionophore and OAG induced the thioredoxin system. However, a selectivity was seen with respect to glutaredoxin activity. Phorbol and 4-O-MeTPA, which are weak PKC activators, failed to influence glutaredoxin activity.





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Fig. 2. Effect of a calcium ionophore and other tumor promoters on mouse skin (a) thioredoxin, (b) thioredoxin reductase and (c) glutaredoxin activities 18 h after a single topical application. The biologicaly effective doses of the compounds were employed (TPA, 8 nmol; OAG, 5000 nmol; A23187, 200 nmol; mezerein, 3.4 nmol). A dose quantitatively equal to TPA was used in the case of the phorbol compounds (phorbol, 4-O-MeTPA and RPA, 8 nmol). The conditions for enzyme assay and treatment of animals are described in Materials and methods. The values are means ± SEM from more than four observations in each group.

 
In view of the tumor-promoting effect of TPA seen only in tumor initiated skin, the observed effects of multiple applications of TPA on the thioredoxin and glutaredoxin systems was investigated and compared in normal and tumor initiated (DMBA-treated) mouse skin. Activities were measured 6 days after the last application of TPA (Tables IV and VGoGo). This time point was selected due to the stability of the observed change for 6 days (see Figure 1Go). The results again showed elevated levels of glutaredoxin, thioredoxin and thioredoxin reductase activities in both normal and tumor initiated epidermis. Interestingly, however, a statistically significant increase in the activity of thioredoxin reductase was noted in tumor initiated skin even 28 days after the last treatment with TPA. The glutaredoxin activity had returned to control levels after this period. Treatment with DMBA alone also mimicked in part the effects of TPA (Tables IV and VGoGo). However, the effect was relatively distinct on thioredoxin and thioredoxin reductase activity.


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Table IV. Effect of multiple treatment with TPA on thioredoxin and glutaredoxin activities in normal and tumor initiated mouse skin
 

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Table V. Effect of multiple treatments with TPA on thioredoxin reductase activity in normal and tumor initiated mouse skin
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our experiments have shown that the topical application of TPA to mouse skin increased the specific activity of thioredoxin, thioredoxin reductase and glutaredoxin in epidermis. The change was stable for 1 week. Multiple applications of phorbol ester also resulted in similar effects but in addition resulted in a large increase in glutaredoxin activity. These results show up-regulation of protein disulfide and mixed disulfide oxidoreductases by the action of TPA and add a new perspective on mechanisms operating in the process of tumor promotion. The results agree with the reported inhibitory effects of the GSH-depleting agent diethylmaleate on mouse skin tumor promotion (30). A convincing amount of evidence is accumulating to support the role of redox regulation in the pathogenesis of cell transformation and growth. Redox modulation of p21 ras and the PKC isoforms has been reported recently (17,31) and a NADPH-dependent protein disulfide reductase activity is suggested for restoration of the kinase function of oxidant-inactivated PKC isoforms (32), which by all criteria is the thioredoxin system.

We observed that not only TPA, but other growth-affecting compunds like RPA, mezerein, OAG and the calcium ionophore A23187 also induced thioredoxin, thioredoxin reductase and glutaredoxin activities. Expression of these NADPH-dependent protein disulfide or mixed disulfide oxidoreductases thus seems to be associated with mitogen-activated protein kinases and intracellular calcium influx. However, phorbol and 4-O-MeTPA do not up-regulate glutaredoxin, but do thioredoxin and thioredoxin reductase. Thus, the mechanism of up-regulation of glutaredoxin appears to be uncoupled from the membrane intercalating function of TPA, however it is associated with PKC activation, intracellular calcium mobilization and the hyperplasiogenic function of the tumor promoting phorbol ester. A sub-carcinogenic dose of DMBA also stimulated induction of not only thioredoxin and thioredoxin reductase but also, marginally, glutaredoxin (Tables IV and VGoGo). More studies are needed to understand the mechanism of this effect.

Tagaya et al. (33) discovered adult human T cell leukemia-derived factor (ADF) in the conditioned medium of HTLV-1 transformed T cells. ADF (12 kDa) augments expression of the interleukin receptor IL-2R/p55 (Tac) and is identical to thioredoxin. Stimulation of expression of thioredoxin mRNA in human resting lymphocytes is also strongly enhanced by TPA (33,34) and thioredoxin is secreted into the medium, where it acts as a co-cytokine synergizing with interleukins (35). Thioredoxin is also secreted from activated T cells (36). Thus, inflammation may result in lymphocyte infiltration with increased expression of thioredoxin and its secretion, which may add to the observed biological effects of TPA.

Apfell and Walker (37) first described a NADPH-dependent protein disulfide reductase activity which was present in increased amounts in tumor tissue, ascites fluid and the serum of tumor-bearing mice in contrast to the normal animals. In this study, free SH groups and disulfide reduction were found to be obligatory for tumor cell proliferation and the maintenance of tumor cell antigenicity. This protein disulfide reductase activity (37) is by a number of criteria identical to the thioredoxin system (38). Another report has described abundant thioredoxin mRNA in Rous sarcoma virus-transformed embryonal chicken fibroblasts (39). High levels of thioredoxin and thioredoxin reductase are thus found in tumor tissue.

The thioredoxin and glutaredoxin systems catalyze the reduction of protein disulfides, enzymes or receptors (23,38). A small change in their intra- or extracellular levels produced by any effector molecule (e.g. the hyperplasiogen TPA) can trigger thiol-dependent redox changes which are amplified by orders of magnitude via enzymes, transcription factors and receptor proteins.

Reports on the role of thioredoxin in regulation of the steroid receptor (40), protein tyrosine kinase, protein kinase C, janus kinase, MAP kinase kinase kinase (17,31,32,41,42) and several transcription factors (4347) make the changes seen in the present studies in the thioredoxin and glutaredoxin systems more relevant to cell differentiation and growth. Convincing evidence is available that thioredoxin activates the glucocorticoid receptor (40), the DNA-binding activity of NF-kB (45) and the Ref-1 enzyme, modulating the DNA-binding activity of the fos–jun protein dimer (46). The oxidative stress inducible protein regulator (oxy-R) and the tumor suppression gene product p53 are also redox modulated (47,48). Glutaredoxin has been identified as controlling the activation of oxy-R protein (48). These results provide clues to the epigenetic changes induced by TPA. Induction of the thioredoxin and glutaredoxin systems by hyperplasiogens and tumor promoters may be related to their requirements in the expression of certain essential or regulatory sets of genes vital for growth or differentiation of cells.

Cellular expression of the thioredoxin and glutaredoxin systems appears to result following activation of the mitogen-activated calcium-dependent protein kinase family of proteins, especially in view of our observations that the intracellular changes in diacylglycerol and calcium homoeostasis produced by OAG and the ionophore A23187, respectively, increase the amounts of thioredoxin, thioredoxin reductase and glutaredoxin activities. Further investigations are needed to substantiate this view. A direct effect on thioredoxin reductase is unlikely since the enzyme from human placenta has been found to be insensitive to calcium in vitro (49).

TPA is known to give rise to reactive oxygen species (ROS) as an early and critical event in tumor promotion that can be blocked by superoxide dismutase (50,51). Addition of 0.1 µM TPA also results in a sharp decline in the intracellular ratio GSH:oxidized glutathione (GSSG) in isolated mouse epidermal cells (52). Normally these cells have very low levels of GSSG (52). This may in fact account for the rapid inhibition of DNA synthesis noted after TPA treatment. Addition of excess extracellular GSH or {alpha}-tocopherol inhibits the effect of TPA on skin tumor promotion and prevents the change in GSH:GSSG. Activation of the oxidative stress regulatory protein, oxy-R, and thus the oxy-R-regulated genes by ROS and hydrogen peroxide is known to result in overexpression of a thioredoxin reductase-like protein (alkyl hydroperoxide reductase) (53). Incidently, thioredoxin reductase is known to reduce lipid hydroperoxides (54) and is a selenoprotein (55) and is therefore an antioxidant (56). Thioredoxin is involved in cellular defense mechanisms against oxidative damage in vivo and therefore also acts as an antioxidant (57,58). The changes in the thioredoxin and glutaredoxin systems produced by TPA seen in our studies can thus also be understood to be a manifestation of the onset of oxidative stress in the responsive cells.

We observed that induction of the thioredoxin and glutaredoxin systems was insensitive to the inhibitors of tumor promotion FA, RA and TPCK. Therefore, induction of the redox systems by TPA appears to be insensitive to these inhibitors and is probably upstream of their site of action. It is also important that thioredoxin-catalyzed protein disulfide reduction and ribonucleotide reduction are influenced by the physiological levels of the cancer preventive sodium selenite (59,60) and that thioredoxin reductase has a functional selenocysteine residue.

A relatively long lasting up-regulation of the activities of the thioredoxin and glutaredoxin systems in epidermis stimulated by TPA could be a result of autoactivation of their transcription. Such a possibility has been described for AP-1 (12). Raised levels of the thioredoxin and glutaredoxin systems could target post-translational modifications of proteins important for obligatory cellular events (e.g. altered expression of genes under promoter control of redox-modulated transcription factors required to provoke cell differentiation and growth in responsive cells), with subsequent regenerative hyperplasia, facilitating the clonal expansion of neoplastic cells in tumor initiated mouse skin (2).

We conclude that TPA up-regulates the expression of thioredoxin, thioredoxin reductase and glutaredoxin over a long period of time. The thioredoxin and glutaredoxin systems may redox-regulate protein kinases and modulate enzyme activities, cell surface or intracellular receptors and transcription factors required for induction of regenerative hyperplasia, which is obligatory for clonal selection and growth of the neoplastic cells in skin tumorigenesis. Further studies in isolated keratinocyte systems are required to delineate the expression pattern of thioredoxin, thioredoxin reductase and glutaredoxin in cell differentiation, transformation and growth as effected by calcium, mitogens and different growth factors.


    Acknowledgments
 
This investigation was supported by grants from the Swedish Cancer Society (961), the Swedish Medical Research Council (13X-3529) and the Knut and Alice Wallenberg Foundation. S.K. was on leave from the Industrial Toxicology Research Centre (CSIR), Lucknow, India, and was supported by a fellowship from the Swedish Cancer Society (961-B89-03V).


    Notes
 
1 To whom correspondence should be addressed Email: arne.holmgren{at}mbb.ki.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 30, 1998; revised May 11, 1999; accepted May 24, 1999.