Absence of Dopaminergic Control on Melanotrophs Leads to Cushing’s-Like Syndrome in Mice

Adolfo Saiardi and Emiliana Borrelli

Institut de Génétique et de Biologie Moléculaire et Cellulaire Centre Nationale de la Recherche Scientifique/INSERM/ Université Louis Pasteur BP 163 67404 Illkirch Cedex C.U. de Strasbourg, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dopamine negatively regulates POMC gene expression in melanotrophs of the intermediate lobe of the pituitary gland. The dopaminergic receptor involved in this control is the dopamine D2 receptor (D2R). The principal products of the POMC gene in melanotrophs are ß-endorphin and {alpha}-MSH. POMC is differently processed in the corticotrophs, where it is not regulated by dopamine and it is principally processed into ACTH. Here we show that D2R-deficient mice have increased POMC expression and intermediate lobe hypertrophy. Strikingly, D2R-deficient mice have unexpected elevated ACTH levels with a corresponding increase of corticosteroids and consequent hypertrophy of the adrenal gland. This phenotype is reminiscent of Cushing’s syndrome in humans. Interestingly, we show that the elevation in ACTH levels is due to an aberrant processing of POMC in melanotrophs. Indeed, we demonstrate that in addition to controlling POMC gene expression in these cells, dopamine, by modulating the expression of the convertases involved in the cleavage of the POMC prohormone, strictly regulates its processing. These results reveal a key role for dopamine in the control of POMC-derived peptides and furthermore indicate an implication of the dopaminergic system in the genesis of Cushing’s syndrome.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In parallel to its functions in the central nervous system, dopamine also exerts an inhibitory neuroendocrine control of hormone synthesis and release in the pituitary gland (1, 2). The pathway involved in such function is the tubero-infundibular pathway. The effects of dopamine on pituitary cells are elicited through its interaction with dopamine D2 receptors (D2Rs) (3). D2R is a member of the seven-transmembrane domain G protein-coupled receptor family. The best characterized signal transduction pathway of D2Rs is the inhibition of adenylyl cyclase, which results in the decrease of intracellular cAMP levels. In addition, other signal transduction pathways have also been shown to be activated by this receptor (3). Two distinct isoforms of D2R have been identified, D2L and D2S, issued from alternative splicing of the same gene (4, 5). D2L and D2S present similar pharmacological properties and anatomical distribution (6). In the pituitary gland D2Rs are present on the membrane of lactotrophs and melanotrophs (7) where it regulates the synthesis of PRL in lactotrophs (2) and of the POMC gene products in melanotrophs (1, 8).

The regulation of the POMC gene transcription deserves special attention in that it is differentially controlled in the two pituitary cell types: the corticotrophs of the anterior lobe (AL) and the melanotrophs of the intermediate lobe (IL). In both cell types POMC synthesis is ensured by the hypothalamic corticotropin releasing factor (CRF) (9, 10), whereas the negative control upon POMC expression is ensured in a cell type-dependent manner. Indeed, down-regulation of POMC transcription is controlled by glucocorticoids in the corticotrophs (11) and by dopamine in the melanotrophs (8). This differential control reflects the physiological specialization of the two POMC-producing cell types. Corticotrophs are in fact the cells devoted to the production of ACTH, which acts on adrenals to stimulate corticosteroid synthesis (12). Corticosteroids, in turn, blunt POMC expression in these cells by an inhibitory feedback loop. In contrast, POMC in rodent melanotrophs produces essentially {alpha}-MSH and ß-endorphin (ß-end), and it is inhibited by dopamine (13). ACTH, {alpha}-MSH, and ß-end derive from the cleavage of the POMC prohormone by the activity of the prohormone convertases PC1 and PC2 (14, 15, 16). PC1 is strongly expressed in corticotrophs and PC2 in melanotrophs (17). This suggests that a strict cell-specific control of POMC transcription and processing is required to keep normal physiological responses.

The generation of D2R-deficient mice (18) has allowed the study of the involvement of this receptor in the control of pituitary gland functions. Interestingly, loss of D2R expression leads to pituitary hyperplasia of AL (19) due to an aberrant increase of PRL levels and consequent overgrowth of lactotrophs. Thus, dopamine is an antiproliferative signal for lactotrophs (19). At the melanotrophs level we have found that lack of dopamine signaling via D2R results in a striking enlargement of the IL. Thus, the antiproliferative effect of dopamine is also observed at the level of the melanotrophs. This is in support of the basic idea that lack of tonic inhibition on endocrine cells, possibly mediated through the inhibition of the cAMP pathway, leads to aberrant growth. In addition, here we show that, in the absence of D2R, melanotrophs lose their cell identity and start to produce ACTH. This aberrant production of ACTH leads to adrenal hyperplasia. Taken together, these results indicate that absence of dopaminergic control on melanotrophs leads to a Cushing’s-like syndrome and suggest that dopamine deficiency might be one of the causes of this disease in humans.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lack of D2R Leads to POMC mRNA Increase and IL Hyperplasia
Histological analyses of the pituitary gland of D2R-null mice have revealed a hyperplasia of the IL with a 40% increase of the number of cells in the lobe (19). To better characterize these effects at the cellular and molecular levels, we first performed an in situ hybridization analysis (Fig. 1AGo) using the mouse POMC probe. As expected, a robust labeling of both the corticotrophs in the AL and melanotrophs in the IL was obtained in the pituitaries of wild-type (WT) and mutant mice. However, a 2-fold increase in POMC mRNA expression was observed in the melanotrophs of D2R-deficient mice as compared with WT siblings (Fig. 1Go). In Fig. 1AGo it is also possible to appreciate that the enlargement of the IL in mutant mice is due to the proliferation of POMC-producing cells (19). Importantly, the observed increase of POMC expression is specifically restricted to melanotrophs and does not extend to corticotrophs, as shown by ribonuclease (RNAse) protection experiments in which we separated the AL from the IL (Fig. 1BGo). In agreement with the in situ hybridization experiments, quantification of POMC mRNA augmentation by RNAse protection demonstrates a 2-fold increase in D2R-null mice as compared with WT animals using similar amount of RNA (Fig. 1Go, B and C). Thus, lack of D2R in the IL results in the appearance of two phenomena, the first corresponding to an up-regulation of POMC expression and the second to the proliferation of melanotrophs.



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Figure 1. POMC Gene Expression in the Pituitary Gland of WT and D2R -/- Mice

A, In situ hybridization using a mouse POMC antisense RNA probe on sections from 4-month-old WT and D2R -/-mice pituitaries. Scale bar, 75 µm. PL, Posterior lobe. B, RNAse protection analysis of POMC mRNA in the AL and IL of WT and D2R -/- mice. Histone H4 expression was used as internal control for RNA quantity in each sample. Results are representative of three independent experiments using different animals (n = 3). C, Quantification of POMC mRNA expression in RNAse protection experiments using D2R -/- mice and WT littermates. Values are expressed as mean ± SD; quantification of the intensity of the band corresponding to the WT was arbitrarily fixed to 100. **, P < 0,01, unpaired Student’s t test.

 
D2R Knockout Does Not Affect Hypothalamic CRF Expression
It is well known that positive hypothalamic signals are needed to induce synthesis and secretion of POMC-derived peptides (9). Corticotropin-releasing factor (CRF), produced at the level of the hypothalamic paraventricular nucleus, is the major releasing factor acting on corticotrophs (11) and melanotrophs (11, 20). Thus, we analyzed whether the increased size of the IL and POMC expression might be secondary to an aberrant hypothalamic control over CRF expression. In situ hybridization of D2R-null and WT brain sections showed no differences in the expression of the CRF gene between the two groups (Fig. 2Go). This indicates that lack of D2Rs does not affect the expression of the hypothalamic releasing factor involved in the regulation of POMC synthesis. Consequently, CRF does not seem to be directly involved in the generation of the IL phenotype of D2R-null mice.



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Figure 2. In Situ Hybridization Analysis of CRF mRNA Expression in the Hypothalamus of WT and D2R -/- Mice

A, Serial sections from the brains of WT and D2R -/- mice were hybridized with a CRF antisense riboprobe. Similar results were obtained in three independent experiments using different animals (n = 3). PVN, Paraventricular nucleus. B, Quantification of CRF mRNA expression in the hypothalamus of WT vs. D2R -/- mice. Grain density/0.01 mm2 was measured in comparable sections of three animals of each genotype in three different experiments. Values are expressed as mean ± SD.

 
Abnormal Increase of ACTH in D2R-Null Mice
Next we verified by RIA whether the increased expression of POMC in D2R-null mice corresponded to a similar elevation of POMC-derived hormones in the sera of these animals. In agreement with the mRNA increase, the results of these analyses clearly showed that ß-end and {alpha}-MSH serum levels were increased 5.7- and 2.7-fold, respectively, in mutant mice, as compared with WT controls (Fig. 3Go). During quantification of POMC-derived peptides, we also measured ACTH levels, which should have been not affected in D2R-null mice. Strikingly, a 4-fold increase of this hormone was measured in D2R-null mice with respect to control littermates (Fig. 3Go). This result was completely unexpected since antagonism of D2R has never been shown to lead to ACTH increase. In addition, ACTH is normally produced by the corticotrophs of the AL and not by melanotrophs in normal pituitary glands. Indeed, it is well established that corticotrophs do not express D2Rs and, in fact, dopamine does not have inhibitory effects on these cells (12). In agreement with this finding, both in situ and RNAse protection analyses of POMC expression show an increase of this mRNA only in RNAs extracted from the IL and not from the AL (Fig. 1Go, A and B). Thus, absence of dopaminergic control in the melanotrophs results in an increase of all POMC-derived peptides and consequently in the loss of melanotrophs’ cell specialization.



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Figure 3. Blood Levels of POMC-Derived Peptides in WT and D2R -/- Mice

The number of animals used for the determination of each hormone is indicated above the corresponding columns. Values are means ± SD. **, P < 0.01, unpaired Student’s t test.

 
Cushing’s-Like Syndrome in D2R-Null Mice
POMC expression in corticotrophs is inhibited by glucocorticoids as an adrenal feedback mechanism to reduce ACTH levels and consequently steroids synthesis. In humans, Cushing’s syndrome refers to the disorder caused by pituitary ACTH hypersecretion (21, 22) and consequent glucocorticoid excess. In light of the observed increase of ACTH levels in D2R-null mice, we analyzed the level of serum corticosterone, the most abundant steroid in rodents, in WT and D2R mutant animals. Interestingly, measurement of plasma corticosterone levels in D2R-null vs. WT animals showed a significant 1.5-fold increase of this steroid (mean ± SD: WT, 290±150 ng/ml, n = 10; D2R -/-, 440 ± 250 ng/ml, n = 11; P < 0.05 Student’s t test). Chronic ACTH hypersecretion in Cushing’s syndrome results in a hyperstimulation of the adrenal cortex that leads to adrenocortical hypertrophy and hyperplasia. Thus, we performed a histological analysis of the adrenal glands of WT and D2R-null mice. We found a remarkable enlargement of the adrenal cortex in glands from D2R-null animals, as compared with WT mice. The histological analysis demonstrates the presence of the characteristic fusion of the zona fasciculata and reticularis (Fig. 4Go), a feature known to be present in patients with Cushing’s syndrome upon ACTH overstimulation. In addition, D2R-null mice presented a hyperpigmentation of their coat (data not shown) due to high {alpha}-MSH and ACTH levels. Again, this is in line with the pathological signs of Cushing’s syndrome.



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Figure 4. Adrenal Hypertrophy in D2R -/- Mice

Hematoxylin-eosin staining of adrenal serial sections from 4-month-old WT (n = 3) and D2R -/- mice (n = 3). Zonae glomerularis (G), fasciculata (F), and reticularis (R) are indicated. In the D2R -/- section the fusion of the zonae fasciculata and reticularis is evident.

 
These results are of a remarkable medical interest since defects of the dopaminergic system have never been associated with Cushing’s disease, due to the knowledge that ACTH is normally not produced by melanotrophs and that corticotrophs do not express D2Rs.

Overexpression of PC1 in the IL of D2R-Null Mice
Importantly, the pituitary cell-specific processing of the POMC prohormone is ensured by two members of the furin family of proteases, PC1 and PC2 (15, 16, 23). Notably, expression of the melanotroph-specific PC2 convertase in AtT-20 cells, a corticotroph-derived cell line, results in a POMC processing similar to melanotrophs (15). To study the mechanism by which dopamine might control POMC prohormone processing, we checked whether the expression of PC1 and PC2 might have been altered in the pituitaries of D2R-null mice. We thus analyzed the expression of these genes in WT and mutant mice by in situ hybridization. In WT animals, PC1 expression is mainly confined to the corticotrophs, while PC2 predominates in the melanotrophs (Fig. 5AGo) (17, 23). It was previously reported that haloperidol treatment (a D2R antagonist) in rats results in an increase in the pituitary expression of PC1 and PC2 (15, 16). Accordingly, the absence of the D2R in the mutant animals provokes a 4- to 5-fold increase of PC1 expression in the melanotrophs of these animals (Fig. 5BGo). This result is consistent with the observed ACTH serum level elevation in mutant animals (Fig. 3Go). A 3- to 4-fold increase in the expression of PC2 in melanotrophs is also observed in D2R-null animals as compared with WT littermates (Fig. 5Go).



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Figure 5. In Situ Analysis of PC1 and PC2 mRNA Expression in the Pituitary Gland of WT and D2R -/- Mice

A, Serial sections from pituitaries of 4-month-old WT and D2R -/- mice were hybridized with mouse PC1 and PC2 antisense riboprobes. Results are representative of three independent experiments on three different animals. Scale bar, 75 µm. B, Quantification of the expression of PC1 and PC2 was performed as described in Materials and Methods. Values are means ± SD. **, P < 0,01, unpaired Student’s t test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dopamine controls physiological functions both at the level of the central nervous system (CNS) and of the pituitary gland. In the pituitary, dopamine acts through the binding to D2Rs, which are localized in two different cell types, the lactotrophs and the melanotrophs. The best characterized function of dopamine on these pituitary cells is the regulation of the synthesis and secretion of PRL and POMC-derived peptides. The knockout of the dopamine D2R gene has allowed a more detailed analysis of the dopaminergic control of hormonal response and of general functions such as the control of cell proliferation and identity. Indeed, absence of dopaminergic receptors in the pituitary leads to aberrant proliferation of the lactotrophs, which in older animals gives rise to tumors (19). This finding indicates a more general function for dopamine in the neuroendocrine system. Indeed, a neuromodulator can act outside of the CNS as a maintenance factor for a particular cellular phenotype. Concomitant with the increase of the PRL-producing cells we observed a decrease of the cells that produce GH. These data are of interest since these two cell types belong to a common cell lineage, and the somatotrophs appear before the lactotrophs during pituitary development. Thus, dopamine might act as a hypothalamic factor involved in the determination and establishment of lactotroph identity.

This notion is strengthened by the phenotype of the IL of D2R-null mice. Indeed, absence of D2Rs in the IL results in hyperplasia of melanotrophs and in aberrant POMC expression and peptide processing. Interestingly, lack of D2R has different impact on the proliferation of the cells in the AL or IL. The hypertrophy and hyperplasia of the IL is one of the clearest features of D2R-null pituitaries, even in young animals (7–8 weeks old). By contrast, the hyperplasia of the AL is delayed in time (3–4 months) but progressive, leading to tumors in aged mice (19). The hyperplasia of the IL is instead contained, even in aged animals. This is likely to be due to the different growth characteristics of lactotrophs vs. melanotrophs. Indeed, lactotrophs belong to a lineage more prone to proliferate under specific stimuli, such as during pregnancy and lactation. It will be important in future studies to assess whether the regulation of the cell cycle might be different in lactotrophs vs. melanotrophs.

The lack of dopaminergic control over POMC expression leads to an increase in POMC-derived peptides. POMC is produced by two pituitary cells, the corticotrophs in the AL, and the melanotrophs in the IL (9). Despite the expression of the same gene, these two cell types differ in the processing of the prohormone peptide, which results in a specialization of the corticotrophs in the production of ACTH and of the melanotrophs in that of {alpha}-MSH. Strikingly, the analysis of D2R-null mice revealed an unexpected alteration of the processing of the POMC propeptide in melanotrophs. There is a significant increase in ACTH serum levels, due to an inappropriate expression of the convertase PC1 in the IL of these mice. Thus, loss of D2R dramatically affects both melanotroph cells’ proliferation and cellular identity, as shown by the alteration of their normal hormone production. This leads to animals with impaired endocrine responses. It is known that melanotrophs do not express glucocorticoid receptors (24) and that ACTH production cannot be fully controlled by the negative feedback loop of corticosteroids on the pituitary, as happens in corticotrophs.

The adrenal phenotype exhibited by D2R-null mice is strikingly reminiscent of Cushing’s syndrome (21, 22), showing the hypertrophy of the adrenal cortex and the fusion of the zonae fasciculata and reticularis. Interestingly, past reports described elevated ACTH levels and Cushing’s syndrome in horses with IL pituitary tumors (25, 26); unfortunately it is not known whether the dopaminergic system was affected in these animals. Whether dopaminergic deregulation might contribute to the genesis of human Cushing’s syndrome has not been elucidated, because in humans the role of dopamine on POMC-producing cells is not clear. The human pituitary lacks a well defined IL, and the POMC-producing cells are intermingled with those of the adenohypophysis. Importantly, earlier studies have shown that dopamine agonists lower ACTH in some, but not all, Cushing’s syndrome patients (27). Thus, dopamine might contribute, in some cases, to the etiology of this disease in humans.

In conclusion, this study shows that dopamine has a key role outside the CNS. It is involved in the regulation of hormone synthesis and secretion, but most importantly in the control of cell proliferation and in the maintenance of the melanotroph phenotype.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice
D2R mutant animals were generated and identified by genomic Southern blot analysis (18). All animals used in the experiments had a mixed 129SVxC57Bl/6 genetic background, with a 75% contribution of C57Bl/6 background; the WT mice used were littermates of D2R-null mice. Animals were bred under standard animal housing conditions, in a 12-h light/12-h dark cycle. Food and water were available ad libitum. All experiments were conducted in conformity with the French publication on animal experimentation (No. 87–848) and the European Communities Council Directive of November 1986 (86/609/EEC).

Histological Analyses
Adrenals from WT and D2R-deficient mice were fixed in Bouin’s fixative for at least 1 day and embedded in paraffin. Serial microtome sections (10 µm) were stained by hematoxylin-eosin. Three animals of each genotype were analyzed.

In Situ Hybridization
WT and mutant mice were killed by decapitation after Rompun (Bayer AG, Leverkusen, Germany) anesthesia. Brains and pituitaries were rapidly removed and frozen in dry ice after embedding in Tissue Tek O.C.T. compound (Sakura Finetek USA, Inc., Torrance, CA). Cryostat sections (10 µm) were thaw-mounted on gelatin-coated slides and stored at -80 C. Before hybridization, sections were rinsed in ice-cold acetone, fixed in 4% formaldehyde, and washed in PBS. Finally, slices were acetylated for 10 min in 0.0265 M acetic anhydride/0.1 M triethanolamine to reduce background, washed in 1x saline sodium citrate (SSC), incubated for 10 min at 60 C in 50% formamide, 1x SSC, and dehydrated. Sections were hybridized overnight at 52 C with 35S-labeled antisense riboprobes specific for pituitary markers in hybridization buffer (300 mM NaCl, 20 mM Tris-Cl, pH 7.5, 10% dextran sulfate, 1x Denhardt’s solution, 5 mM EDTA, pH 8.0, 10 mM Na2PO4, pH 7.0, 50% formamide, 0.5 mg/ml yeast tRNA, 10 mM dithiothreitol). After hybridization, sections were washed twice for 1 h at 55 C in 50% formamide, 2x SSC and treated for 30 min at 37 C with RNAase A (20 mg/ml in 4x SSC). Sections were then extensively washed at 55 C under increasing stringency up to 0.1x SSC and dehydrated in graded ethanols. For autoradiography, dried sections were first exposed to Kodak XAR-OMAT films and subsequently dipped in Kodak NTB2 nuclear emulsion. Slides were exposed at 4 C for 1–4 days, developed, and counterstained with toluidine blue. The specificity of the in situ hybridization results was confirmed by the use of sense strand riboprobes that showed no detectable signals (results not shown). Experiments were analyzed using a Hamamatsu camera (Hamamatsu Photonic Systems, Bridgewater, NJ) with a controller C2400; data were quantified using an Imaging Technology 151 System (Hamamatsu Photonic System, Bridgewater, NJ). The reported increase in POMC, CRF, PC1, and PC2 were obtained by measuring grains density/0.01 mm2 in 10–15 different areas of the analyzed tissue in at least three independent experiments using different animals (n = 3). Data were analyzed by the Student’s t test.

RNAse Protection
Total RNAs were prepared from the AL and IL by the LiCl method (28). RNAse protections were performed as described (28). Three animals of both sexes and genotypes were used in three independent experiments. Equal amounts of RNA (0.1 µg for the IL and 0.2 µg for the AL) were hybridized overnight at 45 C with a molar excess of 32P-labeled mouse POMC and H4 histone riboprobes. Samples were treated with RNAase A (40 µg/ml) and T1 (2 µg/ml), incubated with Proteinase K (150 µg/ml), extracted with phenol-chloroform, and precipitated with ethanol. Protected fragments were run on a 6% polyacrylamide/8 M urea gel. Autoradiograms were analyzed and data quantified using a Bio-Rad GS-700 Imaging Densitometer (Bio-Rad, Hercules, CA).

Hormone Analysis
Blood samples were collected from decapitated animals and centrifuged at 4000 rpm for 15 min in an Eppendorf microfuge. Sera were then removed and stored at -20 C. Hormone levels in serum samples were determined by RIA; each determination was always performed in parallel on both test populations (D2R-null mice and WT littermates), and the results were deduced from their comparison. ACTH, ß-end, and {alpha}-MSH were measured by RIA kits from Peninsula Laboratories, Inc. (Belmont, CA) and corticosterone by an ICN Biomedical (Costa Mesa, CA) kit. The ACTH kit has 0% cross-reactivity for {alpha}-MSH, LH-RH, PACAP 1–38, and ß-end. The ß-end kit has 0% cross-reactivity for ACTH, Met-Enkephalin, {alpha}-MSH, and PACAP 38. The {alpha}-MSH kit has 0% cross-reactivity for ß-end, {alpha}-endorphin, {gamma}-endorphin, Met-Enkephalin, and 0.02% ACTH (human). EDTA and aprotinin were added to the blood samples following manufacturers suggestions. One hundred microliters of serum were used for the {alpha}-MSH assay; 20 µl were used for the ß-end and ACTH tests; 10 µl were used for corticosterone evaluation. Statistical analysis was performed by unpaired Student’s t test.


    ACKNOWLEDGMENTS
 
We acknowledge Drs. P. Sassone-Corsi, Yuri Bozzi, Tarek A. Samad, and members of the laboratory for discussions and critical reading of the manuscript. We thanks Drs. K. E. Mayo (Northwestern University, Evanston, IL), E. Jansen (Leuven, Belgium), and C. Mazzucchelli for cDNA probes. We are grateful to V. Giroult for technical assistance, T. Ding for paraffin sections, S. Falcone for animal care, B. Boulay and J. M. Lafontaine for artwork, and to J.-L. Vonesch for quantification of the in situ data.


    FOOTNOTES
 
Address requests for reprints to: Emiliana Borrelli, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre Nationale de la Recherche/INSERM/Université Louis Pasteur, BP163, 67404 Illkirch Cedex, C.U. de Strasbourg, France. E-mail: eb{at}igbmc.u-strasbg.fr

A.S. was supported by fellowships from the European Economic Community and Fondation pour la Recherche Médicale. This work was supported by funds from Centre Nationale de la Recherche Scientifique, Institut Nationale de la Santé et de la Recherche Médicale, Hôpital Universitaire de Strasbourg, and from a grant from the Association pour la Recherche sur le Cancer to E.B.

Received for publication December 5, 1997. Revision received March 31, 1998. Accepted for publication April 13, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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