(The FASEB Journal. 2003;17:829-835.)
© 2003 FASEB
Direct gene transfer into rat articular cartilage by in vivo electroporation
LAURENT GROSSIN1,
CHRISTEL COURNIL-HENRIONNET1,
LLUIS M. MIR
,
BERTRAND LIAGRE,
DOMINIQUE DUMAS*,
STÉPHANIE ETIENNE,
CORINNE GUINGAMP,
PATRICK NETTER2 and
PIERRE GILLET
Unité Mixte de Recherches 7561, Centre National de la Recherche Scientifique-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP184, F-54505 Vandoeuvre lès Nancy, France;
* Unité Mixte de Recherches 7563 "Mécanique et Ingénierie Cellulaire et Tissulaire" (LEMTA), Centre National de la Recherche Scientifique, Université Henri Poincaré Nancy 1, Faculté de Médecine, BP184, F-54505 Vandoeuvre lès Nancy, France; and
Unité Mixte de Recherche 8121 "Vectorologie et transfert de gènes" Centre National de la Recherche Scientifique, Institut Gustave-Roussy, F-94805 Villejuif Cedex, France
2Correspondence: Unité Mixte de Recherches 7561, Centre National de la Recherche Scientifique-Université Henri Poincaré Nancy 1, Faculté de Médecine, Avenue de la Forêt de Haye, BP184, F-54505 Vandoeuvre lès Nancy, France. E-mail: Patrick.Netter{at}medecine.uhp-nancy.fr
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ABSTRACT
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To establish a system for efficient direct in vivo gene targeting into rat joint, we have evaluated a strategy of gene transfer by means of the delivery of external electric pulses (EP) to the knee after intra-articular injection of a reporter gene (GFP). Rats were killed at various times after the electro gene-therapy to analyze GFP gene expression by immunohistochemistry. GFP staining was detected in the superficial, middle, and deep zones of the patellar cartilage at days 2 and 9, and thereafter only in the deep zone (months 1 and 2). The average percentage of GFP-positive cells was estimated at 30% both one and 2 months after the gene transfer. Moreover, no pathologic change caused by the EP was detected in the cartilage. The level and stability of the long-term GFP expression found in this study demonstrate the feasibility of a treatment of joint disorders (inflammatory or degenerative, focal or diffuse) using electric gene transfer.—Grossin, L., Cournil-Henrionnet, C., Mir, L. M., Liagre, B., Dumas, D., Etienne, S., Guingamp, C., Netter, P., Gillet, P. Direct gene transfer into rat articular cartilage by in vivo electroporation.
Key Words: electropermeabilization • in vivo gene transfer • electro gene therapy • GFP • patellar cartilage
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INTRODUCTION
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METHODS TO TRANSFER GENES of interest into cells in vivo are powerful tools for research on the function and behavior of biological macromolecules and may have application in gene therapy for treatment of a large panel of diseases. Several nonviral methods have been proposed to perform gene transfer in vivo by means of different vectors (1)
. A promising approach to achieve nonviral gene transfer (2)
in mammalian cells is based on the delivery of electric pulses that improve DNA translocation inside the cells. Electroporation (EP), also termed electropermeabilization, has thus been widely used in vitro to introduce foreign DNA sequences in many different cell types (3)
. Moreover, electric pulse-mediated DNA transfer has been applied successfully in vivo in living animals (4)
to target genes to various tissues like skin (5)
, e.g., melanoma (6)
and brain (7)
.
In the case of the skeletal muscle, myocytes, as nondividing cells in basal conditions, allow stable expression of the transfected genes, although the transferred DNA does not usually undergo chromosomal integration. In fact, previous in vivo studies of skeletal muscle have demonstrated that combining EP with intramuscular DNA injection offers the major advantages of 1) enhancing its expression by 100- to 500-fold (8)
, 2) minimizing variability between experiments, 3) resulting in long-lasting gene expression (9
, 10)
and 4) restricting the transfer to a specific area (volume), specially when tissue-specific promoters are used. Of all the nonviral techniques for in vivo gene transfer, EP is simple, inexpensive, and apparently safe (11)
.
Concerning the field of rheumatic diseases, pilot studies (12)
suggest that EP appears as a potential in vivo approach to achieve gene expression in the synovium of rat knees after the intra-articular (i.a.) injection of naked DNA (pDNA), followed by electric pulses delivery. In contrast, in vivo gene transfer in hyaline cartilage has to face its peculiar structure due to a particular volume ratio of the ECM (ECM)/cell, adapted to the articular biomechanical constraints in weight-bearing joints. In fact, articular cartilage is composed of a low number of chondrocytes embedded in a large and compact ECM, consisting of rigid collagenous and noncollagenous components (proteoglycans) that are potential physic barriers to the contact of the cells with foreign particles. The mechanisms of pDNA electrotransfer are the electro-permeabilization of the cells and the electrophoretic transport of the DNA in the tissue (13)
. Therefore, EP offers the possibility that naked DNA could cross the ECM barrier because of its reduced size with respect to that of virus and because of the electrophoretic effects of the delivered pulses on DNA charges, thus permitting DNA access to the chondrocytes.
To test this hypothesis, we performed i.a. injections of a plasmid coding for the green fluorescent protein (GFP) as a reporter gene, followed by the delivery of electric pulses using custom-made external electrodes that cover the patella, regarded as a distinguishable superficial anatomical entity. In this pilot study we evaluate the feasibility, usefulness, and safety of this new approach of in situ gene transfer to the rat patellar chondrocytes.
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MATERIALS AND METHODS
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Plasmid DNA preparation
The plasmid pcDNA3.1/CT-GFP-TOPO (Invitrogen, Cergy-Pontoise, France) containing the cytomegalovirus promoter inserted upstream of the coding sequence of the GFP was amplified using standard procedures and purified using the EndofreeTM Plasmid Maxi Kit (Qiagen, Courtaboeuf, France). Just before the electric pulse delivery, DNA (30 µg) resuspended in 50 µL of NaCl 0.9% was injected i.a. into both shaved rat knees through the infrapatellar ligament (day 0).
Electric pulse delivery
Experiments with animals were conducted in accordance with institutional guidelines following recommendations of the National Institutes of Health for animal experimentation. Six-week-old male Wistar rats (Charles River Laboratories, L’Arbresle, France) were housed in plastic cages with free access to water and standard laboratory chow. Before EP, rats were anesthetized using ketamine hydrochloride (50 mg·kg-1) mixed with acepromazine (1.25 mg·kg-1) injected intraperitoneally in 300 µL. Rat legs were shaved, and the i.a. injection of the plasmid performed. Five minutes later, transcutaneous electric pulses were applied by two custom-made stainless steel parallel plate electrodes (2 cm long, 2 cm large, 1 mm wide, and 12.5 mm apart) placed at each side of the knee, previously covered with a conductive gel (Fig. 1
).
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Figure 1. Principle (a) and mechanism (b) of intra-articular electrotransfer. On day 0, the plasmid is injected intra-articularly, then pulses are delivered by means of 2 electrodes placed on each part of the rat knee (++ on the patella).
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To confirm the safety of EP on rat patellar cartilage preliminary control experiments were performed in three 3 control groups on days 2 and 9: 1) the first group of naive animals were not injected i.a. with pDNA injection or submitted to electric pulses; 2) the second group was only submitted to electric pulses without previous i.a. pDNA injection; 3) the third group was injected i.a. with a single injection of pDNA without applying external electric pulses. Square-wave electric pulses were generated by a PS-15 Jouan electropulsator (GHT 1281, Jouan, St. Herblain, France). Pulses were controlled by a digital oscilloscope (VC 6025, Hitachi, Tokyo, Japan). As described for mice and rat skeletal muscle (8)
, we applied 8 pulses of 20 ms delivered at a repetition frequency of 1 Hz, but we introduced a modification consisting in the delivery of a higher voltage that resulted in a ratio of applied voltage to distance between the electrodes of 250 V/cm.
Histological analysis
At different times after DNA electrotransfer (days 2, 9, 15, 30, and 60), rats were killed by cervical dislocation under anesthesia. Patellae were carefully dissected, fixed, embedded in paraffin, and processed for histological evaluation. Sections of 5 µm were deparaffinized according to standard procedures. To assess the lack of chondrotoxicity due to the delivery of the EP to the patellar cartilage, sections were stained with hematoxylin-eosin-safran, toluidine blue, and picrosirius red, which allow a visualization of the cells, the proteoglycan content, and collagen fibers in the hyaline cartilage.
Measurement of proteoglycan synthesis
Proteoglycan synthesis was assessed in patellar cartilage as described (14
, 15)
. At various key points, rats were killed as described previously and the patellae were carefully dissected out. They were placed in 1 mL of medium consisting of RPMI 1640 with HEPES and sodium bicarbonate supplemented with L-glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100 µg/mL) (Invitrogen), and Na235SO4 (0.7 µCi/mL; Amersham, Les Ulis, France). After incubation for 3 h (37°C, 5% CO2), patellae were washed with 1 mL of cold physiologic saline and fixed overnight in 0.5% cetylpyridinium chloride (Sigma) in phosphate-buffered formalin. After decalcification in 5% formic acid for 6 h, the central area of the patella was sampled with a 2 mm-diameter biopsy punch (Stiefel, Nanterre, France). The punched-out part of the patellae was dissolved overnight in 0.5 mL of Soluene-350 (Packard, Silic, France). The amount of 35S-sulfate incorporated in each compartment was counted using scintillation fluid (Hionic Fluor, Packard) in a beta counter (Packard).
Time course of GFP expression
Fluorescence
Three-dimensional optical sections were analyzed with an Olympus IX-70 epifluorescence inverted microscope equipped with the CellScanTM optical sectioning acquisition system (EPRTM, CSP Inc., Scanalytics, Fairfax, VA, USA) and using a 63xPSF/1.25 NA oil immersion objective. The scanning along the optical axis was performed using a piezo z-axis focus device (z-spacing of 0.25 µm) and images were then collected on a cooled 12 bit Charge-Coupled Device camera (Princeton Instruments, Inc., Monmouth Junction, NJ, USA). An image intensity calibration kit (Inspeck, Molecular Probes, Eugene, OR, USA) was used for calibration and a GFP-specific filter set (Olympus, Melville, NY, USA) selected the integral part of the GFP emission spectrum. For the deconvolution, the blurring function of the optical system was characterized by imaging a through-focus series of optical sections of a 0.17 µm-diameter green fluorescent bead (Microscope Point Source Kit, Molecular Probes) and the deblurring process was applied as described previously (16)
. Both a blank image of the detector dark current and the background were subtracted pixel-by-pixel from each acquired frame.
Immunohistochemistry
Deparaffinized sections were permeabilized with trypsin (Serlabo, Bonneuil-Sur-Marne, France) 0.1% in CaCl2 0.1% for 30 min at 37°C. Joint tissues were incubated with a GFP-specific antibody (1/50, w/v, rabbit anti-GFP antibody, Clontech, Montigny-Le-Bretonneux, France). Slices were then subjected to standard immunodetection procedures with an amplification system (TSA plus DNP HRP System, Perkin-Elmer Life Sciences, Courtaboeuf, France) used according to manufacturer’s recommendations. Color was developed with 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Quentin Fallavier, France) containing hydroxide peroxide and slides were counterstained for 3 min in 1% methyl green, giving green-stained nuclei. The total number of chondrocytes and the number of cells with a positive GFP immunostaining were evaluated in three zones of cartilage under the microscope at x640 magnification (40x16). Results were expressed as the percentage of staining-positive chondrocytes.
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RESULTS
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Feasibility
In a first set of experiments, we undertook optimization of the experimental parameters to obtain efficient transfection of hyaline rat patellar cartilage. The parameters previously used for muscle EP (8 pulses, 200 V/cm, 20 ms, 1 Hz) were initially applied to six rat knees after the i.a. of the DNA. No GFP fluorescence was observed 48 h later in the patella under these experimental conditions. We thus investigated the effects of pulses of larger field strength: at 250 V/cm a typical GFP signal was detected after tissue slice observation with an epifluorescence microscope (n=14 knees, 2 experiments) (Fig. 2
).
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Figure 2. Fluorescence images of the expressed GFP in the rat patellar cartilage. The rat joint is injected with 30 µg of pcDNA3-CT-GFP plasmid DNA and is submitted to 8 electric pulses (20 ms, 1 Hz, 250 V/cm). Three control groups were realized, in which animals are submitted to pDNA injection without electric pulses and to electric impulsions only. Animals were killed at different times and patellae were prepared for histological analyses. Sections of the patella (from each group) were observed on an epi-fluorescent microscope (equipped with the CellScanTM optical sectioning acquisition system). Each image was captured and processed under the same conditions. Fluorescence pictures of patellar cartilage from A) control group (injection of pDNA without external electric pulses): no GFP signal is detected in the patella of this animal (superficial and deep layers). Electroporated rats B) 48 h after electro gene transfer (EGT); C) 1 month after EGT; and D) 2 months after EGT. Bar scale: 18 µm. NB: GFP positively stained cells (35.8% of total surface cells) were observed in the surface of rat patellar cartilage 48 h after gene transfer. However, after 1 wk the number of stained cell dramatically decreased to <5% of the cells of the patellar cartilage superficial area. Two weeks after gene electrotransfer, only few isolated GFP positively stained cells were detected in the superficial layer (<1%). Note that epi-fluorescence pictures represent only some areas of the patellar cartilage, not the entire thickness.
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Localization of the GFP expressing cells was heterogeneous. Cells were not uniformly widespread in the whole cartilage, probably due to 1) the site of i.a. injection or 2) the heterogeneity of the electric field distribution resulting from some variability in the electrode location. Nevertheless, expression of the recombinant protein was found in the whole cartilage, from the superficial layer to the deepest part. On the contrary, no GFP spot was observed after 48 h, when the reporter gene was injected i.a. without the electric pulse delivery. To confirm that the signal observed in the treated rats is not linked to autofluorescence and is the direct consequence of the expression of the electrotransferred GFP gene, immunostaining was performed on adjacent sections with an antibody able to specifically detect the GFP: the results led to the same conclusions (data not shown). No staining against GFP was detected in naive rats or rats exposed to the EP in the absence of pDNA injection.
Safety
In a second type of experiments, we assessed the safety (the so-called "chondroneutrality") (17)
of the electric pulse delivery to the patellar cartilage. Compared with age-matched naive untreated rats, histological examination of the electropulsed patella suggested that the EP, with or without pDNA, produced no tissue damage from day 2 to day 60, as shown from analysis of the cartilage surface, cellularity, and ECM staining using normal and polarized light microscopy (n=8 knees/experiment, 2 experiments). The histological analysis also revealed that EP had no influence on the physiological maturation process of the tissue (data not shown). As the hypertrophic layer is mainly involved in patellar cartilage maturation (18)
, particular attention was paid to this area: no morphological differences of hypertrophic chondrocytes in the deep layer of patellar cartilage were observed at the various times of death in the rat batches (EGT, EGT+pDNA, pDNA).
Moreover, radio sulfate intake in rat patellar cartilage assessed on days 2 and 9 (5 rats, i.e., 10 patellae per group) was similar in control and electropulsed groups, indicating that EP did not alter chondrocytes metabolism (1646±93 c.p.m. vs. 1523±209 c.p.m. on day 2 and 1339±235 vs. 1241±170 on day 9, mean ± SD, control vs. electroporated rats respectively, no statistical difference, Student’s t test). Moreover, body weight gain was similar in the control and EP rats throughout the follow-up. Of the 56 rats used, only one death was scored (which occurred before the treatment) at the time of anesthesia induction. No burning was observed on the skin located under the electrodes (112 knees).
Time course of GFP expression
To investigate the long-term effects of electro gene transfer (EGT) in articular cartilage, rats were injected i.a. with pDNA in both knees and exposed to the EP. Two days (48 h) later, >65% of the patellar chondrocytes were positively stained using an anti GFP-antibody and the distribution of GFP stained cells covered the superficial layer of the cartilage as well as the deeper layers. Then the number of positive cells decreased from 65% to 
30% in 1 wk in all the experiments (Fig. 3
). Thus, there was a rapid reduction of GFP expression, but the stable level was maintained throughout the entire follow-up of the experiments (up to 2 months).
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Figure 3. Time course of GFP gene expression in rat patellae after electroporation. Rats received i.a. 30 µg of plasmid pcDNA3-CT/GFP in 50 µL of NaCl (0.9%), followed by electric pulses (8 pulses, 20 ms, 1 Hz, 250 V/cm). At the indicated time after electroporation, rats were killed and joint tissues were classically prepared for immunohistochemistry with a specific antibody directed against GFP. The total number of chondrocytes and the number of chondrocytes staining positive for GFP using the specific antibody were evaluated separately for each zone of cartilage (superficial and deep zones). Histogram represents the cell score for GFP expression in whole patellar cartilage at different times. Bars show the mean and SEM score (n=6 knees per group). At 48 h, GFP-positive cells were detected in the superficial (35.8±8.8%) and deep areas (68.7±5.8%) of the whole cartilage. After 1 wk, cells expressing GFP are mainly found in the deep layer of cartilage (hypertrophic chondrocytes), only a few cells (<5%) in the superficial zone of electroporated animals.
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One interesting reproducible observation in all the sections analyzed was the localization of the GFP-positive cells in the patellar cartilage. The three areas of the cartilage (superficial, middle, and deep zones) presented positively stained cells 48 h after administration of the electric pulses. However, after 1 wk the stained cells were hypertrophic chondrocytes localized mainly in the deep zone (Fig. 4
), and this particular distribution was still clearly seen after 1 or 2 months. We examined the rest of the joint to detect a possible dissemination of the pDNA to other compartments of the knee. Analyses by fluorescence revealed that GFP signals were detectable in some areas of the joint, but the number of cell was always significantly lower (5% of GFP-positive cells) than in the patellae, indicating that EGT actually allowed the local delivery of the DNA mainly to the targeted tissue.
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Figure 4. Detection of GFP in rat patellar cartilage by immunohistochemistry after in vivo electro gene transfer (EGT). Plasmid pcDNA3.1/CT-GFP (30 µg in 50 µL of 0.9% NaCl) was injected in the intra-articular space joint of rats and animals were submitted to electroporation (8 pulses, 20 ms, 1 Hz, 250 V/cm). Rats were killed after transfection and tissues were prepared for immunohistochemistry with a GFP-specific antibody (1/50). Cells expressing the reporter gene (GFP) developed brown staining (black arrows), whereas no signal was observed in sections from control group. (Original magnification: x640.) Patellar cartilage harvested 48 h after EGT. A) Superficial area from control group. B) Deep layer harvested from control group. C) Superficial area from electroporated rats. D) Deep layer from electroporated rats. Patellar cartilage harvested 2 months after EGT. E) Superficial area from control group. F) Deep layer from control group. G) Superficial area from electroporated rats. H) Deep layer from electroporated rats.
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DISCUSSION
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We report an original approach for nonviral gene delivery in the cartilage that consists of i.a. injection of plasmid DNA, followed by the delivery of electric pulses through external electrodes. The results presented here demonstrate that in vivo EP provides an efficient approach for tissue-targeted local cartilage expression with a relatively high and stable level of expression in spite of the density of the cartilage ECM. To the best of our knowledge, such an approach using EGT has never been reported for targeting cartilage in vivo.
Most authors have restricted their research to in vivo viral methods and strategies that target the synovium in a chondroprotective mode (reviewed in ref 19
). In contrast, there are few data concerning in vivo viral gene transfer in articular chondrocytes using adeno-associated virus (20)
or Sendai virus vectors (HVJ-liposome) (21)
in rats, resulting in the transformation of the cells of the superficial and middle layers. However, these vectors present the risk of systemic dissemination of the virus and its transgene, as well as production of neutralizing antibodies that limit the ability to readminister these viruses (22)
. Therefore, the indirect ex vivo transfection of autologous chondrocytes (23)
or progenitor cells (24
, 25)
with genes coding for growth factors [e.g., TGF-ß1 (26)
, IGF-1 (27)
], followed by their expansion in culture and surgical reimplantation into a focal chondral lesion has also been developed.
One of the main limitations of other nonviral approaches is the lack of an efficient delivery method to focus the genes of interest into the targeted tissues, particularly to the deepest parts of the tissues. Our results show that a strong expression of the reporter gene was observed in 65% of the cells in the patella 48 h after the EGT, but that this level decreased rapidly to 20–30% 1 wk later. However, it is noteworthy that this level of transgene expressing patellar chondrocytes was maintained for at least 2 months. During this period the GFP signal was detected mainly in the hypertrophic chondrocytes of the deepest layer and not in the superficial area of the patellar cartilage. This is not due to a difference of intensity of the electric field in the deeper layer but, in contrast, inherent to the fact that the large cells are more susceptible to the external electric fields: the change in the cell transmembrane potential change (deltaVm, 
Vm) induced by the external fields (of field intensity E) is the primum movens of the cell electroporation. At the point M of the cell surface, defined by its polar angle tétha (
) with respect to the direction of the field, the Vm is given by the equation of Schwann:
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where R is the radius of the cell. Therefore, the larger the radius of the cell will be, the larger will be the Vm (thus the larger the effects) obtained for an identical external field E.
Additional studies are needed to determine whether the chondrocytes localized in the superficial layer of cartilage have lost the transgene or whether its expression is just repressed (or silenced) (28)
. Our preliminary results (internal data) clearly suggest such a silencing process, because in situ hybridization demonstrates the persistence of pDNA in superficial and middle areas even 2 months after EP (Fig. 5
). Further studies with laser microdissection are needed to demonstrate that chondrocytes have kept the construction. This method allows sampling of specific regions of interest of the patella and ancillary genetic analysis with PCR.
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Figure 5. Detection of pDNA in rat patellar cartilage by in situ hybridization 2 months after in vivo electro gene transfer (EGT). Rats received, intra-articularly, 30 µg of plasmid pcDNA3-CT/GFP in 50 µL of NaCl (0.9%), followed by electric pulses (8 pulses, 20 ms, 1 Hz, 250 V/cm). At month 2, rats were killed and joint tissues were classically prepared for in situ hybridization (see ref 29
). A digoxigenin DNA-labeled probe was generated by PCR reaction (PCR Dig probe synthesis Kit, Roche) according to manufacturer’s recommendations with minor modifications, using CMV promoter specific primers. (Magnification x 640.) A) Superficial zone of patellar cartilage. B) Deep zone (hypertrophic chondrocytes) of patellar cartilage. No immunostaining is present in control rats.
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Our present results on normal rat joints also demonstrate that no direct chondrotoxicity is observed after the injection of naked DNA, followed by the pulsed electric fields delivery. The internal structure of the articular cartilage, as well as its biochemical composition, is not modified by the electric pulses, an absolute requirement for the development of a therapeutic approach based on the electro gene transfer. Studies of other tissues or compartments of the joint reveal a slight dissemination of pDNA, generally in the fibrocartilage, the synovial membrane, and the tendon. After 1 wk, and even 2 months later, >95% of the GFP staining is observed in the patella and only a few cells still express GFP in the rest of the joint. The use of EGT to deliver genetic information permits transfection of the whole tissue and not only of the superficial area, as observed when virus vectors or lipoplexes are used.
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CONCLUSIONS
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In rheumatic diseases, chondrocytes are scanty and nonreplicant, particularly in the degenerative hyaline cartilage. For treatment of these arthropathies by gene therapy, it is necessary to achieve a continuous expression of the therapeutic gene by a large number of the remaining chondrocytes. With this in mind, as a consequence of the data reported here, EP targeting of chondrocytes with therapeutic genes appears potentially useful and can represent a nonimmunogenic, eventually repeatable treatment of focal chondral defects or diffuse degenerative diseases of the joint. This kind of gene delivery method could be suitable to develop a "tissue engineering" approach to maintain cartilage homeostasis or to promote cartilage regeneration (e.g., with growth factors) by introducing the appropriate transgene in the cartilage cells. Moreover, this simple and safe method allows the development of new therapies for joint diseases, relying on local delivery and long-term expression of therapeutic transgenes, which could be delivered at the same time in the targeted tissue, thus leading to a genetically engineered tissue used as an artificial factory to produce therapeutic proteins.
Despite its limited use at present, in certain aspects EP is superior to other nonviral gene transfer methods, including lipofection, gene gun, and, of course, direct injection of DNA. A specific feature of EP, particularly interesting in articular disease, is that electrodes of corrected size must be placed along the joint in an adapted way to deliver well-designed electric pulses after intra-articular injection of the gene of interest. This kind of nonviral delivery approach should be improved by using site-specific promoters to restrict transgene expression to the targeted tissue, increasing the safety of this method. Finally, the proof of concept here reported encourages further development of EGT approaches to the direct treatment of chondral diseases.
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ACKNOWLEDGMENTS
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This study was supported by grants from the GIP Fonds de Recherches Aventis (FR99RHU037), the CNRS (Jeune Equipe 2000), the Région Lorraine, Institut Gustave-Roussy, and the EU commission (grant QLK3-CT-1999-00484). The authors acknowledge Bernard Terlain and Jean Yves Jouzeau for their expert comments.
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FOOTNOTES
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1 Both authors contributed equally to this work. 
Received for publication May 31, 2002.
Accepted for publication January 10, 2003.
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ABSTRACT
INTRODUCTION
