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Intracellular Localization of Ornithine Decarboxylase and Its Regulatory Protein, Antizyme-1

Raymond G. Schipper, Vincent M.J.I. Cuijpers, Linda H.J.M. de Groot, Marco Thio and Albert A.J. Verhofstad

Department of Pathology, University Medical Centre Nijmegen, Nijmegen, The Netherlands

Correspondence to: Dr. A.A.J. Verhofstad, Department of Pathology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: A.Verhofstad{at}pathol.umcn.nl


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocytoplasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function.

(J Histochem Cytochem 52:1259–1266, 2004)

Key Words: ornithine decarboxylase • antizyme-1 • polyamine • localization • immunocytochemistry • green fluorescent protein


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
ORNITHINE DECARBOXYLASE (ODC), EC 4.1.1.17, the initial and rate-limiting enzyme in the biosynthetic pathway of polyamines, is involved in the conversion of ornithine to putrescine. Polyamines are multifunctional organic bases that play an essential role in cell growth, cell differentiation, apoptosis, and malignant development (for reviews, see Pegg 1986Go; Cohen 1998Go; Schipper et al. 2000Go). Rapid and dramatic regulation of ODC expression is possible because of its extremely short half-life of several minutes to 1 hr (the shortest half-life of any known enzyme). Its degradation is regulated by a unique regulatory protein named antizyme (AZ), which is induced by polyamine-mediated translational frameshifting (for reviews, see Ivanov et al. 2000Go; Coffino 2001Go). AZ binds reversibly to monomers of ODC and promotes its proteolytic degradation by the 26S proteasome. Four independent isoforms of AZ have been described, of which AZ1 and AZ2 have a wide tissue distribution, while AZ3 is testis specific (Ivanov et al. 2000Go). AZ4 is presently known only as an expressed sequence tag (Coffino 2001Go). AZ1 is the most-studied and ubiquitous member of the AZ gene family.

Despite extensive research, the precise role of the ODC/polyamine system in cellular physiology remains to be clarified. Lack of knowledge of the exact intracellular localization of ODC and/or polyamines is one of the main obstacles to a more precise interpretation of the biological role of the ODC/polyamine system. Previous localization studies of ODC in various cell types showed different patterns that varied from exclusively cytoplasmic to both cytoplasmic and nuclear (Schipper and Verhofstad 2002Go). This might indicate that the (sub)cellular localization of ODC is cell type–specific and/or depends on the physiological status (growth, differentiation, malignant transformation, apoptosis) of cells. Furthermore, intracellular transport of ODC may be a prerequisite for its regulation and function.

To study the intracellular distribution of ODC and AZ1 in more detail, we performed immunocytochemical studies of ODC and AZ1 in cultured cells of human cervix carcinoma (HeLa) and human prostatic carcinoma (PC-346C) cells. In addition, fusion proteins consisting of ODC or AZ1 and enhanced green fluorescent protein (EGFP) were constructed and transiently expressed in HeLa and PC-346C cells. To investigate whether the localization pattern of ODC and AZ1 is subject to change during progression through the cell cycle, we performed immunocytochemical studies in HeLa and PC-346C cells that were synchronized in the mitotic phase. In addition, we studied the effect of putrescine and the polyamine analog SL-11093, known inducers of AZ1 expression, on the localization pattern of AZ1 and ODC.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Cell Culture
Human cervix carcinoma (HeLa) or human prostatic carcinoma (PC-346C) cells were cultured in Dulbecco's modified Eagle's medium (BioWhittaker Europe; Verviers, Belgium) supplemented with 10% fetal calf serum (FCS) (BioWhittaker Europe) and the antibiotic agent gentamycin (40 µg/ml) (Centrafarm; Etten-Leur, The Netherlands). Cultures were maintained in a humidified atmosphere of 5% CO2 in air at 37C. For cell passages, cells were washed with a physiological salt solution (0.9% NaCl) and detached by adding a solution of 0.5 mg/ml trypsin (Sigma Chemicals; St. Louis, MO) and 0.2 mg/ml ethylene diamino tetra-acetic acid in phosphate-buffered saline (PBS).

For cytochemical purposes, cells were grown on 12-well glass slides (Carl Roth; Karlsruhe, Germany). Slides were sterilized with ethanol, washed three times with a physiological salt solution (0.9% NaCl), and transferred to culture dishes. Suspended cells were added to each well (35 µl/well) of the glass slides and allowed to attach at room temperature. Subsequently, cell culture medium was added to the culture dish to cover the glass slides. Dishes were kept at 37C for at least 24 hr before further procedures.

Cell Synchronization
For cell synchronization, HeLa cells were treated for 16 hr with nocodazole (0.4 µg/ml) (Sigma Chemicals), a microtubule-targeted inhibitor that arrests cells at the prometaphase of mitosis (Zieve et al. 1980Go). According to earlier studies, cells can progress through mitosis to G1 in ~2–3 hr after removal from nocodazole (Knehr et al. 1995Go). Cells were harvested at 0 min, 30 min, 1 hr, 2 hr, and 10 hr after removal from nocodazole.

Enhanced Green Fluorescent Protein
Human cDNA of ODC was subcloned into expression vectors pEGFP-N3 and pEGFP-C1 (Clontech Laboratories; Woerden, The Netherlands) with EGFP attached to the 3' end or 5' end of ODC, respectively. To generate EGFP-AZ1 fusion proteins, a cDNA fragment of deltaAntizyme-1 (dAZ1) was used. In this dAZ1, nucleotide 248 is deleted to join ORF1 and ORF2. Because of this, no frameshift by polyamines is needed for the expression of AZ1. dAZ1 was amplified from pAlter-Ex1 cloning vector (Promega; Leiden, The Netherlands) and subcloned into expression vectors pEGFP-C1 and pEGFP-N3. Cells were grown in 6-well culture plates 1–3 days prior to transfection. When the monolayer culture reached ~70% confluency, cells were washed prior to transfection with a physiological salt solution and fed with medium without serum. Cells were transfected with 2 µg of either EGFP-ODC, ODC-EGFP, EGFP-dAZ1, dAZ1-EGFP, or control vector encoding only EGFP using FuGENE 6 Transfection Reagent (Roche; Mijdrecht, The Netherlands) according to the manufacturer's instructions. As a control, cells were included that underwent the same transfection procedure using sterile milliQ water (MilliQ Plus; Millipore, Molsheim, France) instead of 2 µg plasmid.

Approximately 18 hr after transfection, the medium was replaced with fresh medium supplemented with 10% FCS after transfected cells had been washed twice with physiological salt solution. One or two days after transfection, cells were trypsinized and cultured on glass slides. After culturing for at least 24 hr, slides were washed in PBS and immediately analyzed for the detection of EGFP by fluorescence microscopy or fixed for further immunocytochemical analysis.

Immunocytochemistry
After cross-linking fixation with phosphate buffered, pH neutral, 4% paraformaldehyde for 1 hr at 4C, cells were permeabilized with methanol (100% and 50%), for 15 min at –20C at each step.

Localization of ODC was studied using an indirect immunofluorescence method as described previously (Schipper et al. 1999Go). Monoclonal anti-ODC MP16-2 (Neomarkers; Fremont, CA) against epitope P16, consisting of amino acids 355–360 of ODC (Schipper et al. 1993Go), was diluted in PBS containing 2% BSA and 0.1% Triton X-100. The same buffer was used for the dilution of rabbit polyclonal anti-AZ1 antiserum (obtained from O. Heby and J. Nilsson, Umeå University, Sweden). Slides were washed for 15 min in PBS and incubated with primary antibodies overnight at 4C. Monoclonal MP16-2 antibody was detected with a secondary antibody, i.e., goat anti-mouse immunoglobulin conjugated with Alexa 488 or Alexa 594 (Molecular Probe; Eugene, Oregon). Bound AZ1 antibodies were subsequently demonstrated with donkey anti-rabbit immunoglobulin conjugated with Alexa 594 or Alexa 488. All fluorescent secondary antibodies were diluted (1:100) in the same solutions as used for the primary antibodies and incubated for 30 min in the dark at room temperature after slides were washed for at least 20 min in PBS. To stain the nuclei, cells were then covered for 15 sec with a 4',6-Diamidino-2-phenylindole (DAPI) solution. Slides were mounted in Fluorteck (Euro-Diagnostica; Arnhem, The Netherlands). To identify the antibodies conjugated with Alexa fluorchrome 488 or 594 and nuclei stained with DAPI solution, we used a fluorescent microscope with bandpass (512/542 nm) and longpass (590 nm, 425 nm) filters (Leica; Solms, Germany), respectively.

Effects of Polyamines and Polyamine Analogs on AZ1 Induction
To investigate the induction of immunoreactive AZ1 by polyamines, we added putrescine or the polyamine analog SL-11093 (S'LIL; Madison,WI) to cell cultures to induce AZ1 expression. Putrescine (1 mM) (Sigma Chemicals) was added for 24 hr to the culture medium, followed by fixation and immunocytochemical detection of AZ1. Earlier studies showed a cytotoxic effect of polyamines on cells in culture medium containing ruminant serum (Schipper et al. 2000Go and references therein). Therefore, as a standard practice, polyamines in culture medium were supplemented with aminoguanidine (1 mM) (Sigma Chemicals), an inhibitor of serum amine oxidases, to prevent oxidation. Polyamine analog SL-11093 (5 µM) was added for 3 hr, 24 hr, or 48 hr to the culture medium to analyze its effect on AZ1 expression by immunocytochemistry.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Immunocytochemistry
Immunostaining of formaldehyde-fixed cultured HeLa cells showed ODC localization patterns varying from predominantly cytoplasmic to both cytoplasmic and nuclear. In both cases, an intense perinuclear staining was observed (Figure 1A) . AZ1 localization varied from cytoplasmic, with intense (peri)nuclear staining, to exclusively nuclear (Figure 1B). Notably, the perinuclear signal was especially strong when the signal of both ODC and AZ1 was of low intensity in both nucleus and cytoplasm. The majority of cells showed a cytoplasmic staining of ODC, whereas AZ1 immunoreactivity was most frequently found in the nucleus. Immunostaining of PC-346C cells showed distribution patterns similar to those in HeLa cells, although less nuclear staining of ODC and AZ1 was found in PC-346C cells, compared with HeLa cells (data not shown).



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Figure 1

Localization of ODC and AZ-1 in cultured HeLa cells. Immunostaining of ODC (A) and AZ1 (B) shows that ODC is located predominantly in the cytoplasm, whereas AZ1 immunoreactivity is most frequently found in the nucleus of cells. Note the intense perinuclear staining in both ODC- and AZ1-stained cells. Cells transfected with ODC-EGFP (C,E) and AZ1-EGFP (D,F) show identical results. Bar = 5 µm.

 
Transfection Studies with EGFP Constructs of ODC or dAZ1
Cells that were transfected with EGFP displayed homogeneous EGFP expression throughout cytoplasm and nucleus (results not shown).

HeLa cells expressing ODC-EGFP (Figures 1C and 1E) displayed cytoplasmic and/or nuclear localization patterns comparable to the distribution of ODC observed in the immunocytochemical studies. dAZ1-EGFP appeared primarily in the nucleus (Figures 1D and 1F), which was also the predominant site of AZ1 in the immunocytochemical studies. In both ODC-EGFP- and dAZ1-EGFP-transfected cell cultures, a subset of cells displayed a strong, exclusively nuclear, fluorescence signal (Figures 1E and 1F). Some regions in the nucleus, probably representing the nucleosomes, were devoid of signal.

Transfection with constructs containing EGFP fused at the 3' end of ODC and dAZ1 (EGFP-ODC and EGFP-dAZ1) did not produce any fluorescent signals in the cells. Transfection problems may be responsible for this, but it is more likely that the translation of both proteins is inhibited when EGFP is attached at the N terminus.

Immunocytochemistry of Cells Transfected with EGFP Constructs
Immunostaining of ODC in HeLa cells that were transfected with ODC-EGFP clearly showed an increased ODC immunoreactivity. In cells that displayed only the cytoplasmic ODC-EGFP signal, an increased cytoplasmic ODC immunostaining was also found (results not shown). Problematically, the subset of cells in which the green fluorescence signal of ODC-EGFP was predominantly localized in nuclei (Figure 2A) showed also an increased but more perinuclear and cytoplasmic immunostaining of ODC (Figures 2B and 2C).



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Figure 2

Immunocytochemical studies of ODC and AZ1 in HeLa cells transfected with ODC-EGFP (A–E) or dAZ1 (F,G). ODC immunoreactivity (B,C,G) is only increased in ODC-EGFP-transfected cells (B,C versus G). ODC immunostaining patterns differ from ODC-EGFP (B,C versus A) or dAZ1-EGFP (G versus F). Immunostaining patterns of AZ1 in ODC-EGFP-transfected cells show a colocalization of immunoreactive AZ-1 protein and the ODC-EGFP construct (E versus D). In (C), nuclei are stained with DAPI. Bar = 5 µm.

 
AZ1 immunoreactivity was not increased in dAZ1-EGFP-transfected cells (results not shown) probably due to the inability of the AZ1 antiserum to recognize the AZ1-EGFP fusion protein.

Immunostaining of AZ1 (Figure 2E) in ODC-EGFP-transfected cells (Figure 2D) showed colocalization of AZ1 and ODC in the nuclei. In contrast, immunolocalization of ODC in dAZ1-EGFP-transfected cells showed that AZ1-EGFP was located primarily in the nuclei (Figure 2F), whereas ODC localization was predominantly perinuclear (Figure 2G).

According to earlier studies (Coffino 2001Go and references therein), incubating cells with high levels of polyamines or polyamine analogs induces polyamine downregulation by stimulation of AZ1 expression. In the present study, AZ1 expression was clearly induced in the nucleus of PC-346C cells treated with putrescine (Figure 3B) or polyamine analog (Figures 3D and 3F) and colocalized with ODC-EGFP (Figure 3G).



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Figure 3

Immunostaining of AZ1 in PC-346C cells treated without (A,C,E) or with (B) polyamine or polyamine analog (D,F) for 24 hr. AZ staining is increased in the nucleus after treatment of polyamine or polyamine analog and colocalizes with ODC-EGFP (F versus G). Bar = 5 µm.

 
Synchronization Studies
After nocodazole was eliminated from the culture media, ~90% of the cells were rounded up, which indicated that these cells were in mitosis. Typical results of mitotic HeLa cells are shown in Figure 4 . PC-346C cells displayed more or less the same distribution patterns.



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Figure 4

Immunocytochemical studies of ODC and AZ1 in semisynchronized cultures of HeLa cells. Cells were blocked in cell proliferation at (pro)metaphase of cell cycle with nocodazole. At time points between 0 and 3 hr after release from nocodazole, cells were transferred from culture medium to fixative solution, followed by immunocytochemical staining of AZ1 (green boxes) or ODC without (red boxes) or with (blue boxes) DAPI staining. ODC and AZ1 showed similar patterns. At the beginning of (pro)metaphase, an increased perinuclear localization was observed, followed by moderate, more-even staining between chromosomes. During meta-, ana- and telophase, immunoreactivity was located around the chromosomes. Finally, at the end of telophase and during cytokinesis, ODC and AZ1 were detected outside the newly synthesized nuclear envelope.

 
Results indicate that at the time cells were released from nocodazole, ODC and AZ1 were localized primarily in perinuclear regions. In the metaphase, ODC and AZ1 were found to be distributed equally among the chromosomes. Strong positive signals in the vicinity of the chromatids were also observed during anaphase. At the beginning of telophase, we observed two equally brightly stained spherical shapes still connected to each other. When the nuclear envelope was synthesized again during telophase and cytokinesis, ODC and AZ1 were not present in the new nuclei but were located predominantly in perinuclear regions.

Although ODC and AZ1 displayed similar patterns, ODC staining was more intense than AZ1 staining.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Localization Patterns of ODC and AZ1
The exact intracellular localization of ODC and its regulatory proteins is still a matter of debate, because previous studies using various methods (biochemistry, enzyme cytochemistry, immunocytochemistry, and RNA in situ hybridization), showed strikingly variable results (Schipper and Verhofstad 2002Go).

In the present study, we used immunocytochemistry as well as GFP tagging to localize ODC and its regulatory protein, AZ1, in cultured cells. Results show that in most cases, ODC was present in the cytoplasm, whereas AZ1 was found primarily in the nucleus. Remarkably, as revealed by immunocytochemistry, an intense perinuclear staining was observed for both ODC and AZ1 in most cells.

The predominant presence of ODC in the cytosol is in agreement with the vast majority of earlier studies, which showed a cytoplasmic localization of ODC (Schipper and Verhofstad 2002Go and references therein). The role of the ODC/polyamine system in the cytoplasm still needs to be elucidated, but numerous studies suggest that polyamines play essential roles in the machinery of protein biosynthesis. This hypothesis is further supported by immunoelectron microscopical studies of ODC and polyamines, in which the label was closely associated with the rough endoplasmic reticulum and polyribosomal structures, respectively (Morisset et al. 1986Go; Fujiwara et al. 1998Go).

A subset of cells showed a strong ODC-EGFP signal (Figures 1E, 2A, and 2D) or intense immunostaining of ODC (Figure 1A) in the nucleus. Our earlier studies on ODC localization in other cell lines showed that the strongest staining for ODC was found in the nucleoplasm of mitotic cells (Schipper et al. 1999Go). Therefore, this subset of cells may actually represent the mitotic fraction of the cell culture, suggesting a role of ODC in mitosis, as will be discussed later.

Information on the intracellular localization of AZ1 is scarce, but recent studies have indicated an accumulation of AZ1 in the nucleus after inhibition of protease activity (Gritli-Linde et al. 2001Go) or nuclear export (Murai et al. 2003Go). In addition, the amino acid sequence of AZ1 exhibits signals that are involved in nucleocytoplasmic shuttling (Gritli-Linde et al. 2001Go; Murai et al. 2003Go).

Immunocytochemistry versus GFP-tagged Expression
We found a discrepancy when we used the two different localization methods (immunocytochemistry, GFP-tagged expression) to detect ODC within the same cell. Immunocytochemical studies of ODC in cells that were transfected with ODC-EGFP showed that the ODC antibodies were able to detect the ODC fusion protein in the cytoplasm but not in the nucleus. This discrepancy may mean that nuclear ODC exists in a complexed or cryptic form that is less accessible for antibodies to bind. An obvious candidate for forming a complex with ODC is AZ1, which colocalizes with nuclear ODC, as discussed in the next paragraphs.

Localization of ODC and AZ1 during Mitosis
The distinct localization patterns of ODC and AZ1 that were observed in asynchronously growing cell cultures may actually reflect different time points during the cell cycle. Our immunocytochemical studies in semisynchronized cells revealed a nuclear (co)localization of ODC and AZ1 during mitosis, whereas just before and immediately after mitosis, translocation to a perinuclear/cytoplasmic site occurs.

The observation that ODC and AZ1 are located perinuclear in the G1 phase (which is approximately half the cell cycle time) confirms our immunocytochemical data with nonsynchronized cells showing that the perinuclear localization of AZ1 and ODC is the most predominant location compared with all other patterns. In addition, this also may explain why we detected more nuclear staining of ODC and AZ1 in HeLa cells than in PC346 cells (data not shown), inasmuch as HeLa cells proliferate at a higher rate.

Our data show a generally increased expression of ODC in mitotic cells. Recent studies indicate that ODC translation in the G2/M transition is regulated in a cap-independent way by using a ribosomal entry site (Pyronnet et al. 2000Go). Therefore, general inhibition of ODC translation in the G2/M phase of the cell cycle can be overcome and polyamines can be produced when needed for mitotic spindle formation and chromatin condensation. We observed a strong expression of ODC at the periphery of the chromosomes during the second half of mitosis and during cytokinesis, a cell phase in which microtubules play essential roles. These results are in close agreement with those of previous studies (Pomidor et al. 1995Go; Heiskala et al. 1999Go) that indicated that changes of ODC activity and translocations are associated with rearrangements of the cytoskeleton, suggesting attachment of ODC to structural elements and/or a role of ODC in cytokinesis.

On the other hand, its colocalization with AZ1 implies that ODC may actually be degraded during mitosis. Biochemical studies of polyamine regulatory proteins during the cell cycle of normal human dermal fibroblasts show an accumulation of AZ1 transcripts in the G2/M transition, whereas the level of ODC transcripts, as well as ODC activity, diminishes in the mitotic phase (Bettuzzi et al. 1999Go). Inhibition studies of these genes in human prostatic epithelial cells indicate that cyclic polyamine depletion is important for cell cycle progression (Scorcioni et al. 2001Go). The authors of these studies hypothesize that downregulation of polyamine biosynthesis is needed for completion of the mitotic phase, enabling cells to enter a new round of replication.

Is AZ1 Involved in the Nuclear Translocation of ODC?
Our studies show that nuclear localization of ODC is closely related to (co)localization of AZ1. First, induction of AZ1 by polyamines or polyamine analogs promotes an accumulation of ODC as well as AZ1 in the nucleus. ODC is possibly trapped by AZ1 in the cytoplasm and translocated into the nucleus for degradation by nuclear proteases. Second, ODC and AZ1 colocalize in mitotic cells. These observations imply that AZ1 is involved in the intracellular translocation of ODC, which may be a prerequisite for its regulation and function.

In conclusion, intracellular localization of ODC and its regulatory protein, AZ1, is subject to highly dynamic processes. Translocation of ODC by AZ1 may be an important mechanism in the control of the expression and/or function of ODC.


    Acknowledgments
 
We thank Prof. Dr. Lo Persson (Department of Physiological Sciences, University of Lund, Sweden) and Prof. Dr. Olle Jänne (Department of Pathology, University of Helsinki, Finland) for generously providing us with the human ODC cDNA. We gratefully acknowledge Prof. Dr. Olle Heby and Dr. Jonas Nilsson (Department of Molecular Biology, Umeå University, Sweden) for kindly supplying the AZ1 cDNA and AZ1 antiserum. We also thank Dr. F. van Kuppenveld (Department of Medical Microbiology, University Medical Centre Nijmegen) for preparing the ODC-EGFP constructs.

Supported by the Dutch Cancer Society (grants NKB-93-599 and NKB-98-1807) and the Nijbakker Morra Foundation.


    Footnotes
 
Received for publication May 14, 2004; accepted July 10, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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