RAPID COMMUNICATION |
Intracellular Localization of Ornithine Decarboxylase and Its Regulatory Protein, Antizyme-1
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
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Summary |
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(J Histochem Cytochem 52:12591266, 2004)
Key Words: ornithine decarboxylase antizyme-1 polyamine localization immunocytochemistry green fluorescent protein
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Introduction |
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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 2002). This might indicate that the (sub)cellular localization of ODC is cell typespecific 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.
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Materials and Methods |
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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. 1980). According to earlier studies, cells can progress through mitosis to G1 in
23 hr after removal from nocodazole (Knehr et al. 1995
). 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 13 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. 1999). Monoclonal anti-ODC MP16-2 (Neomarkers; Fremont, CA) against epitope P16, consisting of amino acids 355360 of ODC (Schipper et al. 1993
), 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. 2000 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.
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Results |
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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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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 2001 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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Although ODC and AZ1 displayed similar patterns, ODC staining was more intense than AZ1 staining.
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Discussion |
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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 2002 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. 1986
; Fujiwara et al. 1998
).
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. 1999). 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. 2001) or nuclear export (Murai et al. 2003
). In addition, the amino acid sequence of AZ1 exhibits signals that are involved in nucleocytoplasmic shuttling (Gritli-Linde et al. 2001
; Murai et al. 2003
).
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. 2000). 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. 1995
; Heiskala et al. 1999
) 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. 1999). Inhibition studies of these genes in human prostatic epithelial cells indicate that cyclic polyamine depletion is important for cell cycle progression (Scorcioni et al. 2001
). 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.
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Acknowledgments |
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Supported by the Dutch Cancer Society (grants NKB-93-599 and NKB-98-1807) and the Nijbakker Morra Foundation.
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Footnotes |
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Literature Cited |
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