Distribution Patterns of Ornithine Decarboxylase in Cells and Tissues: Facts,Problems,and Postulates

Fractionated Cells

Activity of ODC can be determined by measuring production of ¹⁴CO₂ (Pegg and Williams-Ashman 1968) or [1-¹⁴C]-putrescine (Tabib 1998) from [1-¹⁴C]-ornithine. These methods allow a linear and quantitative estimate of active ODC protein in homogenates of cells and tissues.

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Table 1

Methods for in situ detection of ornithine decarboxylase

Method	Marker	Specificity	Sensitivity	Application	Reference
Biochemistry Activity measurement	14CO2	High	High	Fractionated cells	Pegg and Williams-Ashman 1968; Snyder and Russell 1970; Ono et al. 1972; Raina and Jänne 1968; Murphy and Brosnan 1976; McCormick 1977; Bitonti and Couri 1981; Bartholeyns 1983; Grillo and Fossa 1983; Pomidor et al. 1999
Enzyme cytochemistry Cytochemical CO2 trapping	CO2	Not clear	High	Tissues	Dodds et al. 1990
Labeling with irreversible enzyme inhibitor	[14C]-DFMO	High	Low	Tissues	Pegg et al. 1982; Zagon et al. 1984
	[3H]-DFMO	High	Moderate	Cells/tissues	Emanuelsson and Heby 1982; Anehus et al. 1984; Zagon et al. 1984; Morris et al. 1986; Schweitzer et al. 1988; Gritli-Linde and Linde 1994
	Rhodamine-DFMO, Biotin-DFMO	Not clear	Moderate	Tissues	Gilad and Gilad 1981
Immunocytochemistry Labeling with anti-ODC antibodies	Polyclonal antiserum	Variable	High	Cells/tissues	Persson 1982; Isomaa et al. 1983; Seely and Pegg 1983; Greenfield et al. 1986; Kahana and Nathans 1984; Pomidor et al. 1995
	Monoclonal antibody	High	High	Cells/tissues	Matsufuji et al. 1984; Nishiyama et al. 1989; Schipper et al. 1993
In situ hybridization Labeling with cDNA or cRNA probe	[3H]-UTP,[35S]-UTP, DiG-UTP	High	High	Tissues	Blackshear et al. 1989; Kaipia et al. 1990; Jänne et al. 1991; Bertuzzi et al. 1992; Gan et al. 1992; Crozat et al. 1992; Koibuchi et al. 1993b; Wright et al. 1995; Keinanen et al. 1997; Takada et al. 1998; Ohtsuka et al. 1998

The CO₂-trapping technique has been used to determine ODC activity in subcellular fractions of rat prostate (Pegg and Williams-Ashman 1968), rat liver (Ono et al. 1972; Murphy and Brosnan 1976; Bitonti and Couri 1981; Grillo and Fossa 1983; Bartholeyns 1983), and mouse fibroblasts (McCormick 1977). Most studies showed that ODC activity is mainly or exclusively found in the cytosolic fraction (Pegg and Williams-Ashman 1968; McCormick 1977; Grillo and Fossa 1983), with or without induction by growth stimuli (Ono et al. 1972; Murphy and Brosnan 1976). However, some studies demonstrated the presence of ODC in the nucleus (Snyder and Russell 1970; Bitonti and Couri 1981). On the basis of an improved fractionation procedure, Bartholeyns (1983) established that ODC activity is equally distributed over nuclear and cytoplasmic fractions in non-stimulated liver. Nuclear ODC, which was mainly found in the nucleoli, was not induced by growth stimuli, in contrast to cytoplasmic ODC. Subcellular fractionation of human keratinocytes after detergent and salt extractions revealed that ODC protein and activity were detectable in both soluble and insoluble fractions (Pomidor et al. 1999). The insoluble ODC pool was enriched with phosphorylated ODC and appeared to be associated with the keratin cytoskeleton.

The results of fractionation studies to localize ODC activity are rather conflicting and show ODC activity present in both cytoplasm and nucleoplasm or nucleoli. Nuclear and cytoplasmic ODC may be differently regulated because ODC activity induced by growth-promoting stimuli is increased in the cytoplasm only. On the other hand, either contamination of the nuclear fraction with cytosolic ODC or leakage of nuclear ODC to the cytoplasm may have occurred during homogenization of tissues, which led to false conclusions.

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Table 2

Localization of ornithine decarboxylase in cells a

Cell type	Species	Method	Subcellular localization	Reference
Fractionated cells
Prostate cells	Rat	B	Cyt	Pegg and Williams-Ashman 1968
Liver cells	Rat	B	Cyt/Nuc	Snyder and Russell 1970
			Cyt	Ono et al. 1972
			Cyt	Raina and Jänne 1968
			Cyt	Murphy and Brosnan 1976
			Cyt/Nuc	Bitonti and Couri 1981
			Cyt	Grillo and Fossa 1983
			Cyt/Nuc	Bartholeyns 1983
Fibroblasts	Mouse	B	Cyt/Nuc	McCormick 1977
NHEK, keratinocytes	Human	B	Pnuc/Nuc	Pomidor et al. 1999
Cultured cells
RAW 264, macrophage-like	Mouse	ICc	Cyt
JB6, epidermal			Cyt/Nuc	Greenfield et al. 1984, 1986
Mutant ovary cells	Chinese	ICa	Cyt/Pnuc
	Hamster	EC	Cyt/Nuc	Anehus et al. 1984
KB1, epithelial carcinoma cells	Human	ICe	Cyt
Transformed NIH 3T3 fibroblasts	Mouse			Shayovits and Bachrach 1994
NHEK, keratinocytes	Human	ICb,h	Pnuc/Nuc	Pomidor et al. 1995
Ts RSV-infected fibroblasts	Rat	B;ICd,f	Cyt/PMem	Heiskala et al. 1999
Lymphocytes	Human	ICe	Cyt	Wang et al. 1999
WISH-amnion cells	Human	ICg	Cyt/Pnuc
HCT 116 colon cancer cells	Human
Transformed NIH 3T3 fibroblasts	Mouse			Schipper et al. 1999

^a

B, biochemistry; EC, enzyme cytochemistry; ICa-h, immunocytochemistry using (a) polyclonal antiserum from Persson 1982; (b) polyclonal antiserum from Isomaa et al. 1983; (c) polyclonal antiserum from Greenfield et al. 1986; (d) monoclonal antibody from Matsufuji et al. 1984; (e) polyclonal antiserum of Kahana and Nathans 1984; (f) monoclonal antibody from Nishiyama et al. 1989; (g) monoclonal antibody of Schipper et al. 1993; (h) polyclonal antiserum of Pomidor et al. 1995; Cyt, cytoplasmic; Nuc, nuclear; PMem, plasma membrane; Pnuc, perinuclear.

Cultured Cells

Intracellular localization of ODC in cultured cells was studied for the first time in stimulated murine macrophage-like RAW 264 cells using a polyclonal antiserum against ODC that had been purified from this cell line (Greenfield et al. 1984, 1986). ODC immunoreactivity was found exclusively in the cytoplasm, whereas in a cell line of epidermal origin (JB6), ODC protein was found in both cytoplasm and nucleoplasm but not in nucleoli.

Anehus et al. (1984) compared an immunocytochemical and an enzyme cytochemical method using radiolabeled difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC (Metcalf et al. 1978), to determine the intracellular localization of ODC in ODC-overproducing CHO cells. The immunocytochemical method using polyclonal antibodies of Persson (1982) showed considerably more ODC in the cytoplasm than in the nucleus. In contrast, ODC detected by autoradiography using radiolabeled DFMO was evenly distributed over the cell.

Bachrach and co-workers immunocytochemically detected ODC in transformed mouse fibroblasts, human epithelial carcinoma cells, and lymphoblasts from leukemia patients (Shayovits and Bachrach 1994; Wang et al. 1999). ODC immunoreactivity was decreased after treatment with cell growth-inhibiting drugs. In the growing cell fraction of human keratinocytes, ODC showed a perinuclear/nuclear distribution pattern, but it became more diffusely localized in the cytoplasm when cell growth was arrested (Pomidor et al. 1995). Recently, a combined biochemical and immunocytochemical study demonstrated translocation of a fraction of cytosolic ODC to the plasma membrane during cell activation and transformation of rat fibroblasts (Heiskala et al. 1999).

With our monoclonal antibody MP16-2, we examined localization of ODC in various human and murine cell lines (Schipper et al. 1999). Strong immunoreactivity of ODC was detected in the proliferating fraction of cultured cells, especially in cells showing mitotic figures. In confluent cells, ODC staining was faint and diffusely distributed throughout the cell, predominantly in the cytoplasm with the highest density in the perinuclear region, as revealed by confocal laser scanning microscopy (Figure 2A).

We have investigated the subcellular distribution patterns of ODC in more detail by using fusion proteins consisting of ODC and enhanced green fluorescent protein (EGFP). The expression vector pEGFP-N1 was subcloned with or without a human ODC cDNA fragment. Subsequently, EGFP and ODC-EGFP were expressed in cultured HeLa cells. Typical results of expression patterns of EGFP alone or the fusion protein are shown in Figures 2B–2D and 2G. Expression of EGFP alone in these cells was homogeneous throughout the cytoplasm and nuclei of the cells (Figure 2B). In contrast, expression patterns of ODC-EGFP constructs were heterogeneous (Figures 2C and 2D). Confluent cells showed cytoplasmic perinuclear staining, whereas dividing cells showed heterogeneous staining of the nucleus. When cells were treated with polyamines to induce antizyme-regulated ODC degradation, ODC was predominantly localized in the nucleus (Figure 2G). Immunostaining of antizyme in these cells showed that antizyme co-localizes with ODC in the nucleus (Figures 2F and 2G). These results suggest that ODC accumulates in the nucleus after antizyme induction. Further experiments using inhibitors of proteasomal degradation and/or nuclear translocation are needed to clarify the role of ODC translocation to the nucleus in antizyme-induced degradation of ODC.

It is not yet clear whether the distinct changes in ODC expression or localization patterns actually reflect progression through the cell cycle. The ODC gene has been described as a cell cycle-dependent gene that is only weakly expressed in quiescent cells. Its expression is strongly increased in proliferating cells (Kaczmarek 1986). ODC is an “early gene,” i.e., its transcription is increased early in the cell cycle (Kaczmarek et al. 1987; Pena et al. 1995). ODC mRNA increases at the beginning of the G₁-phase both in cycling and stimulated quiescent smooth muscle cells (Campan et al. 1992). ODC activity increases in late G₁ and peaks in G₂ before the onset of mitosis, suggesting that ODC expression is biphasic during the cell cycle (Heby et al. 1976; Fredlund et al. 1995). Recent studies show that ODC translation in the G₂/M transition is regulated in a cap-independent way by using a ribosomal entry site (IRES) (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.

In general, higher levels of ODC were detected in most studies in smaller, more rapidly growing cells. ODC expression appears to be correlated with cell proliferation and cell transformation. Translocation of ODC in conjunction with cytoskeletal systems might be an important regulatory event in these cellular processes. In situ detection of ODC may therefore be useful as a biomarker for (malignant) cell proliferation or, conversely, as an indicator for sensitivity to anti-proliferative drugs, as suggested by Shayovits and Bachrach (1994).

Methodological Problems

Early studies focused on biochemical measurement of ODC activity in fractionated cell preparations. Activity of the enzyme was found in cytoplasmic as well as nuclear fractions, and particularly in nucleoli. This biochemical method is highly sensitive, but care must be taken lest improper homogenization procedures or storage of frozen supernatants cause artifacts due to the metabolism of ornithine via the mitochondrial pathway (Gaines et al. 1988). Furthermore, redistribution of ODC during isolation and fractionation procedures may have affected accurate localization.

Development of the highly specific and irreversibly binding ODC inhibitor DFMO enabled cytochemical localization of ODC activity using fluorescent or radioactively labeled derivatives of DFMO. Most of these studies report that ODC is localized in both cytoplasm and nuclear compartments. Because DFMO is highly specific for ODC and does not react with the inactive ODC-antizyme complex (Kitani and Fujisawa 1986a), reliable labeling of catalytically active ODC can be obtained. However, the use of DFMO to localize ODC in vivo has some drawbacks. The accuracy and sensitivity of ODC localization strongly depend on in vivo binding of the labeled inhibitor to ODC. Low levels of ODC are not detected, and it is not clear whether DFMO is taken up in a similar way by the different cell types or subcellular compartments that have been investigated (Seely et al. 1982; Kitani and Fujisawa 1986b).

Immunocytochemical detection of ODC revealed different localization patterns, depending on the cell system studied and the antibody used. These patterns varied from cytoplasmic or (peri)nuclear localization to localization in both cytoplasm and nucleus. A problem in ODC immunocytochemistry is that information on the nature or activity of the detected ODC protein cannot be obtained. ODC antibodies may also react with catalytically inactive forms of ODC, e.g., monomeric subunits of ODC or monomers bound to the ODC-inhibitory antizyme protein. Furthermore, ODC antibodies may crossreact with the antizyme inhibitor, which shows a high homology with ODC protein but lacks its catalytic activity (Murakami et al. 1996). The existence of multiple or modified forms of ODC may further complicate the immunocytochemical localization of ODC. In homogenates of mouse kidney, two species of active ODC protein were found, which might represent true isoenzymes (Kitani and Fujisawa 1988). ODC might also be post-translationally modified by arginylation or phosphorylation, producing heterogeneous ODC molecules that are differently charged and/or have different enzymatic properties (Kopitz et al. 1990; Brown et al. 1994; Reddy et al. 1996; Heiskala et al. 1999; Pomidor et al. 1999). Phosphorylation has been shown to mask an epitope of mouse ODC that is recognized by a monoclonal antibody (Donato et al. 1986).

In addition to differences in specificity of antibodies against ODC, effects of cell and tissue preparation procedures, such as fixation, permeabilization, dehydration, and embedding, can greatly affect preservation and/or accessibility of the ODC protein for the antibodies. We recently attempted to systematically develop a reliable method for immunocytochemical detection of ODC (Schipper et al. 1999). In these studies we observed that fixation and antigen retrieval methods have substantial effects on the intensity and specificity of ODC immunostaining.

In contrast to the findings obtained with biochemical and enzyme cytochemical studies, immunocytochemical studies of ODC showed that ODC is present mainly in the cytoplasm. This discrepancy may mean that catalytically active nuclear ODC exists in a complexed or cryptic form or is less accessible for antibodies to bind. On the other hand, cytosolic ODC may exist in an immunologically active but enzymatically inactive form. Immunocytochemical detection of ODC in ODC-overproducing CHO cells showed considerably more ODC in the cytoplasm than in the nucleus. In contrast, ODC detected by autoradiography using radiolabeled DFMO was evenly distributed over the cell (Anehus et al. 1984). This difference in distribution patterns suggests the existence of a cytoplasmic pool of catalytically inactive ODC protein, which might represent post-translationally modified ODC. Recent evidence for this “dormant” pool of ODC has been found in fractionated human keratinocytes in which a phosphorylated form of ODC was demonstrated in the insoluble fractions (Pomidor et al. 1999). This may be an important post-translational regulation mechanism of the activity of ODC, as is known for other enzymes as well, such as glucose-6-phosphate dehydrogenase (Swezey and Epel 1986; for review see Van Noorden and Jonges 1995). Another possibility is that ODC largely exists as an inactive complex with antizyme protein in the cytoplasm, as was reported in adult mouse brain (Laitinen 1985).

In situ hybridization studies using a labeled cRNA or cDNA probe specifically hybridizing with ODC mRNA have been performed on mouse, rat, and human tissues (Table 1). The localization patterns of ODC mRNA were, at least partially, consistent with ODC localization patterns obtained in biochemical and immunocytochemical studies. However, many other experiments have provided evidence for translational and post-translational control of ODC (Heby and Persson 1990). Therefore, localization and concentration of mRNA may not actually reflect ODC expression at the level of ODC protein and/or enzyme activity.

Cell Physiological Aspects

Technical aspects may be responsible for the variety of staining patterns, but it may also be possible that the localization of ODC in cytoplasm and nucleus varies depending on the physiological state of the cell. ODC may be expressed in a particular cell type or subcellular compartment when its products, the polyamines, are needed there. Although intracellular transport of polyamines cannot be ruled out, targeting of ODC to the functional site of polyamines may be the actual transport mechanism.

Polyamines are implied to have an essential role in mitotic spindle organization and modulation of DNA structure and stability. Therefore, nuclear ODC may be an important source for nuclear polyamines. An argument against this proposition is that, to produce nuclear spermidine and spermine in addition to their precursor putrescine, the other biosynthetic enzymes of the ODC/polyamine system also must be translocated/present in the nucleus, for which no evidence (yet) exists. However, putrescine may not serve solely as a precursor for the production of spermidine and spermine but may exert cellular effects of its own, as found recently in intestinal epithelial cells (Bardocz et al. 1998; Löser et al. 1999; Pignata et al. 1999; Farriol et al. 2001).

Localization studies of polyamines using autoradiographic, cytochemical, and immunocytochemical techniques have confirmed the presence of polyamines in the nucleus (Roch et al. 1997, and references therein). A few studies have reported nucleolus-associated localization of ODC (Emanuelsson and Heby 1982; Müller et al. 1991b). It has been suggested that post-translationally modified ODC is translocated to the nucleolus, where it may regulate RNA polymerase I activity (Manen and Russell 1977; Russell 1985). However, more purified fractions of ODC did not affect the latter enzyme activity (Urata et al. 1987; Eichler and Corr 1992), and DFMO was not able to inhibit rRNA transcription (Iapalucci et al. 1984). Therefore, the suggested involvement of ODC in modulating RNA poly-merase activity may actually be untrue, but a possible role of the ODC/polyamine system in other nucleoli-associated functions (e.g., ribosomal RNA synthesis, ribosome biogenesis) cannot be excluded.

The vast majority of the results presented in Tables 2 and 3 show a cytoplasmic localization of ODC. Furthermore, most studies in which ODC localization was examined before and after stimulation report on a cytoplasmic rather than a nuclear localization of stimulated ODC.

The precise functional role of the ODC/polyamine system in the cytoplasm still needs to be elucidated, but many studies suggest that polyamines play essential roles in the machinery of protein biosynthesis. This hypothesis is further supported by immunoelectron microscopic studies of ODC and polyamines in which the label was closely associated with rough endoplasmic reticulum and polyribosomal structures, respectively (Morisset et al. 1986; Fujiwara et al. 1998).

Recent studies in cultured cells using confocal laser scanning microscopy (Pomidor et al. 1995, 1999; Heiskala et al. 1999; Schipper et al. 1999; Figure 2) have revealed the presence of ODC at the plasma membrane and/or nuclear envelope. The plasma membrane as a functional site for ODC is plausible because ODC has been implicated in the formation of signaling complexes in focal adhesion sites (Auvinen et al. 1995), transmembrane calcium and/or calmodulin signaling (Ginty and Seidel 1989), and membrane translocation of protein kinase C (Groblewski et al. 1992). There is no direct evidence that ODC is exported out of the cell, although ODC activity has been found in human prostatic fluid, seminal plasma, and sperm (Jänne et al. 1973; Mohan et al. 1999).

Membrane localization of ODC appeared to be related to structural elements, i.e., actin or keratin cytoskeletal elements (Pomidor et al. 1995, 1999; Heiskala et al. 1999). In activated or transformed fibroblasts, translocation of ODC to the plasma membrane was mediated by a p47^phox membrane-targeting motif-related sequence (amino acids 165-172) and was dependent on phosphorylation (Heiskala et al. 1999). It was suggested that cell shape changes transduced via the cytoskeleton translocate ODC and that concomitantly altered ODC activity affects mitotic cytosketal rearrangement events. These findings also show that translocation of ODC is mandatory for regulation of physiological function of ODC because inhibition of ODC activity or irrelevant membrane targeting of the active enzyme led to defective cytokinesis.

Translocation of ODC may also have a functional role in its degradation. It is well established that antizyme binds to ODC and targets it to the proteasome for degradation. Antizyme has been localized in the cytoplasm but also in the nucleus, especially in cells with high ODC expression (Junttila et al. 1995; Kilpeläinen et al. 2000; Gritli-Linde et al. 2001). When turnover of ODC in the nucleus by antizyme was blocked by a proteasome inhibitor, ODC was expressed in the nucleus (Gritli-Linde et al. 2001). In our GFP experiments, induction of antizyme caused accumulation of antizyme and ODC in the nucleus (Figure 2D). These data suggest an antizyme-mediated translocation of ODC to the nucleus for degradation by nuclear proteasomes.

The variety of ODC distribution patterns as observed in different tissue types may be explained by the various roles of polyamines in cell metabolism and growth. As expected, ODC expression is prominent in the proliferating fraction of rapidly growing tissues. For example, ODC activity is particularly high in tissues with high cell turnover, e.g., intestinal epithelium, which require a constant supply of polyamines for growth, renewal, and metabolism. In contrast, highest activity of ODC has been found in the prostate, in which polyamines are synthesized for export rather than for intracellular use. In transgenic mice that over-express human ODC due to a ubiquitous promoter, abnormalities were found only in testis and brain (Halonen et al. 1993). Targeted ODC overexpression using cell type-specific promotors had no phenotypic effects in liver (Alhonen et al. 1996) and intestine (unpublished observations) but resulted in an increased frequency of papillomas in skin (Megosh et al. 1995).

ODC distribution patterns may depend not only on the cell type but also on cell kinetic behavior, because ODC activity and polyamine pools change during cell growth, differentiation, and cell death in a transient manner. Therefore, the onset of ODC expression can vary greatly among cells, depending on their physiological status, creating heterogeneous and apparently inconsistent results in cytochemical studies.

In conclusion, intracellular localization of ODC is not static but is subject to highly dynamic processes. Future studies in living cells using real-time analysis of fluorescence-tagged ODC and ODC regulatory proteins (antizyme, antizyme inhibitor) are needed to further unravel the complexity of the regulation and function of the ODC/polyamine system.