Endogenous Ornithine Decarboxylase Research Articles

Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney.

We have investigated and characterized a novel ornithine decarboxylase (ODC) related protein (ODCrp) also annotated as gm853. ODCrp shows 41% amino acid sequence identity with ODC and 38% with ODC antizyme inhibitor 1 (AZIN1). The Odcrp gene is selectively expressed in the epithelium of proximal tubuli of mouse kidney with higher expression in males than in females. Like Odc in mouse kidney, Odcrp is also androgen responsive with androgen receptor (AR)-binding loci within its regulatory region. ODCrp forms homodimers but does not heterodimerize with ODC. Although ODCrp contains 20 amino acid residues known to be necessary for the catalytic activity of ODC, no decarboxylase activity could be found with ornithine, lysine or arginine as substrates. ODCrp does not function as an AZIN, as it neither binds ODC antizyme 1 (OAZ1) nor prevents OAZ-mediated inactivation and degradation of ODC. ODCrp itself is degraded via ubiquination and mutation of Cys363 (corresponding to Cys360 of ODC) appears to destabilize the protein. Evidence for a function of ODCrp was found in ODC assays on lysates from transfected Cos-7 cells where ODCrp repressed the activity of endogenous ODC while Cys363Ala mutated ODCrp increased the enzymatic activity of endogenous ODC.

ROLES OF ORNITHINE DECARBOXYLASE (ODC) IN REGULATION OF AMYLOID β-INDUCED MICROGLIAL NEUROINFLAMMATION

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by amyloid β (Aβ) deposition. Increasing evidence supports neuroinflammation is one of primarily concerns in AD. Particularly, microglia are the primary immune cells contribute to neuroinflammation by releasing various proinflammatory cytokines and neurotoxins in the brain. Therefore, inhibition of microglia shows potential value of therapeutic or preventive intervention. Interestingly, it has been demonstrated that arginine metabolism pathway may contribute to the balance between M2 and M1 phenotypes of microglia. In particular, ornithine decarboxylase (ODC) is a rate-limiting enzyme to the regulation of arginine catabolism. As a result, blocking ODC activity may result in an alternative metabolic pathway of arginine in stimulation of inducible NO synthase (iNOS). This is believed to drive neuroinflammation in AD pathogenesis. However, the detailed mechanism remains unclear. In this study, we investigated whether Aβ disrupts ODC homeostasis and contribute to microglial over-activation and neurotoxicity by BV2 and EOC13.31 microglial cell lines. Our results indicated that Aβ interferes endogenous ODC homeostasis and its downstream effects including altered arginine metabolism pathway resulting in microglial over-activation and neurotoxicity. On the contrary, restoration of ODC activity was associated with an increase in M2/M1 microglia ratio, indicating that Aβ-inhibited ODC may contribute to microglial inflammation. Our results demonstrated the modulation of ODC activities in microglia may be a workable strategy for therapeutics against Aβ-induced microglial neuroinflammation.

Novel Interaction of Ornithine Decarboxylase with Sepiapterin Reductase Regulates Neuroblastoma Cell Proliferation

Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein–protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC–SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan–Meier analysis (log-rank test, P=5×10−4), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.

Insect ornithine decarboxylase (ODC) complements SPE1 knock-out of yeast Saccharomyces cerevisiae.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth, differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. Mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%, respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of > 4% of the wild-type strain in polyaminefree media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore, its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system.

Characterization of antizyme function in a yeast complementation system

Ornithine decarboxylase (ODC) is an important enzyme in the synthesis of polyamines. Polyamines are charged organic molecules that are essential for cell growth. Antizyme is an enzyme that aids in the regulation of polyamine levels within the cell by targeting ODC for degradation. A yeast strain has been established that involves the complementation of a null mutant endogenous ODC gene with expression of human ODC. The ODC gene was constitutively expressed while antizyme expression was controlled by galactose induction. In the presence of galactose, induced antizyme expression resulted in eventual cell death due to ODC degradation and depletion of polyamines. Using this system we will transform the strain with a yeast cDNA library controlled by a GAL promoter. We will screen for overexpressed yeast proteins that may inhibit antizyme function. The inhibition of antizyme will allow the yeast to grow since ODC will no longer be degraded. Identifying yeast genes that functionally interact with antizyme will lead to an increased understanding of the activity and function of antizyme in polyamine synthesis. The ability to understand the function and mechanism of antizyme’s ability to target ODC for degradation could help in the control of cell growth. Polyamine regulation is highly conserved in all organisms thus the study of AZ function in yeast can have direct applications to higher vertebrates. Supported by BRIN/INBRE Grant # P20 RR016457 from the NIH.

Immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype

Ornithine decarboxylase (ODC) is a cytosolic enzyme that catalyses the direct decarboxylation of l-ornithine to putrescine, one of the rate-limiting steps of polyamine biosynthesis in plants. In the present study, an ODC-specific murine single-chain antibody fragment (scFvODC1) was generated by phage display technology. To evaluate the effect of the recombinant antibody fragment on ODC activity and polyamine levels, we produced transgenic tobacco plants that accumulated scFvODC1 in the cytosol. Expression levels of up to 4% total soluble protein (TSP) were achieved, resulting in the inhibition of up to 90% of endogenous ODC activity. A significant reduction in putrescine, spermidine and spermine levels was observed in transgenic lines producing high levels of scFvODC1. Furthermore, these lines showed developmental abnormalities and a dwarf phenotype. We show that the immunomodulation of enzyme activity is a powerful approach that can be used to alter complex and tightly controlled metabolic pathways, allowing specific steps in the pathway to be blocked and the resulting physiological effects to be investigated.

Ubiquitin-independent Mechanisms of Mouse Ornithine Decarboxylase Degradation Are Conserved between Mammalian and Fungal Cells

The polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is degraded by the 26 S proteasome via a ubiquitin-independent pathway in mammalian cells. Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1 (AZ1). Mouse ODC (mODC) that is expressed in the yeast Saccharomyces cerevisiae is also rapidly degraded by the proteasome of that organism. We have now carried out in vivo and in vitro studies to determine whether S. cerevisiae proteasomes recognize mODC degradation signals. Mutations of mODC that stabilized the protein in animal cells also did so in the fungus. Moreover, the mODC degradation signal was able to destabilize a GFP or Ura3 reporter in GFP-mODC and Ura3-mODC fusion proteins. Co-expression of AZ1 accelerated mODC degradation 2-3-fold in yeast cells. The degradation of both mODC and the endogenous yeast ODC (yODC) was unaffected in S. cerevisiae mutants with various defects in ubiquitin metabolism, and ubiquitinylated forms of mODC were not detected in yeast cells. In addition, recombinant mODC was degraded in an ATP-dependent manner by affinity-purified yeast 26 S proteasomes in the absence of ubiquitin. Degradation by purified yeast proteasomes was sensitive to mutations that stabilized mODC in vivo, but was not accelerated by recombinant AZ1. These studies demonstrate that cell constituents required for mODC degradation are conserved between animals and fungi, and that both mammalian and fungal ODC are subject to proteasome-mediated proteolysis by ubiquitin-independent mechanisms.

Overexpression of a dominant-negative ornithine decarboxylase in mouse skin: effect on enzyme activity and papilloma formation.

A transgenic mouse line expressing a truncated form of the ornithine decarboxylase (ODC) dominant-negative mutant K69A/C360A under the control of the keratin 6 promoter has been established (K6/ODCdn mice). These mice were backcrossed onto both the DBA/2J and C57BL/6J backgrounds for subsequent tumorigenesis experiments utilizing an initiation/promotion protocol. In short-term experiments, expression of the ODCdn protein product was induced in the epidermis within 24 h after application of the tumor promoter tetradecanoyl phorbol acetate (TPA) to the skin, and ODC activity in the epidermis of K6/ODCdn mice was reduced by at least 75% compared with littermate controls. However, in tumorigenesis experiments utilizing a variety of initiator (7,12-dimethylbenz[a]anthracene; DMBA) and promoter (TPA) concentrations, K6/ODCdn mice formed at least as many tumors as their littermate controls regardless of background strain. In experiments utilizing chrysarobin, a tumor promoter with a different mechanism of action than TPA, again there was no significant difference in tumor formation between K6/ODCdn mice and littermate controls. Similarly, when K6/ODCdn mice were crossed with K5/ODC mice, a transgenic line described previously which forms tumors without application of a promoting agent, double transgenic mice formed as many tumors as mice expressing the K5/ODC transgene alone. Analysis of epidermis following multiple TPA applications revealed a dramatic spike in ODC activity in both K6/ODCdn mice and non-transgenic mice after six applications, and western blot analysis suggested a stabilization of endogenous wild-type ODC in K6/ODCdn transgenic mice. ODC activity, endogenous protein and polyamines were also elevated in tumors from K6/ODCdn mice. The accumulation of endogenous ODC protein is most probably the result of competition from the transgene-derived ODCdn protein for binding of antizyme, which is known to regulate ODC activity by stimulating degradation of the ODC protein.

Decreased ornithine decarboxylase activity in the kidneys of Dahl salt-sensitive rats.

To assess the roles of polyamines (putrescine, spermidine, and spermine) and ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, in the development of salt-sensitive hypertension, we evaluated activity and expression of ODC, urinary polyamine excretion, and antizyme (endogenous ODC inhibitor protein) expression in Dahl salt-sensitive (SS) and salt-resistant (SR) rats after they were fed on a low (0.3%) or high (4%) salt diet for 4 weeks. We also examined the effects of spermidine and difluoromethylornithine (DFMO: a specific inhibitor of ODC) on the systolic blood pressure and ODC protein expression in SS rats fed a high salt diet. Renal ODC activity and urinary polyamine excretion in SS rats were lower than those in SR rats after 4 weeks treatment with a low or high salt diet. The renal ODC protein expression of SS rats was paradoxically increased as compared to the SR group. A high salt diet did not alter ODC activity but increased ODC protein only in SS rats. ODC mRNA and antizyme protein expressions were not significantly different among the four groups. Spermidine supplementation attenuated and DFMO exaggerated hypertension in SS rats fed a high salt diet. Spermidine down-regulated and DFMO up-regulated renal ODC protein in SS rats on a high salt diet. ODC activity was decreased but protein was paradoxically increased in kidneys of SS rats. ODC protein was suggested to increase in compensation for the inhibition of its activity. Impaired ODC activity and polyamine production in the kidney may exaggerate salt-sensitive hypertension in SS rats.

Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy

Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac α-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10– 39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30–32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.

Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy

Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac alpha-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10-39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30-32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.

Induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc: transrepression correlates with acceleration of apoptosis.

Analysis of amino-terminus mutants of c-Myc has allowed a systematic study of the interrelationship between Myc's ability to regulate transcription and its apoptotic, proliferative, and transforming functions. First, we have found that c-Myc-accelerated apoptosis does not directly correlate with its ability to transactivate transcription using the endogenous ornithine decarboxylase (ODC) gene as readout for transactivation. Furthermore, deletion of the conserved c-Myc box I domain implicated in transactivation does not inhibit apoptosis. Second, the ability of c-Myc to repress transcription, using the gadd45 gene as a readout, correlates with its ability to accelerate apoptosis. A conserved region of c-Myc implicated in mediating transrepression is absolutely required for c-Myc-accelerated apoptosis. Third, a lymphoma-derived Thr58Ala mutation diminishes c-Myc-accelerated apoptosis through a decreased ability to induce the release of cytochrome c from mitochondria. This mutation in a potential phosphorylation site does not affect cell cycle progression, providing genetic evidence that induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc. Finally, we show that the increased ability of Thr58Ala mutant to elicit cellular transformation correlates with its diminished ability to accelerate apoptosis. Bcl-2 overexpression blocked and the lymphoma-associated Thr58Ala mutation decreased c-Myc-accelerated apoptosis, and both led to a significant increase in the ability of Rat1a cells to form colonies in soft agar. This enhanced transformation was greater in soft agar containing a low concentration of serum, suggesting that protection from apoptosis is a mechanism contributing to the increased ability of these cells to proliferate in suspension. Thus, we show here for the first time that, in addition to mutations in complementary antiapoptotic genes, c-Myc itself can acquire mutations that potentiate neoplastic transformation by affecting apoptosis independently of cell cycle progression.

Ornithine Decarboxylase Is a Transcriptional Target of Tumor Suppressor WT1

The product of the Wilm's tumor suppressor gene, WT1, is a zinc-finger DNA-binding protein, which is thought to be a transcription factor. Two genes, those encoding epidermal growth factor receptor and syndecan-1, are known to be endogenous targets of WT1. Previous studies had identified binding sites for WT1 in the promoter of the ornithine decarboxylase (ODC) gene. In this paper, we tested whether the endogenous ODC gene might be a target of WT1 by establishing lines of baby hamster kidney (BHK) cells that expressed WT1 isoform A under control of a tetracycline-regulated expression system. When expression of WT1 was activated in BHK cells, the cellular level of ODC mRNA declined, with kinetics that correlated with the increase in WT1 level, demonstrating that the endogenous ODC gene was indeed responsive to cellular level of WT1. WT1 isoforms A and B inhibited the activity of the ODC promoter by approximately fivefold in transiently transfected BHK cells, while isoforms C and D, which have altered DNA binding domains, had no significant effect. The sequence CTCCCCCGC, located at nucleotides −106 to −98 relative to the site of transcriptional initiation in the ODC gene, interacted with the zinc-finger domain of isoforms A and B of WT1 with high affinity and specificity. A mutation in the binding site that disrupted this interaction partially removed the inhibition of ODC promoter activity by WT1, as did mutation of the two E-box sequences in intron I of the ODC gene. Simultaneous mutation of the WT1-binding motif and the two E-boxes completely abolished inhibition by WT1 of ODC promoter activity. These results, taken together, implicate the ODC gene as a downstream target of the tumor suppressor WT1.

Induction of ornithine decarboxylase by IL-3 is mediated by sequential c-Myc-independent and c-Myc-dependent pathways.

Enforced c-Myc expression promotes continuous, growth factor-independent, cell cycle progression and activates expression of the ornithine decarboxylase (ODC) gene and its promoter. c-Myc-responsiveness of murine ODC is mediated by two conserved c-Myc:Max E-boxes in ODC intron 1. c-Myc and ODC are both required for cell growth and their expression is sequentially induced in G0/G1 cells stimulated with mitogens, yet their expression is not modulated by the cell cycle in proliferating cells. Here we demonstrate that regulation of ODC and its promoter by Interleukin-3 (IL-3) in murine myeloid cells is mediated in part by c-Myc, c-Myc induced ODC through the same transcription start site as IL-3 and, in asynchronously growing cells, maximal activity of the ODC promoter required the intronic c-Myc binding sites. However, induction of ODC following IL-3 stimulation of quiescent cells is mediated by at least two pathways. The first phase of this response was independent of the intronic c-Myc:Max E-boxes and de novo protein synthesis. Sustained induction of the ODC promoter however required the c-Myc:Max binding sites and protein synthesis. Accumulation of c-Myc following stimulation of quiescent cells with IL-3 correlated with the delayed phase of the response. Consistent with a two pathway model of ODC regulation, inducible overexpression of dominant negative form of c-Myc (In373-Myc), which specifically inhibits the c-Myc-Max network, inhibited the delayed, but not immediate, induction of ODC promoter activity in response to IL-3. Dominant negative c-Myc protein also effectively suppressed induction of the endogenous ODC gene by IL-3. Therefore, c-Myc functions as a direct and required-regulator of ODC. These results also suggest a model whereby c-Myc's role in regulating its targets may be to convert a transient, immediate-early, activation event into the persistent induction of gene expression.

Co-operation of the 5' and 3' untranslated regions of ornithine decarboxylase mRNA and inhibitory role of its 3' untranslated region in regulating the translational efficiency of hybrid RNA species via cellular factor.

The 5' untranslated region (UTR) has an inhibitory role in the translatability of ornithine decarboxylase (ODC) mRNA and of hybrid mRNA species, whereas the ODC 3' UTR causes a partial release of this inhibition. We designed experiments to explore whether the co-operation between ODC 5' UTR and 3' UTR in the translational regulation is due to a direct interaction of those sequences or whether it is mediated by their interaction with cellular factor(s). We stably transfected Chinese hamster ovary (CHO)-K1 cells and transiently transfected COS-1 cells with expression vectors carrying different chimaeric DNAs having the luciferase (LUC) coding sequence as reporter gene, the ODC 5' UTR or the ODC 3' UTR, or both, in the appropriate positions. We compared the results obtained by assaying the LUC activities of both transfected cell lines with each chimaeric DNA with those observed by translating the hybrid RNAs in a translation system in vitro. When the ODC 3' UTR was present, we observed a partial release of the translation inhibition owing to the ODC 5' UTR only in vivo. The releasing effect was restored in vitro by the addition of cytoplasmic extracts from wild-type CHO-K1 or COS-1 cells, prepared 2 and 8 h after their release from serum starvation. We also observed a partial inhibition of the translatability of the hybrid RNA owing to the presence of the ODC 3' UTR itself; the translational efficiency could be rescued by cell extract from 8 h serum-stimulated cells. The co-operation between the ODC-UTRs might be mediated by factors expressed by cells during particular phases of the cell cycle. Excess copies of the ODC-UTRs, expressed in trans, could compete in binding limited amounts of such regulatory factors and remove them from interaction with the endogenous ODC mRNA. This phenomenon should be reflected by modifications of the kinetics of ODC and/or LUC activities during serum stimulation. The overexpression of the ODC 3' UTR determined an increase in both endogenous ODC activity and LUC activity. Moreover, in the transfectants expressing the hybrid RNA species bearing the ODC 3' UTR the basal ODC activity is higher than that observed in control cells. We suggest that excess copies of the ODC 3' UTR mis-regulate the endogenous ODC translatability, probably by tying up regulatory molecules expressed by cells in limited amounts and sequestering them from the ODC mRNA species they should interact with.

Reversion of malignant phenotypes of human lung squamous carcinoma cells by ornithine decarboxylase antisense RNA

Abnormally elevated activity of ornithine decarboxylase (ODC), and subsequent polyamine accumulation are intimately associated with the genesis, development and metastasis of cancer. In the present study, to control the growth of tumor cells, ODC antisense RNA was used to transfect human lung squamous carcinoma cell line LTEP-78. Compared with the parental cells, growth of the antisense transfected LTEP-78 cells arrested in G0/G1 phase and colony formation in soft agarose and tumorigenicity in nude mice were significantly reduced. Nucleic acid hybridization demonstrated that the transfectants expressed a high level of ODC antisense RNA and a significantly reduced level of endogenous ODC mRNA. The results suggest that the reversion of malignant phenotypes of human lung squamous carcinoma cells transfected with ODC antisense RNA is associated with the inhibition of polyamine biosynthesis.

Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex.

The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

Retinoic acid regulates ornithine decarboxylase gene expression at the transcriptional level

Retinoic acid (RA) is important for normal mammalian development and growth. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the biosynthesis of the polyamines, and we have previously shown that ODC mRNA levels are suppressed by RA in human skin cells. Using HeLa cells, we now show that treatment with 0.5 microM RA for 24 h suppresses endogenous ODC mRNA levels and the expression of a transfected ODC/chloramphenicol acetyltransferase plasmid (Kpn-ODCCAT), containing sequences from -1450 to +810 of the human ODC gene. Co-transfection with either the alpha-RA receptor (alpha-RAR) or a chimeric alpha-RA/oestrogen receptor (alpha-RAER) followed by treatment with the cognate hormone suppresses expression of Kpn-ODCCAT and Not-ODCCAT, which contains sequences from -250 to +514. Liganded alpha-RAR suppresses the activity of Kpn-ODCCAT more markedly than does liganded alpha-RAER (98% and 80% suppression, respectively), whereas both receptors have very similar effects on Not-ODCCAT expression (73% and 67% suppression, respectively). The unliganded alpha-RAR suppresses Kpn-ODCCAT by 76%, whereas unliganded alpha-RAER has no significant effect. These data show that RA regulates ODC-gene expression at the transcriptional level, and that alpha-RAR, but not alpha-RAER, can confer full hormonal responsiveness. This suggests that the activating function present in the alpha-RAR ligand-binding domain is required for full transcriptional regulation.

Direct Transcriptional Stimulation of the Ornithine Decarboxylase Gene by Fos in PC12 Cells but Not In Fibroblasts

We have established rat PC12 pheochromocytoma cell lines stably expressing the estrogen-activatable transcription factor FosER to identify genes that can be regulated by c-Fos in this neuronal cell type. Induction of ectopic c-Fos activity in PC12 cells increased the mRNA levels of the ornithine decarboxylase (ODC) and tyrosine hydroxylase genes with similar kinetics and to the same maximal level as nerve growth factor treatment. In both cases the rate of transcription initiation was increased. Induction of the ODC gene occurred even in the absence of protein synthesis, indicating direct regulation by FosER. ODC expression, however, was not induced by a mutant FosER protein containing a proline insertion in the basic region of the c-Fos moiety, demonstrating the requirement for a functional DNA-binding domain. These data show that FosER, and by extrapolation c-Fos, can directly activate transcription of the endogenous ODC gene in PC12 cells by binding to cis-regulatory sequences. Activation of the ODC gene was unexpectedly transient, as transcripts returned to the basal level after prolonged exposure of PC12 cells to FosER activity. Furthermore, ODC transcription was not at all induced by FosER in rat fibroblasts. To account for this cell-specific action of FosER, we propose that stimulation of the ODC gene by FosER requires either (i) cooperation with another transcription factor(s) or (ii) a specific pattern of modification which is present in PC12 cells but not in otherwise unstimulated fibroblasts. One or both of these mechanisms may be employed by cells to achieve selective gene activation in response to apparently stereotyped induction of c-fos.

Direct transcriptional stimulation of the ornithine decarboxylase gene by Fos in PC12 cells but not in fibroblasts.

We have established rat PC12 pheochromocytoma cell lines stably expressing the estrogen-activatable transcription factor FosER to identify genes that can be regulated by c-Fos in this neuronal cell type. Induction of ectopic c-Fos activity in PC12 cells increased the mRNA levels of the ornithine decarboxylase (ODC) and tyrosine hydroxylase genes with similar kinetics and to the same maximal level as nerve growth factor treatment. In both cases the rate of transcription initiation was increased. Induction of the ODC gene occurred even in the absence of protein synthesis, indicating direct regulation by FosER. ODC expression, however, was not induced by a mutant FosER protein containing a proline insertion in the basic region of the c-Fos moiety, demonstrating the requirement for a functional DNA-binding domain. These data show that FosER, and by extrapolation c-Fos, can directly activate transcription of the endogenous ODC gene in PC12 cells by binding to cis-regulatory sequences. Activation of the ODC gene was unexpectedly transient, as transcripts returned to the basal level after prolonged exposure of PC12 cells to FosER activity. Furthermore, ODC transcription was not at all induced by FosER in rat fibroblasts. To account for this cell-specific action of FosER, we propose that stimulation of the ODC gene by FosER requires either (i) cooperation with another transcription factor(s) or (ii) a specific pattern of modification which is present in PC12 cells but not in otherwise unstimulated fibroblasts. One or both of these mechanisms may be employed by cells to achieve selective gene activation in response to apparently stereotyped induction of c-fos.