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Metabolic changes in immune cells contribute to both physiological and pathophysiological outcomes of immune reactions. Here, by comparing protein expression, transcriptome, and salivary metabolome profiles of uninfected and HIV+ individuals, we found perturbations of polyamine metabolism in the oral mucosa of HIV+ patients. Mechanistic studies using an in vitro human tonsil organoid infection model revealed that HIV infection of T cells also resulted in increased polyamine synthesis, which was dependent on the activities of caspase-1, IL-1β, and ornithine decarboxylase-1. HIV-1 also led to a heightened expression of polyamine synthesis intermediates including ornithine decarboxylase-1 as well as an elevated dysfunctional regulatory T cell (T regDys )/T helper 17 (Th17) cell ratios. Blockade of caspase-1 and polyamine synthesis intermediates reversed the T regDys phenotype showing the direct role of polyamine pathway in altering T cell functions during HIV-1 infection. Lastly, oral mucosal T regDys /Th17 ratios and CD4 hyperactivation positively correlated with salivary putrescine levels, which were found to be elevated in the saliva of HIV+ patients. Thus, by revealing the role of aberrantly increased polyamine synthesis during HIV infection, our study unveils a mechanism by which chronic viral infections could drive distinct T cell effector programs and T reg dysfunction., (© 2023. The Author(s).)

Five (5) HLA-A 0201 restricted epitopes of ornithine decarboxylase derived from Leishmania donovani ( Ld- ODC) were examined by reverse vaccinology to develop prophylactics against visceral leishmaniasis (VL). These consensus epitopes comprising (P1: RLMPSAHAI, P2: LLDQYQIHL, P3: GLYHSFNCI, P4: AVLEVLSAL and P5: RLPASPAAL) were observed and presented by diverse HLA alleles screened by immune-informatics tools. These epitopes were also observed for strong stability for appropriate immune response in in silico screening and molecular dynamics. Top five selected epitopes filtered from population coverage analysis and TAP binding affinity were identified and evaluated against treated cases of VL subjects. Experiments were run individually with synthetic peptides or as the cocktail of peptides. A major population of CD8+ T cells were predominantly IFN-γ producers but not the IL-10 cytokines and shown with granzyme-B activity. Therefore, it can be concluded that the screened HLA-A0201 restricted epitope hotspots derived from Leishmania ODC can trigger CD8+ T cells, which can skew other immune cells functions toward protection. However, a detailed analysis can explore its potentiality as a vaccine candidate.Communicated by Ramaswamy H. Sarma.

In a bid to develop a novel immunoprophylactic measure against visceral leishmaniasis (VL), MHC class-II-restricted epitopes of LdODC were identified by reverse vaccinology approach. Five consensus HLA-DRB1*0101-restricted epitopes were screened. The analysis revealed that the set of epitopes was presented by at least 54 diverse MHC class-II alleles. Based on in silico screening, followed by molecular dynamics simulation, population coverage analysis, and HLA cross-presentation ability, five best epitopes were evaluated. PBMCs isolated from treated VL subjects, when stimulated with synthetic peptide alone or as a cocktail of peptides, triggered a secretory IFN-γ, but not the IL-10 level. Support in this notion came from intracellular cytokine level with a considerable up-regulated IFN-γ produced by CD4 + T cells. Also, the enhanced IFN-γ seemed to be augmented with the activation of macrophages with prominent IL-12 production. Therefore, it can be concluded that the screened MHC class-II-restricted epitope hotspots derived from Leishmania ODC can trigger CD4 + T cells, which can skew macrophage functions towards protection. However, a detailed analysis can explore its potentiality as a vaccine candidate., (© 2020 John Wiley & Sons Ltd.)

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis and restricts M1 macrophage activation in gastrointestinal (GI) infections. However, the role of macrophage ODC in colonic epithelial-driven inflammation is unknown. Here, we investigate cell-specific effects of ODC in colitis and colitis-associated carcinogenesis (CAC). Human colonic macrophages expressed increased ODC levels in active ulcerative colitis and Crohn's disease, colitis-associated dysplasia, and CAC. Mice lacking Odc in myeloid cells ( Odc Δmye mice) that were treated with dextran sulfate sodium (DSS) exhibited improved survival, body weight, and colon length and reduced histologic injury versus control mice. In contrast, GI epithelial-specific Odc knockout had no effect on clinical parameters. Despite reduced histologic damage, colitis tissues of Odc Δmye mice had increased levels of multiple proinflammatory cytokines and chemokines and enhanced expression of M1, but not M2 markers. In the azoxymethane-DSS model of CAC, Odc Δmye mice had reduced tumor number, burden, and high-grade dysplasia. Tumors from Odc Δmye mice had increased M1, but not M2 macrophages. Increased levels of histone 3, lysine 9 acetylation, a marker of open chromatin, were manifest in tumor macrophages of Odc Δmye mice, consistent with our findings that macrophage ODC affects histone modifications that upregulate M1 gene transcription during GI infections. These findings support the concept that macrophage ODC augments epithelial injury-associated colitis and CAC by impairing the M1 responses that stimulate epithelial repair, antimicrobial defense, and antitumoral immunity. They also suggest that macrophage ODC is an important target for colon cancer chemoprevention. Significance: Ornithine decarboxylase contributes to the pathogenesis of colitis and associated carcinogenesis by impairing M1 macrophage responses needed for antitumoral immunity; targeting ODC in macrophages may represent a new strategy for chemoprevention. Cancer Res; 78(15); 4303-15. ©2018 AACR ., (©2018 American Association for Cancer Research.)

We report here a Leishmania donovani ornithine decarboxylase (Ld-ODC) gene used as a DNA vaccine against visceral leishmaniasis in a murine Balb/c mouse model. This study also evaluated the possible mechanism of action directed by this candidate. We found a Th1 immune response after immunization using an Ld-ODC DNA vaccine, with results based on the rearrangement of TCR-V-α-2, proliferation of Carboxy fluorescein Succinimidyle ester positive T cells, which were able to produce cytokines such as TNF-α, IFN-γ, IL-12 and IL-2, but not IL-4, IL-5, IL-6 and IL-10, and modulations of the STAT-1 and p38 MAP kinase signaling pathways. The results were corroborated with the reduction in the amastigote proliferation and parasite killing in spleens after infection in vitro. We conclude this study suggesting that the Ld-ODC DNA construct could be a new vaccine candidate against visceral leishmaniasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms., Competing Interests: The authors declare no conflict of interest.

α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1(-/-) mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8(+) T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8(+) T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Ornithine decarboxylase, the rate limiting enzyme of the polyamine biosynthesis pathway, is significant in the synthesis of trypanothione, T(SH)2, the major reduced thiol which is responsible for the modulation of the immune response and pathogenesis in visceral leishmaniasis. Data on the relationship between ornithine decarboxylase and the cellular immune response in VL patients are limited. Therefore, we purified a recombinant ornithine decarboxylase from Leishmania donovani (r-LdODC) of approximately 77kDa and examined its effects on the immunological responses in peripheral blood mononuclear cells of human visceral leishmaniasis cases. For these studies, α-difluoromethylornithine was tested as an inhibitor and was used in parallel in all experiments. The r-LdODC was identified as having a direct correlation with parasite growth and significantly increased the number of promastigotes as well as axenic amastigotes after 96h of culture. The stimulation of peripheral blood mononuclear cells with r-LdODC up-regulated IL-10 production but not IFN-γ production from CD4(+) T cells in active as well as cured visceral leishmaniasis cases, indicating a pivotal role for r-LdODC in causing strong immune suppression in a susceptible host. In addition, severe hindrance of the immune response and anti-leishmanial macrophage function due to r-LdODC, as revealed by decreased IL-12 and nitric oxide production, and down-regulation in mean fluorescence intensities of reactive oxygen species, occurred in visceral leishmaniasis patients. There was little impact of r-LdODC in the killing of L. donovani amastigotes in macrophages of visceral leishmaniasis patients. In contrast, when cultures of promastigotes and amastigotes, and patients' blood samples, were directed against α-difluoromethylornithine, parasite numbers significantly reduced in culture, whereas the levels of IFN-γ and IL-12, and the production of reactive oxygen species and nitric oxide, were significantly elevated. Therefore, we demonstrated cross-talk with the use of α-difluoromethylornithine which can reduce the activity of ornithine decarboxylase of L. donovani, eliminating the parasite-induced immune suppression and inducing collateral host protective responses in visceral leishmaniasis., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Our previous data showed that the Momordica grosvenori Swingle extract (MSE) exhibited the anti-inflammatory effect through markedly suppressed LPS-induced up-regulation of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and ODC (ornithine decarboxylase) gene expression in RAW 264.7 cells. Regarding the link between inflammation and carcinogenesis, we further investigated the bio-molecular mechanisms of both anti-inflammatory and anti-tumor activities in vivo using a TPA (12-O-tetradecanoyl phorbol 13-acetate)-stimulated mouse skin model. Pretreatment with MSE in mouse skin has led to the reduction of TPA-induced nuclear translocation of the nuclear factor-κB (NFκB) subunits as well as phosphorylation of IκBα and p65 subsequent reduction of IκBα degradation. In addition, the MSE inhibitory effect on upstream of NFκB was found to involve the transcriptional effects of MAPK signaling as indicated by strong suppression on TPA-induced activation of extracellular signal regulate kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK)1/2, phosphatidylinositol 3-kinase (PI3K) and Akt. Moreover, MSE significantly inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-TPA-induced skin tumor formation in mice measured by the tumor multiplicity of papillomas at 20 weeks. The results suggested that MSE contained promising functional ingredients capable of preventing inflammation-associated tumorigenesis.

Hepatocellular carcinoma (HCC) is among the most frequent malignancies in humans. HCC therapy is not efficient and the usual outcome is poor. In this regard, novel approaches to treat and prevent HCC are urgently needed. The Alpha-fetoprotein (AFP) is a serological marker of HCC. Recently it has been shown, that the DNA vaccines expressing AFP are capable in generating immune response against AFP. However, both, the immunization procedures and DNA vaccines used before were complex and not always very effective. We have shown that DNA vaccine encoding HIV-1 reverse transcriptase (RT) with fused ornithine decarboxylase (ODC) degradation signal induced a strong Th-1 immune response against RT in mice. Using this approach we designed a set of novel DNA vaccines bearing AFP and ODC degradation signal. Results obtained on transfected cells demonstrated efficient expression and fast proteasomal degradation of the recombinant AFP. The anti-tumor immune response stimulation was shown in immunized animals and most importantly a notable retardation of tumor growth was observed as a result of protective vaccination.

Antigenic molecules are modified for targeting to the proteasome by ubiquitin (Ub) or by a Ub-independent system such as ornithine decarboxylase (ODC) to be presented by MHC class I molecules. In this study, we compared the immunogenicity of human cytomegalovirus pp65 antigen fused with Ub and/or ODC, using RNA electroporation of human dendritic cells. Among the C-terminal mutants of Ub (G76, A76, and V76), Ub(G) showed the best ability to enhance the degradation of a target protein and stimulate T cells. The pp65 antigens fused with either Ub(G) or ODC enhanced the stimulation to CD8(+) T cells, and the effects of Ub(G) and ODC were similar. Furthermore, the fusion of both Ub and ODC additively increased immunogenicity compared with the single-fusion proteins. The fusion of Ub(G) and ODC enhanced primarily the stimulation of CD8(+) rather than CD4(+) T cells and more efficiently induced pp65-specific T cells in vitro. These additive effects of Ub and ODC in antigen processing may provide improved strategies to stimulate CD8(+) T cells for the development of immunotherapies against the variety of viral diseases and cancers.

Pneumocystis pneumonia (PcP) is the most common opportunistic disease in immunocompromised patients. Alveolar macrophages are responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PcP due to increased intracellular polyamine levels. In this study, the sources of polyamines and mechanisms of polyamine increase and polyamine-induced apoptosis were investigated. The level of ornithine decarboxylase (ODC) was elevated in alveolar macrophages, and the number of alveolar macrophages that took up exogenous polyamines was increased 20-fold during PcP. Monocytes, B lymphocytes, and CD8+ T lymphocytes that were recruited into the lung during PcP expressed high levels of ornithine decarboxylase, suggesting that these cells are sources of polyamines. Both protein and mRNA levels of antizyme inhibitor (AZI) were increased in alveolar macrophages during PcP. This AZI overexpression correlated with increased polyamine uptake by alveolar macrophages, because AZI expression knockdown decreased the polyamine uptake ability of these cells. AZI expression knockdown also decreased the apoptosis rate of alveolar macrophages. Pneumocystis organisms and zymosan A were found to induce AZI overexpression in alveolar macrophages, suggesting that beta-glucan, which is the major component of the Pneumocystis cell wall, induces AZI overexpression. The levels of mRNA, protein, and activity of polyamine oxidase were increased in alveolar macrophages during PcP, indicating that the H(2)O(2) generated during polyamine catabolism caused alveolar macrophages to undergo apoptosis. Taken together, results of this study indicate that Pneumocystis organisms induce AZI overexpression in alveolar macrophages, leading to increased polyamine synthesis and uptake and apoptosis rate of these cells.

Targeting of a DNA vaccine encoded protein for degradation via the proteasome is attempted since it may enhance the immunogenicity of the vaccine. We have fused HIV-1 reverse transcriptase (RT) to mouse ornithine decarboxylase (ODC), a protein rapidly degraded by proteasome in an ubiquitine-independent fashion, to enhance the introduction of RT into the MHC class I pathway. We also designed a fusion of RT with two short signals from the C-terminus of ODC (ODCsig) representing a minimal proteasome-targeting moiety of ODC (PEST signal). Fusion to ODC or ODC signal domain led to a marked enhancement of RT degradation. Plasmids encoding RT-ODC and RT-ODCsig chimera were used to immunize BALB/c mice. The administration of the plasmids was not associated with autoimmune disease. Moreover, mice receiving RT-ODCsig gene mounted a mixed Th1/Th2 response characterized by the in vitro secretion of IFN-gamma, IL-2, TNF-alpha, IL-4, and IL-10 upon stimulation of splenocytes with RT protein or RT derived peptides. Serum titers of 10(2) to 10(3) were observed in more than 50% of animals in that group, whereas fewer animals mounted an anti-RT response in the RT-ODC gene immunized group. Chimeras of the type described here can, therefore, be used in vaccinations aiming to induce HIV-1 RT-specific immune response.

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.

Aim: To investigate the ornithine decarboxylase (ODC) gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology., Methods: Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR. ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay., Results: A cell line, which could steadily secrete anti-ODC mAb, was selected through subcloning four times. Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6+/-1.03% vs 5.26+/-5%, P<0.01)., Conclusion: ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.

The Epstein-Barr virus thwarts immune surveillance through a Gly-Ala repeat (GAr) within the viral Epstein-Barr virus-encoded nuclear antigen 1 protein. The GAr inhibits proteasome processing, an early step in antigen peptide presentation, but the mechanism of proteasome inhibition has been unclear. By embedding a GAr within ornithine decarboxylase, a natural proteasome substrate that does not require ubiquitin conjugation, we now demonstrate inhibition in a purified system, excluding involvement of ubiquitin conjugation or of proteins extraneous to substrate and proteasome. We show further that the GAr acts as a stop-transfer signal in proteasome substrate processing, resulting in vivo in partial proteolysis that halts just short of the GAr. Similarly, introducing a GAr into green fluorescent protein destabilized by the ornithine decarboxylase degradation domain also stops the progress of proteolysis, leading to the accumulation of partial degradation products. We postulate that the ATP motor of the proteasome slips when it encounters the GAr, impeding further insertion and, in this way, halting degradation.

A Trypanosoma brucei bloodstream mutant in which both copies of the ornithine decarboxylase (ODC) gene were knocked out (ODC mutant) was used to determine the biological functions of ODC in T. brucei. Growth of the mutant cells ceased within 12-24 h in regular culture medium deficient in polyamines, but could be rescued by supplementation with 1 mM putrescine. A mouse model of T. brucei infection was used to determine whether the mutant was still infective and was found to develop either extremely low or undetectable levels of parasitemia, suggesting that in T. brucei, ODC activity is essential for establishing an infection. Furthermore, when these mice were subsequently challenged with wild-type T. brucei cells expressing the same variant surface glycoprotein (VSG), they did not develop any parasitemia, indicating that inoculating the mice with the attenuated ODC mutant had conferred protection against challenge by wild-type cells. These results were reproduced in C57BL/6J mice deficient in alpha-beta and gamma-delta T-cell receptors. However, no protection was observed in rag-2 knockout mice deficient in both B and T lymphocytes or in C57BL/10J mice deficient only in B lymphocytes. The results thus suggest that the ODC mutant could induce a T-lymphocyte-independent but B-lymphocyte-dependent immunity against wild-type cells of the same VSG. Such a mechanism of immunity has been elicited only by live T. brucei cells, but not by isolated VSGs or radiation-killed trypanosomes. This ODC mutant may thus represent a genuinely attenuated T. brucei bloodstream form capable of immunizing mammals against infections by African trypanosomes of the same VSG subtype without causing detectable infection by itself. The observation also raises the interesting likelihood that the in vivo treatment of T. brucei bloodstream forms with alpha-DL-difluoromethylornithine is a de facto attenuation of the parasitic organisms, which may very well result in B-lymphocyte-dependent host immune responses to subsequent infections by parasites of the same VSG subtypes.

Addition of spermidine to culture medium of insect cells, Trichoplusia ni 5, at a low cellular density suppressed ornithine decarboxylase (ODC; EC 4.1.1.17) activity and induced ODC inhibitory activity. The inhibitory factor was non-dialyzable, temperature-sensitive, and could reversibly form an inactive complex with ODC. It showed a time-independent and non-stoichiometric pattern of inhibition. Upon addition of spermidine to cultured cells with preinduced ODC, the enzyme decayed more rapidly than after addition of cycloheximide. These data strongly suggested that ODC of Tn5 cells is under negative feedback control by polyamines, in which an antizyme-like regulatory protein plays an essential role.

Using a new anti-human ornithine decarboxylase (anti-hODC) monoclonal antibody, the relationship between the immunoreactivity of ODC and its activity was analyzed in 21 human colorectal cancer tissues, 42 adjacent non-tumorous mucosa specimens, and 10 normal rectal mucosa samples from frozen sections and paraffin-embedded samples. A statistical significant correlation was found between the antibody reaction and the enzymic activity (P < 0.01). The immunohistochemical staining for ODC provides a new and simplified procedure for studying the activity of ODC as compared to previous methods using radioisotopes. It offers the advantages of retrospectively determining the amount of ODC in samples previously embedded in paraffin.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cochlear development. Whereas tissue specific differences in cochlear ODC activity have been demonstrated, cellular localization of ODC protein in the inner ear of the immature rat has not. ODC was localized in inner ear structures using an ODC polyclonal antibody and the effects of cycloheximide on ODC immunoreactivity and enzymatic activity were determined. Tissues demonstrating elevated enzymatic activity contained cells with the strong immunoreactivity. ODC activity was highest in the organ of Corti and lateral wall followed by the cochlear nerve. Immunoreactivity was demonstrated throughout the cochlea with intense staining of the hair cells, pillar cells, Deiter's cells, inner sulcus cells, basilar membrane, stria vascularis, spiral ganglion cell bodies and cochlear nerve fibers. Cycloheximide rapidly diminished cochlear ODC activity and expression of ODC protein. The half-life of cochlear ODC was 30 min. Localization of cellular sites of ODC is important in understanding the role of the ODC-polyamine pathway in cochlear development and will be a valuable marker for tissue damage from ototoxic agents.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosynthesis. We investigated the transcriptional regulation of the rat ODC gene using transient expression assays. The 5'-flanking region (-1156 to +13) of the ODC gene was sufficient to mediate strong basal expression of a luciferase reporter. Sequences between -345 and -93 contributed to basal promoter activity. This region, containing five potential Sp1 binding sites, was analyzed by electrophoretic mobility shift assays. Three specific DNA-protein complexes were identified using H35 nuclear extracts and the -345/-93 ODC probe. Binding to all three was eliminated by competition with an oligonucleotide containing an Sp1 binding site, but not by a mutant Sp1 oligonucleotide. Preincubation with an antibody against Sp1 supershifted complexes associated with one or more of Sp1 binding sites 1-4 as well as with site 5. DNase I footprinting revealed two protected regions: PR-I (-92 to -130) and PR-II (-304 to -332). PR-I contains a putative binding site for Sp1 that was protected by recombinant Sp1 protein. Transfection studies in Schneider SL2 cells demonstrated that the ODC promoter is transactivated up to 350-fold by Sp1 and that this transactivation is dependent on the presence of Sp1 binding sites 1-4. Thus, although the ODC promoter binds multiple nuclear proteins, Sp1 or a related protein appears to be a critical determinant of ODC transcription, possibly through cooperative interactions between Sp1 and additional transcription factors.

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC (Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345-360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355-360) portion whereas MP16-3 reacted with the ODC-(345-350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the formation of the polyamines putrescine, spermidine and spermine. In the present study ornithine decarboxylase-like immunoreactivity (ODC-LI) was localized immunocytochemically in rat spinal motoneurons, motoric nerves and myoneural junctions in several muscles. In the spinal cord ODC-LI was expressed in most of the large multipolar neurons located in the ventral horn at cervical and lumbar levels. ODC-LI was localized in the cytoplasm, dendrites and axons of the labelled neurons. The nuclei of motoneurons were unlabelled; however, the nuclear membranes and the surrounding cytoplasm were strongly stained. ODC-immunoreactive (IR) axons could be traced through the white matter entering the ventral roots. The myelinated axons in the ventral roots and in the nerve bundles among the muscles were intensely stained with ODC antiserum. The myoneural junctions apposing individual muscle fibers showed ODC-LI with slightly less intensity. Some ODC-IR nerve fibers were seen in the muscle spindles. The present results show that motoneurons in adult rat spinal cored express ODC-LI and that OCD-LI is transported to motoric nerves and myoneural junctions. This suggests that polyamines can be synthesized both in the motoneuron somata and in their peripheral projections. Polyamines may thus regulate cellular functions in all parts of motoneurons. In addition, polyamines may be secreted from their distal projections and have tropic effects on Schwann cells and/or muscular tissue.

L-Ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis and thus a unique marker of the proliferation and maturation processes, has been localized by means of immunohistochemistry in the human central nervous system. The enzyme protein has not only been found in the developing nervous system but also in the adult brain. The first immunopositive neuroblasts occurred in the 15th week of gravidity, whereas an increasing number of structures in the medulla oblongata and cerebellum showed ODC immunoreactivity in weeks 18-24 of gravidity. ODC immunoreactivity was revealed in multiple neurons and in the ependymal lining of ventricles. In the adult human brain ODC immunoreactivity was found in the hippocampus and in the spinal cord. In no case were immunoreactive glial cells observed.

Based on methods for ornithine-decarboxylase purification published previously we developed an improved procedure for purification of the enzyme from the kidneys of testosterone-treated NMRI mice. Advantages of the new procedure are, that inactivation of the enzyme during purification is largely reduced by fast methods for purification and by the use of proteinase inhibitors. That way we got pure ornithine decarboxylase within 60 h with a yield of about 70%. A part of the highly purified ornithine decarboxylase was used for the generation of monoclonal antibodies.

Using flow cytometry in combination with membrane permeabilization techniques to enhance binding of antibodies with immunoreactive protein within the cytoplasm, we have developed a method to examine the ornithine decarboxylase (ODC) activity present within subpopulations of epidermal cells following acute and chronic exposure to the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). The method described has the sensitivity to detect basal levels of ODC as well as increases in ODC at early time points following treatment with TPA and has the additional advantage of allowing subpopulation identification and characterization.

An affinity chromatography column with antibody specific to ornithine decarboxylase (ODC) was employed successfully for the retention and subsequent partial elution of ODC with enhanced activity and retention of antigenicity. This may be a useful procedure for further attempts to produce antiserum for identification and characterizations of ODC in vitro and in vivo.

A monoclonal antibody was obtained against rat liver ornithine decarboxylase by using hybridoma technology with a small amount of partially purified enzyme. The antibody, IgG1 of kappa-type, was affinity-purified to homogeneity from culture supernatants of hybridoma cells. While the antibody had no inhibitory effect on ornithine decarboxylase activity when tested alone, it precipitated up to 87 units (60 ng) of the enzyme per microgram in the presence of formalin-fixed Staphylococcus aureus Cowan I bacteria. Immunoadsorption on a column of the monoclonal antibody-Sepharose 4B was shown to be useful for the removal of ornithine decarboxylase from antizyme inhibitor preparations, an essential procedure for the accurate assay of either ornithine decarboxylase-antizyme complex or antizyme inhibitor. It was also shown that antizyme could be affinity-purified by using a column of the monoclonal antibody-Affi-Gel 10 to which ornithine decarboxylase had been bound.

Specific antiserum to a purified preparation of ornithine decarboxylase was prepared and successfully labeled with 125I. Monospecificity of this antiserum was attained by repeated precipitation with normal rat liver. The conditions for optimal antigen/antibody reaction were investigated. When the antibody was tested against either crude or purified regenerating rat liver enzyme a single precipitin line was observed both with Ouchterlony double diffusion plates and with immunoelectrophoresis. The specificity of the purified antiserum was also evaluated by sodium dodecyl sulfate gel electrophoresis of the 14C-labeled enzyme.antibody complex, the radioactivity of which appeared as a sharp peak in the 90,000 molecular weight region. Finally, an approximate antigen/antibody molar ratio was determined using labeled enzyme and labeled antiserum.

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.

This paper presents evidence that a polyamine-dependent protein kinase (EC 2.7.1.37) purified from nuclei of the slime mold Physarum polycephalum catalyzes phosphorylation of ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17). The protein kinase had properties similar to OrnDCase antizyme. Phosphocellulose chromatography of nuclear preparations from P. polycephalum yielded the polyamine-dependent protein kinase of subunit Mr 26,000 that was resolved from a second fraction in which the protein kinase copurified with a phosphate-acceptor protein of subunit Mr 70,000. At Na+ concentrations less than approximately 150 mM, a complex formed between the protein kinase and the phosphate-acceptor protein. The complex did not demonstrate protein kinase or OrnDCase activity. The complex was dissociated by greater than 150 mM Na+ into its constituent proteins. The dissociated complex catalyzed phosphorylation of the Mr 70,000 component in the presence of spermidine and spermine, and it also demonstrated OrnDCase activity. The purified Mr 70,000 component from the complex and authentic OrnDCase, purified by procedures previously reported, were virtually identical with respect to OrnDCase activity, capacity to be phosphorylated by the polyamine-dependent protein kinase, amino acid composition, and immunological crossreactivity. Phosphorylation of OrnDCase by the polyamine-dependent protein kinase sharply inhibited OrnDCase activity. Thus, this is an example of posttranslational covalent modification of OrnDCase with concurrent alteration of its catalytic function. It is also an unusual example of control of the first enzyme in a biosynthetic pathway by a protein kinase that is, in turn, modulated by the immediate end products of the pathway.

We previously described an arginase-deficient, polyamine-dependent Chinese hamster ovary cell line which grows in serum-free medium. From this strain we isolated a new mutant strain that has no detectable catalytic ornithine decarboxylase activity. The mutant cells contain, however, immunoreactive ornithine decarboxylase-like protein roughly in the same quantity as the parent strain. The mutant and the parent cell line strains also contain similar amounts of ornithine decarboxylase-mRNA hybridizable to a specific cDNA. If polyamines are omitted from the medium, proliferation of the mutant cells is considerably retarded and ceases in 6 to 10 days. Addition of ornithine or alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, has no effect on these cells. Putrescine and spermidine decreased in the mutant cells to undetectable levels during polyamine starvation, whereas spermine was reduced to 1/5th of that found in the control cultures. Polyamines appear to be indispensable for the mutant strain, but this was obvious only after the amount of polyamines, found as impurities in bovine serum albumin used in the medium, was reduced by dialysis to 10(-12) M. Because sera contain polyamines, the ability of the mutant strain to grow in serum-free medium is a great advantage in elucidation of the mechanisms of polyamine function.

Incubation with protein kinase NII did not result in phosphorylation or inactivation of mouse kidney ornithine decarboxylase. Partially purified ornithine decarboxylase preparations contained a protein kinase activity and stimulated the activity of RNA polymerase I. However, these properties were due to contaminating protein(s) since further purification reduced the kinase activity and removal of the ornithine decarboxylase with a specific antiserum did not abolish the ability to stimulate RNA polymerase I. Antibodies to RNA polymerase I did not interact with ornithine decarboxylase and antibodies to ornithine decarboxylase did not interact with RNA polymerase I. These results indicate that: a) mammalian ornithine decarboxylase activity is not regulated by phosphorylation by protein kinase NII or the contaminating kinase, and b) the ability of impure preparations of ornithine decarboxylase to stimulate RNA polymerase I is due to a contaminating unrelated protein.

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.

The immunological properties of thymidine kinase from a variety of human tumors suggest that the form of the tumor enzyme resembles that found in the placenta and in the nondividing colonic flat mucosa. To examine the placenta-like characteristics of tumor thymidine kinase, the jejunum and colon from rats ranging in age from fetal to old and from animals treated with dimethylhydrazine (DMH), an intestinal carcinogen, have been studied. In normal jejunum, thymidine kinase activity decreased rapidly with age. Both the activity and the response to phospholipase C and to mercaptans in DMH-induced tumors resembled that of fetal gut, while those in abnormal appearing DMH-treated jejunum were intermediate between normal control of the same age and tumor. Similar but less pronounced changes were seen in the colon. In the jejunum, the level of another enzyme normally associated with rapid cell division, ornithine decarboxylase, was found to be over 100 times that of the liver, colon, and stomach. Treatment of the animals with acetylaminofluorene and with DMH resulted in elevated levels of the enzyme in liver and in colon, respectively, but had little effect on this enzyme in other tissues. The data presented indicate that there were premalignant changes in the levels of both of these enzymes in target tissues of animals treated with carcinogens.

A sensitive enzyme immunoassay (EIA) was developed for the determination of ornithine decarboxylase (ODC, EC 4.1.1.17), in the range of 0.02-10 ng, using an affinity-purified anti-ODC-Fab'-peroxidase conjugate. The amount of ODC protein was determined in crude extracts from the kidney of testosterone-treated mice, regenerating rat liver and human thyroid carcinoma, with purified mouse kidney or rat liver enzyme as standard. In all these tissues, similar activity/protein ratios were found for ODC: 1.2 x 10(6)-1.9 x 10(6) nmol CO2/h/mg of ODC protein, which were roughly equivalent to the final specific activity of purified enzymes. ODC inactivated by alpha- difluoro-methylornithine (DFMO) could also be assayed with this method similarly to active ODC protein. However, ODC-antizyme complex gave a somewhat lower value than free ODC protein.

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) were purified from the livers of rats which had been treated with thioacetamide for 16 h (for details, see miniprint to Obenrader, M.F., and Prouty, W. F. (1977) J. Biol. Chem. 252, 2860-2865). The enzyme was purified over 7,000-fold from liver cytosol with an overall yield of 8%. Enzyme activity was eluted finally in two distinct fractions by chromatography on activated thiol-Sepharose 4B. Both forms appear to be dimeric proteins having molecular weights of approximately 100,000 by equilibrium sedimentation and analysis on a calibrated Sephadex G-200 column. The apparent subunits are approximately 50,000 daltons as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Since electrophoresis in the presence of detergent is the only method used here to indicate subunits, the possibility that conditions of sample preparation resulted in splitting of a labile protein cannot be excluded from consideration. Ornithine decarboxylase has a very broad pH-activity curve with an optimum that shifts from pH 7.0 to pH 7.8 as the enzyme is purified. The apparent Km values for a highly purified mixture of the two forms of enzyme for L-ornithine and pyridoxal 5'-phosphate were determined to be 0.13 mM and 0.25 micronM, respectively. Both sodium and potassium chloride were shown to inhibit enzymatic activity; 50% inhibition occurred at 270 mM for each when Km amounts or ornithine were used. Rat liver ornithine decarboxylase antiserum was prepared in rabbits using Form I of the enzyme as the antigen. The antibody was shown to precipitate quantitatively the ornithine decarboxylase activity isolated from induced rat liver and rat ventral prostate. The specificity of the antiserum was demonstrated by rocket immunoelectrophoresis and by gel electrophoresis in the presence of sodium dodecyl sulfate using immunoprecipitates obtained from enzyme preparations labeled either in vivo, with [3H]leucine, or in vitro, by reductive methylation using formaldehyde and sodium [3H]borohydride. The antibody preparation has been used in a titration method to assess the half-life of antigen in livers of rats induced for ornithine decarboxylase by injection of thioacetamide. In two experiments, the t1/2 of activity at the height of induction, following injection of cycloheximide, was 19 and 24 min, while the t1/2 of disappearance of antigen was 28 and 33 min, respectively. In each experiment the t1/2 for antigen was significantly longer than the t1/2 for loss of enzyme activity. Enzyme levels appear to be modulated primarily by synthesis and degradation of antigen. Furthermore, the observation that enzyme activity is lost with a shorter t1/2 than antigen is consistent with the idea that denaturation is an initial step in the degradation of this enzyme...

Repeated injections of 1,3-diaminopropane, a potent inhibitor of mammalian ornithine decarboxylase, induced protein-synthesis-dependent formation of macromolecular inhibitors or ;antienzymes' [Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A.73, 1858-1862] to ornithine decarboxylase in normal rat liver. Addition of the macromolecular inhibitors, produced in response to repeated injections of diaminopropane, to active ornithine decarboxylase in vitro resulted in a profound loss of the enzyme activity, which, however, could be partly recovered after passage of the enzyme-inhibitor mixture through a Sephadex G-75 columin in the presence of 0.4m-NaCl. This treatment also resulted in the appearance of free inhibitor. In contrast with the separation of the enzyme and inhibitory activity after combination in vitro, it was not possible to re-activate, by using identical conditions of molecular sieving, any inhibited ornithine decarboxylase from cytosol fractions obtained from animals injected with diaminopropane. However, the idea that injection of various diamines, also in vivo, induces acute formation of macromolecular inhibitors, which reversibly combine with the enzyme, was supported by the finding that the ornithine decarboxylase activity remaining after diaminopropane injection appeared to be more stable to increased ionic strength than the enzyme activity obtained from somatotropin-treated rats. Incubation of the inhibitory cytosol fractions with antiserum to ornithine decarboxylase did not completely abolish the inhibitory action of either the cytosolic inhibitor or the antibody. A single injection of diaminopropane produced an extremely rapid decay of liver ornithine decarboxylase activity (half-life about 12min), which was comparable with, or swifter than, that induced by cycloheximide. However, although after cycloheximide treatment the amount of immunotitrable ornithine decarboxylase decreased only slightly more slowly than the enzyme activity, diaminopropane injection did not decrease the amount of the immunoreactive protein, but, on the contrary, invariably caused a marked increase in the apparent amount of antigen, after some lag period. The diamine-induced increase in the amount of the immunoreactive enzyme protein could be totally prevented by a simultaneous injection of cycloheximide. These results are in accord with the hypothesis that various diamines may result in rapid formation of macromolecular inhibitors to ornithine decarboxylase in vivo, which, after combination with the enzyme, abolish the catalytic activity but at the same time prevent the intracellular degradation of the enzyme protein.

Stepwise increments of the concentration of 2-difluoromethylornithine (DFMO), a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (ODC), resulted in a selection of cultured human IgG-myeloma cells (Sultan cell line) capable of growing in the presence of up to 3 mM-DFMO. This capacity was associated with 10-fold increase in ODC activity in the dialysed extracts of drug-resistant myeloma cells, markedly enhanced synthesis rate for ODC enzyme molecules, as revealed by a 20 min [35S]methionine labelling of cellular proteins, followed by specific immunoprecipitation and SDS/polyacrylamide-gel electrophoresis, dose-dependently increased expression of ODC mRNA in resistant cells (effective dose causing 50% inhibition), dose-dependent amplification of ODC gene sequences in a 9-kilobase-pairs EcoRI genomic DNA fragment, and (v) a 10-fold increase in the ED50 (effective dose causing 50% inhibition) for the anti-proliferative action of DFMO in these myeloma cells. These results represent one of the few gene amplifications described in cultured human cells.

Ornithine decarboxylase was purified from androgen-treated mouse kidney to homogeneity and high specific activity. The purified enzyme was utilized for production and screening of rat monoclonal and polyclonal antibodies. A rat monoclonal antibody was isolated which was capable of immunoprecipitation of native mouse kidney ornithine decarboxylase activity or the [3H]difluoromethylornithine-inactivated enzyme. Phosphorylation of mouse ornithine decarboxylase by casein kinase-II prior to immunoprecipitation led to complete loss of the epitope recognized by the monoclonal antibody but did not alter recognition by polyclonal antibody. Mammalian ornithine decarboxylase activity obtained from several species, in crude or partially purified extracts, was subjected to quantitative immunoprecipitation with monoclonal and polyclonal antibody. Polyclonal antibody immunoprecipitated all of the ornithine decarboxylase activity from every extract tested, while monoclonal antibody was capable of only limited immunoprecipitation (60-80%). Due to the inability of the monoclonal antibody to recognize ornithine decarboxylase phosphorylated in vitro by casein kinase-II and the partial immunoprecipitation of ornithine decarboxylase activity from cell extracts, a portion of the ornithine decarboxylase molecule population must exist in a phosphorylated state. This immunological evidence further confirms existing data that the enzyme exists in at least two distinct forms.

L-Ornithine decarboxylase was purified to apparent homogeneity from the kidneys of testosterone-treated mice. Antibodies to ornithine decarboxylase were raised in a rabbit using the purified enzyme. Ouchterlony double diffusion technique revealed a single precipitin line between the antiserum and purified mouse kidney ornithine decarboxylase. The antibodies inhibited ornithine decarboxylase from various tissues of mice and rats to the same extent, indicating a close immunological relationship. S-Adenosyl-L-methionine decarboxylase and L-histidine decarboxylase from mouse kidney as well as ornithine decarboxylase from Escherichia coli were unaffected by the antibodies.

A macromolecular inhibitor to ornithine decarboxylase (ODC) present in mouse brain was identified as ODC antizyme [Fong, Heller & Canellakis (1976) Biochim. Biophys. Acta 428, 456-465; Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1858-1862] on the basis of kinetic properties, Mr and reversal of its inhibition by antizyme inhibitor. The brain antizyme, however, did not cross-react immunochemically with any of seven monoclonal antibodies to rat liver antizyme. ODC activity in mouse brain rapidly decreased after birth, in parallel with putrescine content, and almost disappeared by 3 weeks of age. Free antizyme activity appeared shortly after birth and increased gradually, whereas ODC-antizyme complex already existed at birth and then gradually decreased. Thus total amount of antizyme remained about the same throughout the developmental period in mouse brain. In addition to ODC-antizyme complex, inactive ODC protein was detected by radioimmunoassay in about the same level as the complex at 3 weeks of age. Upon cycloheximide treatment, both free ODC activity and ODC-antizyme complex rapidly disappeared, although free antizyme and the inactive ODC protein were both quite stable.

Antiserum against ornithine decarboxylase (EC 4.1.1.17) was prepared in rabbits using purified ornithine decarboxylase from rat liver as the antigen. Immunoglobulins from the immune sera were covalently coupled to agarose by cyanogen bromide activation. With the aid of this immunoadsorbent against the enzyme it has been shown that following partial hepatectomy and growth hormone administration, the ornithine decarboxylase activity is elevated concomitantly with the increase in the immunoreactive enzyme protein. In addition, the rapid decay in ornithine decarboxylase activity in regenerating rat liver after cycloheximide injection is accompanied by a decrease in the immunoreactive protein. These results suggest that the activity of ornithine decarboxylase in rat liver is regulated through rapid changes in de novo synthesis and degradation of the enzyme protein.

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.

Antiserum elicited to ornithine decarboxylase (ODC) purified from murine RAW 264 macrophage-like cells has been employed to localize ODC in cultured murine cells. The antiserum immunoprecipitated 100% of the ODC activity from the cultured cells. The specificity of the antiserum was demonstrated by the immunoprecipitation from 35S-methionine metabolically-labeled cell extracts of a single protein which migrated upon SDS-gel electrophoresis coincident with authentic ODC. Indirect immunofluorescence experiments were performed on paraformaldehyde-fixed RAW 264 cells and JB6 epidermal cells using the rabbit anti-ODC antiserum and FITC-conjugated goat anti-rabbit IgG. Little immunofluorescence was apparent in non-stimulated cells. Intense immunofluorescence was detectable in stimulated cells at times of peak cellular ODC activity. Antigenically-reactive ODC was localized diffusely in the cytoplasm and was absent in the nuclei of RAW 264 cells, whereas in the JB6 cells the immunodetectable enzyme protein was localized in a punctate pattern in both the cytoplasm and nucleoplasm and was absent in the nucleolus. The appearance and disappearance of immunoreactive ODC in both cell types after stimulation was consistent with the alterations in ODC activity.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.