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A transgenic mouse line carrying ornithine decarboxylase cDNA as the transgene under the control of a mouse mammary tumor virus long terminal repeat (MMTV LTR) promoter was generated in order to study whether ornithine decarboxylase transgene expression will have any physiological or pathological effect during the entire life of a transgenic mouse. The high frequency of infertile animals and the loss of pups made the breeding of homozygous mice unsuccessful. However, a colony of heterozygous transgenic mice was followed for 2 years. In adult heterozygous transgenic mice, ornithine decarboxylase activity was significantly increased in the testis, seminal vesicle and preputial gland when compared to non-transgenic controls. In contrast, ornithine decarboxylase activity was decreased in the kidney and prostate of transgenic mice. No significant changes in ornithine decarboxylase activity were found in the ovary and mammary gland and only moderate changes in ornithine decarboxylase activity were detected in the heart, brain, pancreas and lung. The most common abnormalities found in adult animals (12 males and 20 females) of the transgenic line were inflammatory processes, including pancreatitis, hepatitis, sialoadenitis and pyelonephritis. Spontaneous tumors were observed in eight animals, including two benign tumors (one dermatofibroma, one liver hemangioma) and six malignant tumors (one lymphoma, one intestinal and three mammary adenocarcinomas and one adenocarcinoma in the lung). No significant pathological changes were found in 17 nontransgenic controls.

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Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).

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The distribution of ornithine decarboxylase antizyme messenger ribonucleic acid (AZ mRNA) and AZ-like immunoreactivity (LI) was studied in the brainstem and spinal cord motoneurons and in the extraocular and triceps surae muscles of rat. In situ hybridization showed AZ mRNA in the gray matter of the spinal cord at different levels of spinal cord with highest AZ mRNA levels in the ventral horn of the spinal cord. No apparent changes in AZ mRNA contents were seen after unilateral transection of the sciatic nerve in lumbar motoneurons. AZ-immunoreactive (IR) motoneurons were observed in the nucleus of the VI cranial nerve and in the ventral horn of the spinal cord. These motoneurons also showed ornithine decarboxylase (ODC)-LI. Subcellularly, AZ-LI was observed both in the nuclei and cytoplasm of labeled motoneurons. Heavily stained AZ-IR nerve fibers and myoneural junctions were observed among muscle fibers in different muscles. In addition, the nuclei of muscle fibers showed AZ-LI.

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Several studies have documented increased expression of ornithine decarboxylase (ODC) in neoplastic colorectal tissue versus normal-appearing colonic mucosa. The present study was undertaken to determine whether there is an association between the degree of overexpression of ODC in colorectal carcinomas and survival in a series of 74 patients. A high level of tumor ODC expression was found to be significantly associated with greater survival in our patient series. Patients with tumor ODC activities greater than the median and especially in the highest quartile experienced a more favorable outcome than those patients with ODC values below the median or in the lowest quartile (P = 0.03 and 0.02, respectively). The presence of a GTP-activatable isoform of ODC was also significantly associated with a favorable prognosis but only in tumors of the right colon (P = 0.01). There was no association found between ODC activity and tumor grade, tumor size, or patient age, sex, or race. Our results demonstrate that high levels of ODC expression (and presence of a GTP-activatable isoform for right-sided colon tumors) predict a favorable prognosis in human colorectal carcinoma. Knowledge of a patient's ODC status at the time of surgery may be useful in decisions regarding adjuvant therapy. Understanding the mechanism(s) involved should lead to new therapeutic approaches for advanced colorectal carcinoma.

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The activity of ornithine decarboxylase (ODC) measured in different regions of rat brain was highest in the hippocampus and lowest in the cerebellum. The ODC activity of a crude extract of the cerebellum was increased by the addition of GTP to the enzyme assay. Following dissociation of the ODC-antizyme complex by Sephadex G-75 chromatography in buffer containing 0.25 M NaCl, the GTP-activatable ODC was found in every brain region analysed. This GTP-activatable brain ODC has greater affinity for antizyme than the non-GTP-activatable brain ODC or the kidney ODC. The irreversible inhibitor of ODC, alpha-difluoromethylornithine (DFMO), inhibited approx. 60% of the ODC activity of all brain regions, whereas kidney ODC was inhibited totally by DFMO. When extracts of brain and kidney were incubated at 55 degrees C, kidney ODC was rapidly inactivated, but brain ODC was more heat-stable. Brain ODC, but not kidney ODC, was activated by GTP and ATP, and also by their deoxy forms. The K1/2 for activation of the enzyme was 2 microM for GTP and 40 microM for ATP. Using partially purified brain ODC, the activation by GTP was irreversible. These results demonstrate for the first time that the GTP-activatable ODC exists in the brain and is associated with the antizyme. The possible mechanisms of activation by GTP, the significance of this finding for the regulation of brain ODC, and the similarities to and differences from the GTP-activatable ODC found in certain rodent and human tumours are all discussed.

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Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.

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Ornithine decarboxylase (ODC) activity was measured in colon adenocarcinomas and adjacent normal-appearing colon mucosa from a total of 40 patients undergoing surgical resections. The enzyme activity was measured in the presence and absence of GTP, since recent work has demonstrated a GTP-activatable form of ODC in some murine and human tumors. In general, ODC specific activity was higher in adenocarcinomas than in adjacent normal-appearing mucosa. Of greater interest, however, was the finding that 13 of 40 tumors and 3 of 40 mucosae contained a GTP-activatable form of ODC. These are minimal estimates of the proportion of tissues positive for this enzyme form, since a multiple sampling protocol indicated that expression of a GTP-activatable ODC was not uniform throughout a given tumor. Chromatographic analyses of tumor extracts revealed the presence in some tumors of multiple size forms of ODC, only some of which were activated by GTP. Enzyme kinetic data indicated that the multiple forms of ODC can have different affinities for L-ornithine and that GTP can "normalize" the aberrant kinetic properties of these forms. While there was no statistically significant correlation of the presence of a GTP-activatable ODC with stage of disease, analysis of our data revealed a positive association of a GTP-activatable ODC with tumor site; a much higher percentage of tumors of the cecum contained this ODC isoform than tumors of other colonic segments (64% versus less than or equal to 25% for other sites). These results demonstrate (a) the presence of a functionally distinct form of ODC in some human colon adenocarcinomas and (b) a distinct regional distribution of this ODC form within the colon. We suggest this alteration in a key enzyme in the growth-associated pathway of polyamine biosynthesis may play a role in colon tumor progression.

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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.

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Mouse brain ornithine decarboxylase (ODC) was purified to near-homogeneity by using (NH4)2SO4 precipitation and chromatography on heparin-Sepharose, pyridoxamine phosphate-agarose and DEAE-cellulose. On SDS/polyacrylamide-gel electrophoresis, the final preparation gave one protein band similar to that obtained for purified mouse kidney enzyme, corresponding to an Mr of 53.000. The overall yield of the purification exceeded about 50-fold the total activity of the enzyme in the starting material. By affinity chromatography on ODC-bound Sepharose, the extra enzyme activity was shown to originate, at least partly, from the enzyme-antizyme complex. These results demonstrate that ODC in mouse brain occurs mainly in an inactive form and is activated during purification.

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Mouse brain ornithine decarboxylase activity is about 70-fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immunoreactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme-mediated mechanism in adult brain.

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The effects of chlorpromazine, imipramine, thioridazine, chlorprothixene, amitriptyline, desipramine and triflupromazine on adenosylmethionine decarboxylase purified from rat liver have been studied. The compounds caused competitive inhibition of the enzyme at 10(-5) - 10(-3) M concentrations. For chlorprothixene and triflupromazine the inhibition was linear, while the other drugs showed increasing, nonlinear inhibition at higher concentrations. Apparent Ki's for the compounds were between 6.8 X 10(-5) M (for chlorprothixene) and 6.4 X 10(-4) M (for desipramine). Inhibition of 50% under optimal assay conditions was achieved between drug concentrations of 1.3 X 10(-4) M (thioridazine) and 1.3 X 10(-3) M (imipramine).

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Intraperitoneal injection of chlorpromazine and imipramine increases mouse brain ornithine decarboxylase but decreases S-adenosyl-L-methionine decarboxylase activity. Maximal effect was obtained 6-8 hr after treatment at which time single dose of chlorpromazine (50 mg/kg) stimulated ornithine decarboxylase activity 7-fold and decreased S-adenosylmethionine decarboxylase activity to 50% from the control level. Correspondingly, ornithine decarboxylase activity was 5.5 times higher than the control value and S-adenosylmethionine decarboxylase activity about 40% from that after imipramine injection (80 mg/kg). The possible dependence of the enzyme responses on adrenergic receptors was studied using alpha-adrenoceptor antagonist, phentolamine, and beta-adrenoceptor antagonist, propranolol, concurrently with chlorpromazine and imipramine. The stimulation of ornithine decarboxylase but not the inhibition of S-adenosylmethionine decarboxylase could be abolished by propranolol (10 mg/kg), whereas phentolamine (10 mg/kg) slightly increased ornithine decarboxylase activity even when given alone. This suggests that beta- but not alpha-adrenergic mediation is involved in the stimulation of mouse brain ornithine decarboxylase activity and that brain ornithine and S-adenosylmethionine decarboxylase activities are independently regulated. When chlorpromazine and imipramine were tested in vitro, both of them turned out to have an inhibitory effect on S-adenosylmethionine decarboxylase. The former caused 50% inhibition at a concentration of 1 mM and the latter at 2 mM. Preliminary tests suggest that the type of inhibition is noncompetitive for both of them.

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Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, is known to be regulated by a macromolecular inhibitor, termed antizyme, in a number of cellular systems. The present results show that the antizyme is also a functional component of polyamine metabolism in the brain. It could be demonstrated both in normal randomly selected mice and in animals which had been subjected either to intracerebroventricular injection of saline, which is known to cause a transient activation of ornithine decarboxylase, or to 1,3-diamino-2-propanol, an antizyme-inducing agent. When compared to tissues or cell systems studied so far, the cytosol fraction from mouse brain homogenate appeared to contain an exceptionally high amount of antizyme, that was bound to some material other than active ornithine decarboxylase. This feature was seen in all the animal groups studied, being most prominent after saline injection, when the amount of dissociable antizyme exceeded 14-fold the corresponding released ornithine decarboxylase activity. In untreated animals the excess was about eightfold and after 1,3-diamino-2-propanol about fivefold.

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Mouse brain ornithine decarboxylase (ODC) activity is high at the time of birth, whereas S-adenosyl-L-methionine decarboxylase (SAM-DC) activity is low. ODC activity, and putrescine, spermidine and spermine concentrations decline rapidly during postnatal development to the low level characteristic of mature brains, while SAM-DC activity behaves in the opposite manner. The fluctuations in mouse brain polyamine metabolism are in accord with those found in the rat. The apparent Km values of ODC and SAM-DC for their substrates decline parallel with the decrease of substrate and product concentrations during ontogeny suggesting substrate and/or product dependent regulation of polyamine synthesis in the developing brain.

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The levels of putrescine and spermine in mouse brain were rather constant at different times of day, as were the activities of ornithine and S-adenosyl-L-methionine decarboxylases. Contrary to an earlier report, the level of spermidine was found to be relatively constant. A possibly significant feature in the present results was the steady decline during the light period and rise during darkness of cerebral spermidine and spermine levels, the differences between maximum and minimum being about 15% for both compounds.

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Injections of beta-(p--chlorophenyl)-gamma-aminobutyric acid caused a monophasic stimulation of the activity of neural L-ornithine decarboxylase, to reach a maximum of 9-fold compared with the control values 3 h after treatment. Stimulation of hepatic L-ornithine decarboxylase was biphasic, the activity reaching its first peak, 48-fold compared with the control values, similarly at about 3 h after administration, and returning to its initial level by 4 h, and rising to a second peak, about one-third of the magnitude of the first, about 25 h after the injection. The effect in the adrenal gland of the mouse was multiphasic, reaching its maximum, 94-fold enzyme activity compared with the control values, 7--8 h after treatment. There were also marked fluctuations in the activity of S-adenosyl-L-methionine decarboxylase in the tissues examined.

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