Androgen receptor, testosterone uptake and karyotype in androgen-dependent mouse tumor (SC 115) and its androgen-independent subline (CS 2)

Content of androgen receptor, retention of injected testosterone and karyotype of SC 115, androgen-dependent tumor, were compared with those of CS 2, an androgen-independent subline derived from SC 115. Although Bmax was less than that of SC 115, androgen receptor was present in the cytosol and the nuclear extract from CS 2. To examine the ability for androgen retention, a large amount of testosterone was injected into tumor-bearing mice, and the amount of androgen in the crude nuclear and postnuclear fractions of tumors was compared. In both fractions, retention of injected androgen was higher in the SC 115 than in the CS 2. Since most of the injected testosterone was not metabolized in the tissues and the injection of testosterone 5 alpha-reductase inhibitor showed no significant influence on the growth rate of the SC 115, intracellular active androgen was assumed to be testosterone in these tumor cells. As the CS 2 was tetraploid, the androgen independency of the CS 2 seems to be related to chromosomal changes.     

Effects of testosterone on renal ornithine decarboxylase and kidney function

Testosterone injection caused a 2,000% increase in renal ornithine decarboxylase activity in intact male mice. A single injection of testosterone produced the same effect as repeated injections. The response was dose-dependent and could be blocked by actinomycin, diaminopropane, and cadaverine. Cycloheximide and putrescine had no inhibitory effect. Renal ODC response to arginine vasopressin was altered after castration; however, urine specific gravity and serum osmolality were unaffected by changes in renal ornithine decarboxylase activity.     

Changes in ornithine decarboxylase activity in normal tissues of tumor-bearing mice during tumor growth.

The ornithine decarboxylase [EC 4.1.1.17] activities in the liver and spleen of tumor-bearing mice increased remarkably, reaching a peak 4 to 6 days after inoculation of tumor cells. On the contrary, the enzyme activity in the kidney decreased during tumor growth and had almost disappeared on day 6 after tumor inoculation. Injection of cell-free tumor homogenate also raised the enzyme activities in the liver and spleen, but did not change the activity in the kidney. No increase in enzyme activity in the liver of mice was observed on injection of homogenates of normal tissues, such as liver, spleen, kidney, and muscle.     

In vitro effect of androgen on RNA synthesis in nuclei from androgen-independent subline of Shionogi carcinoma (CS 2)

By serial transplantation of CS 1, a subline of Shionogi carcinoma SC 115, to female mice, another subline was obtained and designated CS2. The subline showed a complete loss of androgen dependency on the growth of the tumor. When male mice bearing the tumor were castrated and treated with testosterone, the activity of RNA polymerase I in isolated nuclei from the tumor hardly varied during the period of the experiments (36 h), while the activity of RNA polymerase II exhibited a transient increase (about 40%) at 6 h after the testosterone injection. The results, together with the previous ones showing 80% and 40% increases in RNA polymerase I activity at 24 h after testosterone administration in the case of SC 115 (androgen-dependent tumor) and CS 1 (less androgen-dependent tumor), respectively, indicate that the stimulation of RNA polymerase I activity by androgen in the tumor tissues is closely related to the androgen dependency on the growth of the tumors.     

Transgenic mice aberrantly expressing human ornithine decarboxylase gene.

We have generated transgenic mice carrying human ornithine decarboxylase gene. Two different transgene constructs were used: (i) a 5'-truncated human ornithine decarboxylase gene and (ii) an intact human ornithine decarboxylase gene. Transgenic mice carrying the 5'-truncated gene did not express human ornithine decarboxylase-specific mRNA. Transgenic mice carrying the intact human ornithine decarboxylase gene expressed human-specific ornithine decarboxylase mRNA in all tissues studied. However, as indicated by actual enzyme assays, the expression pattern was highly unusual. In comparison with their wild-type littermates, the transgenic mice exhibited greatly elevated enzyme activity in almost every tissue studied. Ornithine decarboxylase activity was moderately elevated in parenchymal organs such as liver, kidney, and spleen. Tissues like heart, muscle, lung, thymus, testis, and brain displayed an enzyme activity that was 20 to 80 times higher than that in the respective tissues of nontransgenic animals. The offspring of the first transgenic male founder animal did not show any overt abnormalities, yet their reproductive performance was reduced. The second transgenic founder animal, showing similar aberrant expression of ornithine decarboxylase in all tissues studied, including an extremely high activity in testis, was found to be infertile. Histological examination of the tissues of the latter animal revealed marked changes in testicular morphology. The germinal epithelium was hypoplastic, and the spermatogenesis was virtually totally shut off. Similar examination of male members of the first transgenic mouse line revealed comparable, yet less severe, histological changes in testis.     

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.     

Two variants of ornithine decarboxylase activity in the mouse liver. The nature of enzyme induction

Ornithine decarboxylase activity in mouse liver is predominantly located in the cell nuclei. After injection of some inducing agents (thioacetamide, diethylnitrosamine, hydrocortisone) the enzyme leaves the nucleus for cytosol. A circadian rhythm of ornithine decarboxylase activity has been observed in nucleus and cytosol, the decrease of enzyme activity in the nucleus being accompanied by its increase in cytosol. The enzyme obtained from intact mice with a minimal level of ornithine decarboxylase activity in the cytosol differs in ion-exchange properties, pH-optimum and Km for ornithine from the thioacetamide stimulated (nucleus enzyme).     

Stimulation of ornithine decarboxylase activity and inhibition of S-adenosyl-l-methionine decarboxylase activity in leukaemic mice by methylglyoxal bis(guanylhydrazone) (Short Communication)

Administration of methylglyoxal bis(guanylhydrazone) to leukaemic mice results in an early depression followed by a marked elevation of S-adenosyl-l-methionine decarboxylase activity. Further, there is an early prolonged increase in the activity of ornithine decarboxylase, the initial enzyme in the polyamine biosynthetic pathway. Because of the profound effects of methylglyoxal bis(guanylhydrazone) in vivo on the polyamine biosynthetic pathway, the drug can no longer be considered a specific inhibitor of spermidine synthesis.     

Concentrations of putrescine and polyamines and their enzymic synthesis during androgen-induced prostatic growth

1. Castration of adult rats resulted in marked decreases in the amounts of putrescine, spermidine and spermine in the ventral prostate gland. Spermidine concentrations decline rapidly over the first 11 days after androgen withdrawal, reaching a value of only 12% of normal controls. Spermine concentrations diminish more slowly, reaching 24% of normal within 11 days. The spermidine/spermine molar ratio falls from 0.9 to 0.46 under these conditions. Putrescine concentrations decrease by 70% at 7 days after castration and then remain constant for some days. 2. After daily injections of testosterone propionate to rats castrated 7 days previously, prostatic spermidine and putrescine concentrations increase significantly within 24h; normal or even greater values are observed within 8 and 4 days respectively. In contrast, the spermine concentration does not increase until 5 days after commencement of androgen treatment. 3. The activities of two enzymes involved in polyamine biosynthesis (ornithine decarboxylase and a putrescine-activated S-adenosyl-l-methionine decarboxylase system) were greatly decreased soon after castration: after 7 days the respective values were 15% of normal for ornithine decarboxylase and 7% of normal for putrescine-dependent decarboxylation of S-adenosyl-l-methionine. Injection of testosterone propionate into animals castrated 7 days previously induced a rapid increase in both enzymic activities: ornithine decarboxylase was doubled in 6h, and increased three- to four-fold within 48h, whereas the putrescine-dependent decarboxylation of S-adenosyl-l-methionine doubled in 3h and increased tenfold within 48h of commencement of daily androgen treatments. 4. The activity of these enzyme systems was very low in the ventral prostates of hypophysectomized rats and was increased by administration of testosterone in a manner similar to that found in castrated rats. 5. Alterations in the activity of two ventral-prostate enzymes involved in ornithine production (arginase) and utilization (ornithine–2-oxoglutarate transaminase) that result from changes in the androgenic status of rats are described. 6. The findings presented suggest that the activities of ornithine decarboxylase and the putrescine-dependent S-adenosyl-l-methionine decarboxylase system, rather than ornithine concentrations, are rate-limiting for the formation of putrescine and polyamines in rat ventral prostate. 7. The relation of polyamines to androgen-induced prostatic growth is discussed with particular reference to the biosynthesis of proteins and nucleic acids.     

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney

An immunoblotting technique was used to study the forms of ornithine decarboxylase present in androgen-induced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (approximately 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive alpha-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr approximately 53 000) without substantial loss of enzyme activity. The synthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S]methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fraction of total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1,3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity did not greatly selectively inhibit the synthesis of the enzyme. However, 1,3-diaminopropane did produce an increase in the rate of degradation of ornithine decarboxylase and a general reduction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1,3-diaminopropane.     

Multiple species of ornithine decarboxylase in mouse kidney. Effect of testosterone

Multiple species of ornithine decarboxylase were separated by chromatography of mouse kidney extract on DEAE-Sepharose CL-6B. The elution patterns of ornithine decarboxylase activity and immunoreactive enzyme protein in the kidneys of untreated and testosterone-treated male mice did not differ otherwise than in order of magnitude. The immunoblots of the chromatography fractions neither revealed any differences in enzyme subunit size between two experimental groups. These findings suggest that the stabilization of ornithine decarboxylase by androgens is not due to the molecular changes of enzyme protein.     

Changes in mouse kidney ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein as measured by radioimmunoassay

Antibodies were produced in rabbits to homogeneous mouse kidney ornithine decarboxylase and used to determine the amount of this protein present in kidney extracts by a competitive radioimmunoassay procedure. The labeled ligand for this assay was prepared by reacting renal ornithine decarboxylase with [5-3H] alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor. The sensitivity of the assay was such that 1 ng of protein could be quantitated and the binding to ornithine decarboxylase of a macromolecular inhibitor (antizyme) or alpha-difluoromethylornithine did not affect the reaction. It was found that treatment of female mice with testosterone produced a 400-fold increase in ornithine decarboxylase protein in the kidney within 4-5 days. Exposure to cycloheximide or to 1,3-diaminopropane led to a rapid disappearance of the protein which paralleled the loss of enzyme activity. There was no sign of any immunoreactive but enzymatically inactive form of mouse kidney ornithine decarboxylase under any of the conditions investigated. The results indicate that fluctuations of the enzyme activity in this organ are mediated via changes in the amount of enzyme protein rather than by post-translational modifications or interaction with inhibitors or activators.     

Stabilization of ornithine decarboxylase and N1-spermidine acetyltransferase in rat liver by methylglyoxal bis(guanylhydrazone)

The activities of ornithine decarboxylase and spermidine N1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Inhibition of ornithine decarboxylase induction of interferon (alpha + beta) and its reversal by dibutyryl adenosine 3',5'-monophosphate

Interferon (alpha + beta) given to C3H/HeN mice intraperitoneally inhibited increases in the activities of adenylate cyclase and ornithine decarboxylase after partial hepatectomy. The inhibition of ornithine decarboxylase was prevented by administration of dibutyryl cAMP. Core (2'-5')oligo(adenylate), i.e. A2'p5'A2'p5'A or (A2'p)2A, as well as interferon inhibited the increases in these two enzymes caused by partial hepatectomy. The inhibition by (A2'p)2A of ornithine decarboxylase activity was reversed by dibutyryl cAMP. These results suggested that the activity of interferon was similar to that of (A2'p)2A and that the inhibition of ornithine decarboxylase induction caused by these agents resulted from the inhibition of adenylate cyclase activity.     

In vivo 13C nuclear magnetic resonance spectroscopy using a solenoid transmit/receiver coil

The administration of cadmium (1.25 mg as Cd2+/kg, ip.) to male rats resulted in a significant increase of hepatic and renal ornithine decarboxylase activity. The maximum increase of ornithine decarboxylase activity to about 10-fold of the controls was seen at 4 hr after the administration of cadmium, and the increased enzyme activity was returned to control levels by 12 hr. Cadmium produced somewhat dose-dependently the increase of ornithine decarboxylase activity. The increase of ornithine decarboxylase seen on the administration of cadmium was cancelled by pretreatment of rats with cycloheximide. The treatment of female rats with cadmium also caused the increase of hepatic ornithine decarboxylase activity, but not renal enzyme activity.     

A rat monoclonal antibody which interacts with mammalian ornithine decarboxylase at an epitope involved in phosphorylation

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.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

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.     

Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme

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.