Effects of agmatine accumulation in human colon carcinoma cells on polyamine metabolism, DNA synthesis and the cell cycle

Putrescine, spermidine and spermine are low molecular polycations that play important roles in cell growth and cell cycle progression of normal and malignant cells. Agmatine (1-amino-4-guanidobutane), another polyamine formed through arginine decarboxylation, has been reported to act as an antiproliferative agent in several non-intestinal mammalian cell models. Using the human colon adenocarcinoma HT-29 Glc(-/+) cell line, we demonstrate that agmatine, which markedly accumulated inside the cells without being metabolised, exerted a strong cytostatic effect with an IC50 close to 2 mM. Agmatine decreased the rate of L-ornithine decarboxylation and induced a 70% down-regulation of ornithine decarboxylase (ODC) expression. Agmatine caused a marked decrease in putrescine and spermidine cell contents, an increase in the N1-acetylspermidine level without altering the spermine pool. We show that agmatine induced the accumulation of cells in the S and G2/M phases, reduced the rate of DNA synthesis and decreased cyclin A and B1 expression. We conclude that the anti-metabolic action of agmatine on HT-29 cells is mediated by a reduction in polyamine biosynthesis and induction in polyamine degradation. The decrease in intracellular polyamine contents, the reduced rate of DNA synthesis and the cell accumulation in the S phase are discussed from a causal perspective.     

Inhibition of human colon carcinoma cell growth by ammonia: a non-cytotoxic process associated with polyamine synthesis reduction

Ammonia, produced by bacterial degradation of unabsorbed and endogenous nitrogenous compounds, is found to be present at millimolar concentrations in the colon lumen. From in vivo animal experiments, this metabolite has been shown to alter colonic epithelial cell morphology and to increase compensatory cell proliferation when present in excess. In this in vitro study, using the human colon adenocarcinoma HT-29 Glc(-/+) cell line treated with increasing doses of NH(4)Cl, we found that 20 mM NH(4)Cl, a concentration close to that found in the large intestine lumen, was able to increase the volume of vacuolar lysosomes and to repress HT-29 Glc(-/+) cell proliferation. This growth-inhibitory effect was not correlated with decrease of cell viability, with modification of cell differentiation and change of the cell distribution in the different cell cycle phases, thus indicating a proportional slowdown in all cell cycle phases. In contrast to what is found in healthy colonocytes, ammonia was not metabolized by HT-29 cells into carbamoyl-phosphate (carbamoyl-P) and citrulline, indicating that ammonia was likely acting on cells by itself. This agent was shown to markedly reduce cellular ornithine decarboxylase (ODC) activity resulting in a threefold decrease in the capacity of HT-29 cells to synthetize polyamines, these latter metabolites being strictly necessary for cell growth. The unexpected finding that ammonia is acting as an antimitotic agent against tumoral HT-29 colonic cells may be related to the inability of these cells to metabolize this compound.     

De novo synthesis of arginine and ornithine from citrulline in human colon carcinoma cells: metabolic fate of l-ornithine

In human colon carcinoma cells (HT-29 cells), l-arginine is the common precursor of l-ornithine which generates polyamines strictly necessary for cellular growth, and nitric oxide which has a strong antiproliferative activity. We show here that proliferative HT-29 cells possess the capacity for de novo synthesis of l-arginine from l-citrulline, but not from l-ornithine. l-Ornithine is apparently not an l-arginine precursor due to the absence of any detectable ornithine carbamoyltransferase activity. In contrast, the newly synthesized l-arginine was competent for urea and thus l-ornithine production in a context of a high putrescine production in the ornithine decarboxylase pathway and a low degradation of this polyamine in the diamine oxidase pathway. However, cells grown in an arginine-free culture medium containing added l-citrulline were unable to reach confluency. Furthermore, the low amount of nitric oxide produced from l-arginine by these cells was apparently not involved in the control of cell growth since inhibition of nitric oxide synthase activity was without effect. On the other hand, the capacity of more differentiated and less proliferative HT-29 cells for de novo l-arginine synthesis from l-citrulline was increased. It is concluded that l-citrulline is a precursor of l-arginine and l-ornithine in proliferative HT-29 cells and that the metabolic fate of l-ornithine in these cells is mainly devoted to polyamine synthesis. The similarity between differentiated HT-29 cells and the enterocytes of newborn animals in terms of l-arginine metabolism is finally discussed.     

Polyamine depletion by ODC-AdoMetDC antisense adenovirus impairs human colorectal cancer growth and invasion in vitro and in vivo

BACKGROUND: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Polyamine concentrations are elevated in colorectal cancer. Depletion of polyamine content in colorectal cancer by chemotherapy is related to tumor regression and impaired tumorigenicity. The current study evaluates the therapeutic effects of antisense ODC and AdoMetDC sequences on colorectal cancer in vitro and in vivo. METHODS: Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). The human colon cancer cell lines, HT-29 and Caco-2, were infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore, the antitumor effects of Ad-ODC-AdoMetDCas were also evaluated in vivo in a nude mouse tumor model. RESULTS: Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. The adenovirus also induced tumor regression in established tumors in nude mice. CONCLUSIONS: Our study suggests that Ad-ODC-AdoMetDCas has antitumor activity and therapeutic potential for the treatment of colorectal cancer.     

Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis.     

Effects of cyclosporine and alpha-difluoromethylornithine on the growth of mouse colon cancer in vitro

The growth and survival of mouse (MC-26) colon carcinoma in vitro and in vivo are significantly reduced by inhibitors of polyamine biosynthesis. alpha-Difluoromethylornithine (DFMO), is a specific and irreversible inhibitor of ornithine decarboxylase (ODC); the rate-limiting enzyme in polyamine biosynthesis. DFMO treatment inhibits the growth of MC-26 colon cancer cells and decreases MC-26 cell survival both in vitro and in vivo. In the present study, we examined the effects of cyclosporine (CsA) on growth, survival, and polyamine levels in MC-26 colon cancer in vitro. CsA had inhibitory effects on MC-26 colon cancer growth which were similar to DFMO; these effects were blocked by the addition of the polyamine, putrescine. The combination of CsA (8.3 X 10(-4) mM) and DFMO (0.5 mM or 1.0 mM) inhibited MC-26 cell survival to a greater extent than either agent alone. These results suggest that CsA given in combination with other agents which inhibit polyamine synthesis may be useful for the treatment of colon cancer.     

The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.     

Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.

This study analyzes the effects of polyamine starvation on cell cycle traverse of an arginase-deficient CHO cell variant (CHO-A7). These cells grow well in serum-free medium, provided that it contains ornithine or polyamines or both. In the absence of ornithine or polyamines or both, the CHO-A7 cells develop severe polyamine deficiency and, as a consequence, grow more slowly. When grown to a stationary phase in the presence of ornithine or putrescine or both, the CHO-A7 cells became arrested in G0/early G1. However, when starved for ornithine and polyamines, they accumulated in the S and G2 phases. Ornithine and polyamine starvation of CHO-A7 cells causes an increase in ornithine decarboxylase activity. When this increase was prevented by treatment with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, growth was further suppressed, and a greater fraction of cells were found in the S and G2 phases of the cell cycle.     

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10-40 microM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6-24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E(2) by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.     

The role of polyamines in animal cell physiology

The ubiquitous distribution of polyamines in nature suggests that they fulfil some fundamental role(s) in living organisms. In animal cells, polyamine content closely parallels changes in the rate of cell proliferation so that the highest content is always observed in rapidly growing cells. The activity of ornithine decarboxylase (which is the first enzyme in the polyamine biosynthetic pathway) has been found to increase significantly in many systems shortly after exposure to hormones. Also, addition of polyamines greatly stimulates cell-free macromolecular synthesis. Observations such as these have suggested that polyamine accumulation stimulates cell growth and is important in the regulation of macromolecular biosynthesis. However, it is also possible to interpret such data as evidence that polyamine accumulation is the result, not the cause, of increased cell growth. This review supports the latter concept and re-examines the significance of the early induction of ornithine decarboxylase activity and of the stimulatory effects of exogenous polyamine on macromolecular synthesis. It is proposed that the polyamines are important only in maintaining cell growth that has already been stimulated by other factors and that their biosynthesis is to a large extent determined by the accumulation of RNA in the cell.     

Chemopreventive effect of chitosan oligosaccharide against colon carcinogenesis

Chitosan oligosaccharide (COS, 3 kDa相似文献   

Human lung tumor sensitivity to difluoromethylornithine as related to ornithine decarboxylase messenger RNA levels

The differential response to polyamine depletion has been studied in two types of human lung tumor cells. Small cell lung carcinoma cells die following polyamine depletion by difluoromethylornithine treatment while non-small cell lines demonstrate a typical cytostatic response. We now report that a small cell line, NCI H82, has a lower apparent capacity for polyamine biosynthesis than does a representative non-small cell, NCI H157. In subconfluent cultures, the ornithine decarboxylase activity is 25 times lower in the small cell than the non-small cell and by comparison, the polyamines in the small cell line are markedly reduced. Most significantly, levels of mRNA coding for ornithine decarboxylase are approximately 100-fold lower in the small cell than the non-small cell line, and this difference does not appear to be a result of gene rearrangement. These results suggest that differential sensitivity to polyamine depletion is related to different steady-state levels of ornithine decarboxylase mRNA.     

Effects of adrenaline in human colon adenocarcinoma HT-29 cells

AimsStress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved.Main methodsThe effect of adrenaline on HT-29 cell proliferation was determined by [³H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E₂ (PGE₂) release were determined by zymography and enzyme immunoassay, respectively.Key findingsAdrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE₂ release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β₁- and β₂-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE₂ release in HT-29 cells.SignificanceThese results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β₁- and β₂-adrenoceptors by a COX-2 dependent pathway.     

N-glycosylation modification of proteins is an early marker of the enterocytic differentiation process of HT-29 cells

The human colon cancer cell line HT-29 remains totally undifferentiated when glucose is present in the culture medium (HT-29 Glc+), while the same cells may undergo typical enterocytic differentiation after reaching confluence when grown in glucose-deprived medium (HT-29 Glc-). Recently, we demonstrated a deficiency in the overall N-glycan processing in confluent undifferentiated cells, whereas differentiated cells follow a classical pattern of N-glycosylation. The main changes in N-glycosylation observed in confluent undifferentiated cells may be summarised as follows: 1) the conversion of high mannose into complex glycopeptides is greatly decreased; 2) this decreased conversion could be a consequence of an accumulation of Man9-8-GlcNAc2-Asn high mannose species. Whether these changes in N-glycan processing appear progressively during cell culture or are already present from the beginning of the culture was investigated in this study by comparing the actual status of N-glycan processing in exponentially growing HT-29 Glc- and HT-29 Glc+ cells. Under these conditions, HT-29 Glc- cells do not exhibit any characteristics of differentiation. The conversion of high mannose into complex glycoproteins is severely reduced in HT-29 Glc+ cells, regardless of the growth phase studied. In contrast, HT-29 Glc- cells display a normal pattern of N-glycan processing in both growth phases. We therefore conclude that N-glycan processing may be used as an early biochemical marker of the enterocytic differentiation process of HT-29 cells.     

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

Lack of cyclooxygenase-2 activity in HT-29 human colorectal carcinoma cells

Induction of cyclooxygenase-2 (COX-2) is an early event in the sequence of polyp formation to colon carcinogenesis. COX-2 is at elevated levels in human colorectal cancers and in tumors and polyps of mouse models of colorectal cancer. Mutation of the adenomatous polyposis coli (APC) gene is the initial event leading to colorectal cancer. Colorectal cells in culture which express mutant APC are often used to examine the association of COX-2 expression and apoptosis. The expression of full-length APC in HT-29 cells, a human colorectal carcinoma cell line which normally expresses truncated APC and highly expresses COX-2, inhibits cell growth through increased apoptosis and results in a down-regulation of COX-2 protein. In this report, we examine whether down-regulation of COX-2 is directly linked to the increase in apoptosis observed in these HT-29-APC cells. We present evidence that COX-2 and apoptosis are not linked since COX-2, although expressed, is catalytically inactive. Interestingly, the COX-2 cloned from HT-29 cells is catalytically active when transfected into HCT-116 cells, a colorectal cell line which normally does not express COX-2, but is not active in the HT-29 cell line itself.     

Regulation of polyamine biosynthesis in normal and transformed hamster cells in culture

Several aspects of polyamine biosynthesis were compared in low-passage hamster embryo fibroblasts and transformed hamster fibroblasts. Earlier studies had demonstrated a larger and longer-lasting induction of ornithine decarboxylase activity in transformed cells than in hamster embryo fibroblasts. The increases in intracellular polyamine concentrations after serum stimulation were much greater in chemically transformed HE68BP cells than in normal hamster fibroblasts. Treatment of confluent cultures with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, greatly potentiated ornithine decarboxylase induction by fresh medium in HE68BP cells, but not in hamster fibroblasts. A similar synergistic effect was observed when transformed cells, but not normal cells, were treated with the combination of insulin and promoter. HE68BP cells were capable of growth in medium containing serum concentrations as low as 0.5%, whereas only concentrations of 5% or more supported the growth of hamster embryo fibroblasts. Low serum concentrations induced ornithine decarboxylase in HE68BP cells but not in normal cells, and a given serum concentration always produced a greater induction of ornithine decarboxylase in transformed than in normal cells.Another enzyme involved in polyamine synthesis, $\text{S-}\text{adenosyl-}\text{L}\text{-methionine}$ decarboxylase was induced in normal and transformed cells by serum-containing medium or tetradecanoylphorbol acetate, but in contrast to ornithine decarboxylase, no synergistic effect was seen in transformed cells exposed to the combination of fresh medium and the tumor promoter. A macromolecular inhibitor of ornithine decarboxylase was readily detected in hamster fibroblast cultures treated with high concentrations of putrescine, but little or none of this inhibitor was found in HE68BP cultures. In both cell types, however, serum induction of ornithine decarboxylase was inhibited under conditions of excess putrescine.The results demonstrate several differences between normal and transformed hamster cells in the regulation of polyamine synthesis.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.