Regulation of ornithine decarboxylase during oncogenic transformation: mechanisms and therapeutic potential.

The activity of ornithine decarboxylase (ODC(1)), the first enzyme in polyamine biosynthesis, is induced during carcinogenesis by a variety of oncogenic stimuli. Intracellular levels of ODC and the polyamines are tightly controlled during normal cell growth, and regulation occurs at the levels of transcription, translation and protein degradation. Several known proto-oncogenic pathways appear to control ODC transcription and translation, and dysregulation of pathways downstream of ras and myc result in the constitutive elevation of ODC activity that occurs with oncogenesis. Inhibition of ODC activity reverts the transformation of cells in vitro and reduces tumor growth in several animal models, suggesting high levels of ODC are necessary for the maintenance of the transformed phenotype. The ODC irreversible inactivator DFMO has proven to be not only a valuable tool in the study of ODC in cancer, but also shows promise as a chemopreventive and chemotherapeutic agent in certain types of malignancies.

Transgenic mouse models for studies of the role of polyamines in normal, hypertrophic and neoplastic growth.

Transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner have considerable promise for evaluation of the roles of polyamines in normal, hypertrophic and neoplastic growth. This short review summarizes the available transgenic models. Mice with large increases in ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase or antizyme, a protein regulating polyamine synthesis by reducing polyamine transport and ODC in the heart, have been produced using constructs in which the protein is expressed from the alpha -myosin heavy-chain promoter. These mice are useful in studies of the role of polyamines in hypertrophic growth. Expression from keratin promoters has been used to target increased synthesis of ODC, spermidine/spermine-N(1)-acetyltransferase (SSAT) and antizyme in the skin. Such expression of ODC leads to an increased sensitivity to chemical and UV carcinogenesis. Expression of antizyme inhibits carcinogenesis in skin and forestomach. Expression of SSAT increases the incidence of skin papillomas and their progression to carcinomas in response to a two-stage carcinogenesis protocol. These results establish the importance of polyamines in carcinogenesis and neoplastic growth and these transgenic mice will be valuable experimental tools to evaluate the importance of polyamines in mediating responses to oncogenes and studies of cancer chemoprevention.

Targeted antizyme expression in the skin of transgenic mice reduces tumor promoter induction of ornithine decarboxylase and decreases sensitivity to chemical carcinogenesis.

To directly evaluate the role of increased ornithine decarboxylase (ODC) and polyamines in mouse skin carcinogenesis, we used bovine keratin 5 (K5) and keratin 6 (K6) promoter elements to direct the expression of antizyme (AZ) to specific skin cell populations. AZ is a multifunctional regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. K5-AZ mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 0 and 24 h exhibit increases in epidermal and dermal ODC activity that are reduced in magnitude. K6-AZ mice treated similarly do not show any increased ODC activity or protein after a second application due to TPA-induced expression of AZ protein. Epidermal and dermal polyamine content, particularly spermidine, is reduced in untreated K5-AZ mice and TPA-treated K5-AZ and K6-AZ mice. Susceptibility to 7,12-dimethylbenz(a)anthracene/TPA carcinogenesis was also investigated for two K6-AZ transgenic lines [K6-AZ(52) and K6-AZ(18)] and a single K5-AZ line. K6-AZ(52) mice had a substantial delay in tumor onset and a >80% reduction in tumor multiplicity compared with normal littermates. K6-AZ(18) and K5-AZ mice also developed fewer papillomas than littermate controls (35% and 50%, respectively), and the combination of these lines to produce double transgenic animals yielded an additive decrease (70%) in tumor multiplicity. These mice demonstrate for the first time that AZ suppresses tumor growth in an animal cancer model and provide a valuable model system to evaluate the role of ODC and polyamines in skin tumorigenesis.

Targeted overexpression of ornithine decarboxylase enhances beta-adrenergic agonist-induced cardiac hypertrophy.

These studies were designed to determine the consequences of constitutive overexpression of ornithine decarboxylase (ODC) in the heart. Induction of ODC is known to occur in response to agents that induce cardiac hypertrophy. However, it is not known whether high ODC levels are sufficient for the development of a hypertrophic phenotype. Transgenic mice were generated with cardiac-specific expression of a stable ODC protein using the alpha-myosin heavy-chain promoter. Founder lines with >1000-fold overexpression of ODC in the heart were established, resulting in a 50-fold overaccumulation of putrescine, 4-fold elevation in spermidine, a slight increase in spermine and accumulation of large amounts of cadaverine compared with littermate controls. Despite these significant alterations in polyamines, myocardial hypertrophy, as measured by ratio of heart to body weight, did not develop, although atrial natriuretic factor RNA was slightly elevated in transgenic ventricles. However, stimulation of beta-adrenergic signalling by isoproterenol resulted in severe hypertrophy and even death in ODC-overexpressing mice without further altering polyamine levels, compared with only a mild hypertrophy in littermates. When beta1-adrenergic stimulation was blocked by simultaneous treatment with isoproterenol and the beta1 antagonist atenolol, a significant, although reduced, hypertrophy was still present in the hearts of transgenic mice, suggesting that both beta1 and beta2 adrenergic receptors contribute to the hypertrophic phenotype. Therefore these mice provide a model to study the in vivo co-operativity between high ODC activity and activation of other pathways leading to hypertrophy in the heart.

Modulation of potassium channels in the hearts of transgenic and mutant mice with altered polyamine biosynthesis.

Inward rectification of cardiac I(K1)channels was modulated by genetic manipulation of the naturally occurring polyamines. Ornithine decarboxylase (ODC) was overexpressed in mouse heart under control of the cardiac alpha -myosin heavy chain promoter (alpha MHC). In ODC transgenic hearts, putrescine and cadaverine levels were highly elevated ( identical with 35-fold for putrescine), spermidine was increased 3.6-fold, but spermine was essentially unchanged. I(K1)density was reduced by identical with 38%, although the voltage-dependence of rectification was essentially unchanged. Interestingly, the fast component of transient outward (I(to,f)) current was increased, but the total outward current amplitude was unchanged. I(K1)and I(to)currents were also studied in myocytes from mutant Gyro (Gy) mice in which the spermine synthase gene is disrupted, leading to a complete loss of spermine. I(K1)current densities were not altered in Gy myocytes, but the steepness of rectification was reduced indicating a role for spermine in controlling rectification. Intracellular dialysis of myocytes with putrescine, spermidine and spermine caused reduction, no change and increase of the steepness of rectification, respectively. Taken together with kinetic analysis of I(K1)activation these results are consistent with spermine being a major rectifying factor at potentials positive to E(K), spermidine dominating at potentials around and negative to E(K), and putrescine playing no significant role in rectification in the mouse heart.

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

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

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway.

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.

Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6.

Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5'-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5'-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E.4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70(S6k), resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucine-induced effects. Thus, leucine acting through an mTOR-dependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.

Ornithine decarboxylase induction in transformation by H-Ras and RhoA.

The objective of these studies has been to develop a better understanding of the regulation of ornithine decarboxylase (ODC) during the neoplastic process, and to determine whether induction of ODC is a necessary component in the action of the ras oncogene. Specifically, we have studied the role of ODC overexpression in signaling pathways mediated by Raf or RhoA. Cells transformed by ras are known to have constitutively high levels of ODC activity that correlate with oncogenic transformation. To determine which pathways downstream of Ras contribute to the regulation of ODC activity, NIH-3T3 cells were transfected with plasmids coding for activated mutants of either H-Ras or RhoA, or oncogenic nu-Raf. There was a good correlation between increasing ODC specific activity and change in morphology from normal to transformed in the nu-Raf, HRas(61L), and RhoA(63L) clones. Increasing ODC activity also correlated positively with the ability to grow in soft agar in both the H-Ras- and RhoA-expressing cells. In stable transfections, coexpression of the ODC dominant negative mutant K69A/C360A with either HRas(61L) or RhoA(63L) both inhibited intracellular ODC activity and caused a reversion of the transformed phenotype, as measured by a dramatic reduction in the ability of these cells to grow in soft agar and form foci on a monolayer. These results suggest strongly that ODC induction is necessary for transformation by oncogenic Ras. In contrast, expression of K69A/ C360A had no effect on the ability of nu-Raf-transformed cells to grow in soft agar, although intracellular ODC levels were inhibited. When grown on a monolayer, these cells also maintained their transformed appearance. Furthermore, expression of the ODC dominant negative mutant did not affect the phosphorylation of mitogen-activated protein kinase in nu-Raf-transformed cells. These experiments provide strong support for the concept that transformation by activated ras is accompanied by an induction of ODC. The results using RhoA(63L) and nu-Raf suggest that this increase in ODC activity is mediated at least in part through a Raf/ mitogen-activated protein kinase independent pathway.

The upstream open reading frame of the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive translational control element.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the pathway of polyamine biosynthesis. The cellular levels of the polyamines specifically regulate AdoMetDC translation through the 5'-leader of the mRNA, which contains a small upstream open reading frame (uORF) 14 nucleotides from the cap. Mutating the initiation codon of the uORF, which encodes a peptide product with the sequence MAGDIS, abolished regulation. In addition, the uORF is sufficient, by itself, to provide polyamine regulation when inserted into the 5'-leader of the human growth hormone mRNA. Changing the amino acid sequence at the carboxyl terminus of the peptide product of the uORF abolished polyamine regulation. In contrast, altering the nucleotide sequence of the uORF at degenerate positions, without changing the amino acid sequence of the peptide, did not affect regulation. Extending the distance between cap and uORF, thereby changing the rate of initiation at the initiator AUG of the uORF, did not alter polyamine regulation. When the uORF was extended so as to overlap, out of frame, the downstream major cistron, polyamine regulation was abolished. We propose that polyamines do not modulate the rate of recognition of the uORF but rather regulate interaction of the peptide product of the uORF with its target.

Expression of an ornithine decarboxylase dominant-negative mutant reverses eukaryotic initiation factor 4E-induced cell transformation.

pMV7-4E cells (4E-P2), which overexpress translation initiation factor eIF-4E, contain elevated levels of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis. We have shown previously that this induction appears to be related to the transformed phenotype of these cells (L. M. Shantz and A. E. Pegg, Cancer Res., 54: 2313-2316, 1994). To test whether increased ODC activity is responsible for the transformation of 4E-P2 cells, a dominant-negative mutant of ODC was used to reduce the intracellular ODC activity in 4E-P2 cells, and the resulting phenotypic changes were examined. The mutant K69A/C360A contains mutations to alanine of two key active site residues, lysine 69 and cysteine 360, and is truncated at 425 amino acids. Combination of purified K69A/C360A and purified wild-type ODC resulted in a dose-dependent decrease in specific activity compared with wild-type ODC alone, with a 71% reduction at equimolar concentrations. This mutant was transfected into 4E-P2 cells, and stable clones that expressed the truncated K69A/ C360A were isolated. Several clones were tested for their ability to form transformed foci on a monolayer, grow in soft agar, and form tumors in nude mice. When ODC activity was reduced by 60%, the transformed phenotype of 4E-P2 cells was abolished, suggesting strongly that high ODC levels are critical to the transformation of these cells. In addition, K69A/C360A can be used to determine the ODC activity associated with transformation in both in vitro and in vivo systems.

Regulation of ornithine decarboxylase in a transformed cell line that overexpresses translation initiation factor eIF-4E.

pMV7-4E cells (4E-P2), derived from NIH-3T3 cells, overexpress eIF-4E and exhibit characteristics of transformation, possibly due to translational relief of mRNAs encoding proteins that regulate cell growth. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is induced in 4E-P2 cells, and this induction appears to be related to the transformed phenotype of these cells. ODC mRNA contains extensive secondary structure in its 5' untranslated region (5'UTR) and may be regulated by eIF-4E, which melts mRNA secondary structure. To better understand this regulation, cDNA constructs containing the wild-type 5'UTR of ODC or deletion mutants inserted ahead of the luciferase gene were transfected into 4E-P2 and 3T3 cells. Expression of luciferase was higher in 4E-P2 cells in all cases, suggesting that the secondary structure of the ODC 5'UTR inhibits expression in 3T3 cells, and this inhibition is overcome by the high eIF-4E levels in 4E-P2 cells. When a small open reading frame present in the 5'UTR of ODC was destroyed by a point mutation, this luciferase construct was expressed about 6-fold over that containing the wild-type 5'UTR in both cell lines, although both of these 5'UTRs contain the same predicted secondary structure. Thus, factors in addition to eIF-4E may be involved in the regulation of ODC. To examine the differences in ODC regulation by polyamines in normal and transformed cells, the effect of N1,N12-bis(ethyl)spermine (BE-3-4-3) on the synthesis and degradation of ODC was examined. ODC activity in 4E-P2 cells was 10 times less sensitive to reduction by BE-3-4-3 compared to 3T3 cells, suggesting that high ODC levels in eIF-4E-overexpressing cells are the result of decreased regulation by polyamines as well as relief of translational regulation by eIF-4E.

Ornithine decarboxylase as a target for chemoprevention.

l-Ornithine decarboxylase (ODC) is essential for polyamine synthesis and growth in mammalian cells; it provides putrescine that is usually converted into the higher polyamines, spermidine and spermine. Many highly specific and potent inhibitors of ODC are based on the lead compound alpha-difluoromethylornithine (DFMO), which is an enzyme-activated irreversible inhibitor. DFMO is accepted as a substrate by ODC and is decarboxylated, leading to the formation of a highly reactive species that forms a covalent adduct with either cysteine-360 (90%) or lysine-69 (10%). Both modifications inactivate the enzyme. ODC activity is normally very highly regulated at both transcriptional and post-transcriptional levels according to the growth state of the cell and the intracellular polyamine content. Experimental over-production of ODC can be caused by either transfection with plasmids containing the ODC cDNA with part of the 5'-untranslated region (5'UTR) deleted under the control of a very strong viral promoter, or transfection of plasmids that cause the overproduction of eIF-4E, reported to be a limiting factor in the translation of mRNAs with extensive secondary structures in the 5'UTR. In both cases, unregulated overexpression of ODC transforms NIH 3T3 cells to a neoplastic state. Along with studies showing that many tumor promoters increase ODC activity and that a number of preneoplastic conditions and tumor samples show high levels of ODC, these results suggest that ODC may act as an oncogene in an appropriate background. This provides a rationale for the possible use of ODC inhibitors as chemopreventive agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the 5'-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine.

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5'-untranslated region (5'UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5'UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5'UTR, as well as chimaeric constructs in which the AdoMetDC 5'UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5'UTR, the AdoMetDC protein-coding region and the 3'UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5'UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5'UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5' end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5'UTR that may include but is not entirely due to the internal ORF.

Overproduction of ornithine decarboxylase caused by relief of translational repression is associated with neoplastic transformation.

The mRNAs for two key enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), both long 5' untranslated regions (5'UTRs) that could be important in the regulation of enzyme levels by affecting the translation of these mRNAs. In order to test this hypothesis, ODC and AdoMetDC activities were measured in 3T3 cells and in 3T3 cells overexpressing eIF-4E (P2 cells). eIF-4E has been reported to be a limiting factor in the translation of mRNAs with extensive secondary structures in the 5'UTR. AdoMetDC activity was not greatly different in the two cell lines, but ODC activity was much greater in the P2 cells. These results were confirmed by transfecting these cells with plasmids containing a luciferase complementary DNA fused to follow the 5'UTR from ODC or AdoMetDC. The ODC 5'UTR construct produced a higher luciferase activity in the P2 cells. The high level of expression of ODC may be a critical factor in the transformed phenotype of the P2 cells since the ability of these cells to grow in soft agar was blocked by levels of the ODC inhibitor, alpha-difluoromethylornithine, that reduced the ODC activity to values comparable to those of the parent 3T3 cells. These results provide more evidence for a critical role of ODC activity in neoplastic transformation and for the importance of its translational regulation in cell growth and transformation.

Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli. Determination of sites for putrescine activation of activity and processing.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity. To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine. Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing. The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography. Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.