independent regulation of ornithine decarboxylase and s...

[CANCER RESEARCH 45. 3567-3572, August 1985]

Independent Regulation of Ornithine Decarboxylase and S-Adenosylmethionine

Decarboxylase in Methylthioadenosine Phosphorylase-deficient MalignantMurine Lymphoblasts1

Masaru Kubota, E. Olavi Kajander, and Dennis A. Carson2

Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037

ABSTRACT

The control of polyamine synthesis in neoplastia cells is complex and incompletely understood. Using murine lymphoma cellsdeficient in methylthioadenosine (MTA) phosphorylase, we haveanalyzed the role of MTA in the regulation of ornithine decarbox-ylase and S-adenosylmethionine (SAM) decarboxylase, the tworate-limiting enzymes in the polyamine-biosynthetic pathway.The addition of MTA to the enzyme-deficient lymphoblasts in

duced within 1 to 3 h an increase in the activities of bothdecarboxylases and an accompanying rise in putrescine anddecarboxylated SAM levels. The ornithine decarboxylase inhibitor a-difluoromethylomithine blocked the MTA-triggered accu

mulation of putrescine but not decarboxylated SAM. In a reciprocal manner, the SAM decarboxylase inhibitor methylglyoxalbis(guanylhydrazone) prevented the accretion of decarboxylatedSAM but not putrescine. The MTA-induced rise in SAM decar

boxylase and ornithine decarboxylase activities preceded byseveral hours changes in spermidine or spermine pools. However, MTA decreased the flux through the polyamine-synthetic

pathway, as estimated by the incorporation of radioactive ornithine into spermine. Similar changes in polyamine metabolismwere observed in a secondary mutant deficient in MTA phosphorylase, but resistant to MTA toxicity. These results suggest thatthe velocity of polyamine synthesis, or the concentration of MTAitself, may regulate ornithine decarboxylase and SAM decarboxylase activities through separate, growth-independent mecha

nisms.

INTRODUCTION

Polyamines are organic cations that subserve diverse functions in dividing mammalian cells. States of increased cellularproliferation, such as cancer, are commonly associated withaccelerated rates of polyamine synthesis. The factors regulatingpolyamine synthesis and degradation in normal and malignantcells have been investigated in several laboratories but have notbeen definitively elucidated (1-6).

MTA3 is generated stoichiometrically from S-adenosyl-(5')-deoxy-(5')-3-methylthiopropylamine (commonly called decarbox

ylated SAM) during the synthesis of the polyamines spermidine

1This work is supported by Grants CA31497 and CA35048 from the National

Cancer Institute, and by Grant GM23200 from the NIH. This is Publication 3750BCRfrom the Research Institute of Scripps Clinic, La Jolla, CA 92037.

2To whom requests for reprints should be addressed, at Department of Basic

and Clinical Research, Scripps Clinic and Research Foundation, 10666 North TorreyPines Road, La Jolla, CA 92037.

'The abbreviations used are: MTA, 5'-methylthioadenosine; SAM, S-adenosylmethionine; DFMO, DL-a-difluoromethylomithine; MGBG, methylglyoxal bis-

(guanylhydrazone).Received 1/30/85; revised 4/25/85; accepted 5/1/85.

and spermine. Normally, mammalian cells cleave the thioethernucleoside via a specific enzyme, MTA phosphorylase. The 2reaction products, adenine and 5-methylthioribose-1-phosphate,

are reconverted to adenine nucleotides (7) and methionine (8, 9),respectively. In 1977, Toohey (10) reported the absence of MTAphosphorylase in 4 murine malignant hematological cell lines.Subsequent experiments from this laboratory (11, 12) demonstrated that MTA phosphorylase activity was lacking in manyhuman and murine malignant cell lines of diverse origin. Importantly, some leukemic cell clones obtained directly from patientswere also phosphorylase deficient (13). In contrast, MTA phosphorylase was abundant in all normal human tissues and in celllines established from them.

MTA phosphorylase deficiency represents an "experiment ofnature" that distinguishes some malignant cell lines from their

normal counterparts. Understanding the effects of MTA on cellular metabolism could provide insight concerning the overallcontrol of polyamine synthesis in normal and neoplastic states.In the present work, we have analyzed the time-dependent

changes in SAM and polyamine metabolism elicited by MTA inan enzyme-deficient malignant lymphoblastoid cell line (R1.1H).

In dividing cell cultures, MTA rapidly and dose dependentlyaugmented the activities of ornithine decarboxylase and SAMdecarboxylase, the 2 rate-limiting enzymes in the polyamine-

synthetic pathway. The rise in the activities of the 2 decarboxylases in turn caused a progressive accumulation of putrescineand decarboxylated SAM within the cells. The MTA-inducedchanges occurred prior to any observable alterations in intracel-

lular polyamine pools, or in cell growth, but were accompaniedby a decrease in the rate of spermine biosynthesis. These resultssuggest that ornithine decarboxylase and SAM decarboxylasein malignant cells may be regulated by separate mechanismsthat are not solely dependent on the state of cellular proliferation.Portions of this work have been communicated in a preliminaryform (14).

MATERIALS AND METHODS

Cell Lines. The murine T-lymphoma cell line, R1.1, came from Dr.

Robert Hyman (Salk Institute, La Jolla, CA). A mutant (clone H) deficientin MTA phosphorylase was selected from wild-type R1.1 cells by a"tritium suicide" method, as described previously (15). A secondary

mutant clone (H5) was isolated by limiting dilution after clone H cellswere cultured for several months in medium containing increasing MTAconcentrations (from 10 MM to 1 rriM). The H5 cell line proliferatedequivalently, whether or not 500 MMMTA was included in the medium.For metabolic studies, the cells were dispersed in RPMI-1640 mediumsupplemented with 10% heat-inactivated horse serum or with 9% heat-

inactivated horse serum, 1% fetal bovine serum, penicillin (100 units/ml),streptomycin (100 Mg/ml), and 2 mw L-glutamine, all from Microbiological

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REGULATION OF POLYAMINE SYNTHESIS

Associates (Bethesda, MO). The cells grew with an average doublingtime of 13 (clone H) and 16 (H5) h, that was not affected by the type ofserum added. Fetal bovine serum, but not horse serum, contains substantial MTA phosphorylase (11) and spermine oxidase activity (16,17).The inclusion of 1% fetal bovine serum in the growth medium enabledcell growth to proceed to higher cell densities but was not sufficient tosubstantially break down exogenously added MTA under the conditionsstudied. No differences were observed in enzyme induction patterns, orin polyamine and decarboxylated SAM levels, whether only horse serumor combination of horse and fetal bovine sera were used to supplementthe medium. In experiments where spermidine was added to the medium,fetal bovine serum was omitted.

Measurement of Polyamines, SAM, and Decarboxylated SAM. R1.1lymphoblasts at a density of 1 x 106/rnl were incubated using mildagitation (with agitation, cells can grow to a density of 4 x I06/ml) at37Â°Cwith MTA, DFMO, and MGBG in various combinations, as indicatedin "Results." From 1 to 16 h later, the cell pellets were collected by

centrifugation at 700 x g, at 4Â°C.After the removal of the supernatants,

the cells were washed twice in cold phosphate-buffered saline (0.15 M

NaCI:0.01 M phosphate buffer, pH 7.4). Finally, the packed cells wereextracted with ice-cold 0.4 M perchloric acid and were frozen at -20Â°C

until analyzed. Polyamine concentrations were measured fluorometri-cally, after separation by reverse-phase ion-pair high-performance liquidchromatography and postcolumn derivatization with o-phthaldehyde,

according to the method of Seiler and KnÃ¶dgen(18).The effects of MTA on polyamine synthesis were assessed as de

scribed previously (11). Briefly, 2.5-ml aliquots containing 5 x 10s cells/ml were cultured for 1 to 6 h (or 2.5 x 105 cells/ml when cells wereincubated for 22 h) at 37Â°Cwith 5 /Â¿Ciof L-[3H]omithine per ml (15.2 Ci/

mmol; New England Nuclear, Boston, MA). The cells were pelleted andwashed twice in phosphate-buffered saline, and intracellular polyamines

were analyzed as described above, except that the radioactivity in theputrescine, spermidine, and spermine fractions was also measured.

To measure SAM and decarboxylated SAM, cell pellets were washedand extracted as described for polyamine determinations, but they wereimmediately analyzed by high-performance liquid chromatography ex

actly as described by Wagner ef al. (19).Enzyme Assays. Ornithine decarboxylase activity was measured by

the radiochemical method of Demetriou et al. (20), except that bovineserum albumin (1 mg/ml) was included in the reaction buffer. The activityof SAM decarboxylase was determined by the method of Pegg and Poso(21), again supplementing the reaction medium with bovine serum albumin (1 mg/ml). Enzyme activities were measured using 100,000 x gsupernatant fractions of cell extracts as samples.

Chemicals. A decarboxylated SAM standard was kindly supplied byDr. Anthony Pegg (Pennsylvania State University, State Park, PA). DFMOwas the gift of Dr. D. J. Wilkins (Merrell/Dow Research, Strasbourg,France). Other chemicals were purchased from Sigma (St. Louis, MO) atthe highest grade available.

RESULTS

MTA-induced Changes in Polyamine Synthesis and Intra

cellular Polyamine Pools. Although MTA Â¡sa potent inhibitorofpolyamine synthesis, its rapid degradation by MTA phosphorylase prevents unambiguous evaluation of its possible role in theregulation of this pathway. We isolated a MTA phosphorylase-deficient clone (H) containing less than 1% of wild-type activity.

All other investigated enzyme activities related to purine andpolyamine metabolism were comparable to wild type (15). In themutant, the intracellular MTA levels reflected the added MTAconcentrations in the medium (data not shown). The uptakemechanism for MTA is probably facilitated diffusion (22). The

intracellular degradation of MTA in the mutants has been shownto be insignificant (22). The addition of MTA to the medium ofproliferating MTA phosphorylase-deficient murine lymphoma

cells induced a rapid and progressive rise in the activities of SAMdecarboxylase and omithine decarboxylase. As shown in Chart1, SAM decarboxylase activity doubled after 1 h and had increased 8-fold by 6 h after the addition of 0.5 mM MTA. The

activity of ornithine decarboxylase rose significantly by 2 h afterthe addition of MTA and thereafter increased in parallel withSAM decarboxylase.

During the first 6 h after the addition of MTA, the enzyme-

deficient lymphoblasts continued to proliferate at the same rateas untreated cells, as estimated by direct cell counting, and byradioactive thymidine uptake at 2-h intervals (results not shown).

The very early effects of MTA on polyamine metabolism werenot a nonspecific consequence of alterations in cell growthkinetics. Thus, the same changes occurred in clone H5, thatgrew normally in 500 pM MTA.

The increased activity of SAM decarboxylase was accompanied by a concomitant rise in decarboxylated SAM pools. Asillustrated in Chart 2, decarboxylated SAM levels rose fromundetectable levels (<1 pmol/106 cells) in untreated lymphomacells to 20 pmol/106 cells at 1 h. By 3 h after the addition of

MTA, decarboxylated SAM and SAM pools were nearly equivalent. These changes were similar in all R1.1 sublines. In a

BOO

700

Â± 600

* 500

Â£ 400

101

S-ADENOSYL METHIONINE ,DECARBOXYLASE

ORNITHINE'DECARBOXYLASE

2 3 4HOURSAFTERADDITIONOFMTA

Chart 1. Inductionof SAM decarboxylaseand omithine decarboxylaseby MTA.Proliferating MTA phosphorylase-deficient R1.1 lymphoma cells (clone H) werecultured at a density of 10Â°cells/ml in regular medium supplementedwith 500 pÂ»

MTA and utilizing mild agitation. From 1 to 6 h thereafter, aliquots of cells wereremoved, and the activities of the 2 decarboxylases were measured radiochemi-cally. The percentages of control enzyme activities refer to the activities at thesame time points in cultures lacking exogenous MTA, which remained practicallyconstant during the 6-h incubation period. The zero-time percentage of controlenzyme activity for SAM decarboxylase was 87 pmol/min/mg of protein, and foromithine decarboxylase, 69 pmol/min/mg of protein. In 3 replicate experiments,the same basic pattern was observed.


3568




Effect of MTA on dcSAM Levels

CO

Chart 2. MTA-induced changes in decar-boxylated SAM (dcSAM) levels. R1.1 wild type(A and A), MTA phosphorylase-deficient cloneH (O and â€¢),and MTA phosphorylase-deficient,MTA-resistant clone H5 (Q and â€¢)cells wereincubated with 500 Â»MMTA as described inChart 1. The intracellular contents of decarbox-

ylated SAM (solid symbols) and the ratio ofdecarboxylated SAM to SAM (open symbols)were determined after separation by high-performance liquid chromatography as describedin "Materials and Methods." This experiment

was repeated 3 times with similar results.

Hours

complementary fashion, intracellular putrescine content changedin concert with the activity of omithine decarboxylase. Putrescinecontent increased to 150% of control by 3 h and was elevated3-fold at 5 h, as shown by the data of Chart 3 for clone H cells.By contrast, short-term exposure to MTA did not alter intracel

lular spermidine or spermine levels (Chart 3). Nonetheless, theMTA treatment did inhibit spermine biosynthesis, as estimatedby the incorporation of radioactive ornithine into spermine (Chart4). MTA did not block severely ornithine incorporation into spermidine. Supplementation of the culture medium with spermidinecould partially prevent MTA effects. When spermidine was addedat 100 Â¿Â¿Msimultaneously with 0.5 ITIMMTA, ornithine decarboxylase activity dropped to unmeasurable levels by 1 h. SAMdecarboxylase activity initially rose slightly (130% of controlactivity at 2 h), but it fell to 71% of control by 4 h. At this lattertime point, the decarboxylated SAM:SAM ratio was 0.15, compared to 1.0 in MTA-supplemented medium lacking exogenous

spermidine.Table 1 summarizes the dose-related effects of 16-h exposure

to exogenous MTA on the levels of polyamines, SAM, anddecarboxylated SAM in the MTA phosphorylase-negative as wellas -positive lymphoma cells. As little as 10 MM of the thioether

nucleoside elevated putrescine and decarboxylated SAM levels.Consistent with the MTA-mediated block in de novo spermineformation, 16-h incubation with the nucleoside dose dependently

reduced intracellular spermine concentrations.Effect of Inhibitors of Polyamine Synthesis. DFMO is an

enzyme-activated, irreversible inhibitor of ornithine decarboxyl

ase (23). MGBG is a competitive inhibitor of SAM decarboxylase(24). The inclusion of DFMO in the culture medium blocked the

300Putrescine

Spermidine

Spermine

HoursChart 3. Effect of MTA on putrescine and polyamine levels. MTA phosphorylase-

deficient R1.1 lymphoma cells (clone H) were incubated with MTA as described inChart 1. At the indicated times, the percentages of control levels of putrescine (0h = 100% = 0.51 nmol/10* cells), spermidine (100% = 2.69 nmol/10" cells), andspermine (100% = 0.64 nmol/106 cells) were determined. The chart shows the

results from duplicate cultures from one of 3 replicate experiments with similarresults.

MTA-induced increase in putrescine pools but further enhancedthe elevation in decarboxylated SAM levels (Table 2). In a reciprocal manner, MGBG inhibited the MTA-induced rise in decar-


3569




boxylated SAM levels but did not prevent the increase in putres-

cine pools. These data indicate that the effects of MTA onputrescine and decarboxylated SAM concentrations occurredindependently and that the rise in putrescine and decarboxylatedSAM levels depended upon the ongoing activity of ornithinedecarboxylase and SAM decarboxylase, respectively.

DISCUSSION

The addition of MTA to cultured lymphoma cells severelyperturbed SAM and polyamine metabolism. The earliest changesinduced by MTA were (a) at 1 h, an increase in SAM decarboxylase activity and a concomitant rise in decarboxylated SAMpools and (b) at 2 to 3 h, an elevation in ornithine decarboxylaseactivity and a secondary expansion of putrescine pools. Theactivities of the 2 decarboxylases continued to increase for atleast 6 h in MTA-supplemented medium. At that time, decarbox

ylated SAM levels were equivalent to, or even greater than, total

200

150

100

so

22

HOURS

Chart 4. Inhibition of spermine biosynthesis by MTA. MTA phosphorylase-deficient lymphoma cells (clone H) were cultured in regular medium supplementedwith [3H]omithine (5 ^Ci/ml) and 100 >M MTA. At the indicated time points, the

incorporated radioactivity was measured and compared to the control at the sametime point. In control experiments at 6 h, the following incorporations of radiolabelin 100-nl high-performance liquid chromatography samples were measured: putrescine, 16,394 cpm; spermidine, 7,490 cpm; and spermine, 220 cpm.

SAM concentrations. Importantly, these metabolic changes occurred independently of detectable alterations in spermidine orspermine content and several h before cell growth declined.These results establish (a) that MTA is a rapid inducer of SAMdecarboxylase and ornithine decarboxylase in the enzyme-defi

cient lymphoma cells, (b) that the stimulation of enzyme activityis one cause for the MTA-induced increase in putrescine and

decarboxylated SAM pools, and (c) that these effects are notmediated by a product of MTA catabolism.

The regulation of putrescine and polyamine synthesis in mammalian cells is exceedingly complex and has been reviewed (4,6). Both ornithine decarboxylase and SAM decarboxylase areminimally active in nonproliferating cells but are induced rapidlyafter application of a growth-promoting stimulus. In dividing cells,the half-lives of the 2 enzymes are extremely short. It has been

pointed out that, when general protein synthesis increases,enzymes that have a high rate of synthesis and degradationrespond with a greater and more rapid rise in activity than doenzymes with a slow turnover rate (6). The rapid induction of the2 enzymes by MTA distinguishes the regulation of the 2 decarboxylase activities from nonspecific changes in growth kinetics.MTA is a cytostatic agent toward normal and malignant lymphocytes, despite its ability to augment ornithine decarboxylase andSAM decarboxylase activities. Importantly, MTA exerted similareffects on decarboxylated SAM and polyamine synthesis inlymphoma cells sensitive or resistant to the antiproliferativeeffects of the nucleoside.

In cultured cells, the functional activities of ornithine decarboxylase and SAM decarboxylase reflect a balance between thesynthetic and degradative rates of the 2 enzymes and theirrespective states of activation (6, 25-27). The content of putres

cine and polyamines may influence one or several of theseparameters. In model systems, the addition of spermidine togrowing cells has been shown to reduce ornithine decarboxylaseand SAM decarboxylase activities (25, 28, 29). In the presentstudies, exogenous spermidine prevented the MTA-induced

changes in the 2 decarboxylases and the associated increase inputrescine and decarboxylated SAM levels. Thus, the MTA treatment did not preclude the negative regulation by spermidine onputrescine and decarboxylated SAM synthesis.

TabtelMTA-induced changes in SAM and polyamine metabolism in MTA phosphorylase-positive and -deficient lymphoma cells

MTA phosphorylase-deficient R1.1 lymphoma cells (clone H), secondary mutant cells resistant to MTA (H5), and MTA phosphorylase-positivewild-type cells were incubated for 16 h with the indicated concentrations of MTA, and the intracellular contents of polyamines, SAM, anddecarboxylated SAM were determined.

Polyamines (nmol/1 06cells)Cell

lineHH5Wild

typeTreatmentNoneMTA,

10Â»/MMTA,100MMMTA,500IMNoneMTA,

100MMMTA,500MMNoneMTA,

100MMMTA,500 MMPutrescine0.46

Â±0.05"0.84

Â±0.031.60 Â±0.201.60

Â±0.330.45

Â±0.041.57 Â±0.711.62

Â±0.160.50

Â±0.021.00 Â±0.041.43

Â±0.03Spermidine2.79

Â±0.172.73Â±0.112.83

Â±0.242.30Â±0.212.80

Â±0.252.86Â±0.321.47

Â±0.162.58

Â±0.412.45Â±0.382.1

2 Â±0.46Spermine0.60

Â±0.070.47Â±0.050.32Â±0.080.28Â±0.080.58

Â±0.140.33Â±0.020.21Â±0.070.54

Â±0.080.33Â±0.020.24Â±0.10CAfJt(pmol/10Â»

cells)43.1645.050.443.932.746.056.442.556.042.6Decarboxyl-atarl

QAMillcuOHM(pmol/10Â«cells)<1.0"4.128.444.6<1.027.541.3<1.035.451.5

8 Mean Â±SD of 3 experiments.' Mean of 2 to 3 experiments.


3570




Table2Effect of inhibitors of polyamine synthesis on MTA-inducedchanges in SAM and polyamine metabolismin MTAphosphorylase-deficient

lymphoma cellsThe lymphoma cells (clone H) were incubated for 16 h with the indicated agents, and the intracellular contents of polyamines, SAM, and

decarboxylated SAM were measured. Finalconcentrations of added compounds were MTA, 500 MM;MGBG, 100 >IM;and DFMO, 1 mw.Polyamines(nmol/10* cells)

TreatmentNone

MTAMGBGDFMOMTA + MGBGMTA + DFMOPutrescine0.50

Â±0.06a

1.33 Â±0.160.66 Â±0.250.05 Â±0.020.97 Â±0.170.06 Â±0.04Spermidine2.47

Â±0.292.39 Â±0.631.49 Â±0.230.80 Â±0.251.68 Â±0.161.73 Â±0.72S

perniine0.48

Â±0.070.33 Â±0.060.35 Â±0.070.48 Â±0.110.18 Â±0.010.40 Â±0.12SAM

(pmol/106cells)40.0

Â±3.558.9 Â±3.6

119.2*

30.9 Â±4.8116.2"

48.9 Â±9.0Decarboxylated

SAM(pmol/10Â°cells)<1.0

48.6 Â±7.5<1.0

8.7 Â±0.8<1.0

115.8Â±14.1a Mean Â±SD of 3 experiments.6 Two experiments only.

MTA is a powerful inhibitor of mammalian spermine synthase(30-31) and, to a lesser degree, of spermidine synthase (32). Inthe MTA phosphorylase-deficient lymphoma cells, MTA did not

substantially reduce spermidine and spermine levels, at leastduring short-term incubations. However, the thioether nucleoside

did block the incorporation of radioactive ornithine into spermine,suggesting that MTA did inhibit spermine synthase activity. Thenet rate of spermine formation reflects both its synthesis anddegradation (4). Possibly, the addition of MTA was followed bya reduction in spermine catabolism. This may explain why, inshort-term experiments, no fall of spermine levels was evident,in spite of the greatly reduced synthetic rate. The MTA-induced

augmentation of putrescine and polyamine levels may be attributed not only to an increase in ornithine decarboxylase and SAMdecarboxylase activity but also to a lack of their utilization forspermine synthesis.

Putrescine has been shown to stimulate SAM decarboxylaseactivity by stabilizing the enzyme and by lowering the Km forSAM (33). The rise in decarboxylase activity induced by MTAmust be largely independent of this mechanism. Thus, SAMdecarboxylase activity increased prior to any observable changesin putrescine pools. Furthermore, the ornithine decarboxylaseinhibitor DFMO prevented the MTA-triggered changes in putres

cine levels, without altering the effects of the nucleoside ondecarboxylated SAM levels.

The exact mechanism by which MTA enhances ornithine decarboxylase and SAM decarboxylase activities remains unclear.The data suggest that nonspecific effects on protein synthesis,as well as alterations in intracellular polyamine pools, do not playan important role. Nonetheless, MTA did inhibit the flux throughthe polyamine-biosynthetic pathway. Conceivably, ornithine de

carboxylase and SAM decarboxylase activities are regulated bythe velocity of polyamine synthesis, rather than by the intracellular concentrations of polyamines.

Pegg ef al. (34) and Raina ef al. (35) have demonstrated thatthe nondegradable MTA analogue methylthiotubercidin elevatedornithine decarboxylase and SAM decarboxylase activities incultured cells. Methylthiotubercidin is a substantially weakerinhibitor than MTA of spermine formation (35). It is possible thatMTA, and also methylthiotubercidin, directly affects the synthesis, state of activation, or degradation of ornithine decarboxylaseand SAM decarboxylase. A direct analysis of the effects of MTAon the generation and turnover of mRNA species correspondingto the 2 enzymes should clarify these issues.

Collectively, our experiments indicate that MTA is a rapid and

potent inducer of SAM decarboxylase and ornithine decarboxylase in MTA phosphorylase-negative lymphoma cells. The in

creased activity of the 2 decarboxylases, combined with a reduction in polyamine synthesis, causes a progressive rise indecarboxylated SAM and putrescine levels. The MTA-triggeredrise in SAM decarboxylase and ornithine decarboxylase activitiescannot be attributed to alterations in spermidine or sperminelevels, nor to changes in the rate of cellular proliferation. Theseresults support the notion that MTA, the nucleoside by-productof polyamine biosynthesis, may also influence the rate-limitingenzymes in the polyamine-synthetic pathway.

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1985;45:3567-3572. Cancer Res Masaru Kubota, E. Olavi Kajander and Dennis A. Carson Phosphorylase-deficient Malignant Murine Lymphoblasts-Adenosylmethionine Decarboxylase in Methylthioadenosine

SIndependent Regulation of Ornithine Decarboxylase and

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