imbalanceinornithinemetabolisminhepatomasofdifferent...

[CANCER RESEARCH 32, 1924â€”1932,September 19721

has been reported to occur in at least 1 mammalian protein( 33), codons for L-omithine are not accommodated by theuniversal genetic code, and tRNA molecules or tRNAaminoacyl synthetases cognate to this amino acid have neverbeen detected in animal cells; the exceptional finding ofL-ornithine in a urate-binding globulin probably reflects theformation of this amino acid from L-arginyl residues inpeptide linkage after complete assembly of the polypeptidechains (33). Thus L-omithine cannot be regarded as a truebuilding block for protein biosynthesis. The action of arginaseon L-arginine produces L-omithine during the operation of theurea cycle in ureotelic species. In mammals, the activity ofarginase is by far the greatest in the liver, an organ which is theprincipal site of operation of the complete urea cycle; next inrank in arginase activity are organs such as kidney and smallintestine, which are among the few extrahepatic tissues thatcarry out the urea cycle (5, 13, 17, 40, 45, 48, 49). Thelimited tissue distribution of a functional urea cycle appears,at least in part, to reflect an absence of significant levels of themitochondrial enzyme L-ornithine carbamyl transferase (EC2.1 .3.3) from many organs (13, 17, 40). The activities ofornithine carbamyl transferase, arginase, and other urea cycleenzymes have been examined in a variety of tumors of liverand other organs(l3, 17, 40, 48, 49).

Other enzymes besides ornithine carbamyl transferase thatutilize L-orithine are present in many higher animal cells.L-Arginine:glycine amidinotransferase (EC 2.1 .4.1) catalyzesthe reversible formation of L-ornithine and guanidoacetic acidfrom glycine and L-arginine, a key step in creatinebiosynthesis. The facile reversibility of this reaction (K@q 1.1at 37Â°) enables the enzyme readily to promote theconsumption of L-omithine to form L-arginine (26). As far aswe can ascertain, the activity of L-arginine:glycine amidinotransferase in malignant tissues has not been scruntinized.Another enzyme , ornithine transaminase (L -0rithine :ketoacid aminotransferase (EC 2.6.1 .13), catalyzes the productionof glutamic-'y-semialdehyde L@'-pyrrolidine-5 -carboxylate)from L-ornithine and 2-ketoglutarate, or certain other t-ketoacids (36). Ornithine transaminase, which is localized in themitochondria of many different mammalian tissues, catalyzesthe back reaction (i.e., synthesis of L-ornithine fromglutamic.'y-semialdehyde and glutamate) at significant rates;nevertheless the main function of the enzyme appears to be inthe utilization of L-omithine for the synthesis of glutamic7-semialdehyde which can in turn be converted enzymically

SUMMARY

In comparison with normal livers from rats of the same age,sex, and dietary regimen, the L-omithine decarboxylase (EC4.1 .1 .17) activities of fast-growing Morris hepatomas (e.g.,lines 3924A and 7777) were very high, whereas a number ofslow-growing and more differentiated hepatomas exhibitedL-omithine decarboxylase activities which were considerablyless elevated.

None of the hepatomas examined, regardless of their speedof growth, had significantly elevated S-adenosylmethioninedecarboxylase activities in comparison with that of liver.

The steady-state concentration of putrescine in some butnot all of the fast-growing neoplasms was much greater thanthat in the corresponding normal liver controls. The putrescinecontent of all slow-growing hepatomas was usually abovenormal hepatic range.

The spermidine/spermine ratio tended to be higher in all ofthe hepatomas studied than in the corresponding normal livers;the overall concentrations of these two polyamines in alltumors were, however, within the range seen in the normallivers (0.6 to 1.5 j.zmoles/g.)

The utilization of L-ornithine for aliphatic polyaminesynthesis versus the operation of the urea cycle in rat liver andits neoplasms is discussed.

INTRODUCFION

In this and the accompanying paper (40), it will be shownthat, among various Morris rat hepatomas of vastly differentgrowth rates, there exist profound differences in the activityof certain enzymes involved in metabolic pathways that utilizeL-ornithine.

There are a number of reasons why particular interestattaches to the formation and utilization of L-ornithine byhepatic neoplasms of ureotelic animals. Although L-ornithine

1 This is Paper 1 in a series of publications on â€œMetabolic Imbalance

in L-Omithine Utilization in Hepatomas of Different Growth Rates.â€•2 Recipient of USPHS Grant HD-04592. To whom requests for

reprints should be addressed, at the Ben May Laboratory for CancerResearch University ofChicago, Chicago, Ill. 60637.

3 Recipient of USPHS Grant CA-05034 and Grants from the

American Cancer Society and Damon Runyon Memorial Fund, Inc.Received March 24, 1972; accepted June 1 , 1972.

1924 CANCER RESEARCH VOL. 32

Imbalance in Ornithine Metabolism in Hepatomas of DifferentGrowth Rates as Expressed in Formation of Putrescine,Spermidine, and'

H. Guy Williams-Ashman,2 Gordon L. Coppoc, and George Weber3

The Ben May Laboratory for Cancer Research, Unive,rity of Chicago, Chicago, Illinois 60637 fH. G. W.-A., G. L. C.J , and Department ofPharmacology,Indiana University School ofMedicine, Indianapolis, Indiana 46202 fG. W.J

Research. on September 11, 2018. © 1972 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Polyamine Formation in Hepatomas

into either L-glutamate or L-proline (36, 45). Herzfeld andKnox (7) have studied ornithine transaminase in variousnormal and cancerous rat tissues; it appears that the activity ofornithine transaminase varies inversely with the growth ratesofsome Morris rat hepatomas.

An important avenue for utilization of L-orinithine inhigher animal tissues is in the biosynthesis of the basesspermidine and spermine. The only known series of reactionsfor the synthesis of these polyamines in animal cells involveutilization of the constituent base putrescine (1 ,4-diaminobutane). The enzyme L-ornithine decarboxylase (EC 4.1.1.17)catalyzes the synthesis of putrescmne and carbon dioxide fromL-ornithine (19). The sequential actions of arginase andornithine decarboxylases thus provide a pathway for thebiological formation of putrescine from L-arginine. Arginase istherefore of potential importance in the biosynthesis ofputrescine (and hence of spermidine and spermine), as well asin the urea cycle (45). Although an alternate pathway forputrescine synthesis from L-arginine involving the intermediary formation of the base agmatine is known to takeplace in certain bacteria and higher plants (19, 34), this seriesof reactions do not seem to operate in mammalian tissues,from which L-arginine decarboxylase appears to be absent (19,

43, 45).An enzyme (spermidine synthase) present in most animal

tissues catalyzes a transfer of a propylamino group fromdecarboxylated S-Ado-Met4 to putrescine to yield spermidineand 5'-methylthioadenosmne (9, 10, 20, 25). Another enzyme,designated spermine synthase, promotes the formation ofspermine and 5'-methylthioadenosine from decarboxylatedS-Ado-Met and spermidine (21, 25, 43, 45). The decarboxylated S-Ado-Met that serves as a propylamino group donor inboth the spermidine and the spermine synthase reactions issynthesized from S-Ado-Met by the enzyme S-Ado-Metdecarboxylase. Contrary to an earlier view (20), it is nowestablished that S-Ado-Met decarobxylase in mammalian (3, 9,10, 25) and yeast (12) cells is separable from spermidine andspermine synthase, the decarboxylase from both of the latterbiological sources being markedly and specifically stimulatedby putrescine (3, 9, 10, 20, 25) and to a lesser degree byspermidine (20, 25) and a few other aliphatic diamines (47). Inthe sequence of reactions in animal cells that ultimately resultin the formation of spermidine and spermine, putrescine servesas a product and weak competitive inhibitor of the 1streaction, omithine decarboxylase (1 1, 19); an activator of the2nd reaction, S-Ado-Met decarboxylase; a substrate forspermidine synthase; and a competitive inhibitor of sperminesynthase (21).

Spermidine and spermine are present in all nucleated animalcells (2, 35, 43, 45). The overall concentrations of thesepolyamines in most rat tissues varies between roughly 0.2 and1.5 pmoles/g, fresh weight (8, 23, 27), with spermidine/spermine quotients being in the range of 0.4 to 1.5 (8); inmany but not all rat tissues, the spermidine/spermine ratiodeclines with the age of the animals (2, 8). A few rat tissues,notably the ventral lobe of the prostate gland, contain verymuch larger amounts (5 to 7 pmoles/g) of spermidine and

4 The abbreviations used is: S-Ado-Met, S-adenosyl-L-methionine.

spermine (18), although in the latter instance a considerableproportion of the total polyamines are present extracellularlyin prostatic secretion stored in the lumina of the gland (cf.Ref. 44). The steady-state concentrations of putrescine inmost mammalian tissues are nearly always much lower thanthose of spermidine and/or spermine (43, 45). The proclivityof spermidine and spermine to interact with and to influencethe enzymic synthesis and degradation of polynucleotides, andalso to stabilize certain biological membranes (2, 35, 43, 45),has naturally led to many speculations that these aliphaticpolyamines may function as important intracellular regulatorsof many physiological processes. As reviewed in detail andcomprehensively documented elsewhere (2, 4, 18, 23, 25, 35,43â€”46), in regenerating liver, and during the growth of othertissues induced by hormones and other treatments, thereoccurs an increased synthesis of spermidine and enhancedornithine decarboxylase activities, and sometimes elevations inputrescine.activated S-Ado-Met decarboxylase as well.

Surprisingly little systematic attention has been given topolyamines and their biosynthetic enzymes in malignant cells.The literature records some sporadic observations on the levelsof putrescine, spermidine, and spermine in certain experimental animal neoplasms (1 , 2, 15, 16, 24, 27, 28, 32) and, ina few instances, of their omithine decarboxylase (28, 30, 32)and S-Ado-Met decarboxylase (28) activities. TIus papersummarizes measurements of the levels of putrescine,spermidine, and spermine, and of omithine and S-Ado-Metdecarboxylases, in a number of Morris rat hepatomas of verydifferent growth rates. The findings are discussed in relation toparallel studies on the omithine carbamyl transferase activitiesof the same spectrum of tumors that are recounted in the

companion paper (40).

MATERIALS AND METHODS

Biological. All rats were adult males and were housed inseparate cages in an air-conditioned room that was illuminateddaily from 6 a.m. to 7 p.m. Purina laboratory chow and waterwere available ad libitum. All rats were killed between 9 a.m.and 10 a.m. Examination of stomach contents at autopsyrevealed that all the rats used were well fed during the nightbefore death. Control and tumor-bearing rats of the Buffaloand ACI/N strains were shipped by air express from Dr. H. P.Morris of Howard University, Washington, D. C., to IndianaUniversity School of Medicine, Indianapolis, Ind. Thetransplantable hepatomas used were the slowly growing 7787,8999, 9618A, and 7800; the intermediate 7777; and thefast-growing 3924A and 3683F varieties. The tumors of slowor intermediate growth rates were carried in Buffalo ratswhereas those exhibiting rapid growth rates were carried inACI/N rats. Hepatomas 7787, 8999, and 96l8A weretransplanted bilaterally in the thigh muscles; all the othertumor strains were transplanted s.c.

Materials. DL-Ornithine-l-'4C (26.4 mCi/mmole) andputrescine dihydrochloride-l,4-'4C (50 mCi/mmole) werepurchased from Amersham-Searle Corp., Arlington Heights, Ill.DL-Methionine-l -â€˜â€˜@C (5 .3 mCi/mmole), aminopropyltetramethylene diamine-l ,4-' 4C trihydrochloride (spermidine,

SEPTEMBER 1972 1925



H. Guy Williams-Ashman, Gordon L. Coppoc, and George Weber

10.6 mCi/mmole) and bis(aminopropyl)tetramethyl.enediamine-l ,4i 4C tetrahydrochioride (spermine, 9.67mCi/mmole) were obtained from New England Nuclear,Boston, Mass. S-Ado-Met-'4COOH was synthesized by theprocedures described by Pegg and Williams-Ashman (14) andstored at â€”20Â°in solutions adjusted to pH 2 .0 with HC1.Unlabeled S-Ado-Met was prepared in the same fashion. Thespecific radioactivity of the labeled S-Ado-Met was adjustedapproximately 3.5 cpm/pmole. In most of the experimentsreported, DL-omithine-l-'4C was diluted with the unlabeledL-amino acid to give a specific radioactivity of about 1cpm/pmole. The D isomer was assumed not to be attacked byornithine decarboxylase in calculations of the rate ofdecarboxylation of L-ornithine (19).

Preparation of Tissue Extracts. The rats were stunned,decapitated, and exsanguinated. Livers and tumor tissue wereremoved rapidly and blotted. Hemorrhagic, necrotic, and hostfibrous tissue was dissected away from the tumors. Allmaterial was kept on iced glass vessels. The iced tissue wasminced, weighed, and homogenized in 3 to S volumes of cold10 mM sodium phosphate, pH 7.2, containing 0.1 mMdisodium EDTA, within 10 mm after removal of the samplesfrom the animals. Homogenization was for 1 mm and about 20strokes in a glass homogenizer equipped with a Teflon pestle.An aliquot of the homogenate equivalent to about 0.5 g, wetweight, of tissue was immediately removed and placed in atube containing 0.1 ml of a mixture of labeled putrescine,spermidine, and spermine for checks on the recovery of theseamines (the amounts of the labeled amines added werechemically negligible). After mixing, the homogenates weredeproteinized by addition of an equal volume of 10% (w/v)trichloroacetic acid. Further handling of this material foramine analyses is described below . The remainder of theoriginal homogenate was made 5 mM with respect todithiothreitol. After centrifugation for 10 mm at 37,000 X gat 0Â°, the supernatant fluid was removed with a Pasteur pipetand centrifuged for 90 mm at 43,500 X g. The finalsupernatant fluid was used for assays ofenzyme activities. In afew experiments, the soluble extracts of homogenates weretreated with ammonium sulfate (Mann Research Laboratories,New York, N. Y.; enzyme grade) to give a final saturation at

00 of 80%. The insoluble material was harvested aftercentrifugation and dialyzed against 3 changes of at least 30volumes of the original homogenizing medium containing 5mM dithiothreitol.

Determination of Ornithine Decarboxylase Activity. Thereaction was followed by release of@@ CO2 from L-ornithinel-'4C (7, 13). The reaction mixture contained 50 j.zmolesglycylglycine buffer (pH 7.2), 0.1 pmole pyridoxal5-phosphate, 3 pmoles dithiothreitol, 0.5 pmole L-ornithine,and approximately 500,000 cpm DL-ornithine-l-'4C, plustissue extract in a total volume of 0.5 ml. The reactions wereallowed to proceed for the times indicated at 37Â°and werehalted by placing of the tubes on ice and immediate additionof 0.5 ml of 40% (w/v) trichloroacetic acid via a 22-gaugeneedle through the rubber stopper. The tubes were incubatedfor another 30 mm at 37Â°so as to ensure that all@@ CO2released during the enzymic reaction was trapped in theHyamine-HC1 present in the polypropylene center well (1 1,19).

Determination of Putrescine-activated S-Ado-MetDecarboxylase Activity. The reaction was followed by releaseof â€˜4C02 from S-Ado-Met-'4COOH in the absence orpresence of saturating levels (2.5 mM) of putrescine (6, 9, 14).Each tube contained 50 pmoles of sodium phosphate, pH 7.1;S jimoles of dithiothreitol; 0.1 j.zmole S-Ado-Met-'4COOH(approximately 2.5 cpm/pmole); tissue extract; and, if added,I .25 j.zmoles of putrescine in a total volume of 0.5 ml. Thetubes were incubated for the specified times at 37Â°and thenprocessed as in the assay for ornithine decarboxylase activity.

Determination of Putrescine, Spermidine, and Spermine inTissues. The methods used involved slight modification of theprocedures described by Pegg et a!. (18), involving extractionof the amines into 1-butanol; electrophoresis on paper; andestimation of the bands containing putrescine, spermidine, orspermine with the cadmium-ninhydrin reagent. Radioactivestandards containing chemically negligible amounts of the 3amines were routinely added to the initial tissue homogenatesin order to correct for recoveries of endogenous putrescine,spermidine, and spermine, which were on the average of 76%in a large number of experiments. Butanol extracts ofdeproteinized homogenates of rat hepatomas 7800, 7787, and8999 contained appreciable amounts of a substance(s) thatmigrated on paper electrophoresis at either pH 3.6 and pH 4.3to a position between putrescine and spermidine; the materialwas stained blue-grey immediately after application of thecadmium-ninhydrin reagent and brief heating. In many of itsproperties, this material resembled histamine. No attempt toestimate the amounts of this substance quantitatively wasundertaken, however, because the substance was notrigorously identified from a chemical standpoint.

Other Methods. The methods described by Weber et al. (42)were used for the freeze-clamping of tissues in situ. Proceduresfor the determination of radioactivity and of protein, and allother methods, are described elsewhere (3, 8â€”12,18â€”21).

RESULTS

Concenfrations of Putrescine, Spermidine, and Spermine inMorris Hepatomas and Normal Rat Livers. Table 1 summarizesmeasurements of the concentrations of 3 aliphatic amines invarious lines of Morris rat hepatomas and in the livers of

control animals of the same strain, age, and sex, and subject tothe same dietary regimen. When expressed in terms of nmolesof amine per g, fresh weight, of tissue, the concentration ofspermidine were not significantly different from those in thecorresponding control animals in the cases of hepatomas 7777and 7800. However, in every other instance, the spermidineconcentrations were significantly higher in the hepatoma ascompared with normal liver tissue. Table 1 also indicates thatthe spermine concentrations in many of the hepatomasapproximated to the range found in normal liver controlspecimens. It is evident, however, that the spermidine/spermine quotients were significantly larger in the tumors ascompared with normal liver with regard to all of the Morrishepatoma lines studied with the exceptions of hepatomas7777 and 7800, in which instances the mean spermidine/spermine ratios were pratically the same as in the controllivers. Nevertheless, the degree of significant increases in the





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SEPTEMBER 1972 1927




spermidine/spermine quotients found in many of the the standard procedure described above and then allowed tohepatoma strains were not directly proportional to the speed stand on iced beakers for 90 min. With the exception of anofgrowth of the tumors. apparent rise in the normal liver putrescine levels from 11 to

In normal rat liver and many other mammalian tissues, the 29 nmoles per g, fresh weight (for which no explanation canbe provided at present), the concentrations of the amines inthe freeze-clamped liver and hepatoma were almost identicalwith those found in tissues that had been dissected at roomtemperature from dead rats and then allowed to stand on icefor 90 mm. In other experiments with male Sprague-Dawleyrats weighing roughly 350 g, livers from exsanguinated ratswere allowed to autolyze for 4 hr at 25Â° prior tohomogenization, deproteinization, and the performance ofamine estimations. In the latter instance also, hardly anychange in the concentrations of putrescine, spermidine, andspermine were observed in comparison with liver samples thatwere homogenized and deproteinized within 2 min after theirremoval from the animals. These findings fit in with reportsthat, in rat tissues, spermidine and spermine turn over onlyvery slowly (29), apparently because of a lack (or only veryfeeble activity) of enzymic machinery to degrade polyamines.The results also suggest that the extremely high steady-statelevels of putrescine found in hepatoma 3924A are almostcertainly not due to experimental artifacts. The concentrationsof polyamines in mammalian blood are so low as to bevirtually undetectable by methods similar to those used in thisstudy (2, 23).

Decarboxylases of Polyamine Biosynthesis. Suitableexperiments indicated that release of â€˜â€˜@CO2 from appropriatesubstrates in the omithine decarboxylase and putrescineactivated S-Ado-Met decarboxylase standard assay systemswere proportional to the time of incubation over an initial60-mm period and to the amounts of soluble liver or tumorextracts added. Since nearly all ofthe ornithine decarboxylase(1 1, 19, 25) and S-Ado-Met decarboxylase (20, 25) activity ofthe livers and many other oi@gansof normal rats is localized inthe supernatant fluid resulting from high-speed centrifugationof tissue homogenates, it was assumed that most of theactivity of these 2 decarboxylases was present in the solublefraction of hepatoma homogenates. Table 2 shows that theornithine decarboxylase activity of soluble extracts of all ofthe Morris hepatomas examined except hepatoma 7800 weresignificantly greater than those of livers of correspondingcontrol animals. Remarkably high ornithine decarboxylaseactivities were observed with hepatomas 7777 and 3924A.Although no direct linear relationship between orithinedecarboxylase activities and the rates of tumor growth wasapparent, the decarboxylation of ornithine tended to behighest in tumors of the fastest growth rate.

Table 2 also shows that the S-Ado-Met decarboxylaseactivities (determined in the presence of saturating levels ofthe putrescine activator) in all of the Morris hepatomas studiedwas decreased in comparison with that of normal livercontrols, with the exception of hepatoma 7777. Although theS-Ado-Met decarboxylase activities of the normal liver controlspecimens were, on a comparable molar basis, in nearly everyinstance substantially higher than the rate of decarboxylationof L-omithine, in nearly all the Morris hepatomas the activityof ornithine decarboxylase was greater than that of S-Ado-Metdecarboxylase. In every tumor line examined, the production

steady.state concentrations of putrescine are strikingly lowerthan those of spermidine and spermine (8, 23, 27, 43, 45).This point is illustrated in Table 1, from which it is alsoevident that in the Morris hepatomas studied, putrescineconcentrations were significantly increased above the upperlimits of the range seen in normal liver controls, with theexception of hepatoma 7800. Extraordinarily large amounts ofputrescine were found to accumulate in hepatoma lines 3924Aand 3683F. In the 2 latter neoplasms, the putrescine/spermidine quotient is much higher than that of all of thenormal livers and most of the other hepatomas examined. Inhepatoma 3924A, where the putrescine/spermidine ratio wasabout 8 times that seen in the corresponding control normalliver, the concentration of spermine per unit, wet weight, oftissue is noticeably lower than the value for spermine found innormal livers or other Morris hepatomas of this series. Thisaccords with previous findings that putrescine serves as acompetitive inhibitor against the spermidine substrate in thespermine synthase reaction (2 1), and supports the prediction(21 , 43, 45) that spermine synthesis in living mammalian cellsmay depend, among other things, on the putrescine/spermidine quotient. Two other conclusions that emerge fromthe data in Table 1 are: (a) putrescine concentrations tendedto be highest in the most rapidly growing Morris hepatomas;and (b) the steady-state concentration of putrescine did notalways parallel exactly the orithine decarboxylase activitiesof the same samples of tumor tissue from different strains ofMorris hepatomas, although all the neoplasms for whichputrescine content was abnormally high did exhibit enhancedornithine decarboxylase activity (cf. Table 2).

Stability of Aliphatic Ammes in Normal Liver and MorrisHepatomas. The standard procedures used for measurement ofaliphatic amine concentrations in normal liver and Morrishepatomas involved careful dissection of healthy nonnecrotictumor masses away from adjacent connective and other tissuesimmediately after death of the animals by stunning. Thesemanuevers involved bits of the tumor tissue being allowed tostand on iced beakers for as long as 15 min prior tohomogenization of the tissue, followed by immediatedeproteinization for the amine analyses. The control livertissue was handled in exactly the same fashion. Thus it isconceivable that the amine analyses might have been subject toconsiderable errors due to rapid formation or utilization ofputrescine, spermidine, and spermine during the time when thetissue was dissected from the dead animals and allowed tostand on ice for a minimum of 5 mm. As a check of this point,samples of hepatoma 3924A obtained from rats anesthetizedwith ether were free-clamped with tongs cooledto the temperature of liquid nitrogen within 1 to 2 sec afterremoval of the tumors from the living animals. Thesespecimens were pulverized while frozen and then processed foramine analyses. The concentrations of putrescine, spermidine,and spermine in such freeze-clamped 3924A tumor and normalliver from corresponding control animals were compared withthose found in tissue samples that were obtained according to




Ornithine decarboxylase/ S-AdoMetdecarbox

No.ofBodyOrnithine

decarboxylase activity S-Ado-Met decarboxylaseactivityylasePer

PerTissuesexperiments@vt(g)Per g % cell 7 Per g % cell %Ratios@Normal

liter(Bid/jab)Control

for8999535720Â±3 100 0.93 100 42 Â±4 100 0.20 1000.481007787439421Â±6 100 0.93 100 68 Â±16 100 0.30 1000.311007800535720Â±3

100 0.95 100 42Â±4 100 0.20 1000.481009618A448425Â±0.3100 1.13 100 36Â±3 100 0.16 1000.691007777412617

Â±3 100 0.76 100 17Â±1 100 0.08 1001.00100Normal

bis'er (ACl/N)Control

for3924A52749Â±1 100 0.41 100 41 Â±5 100 0.18 1000.221003683F52749Â±I 100 0.40 100 41 Â±5 100 0.18 1000.22100Hepatomas8999532039

@ 3 195Â° 1.25 134 23 Â±1 55â€• 0.07 351.703547787532647Â±4 224Â° 1.49 160 44 Â±3 65Â° 0.14 471.073459618A543654Â±4

216a 3.14 278 10Â±1 28Â° 0.06 385.407837800522613Â±2 65 0.49 52 16Â±1 38Â° 0.06 300.8116977775172132Â±12 776Â° 5.76 758 39Â±4 229Â° 0.17 2133.383383924A524274Â±9 822Â° 5.61 1368 10Â±1 24Â° 0.08 447.4033643683F524434Â±7 378@ 2.38 595 17 Â±1 41Â° 0.12 672.00 909


Table 2Decarboxylation of L-ornithine and S-Ado-Met by extracts of normal and neoplastic liver

The means Â±SE. of the â€˜4C02 formed from L-ornithine-l-' 4C and S-Ado-Met-' 4COOH are expressed in nmoles/hr/g of tissue, wet weight, ornmoles/hr/cell X 106

a Statistically significantly different from values of respective control normal livers (p < 0.05).

of â€˜â€C̃O2 from@ COOH was markedly stimulated,as in normal liver, by addition of saturating (2.5 mM)concentrations of putrescine. In some of the neoplasms thatexhibited very high ornithine decarboxylase activities, andcorrespondingly elevated steady-state levels of putrescine, theextent of enhancement of S-Ado-Met decarboxylation byexogenous putrescine was less than that observed with solubleextracts of normal control livers, or of other tumors in whichputrescine contents were low (e.g., 7787, 96l8A) Suchfindings, illustrated by the representative experiment shown inChart 1, presumably reflect the presence of larger amounts ofendogenous putrescine in the tumor extracts used todetermine the decarboxylase activities.

The experiments in Table 3 show that 1 hr after injection ofthe protein biosynthesis inhibitor cycloheximide, thereoccurred a precipitous fall in both the ornithine andS-Ado-Met decarboxylase activities of hepatoma 7800. Thedecline in ornithine decarboxylase was significantly greaterthan the decline in S-Ado-Met decarboxylase. These findingssuggest that, as in normal rat tissues (25, 31) these 2decarboxylases in a hepatic neoplasm may turn over veryquickly, with apparent half-lives ofless than 30 mm. However,unequivocal estimations of the half-life of an enzyme ineukaryotic cells must rest on actual determinations of theamount of enzyme protein by suitable radioimmunological or

other procedures rather than only on estimations of enzymeactivities. Until the latter sort of experiments have beencarried out, the conclusion that ornithine and S-Ado-Metdecarboxylases in Morris hepatoma 7800 turn over veryrapidly can be regarded only as a tentative one. Neither theproduction of lactate by tissue slices aerobically (39) nor thepyruvate kinase activity (41) of the same hepatoma 7800tissue used in the experiments shown in Table 3 weresignificantly influenced by administration of cycloheximideunder these experimen tal conditions.

DISCUSSION

The foregoing results indicate that, in comparison withnormal livers from well-fed control rats of the same strain andage, the concentration of putrescine was increased in all exceptone of the Morris hepatoma lines studied in this series ofexperiments and that in the most rapidly growing tumors theputrescine concentrations were 5 to 10 times those observed inthe control livers. The general trend in spermidineconcentrations with respect to tumor growth rate was in thesame direction, although smaller in magnitude, in 5 of the 7hepatoma lines examined. On the other hand, spermineconcentrations did not vary in any systematic way with

SEPTEMBER 1972 1929



ReactionEnzyme

activityNaCl

solutioninjectionCycloheximideinjectionOrnithine

decarboxylasePutrescine-activated S-Ado-Metdecarboxylase14.2

Â±1.621.0 Â±2.63.4

Â±0.48.9 Â±0.7


decline very rapidly after injection of the protein synthesisinhibitor cycloheximide. Although this finding, as alreadyconsidered above, does not rigorously prove that these 2decarboxylases of polyamine biosynthesis have very fast ratesof turnover in both normal liver and in hepatomas, the resultscertainly indicate that this is likely to be the case. Obviouscomplexities arise in the interpretation of the activities ofenzymes in animal cells that may have extremely shorthalf-lives. In this study, we did not systematically search fordiurnal oscillations in the activity of these decarboxylases,possibly of the type previously found with other enzymes(including certain ones with relatively fast turnover rates) inliver and Morris hepatomas by Potter etaL (22). Nevertheless,for reasons discussed above, it seems unlikely that alterationsin food intake could account for the differences in orithinedecarboxylase and S-Ado-Met decarboxylase in hepatomasversus normal animals that were observed in the well-fedanimals used in experiments described in this paper. Also it isevident from Tables 1 and 2 that the scatter of values forconcentrations of aliphatic amines and activities of theirbiosynthetic decarboxylases was nearly always quite small inany given series of control livers.

Over the 1st day or so after partial hepatectomy of adultrodents, there are increases in ornithine decarboxylase activityand the spermidine/spermine quotient, as well as transitoryrises in tissue putrescine concentrations, in the regeneratinglivers (6, 25, 29â€”31) that are in most instances of greatermagnitude than the enhancements of these parameters whichwe observed in many Morris hepatomas as compared withnormal rat liver. In contrast, there occur very large decreases inornithine carbamyl transferase activity as a function ofincreasing hepatoma growth rate and dedifferentiation but nostriking changes in the regenerating liver (40).

Pertinent to these considerations is the fact that, whenexpressed in terms of moles of substrate transformed at 37Â°,the activities of ornithine and S-Ado-Met decarboxylases areextremely low in comparison with the activities of all of theenzymes of the urea cycle in normal rat liver. For example, ona comparable molar basis, the activities of L-ornithine andS-Ado-Met decarboxylases in normal rat liver are of the same

Table 3

Effect of cycboheximide administration on the decarboxybation ofL-ornithine and S-Ado-Met by extracts ofhepatoma 7800

Well-fed rats were given i.p. injections of cycloheximide, 75 mg/kgbody weight, dissolved in 0.15 M NaC1, or with an equivalent volume ofthe NaCl solution. After 1 hr, the animals were sacrificed and the tumorwas removed, minced, weighed, and homogenized in 5 volumes of 10mM sodium phosphate buffer (pH 7.2) containing 0.1 mM EDTA and 5mM dithiothreitol. A high-speed supernatant fraction was prepared(39,000 x g for 90 mm) and used for the assays, which were performedas described in the text. The data are expressed as the mean Â±SE. of

nmoles of â€4̃C02 formed per 60 mm per g tissue, wet weight. Five ratswere used in each group and the assays were performed in duplicate.

L&J0@cr

LL@

C1J@)ow

,tâ€”@

0C.)

Chart 1. Time course of release of carbon dioxide fromS-Ado-Met-' 4COOH by centrifuged tissue extracts in the presence (+PU) or absence of putrescine (2.5 mM). The composition of thereaction mixtures is described in the text. The extract from normalACI/N rat liver (A) contained 1 mg of protein (PROT.), and the extractfrom hepatoma 3924A (B) contained 2.8 mg of protein.

growth rates of the tumors. Thus, according to the MolecularCorrelation Concept proposed by Weber (37, 38), the behaviorof tissue concentrations of putrescine and spermidine falls intoClass 2 (in which are grouped biochemical parameters that arealtered in the same direction in the tumor spectrum), whilethe spermine values can be characterized by Class 3 , i.e., theirconcentrations show no simple correlation with tumor growthrates. In 5 out of the 7 hepatoma strains the spermidine/spermine ratio was significantly increased in comparison withthe normal liver controls. The differences between normal liverand hepatoma tissue that we observed with regard toconcentrations of putrescine, spermidine, and spermine weresimilar whether the results were expressed on the basis of wetweight of tissue, or per cell asjudged by nuclear counts (Table1), or on the basis of total tissue DNA or protein (data notshown). Thus the results do not simply reflect differences inthe water content of the hepatomas versus their normal livercontrols.

The observed variations in ornithine decarboxylase activitieswere compatible with the values we obtained for the tissueconcentrations of the 3 aliphatic amines. Ornithinedecarboxylase is the only enzyme known to be present inmammalian tissues that can promote the formation ofputrescine, the key substrate for the biosynthesis of bothspermidine and spermine (1 1, 43). The activity of ornithinedecarboxylase was significantly increased in all of the tumorsexcept hepatoma 7800, which was in fact the only Morrishepatoma examined that exhibited a putrescine content thatwas within the range seen in the normal liver controls. Incontrast, S-Ado-Met decarboxylase activities were eitherwithin the normal range or actually decreased in all of thehepatomas except hepatoma 7777 , in which there was asomewhat heightened S-Ado-Met decarboxylase activity. Inevery one of the hepatomas examined, the ratio of ornithinedecarboxylase to S-Ado-Met decarboxylase was significantlyhigher than the quotient seen in normal livers. The latterrelationship may thus be grouped in Class 2 of the MolecularCorrelation Concept (37, 38).

The activities of both ornithine decarboxylase andS-Ado-Met decarboxylase in at least 1 Morris hepatoma behavelike the corresponding normal liver enzymes in that they

MINUTES





order of magnitude (Table 2) but are roughly 1,000,000 timesless than the activity of ornithine carbamyl transferase whenall enzyme activities were determined at pH 7.1 to 7.4 [seeTable 4 of the accompanying paper (40)] . Elsewhere (45 , 47)it has been suggested that the activities of ornithine andS-Ado-Met decarboxylases may be important rate-limitingfactors in the overall synthesis of spermidine and spermine inliver and some other rat tissues, especially in view of the factthat the Km values for the substrates in the first (Km forL-ornithine, approximately 0.1 mM) and second (Km forS-Ado-Met, approximately 0.01 mM) of these reactions in rattissues are near the range of their actual steady-state tissueconcentrations.

Our experiments indicate that, in the transitions fromnormal liver through highly differentiated and slowly growingMorris hepatomas to the end point of very rapidly dividing andanaplastic tumors of this class, there is no loss in the ability ofthe neoplasms to synthesize spermidine and spermine. Theseobservations emphasize that these polyamines are likely toserve essential roles in the metabolic economy of all normaland cancerous nucleated animal cells. The continued synthesisof aliphatic polyamines in malignant cells occurs despitevery rapid protein biosynthesis that entails utilization ofmethionine and arginine (a precursor of ornithine), which alsoserve as precursors for the formation of spermidine andspermine. The retention or increase in aliphatic amines andornithine decarboxylase activities in the hepatoma spectrumoccurs in the face of a pronounced decrease in ornithinecarbamyl transferase that paralleled increases in growth ratesof the various hepatomas, as considered in Ref. 40. Asdiscussed in detail in Ref. 40, this results in a strikingimbalance in the ratio of ornithine decarboxylase/orithinecarbamyl transferase activities that is compatible with theobserved increases in tissue concentrations of putrescine andspermidine and the corresponding decline in activity of theurea cycle.

It appears from a recent publication (28), although thepertinent data reported were not treated statistically and aretherefore hard to evaluate, that a few drugs that increase thesurvival time of mice bearing the Li 2 10 leukemia cansubstantially lower the concentrations of putrescine,spermidine, and spermine in this murine tumor. Recently, ithas also been shown that another drug, methyiglyoxalbis(guanylhydrazone), which is known to inhibit theproliferation of a variety of neoplasms is a very potent andapparently a rather specific in vitro inhibitor of putrescineactivated S-Ado-Met decarboxylases isolated from mammalianprostate and liver and from yeast (47). It would be of interestto examine the effects of this and related antineoplastic drugsthat may inhibit enzymes of polyamine biosynthesis on thelevels of spermidine and spermine in malignant tissues growingin living animals.

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1972;32:1924-1932. Cancer Res H. Guy Williams-Ashman, Gordon L. Coppoc and George Weber Spermidine, and SpermineGrowth Rates as Expressed in Formation of Putrescine, Imbalance in Ornithine Metabolism in Hepatomas of Different

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