(CANCER RESEARCH 37, 911-917, March 1977]

SUMMARY

A selective deficiency of umidine triphosphate (UTP) wasinduced in AS-30D matascites hepatoma cells by the synemgistic action of D-galactosamine and 6-azaunidine. The mesistance of these hepatoma cells to low concentrations of Dgalactosamine (<2 mM) was due to their active de novopynimidine synthesis which compensated the trapping ofumidylate in the form of umidine diphosphate-amino sugarsderived from D-galactosamine. The additional blockage ofde novo pynimidine synthesis led to noncompensated unidylate trapping with a UTP content of less than 0.05 mmole/kgof cell wet weight as compared to the control level of 0.66mmole/kg. The induction of UTP deficiency by incubatingthe cells with low concentrations of D-galactosamine and 6-azaunidine (0.5 mM each) was not accompanied by significant changes in the content of adenine and guanine nucleotides, unidine diphosphate glucose, and unidine diphosphate galactose. The depletion of UTP pools could be meversed within 10 mm by the addition of unidine; orotate orumacilwere completely ineffective in these hepatoma cells.

A UTP content in the range of 0.1 to 0.4 mmole/kg,induced by either 6-azaumidine or D-galactosamine, was associated with a reversible depression of cell growth in suspension culture. A UTP content below 0.05 mmole/kg led toirreversible growth inhibition and to necrocytosis in culture,as well as to a loss of transplantability in vivo. Unidinereversal studies indicated that the percentage of cells ableto resume growth in culture decreased with an increasingtime period of UTP deficiency. The deficiency period mequired for irreparable or lethal damage in these hepatomacells ranged from 3 to 20 hr.

The principle of noncompensated unidylate trapping canbe extended to other inhibitors of nucleotide synthesiscombined with various nucleotide-trapping sugar analogs.Noncompensated nucleotide trapping may be useful for aninduction of selective nucleotide deficiencies in tumor cells.

INTRODUCTION

A depletion of nucleotide pools can serve as an efficienttool to inhibit cellular growth and to induce cell death under

,ThisworkwassupportedbytheDeutscheForschungsgemeinschaft,Bonn-Bad Godesberg , through “Forschergruppa Labarerkrankungen ,“Freiburg, Germany.

Received August 17, 1976; accepted December 14, 1976.

some circumstances. The consequences of nucleotide deficiency depend not only on the type or group of nucleotidesinvolved, but also on the extent and the time period of theirdepletion (9, 13). The mechanisms available for an induction of nucleotide deficiency include (a) inhibition of denovo synthesis, , by analogs (4, 36); (b) acceleration ofcatabolism, e.g., by phosphate trapping (10, 40); (c) interfenence with the generation of high-energy phosphatebonds (10, 29, 33); (d) trapping of the nucleoside monophosphate moiety (20, 21); and (e) trapping of the nucleoside portion of nucleotides (10).

A selective depletion of the UTP pool leading to cellnecrosis has been observed in liven after a galactosamineinduced trapping of unidylate (8, 16, 20). In ascites hepatoma cells, however, galactosamine concentrations higherthan 2 mM were required for a depression of cellular UTP tolevels below a critical concentration range onto a content ofless than 0.1 mmole/kg (23). In addition, a marked depression of the ATP level was associated with this reduction ofthe UTP pool (23). The approach of this study was thecombination of a blockage ofde novo pymimidine nucleotidebiosynthesis with an amino sugar-induced trapping of undylate. This procedure made possible the use of low galactosamine concentrations and resulted in a severe and selective deficiency of UTP in hepatoma cells. The combinationof an inhibition of nucleotide biosynthesis with a trapping ofnucleotides induced by sugar analogs can serve as a tool bywhich selective nucleotide deficiencies can be induced invarious cells or tissues. The choice of the inhibitor and ofthe sugar analog enables cell specificity of nucleotide depletion. A preliminary report on part of this work was givenearlier (16).

MATERIALS AND METHODS

Ascltes Hepatoma Cells. The transplantable rat asciteshepatoma AS-30D (37) was carried in 7- to 10-week-oldfemale Spnague-Dawley mats(Voss, Tuttlingen, Germany).The tumor cells were transplanted at 7-day intervals by i.p.injection of 0.2 ml of ascitic fluid collected under sterileconditions. Transplant generations 315 to 365 were used inthis study.

Chemicals, Enzymes, and Isotopes. D-Galactosamine HCI was purchased from C. Roth, Kanlsmuhe, Gemmany; 6-azaunidine, umidine, all other nucleosides, nucleo

911MARCH 1977

Uridine Triphosphate Deficiency, Growth Inhibition, and Deathin Ascites Hepatoma Cells Induced by a Combination ofPyrimidine Biosynthesis Inhibition with UridylateTrapping1

Dietrich 0. R. Keppler

Biochemisches Institut, Universitat Freiburg im Breisgau, 0 7800 Freiburg, Germany

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AS-30Dcells were incubatedat a concentration of 2.5 x109/Iiterat37°in standard medium (23) supplemented with 0.35 mM[2-14C]uridine

(0.8 mCi/liter) and, in the experimental flask, with 2mMD-galactosamine.The cells were collected and frozen (23)after60mm;

nucleotideswere extractedwith 0.6 M perchlonicacid.Uracilnucleotideswereseparatedby paperchromatographyin 2differentsolvents

(11). The relative radioactivities were determined bythecountingof I -cm segmentsof the paper(11) andwereexpressedasthe

percentage of total radioactivity in uracil nucleotides.Meanvaluesfrom 3 experiments ±S.D.Radioactivity

in uracil nucleotides(%

oftotal)Uracil

nucleotide ControlGalactosamineUTP

68±825±4UDP8±28±2UMP4±13±1UDP-glucuronate7 ±2 5 ±1UDP-glucose

+ UDP-galactose 2 ±1 2 ±1UDP-hexosamines0 30±4UDP-N-acetylhexosamines

11 ±3 27 ±5

0. 0. R. Kepp!er

tides, and cofactors were from Boehmingen Mannheim,Mannheim, Germany. Powdered Swim's S-77 medium andSwim's 67-G medium were from Grand Island BiologicalCompany, Grand Island, N. Y. UDP-galactose 4-epimeraseand lactate dehydrogenase were purchased from SigmaChemical Co. , St. Louis, Mo. All other enzymes used in thisinvestigation were from Boehninger Mannheim, Mannheim,Germany. [2-14C]Uridine and D-[1-‘4C]galactosamme werefrom Amersham Buchler, Bmaunschweig, Germany.

Incubation of Hepatoma Cells. The AS-30D ascites hepatoma cells (37) were collected on the 7th day after transplantation and were washed and suspended in standardmedium as described previously (23). Incubations for up to6 hr were performed at cell concentrations of 1.5 to 2.5 x109/Iiten in closed Erlenmeyer flasks under COIaim (1/20) ona gyratory shaker at 130 rpm. The temperature of the suspension was 37 ±0.4°;the pH was kept between 7.45 and7.25, if necessary, by addition of small amounts of solidNaHCO3. Duplicate 10-mi aliquots were removed for cell wetweight determinations (23) at least twice, usually at 30 and150 mm afterthebeginningof incubation.The procedurefor cooling, freezing, and deproteinization of the cells fornucleotide and metabolite analyses has been described(23).

Suspension Culture of AS-30D Cells. Cells were collected under sterile conditions on the 5th or 6th day aftertransplantation and were suspended at a cell concentrationof 2 to 4 x 108/liter in Swim's 67-G medium containingpancreatic autolysate (1/25 by volume) (31). This mediumwas supplemented with 2 mM glutamine and ascitic fluid (1/50 by volume) sterilized by filtration of AS-30D ascites hepatoma fluid . The cells were grown during the late exponentialphase at 37°on a gyratory shaken at 120 rpm in siliconizedErlenmeyen flasks under CO@/aim(1/20). Proliferation andgrowth were monitored by cell counts in a blood-countingchamber and by measurement of the absombance of thesuspensions at 623 nm; cellular DNA (5) and protein contents (1) were determined at 24-hr intervals. Morphologicalexaminations, including the exclusion of trypan blue bythese cells, were performed by phase-contrast microscopy.

EnzymatIc Analyses of Nucleotides. UTP (18), UDP-glucose, and UDP-galactose (22) were determined spectrophotometmically using UDP-glucose dehydrogenase as indicatorenzyme. The lUMP2 was measured as UMP after snakevenom phosphodiesterase hydrolysis of 5'-nucleotides (15).6-Azauridine 5'-monophosphate did not interfere with thespecific assay for UMP. The enzymatic determination of

@GMPand ICMP has been described (15). ATP was measured with yeast hexokinase and glucose-6-phosphate dehydrogenase (26). Pyruvate kinase and adenylate kinasewere used for the analysis of ADP and AMP (14).

RESULTS

Metabolism of D-Galactosamine in AS-30D Cells. TheAS-30D ascites hepatoma line has kept the capacity to me

2 The abbreviations used are: lUMP, sum of all acid-soluble uracil 5'-

nucleotides; @GMP,sum of all acid-soluble guanine 5'-nucleotidas; @CMP,sum of all acid-soluble cytosine 5'-nucleotides; UDP-hexosamines, sum ofUDP-galactosamine and UDP-glucosamina.

tabolize D-galactose. The galactose elimination matewas 9.1mmoles/kg cells x hr when the cells were incubated in thepresence of 1 mM galactose. Enzymes of the galactosepathway also metabolize the galactose analog D-galactosamine (for review, see Refs. 8 and 9). A marked accumulationof galactosamine 1-phosphate to a cellular content of morethan 8 mmoles/kg was observed when AS-30D cells wereincubated in the presence of 2 mM galactosamine (16). Theformation of UDP-hexosam ines (UDP-galactosam me +UDP-glucosamine) and the accumulation of UDP-N-acetylhexosamines were demonstrated by biosynthetic labeling ofthese amino sugar nucleotides with [1 @14C]galactosam me(16) and with [2-―C]unidine(Table 1). The diversion of thelabeled unidine to the synthesis of UDP-hexosamines andUDP-N-acetylhexosamines was associated with a stronglyreduced label in UTP (Table 1). The accumulation of UDPamino sugars caused an increase in LUMP when the cellswere incubated or grown in the presence of galactosamine(Chart1).

The Role of de Novo Pyrimidine Biosynthesis in theGalactosamine-induced Depression of UTP Content. Thetrapping of umidylate by formation and accumulation ofUDP-amino sugars derived from galactosamine can becounterbalanced by an increased synthesis of unidylateeither de novo or on the salvage pathway (16, 20). An activede novo pymimidine nucleotide biosynthesis in AS-30D hepatoma cells was indicated by the increase of @UMPat a mateof 0.65 mmole/kg cells/hr between 1 and 4 hr after additionof a low galactosamine concentration (0.5 mM) to the medium (Chart 1). This rise of @UMPwas completely suppressed after a selective inhibition of the formation of umidylate from omotidine 5'-phosphate (7) by use of 0.1 to 1 mM 6-azaumidine (Chart 1). The increased de novo synthesis ofuridylate was elicited by only a limited depression of theUTP content to about 0.45 mmole/kg cells (Chart 2B). Therise of LUMP in AS-30D cells showed a lag phase of about30 mm, however, in comparison with the drop in the UTPlevel (Charts 1 and 2). Higher galactosamine concentrations

Table 1Galactosamine-induced change of the labeling pattern of uracil

nucleotides

912 CANCER RESEARCH VOL. 37

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@:::__________@ 0.3p •

8.

______________@ 0.1A

U@ 2 3 4 5

INCUBATIONTIME(he)

Chart 2. Induction of UTP deficiency and

Noncompensated Uridylate Trapping in Hepatoma Cells

the UTP pool induced by this “noncompensated unidylatetrapping.―

Selectivity of UTP Deficiency in Hepatoma Cells. Thedepletion of UTP pools in AS-30D cells was associated witha general depression of pumine and pyrimidine nucleotidecontent when galactosamine concentrations higher than 1mM were used (23). This lack of selectivity in the depletionof nucleotide pools was no longer observed when the concentration of galactosamine was lowered to 0.5 mM. Adenosine phosphates, the adenylate energy charge, and @GMPremained in the control range, and the @CMPdecreased toabout 70% as compared to the controls when UTP droppedto less than 5% (Chart 3). The low levels of UDP-glucose andUDP-galactose in AS-30D cells (23) remained, in contrast toliver (21), within the control range when the low concentration of galactosamine was used (Charts 3 and 4). This condition made possible a discrimination between the consequences of a deficiency of UDP-hexoses and of UTP, mespectively, in these hepatoma cells.

Induction of UTP Deficiency for Defined Time Periods. Areversal of UTP deficiency within 10 mm was accomplishedby means of unidine (Chart 4, left). The early matesof uptakeand phosphorylation of 0.5 mM unidine were measured bythe increase of @UMPwith time (Chart 4, right) andamounted to 0.6 and 6.0 mmoles/kg cells x hr in untreatedand UTP-deficient AS-30D cells, respectively. @UMPcontinued to rise at a lower mateof 0.8 mmole/kg cells x hr afterreplenishment of the UTP pools (Chart 4). This rise comesponded to the formation of UDP-amino sugars derivedfrom galactosamine. Addition of umidine to UTP-deficientcells resulted in an increase of UDP-glucose to levels abovethe control values (Chart 4, left). Uptake and phosphorylation of unidine were not inhibited to a significant extent byequimolam concentrations of 6-azaunidine. This is consistentwith the low Km/Ki ratio for the inhibition of unidine phosphorylation in whole cells by 6-azaunidine (25). Cytidine alsoserved as a source of uracil nucleotides in UTP-deficienthepatoma cells. A linear increase of LUMP at a nate of 0.5mmole/kg cells x hr in the presence of 0.5 mM cytidine(Chart 4, right) indicated that the deamination was rate

a'

a'

a'.x

0EE

0.ID

U I I .5 4

INCUBATIONTIME (he)

adjustment of UTP levels inascitas hepatoma cells. AS-30D cells were incubated at 37―in standardmedium (23)supplemanted with 2 (A) or 0.5 (B) mM o-galactosamine, 1 (A) or(B) 0.5 mM 6-azaunidina, and 1 mM unidine (A ). Freezing of the cells in liquidnitrogen (23) preceded the enzymatic analysis of UTP content (18). Meanvalues ware from 4 to 8 separate experiments with all standard deviations atlass than 20% of the mean. The same changes in UTP content were foundwhen tissue culture medium (Swim's 67-G) instead of standard medium (23)was used. 0, controls; 0, 6-azaunidina; A, 6-azaunidine + galactosamina; S,galactosamine; •,6-azauridine + galactosamine + unidine.

induced a stronger depression of the UTP content (Chart2A) and a higher rate of de novo uridylate formation (16). Itshould be noted, in comparison to liver (20, 21), that the risein LUMP occurred in the absence of significant changes ofthe levels of UDP-glucose and UDP-galactose. Inhibition ofde novo pyrimidine nucleotide biosynthesis by 6-azaumidine(28) was followed by a slight decrease in LUMP (Chart 1)and by a depression of the UTP content to 0.13 mmole/kgcells (Chart 2B). Neither the inhibition of uridylate biosynthesis nor a trapping of unidylate induced by galactosamine(<2 mmoles/liter) resulted in a depletion of UTP pools toless than 0.1 mmole/kg cells. Only a combination of bothmechanisms led to a severe deficiency of UTP. Under thiscondition, a UTP content of less than 0.04 mmole/kg cellswas reached after 1 and 2 hr when galactosamine concentrations of 2 and 0.5 mM were used, respectively (Chart 2). Amajor difference in the effects of these galactosamine concentrations was thus related to the time course of inductionof UTP deficiency rather than to the extent of depletion of

INCUBATION TIME (hr)

Chart 1. Acid-soluble uracil nucleotides and the changes in them inducedby galactosamine and 6-azauridine. The incubation of AS-30D cells and thedetermination of cell wet weights ware performed as described under “Materials and Methods.―the WMP was determined anzymatically as UMP aftersnake venom phosphodiasterase hydrolysis of call extracts (15). Each pointrepresents the mean from 6 to 8 separate experiments with standard deviations of less than l2% of the mean. The concentrations of D-galactosamineand 6-azaunidina were 0.5 mM each. 0, control;@ 6-azauridine; A, 6-a.zauridina + galactosamina; •,galactosamine.

®

@E@EE@

INCUBATIONTIME(hr)

)8

I'U

a,

I

Chart 3. Nucleotide content and energy charge (E.C. @)during the induction of UTP deficiency in AS-30D cells. The concentrations of D-galactosamine and 6-azauridina in the incubation medium were 0.5 mM each. Eachpoint represents the mean from 4 to 10 separate experiments; all standarddeviations were below 18% of the mean.

913MARCH 1977

0.7

@ 0.5

@ 0.3

E8.; o.i

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Addition to incubation mediumAscitestumorgrowth60-day

survivors

(%)None15/166Galactosamine13/1476-Azaunidine13/147Galactosamine

+6-azauridine0/14100Galactosamine+6-azauridine12/120+

uridine

D. 0. R. Kepp!er

Table 2

Effect of UTP deficiency on transplantability and in vivo growth ofAS-30D cells

The cells were incubated for 6 hr as described under “Materialsand Methods―in standardmedium(23)supplementedwith 2 mMDgalactosamine, 1 mM 6-azauridine, 1 mM uridine, or combinationsof these compounds. The cells were collected by centrifugation for5 mm at 120 x g and resuspended at a concentration of 10―cells/liter in cold Swim's S-77 medium supplemented with 25 mMNa2HPO4,pH 7.45. The UTPdeficiency period in the cells treatedwith galactosamineand 6-azauridinewas much longer than 6 hr,since the intracellular metabolites of the drugs were still present.FemaleSprague-Dawleyrats,weighing 210 ±30 g and 82 ±8 daysof age,weregiven i.p. injectionsof 3 x 10@cells/animal. The meansurvival time of rats with ascites tumor growth was 23 days. Ascitestumor growth is expressedby the numberof tumor-bearing rats ascompared to the number of rats receiving injections of AS-30Dcells.

TIMEAFTERGo(N+6-AzoUrd(hr) TIMEAFTERGoLN+ 6-AzaUrd(hr)

Chart 4. Reversal of UTP deficiency by unidine. AS-30D cells were incubated as described under “Materialsand Methods.―After a praincubationperiod of 15 mm, D-galactosamine (Gal N) and 6-azaunidine (6-AzaUrd) wereadded (1/200 by volume) to give a final concentration of 0.5 mM for each.Two hr later, unidine, cytidine, or uracil ware added at final concentrations of0.5mM.Eachpoint representsthemeanfrom 4to 5separateexperiments.A,UTP, and UDP-glucose (UDP-Glc) contents; B, changes in LUMP.

limiting since @CMPincreased about 6 times faster thanLUMP under this condition. Cytidine was insufficient, however, for a complete reversal of UTP deficiency within 2 hr.Pynimidine precursors without any detectable effect on theuracil nucleotide content included umacil(Chart 4, right) andorotate. The lack of increase in pynimidine nucleotide content after addition of 0.5 mM orotate to the medium wasobserved in both untreated and galactosamine-treated AS30D cells. This is in contrast to liver (22) and may be usefulfor a selective protection of the liver against UTP deficiency(8).

Loss of Transplantability and Tumor Growth in Vivo. Theinduction of UTP deficiency in AS-30D cells by galactosamine + 6-azaumidine (Chart 2A) caused a loss of transplantability and ascites hepatoma growth (Table 2). This effectwas not observed when UTP deficiency was prevented byaddition of unidine to the incubation medium. The limiteddepression of the UTP content by 6-azauridine or by galactosamine alone and the intracellular accumulation of galactosamine metabolites had no effect on transplantability under the conditions studied (Table 2).

InhibitIon of Cell Proliferation In Suspension Culture byReversible UTP Deficiency. Cell number, cell protein, andDNA content doubled in 21 hr when AS-30D cells weregrown in primary suspension cultures at cell concentrationsbetween 1 and 9 x 108/litem(Charts 5 and 6). This rate of cellgrowth and proliferation was depressed by less than 13% insuspensions supplemented with low concentrations of galactosamine (@0.5 mM) or with uridine + galactosamine +6-azaumidine (Chart 5). A significant depression of cellgrowthwas thusnotcausedbythelimitedreductionofUTPpools in the presence of galactosamine alone (Chart 2B), orby an intracellular formation of galactosamine metabolites,or by 6-azaunidine 5'-monophosphate as the metabolite of6-azauridine (25, 28). A reduction in the cellular UTP content to about 0.13 mmole/kg induced by 6-azaumidine alone(Chart 2B) led to a marked depression of cell proliferation(Chart 5). The increase in cellular protein during the initial24 hr of a culture in the presence of 6-azaumidine wasreduced by 32% when compared to the gain in protein of anuntreated cell suspension. Uridine readily reversed the in

LU8-

-aU,..aLUU

Chart 5. Effect of different UTP levels on hepatoma cell proliferation insuspension culture. Cultures of 50 ml each were grown as described under“Materialsand Methods.―Points represent the mean from cell counts of atleast 2 flasks kept simultaneously under the same conditions. The cell numbars include viable, nonviable, and necrotic cells. The concentrations of Dgalactosamine, 6-azauridine, and uridine were 0.5 mM each. 0, control (noaddition); L@,6-azaunidine; 0, galactosamine; U, galactosamine + 6-azauridine; •,galactosamine + 6-azauridine + unidine.

hibitomy effects of 6-azaumidine on AS-30D cell growth.A complete cessation of cell proliferation induced by ga

lactosamine + 6-azaunidine (Charts 5 and 6) was associatedwith a lack of increase in cellular protein and DNA at 24 and48 hr. Examination of these cells by phase-contrast microscopy revealed a loss of all cell clusters after 24 hr. Thenumber of single cells that were altered by a rupture of theplasma membrane with cytoplasmic protrusions and cellshrinkage increased between 16 and 48 hr. The appearanceof necrotic cells was associated with an increasing amountof cellular debris. Prevention of UTP deficiency by addition

6TIME (hr)

914 CANCER RESEARCH VOL. 37

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Noncompensated Uridylate Trapping in Hepatoma Ce!!s

LU

-ILUU

P0

Chart 6. Reversal by unidine of the growth inhibition induced by UTPdeficiency in AS-30D cells. The experimental conditions and drug concentrations werethe sameasdescribedfor Chart5. •,control cells (no addition);.,galactosamine+6-azaunidine.Opensymbols,culturesinthepresenceofgalactosamine + 6-azaunidine with unidine added at the following times: 0, 6hr; 0, 9 hr; L@,16 hr; 0, 20 hr.

of uridine completely protected against necrosis of AS-30Dcells. Necrocytosis was also prevented by the addition ofcytidine.

The point of irreversible or irreparable cell damage wasStudied by means of UTP deficiency periods of differentlengths. Uridine was added after 3, 5, 6, 9, 16, 20, and 24 hr,respectively, to separate cell cultures containing galactosamine + 6-azaunidine. The potential to resume exponentialcell growth was lost after about 20 hr (Charts 6 and 7).Reversal of UTP deficiency after 3 to 16 hr resulted in anincreasing amount of necrotic cells and cellular debris associated with a decreasing number of cell clusters andviable cells when examined between 24 and 48 hr (Chart 6).The comparison of the growth rates at a given cell concentration with the slope of the control curve indicated that thepercentage of cells able to resume growth after unidineaddition decreased with an increasing length of the UTPdeficiency period (Chart 7).

(U

DEFICIENCYPERIOD(UTP<aO5mmote/kg)(hr)

Chart 7. Late consequences of different time periods of UTP deficiency.The experimental conditions ware the same as those described for Chart 6.Uridine was added to cultures containing D-galactosamine + 6-azauridine(0.5 mM each) after 3, 5, 6, 9, 16, and 20 hr; this corresponds to periods ofdepression of UTP content below 0.05 mmole/kg of 1, 3, 4, 7, 14, and 18 hr,respectively (see Chart 28). The growth rates were monitored by measurements of the absorbanca of the suspensions at 623 nm. Growth rates betwean 42 and 48 hr were expressed as percentage of the growth rate in thecontrol culture at the respective cell concentration.

TIME (hr)

MARCH 1977 915

DISCUSSION

A severe and selective depletion of the UTP pool can beinduced only if the mateof unidylate-consuming processesclearly exceeds the rate of umidylate production by de novosynthesis and regeneration (20). The unidylate-trapping action of galactosamine was compensated for largely by theactive de novo pynimidine nucleotide biosynthesis of theAS-30D ascites hepatoma cells when concentrations of theamino sugar below 2 mM were used (Charts 1 and 2). Theadditional blockage of de novo pynimidine nucleotide biosynthesis led to a noncompensated umidylate trapping withsevere UTP deficiency as the primary consequence. Theselective inhibition of uridylate biosynthesis by 6-azaunidinehas been shown to cause a reversible depression of cellgrowth (34) (Chart 5). The depression of UTP content (Chart2B) and the inhibition of nucleic acid synthesis by 6-azaumidine (25) in AS-30D and Novikoff hepatoma cells in suspension were only moderate in comparison with the synergisticeffect of the noncompensated umidylate trapping. It is memarkable that the blockage of de novo pynimidine nucleotide biosynthesis by 6-azaumidine in liver in vivo was completely compensated for, probably by salvage pathway synthesis, and there is no depression of hepatic UTP content(21). The rate of net umidylate biosynthesis in the presenceof galactosamine is only one-third as high in liver as in AS30D cells (16). It is consistent that an additional inhibition ofunidylate synthesis is not required in liver for an induction ofsevere UTP deficiency by galactosamine. The blockage ofpynimidine biosynthesis in hepatoma cells, on the otherhand, allows the use of a galactosamine concentration (0.5mM) which is lower than that required for a necrogenicaction in hepatocytes [1 mM (24)].

The inhibitory effects of D-galactosamine and D-glucosamine on various tumor lines have been studied extensively(2, 27, 32, 38). The effective amino sugar concentrationsused in most studies were between 20 and 125 mM (2, 38).These concentrations would cause severe toxicity in vivo , atleast with galactosamine, which induces a spotty necrotichepatitis at a dose of 1 to 2 mmoles/kg body weight (8, 19).The available evidence indicates that the cytotoxicity ofgalactosamine and of glucosamine, at least at low concentrations, is mediated by an interference of uracil nucleotidemetabolism with subsequent induction of UTP deficiency(3, 9, 21). An additional general loss and depression ofnucleotides caused by phosphate trapping in the form ofamino sugar monophosphates has been observed only athigh concentrations of galactosamine (23) and glucosamine(3, 30). One may expect that the tumor growth-inhibitoryeffects of the umidylate-trapping amino sugars can be induced with relatively low concentrations (<1 mM) when thesugar analogs are combined with an inhibitor of unidylatesynthesis. This relationship has been established for theeffect of galactosamine in AS-30D ascites hepatoma cells(Charts 5 and 6); a complete inhibition of cell growth bygalactosamine alone required a concentration of about 8mM (not shown).

The selective and reversible adjustment of UTP levels inascites hepatoma cells (Charts 2 to 4) allowed an analysis ofthe extent and time period of UTP depression as related tothe growth-inhibitory on the lethal consequences to the

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D. 0. A. Keppler

cells. A UTP content of between 0.4 mmole/kg and thecontrol mange(0.66 ±0.05 mmole/kg) was without a significant effect on AS-30D cell growth, viability, or transplantability (Charts 2 and 5; Table 2). A UTP content of between0.1 and 0.4 mmole/kg was associated with a reversibledepression of cell growth (Charts 2 and 5), but transplantability was not affected (Table 2). A UTP content of below0.05 mmole/kg (Charts 2 and 3) led to lethal cell damageindicated by an irreversible growth inhibition (Chart 7), necrocytosis, and a loss of transplantability (Table 2). The timeperiod of UTP deficiency required for irreparable or lethaldamage in the hepatoma cells was not in a similar mangeforall cells in the nonsynchronized suspension culture (Chart7). The uridine reversal studies indicated that the lethal UTPdeficiency period may be as short as about 3 hr in somecells and as long as about 20 hr in others. These observations would be compatible with a cell cycle-dependent sensitivity of the cells to UTP deficiency. Alternatively, onecould also consider a varying susceptibility of the cells tothe induction of UTP deficiency. A comparable time-dependent increase in the percentage of killed cells has beendescribed after exposure of LS178Y cells to cytosine amabinoside for different periods of time (6).

The present study with ascites hepatoma cells supportsthe view (9, 16) that neither the metabolites of galactosamine nor the low levels of UDP-glucose and UDP-galactoseare growth inhibitory or cytotoxic (Charts 3 to 5). The eventsleading from UTP deficiency to growth inhibition most likelyinclude inhibition of ANA and protein synthesis, both ofwhich have been studied in detail as consequences of hepatic UTP deficiency (9, 20, 35). Our understanding of thesequence of events leading from UTP deficiency to cellulardeathisincompleteatpresent(17).The differentialeffectofthis selective substrate deficiency on the various forms ofANA-polymerases may result in a lethal imbalance of proteinsynthesis.

The combination of galactosamine-induced umidylatetrapping with 6-azaunidine-induced inhibition of the de novosynthesis of uridylate demonstrates a new principle for aninduction of nucleotide deficiency in tumor cells (Chart 8).Replacement of galactosamine by glucosamine as the un

NDP—SUGAR(ANALOG)I SUGAR(ANALOG)

dylate-tnapping sugar analog alters the cell specificity. Inanalogy to noncompensated unidylate trapping, a noncompensated trapping of guanylate or cytidylate may be aneffective means for tumor cell kill as well as for inhibition ofviral multiplication . Noncompensated guanylate trappingcan be induced by the combination of an inhibitor of inosinate dehydrogenase (12, 39) with guanylate-trapping analogs of D-mannose or L-fucose. The noncompensated nucleotide trapping may be useful in the design of drug combinations inducing selective nucleotide deficiencies by ametabolic synergism. The intensity and duration of nucleotide deficiency can be further influenced by prevention orinhibition of nucleoside uptake or by supply of precursorson the salvage pathway. The latter possibility makes possible the reversal of a nucleotide depletion after a definedtime period.

ACKNOWLEDGMENTS

The author is grateful to Ute Stumpp-Grigat for her excellent technicalassistanceandtoDr.JamesJ.Starlingforhiscriticalreadingofthemanuscript.

REFERENCES

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RNA

DNA-@ 11@

‘•‘%__.._NDP,— 11@

NMP,//@ INHIBITOR

SALVAGE DE NOVOPATHWAY SYNTHESIS

Chart 8. Induction of nucleoside phosphate deficiency by the synergisticaction of an inhibitor of de novo nucleotide synthesis and nucleotide trapping induced by amino sugars and other sugar analogs. NDP, nucleosidediphosphate; NTP, nucleoside tniphosphate; NMP, nucleoside monophosphate.

916 CANCER RESEARCH VOL. 37

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1977;37:911-917. Cancer Res   Dietrich O. R. Keppler  Pyrimidine Biosynthesis Inhibition with Uridylate Trappingin Ascites Hepatoma Cells Induced by a Combination of Uridine Triphosphate Deficiency, Growth Inhibition, and Death

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