[CANCER RESEARCH 39, 4930-4938, December i 979]0008-5472/ 79/0039-0000$02.00

Retinol Inhibition of Ornithine Decarboxylase Induction and G1Progression inChinese Hamster Ovary Cells'

Man K. Haddox,2 Karen F. Frasier Scott, and Diane Haddock Russell3

Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724

sequential events of the growth response under controlledconditions without the influence of a transforming or promotingstimulus. We have previously demonstrated in mitotically synchronized CHO4cells that vitamin A treatment arrested cells inmid-G1 phase (16, 17). The inhibitory effect of vitamin A was afunction of cell cycle position; cell cycle progression wasblocked only when retinol was added to the cells prior to midG1 progression. The arrest was reversible and resulted in a 2-hr delay in the onset of DNA synthesis in cells exposed to a G1pulse of retinol, suggesting that, in the presence of the vitamin,the cells progressed to a point in mid-G1 prior to some keyevent required for G1-S-phase transition. An increase in ODCactivity is a ubiquitouseventmarkingG1progressionof proliferating cells (28, 57). The excursion of ODC activity in retinoltreated cells was used, therefore, as a marker of the extent ofG1 progression to localize the temporal site of vitamin action.It was found that the vitamin-induced arrest blocked the cellsat a site prior to the induction of ODC. These results suggestedthe presence of a retinol-sensitive restriction (50) or commitment point in CHO cells during mid-G1 phase and that ODCinduction was associated with passage through or beyond thispoint. In the present studies, we have examined this relationship between the inhibition of ODC and the inhibition of growthin order to establish any obligatory correlations between the 2vitamin-inhibited processes.

MATERIALS AND METHODS

Cell Culture Techniques. CHO cells were grown in monolayer in McCoy's Medium 5A containing 20% fetal calf serum,penicillin (100 units/mI), and streptomycin (100 zg/mI). Cellcultures were incubated at 37°in a humidified 95% air-S%CO2 atmosphere. Cell-doubling times were 13 to 15 hr underthese conditions. Cell numbers were determined by an electronic particle counter (Coulter Electronics, Hialeah, Fla) onsamples collected by scraping with a rubber policeman intoPuck's Saline A. Cells were synchronized by selective detachment of cells blocked in metaphase by a 2-hr incubation in thepresence of 0.06 @sgcolcemid per ml (67). The mitotic cells(mitotic index, >90%) were washed free of the drug by centrifugation, resuspended in fresh, complete medium (first wash,4°;second wash, 37°),plated (0.5 to 1 x ‘106 cells per S mlmedia) in plastic Petri dishes (Falcon Plastics, Oxnard, Calif.)at 37°,and allowed to progress through the cell cycle. Within60 mm, greater than 90% of the cells had exited mitosis. Flowmicrofluorometric measurements of cellular DNA content wereperformed with a Coulter Model TPS-1 cell sorter on Puck's

4 The abbreviations used are: CHO, Chinese hamster ovary; ODC, ornithine

decarboxylase; SAMD, S-adenosyl-L-methionine decarboxylase; cyclic AMP,cyclic adenosine 3':S'-monophosphate; TPA. 12-O-tetradecanoylphorbol-i 3-

â€acetate.

CANCER RESEARCH VOL. 394930

ABSTRACT

Vitamin A inhibits ornithine decarboxylase (ODC) inductioninG1phaseof thecellcycleof synchronousculturesof Chinesehamster ovary (CHO) cells. The retinol-promoted inhibition ofODC was not the result of an effect on general protein synthesis, was effective only in G1 prior to the time an increase inODC activity had begun, did not involve disruption of the G1-phase increase in cyclic adenosine 3' :5'-monophosphate-dependent protein kinase activity, and required transcription-dependent events for reversal. The inhibition of ODC inductionwas associated with a block of cell cycle progression in G1phase. In the presence of the vitamin, there was no incorporation of [3H]thymidine, no induction of S-phase-dependent Sadenosyl-L-methionine decarboxylase, and no ultimate celldoubling. The inhibition of ODC and the inhibition of S-phasetransition displayed a similar concentration dependence, with60% inhibition occurring in the presence of 80 @Mretinol, asimilar dependence on cell locus in early G1phase for efficacy,and a similar reversal after removal of the vitamin, with theincrease in ODC preceding the increase in S-phase transitionin a parallel fashion. Arrest of the cells at the G1retinol-sensitiverestriction point prior to ODC induction resulted in the inhibitionof the G1-phase-dependentincreasein RNAsynthesis.

Other naturally occurring retinoids also inhibit CHO cellgrowth (retinal > retinol > retinyl acetate > retinoic acid).Retinal, the most potent, displayed a paradoxical effect onCHO cells. At very low concentrations (1 to 5 /zM), retinalstimulated CHO growth parameters; ODC activity was enhanced in a parallel fashion. At higher concentrations, retinalinhibited in a manner similar to that described for retinol.

INTRODUCTION

Vitamin A and several of its analogs have prophylactic andtherapeutic effects on the chemical induction and growth ofbenign and malignant tumors in whole animals (4, 5, 66). Ingeneral, there is a direct, inverse relationship between the levelof retinoid in the diet and the incidence of neoplasms after achemical carcinogen (65). Carcinogenesis is a prolonged, multistage process, and the vitamin apparently acts to inhibit thepromotion phase and does not affect initiation. Whether theaction of vitamin A is directed to an event specific to someaspect of the transforming process or results from inhibition ofan essential aspect of cell growth is unknown.

Synchronous cells in culture provide a model to study the

1 This work was supported by USPHS Research Grant CA-i 4783 from the

National Cancer Institute.2 To whom requests for reprints should be addressed.

3 Recipient of Research Career Development Award CA-00072 from the

National Cancer Institute.Received January i 6, 1979; accepted September i i , i 979.

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Retinol Inhibition of ODC and G1Progression

Saline A-washed, ethanol-fixed (70%, 10 mm) cells suspendedin hypotonic citrate containing 5 mg propidium iodide per 100ml as the fluorescent DNA stain (33). Retinoid solutions wereprepared in dioxane under subdued light and used immediately.Control cell populations received an equivalent amount ofdioxane (0.5%), which was found to have no effect on growthparameters. Survival of cells in the presence of retinol ordioxane was determined by the ability of the cells to formcolonies. Three dilutions of cell number were plated in triplicateinto new plastic Petri dishes containing prewarmed media ingrowth media alone or containing 0.5% dioxane or retinol andincubated for 10 days. Colonies were fixed with 50% aceticacid, stained with crystal violet, and counted. Cell viability wasdetermined by the capacity to give rise to a colony of 50 ormore cells.

Macromolecular Synthesis. Entry into S phase of synchronized cell populations was estimated by pulse measurementsof [3H]thymidine incorporation into acid-insoluble material. Atthe times indicated after mitotic release, 1 x 106 cells in S mlfull medium were incubated with the isotope (S @tCi/ml)for 15to 30 mm. The incubation was terminated by removing themedia and washing the monolayer twice with fresh media priorto harvesting the cells. Cells were pelleted by centrifugationand extracted by sonication (two 1S-sec bursts at 60% power;Kontes Co., Vineland, N. J.) into 10% trichloroacetic acid.Insoluble radioactivity was determined by filtration of the extract through cellulose ester filters (0.45-@sm pore; MilliporeCorp., Bedford, Mass.); the level of cpm/1 06 cells/mm wastaken as an Index of DNA-synthetic activity. RNA- and proteinsynthetic rates were determined by similar pulse measurementsuponincubatingthe cellswith[3H]uridineor [3Hjleucine,respectively.

Polyamine Determinations. Aminoguanidine bicarbonate(1O@M)was included in the medium of cultures (2 x 106cells)to be analyzed for polyamine concentrations to inhibit diamineoxidase present in serum. At the times indicated after mitosis,the medium (5 ml) was poured off into 1 ml 50% trichloroaceticacid, and 2.5 ml of chilled 10% trichloroacetic acid was rapidlyadded to the cell monolayer. The cell precipitate was scrapedinto solution, the dish was rinsed with an additional 2.5 ml acid,and the extract was sonicated (two 30-sec bursts) to ensurecellular disruption. Five hundred pmol of 3,3'-iminobispropylamine were added to both media and cell extracts to serveas an Internal standard for estimation of polyamine recovery,and the samples were stored at —80°until processing foranalysis. Extracts were centrifuged for 15 mm at 20,000 x g,and the supematant was removed and extracted 3 times with3 volumes of water-saturated ether to remove the acid, evaporated to dryness, and finally suspended in 200 @tIof 0.1 NHCI.An 8O-@daliquot of the reconstituted extract was analyzed forputrescine,spermidine,and spermineconcentrationsutilizinga Durrum D-500 automatic amino acid analyzer (41 , 58).

Assay for ODC Activity. ODC activity was measured in cellsupernatants as previously described with minor modifications(59). Cells were harvested by scraping, sedimented and sonicated (two 30-sec bursts) in 0.5 ml SOm@Na2H-KH2P04, pH7.2, containing 0.1 mM EDTA, 1 .0 m@ dithiothreitol, S m@ NaF,1 mM phenylmethylsulfonylfluoride, and 30 @tMpyridoxal phosphate. Two 20O-@tlaliquots of a 10,000 x g (S mm) supernatantof each sample were incubated for 60 mm at 37° in thepresence of 0.25 mM L-[1-14C]ornithine (0.5 @iCi)in 1S-mI

tapered centrifuge tubes fitted with rubber stoppers and centerwells (Kontes Co.). The assay was terminated by the additionof 0.6 ml of 1 Mcitric acid, and the ‘4CO2evolved was collectedon Whatman No. 3MM filter paper (Whatman Inc. , Clifton, N.J.) spotted with 20 @zlNCS present in the center well. The filterpaper was placed in toluene-Omnifluor and the radioactivitydetermined in a liquid scintillation spectrometer.

Assay of SAMD Activity. Enzyme activity was determined bymeasuring the liberation of ‘4CO2from S-adenosyl-L-[Carboxy!14C]methionine(21). Cells (S x 106) were harvested by scraping, sedimented,and sonicatedin 1 ml SOmMNa2H-KH2PO4buffer, pH 7.2, containing 1 m@ dithiothreitol. Two 200-@ilaliquots of a 10,000 x g supernatant from each sample wereincubated for 60 mm at 37°in the presence of 0. 15 mM Sadenosyl-L-[Carboxyl-'4C]methionine (0. 1 zCi) and either 2.5mM putrescine dihydrochloride or S mM spermidine trihydrochloride. The 14CO2evolved was collected and determined asdescribed under the ‘‘Assayfor ODC Activity.―

Cyclic AMP-dependent Protein Kinase Activity Ratio Determination. Cells (2 x 106) were harvested by scraping,pelleted, and sonicated (two 30-sec bursts) at 4°in 500 j@l10mM Na2H-KH2PO4 (pH 7) containing S mr@iNaF, 2mM EDTAand 0.5 mM 3-isobutyl-1 -methylxanthine. A 50-psIaliquot of a10,000 x g (S mm)supernatant was assayed for endogenouslyactivated protein kinase by incubating for S mm in a totalvolume of 75 @lcontaining SO m@Na2H-KH2PO4(pH 7), 0.5mM 3-isobutyl-1 -methylxanthine, S mM NaF, 10 mi@iMgCI2, SOILg F2b histone, 0.1 mt@i ATP, and 1 @zCi [-y-32P]ATP. Total

protein kinase activity was assayed by including 1 @zMcyclicAMP in the reaction. Incorporation of 32P into protein wasdetermined by chromatography of a 50-SIaliquot of the reactionon ITLC silica gel-impregnated glass fiber sheets (GelmanInstrument Co., Ann Arbor, Mich.) (24).

Chemicals. McCoy's Medium SA, Puck's Saline A, fetal calfserum, penicillin, streptomycin, trypsin, and Colcemid werepurchased from Grand Island Biological Co., Grand Island, N.V. Dithiothreitol and pyridoxal phosphate were obtained fromCalbiochem, La Jolla, Calif. S-Adenosyl-L-[carboxyl-14C]methionine and Omnifluor were from New England Nuclear, Boston,Mass. Dioxane, 3,3'-iminobispropylamine, and 3-isobutyl-1 -methylxanthine were purchased from Aldrich Chemical Co.,Milwaukee, Wis. Retinol (trans), retinal, retinoic acid, retinylacetate, phenylmethylsulfonylfluoride, cyclic AMP, ATP, histone F2b, aminoguanidine bicarbonate, L-ornithine hydrochloride, putrescine dichloride, spermidine trihydrochloride, and Sadenosyl-L-methionine (iodide salt) were purchased fromSigma Chemical Co., St. Louis, Mo. L-[1-14C]Ornithine hydrochloride and NCS were obtained from Amersham Corp., Arlington Heights, III.; [methy!-3H]thymidine, [3H]uridine, and [3H]IeucinewerefromICNChemicalandRadioisotopeDivision,Irvine,Calif.; and [‘y-32P]ATPwas from New England Nuclear.

RESULTS

Concentration-dependent Inhibition of ODC and Thymidine Incorporation by Retinol Addition to Mitotically Synchronized CHO Cells. In a previous report, we described thecoincident inhibition of ODC induction and cell cycle progression after the addition of retinol (120 @tM)to mitotically synchronized CHO cells (16, 17). Chart 1 depicts the concentration dependence of these 2 effects of vitamin A. ODC Varied in

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M. K. Haddox et al.

a similar concentration dependence to the inhibition of ODCinduction. G1-S-phase transition, as indicated by [3H]thymidineincorporation, occurred in the control cells at S to 6 hr aftermitotic release (Chart 1, inset). In cultures exposed to increasing concentrations of retinol, the rate of DNA synthesis wasprogressively decreased at all times tested from S to 13 hrafter mitosis. A 65% inhibition was observed with 80 @Mretinol,while in cultures exposed to 160 @sMthe cells failed to enter Sphase, as indicated by the lack of label incorporated at anytime tested. Cell counts conducted after 15 hr of incubationindicated that, while the control cells had doubled in number,those cultures plated in the presence of 120 or 160 @Mretinolremained at the original number plated. These results indicateda block of cell progression prior to entrance into S phase inaccordance with previous findings that exposure of asynchronous cells to the vitamin arrested the cells at some point in G1prior to an increase in cellular DNAcontent as assessedbyflow microfluorimetry (17). Furthermore, the concentration dependence for the inhibition of both ODC induction and S-phaseentryis similarto thatpreviouslyestablishedfor inhibitionofcell number increase in logarithmically growing asynchronousCHO cells. Although some variation in the maximal concentration of retinol required to totally inhibit ODC and S-phase entrywas seen in different experiments (i.e. , from 100 to 160 @sM),the parallelism between the effect on ODC and S-phase entrywas always apparent. The variation may relate to differencesin the composition of either the different lots of retinol or thedifferent batches of media used.

To ensure that the inhibitory effects of retinol on these growthevents were not due to a nonspecific detergent-like action ofthe vitamin, the ability of the cells to form colonies whenincubated for 10 days in the presence of 80 or 160 .tMretinolwas assessed. It was possible to obtain plating efficiencies inthis manner without removing the vitamin, because the cellsare apparently able to metabolize it to an inactive state. Wehave shown previously that CHO cells plated in the presenceof retinol, after a 24- to 40-hr period of incubation in which noincrease in cell number occurs, begin to increase in number atthe same rate as the control cultures (17). The escape frominhibition apparently results from metabolic removal, since asecond addition of retinol after 24 hr preserves the proliferationblock for at least an additional 30 hr. Therefore, the 10-dayincubation period allowed those cells which remained viableduring the initial period of growth inhibition by the vitamin torecover and grow to form visible colonies. The antiproliferativeeffect of the vitamin was not the result of a general toxicity,since the plating efficiency of the dioxane and the 80 @zMretinoltreated cells was the same as that of the control (80%), whilethat of the 160 LMretinol-treated cells was only decreased by24%.

Retinol Inhibition of SAMD Induction. Another index of a G1phase progression block would be the lack of an induction ofan S-phase-dependent enzyme. SAMD, the second enzyme ofthe polyamine biosynthetic pathway, increases in activity durhg 5 phase of the CHO cell cycle (1 3). As shown in Table 1,

the enzyme failed to increase in retinol-treated cells. Bothputrescine-dependent and spermidine-dependent SAMD haveapproximately doubled in the control cultures by mid-S phase(10 hr). The activity of both forms of the enzyme in the vitaminexposed cells, however, remained at the level expressed in theG1phase control cultures.

/ @6‘If

80

4OURS

60

0

S0

0 40

Chart 1. Concentration dependence of retinol inhibition of ODC induction and[3H]thymidine incorporation into mitotically synchronized CHO cells. Mitoticallysynchronized cells were plated in the presence of the indicated concentration ofretinol. The cells were harvested, extracted, and assayed for supernatant ODCactivity after 2 or 6 hr of incubation (0) or for [3Hjthymidine incorporated intoacid-insoluble material after 3 or 11 hr of incubation ØJ).The data presented inthe concentration dependence curve represent the average of 4 experiments.The increment in the retinol-treated cells, in either ODC activity after 6 hr ascompared to 2 hr of incubation, or thymidine incorporation as assayed at i 1 hras compared to 3 hr of incubation, is plotted as a percentage of the incrementduring the same time period in the control cells. Estimations of total cellular[3Hjthymidine in cell cultures washed extensively after the short-term pulseshowed comparable amounts in the control and in the 50 and 80 @.tMretinoltreated cells and 20 and 40% less amounts in the 120 and 160 @Mretinol-treatedcells. This effect of the vitamin to limit transport of the nucleoside into the cellsis not sufficient to account for the total inhibition of incorporation seen aftervitamin addition. Inset, time course from a representative experiment of ODCactivity (pmol/hr/ 106 cells) and thymidine incorporation (cpm x 10@/108 cells)in mitotically synchronized cells plated in the presence of 0.5% dioxane (•.U)or i 20 @Mretinol (0, ED;dThd, thymidine.

activity in the control cultures as a function of the CHO cellcycle (Chart 1, inset). The enzyme activity was maximal inmitotic cells and was rapidly turned over after mitotic exit toachieve a minimal expressed activity at 1 hr, which is maintamed during the first 2 hr of G1 phase. An induction of theenzyme was first apparent at Hr 3, after which a progressiveincrease occurred in a biphasic manner, with the first peak ofactivity at 6 hr, a time corresponding to an increase in[3H]thymidine incorporation. The inhibitory effect of retinol onthe cell cycle-dependent increase in ODC activity was concentration dependent. Enzyme activity was depressed in culturesallowed to progress in the presence of SO @Mretinol. A 60%inhibition was apparent after the addition of 80 jIM, while incultures exposed to 160 @Mthe ODC activity was completelyinhibited and remained at the basal 2-hr control cell level. Thiswas not due to a direct inhibitory effect of retinol on ODC sincein vitro addition of retinol (1 20 @tM)to cell supernatants had no

effect on the enzyme activity. There was no effect of the vitaminon the time course of ODC expression; the submaximal increases in ODC which did occur in response to lower concentrations of retinol displayed a biphasic nature similar to that ofthe control cultures.

The inhibition of cell cycle progression by retinol displayed

20

80[Retinol]x

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Retino! Inhibition of ODC and G, Progression

Effect of Retino! on RNA and Protein Synthesis in Mitoti-cally Synchronized CHO Cells. All of the effects of the vitaminon CHO cells examined thus far could result from a generaleffect to inhibit protein synthesis. Translation is required for theinduction of both the polyamine-biosynthetic enzymes and forthe enzymes involved in DNA replication. Furthermore, proteinsynthesis inhibitors have previously been shown to inhibit Gtprogression (20, 43, 63, 69). Therefore, the rate of proteinsynthesis was examined after retinol addition. Chart 2, left,shows the effects of 120 /¿Mretinol on protein synthesis duringsynchronous CHO cell cycle progression. The rate of [3H]-

leucine incorporation remained fairly constant during the initial8 hr of CHO cell progression, with a maximum occurringbetween Hr 4 and 6 at a rate 20% higher than demonstrated atthe other times tested. At all times in the presence of thevitamin, the rate of amino acid incorporation was inhibited 20to 30%. Therefore, it seems unlikely that the 90% inhibition ofODC expression observed at this vitamin concentration wasthe result of a general inhibition of protein synthesis.

In contrast, the effect of vitamin addition on RNA synthesiswas marked. During G, progression, the rate of [3H]uridine

incorporation increased 2-fold in the control cultures peakingat 6 hr (Chart 2, right). In the retinol-treated cells, there was no

Table 1Inhibition by retino/ of S-phase-dependent SAMD induction

Mitotically synchronized cells were incubated in the presence of 0.5% dioxane(control) or 120 JIM retinol. At the times indicated, cells were harvested andsoluble putrescine-dependent and spermidine-dependent SAMD activities weredetermined as described in 'Materials and Methods."

pmol/h/106 cells

Time after mitosis (hr)Putrescine-dependent SAMD

Spermidine-dependent SAMD

26

10

10 + retinol

200250530249

40308026

increase in RNA synthesis. The rate was 70% of the control at2 hr and remained constant so that uridine incorporation at 8hr was only 30% that of the control.

Ability of Retinol to Inhibit ODC and Block Cell CycleProgression Restricted to G, Phase. As shown in Chart 3, theeffect of retinol to block the cell cycle-dependent increase inODC activity was only apparent when the vitamin was addedprior to Hr 4 of the cell cycle. If retinol was added at the timeof mitotic exit or after 2 hr of Gìprogression, the increase inODC detectable in the control cells was inhibited. If retinol wasadded after 4 or 6 hr of progression, the vitamin was withouteffect, and ODC activity continued to increase as in the controlcells. This lack of effect was not due to a time dependence forvitamin transport or action, since only 2 hr were required forthe inhibitory effect to become apparent after the addition atHr 2, while even 4 hr after the addition at Hr 4 the control levelof activity was maintained. A similar Gi-phase dependence ofthe effect of retinol on cell cycle progression was noted in theprevious report (17). Addition of retinol at 0 or 2 hr blockedany increase in cell number, while cultures to which the vitaminwas added after 4 or 6 hr of progression doubled as did thecontrols.

G,-Phase-specific Expression of Cyclic AMP-dependentProtein Kinase Not Altered by Retinol. The transcription-dependent events required for ODC induction, which are sensitiveto enhancement by cyclic AMP, occur during the initial hoursof G, progression. Cytoplasmic cyclic AMP-dependent protein

kinase is activated during Gìtransition prior to the increase inODC activity (10). If exogenous cyclic AMP is added at the timeof kinase activation, the increase in ODC is potentiated (9).Wertz and Mueller (74) have recently shown that retinoic acidcan inhibit TPA-stimulated membrane events, suggesting thatthe action of the vitamin in CHO cells could be to disrupt theserum factor(s) membrane growth stimulus. Thus, the action ofthe vitamin could be to interfere with the transmission of thecyclic AMP-mediated trophic stimulus responsible for ODC

Chart 2. Retinol inhibition of leucine and uridine incorporation. Control (•)or 120 JIM retinol-treated (C)mitotically synchronized cells were incubated at varioustimes after mitosis for 30 min in the presence of ( 'H|-leucine or ['Hjuridine. The cells were washed and har

vested, and the incorporation of radiolabel into insolublematerial was determined. Estimations of total 'H precur

sor in the cells showed no effect of the vitamin onleucine uptake and a 30% inhibition of uridine uptake atthe 2 later times tested. Bars. S.D.

a.

Leucine

io

Uridine

0- —

8Hours after Mitosis

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Cyclic AMP-dependent protein kinase activation in retinol-treatedcellsMitotically

synchronized cells were incubated in the presence of 0.5%dioxane(untreatedcontrols) or 120 @Mretinol (retinol-treated) for 1, 2, or 3 hraftermitosis.

Cells were harvested and assayed for protein kinase activity intheabsence(activated) or presence (total) of cyclic AMP. The activity ratio istheratio

of “Pincorporated into histone in the absence and in the presence ofcyclicAMP.Activated

kinase (cpm/5 A i imin/ 106 cells) ct v tyratioTime

after Untreated Retinol- Untreated Retinolmitosis (h) controls treated controlstreated1

3550 3500 0.320.3225980 6i75 0.600.6232500 2425 0.25 0.24

been reversible by the addition of exogenous putrescine (26,37). However, putrescine addition (1 mM) to CHO cells had noeffect on preventing or relieving the retinol-induced cell cycleblock as measured by either [3H]thymidine incorporation orultimate cell doubling. Measurements of intracellular polyamines showed putrescine not to be a limiting factor. As shownin Table 3, the diamine level was actually higher in the vitamintreated cells after 4 or 6 hr of progression. Although themechanism in these cells for such an increase in putrescine inthe absence of any induction of ODC is unknown, a similarphenomenon has been shown to occur in several other growingtissues (23, 25, 64). In those studies, the biosynthetic pathwaywas shown to be reversed, and the increased putrescine wassynthesized from spermidine and spermine. Similarly, the concentrations of the polyamines were significantly decreased inthe retinol-treated cells. No significant amounts of any of thepolyamines were detectable in the growth media from eitherthe control or retinol-treated cells.

Effects of Other Naturally Occurring Retinolds on GrowthParameters. The efficacy of the other naturally occurring retinoids to affect asynchronous CHO cell logarithmic growth wasexamined. Chart 5 shows that retinal was the most potentretinoid, completely inhibiting cell doubling at a concentrationof 25 @LM.Retinol was the second most effective, while 200@LMretinyl acetate and 1000 @tMretinoid acid were required tofully inhibit growth. Although retinal was the most potent, it wasnot the main object of study in these experiments because ofits paradoxical effect on CHO cell growth parameters. Asshown in Table 4, very low doses of retinal (1 to 5 @M)oftenstimulated CHO cell proliferation as measured by an increasein the extentof ODCinductionthroughoutG1,by a stimulationof the rate of [3H]thymidine incorporation, and by an enhancement of cell growth rate. Higher doses of retinal (greater thanS @tM)inhibited all aspects of growth in a manner similar to theeffects of retinol previously discussed. Similar contrasting effects of retinoids on DNA synthesis have been demonstrated inlymphocytes (32) and may reflect different functional receptorsfor different forms of the vitamin (49).

Pardee (SO) has suggested that restriction points occurduring the cell cycle which involve events on which all othersthereafter depend. The present study provides further supportto the hypothesis that vitamin A acts to limit CHO cell growth atsuch a target event in G1phase (16, 17). (a) Cells incubated inthe presence of retinol do not enter S phase as shown by flow

6 8

DISCUSSION

M. K. Haddox et a!.

Table 2

200'

ISO'

00'

50'

2 4Hours offer Mitosis

U)

B,

I,,@@.0

—

,__._o—— @-—.- —..

,.

Chart 3. Retinol inhibition of ODCinduction as a function of cell cycle position.Retinol (i 20 /LM)was added to mitotically synchronized cells at 0 (0), 2 (0). 4(L:i),or 6 (0) hr after mitotic exit. At the times indicated, control (•)and retinoltreated cells were harvested and assayed for ODC activity.

induction. Table 2, however, shows that retinol addition did notaffect the increase in cyclic AMP-dependent protein kinaseactivitywhichoccurred2 hr aftermitoticexit.Theratioof activeto total kinase increased 2-fold in both control and retinoltreated cultures.

Recovery of ODC after Retinol Removal Is Blocked byActinomycin D. To determine if transcription of mRNA for ODCwas occurring in the presence of retinol, cells were exposed tothe vitamin for the first 4 hr of G1 progression, and then themonolayers were washed and supplemented with fresh mediacontaining additional retinol, actinomycin D, or dioxane (control). Cells resupplemented with retinol retained the inhibitionof ODC expression (Chart 4A). Those cells removed from thepresence of the vitamin and placed in control media displayedincreased ODC activity within 30 mm which continued to progressively increase, lagging behind the activity of control cellsby about 2 hr. This recovery paralleled and preceded therecovery of S-phase transition after vitamin removal (Chart 4B).However, cultures to which actinomycin D was added afterremoval of the vitamin showed no increase in enzyme activitywhen tested from 30 mm to 4 hr after media replacement.

Increased Putrescine Concentrations In Retinol-blockedCells. Several studies examining the effects of addition ofdirect inhibitors of ODC expression (i.e. , substrate analogs) tocells in culture have shown concomitant inhibition of cell proliferation (6, 26, 37, 38, 68). In some cases, this effect has

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Polyamine concentrations in retinol-treatedcellsMitoticallysynchronized cells were incubated in the presence of 0.5% dioxane (control) or 120 .tM

retinol. At the time indicated, the cells were harvested, and TCA-extracted for analysis of polyamineconcentrations, which are expressed as pmol/1 06cells.Time

Putrescine Spermidine Spermineafter

mitosis(hr) Control Retinol Control Retinol ControlRetinol2

0.71 ±O.03a 0.64 ±0.204 0.71 ±0.08 0.83 ±0.076 0.78 ±0.0i i.i8 ±0.02 3.64 ±0.2i 2.96 ±0.33 2.44 ±0.08 1.82 ±0.28a

Mean ± S.D.

Retinol Inhibition of ODC and G1Progression

400

3O0@

20O@

00'

II

II

II

II

II

I

A ODC B dThd Incorporation

20

@0a)00a0UC

in0

I—r@:@,Io

i:

E0.0

‘I,

a,Uto0

‘C

0Ea

I

II

II

-@--@- p

I I I

2 4 6 8 4 6 8 0 2

HOURS AFTER MITOSIS

Chart4. InhibitIonof recoveryof ODCactivityafterretinolremovalbyactinomycin0. Mitoticallysynchronizedcellswereplatedin thepresenceof 0.5%dioxane(control) (•)or 120 @Mretinol (0). After 4 hr of incubation, the monolayers were washed and resupplemented with prewarmed, pregassed fresh media containing0.5% dloxane, (•,ED. 120 @.iretinol (0) or 0.5% dioxane and 0.04 @gactinomycin 0 per ml (a). At the times indicated after mitosis, cells were harvested andassayed toi' ODC activity (A) or incubated with [‘H)thymidineto assess DNA synthetic rate (B).

Table 3

microfluonmetric analysis of the G1-like DNA distribution inasynchronous cells and by the failure of synchronous cells toincorporate thymidine into DNA or to induce the S-phasedependent enzyme, SAMD. (b) The removal of retinol fromsynchronous cells incubated in its presence during G1 phaseresults In a 2- to 3-hr lag before the initiation of DNA synthesisas assessed by thymidine incorporation kinetics. This lag cannot be solely accounted for by a time requirement to clear thevitamin from the cell, since the synthesis of ODC resumeswithin 30 mm after removal. (C)Retinol is effective in inhibitingcell doubling only when added prior to the midpoint of G1phase. These results suggest that the site of action of thevitamin, representing a restriction point for cell cycle progression, occurs several hr prior to the onset of S phase.

The effect of the vitamin to inhibit G1-dependent ODC induction displays characteristics very similar to its effects on theproposed G1 restriction point. (a) The concentration-dependence of retinol inhibition of the increase in ODC is similar tothat for the inhibition of thymidine incorporation and cell doubling. (b) Addition of the vitamin is only effective to inhibit the

rise in ODC activity when added during the first 2- to 3 hr of G1progression. (c) The recovery of ODC activity after retinolremoval precedes the entry of the cells into S phase, suggesting induction of the enzyme constitutes part of the delayed G1progression events whose requirement for occurrence imposesthe lag in S-phase entry. Vitamin A action to limit growth isbelieved to represent a physiological function in certain celltypes(65).Forexample,increasedepithelialmitoticindicescan be shown in states of retinoid dietary deficiency (18, 19).It is consonant, therefore, that the block demonstrated in thisreport occurs in G1 prior to ODC expression. The control ofcell reproduction in vivo usually occurs by arrest at some sitein this cell cycle phase (52). Nongrowing cells contain virtuallyno ODC, and the induction of the enzyme is a ubiquitous initialresponse to growth stimulants (57). Thus, ODC induction represents a ‘‘landmark'‘event by which the extent of G1progression can be charted (51) and may represent the regulatoryevent on which the antiproliferative action of vitamin A in CHOcells is imposed.

The mechanism by which vitamin A acts to inhibit ODC

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Mitotically synchronized cells (5 x i 0@)were plated in the presence of0.5%dioxane(control), 5@ retinal, or 25 @Mretinal. Determinations were madeforODC

activity, [‘HJthymidineincorporation, and cell number after 6, 9, and 24hrofincubation, respectively.[‘H]Thymi

dine incorpoODC activity ration (cpm Cellnumber(pmolihriio6

x 103/i06 at 24hr(xcells)cells)106)Control

350 61.55@Mretinal 600 20225

@t@iretinal i 00 1 08

M. K. Haddox et a!.

specifically limit translation of the mRNA for the enzyme. SinceODC has an extremely short half-life of 11 to 20 mm (22, 31,60), message must be continually translated during cell cycleprogression to maintain the induction of increasing enzymelevels. An inhibitory effect of retinol on ODC synthesis, therefore, would be apparent after vitamin addition at any time. (C)The addition of actinomycin D to cells at the time of retinolremoval completely blocked the increase in enzy@meactivitywhich was detectable in the control cells within 30 mm. If themRNA for ODC were being transcribed during the time ofretinol exposure and the inhibition resulted from vitamin actionat another locus, some increase in enzyme activity might beapparent upon release in the presence of the actinomycin Dfrom the translation of this preexisting mRNA. This protocol, ofcourse, presumes that any ODC mRNA synthesized during theretinol incubation would be stable enough to survive and betranslated during the 30 mm after retinol removal and actinomycin D addition. Although there is no information available onthe t112of ODC mRNA, this type of experimental protocol hasbeen used successfully to show in a G1-phase temperaturesensitive mutant of Chinese hamster cells that the translationand not the transcription of ODC mRNA was blocked at thenonpermissive temperature (34). In those studies, an increasein ODC was observed 2 hr after shift to the permissive temperature in the presence of a transcriptional inhibitor. In contrast,in thepresentstudythe increaseinenzymeactivityuponretinolremoval was totally blocked by the transcription inhibitor. Itthus appears that no mRNA for ODC is being transcribed in thepresence of the vitamin. (d) The G1 time dependence of theretinol action in limiting ODC induction coincides with the timeof G1progression previously established for the events constituting ODCgeneactivation(9).

Retinoids have been postulated to act like steroid hormones(2, 7, 35), and under certain circumstances the vitamin or Itsbinding protein has been localized to the cell nucleus (53, 61,62). Therefore, the vitamin may act to affect transcriptiondirectly without affecting cytoplasmic events. However, severalother sites of vitamin A action have been reported in other celltypes which could limit nuclear function. For example, Wertzand Mueller (74) have shown that retinol inhibits TPA acceleration of the incorporation of choline into lymphocyte phospholipids and have postulated that such an event may representthe site of action of vitamin A to inhibit subsequent DNAreplication. The inhibition of choline incorporation, however, isapparently not involved in the retinol inhibition of TPA inductionof ODC in lymphocytes, since preincubation is required toinhibit ODC (32) but not to inhibit phospholipid metabolism(74). This suggests there are at least 2 sites at which retinoidscan act to inhibit growth-related events, which may be differentially expressed in different cell types. ODC induction in TPAstimulated epidermis has been shown to be dependent onprostaglandin synthesis (72), another event catalyzed by amembrane enzyme, which has been suggested as a possiblesite of retinoid action (71). However, it appears unlikely thatthis is the mechanism in CHO cells, since the addition of knowninhibitors of prostaglandin synthesis (indomethacin, aspirin,and 5,8, 11,14-eicosatetryenoic acid) have been found to haveno effect on CHO growth parameters.5

A striking consequence of the retinol block of cell cycle

5 M. K. Haddox and 0. H. Russell, unpublished observations.

0

0

0z-J-J0(-)

Table 4Concentration-dependent stimulation and inhibition by retinal of cell growth

parameters

HOURSChart 5. Concentration-dependent inhibition of asynchronous CHO cell

growth by various retinoids. Asynchronous logarithmically growing CHO cellswere plated in the presence of the indicated concentrations of the variousretinoids or in 0.5% dioxane (control) (•).At the times indicated, cell sampleswere collected by scraping with a rubber policeman and counted by a Coulterelectronic particle counter.

appears to involve a block of the transcriptional event requiredfor new biosynthesis of the enzyme. (a) There is no effect ofthe vitamin on inhibiting the intracellular activation of cyclicAMP-dependent protein kinase, the presumed cytoplasmic signaIling event which promotes induction of the enzyme (30, 57).(b) Retinol addition at concentrations which inhibit ODC synthesis 90% only produces an approximately 20% inhibition ofgeneral protein synthesis. This inhibition could reflect the specific inhibitionof the synthesisof certainproteinssuchasODC.However, the cell cycle dependence of the vitamin action ininhibiting ODC is an indication that the effect of retinol is not to

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Retino! Inhibition of ODC and G1 Progression

promotion is unclear, Boutwell et a!. have pointed out thesimilarity between the intracellular events after TPA stimulusand those occurring during the normal growth process. ODCinduction is such a common event. We show here that ODCinduction is a common target site for retinoid inhibition of bothTPA-promoted epidermis and CHO cell cycle. Therefore, thosecells which possess a receptor for retinoid antiproliferativeaction may be inhibited in the sequence of growth events at acommon point involving polyamine biosynthesis.

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17. Haddox, M. K., and Russell, 0. H. Cell cycle-specific locus of vitamin Ainhibition of growth. Cancer Res., 39: 2476—2480,1979.

18. Harris, C. C., Silverman, T., Smith, J. M., Jackson, F., and Boren, H. B.Proliferation of tracheal epithelial cells in normal and vitamin A-deficientSyrian golden hamsters. J. NatI. Cancer Inst., 51: i 059—1062, 1973.

19. Harris, C. C.. Sporn, M. B., Kaufman, D. G., Smith, J. M., Jackson, F. E.,and Saffiotti, U. Histogenesis of squamous metaplasia in the hamster tracheal epithelium caused by vitamin A deficiency or benzo(a)pyrene-ferricoxide. J. NatI. Cancer Inst., 48: 743—761, i 972.

20. Harris, H. The initiation of deoxynucleic acid synthesis in the connectivetissue cell, with someobservations on the function of the nucleolus. Biochem.J., 72: 54—60,1959.

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progression is the inhibition of uridine incorporation into RNA.The G1-phase increase in RNA synthetic rate has been shownin severalcell types to reflect an increasedsynthesisof rRNArather than heterogeneous RNA (1, 29, 42, 54, 70). Inhibitorstudies in which low concentrations of actinomycin D wereused to specifically block rRNA synthesis have shown that thisG1-phase event is essential for entry into S phase (1, 11, 12).Polyamine-biosynthetic events have been shown in many typesof growth systems to be intricately tied to rRNA synthesis (8,56). Furthermore, intact nucleolar function has been shown tobe essential to ODC expression in at least one cell type, theanucleolar mutants of Xenopus laevis (55). The present demonstration, therefore, of the correlation between the block inODC and the block in rRNA synthesis produced by retinol addsto the list of examples of this coupling. Since putrescine concentrations were not found to be limiting during retinol exposure, the putative interaction between the 2 systems demonstrated here may involve the direct action of ODC on RNApolymerase I function (39, 40).

The differences that exist between our findings and those inother cell types in which ODC induction is inhibited by retinoidsprobably reflect differences in the nature of the systems beingstudied. In epidermis, the inhibitory effect of retinoic acid onTPA-promoted epidermal papilloma formation is associatedwith a similar inhibition of TPA-induced epidermal ODC (71,73). Further, the efficacy of different natural and syntheticretinoid analogs to inhibit papilloma formation directly paralleled their inhibition of ODC induction (73). However, in theskin, retinoids had no effect on the TPA-stimulated increase inSAMD, while in CHO cells, the vitamin inhibited the increase inthe enzyme. Possibly the SAMD increase in skin is the directresult of a TPA-promoted event independent of the retinoidsensitive event responsible for ODC induction, while in CHOcells the SAMD increase is a function of cell cycle progressioninto S phase. Since the retinol prevents entrance into S phaseby acting at a point in G1, the lack of SAMD increase occurs asan indirect result of vitamin action. The relative efficacy of theretinoids to inhibit growth in CHO cells and epidermis alsodiffers. This may reflect variations in the cellular distribution ofthe different retinoid-binding proteins (48, 49, 62) or in themetabolism of the vitamin. The effect of retinoic acid, the mostpotent inhibitorof TPAinductionof ODCin both skin (71) andlymphocytes (32), displays a broad-sloped type of concentration dependence, which suggests that it may not be the activeagent and that metabolic activation is required. In contrast, theconcentration dependence in CHO cells is fairly precipitous,and therefore retinol may be the actual effector molecule.

Pardee (50) has speculated that the restriction points essential to cell cycle progression will represent sites of fundamentaldifference in regulation between normal and malignant cells.Therefore, it is interesting to note, in conjunction with theconsideration that ODC induction may represent a G1 restriction point for retinol action, the accumulating examples ofaberrant, less restricted control of ODC expression during cellcycle progression of transformed cells. In general, the characteristics of polyamine biosynthesis in malignant cells reflecta diminished response to normal control mechanisms (3, 14,I 5, 27, 36, 45). Such a loss of regulation of ODC expression

has been postulated by Boutwell and O'Brien (44, 46, 47) tobe essential to the TPA-promoted transformation process inepidermis, which retinoids block. Although the mechanism of

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1979;39:4930-4938. Cancer Res   Mari K. Haddox, Karen F. Frasier Scott and Diane Haddock Russell  Progression in Chinese Hamster Ovary Cells

1Retinol Inhibition of Ornithine Decarboxylase Induction and G

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