(CANCERRESEARCH36,3766-3770,October1976]

SUMMARY

The induction of mouse epidermal omnithine decamboxylase, 1of the earliest and largest phenotypic changesfollowing treatment of mouse skin with the tumor-promotingagent, 12-O-tetnadecanoyl-phonbol-1 3-acetate, can be inhibited by prior administration of colchicine. Maximal inhibition of this enzyme induction was observed when colchicine was injected i.p. 90 or 120 mm before promoter treatment, although time intervals up to 20 hr between colchicine and promoter treatment were effective. The effect ofcolchicine was dose dependent, with a dose as low as 25nmoles/mouse causing an inhibition of 35%. Other microtubule-disnupting agents, vinblastine, vincnistine , and Colcemid , had a similar effect on omnithine decarboxylase activity. However, f3,'y-lumicolchicine, a photochemical denyative of colchicine with no antimitotic or microtubule-disrupting ability, and cytochalasin B, an inhibitor of microfilament-dependent processes, had no effect. N6,021-dibutyryl3' ,5'-cyclic adenosine monophosphate, when administeredjust before colchicine, blocked the inhibitory action of colchicine. The results of these studies suggest that colchicine-sensitive structures, most likely containing microtubules, may be mediating elements between the binding oftumor promoters, perhaps to specific cell surface receptons,and the subsequent induction of omnithinedecanboxylase.

INTRODUCTION

Tumors can be efficiently produced in mouse skin bytreatment with a single, topical application of a subcarcinogenic dose of a compound such as dimethylbenz[a]anthracene followed by frequent, repetitive applications of a promoting agent such as croton oil on its active component,TPA3 (1, 3). This 2-stage system, termed initiation andpromotion, has been utilized by us as a model for determining, in intact animals, which biochemical events might

1 This work was supported in part by Grant BC-14 from the American

CancerSocietyand GrantsCA-07175and CA-05002from the NIH.2 Present address: Wistar Institute, 36th and Spruce Streets, Philadelphia,

Pa. 19104.3 The abbreviations used are: TPA, 1 2-O-tetradecanoyl-phorbol-1 3-ace

tate; cyclic AMP, cyclic adenosine 3':5'-monophosphate; dibutyryl cyclicAMP, N',02'-dibutyryl cyclic adenosine 3':5'-monophosphate.

Received April 22, 1976; accepted July 6, 1976.

be essential for the neoplastic process in mouse skin. Thus,treatment of mouse skin with tumor-promoting agentscausesa rapid, transient induction of onnithinedecarboxylase (L-onnithinecamboxy-lyase,EC 4.1.1.17), the 1st and,probably, rate-limiting step for polyamine biosynthesis inanimal cells (10, 11). The 2nd enzyme in this biosyntheticpathway, S-adenosylmethionine decamboxylase (EC4.1 .1 .50), is also induced by promoting agents but, in addition, is induced by compounds that cause epidenmal hypenplasia but which promote weakly or not at all. It was concluded (11) that the induction of omnithine decarboxylasemay be a unique and specific consequence of treatment ofmouse epidermis with promoting agents. In contrast, theinduction of S-adenosylmethionine decanboxylase comelated with the ability of a given compound, whether it promotes effectively on not, to produce a hyperplastic mesponse.

Several studies suggest that an interaction with theplasma membrane is an essential 1st Step @flproducing themany biochemical effects of promoters. These include thelipophilic nature of the strongly promoting phorbol esterseries (7); the stimulation of 32P@incorporation into membrane phospholipids, both in mouse skin (14) and in cellcultures (17); and the ability of promoters to block theisoprotenenol-stimulated accumulation of epidemmal cyclicAMP through an inactivation of the membrane-bound f3-admenergic receptor (6).

The above evidence suggested to us that an interactionwith the cell membrane may be required for the subsequentpromoter-induced biochemical changes. The question memains how the binding of a promoter at the cell surfacetriggers the known temporal sequence of events in mouseepidermis: increased phospholipid synthesis (14); the induction of omnithine decarboxylase (10); increased incorporation of precursors into RNA, protein, and DNA (2), and astimulation of cell division (13). In recent years, the involvementof microtubules onmicrotubule-contamningassembliesin cellular morphological changes (8), agglutinability ofcells by lectins (4), cell surface receptor mobility (18), andmitogenesis in cells in culture (19) have been postulated. Ithas been suggested that these structures may be importantfor the transmission of signals generated by the binding ofvarious ligands to cell surface receptors into the interior ofthe cell (4, 19). In the present report we have asked whethermicrotubules play a role in the induction of omnithine decarboxylase in mouse epidermis by the potent tumor promoter,TPA. Colchicine, which is known to interfere with microtubule function by binding to its protein subunit and prevent

3766 CANCER RESEARCH VOL. 36

The Effect of Colchicine on the Induction of OrnithineDecarboxylase by I 2-O-Tetradecanoyl-phorbol-1 1

T. G. O'Brien,2R. C. Simsiman,and R. K. BoutwellMcArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706

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Table1The

effect of colchicine pretreatment on the induction of mouse epidermalornithiriedecarboxylasebyTPAGroups

of 4 mice received injections i.p. of 100 @gcolchicine dissolved in 0.2 ml0.9%NaCIsolution or 0.9% NaCIsolution alone (“primary treatment'‘)and at varioustimesthereafter

were treated topically with 17 nmoles TPA dissolved in 0.2 ml acetoneoracetonealone(“secondarytreatment―).“Interval―refersto thetimebetweenprimaryandsecondary

treatments.All mice were killed 4 hr after the secondarytreatment,epidenmalextractswereprepared,andomnithinedecamboxylaseactivitywasdeterminedintriplicateas

described in “Materialsand Methods.―The data represent a combination of3experimentsusingthe sameTPAsolutionand the sameage-groupof mice.TheTPAcontrol

value is the mean ±S.E. of control enzymeactivity from each of the 3 expeniments,while the othervaluesrepresentthe meanof triplicateenzymedeterminationsfrom

a single group from 1 of the experiments. All time intervalswere repeatedatleastonce,andthedifferencesbetweenthe controlandexperimentalgroupsweresimilartothosepresentedin the table.Interval

Secondary Ornithine decar-InhibitionPrimarytreatment (hn) treatment boxylase activity―(%)0.9%

NaCIsolution 2.0 Acetone0.010.9%NaCIsolution 2.0 TPA 2.06 ±0.09Colchicine

0.5 TPA 1 .3335Colchicine1 .0 TPA 1.2639Colchicine1 .5 TPA 0.5076Colchicine2.0 TPA 0.4877Colchicine

12.0 TPA 0.3885Colchicine20.0 TPA 1.00 51

Coichicine and Ornithine Decarboxy!ase Induction

ing their aggregation (20),was used as a meansof answering this question. The induction of omnithine decarboxylasewas chosen for study because it is 1 of the earliest andlargest biochemical effects of promoter treatment, becausenew RNA and protein synthesis is required (hence nuclearcytoplasmic activity is involved), and because there is evidence that the induction of this enzyme is essential to thetumor-promoting ability of TPA (11). It would also be ofgeneral interest to detenmine in intact animals whether colchicine-sensitive processes might be important in the regulation of gene expression following administration of exogenous chemicals.

MATERIALS AND METHODS

Female Charles River CD-i mice were obtained fromCharles River Breeding Laboratories, Wilmington, Mass.,and were used at 8 to 9 weeks of age. The dorsal hair wasremoved with surgical clippers 1 to 2 days before an expemiment, and only mice showing no hair regnowthwere used.All animals received water and Wayne Breeder Blox (Allied

Mills, Chicago, Ill.) ad !ibitum throughout the experimentalperiod. Mice were killed between 1 and 3 p.m. to avoidvariations due to cimcadianrhythms.

Chemicals applied to mouse skin were dissolved in acetone and delivered in a volume of 0.2 ml. Compounds thatwere injected were dissolved in 0.9% NaCI solution andadministered i.p. in a volume of 0.2 ml. TPA was purchasedfrom Consolidated Midland Corp., Brewster, N. V. Colchicine was obtained from Nutritional Biochemicals, Cleveland, Ohio. Colcemid (demecolchicine) was purchasedfrom Calbiochem, Irvine, Calif., and vincristine sulfate was agenerous gift of Eli Lilly Co., Indianapolis, Ind. Vinblastinesulfate, dibutyryl cyclic AMP, and hydnoxyurea were ob

tamed from Sigma Chemical Co. , St. Louis, Mo. Cytochalasin B was obtained from Imperial Chemical Industries,Ltd ., Wilmslow, Cheshire, England . /3,y-Lumicolchicinewas prepared from colchicine by the method of Wilson andFniedkin(21).

DL-[1-14C]Omnithinewas obtained from Amensham/SearleCorp., Arlington Heights, III. (specific activity, 43 mCi/mmole).

In order to obtain sufficient epidermal material, groups of4 to 5 mice constituted each treatment group. Followingtreatment, the mice were killed by cervical fracture, theskins were excised, and the epidermis was isolated free ofdermal material as described previously (10). Following homogenization and centnifugation at 30,000 x g for 30 mm,the epidermal extracts were assayed immediately for omnithine decamboxylase activity.

The onnithine decanboxylase activity was measured by therelease of ‘4CO2from DL-[1-14C]omnithineas described (10).Each incubation flask contained, in a final volume of 2.0 ml,125 @molessodium phosphate, pH 7.2; 0.4 @molepymidoxalphosphate; 1 @moIedithiothmeitol; 0.2 @moleL-ornithine(approximately 0.5 @CiDL-[1-14C]onnithine); and 0.1 to 0.5ml epidermal extract. Enzyme specific activities were expressedas nmoles CO2 evolvedin30 mm/mg solubleprotein. The protein concentration of the epidemmal extractswas measured by the method of Lowry et a!. (9).

RESULTS

The effectof colchicineon the inductionof omnithinedecarboxylase by TPA was determined by injecting 100 @gof colchicine/mouse, i.p., at various times beforeTPAtreatment and measuring onnithine decarboxylase activity 4 hrafter TPA. Table 1 gives the combined results of 3 expemi

a Onnithine decanboxylase activity is expressed as nmoles of CO2 evolved in 30 mm/mgprotein.

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Colchicine dose(pg/mouse)Onnithine

decarboxylaseactivity (nmoles CO2in 30

mm/mg protein)Inhibition(%)01.70101.1035500.63631000.3977

I.

>

,: I00@-U@4

@ 80.@

-4g8

@LL

Chart 1. The effect of various compounds on the induction of mouseepidermal ornithine decarboxylase by TPA. Groups of 4 mice received injections i.p. of 0.2 ml 0.9% NaCI solution or 250 nmoles of 1 of the testcompoundsdissolvedin 0.9%NaClsolution (exceptfor the cytochalasinBexperiment in which dimethyl sulfoxide:H2O (1:9 by volume) was the vehicle.Two hr later, eachanimal receiveda topical applicationof 17 nmolesTPA,and all mice were killed 4 hr after TPA. Epidermal extracts were prepared andornithine decarboxylase activity determined in triplicate as described in“Materialsand Methods.―Eachcompoundwastestedin a separateexperiment, and the effect of each is expressed as a percentage of its control(vehicle+ TPAtreatment).Eachcompoundwas testedat least twice, withsimilar results. The mean ±SE. of the control groups was 2.44 ±0.01.

ase, whereas f3,y-lumicolchicine, a photochemical denivative of colchicine, which does not inhibit mitosis and doesnot bind to microtubule subunits (22), and cytochalasin B,an inhibitor of microfilament-dependent processes, had noeffect on the enzymeinduction. In addition, /3,'y-lumicolchicine does not block the inhibitory effect of colchicine whenadministered simultaneously with and in equimolaramounts to colchicine. In order to determine whetherblocking epidenmal cells at another stage of the cell cyclecould similarly inhibit the induction of this enzyme by TPA,micereceivedinjectionsofa largedose ofhydroxyurea(500mg/kg i.p., 2 hr before TPA), a potent inhibitor of epidenmalsemiconservative DNA synthesis (16). However, this treatment, which blocks the entry of cells into S phase, had noeffect on the TPA-induced enzyme activity. The onnithinedecarboxylase specific activity of the control group was1.96nmoles ofCO2 in30 mm/mg ofprotein,whilethatofthehydnoxyunea-tneatedgroup was 1.86.

The ability of cyclic AMP to promote the assembly ofmicrotubules in cells in culture, which may be the basis forthe morphological changes induced in cells by exogenouscyclic AMP on its dibutymyl derivative (8), suggested to usthat if a similar enhancement of microtubule assemblycould be accomplished in mouse epidermis in vivo, theinhibitory effect of colchicine on the omnithine decanboxylase induction might be reduced or prevented. This wasfound to be true. Administration of dibutymyl cyclic AMP 15mm before colchicine treatment prevented the inhibition ofthe enzyme induction; the induced activity was not signifi

-1

T. G. O'Brien et a!.

ments using the same batch of TPA and colchicine (different batches of TPA tended to vary in the magnitude of theenzyme induction produced, probably due to slight vamiations in the actual dose applied). Near-maximal inhibitionwas reached when the drug was injected 90 or 120 mmbefore TPA, and the extent of inhibition did not appreciablychange even when colchicine was injected 12 or 20 hrbefore promoter treatment. This general pattern has beenobserved consistently in subsequent experiments. Sinceour main interest was in the mechanism of this inhibition,and in order to avoid any possible long-term metaboliceffectsof colchicinetreatment,colchicineor othertestcompounds were administered 2 hr before promoter applicationunlessotherwisestated.Thiseffectofcolchicinetreatmentishighlyreproducible;

in 5 separate experiments performed with the same stock ofTPA, the omnithine decamboxylase activity in the colchicinetreated groups (100 @tg/mouse,i.p., 2 hr before TPA) was0.98 ±0.08 nmole CO2in 30 mm/mg protein (mean ±S.E.),while the control groups (0.9% NaCI solution 2 hr beforeTPA) had a mean specific activity of 2.58 ±0.10 nmoles CO2in 30 mm/mg protein, an average inhibition of 62%. In otherexperiments it has been found that the drug is ineffectivewhen injected at any time after promoter treatment or ifapplied topically at any time relative to TPA treatment. Colchicine did not inhibit omnithine decamboxylase activitywhen added to incubation flasks at a concentration of 10@M and also did not cause the formation of an inhibitor of thisenzyme, since mixing extracts from colchicine-treated andcontrol animals always gave additive specific activities.

The effect of various doses of colchicine, administered 2hr beforeTPA, on the subsequentinductionof omnithinedecarboxylase is illustrated in Table 2. The inhibition isdose dependent, with a dose as low as 10 pg/mouse (25nmoles) still capable of a 35% inhibition.

Having established that colchicine inhibits the inductionof this enzyme, it was of interest to determine whether thiseffectwas a consequence of the abilityof colchicinetodisrupt microtubules (20). Chart 1 demonstrates that yinblastine, vincnistine, and Colcemid, each of which possesses antimitotic and microtubule-disrupting activity, areeffective inhibitors of the induction of omnithine decarboxyl

Table 2

Theeffect of dose of colchicine on the induction of mouseepidermal ornithine decarboxylaseby TPA

Groups of 4 mice received injections i.p. of 0.2 ml 0.9% NaCIsolution or the indicated amount of colchicine dissolved in 0.9%NaCIsolution. Two hr later, all mice receiveda topical applicationof 17 nmoles TPA in 0.2 ml acetone and were killed 4 hr later.Epidermal extracts were prepared, and omnithinedecarboxylaseactivity was determined as described in “Materialsand Methods.―Each value is the mean of triplicate determinations of enzymeactivity (variation <10%). The experiment was done 3 times withsimilar results.

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TreatmentOmnithine

decamboxylaseactivity (nmoles CO2 in 30

mm/mgprotein)0.9%NaClsolution + 0.9%NaCIso 2.69 ±0.16lution

+TPA0.9%NaCI solution + colchicine +1 .10 ±0.09°TPADibutyryl

cyclic AMP + colchicine +2.40 ±0.30―TPADibutynyl

cyclic AMP + 0.9% NaCl1 .96 ±0.37―solution+TPADibutyryl

cyclic AMP + acetone0.02 ±0.01

Co!chicine and Ornithine Decarboxy!ase Induction

Table 3Theeffect of dibutyry! cyclic AMPand colchicine on the induction

of mouseepiderma!ornithinedecarboxy!aseby TPAGroups of 4 mice each received injections i.p. of 0.2 ml 0.9%

NaCIsolution on1.0mgdibutyryl cyclic AMPdissolvedin 0.9%NaCIsolution. Fifteen mm later, 0.2 ml 0.9% NaCIsolution on 100 @gcolchicine dissolved in 0.9% NaCIsolution were injected i.p. , followed 2 hr later by a topical application of 0.2 ml acetone or 17nmoles TPA dissolved in acetone. The mice were killed 4 hr afterTPA, and epidermal extracts were prepared, and omnithine decarboxylase was determined as described in “Materialsand Methods.―Results are expressed as the mean ±SE. of 3 separate expeniments.

this inhibitory effect; near maximal inhibition occurs whenthe interval between the 2 treatments is 2 hr, but the effect isonly slightly diminished when the interval is extended to 20hr. Intervals shorten than 2 hr are progressively less effective, while colchicine treatment concurrent with or afterTPA application is ineffective. In vitro studies on the bindingcharacteristics of colchicine to tubulin, the microtubulesubunit protein, have shown that the rate of colchicinetubulin complex formation is slow, but, once formed, thecomplex is stable (20). It is precisely these characteristicsthat may account for the relatively long pretreatment timerequired and the long-lasting nature of the colchicine effecton this enzyme induction in vivo.

The ability of colchicine and related drugs to modulatethe effect of promoters on gene expression is most likelydue to the binding of these drugs to epidermal microtubularsubunits, thereby interfering, at least partially, with microtubule function. Microtubules are ubiquitous cellular components, and their subunits are known to bind colchicineand other antimitotic drugs with high affinity (20). A recentreport has described the rapid appearance of cytoplasmicmicrotubules in cells exposed only briefly to TPA (5), suggesting that agents capable of preventing the assembly ofmicrotubules (e.g., colchicine) might block the cellularchanges caused by TPA. In addition to colchicine, othermicrotubule-disnupting agents (vinblastine, vincnistine , andColcemid) inhibit omnithine decarboxylase induction due toTPA, whereas a structural analog of colchicine which doesnot possess antimitotic activity (f3,-y-lumicolchicine) was ineffective. Cytochalasin B, which interferes with microfilament function, also was ineffective.

An accumulation of mitotic cells seems unlikely to beresponsible for the colchicine effect. The epidermal mitoticindex is not increased 4 hr after TPA treatment (12), and it isdoubtful that the number of cells blocked in mitosis at 6 hrafter colchicine treatment (the time of our enzyme determinations) could account for the large reduction in ornithinedecarboxylase activity, even assuming these cells were totally unresponsive to TPA. Hydmoxyurea treatment, whichblocks epidermal cells at another point in the cell cycle (theentry to S phase) had no effect on TPA-induced omnithinedecarboxylase activity.

The mechanism whereby dibutyryl cyclic AMP antagonizesthe inhibitory effect of colchicine is unknown, but theability of dibutyryl cyclic AMP to cause morphologicalchanges in cells in culture that can be prevented by Colcemid (8)indicatesthata high intracellularcyclicAMP levelmay promote the assembly of microtubules (which are presumably responsible for the morphological changes observed). A similar phenomenon may occur in mouse epidermis; dibutyryl cyclic AMP may promote the assembly ofmicrotubules, thus reducing the supply of microtubule subunits to which colchicine could bind. Alternatively, dibutyrylcyclic AMP might stabilize existing microtubule assemblieseither directly or through a mechanism involving phosphorylation of a “microtubule-associatedprotein―required forthe assembly or function of microtubules (15).

The results presented here, together with recent reportsof a submembranous assembly containing microtubules capable of modulating receptor movement and mitogenesis

a Differs from TPA control (p < 0.01).

b Does not differ from TPA control (p < 0.1).

cantly different from that of the TPA-treated control (Table3). The cyclic nucleotide did not potentiate the inductioncaused by TPA in the absence of colchicine treatment, nordid dibutyryl cyclic AMP treatment alone induce this enzyme, so it appears that cyclic AMP acts to antagonize theeffect of colchicine, rather than independently influencingthe enzyme induction by TPA.

DISCUSSION

Several lines of evidence, including structural considerations (7) and biochemical studies in intact mouse epidermis(6, 14), as well as cells in culture (17), suggest that promotems,especially the phonbolesters, may interact specifically with the membrane site(s), thus triggering an orderedsuccession of cellular events: induction of omnithinedecarboxylase; stimulation of phospholipid synthesis (14); a sequential stimulation of RNA, protein, and DNA synthesis (2);and, finally, cell division (13). We have suggested that theinduction of epidermal omnithine decarboxylase by tumorpromoters is an essential part of mouse skin carcinogensis(10, 11). The present study is part ofan effort to discover themechanism responsible for the induction of omnithine decamboxylase by tumor promoters. Since TPA may physicallyinteract with cells of the epidermis only at their surface, anunderstanding of how such an interaction triggers the rapidand substantial increase in the synthesis of a specific protein would be of obvious importance to other systems inwhich binding to cell surface receptors leads to changes ingene expression.The resultsreportedhereinestablishthatcolchicinepre

treatment significantly inhibits the induction of omnithinedecamboxylase by TPA. The timing of colchicine treatmentrelative to promoter application is important for obtaining

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T.G. O'Brieneta!.

(4, 19), indicates that colchicine-sensitive structures, possibly in onassociated with the cell membrane, may serve as aninitial regulatory element between the binding of TPA to cellsurface receptors and the subsequent induction of omnithine decanboxylase. Such microtubule-containing structunesmayconstitute a general mechanismfor the tmansmission of signals generated at the plasma membrane into theinterior of the cell where effects on gene expression can bemanifested. More information is needed on the interactionof TPAand colchicine with microfilaments, aswell ason themoleof cyclic AMP to further substantiate the hypothesis.

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8. Hsie, A. W., and Puck, T. T. Morphological Transformation of ChineseHamsterCellsby DibutyrylAdenosineCyclic3' ,5'-MonophosphateandTestosterone. Proc. NatI. Aced. Sci. U. S., 68: 358-361 , 1971.

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1976;36:3766-3770. Cancer Res   T. G. O'Brien, R. C. Simsiman and R. K. Boutwell 

-Tetradecanoyl-phorbol-13-acetateODecarboxylase by 12-The Effect of Colchicine on the Induction of Ornithine

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