effects of hydroxyurea on dna and rna synthesis in mouse skin,...

[CANCER RESEARCH 28, 2217-2227, November 1968]

Effects of Hydroxyurea on DNA and RNA Synthesis in Mouse Skin,

Liver, and Thymus and on Skin Tumorigenesis Initiated by/3-Propiolactone1

Harold C. Smith2, R. K. Boutwell, and Van R. Potter

McArdle Laboratory for Cancer Research, University of Wisconsin Medical Center, Madison, Wisconsin 53 706

SUMMARY

Hydroxyurea was investigated as an inhibitor of DNAsynthesis in the tissues of intact mice for extended periods oftime. During administration of 5 mg of hydroxyurea i.p. every45 min for 12 hr, incorporation of thymidine-3H into DNA of

mouse skin, liver, and thymus was depressed to 5%, 25%, and5%, respectively, of values found in saline-injected controls.Incorporation of cytidine-5-3H into RNA during drug adminis

tration was essentially unaffected by hydroxyurea. After thelast of the multiple injections of the compound, DNA synthetic ability returned to normal levels in the skin and liverwithin 3-5 hours. Recovery of the ability to synthesize DNAwas slower and more complicated in the thymus. Hydroxyureashowed little direct effect on RNA synthesis during the recovery period.

Multiple injections of hydroxyurea which blocked DNAsynthesis for 12â€”15hr had little or no effect on skin tumorincidence in mice initiated with 0-propiolactone before, during, or after administration of the drug.

INTRODUCTION

Hydroxyurea is a potent and selective inhibitor of DNAsynthesis in bacteria (20, 21), in mammalian cells in vitro (12,17, 19, 27, 33, 34), in ascites tumor cells (31), and inregenerating liver (18, 24, 32). In man, hydroxyurea has beenshown to reverse orotic aciduria in leukemia patients receiving6-azauridine (29). In rat and chick embryos and in rat fetushydroxyurea is teratogenic (5, 13). The compound causes celldeath in proliferating tissues of duodenal crypts and bonemarrow in intact rats (18) and in Chinese hamster cellsexposed to hydroxyurea while synthesizing DNA (S phase ofcell cycle) (26).

The site of action of hydroxyurea in the inhibition of DNAsynthesis involves the conversion of ribonucleotides todeoxyribonucleotides (1, 7, 8, 14, 34). However, the findings

This work was supported in part by grants from the American CancerSociety (E-6) and the USPHS (TO1-CA-5002 and CA-07175).

Present address: Division of Plastic Surgery, School of Medicine,University of North Carolina, Chapel Hill, N.C., 27515.

Received March 11, 1968; accepted August 1, 1968.

of Vogler et al. (29) suggest inhibition of de novo pyrimidinebiosynthesis at a step prior to orotic acid formation.

The purpose of this study was to investigate DNA synthesisin mouse skin, liver, and thymus following single injections ofhydroxyurea and both DNA and RNA synthesis following multiple injections of the compound for extended periods up to12 hours. The experimental system thus developed was utilizedto study the relationship of DNA replication and tumor initiation by |3-propiolactone (6).

MATERIALS AND METHODS

Materials

Two strains of female mice 6â€”8weeks of age were used:STS mice obtained from A. R. Schmidt Co., Madison, Wisconsin, and Charles River CD strain obtained from the CharlesRiver Breeding Laboratories, Wilmington, Massachusetts.

Hydroxyurea was obtained from Nutritional BiochemicalsCorporation, Cleveland, Ohio.

Thymidine-methyl-3H (3 c/mmole) and cytidine-5-3H (3

c/mmole) were obtained from Schwarz BioResearch, Inc.,Orangeberg, New York.

Treatment of Animals

The backs of the mice were shaved with electric clippers 1-3days prior to use in an experiment. In single-dose experimentszero time was designated as the time of injection of hydroxyurea or isotonic NaCl (saline), whereas in multiple dose experiments, zero time indicated the time of the final injection ofhydroxyurea or isotonic NaCl.

Hydroxyurea at the appropriate dose level in 0.2 ml of wateror isotonic NaCl was injected i.p. In all experiments, 0.2 ml ofisotonic NaCl was injected i.p. in groups of control animals. Insome experiments additional groups of uninjected controlanimals were also used. Each experimental point was normallydetermined by 4 mice.

Experimental groups were constituted so that no mice werecaged together that had not been together since they were 4â€”5weeks of age. After administration of the appropriate regimenof hydroxyurea or isotonic NaCl, each mouse was injected i.p.with 60 i/c thymidine-3H in 0.2 ml of isotonic NaCl 20 min

before killing when specific activity of DNA was to be

NOVEMBER 1968 2217

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Harold C. Smith, R. K. Boutwell, and Van R. Potter

determined. In experiments for the determination of specificactivity of RNA, each animal was injected i.p. with 10 ÃŸccytidine-5-3H in 0.2 ml isotonic NaCl 1 hour before it was

killed.

Preparation of Tissue

Animals were killed by cervical dislocation and weighed.Skin from the shaved backs was removed, pinned hair sidedown to a cork board, and frozen in liquid nitrogen. Thethymus and liver were excised, wrapped in glassine paper, andfrozen in liquid nitrogen. Tissues were frozen within 3 minafter killing the animal. Lipid and muscle tissue was scrapedfrom the frozen dermis with a scalpel (28), and an area 30 x40 mm was removed for analysis. All tissues were stored at-70Â°C or -20Â°C until analyzed.

Determination of Specific Activity

DNA was isolated by the method of Orlov and Orlova (16).Tissue samples were heated in 2 ml of l N NaOH in a boilingwater bath for 6â€”7min. One-mi aliquots were chilled in an icebath and to each sample was added 0.5 ml of an ice coldsolution of 20% acetic acid saturated with NaCl. After standing in an ice bath 5â€”10min, samples were centrifuged and thesupernatants poured into 6 ml cold 95% ethanol. The pelletswere dissolved in 1 ml IN NaOH, chilled, reprecipitated with0.5 ml of cold acetic acid-NaCl solution, centrifuged, and eachsupernatant added to the 6 ml of cold 95% ethanol containingthe corresponding first supernatant. After standing in an icebath for 2 hrs, the precipitates from the ethanol solution werecollected and washed 3 times with cold 5% trichloroacetic acid(TCA), 2 times with cold 95% ethanol, and 3 times with 3:1solution of ethanol:diethylether at 45-55Â°C for 5 min.

Precipitates were hydrolyzed in 2 ml of 5% TCA for 20 min at95Â°C.Duplicate 0.2-ml aliquots of the hydrolysates were used

for determination of DNA by the diphenylamine method (4)and for liquid scintillation counting. A Packard Tri-carb liquidscintillation spectrometer (model 3375) was used for counting.Correction for quenching was determined by use of automaticexternal standard ratios. Specific activity was calculated by theformula:

dpm//ig DNA or RNA

/ne injected/gm body weightAfter homogenization of the appropriate sample of tissue,

RNA was isolated by a modified Schmidt-Thannhauser procedure (11). Aliquots of the hydrolyzed RNA were used todetermine RNA by the orcinol reaction (22) and for liquidscintillation counting.

Tumor Experiments

Tumor initiation with ÃŸ-propiolactone was carried out aspreviously described (6) at the following times with respect tothe last of 17 injections (5 mg/45 min) of hydroxyurea: â€”20hr, -2 hr, +1 hr, and +24 hr.

RESULTS

The effect of hydroxyurea on the specific activity of DNAafter a single injection of 2.5 mg (100 mg/kg for a 25-gm

mouse) or 12.5 mg per mouse in 2 groups of 20 mice each isshown in Charts 1, 2, and 3 for skin, liver, and thymus DNArespectively. The specific activities of the DNA from thevarious tissues of the group of 19 untreated control mice wereas follows: 12.2 Â±3.3 (S.D.)3 for skin, 6.0 Â±2.2 for liver, and

4.6 Â±1.9for thymus. The effect of administration of hydroxyurea was to depress the incorporation of thymidine-3H into

DNA of skin and thymus to less than 5% of the value found inuntreated control mice at 0.5 and 1 hr after injection of thedrug. In liver the depression was somewhat less with thespecific activity of the DNA reaching 20-40% of controlvalues. Doses of 2.5 or 12.5 mg/mouse were equally effectivein maintaining the depression at 0.5 and 1 hr. Three hoursafter injection of hydroxyurea, animals receiving 2.5 mg hadrecovered the ability to incorporate thymidine-3H into DNA

to a value of 40% of control mice. At the 12.5 mg dose level,recovery was somewhat slower. At 24 hr, specific activities ofDNA from skin and liver were near normal ranges found incontrol animals, whereas in thymus the specific activity ofDNA was 50-60% that of controls.

SKIN DNA

O I 3

Hours

Chart 1. Effect of a single i.p. injection of hydroxyurea (HU) at 0time on incorporation of thymidine-3H into DNA of mouse skin

during 20 min as percent of untreated control specific activity.Nineteen untreated mice with skin DNA specific activity of 12.2 Â±3.3represents 100% Â±27%. Each experimental point was determined with4 animals.

/SX2S-D-=V-â€¢

2218 CANCER RESEARCH VOL. 28



LIVER DNA

24Hours

Chart 2. Effect of a single i.p. injection of hydroxyurea (HU) at 0time on incorporation of thymidine- H into DNA of mouse liverduring 20 min as percent of untreated control specific activity.Nineteen untreated mice with liver DNA specific activity of 6.0 Â±2.2represents 100% Â±37%. Tissues were from the same mice described inChart 1.

Nucleic Acid Synthesis and Skin Tumorigenesis

THYMUS DNA

140

120

60ci

< 40ZO

20

/' ryt ^~12-5m9HU

I 3Hours

24

Chart 3. Effect of a single Lp. injection of hydroxyurea (HU) at 0time on incorporation of thymidine- H into DNA of mouse thymusduring 20 min as percent of untreated control specific activity.Nineteen untreated mice with thymus DNA specific activity of 4.6 Â±1.9 represents 100% Â±41%. Tissues were from the same animals described in Chart 1.

Administration of 2.5 mg/dose i.p. at 1.5-hr intervals wasineffective in maintaining a prolonged depression of DNAsynthesis. After 2-8 injections the specific activity of DNAranged from 50-200% of control values in the 3 tissues studied.In contrast, after 9 injections of 2.5 mg/dose given at 0.5-hrintervals, the specific activity of skin DNA 0.5 hr after the lastinjection of hydroxyurea was less than 2% that of untreatedcontrol mice and under 5% that of controls at 1.5 hr posttreatment. Some recovery of the ability to incorporate thy-midine-3H into DNA was seen at 2.5 hr. Five mg of hydroxyurea given at intervals of 0.75 hr was equally effective incausing depression of DNA synthesis. After 6 injections ofhydroxyurea under this regimen, the specific activity of DNAin skin and thymus was less than 5% of controls at 0.75 and1.75 hr after the last injection, with some recovery evident at2.75 hr. Inhibition of DNA synthesis in liver was somewhatless under either regimen of multiple injections of hydroxyurea, and there was evidence of beginning recovery at theearlier times of 1.5 of 1.75 hr after the last injection of 2.5and 5 mg respectively. The results of these experiments areshown in Table 1.

In subsequent experiments, hydroxyurea at 5 mg/dose injected at 45-min intervals for 12 hr (85 mg/17 injections) wasused to block DNA synthesis in skin during the treatmentperiod. Although DNA synthesis was almost completely inhibited during the 12 hi of this regimen, recovery of synthetic

capacity was rapid. The specific activity of skin DNA was40â€”60%of the value found in control mice by 3 hr after thelast injections as shown in Chart 4. By 5 hr the specific activityof DNA in the drug-treated animals exceeded that of controlsand remained higher until 24 hr after the last injection. From24-72 hr, specific activity of skin DNA declined in hydroxy-urea-treated mice as shown in Chart 5. In contrast, in thesaline-injected animals there was a marked stimulation in DNAsynthesis from 36â€”60hr with a decrease toward normal rangesby 72 hr.

In Charts 6 and 7 the data show that in liver, after the initialrecovery from the hydroxyurea block, there is little differencebetween DNA specific activity of drug-injected and isotonicNaCl-injected mice. From 37â€”72 hr, however, there was adepression in DNA synthesis in the hydroxyurea-treatedanimals. In contrast, the thymus appears to be more sensitiveto the effects of hydroxyurea than does the liver and skin.Charts 8 and 9 show that with few exceptions there was amarked depression in specific activity of thymus DNA in thehydroxyurea-treated mice compared to those receiving isotonic NaCl.

In sharp contrast to the marked inhibition of DNA synthesis,there was little effect of hydroxyurea on the incorporation ofcytidine-5-3H into RNA in mouse skin, liver, and thymus

during the 12 hr period of administration of the drug (shownin Charts 10, 11, and 12 respectively). In skin there is little or

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Table l

Experiment No. ofNo.mice1

35444442

12444444Hydroxyurca/

injection(mg)none2.52.52.52.52.5none2.52.52.55.05.05.0No.ofinjectionsa23478a999666Hoursbetween

injectionsa1.51.51.51.51.5a0.50.50.50.750.750.75Hoursafter finalinjectiona1.51.51.51.51.5a0.51.52.50.751.752.75Specific

activity of DNA(% of saline-injectedcontrol)Skin1006296741005810014Â±32Â±

26Â±44Â±

8Â±39Â±36Â±28.39Â±

0.91Â±1.510.4

Â±9.42155Â±

1.43Â±0.97Â±

5.7Liver10067597416896100142121142415+Â±+Â±Â±Â±Â±0Â±0Â±0Â±0Â±5Â±2Â±322129183234364.77.02.32.35.812.8Thymus100

29861598

2910121138

1371Â±151003.83.818.91.92.55.715.11.91.914.21.10.83.8

The effect of variations in the amount, and in the interval between multiple injections, of hydroxyurea on the incorporation of tritiated thymidine intoDNA of mouse skin, liver and thymus. The specific activity of the DNA of saline-injected control mice was as follows: Experiment 1, skin 14.3 Â±4.6, liver

7.3 Â±2.5, and thymus 7.5 Â±2.7 (average of 35 mice, each organ); Experiment 2, skin 10.6 i 3.0, liver 8.6 Â±3.1, and thymus 5.3 Â±0.8 (average of 12 mice,each organ).

aA group of the saline-injected control mice in each experiment were analyzed in parallel with each of the variations imposed on the hydroxyurea-treated

mice; each experiment was executed as a unit.

60

50

40

aCO

30

20

10

-12 013579 12

Hours18 24

Chart 4. Effect of repeated injections of hydroxyurea (HU) onspecific activity of skin DNA from 1-24 hr after the last injection ofthe drug. Thirty-two mice were injected i.p. with 5 mg HU/0.2 ml waterevery 45 min from -12 to 0 hr (85 mg HU/17 injections/mouse).Thirty-two control mice received 0.2 ml of saline at each injection time.Sixty /Lie thymidine-3H in 0.2 ml saline were given 20 min before

killing each mouse.

no difference in RNA specific activities found in hydroxy-urea-injected mice and saline-injected control animals. Thespecific activity of liver RNA exhibited more variation between mice receiving the different treatments than was foundin skin RNA. In thymus, however, incorporation of cytidine-

5-3H into RNA was 40-60% less in hydroxyurea-treated thanin control mice from â€”10to â€”6hr.

In the period of recovery following a 12-hour treatment withhydroxyurea (5 mg/dose at 45 min intervals), the specificactivity of skin RNA was not far different from that found inisotonic NaCl-injected controls as shown in Chart 13. Therewas a slight stimulation of cytidine-5-3H incorporation intoskin RNA from about 7-18 hr, but from 24-204 hr RNAspecific activity was essentially within the range of valuesfound in control mice. The data in Chart 14 indicate that thereis probably little significant difference in RNA specific activityof livers from drug-treated and saline-treated mice. RNAsynthesis in thymus was more sensitive to the effects ofhydroxyurea. From 48-72 hr there was a stimulation ofapproximately 100% in the drug-treated mice compared tosaline controls, as shown in Chart 15.

Results of the tumor experiment are shown in Charts 16 and17. The number of mice bearing papillomas ranged fromapproximately 85-95% in the hydroxyurea-treated groups andfrom 80-95% in the saline-injected groups, with papillomas-/mouse ranging from 3.5â€”5.5 in hydroxyurea-treated and 2.2to 5.5 in the saline-injected animals. It appears that there wasno effect of treatment with hydroxyurea on the incidence ofskin tumors in mice initiated with 0-propiolactone, whetherthe initiator was applied before, during, or after administrationof the drug.

DISCUSSION

The high specificity of hydroxyurea as an inhibitor of DNAsynthesis suggests its use as a tool in the study of pathologiclesions which may result from alterations in the geneticmaterial, especially if these are dependent upon DNA synthesisas, for example, in the fixation of an alteration in a replicatale





SKIN DNA

-12 12 18 24 36

Hours48 60 72

Chart 5. Effect of repeated injections of hydroxyurea (HU) on specific activity of skin DNA from 12-72 hr after the last injection of the drug.Hydroxyurea (5 mg/0.2 ml H2O)or 0.2 ml of saline and 60 ÃŸ:thymidine-3H were administered to two groups of 28 mice each as described in

Chart 4.

form of DNA. The rapid metabolism and excretion of hydroxyurea by the mouse and the rat after i.p. or oral administration (2, 18) and the concomitant rapid resumption of DNAsynthesis further recommends the drug for the manipulationof DNA synthesis in vivo.

Hydroxyurea added to mammalian cells in vitro causes lethaldamage to cells synthesizing DNA (S phase) without apparentdamage to cells in other phases of the cell cycle (27). Celldeath in proliferating tissues of the rat has been reported by

LIVER DNA

48

40

a.V)

24

16

]iâ€”â€¢Saline

HU

Inj. Period

-12 013579 12

Hours

16 24

Chart 6. Effect of repeated injections of hydroxyurea (HU) onspecific activity of liver DNA in the same mice described in Chart 4.

Philips et al. (18). The almost complete but short-livedinhibition of DNA synthesis in mouse skin after a single i.p.injection of hydroxyurea (100 mg/kg or 500 mg/kg) suggeststhat the drug might be used to kill a small but highly selectedpopulation of cells in skin. By judicious spacing of hydroxyurea injections, it may be possible to kill all cells entering Sphase without appreciable effects on protein and RNAsynthesis (24).

Repeated injections of hydroxyurea (200 mg/kg) at 45-minintervals effectively stopped DNA synthesis in mouse skin andthymus. In skin the quick recovery and early overshoot 5 hrafter the last injection suggests that hydroxyurea inducedsynchronized replication of DNA in intact mice. Adams andLindsay (1) have suggested the use of hydroxyurea as a cellsynchronizing agent for cultured cells. The slower recovery ofDNA synthesis in the thymus is to be expected in view of thelability of this tissue to stress in general. After 12 hr administration of hydroxyurea, the thymus was usually quite small.The recovery of the thymus and the elevated specific activityof thymus DNA from 2â€”3days after the last injection is verysimilar to thymus recovery following X-irradiation (15). Inliver the similarity in DNA specific activity between hydroxy-urea-injected and saline-injected mice from 3â€”72hr indicatesthat after the early resumption of DNA synthesis, this organ isaffected more from stress than from effects of the drug. Insaline-injected control mice, the stimulation of DNA synthesisin all 3 tissues studied from 26-60 hr is indicative of animalresponse to stress of handling and repeated injections.

The response of RNA synthesis to the effects of repeatedinjections of hydroxyurea is in marked contrast to the response of DNA synthesis. During the 12-hr period when DNAsynthesis is essentially stopped, specific activity of RNA fromhydroxyurea-treated mice is near or within the range of values

NOVEMBER 1968 2221




LIVER DNA

48 60 72-I27 0

Chart 1. Effect of repeated injections of hydroxyurea (HU) on specific activity of liver DNA in the same mice described in Chart 5.

12 18 24 36

Hours

found in saline-injected mice. With the exception of the morelabile thymus, which shows some depression in RNA synthesisin the early period of treatment, there is probably no significant difference between RNA specific activity of hydroxy-urea-treated and saline-injected mice. In the period of re

covery from the effects of the drug, fluctuations in RNAsynthesis are probably secondary reflections of body responseto the drug and to stress.

THYMUS DNA

-12 013579 12

Hours18 24

Chart 8. Effect of repeated injections of hydroxyurea (HU) onspecific activity of thymus DNA in the same mice described in Chart 4.

Although it is likely that inhibition of DNA synthesis couldbe maintained for longer periods than were demonstrated inthis report, it was our judgment to stop after 17 injections ofhydroxyurea given over a 12-hour period. Certainly thechanges observed in the nucleic acid metabolism of tissues ofthe saline-treated control mice in the hours following cessationof treatment suggest that the control animals reacted to thestress of the injection regimen. Because the duration ofinhibition of DNA synthesis attributable to hydroxyurea wasabout 12â€”15 hours, it seemed appropriate to test ÃŸ-propiolactone as an initiator rather than a carcinogenic hydrocarbon for the reasons that the former is highly reactive andhas a short half-life in vivo (< 2 hr) and reaches maximumbinding to DNA within 2 hr (6). In contrast, the carcinogenichydrocarbons remain in the skin much longer and do notattain maximal binding to DNA until 24 hours.

Mice treated with an appropriate dose-schedule of actino-mycin D have been found to be protected against the tumor-initiating activity of certain carcinogenic hydrocarbons,urethan, and 0-propiolactone (9, 10). Further studies of thephenomenon revealed that the minimal effective dose scheduleof actinomycin D inhibited the incorporation of thymidineinto skin DNA by 75-90% for at least 2 days, while the effecton RNA synthesis was much less (11). On the other hand, theresponse to an initiator has been reported to be increasedduring periods of increased DNA synthesis (25). Furthermore,Suss and Maurer (28), who also used hydroxyurea to inhibitDNA synthesis in mouse skin, showed that the binding ofhydrocarbon to DNA was proportional to the rate of DNAsynthesis. These and similar observations suggest that skintumor formation may require DNA synthesis (3,11).

At least 3 mechanisms are compatible with the generalization that cells actively synthesizing DNA are more susceptibleto initiator: (a) The initiator may react with DNA precursors,





THYMUS DNA

12 18 24 36

Hours46 60 72

Chart 9. Effect of repeated injections of hydroxyurea (HU) on specific activity of thymus DNA in the same mice described in Chart 5.

and the incorporation of an altered precursor into DNA isdependent on the rate of DNA synthesis, (Â¿)DNA in thereplicating state is more susceptible to reaction with initiatorsthan DNA in the stabilized state, (c) DNA, which has beenaltered by an initiator, must replicate in order to fix the errorin a replicatale form before the change has been repaired.

In order to further investigate the role of DNA synthesis in

SKIN RNA

2 200-coO'S 160h

g 120

t3 100

. 80

40

]iâ„¢â€¢Saline

Ã€-L

> Â± ^

Injection Period

-12 -IO -8 -6 -4

Hours

-2

Chart 10. Effect of repeated injections of hydroxyurea (HU) onspecific activity of skin RNA during administration of the drug. Twogroups of 24 mice each were injected i.p. with 5 mg HU/0.2 ml H2Oorwith 0.2 ml of saline beginning at -12 hr and repeated every 45 minuntil killed. One hr before killing each mouse received i.p. 10 juccytidine-5-3H in 0.2 ml of saline.

the process of initiation, a search for other agents whichselectively affect DNA synthesis in mouse skin without theside effects of actinomycin D (especially cytotoxicity) hasbeen pursued. Hydroxyurea represents a most promising agent;the metabolic studies reported here show that administrationof repeated doses of the drug blocked DNA synthesis in mouseskin for 12-15 hours with little or no effect on RNAsynthesis. However, in contrast to the results with actinomycinD, there was no effect of hydroxyurea on the initiation of

LIVER RNA

10 -8 -6 -4-12

Chart 11. Effect of repeated injections of hydroxyurea (HU) onspecific activity of liver RNA in the same mice described in Chart 10.

NOVEMBER 1968 2223




THYMUS RNA

Ã Â«Wo

o.Vi

200

160

120

100

80

40

1- ' â€¢1

f-"+ Saline

HU

Injection L Reriod

-12 -IO -8 -6

Hours

-4 -2

Chart 12. Effect of repeated injections of hydroxyurea (HU) onspecific activity of thymus RNA in the same mice described in Chart10.

tumors with 0-propiolactone. This result was obtained despitethe fact that hydroxyurea inhibited DNA synthesis, asmeasured by the incorporation of tritiated thymidine, moreeffectively than actinomycin D. However, there was oneimportant difference: DNA synthesis was inhibited for only12-15 hours with hydroxyurea; in the case of actinomycin D,

the duration of inhibition was at least 48 hours. Thus, the lackof inhibition of tumor formation initiated by /3-propiolactonedoes not eliminate the possibility that DNA synthesis or cellreplication may be essential to the initiation of tumor formation. Finally, there was no inhibition of skin RNA synthesisand no apparent skin wounding in the hydroxyurea-treatedmice. Therefore, a possible role of the side reactions, especiallycell killing, remains to explain the inhibition of tumorigenesisby actinomycin D.

ACKNOWLEDGMENTS

The authors acknowledge the valuable technical assistance of Mrs.Julie Corbett and Mrs. Judy Goth. Helpful suggestions were made byDrs. Nancy H. Colburn and Henry Hennings.

SKIN RNA

01 3579 12 18 24 36 48 60 72Hours

108 156 204

Chart 13. Effect of repeated injections of hydroxyurea (HU) on specific activity of skin RNA from 1 hr to 204 hi following administration ofthe drug. Two groups of 60 mice each were treated as described in Chart 10.





LIVER RNA

Ct

01 3579 12 18 24 108 156 20436 48 60 72Hours

Chart 14. Effect of repeated injections of hydroxyurea (HU) on specific activity of liver RNA in the same mice described in Chart 13.

THYMUS RNA

01 3 579 12 18 24 36 " 48 60 72

Hours108 156 204

Chart 15. Effect of repeated injections of hydroxyurea (HU) on specific activity of thymus RNA in the same mice described in Chart 13.

NOVEMBER 1968 2225




-20 -2 +1 +24Time of Initiation â€” Hours

Chart 16. Effect of repeated injections of hydroxyurea (HU) onpapilloma incidence in STS mice. Hydroxyurea (5 mg/0.2 ml H2O)or0.2 ml of saline was injected i.p. every 45 min from -12 hr to 0 time. A

BPL dose of 480 jLtmolesin 0.3 ml of acetone was applied to the shavedback of each mouse at one of the following experimental times: â€”20hr, -2 hr, +1 hr, or +24 hr relative to the last injection of hydroxyureaor saline (0 time). Each experimental group contained 20-25 mice.

REFERENCES

1. Adams, R. L. P., and Lindsay, J. G. Hydroxyurea. Reversal ofInhibition and Use as a Cell-Synchronizing Agent. J. Biol. Chem.,242: 1314-1317, 1967.

2. Adamson, R. H., Ague, S. L., Hess, S. M., and Davidson, J. D. TheDistribution, Excretion, and Metabolism of Hydroxyurea-14C. J.

Pharmacol. Exptl. Therap., 750: 322-327, 1965.3. Bates, R. R., Wortham, J. S., Counts, W. B., Dingman, C. W., and

Gelboin, H. V. DNA Synthesis and Skin Tumorigenesis by 7,12-Dimethylbenz(a)anthiacene: Inhibition by Actinomycin D. CancerRes., 28: 27-34, 1968.

4. Burton, K. A Study of the Condition and Mechanism of theDiphenylamine Reaction for the Colorimetrie Estimation ofDeoxyribonucleic Acid. Biochem. J., 62: 315-323, 1965.

5. Chaube, S., and Murphy, M. L. The Effects of Hydroxyurea andRelated Compounds on the Rat Fetus. Cancer Res., 26:1448-1457,1966.

6. Colburn, N. H., and Boutwell, R. K. The Binding of ÃŸ-Propiolactone to Mouse Skin DNA in Vivo: Its Correlation withTumor-initiating Activity. Cancer Res., 26: 1701-1706, 1966.

7. Frenkel, E. P. and Arthur, C. Induced Ribotide Reductive Conversion Defect by Hydroxyurea and its Relationship to Megaloblas-tosis, Cancer Res., 27: 1016-1019, 1967.

8. Frenkel, E. P., Skinner, W. N., and Smiley, J. D. Studies on aMetabolic Defect Induced by Hydroxyurea (MCS-32065). CancerChemotherapy. Rept., 40: 19-22, 1964.

-20 -2 +1 +24

Time of Initiation â€” Hours

Chart 17. Effect of repeated injections of hydroxyurea (HU) onpercent of mice bearing papillomas at 18 weeks. The same mice aredescribed in Chart 16.

9. Gelboin, H. V., Klein, M. and Bates, R. R. Inhibition of MouseSkin Tumorigenesis by Actinomycin D. Proc. Nati. Acad. Sci. U.S.,53: 1353-1360, 1965.

10. Hennings, H., and Boutwell, R. K. On the Mechanism of Inhibitionof Benign and Malignant Skin Tumor Formation by ActinomycinD. Life Sciences, 6: 173-181, 1967.

11. Hennings, H., Smith, H. C., Colburn, N. H., and Boutwell, R. K.Inhibition by Actinomycin D of DNA and RNA Synthesis and ofSkin Carcinogenesis Initiated by 7,12-Dimethylbenz(a)anthraceneor ÃŸ-Propiolactone.Cancer Res., 28: 543-552, 1968.

12. Kim, J. H., Gelbard, A. S., and Perez, A. G. Action of Hydroxyureaon the Nucleic Acid Metabolism and Viability of HeLa Cells.Cancer Res., 27: 1301-1305, 1967.

13. Murphy, M. L., and Chaube, S. Preliminary Survey of Hydroxyurea(NSC-32065) as a Teratogen. Cancer Chemotherapy. Rept., 40:1-7, 1964.

14. Newhard, J. Studies on the Acid-Soluble Nucleotide Pool inEscherichia Coli IV. Effects of Hydroxyurea. Biochim. Biophys.Acta, 145: 1-6, 1967.

15. Nygaard, O. F. and Potter, R. L. Effect of X-Radiation on DNAMetabolism in Various Tissues of the Rat. II. Recovery afterSublethal Doses of Irradiation. Radiation Res., 12: 120-130,1960.

16. Orlov, A. S., and Orlova, E. I. A Simple Method for the Determination of Deoxyribonucleic Acid in Animal Tissue. Biochem. (USSR)(English TransÃ.),26: 722-726, 1962.

17. Pfeiffer, S. E. and Tolmach, L. J. Inhibition of DNA Synthesis inHeLa Cells by Hydroxyurea. Cancer Res., 27: 124-129, 1967.

18. Philips, F. S., Sternberg, S. S., Schwartz, H. S., Cronin, A. P.,Sodergren, J. E., and Vidal, P. M. Hydroxyurea. I. Acute CellDeath in Proliferating Tissues in Rats. Cancer Res., 27: 61-74,1967.





19. Pollock, R. D., and Rosenkranz, H. S. Metabolic Effects ofHydroxyurca on BHK-21 Cells Transformed with Polyoma Virus.Cancer Res., 27: 1214-1224, 1967.

20. Rosenkranz, H. S. and Carr, H. S. Studies with Hydroxyurea. II.Prolonged Exposure of Escherichia Coli to Hydroxyurea. Bacteriology, 92: 178-185, 1966.

21. Rosenkranz, H. S., Garro, A. J., Levy, J. A. and Carr, H.S. Studieswith Hydroxyurea. 1. The Reversible Inhibition of Bacterial DNASynthesis and the Effect of Hydroxyurea on the BactericidalAction of Streptomycin. Biochem. Biophys. Acta, 114: 501-515,

1966.22. Schneider, W. C. Determination of Nucleic Acids in Tissues by

Pentose Analysis. In: S. P. Colowick and N. O. Kaplan (eds.),Methods in Enzymology, Vol. 3, pp. 680-684, New York: Academic Press, 1957.

23. Schulman, J., and Falkcnheim, M. Review of Conventions inRadio-tracer Studies. Nucleonics, J: 13-23, 1948.

24. Schwartz, H. S., Garofalo, M., Sternberg, S. S., and Philips, F. S.Hydroxyurea: Inhibition of Deoxyribonucleic Acid Synthesis inRegenerating Rat Livers. Cancer Res., 25: 1867-1870, 1965.

25. Shinozuka, H., and Ritchie, A. C. Pretreatment with Croton Oil,DNA Synthesis, and Carcinogenesis by Carcinogen Followed byCroton Oil. Intern. J. Cancer, 2: 77-84, 1967.

26. Sinclair, W. K. Hydroxyurea: Differential Lethal Effects on Cul

tured Mammalian Cells During the Cell Cycle. Science, 750;1729-1731, 1965.

27. Sinclair, W. K. Hydroxyurea: Effects on Chinese Hamster CellsGrown in Culture. Cancer Res., 27: 297-308, 1967.

28. Suss, R., and Maurer, H. R. Reduced Binding of CarcinogenicHydrocarbons to DNA of Mouse Skin During Inhibition of DNASynthesis. Nature, 217: 753-754, 1968.

29. Vogler, W. R., Bain, J. A., and Huguley, C. M., Jr. In Vivo Effect ofHydroxyurea on Orotic Acid Synthesis. Cancer Res., 26:1827-1831, 1966.

30. Wiest, W. G. and Heidelberger, C. The Interaction of CarcinogenicHydrocarbons with Tissue Constituents, I. Methods. Cancer Res.,13: 246-249, 1953.

31. Yarbro, J. W., Kennedy, B. J., and Bamum, C. P. HydroxyureaInhibition of DNA Synthesis in Ascites Tumor. Proc. Nati. Acad.Sei. U. S., 53: 1033-1035, 1965.

32. Yarbro, J. W., Niehaus, W. G. and Barnum, C. P. Effect ofHydroxyurea on Regenerating Rat Liver. Biochem. Biophys. Res.Commun., 19: 592-597, 1965.

33. Young, C. W., and Hodas, S. Hydroxyurea: Inhibitory Effect onDNA Metabolism. Science, 146: 1172-1174, 1964.

34. Young, C. W., Schochteman, G., and Karnofsky, D. A. Hydroxy-urea-induced Inhibition of Deoxyribonucleotide Synthesis: Studies in Intact Cells. Cancer Res., 27: 526-534, 1967.

NOVEMBER 1968 2227



1968;28:2217-2227. Cancer Res Harold C. Smith, R. K. Boutwell and Van R. Potter

-PropiolactoneβbySkin, Liver, and Thymus and on Skin Tumorigenesis Initiated

Effects of Hydroxyurea on DNA and RNA Synthesis in Mouse

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