BIOCHEMICAL

21, 168-181

MEDICINE

(1979)

Effects of Ultraviolet Light on Epidermal Polyamine Metabolism N. Centre

SEILER

de Recherche 67084

AND

B.

KNC~DGEN

Merrell International, 16, rue d’Ankara, Strasbourg Cedex. France

Received

November

8, 1978

There is evidence that the diamine putrescine (1 ,Cdiaminobutane) and its derivatives spermidine EN’-(3-aminopropyl)-1 ,Cdiaminobutane] and spermine [N1JV4-bis-(3-aminopropyl)1,Cdiaminobutane] play an important role in cell growth and cell proliferation (l-6). Increasing interest has been given to the role of these amines in disease states, including proliferative diseases of the skin (7-19) and wound healing (20). The role of putrescine (21) and of ornithine decarboxylase (OrnDC), the enzyme yielding putrescine for spermidine biosynthesis, in epidermal cell proliferation and in skin carcinogenesis has been the topic of numerous publications during the last few years (22-32). Polyamine patterns and the patterns of some of the enzymes involved in polyamine biosynthesis have been compared in normal skin and in involved and noninvolved skin of psoriatic patients in order to establish interrelations between polyamines and epidermal proliferation, and to study effects of psoriasis therapy on epidermal polyamine metabolism (33-37). Ultraviolet irradiation of skin induces increased mitosis, increased DNA synthesis, and epidermal hyperplasia (38). Exposure of hairless mice to uv light under standardized conditions and measurement of [3H] thymidine incorporation into epidermal DNA are considered a model suitable for screening cytostatic drugs for potential use in the therapy of psoriasis (39). We aimed to explore whether this model is also suitable for studying changes of polyamine metabolism accompanying increased rates of cell proliferation and for investigating the effects of compounds which inhibit polyamine biosynthesis in the molecular and cellular events induced in the skin by uv light. 168 0006-2944/79/020168-14$02.00/O Copyright All rights

@ 1979 by Academic F’ress. Inc. of reproduction in any form reserved

EPIDERMAL

POLYAMINE

MATERIALS

METABOLISM

169

AND METHODS

Experimental animals. Hairless mice (HRS/J strain) were originally obtained from CNRS, Orleans, France, and then bred in our animal quarters. In the experiments to be described, both males and females were used (weighing 20 + 2 g). The animals had normal access to water and standard diet (Charles River, France) ad libitum. They were on a 12-hr light, 12-hr dark schedule. Ultraviolet irradiation. Free movement of the animals was limited to a 10 x 20-cm area (stainless steel box). They were exposed for 10 or 50 min to the light of a germicidal lamp (G8T5, General Electric, Cleveland, Ohio, U.S.A.) from a distance of 20 cm, as suggested by Du Vivier and Stoughton (39). No signs of skin irritation and no macroscopically observable changes were observed under these conditions. Preparation of epidermal extracts. The animals were sacrificed by diethylether inhalation in order to avoid bleeding. The dorsal skin was cleaned with ethanol and then an approximately 20 x 30-mm sample was excised from each animal. By exposure of the skin to 55°C for 1 min on a heated glass plate, the epidermis became easily separable from the dermis with a scalpel (39). The epidermis samples were weighed and immediately homogenized with 20 vol of ice-cold 0.2 N perchloric acid using small all-glass homogenizers. The acid insoluble constituents were sedimented by 20-min centrifugation at 3000g. The supernatants were used for polyamine determinations, the precipitates for the measurement of DNA synthesis. Determination of polyamines. The perchloric acid extracts of the epidermis were passed through a filter (Millex filter unit 0.22 pm; Millipore, Molsheim, France) and 50-~1 aliquots were directly applied to the column of a Dun-urn amino acid analyzer. The elution from the ionexchange column and the measurement of the polyamines in the eluate followed published procedures (40,41). For the determination of the specific radioactivities of the polyamines, after their labeling in vivo by repeated injections of [1,4-‘*C]putrestine, the perchloric acid extracts were reacted with an excess of 5-dimethylaminonaphthalene-1-sulfonylchloride (Dns-Cl). The Dnsderivatives were separated by thin-layer chromatography on silica gel using chloroform-triethylamine (5 + 1). The fluorescent bands of the Dns-polyamines were extracted and reseparated on a second silica gel plate with cyclohexane-ethyl acetate (1 + 1) as solvent. Finally, the fluorescent spots were extracted with dioxane. Specific radioactivity was determined by the measurement of fluorescence intensity and radioactivity (by liquid scintillation counting) of the same sample. [For a full description of the method, see (42)].

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KNijDGEN

Determinution of the rate of DNA synthesis. [“HlThymidine, 25 &i, dissolved in 250 ~1 of water was injected intraperitoneally 1 hr before sacrifice. The precipitates of the perchloric acid extracts were submitted to the procedure for DNA purification and measurement described in detail by Du Vivier and Stoughton (39). Specific radioactivity of DNA was estimated in two aliquots of each epidermis sample. Chemicals and radiochemicals. The usual laboratory chemicals were from E. Merck, Darmstadt, Germany. cr-Difluoromethylornithine and Dns-Cl were synthesized in our laboratory using published procedures (43,44). RNAse (free of DNAse) and pronase (fromStreptomyces griseus) were purchased from Serva, Heidelberg, Germany. Calf thymus DNA (Sigma Chemical Co., St Louis, MO., U.S.A.) was used as standard. [ 1,4-rJC]Ptttrescine (specific radioactivity 89 Ci/mole) and 13Hlthymidine (specific radioactivity 20 Ci/mole) were from New England Nuclear Corporation, Boston, Massachusetts, U.S.A. RESULTS

1. The Effect of Ultraviolet Concentrations

Light on Epidermal

Polyamine

A lo-min exposure of hairless mice to the light of a germicidal lamp under standardized conditions induces a rapid five- to eightfold increase of epidermal putrescine levels within about 6 hr. After this time, epidermal putrescine levels decline gradually to reach base levels at about 47-hr postirradiation (Fig. 1). The changes of spermidine and spermine concentrations, following uv irradiation of mouse skin, are also highly significant (Fig. 1). The spermidine level increases gradually over a period of 48 hr to reach at its maximum a net increase of about 0.8 pmole/g; i.e., an amount of spermidine exceeding 50% of its normal pool is newly formed within 2 days in uv-irradiated epidermis. At 72 hr after irradiation epidermal spermidine is still significantly above base level. The increase of spermidine is accompanied by a decrease of spermine. Minimum levels are reached at about 24-hr postirradiation with a slow increase thereafter. At 72 hr spermine levels have nearly normalized. At minimum levels, the epidermal spermine pool is only about 50% of the basal pool, with a net loss of 0.3-0.4 p.mole/g. Prolongation of exposure to uv light from 10 to 50 min had no further effect on epidermal spermidine and spermine concentrations. However, the effect on putrescine levels was enhanced: After irradiation for 50 min, 0.18 _’ 0.003 @mole/g putrescine was found at 24 hr, whereas only 0.085 2 0.02 pmol/g was found at the same time after 10 min of irradiation. Peak levels at 6 hr were about the same for both times of exposure to uv light.

EPIDERMAL

POLYAMINE

ObDS TIME

24 AFTER

171

METABOLISM

UV

47 IARAOIATION

72

FIG. 1. The effect of IO-min exposure to uv light of hairless mice on epidermal putrestine, spermidine, and spermine levels, and on the incorporation of [SH]thymidine into epidermal DNA. The vertical bars indicate SD of the values obtained from three to five animals.

2. The Incorporation

of [W]Thymidine

into Epidermal

DNA

For the measurement of the rate of synthesis of DNA, [3H]thymidine was administered 60 min before sacrifice. The total amount of radioactivity incorporated into epidermal DNA was low. The method is therefore not as precise, as for instance the measurement of the polyamines spermidine and spermine. Nevertheless it is clear from the data that exposure of the skin to uv light under the described conditions increases DNA synthesis rate maximally by a factor of about three (Fig. 1). At 6 hr, when putrescine levels exhibit the first maximum, no change is observed in DNA synthesis rate. Maximal rates seem to be reached at

172

SEILER

AND

KNiiDGEN

about 24 hr, when spermine levels are minimal after which rates of DNA synthesis decrease gradually to almost reach base levels 72-hr postirradiation. From the data summarized in Fig. I it appears that the most suitable time for the determination of the changes of both polyamine and DNA metabolism is probably 24 hr after the exposure of the dorsal skin to uv light. At this time the rate of DNA synthesis is higher than at 54 hr, the time chosen by Du Vivier and Stoughton (39) for DNA synthesis rate measurements. The concentration changes of putrescine, spermidine, and spermine are very considerable at 24 hr, though maximal only in the case of spermine. It should be noted in this context that the spermidinei spermine ratio was increased from 2.39 + 0.38 at time zero to 6.25 2 1.54 at 24 hr. This ratio is a reliable parameter and its increase seems to be an indicator of enhanced cell proliferation, because it coincides with elevated DNA synthesis rates. It was found that 50 min of exposure to uv light yields somewhat better reproducibilities, presumably due to a more even exposure of the skin to irradiation. Most of the results described below were therefore obtained under these experimental conditions. 3. The Effects of cr-Difuoromethylornithine on Epidermal Polyamine Metabolism cY-Difluoromethylomithine (DFMOm) is an enzyme-activated irreversible inhibitor of OrnDC (44). It reduces the OmDC activity of cultured cells (45,46) and of several organs in vivo (47), thereby decreasing putrestine and spermidine levels and the proliferation rate of, among others, cultured rat hepatoma cells. This compound was administered in a first experiment by intraperitoneal injections of 200 mg/kg, every 2 hr, starting 2 hr before the animals were exposed to uv light for 10 min. At 6 hr after uv irradiation (i.e., 2 hr after the fourth dose of DFMOm) the animals were sacrificed by inhalation of diethylether, and the polyamines were determined in the epidermis. Both the uv-induced elevation of putrescine and the typical elevation of the spermidine/spermine ratio were completely prevented by DFMOm (Table 1). For topical application a 1 + 10 w/w mixture of DFMOm with an oil-in-water emulsion’ was prepared. About 50 mg/cm” of this mixture were applied to the dorsal skin of the animals 2 hr before exposure for 10 min to uv light. As can be seen from the data of Table 1, the uv-induced increase of epidermal putrescine was completely prevented by DFMOrn. A significant decrease of the spermidine/spermine ratio in comparison with that of uv-irradiated controls was observed in the case of systemic DFMOrn administration. The cream used as a vehicle for ’ The England.

oil-in-water

emulsion

was kindly

prepared

for us by Vick

International,

Slough,

EPIDERMAL

POLYAMINE TABLE

EFFECT

173

METABOLISM 1

OF SYSTEMICALLY

(DFMOm)

OR TOPICALLY APPLIED CY-DIFLUOROMEYHYLORNITHINE ON THE ULTRAVIOLET-INDUCED FORMATION OF PLJTRESCINE IN MOUSE EPIDERMIS’

Treatment Nonirradiated untreated controls Ultraviolet irradiation without any other treatment 4 x 200 mg/kg DFMOm ip and uv irradiation Topical application of DFMOm (dissolved in an oil-in-water emulsion) before exposure to uv light Topical application of the oil-in-water emulsion before exposure to uv light (control)

Putrescine (pmoleig)

Spermidine/ Spermine

0.026 k 0.009 0.14 rt 0.03 0.020 2 0.006

2.39 +- 0.38 2.92 r 0.25 2.34 k 0.13

0.025 + 0.007

2.81 k 0.03

0.089 t 0.004

3.54 + 0.7

o In these experiments either four ip doses (200 mglkg) of DFMOm were given, starting 2 hr prior. uv irradiation, or about 50 mg/cm* of a (1 + 10, w/w) mixture of DFMOm in an oil-in-water emulsion were applied to the dorsal skin of hairless mice 2 hr before uv irradiation. Polyamine concentrations were measured 6 hr after the IO-min exposure to uv light. The values are the means 2 SD (n = 3 or 4). Since the changes of spermidine and spermine were not highly significant during the first 6 hr (see Fig. 1) only the more reliable spermidine/ spermine ratios are given.

the drug seems to have effect of its own on uv-induced polyamine metabolism (Table 1). OmDC has a turnover rate of the order of 15-30 min (48). This implies that constant (and high) levels of an inhibitor are necessary to keep OrnDC activity low. Repeated injections of DFMOrn seemed to be impractical. Oral administration of the drug as a 3% solution in tap water produced high and sufficiently constant drug levels in liver, brain, and other organs. The net water intake of the drug-treated animals was not significantly lower than that of the controls. Assuming an average water intake of 4 ml per day, the animals received a dose of 6 g/kg/day. This mode of administration was used in the present work to study chronic DFMOm effects on epidermal polyamine metabolism. Groups of mice received the drug for 7 and 21 days, respectively. At 24 hr before sacrifice one half of the animals was exposed to uv light for 50 min. At 1 hr before sacrifice all animals received 25 &i 13Hlthymidine. The DFMOm-treated group showed no weight difference in comparison with the controls even after 2 1 days of treatment. The effects of DFMOm on polyamine metabolism are summarized in Fig. 2. Poiyamine concentrations of the two nonirradiated control groups were within the normal limits. The uv-induced changes of polyamine concentrations were the same as those observed in the preceding experiments: an increase of putrescine and spermidine pools with a concomitant decrease of the spermine pool. Seven days of treatment with DFMOm decreased basal

SEILER AND KNODGEN

174

DRUG-FREE

CONTROLS

0(-01FLUOROMET~LORNITHlNE. TREATED

T

loo

50 6 E t

0

I

l4L 0

PUTRESCINE

ANIMALS

m

SPERMIOINE

0

SPELRYINE

FIG. 2. Histogram demonstrating the effect of chronic (7 and 21 day) oral application of a-difluoromethylomithine on epidermal polyamine levels and on the uv light-induced changes of polyamines. The vertical bars indicate SD of the values obtained from three animals.

levels of epidermal putrescine by about 50%. Prolongation of the treatment with the drug did not enhance this effect on putrescine, but may have caused a slight, though not statistically significant decrease of spermidine. Spermine levels were not changed in the DFMOrn-treated animals. In the DFMOm-treated animals putrescine levels remained low, or were even further lowered upon exposure to uv light. Strikingly, spermidine levels not only did not increase, but were considerably decreased, and spermine levels remained practically unchanged under these conditions, in contrast to the uv-irradiated drug-free controls. There was no statistically significant difference in the uv-induced changes between the animals treated with the drug for a different length of time. In a pilot experiment it was found that uv-induced increase of DNA

EPIDERMAL

POLYAMINE

METABOLISM

175

synthesis rate, as measured by [3H]thymidine incorporation from 53to 54hr postirradiation, was not altered by oral DFMOm, if drug administration started 12 hr before exposure to uv light. Comparable results were obtained by chronic treatment with DFMOm, as can be seen in Table 2. The specific radioactivity of DNA was the same in both control groups, and a significant increase of thymidine incorporation was noted after irradiation, although this was minor in the 7-day experiment. Treatment with DFMOrn for 7 days has no significant influence on DNA synthesis, but after 21 days of treatment, 13Hlthymidine incorporation was less than 50% of the corresponding nonirradiated control, and the uv-induced increase of DNA synthesis was somewhat lower in the DFMOrn-treated than in the untreated groups. Due to the uncertainty of the measurements of the specific radioactivity of DNA and the relatively small number of animals in this experiment it is not possible to draw firm conclusions, but it appears from the present data that DNA formation in the epidermis is either unaffected or not greatly affected even by prolonged treatment with DFMOm. 4. Metabolic

Interrelations

between

Spermidine

and Spermine

As was shown above, uv irradiation of skin produces an increase of spermidine and a decrease of spermine concentrations. Irradiation of the skin of animals with (partially) blocked OmDC activity produces a different pattern of polyamines upon exposure to uv light: spermidine concentrations decrease, and spermine concentrations remain virtually constant. The following questions arise from these findings: What is the effect of the blockade of OrnDC on uv-induced spermidine/spermine interrelations? Is the uv-induced increase of spermidine concentration partially due to formation from spermine by breakdown, as was shown previously to occur after injection of labeled spermine in brain (49)? Is spermine newly formed, or only degraded? At least partial answers to these questions were expected from the following experiment: The epidermal polyamines were labeled by three intraperitoneal injections of 10 @i [ 1,4-[*Clputrescine (specific radioactivity 89 Cilmole) on 3 consecutive days. At 12 hr after the last injection of radioactive putrescine all animals were deprived of drinking water for 17 hr (overnight). One group received the 3% solution of DFMOm, and the other group received tap water. At 24 hr later, one half of each group was exposed to uv light for 50 min. Treatment with DFMOm was continued for a further 24 hr (i.e., for a total of 48 hr). Skin samples were prepared as before, and the specific radioactivities of the polyamines were determined as described in the methods section. The radioactivity present in putrescine was too low to be precisely determined. The values obtained for spermidine and spermine are summarized in Table 3.

DFMOm + uv irradiation

DFMOm

Ultraviolet irradiation

DFMOm + uv irradiation -

DFMOm

Ultraviolet irradiation

-

Treatment

2

1.40 k 0.14 1.84 k 0.36 1.45 + 0.14 1.06 k 0.19 1.27 ‘-c 0.10 1.97 t 0.33 1.13 r 0.11 0.84 -+ 0.04

0.019 k 0.003 0.073 + 0.009 0.009 2 0.004 0.008 k 0.006 0.015 + 0.001 0.093 5 0.02 0.014 ? 0.001 0.009 2 0.004

0.61 + 0.02 0.34 20.08 0.71 2 0.09 0.55 ? 0.04 0.66 k 0.05 0.36 + 0.03 0.73 k 0.05 0.61 + 0.06

Spermine Wnolek)

OF WDIFLUOROMETHYLORNITHINE METABOLISM IN HAIRLESS MICE"

TABLE

Spennidine (wnolek)

APPLICATION POLYAMINE

Futrescine bmolek)

OF CHRONIC ORAL EPIDERMAL

2.29 f 0.18 5.47 k 1.2 2.05 ” 0.30 1.91 k 0.30 1.92 + 0.02 5.59 + 1.3 1.55 k 0.14 1.37 k 0.07

Spermidinei Spermine

(DFM0rn)o~

690 +- 225

165 t 6

1190 + 30

370 e so

520 2 140

360 t 120

730 i- 150

360 + 30

DNA specific radioactivity (cpmi100 Fg)

(’ At 24 hr prior sacrifice, some of the animals were exposed under standardized conditions to uv light. At 1 hr before sacrifice, all animals received 25 &i of 13Hlthymidine (specific radioactivity 20 Ci/mmole) intraperitoneally. The mice were killed with diethylether. Skin samples were collected, and epidermis was prepared and extracted as described in the methods section. The figures in the table are means of the values obtained from three animals -t SD. Hairless mice (HRS/J strain) received instead of drinking water a 3% solution of DFMOrn for 7 and 21 days, respectively.

21

Duration of treatment (days)

EFFECT

EPIDERMAL

POLYAMINE TABLE

ULTRAVIOLET-INDUCED SPERMINE

Treatment Control (no DFMOm, no uv irradiation) No DFMOm, SO-min uv irradiation 48-hr treatment with DFMOm, no uv irradiation 48-hr treatment with DFMOm, 50-min uv irradiation

177

METABOLISM 3

EFFECTS ON THE SPECIFIC RADIOACTIVITIES OF SPERMIDINE IN MOUSE EPIDERMIS AND THE INFLUENCE OF TREATMENT WITH WDIFLUOROMETHYLORNITHINE (DFMOm)

AND

Specific radioactivity Spermine (cprn/nmole)

Spermidine/ Spermine

201 k 56

88 -t 23

2.28 h 0.23

114 t 52

65 -c 31

1.75 + 0.08

251 2 68

99 1?125

2.54 -+ 0.15

157 rf: 57

65 t 28

2.42 !z 0.24

Spermidine

s For experimental details see text and the methods section. The figures in the table are means of the values obtained from three animals f SD.

The specific radioactivity of spermine was lower than that of spermidine by a factor of 2.3-2.5, both in the DFMOm-treated and the nontreated mice. This shows that equilibrium labeling of spermidine and spermine was not achieved under the experimental conditions, if one does not assume a higher turnover rate for spermine than for spermidine. Ultraviolet irradiation decreased the specific radioactivities of spermidine and spermine, the decrease of the specific radioactivity of spermidine being relatively greater than that of spermine in the case of the drug-free controls. DISCUSSION

Exposure of the skin of uv light produces reproducible, time-dependent changes of the epidermal polyamine pattern, concomitant with an increase of DNA synthesis which signals an increase of cell proliferation rate. Although it has not been determined, there is little doubt that the rapid increase of putrescine concentration observed after exposure of skin to uv light is due to a proportional increase of OmDC activity. In many different systems a close parallelism has been shown between putrescine concentration and OmDC activity. For example, the fluctuation of OmDC during the cell cycle is paralleled by corresponding fluctuations of the putrescine concentration (50); in the developing rat brain a close correlation exists between OmDC activity (5 1) and putrescine concentration (52); inhibition of OmDC in rat hepatoma cells with DFMOm is

178

SElLERANDKNijDGEN

accompanied by a proportional decrease of putrescine levels (53). In the present work OrnDC determinations were not undertaken for the following reason: (i) Epidermis was prepared by a method in which no particular steps were taken to exclude enzyme inactivation. (ii) Putrescine concentrations can be measured more precisely than low activities of OrnDC. (iii) Topical application of an irreversible inhibitor prevents enzyme activity determinations, because its complete removal before assay cannot be ensured. The systemic or topical administration of DFMOrn, presumably the most potent irreversible inhibitor of OrnDC presently available, showed significant effects on putrescine formation. It lowered the basal rate of putrescine formation after chronic administration, and it completely blocked its induced formation. This is another example of the effectiveness of this compound in an in vivo system. Previously it had been shown that DFMO inhibits OrnDC and decreases putrescine levels in the spleen of leukemia-inoculated mice (54), and C. Danzin showed that prostatic putrescine concentrations are very considerably lowered by this compound (47). The uv-induced changes of spermidine and spermine metabolism in epidermal cells are quite dramatic. From the data of Tables 2 and 3 it can be seen that the turnover rates of spermidine and spermine were considerably increased. The decrease of the specific radioacitivity of spermine upon uv irradiation shows that spermine is synthesized, despite the significant decrease of the total spermine pool in drug-free animals. It shows further that the newly formed spermine stems mainly from newly formed (nonradioactive) spermidine, rather than from the preexisting (labeled) compound. The considerable decrease of the specific radioactivity of spermidine, both in the drug-treated and drug-free epidermis, is indicative of rapid spermidine formation. To what extent this is achieved via the normal synthetic pathway, or by breakdown of spermine, cannot be answered precisely, because of the relatively low specific radioactivity of spermine, in relation to that of spermidine under the experimental conditions. But one can conclude from the figures of Tables 2 and 3, that the main source of spermidine is the biosynthetic pathway. The somewhat increased specific radioactivity of spermidine and spermine in the epidermis of the DFMOrn-treated animals before uv irradiation (Table 3) and the decrease of spermidine concentration (Table 2) together with a somewhat less marked decrease of its specific radioactivity after uv irradiation (Table 3) indicates that DFMO treatment decreases both normal and ultraviolet-induced spermidine and spermine turnover rates. The restricted availability of putrescine is most probably the limiting factor for spermidine formation in the induced state. Spermine formation from spermidine seems not to be greatly influenced under the experimental condi-

EPIDERMAL

POLYAMINE

METABOLISM

179

tions, and spermine degradation is apparently decreased, so that no net loss of spermine is observed in the DFMOm-treated animals. Although the epidermal changes of polyamine metabolism due to treatment with DFMOrn were significant, they were apparently not sufficient to significantly affect DNA synthesis and cell proliferation rate. From the work with cultured rat hepatoma cells we know (46) that a marked depletion of spermidine is required before growth rates are affected. The simplicity and reproducibility of the in viva model of uv lightinduced cellular proliferation used in the present work recommends itself for further studies of the still not clarified interrelations between polyamines and DNA synthesis and the roles of the polyamines in the regulation cell growth and proliferation. SUMMARY Exposure of the dorsal skin of hairless mice to the light of a germicidal lamp under standardized conditions produces reproducible, timedependent increases of putrescine and spermidine concentrations, and a decrease of spermine levels, concomitant with the increase of the rate of DNA synthesis. Systemic or topical treatment of the animals with a-difluoromethylomithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, completely prevents the uv light-induced rapid formation of putrescine, and diminishes the enhancement of polyamine turnover rates. A significant effect of the drug on the rate of DNA synthesis could not be shown. The model is suitable for the study of interrelations between polyamine metabolism and cell proliferation and for the screening of compounds designed as inhibitors of polyamine biosynthesis. ACKNOWLEDGMENT We appreciate very much the advice and assistance of Dr. P. J. Schechter in establishing a strain of hairless mice in our Center, as well as the preparation of the topical drug. Our thanks are also due to Dr. J. Grove for the determination of polyamines.

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2. Bachrach, U., “Function of the Naturally Occurring Polyamines.” Academic Press, New York, 1973. 3. Raina, A., and Jgnne, J., Med. Bid. 53, 121 (1975). 4. Morris, D. R., in “Advances in Polyamine Research” (R. A. Campbell, D. R. Morris, D. Bartos, G. D., Daves, Jr., and F. Bartos, Eds.), Vol. 1, p. 10.5. Raven Press, New York, 1978. 5. JSinne, J., P&ii, H., and Raina, A., Biochim. Biophys. Acra 473, 241 (1978). 6. Rupniak, H. T., and Paul, D., in “Advances in Polyamine Research” (R. A. Campbell, D. R. Morris, D. Bartos, G. D. Daves, Jr., and F. Bartos, Eds.), Vol. 1, p. 117. Raven Press, New York, 1978.

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Yuspa. S. H., Lichti, U., Ben, T., Patterson, E., Hennings, H., Slaga, T. J., Colbom, N., and Kelsey, W., Nature