[CANCER RESEARCH 50, 2631-2635, May 1, 1990]

Ultraviolet Radiation Induction of Ornithine Decarboxylase in Rat Keratinocytes1

Cheryl F. Rosen,2 Dragan Gajic, and Daniel J. Drucker

Department of Medicine, Women's College Hospital [C. F. R., D. G./, and Departments of Medicine, Clinical Biochemistry, and Genetics, Toronto General Hospital

ID. J. D.J, University of Toronto, Toronto, Ontario, Canada M5S 1B6

ABSTRACT

UV radiation plays an important role in the induction of cutaneousmalignancy, including basal cell and squamous cell carcinomas andmalignant melanoma. In addition to its effects on DNA damage andrepair mechanisms, UV radiation has been shown to modulate theexpression of specific genes, altering the levels of their mRNAs and thesynthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied theregulation of ornithine decarboxylase (ODC) gene expression in responseto UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicatedin carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern

blot analysis of keratinocyte RNA under basal conditions demonstratedthe presence of two ODC mRNA transcripts. Increasing exposure toUVB resulted in a dose-dependent increase in the levels of both ODCmRNA transcripts. The induction of ODC gene expression followingUVB was noted 2 h after UVB exposure, and ODC mRNA levelscontinued to increase up to 24 h after UVB exposure. The UVB-inducedincrease in ODC gene expression was not serum dependent, despite theability of serum alone to induce ODC gene expression. The mRNAtranscripts for actin and hexoseaminidase A were not induced after UVBexposure. These studies show that the UVB-induced increase in ODCactivity is due, at least in part, to an increase in ODC gene expressionand they provide a useful model for the analysis of the molecular effectsof UVB radiation.

INTRODUCTION

UVR3 produces multiple alterations in cutaneous biology,

including inflammation, proliferation of keratinocytes andmelanocytes, and induction and/or exacerbation of diseases ofthe skin. UVR has also been shown to be an environmentalcarcinogen, implicated in the development of cutaneous malignancy, including basal cell and squamous cell carcinoma andmalignant melanoma. UV radiation has been arbitrarily dividedinto three regions according to wavelength: UVA (320-400nm), UVB (290-320 nm), and UVC (200-290 nm). Althoughsolar UV radiation contains UVA, UVB, and UVC, only UVAand UVB are present in terrestrial sunlight. Exposure of skinto these wavelengths can produce significant damage to cutaneous structures. Both UVA and UVB are capable of damagingDNA, resulting in the production of cyclobutane pyrimidinedimers and (6-4)thymine photoproducts. Furthermore, bothUVA and UVB have been shown to be mutagenic in severalmodel systems (1, 2).

Despite the accumulating body of evidence implicating UVRexposure in the pathogenesis of tissue injury and cutaneousdisease, the molecular and cellular basis for the multiple effects

Received 9/11/89; revised 12/7/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by grants from the Cancer Research Society,Ontario Ministry1 of Health Grant to Dermatology. Medical Research Council,and the National Cancer Institute of Canada. D. J. D. is a Career Scientist of theOntario Ministry of Health.

2To whom requests for reprints should be addressed, at Women's College

Hospital, 60 Grosvenor St., Suite 307, Toronto, Ontario, Canada M5S 1B6.3The abbreviations used are: UVR, UV radiation; ODC. ornithine decarbox

ylase; TPA, 12-O-tetradecanoylphorbol-13-acetate; cDNA, complementary DNA.

of UV radiation remains incompletely understood. The resultsof recent experiments have suggested that UVR may exert itseffects in part through the modulation of the transcriptionalactivity of specific genes. UVC, perhaps the best studied of theUVR wavelengths, has been shown to exert its effects at thelevels of both mRNA translation and gene transcription (3-7).Considerably less information is available with regard to thespecific mechanisms of action of UVA and UVB, the regionsof the UV spectrum known to be important in the pathogenesisof cutaneous disease.

In order to gain further information about the moleculareffects of UVB, we have been studying the UVB induction ofthe enzyme ODC in a newborn rat keratinocyte cell line (8, 9).ODC is the first enzyme in the mammalian polyamine biosyn-thetic pathway, forming putrescine by the decarboxylation ofornithine. The polyamines (spermine, spermidine, and putrescine) play an important role in cell growth and differentiationand have been implicated in the process of carcinogenesis (10,11) ODC enzyme activity has been shown to be induced bymany stimuli, including androgens (3), serum (12), UVB radiation (13-15), c-Ha-ras oncogene transfection (16), polyaminedeprivation (17), and phorbol esters (18, 19). The regulation ofODC activity has also been shown to occur at different levels,including ODC gene transcription (3, 4), regulation of mRNAstability, mRNA translation (5-7), and protein stability (16, 20,21), and post-translational interactions and modifications (22,23). We report that exposure of rat keratinocytes to UVB resultsin a dose-dependent increase in ODC activity, which is associated with a specific and dose-dependent increase in the expression of the ODC gene.

MATERIALS AND METHODS

Materials. Tissue culture media and calf and fetal bovine sera wereobtained from GIBCO (Burlington, Ontario) DL-[l-'4C]ornithine wasfrom Amersham Corp. (Oakville, Ontario). [a-32P]-ATP (>800 Ci/

mmol) was from ICN Radiochemicals (Montreal, Quebec). Restrictionenzymes and DNA polymerase were from Pharmacia LKB Biotechnology Inc. (Montreal, Quebec). All chemicals were from Sigma (St. Louis,MO) or Fisher (Markham, Ontario). Nitrocellulose membranes wereobtained from Schleicher and Schuell (Montreal, Quebec). The mouseODC cDNA (pODClo) was kindly provided by Dr. O. Janne, ThePopulation Council and Rockefeller University (New York, NY). Thehexoseaminidase A cDNA was a gift from Dr. R. G. Korneluk and Dr.J. A. Lowden, Hospital for Sick Children, University of Toronto,(Toronto, Ontario). The chicken actin cDNA was obtained from Dr.D. Cleveland, Johns Hopkins University (Baltimore, MD).

Cell Culture. Newborn rat keratinocytes were kindly provided by Dr.H. Baden (Massachusetts General Hospital, Boston, MA) (9). Thesecells continue to produce several macromolecules characteristic ofdifferentiated keratinocytes, such as desmosomes and cornified envelopes. They have not undergone malignant transformation, inasmuchas they did not form colonies when inoculated onto soft agar and didnot produce tumours on inoculation into newborn rats (9). Cells weregrown in Dulbecco's minimal essential medium, supplemented with 1%

hydrocortisone and either 5% fetal bovine serum and 10% calf serum(designated regular serum) or 0.05% fetal bovine serum and 0.1% calfserum (designated low serum). Cells were grown under an atmosphereof 5% CO2 at 37°C.Unless otherwise noted, experiments were carried

out on cells passaged fewer than 20 times.2631

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ULTRAVIOLET B RADIATION INDUCTION OF ODC

UV Irradiation. The light source consisted of two Westinghouse FS-20 bulbs, which emit UV radiation between 280 and 380 nm with aprincipal emission between 290 and 320 nm, peaking at 313 nm.Irradiance measurements were determined prior to each experimentusing an IL-500 radiometer with a SEE-240 DVB probe, with an IL-UVB filter. At least four irradiance measurements were taken in eachfield site to ensure uniform irradiance, within a range of ±10%.beforeeach set of exposures. The mean irradiance was 0.05 mW/cm2. The

distance between the light source and the probe was the same as thatbetween the source and the tissue culture plates during irradiation.Immediately prior to irradiation, the culture medium was removed andreplaced with phosphate-buffered saline. The plastic lids were removedfrom the tissue culture plates for the duration of the irradiation.Measurement of medium temperature demonstrated less than a 0.1 °C

change in temperature following irradiation. After irradiation, thesaline was replaced with culture media containing either regular or lowserum as specified. Sham-irradiated keratinocytes were treated identically but not exposed to UVR.

Assay of ODC Activity. For the determination of ODC activity,keratinocytes were rinsed with phosphate-buffered saline and scrapedwith a rubber policeman into 1.5 ml of ODC assay buffer, consistingof 50 mM Tris-HCl, 1 mivi EDTA, and 15 mivi dithiothreitol, at pH7.8. Cells were sonicated while on ice and centrifuged for l h at 15,000x g at 4°C.ODC activity in the clear supernatant was determined bymeasuring the release of I4CO2from DL-[l-uC]ornithine, as previouslydescribed (14). The final assay mixture contained 50 m\i Tris-HCl, 1.0mM EDTA, 0.1 m.M pyridoxal phosphate, 15 mM dithiothreitol, 0.4mM L-ornithine, and 0.4 pCi DL-jl-'^CIornithine, including 188 n\ of

the supernatant in a final volume of 200 pi. The reaction was carriedout in 15-ml snap-top tubes (Falcon), in which a paper disc (Schleicherand Schuell, Keen, NH) soaked in toluene and NCS (Amersham) wassuspended on a 22-gauge needle. Each sample was incubated at 37°Cfor 60 min, with the released UCO2 being absorbed onto the paper disc.

The reaction was stopped by the addition of 0.5 ml of 2 M citric acid.The incubation was then continued for a further 60 min to allow forcomplete absorption of the 14CO2.The discs were then transferred to

vials containing 10 ml Omnifluor (DuPont). Radioactivity was measured in a Beckman liquid scintillation counter. Assays were alwayscarried out in triplicate. Protein content was determined using theBradford assay (24), with bovine serum albumin as the standard.

RNA Analysis. Total cellular RNA was isolated as previously described (25). RNA was size fractionated by formaldehyde-agarose gelelectrophoresis, stained with ethidium bromide to assess the integrityand migration of the RNA, and transferred to a nitrocellulose membrane (Schleicher and Schuell). The RNA was fixed to the membraneby UV irradiation. cDNA probes for mouse ODC, chicken actin, andhuman hexoseaminidase A were labeled by the random priming technique to a specific activity of approximately 5 x IO8cpm/pg (26). Afterovernight prehybridization in Ix Denhardt's, 4x saline sodium citrate

(20 x stock, 175.3 gm/1 NaCl, 88.2 gm/1 Na citrate, pH 7.0), 200 /ig/ml salmon sperm DNA, 40% deionized formamide, 5% dextran sulfate,0.014 MTris, pH 7.4, hybridization was performed in the same solutionwith 1 x 10' cpm/ml 32P-labeled cDNA probe, for 24 h at 42°C.Final

washing conditions were O.lx saline sodium citrate, 0.1% sodiumdodecyl sulfate, at 50°C.Autoradiography was performed using KodakX-Omat film at —70°C.Autoradiograms were quantified by scanning

with a laser densitometer.

RESULTS

To define an appropriate dose range of UVB exposure forthe analysis of the UVB induction of ODC enzyme activity, weexamined the effects of UVB radiation on the growth of keratinocytes in vitro. Following exposure of keratinocyte culturesto 6 mJ/cm2 or greater, more than 80% of the cells did notsurvive more than 24 h. After exposure to 5 mJ/cm2, 70% ofcells survived, while, after receiving a dose of 2 mJ/cm2, 90%

of cells remained viable. Accordingly, we examined the effectsof a range of UVB doses, varying from 1 to 5 mJ/cm2, on the

induction of ODC activity in subconfluent 10-cm plates of ratkeratinocyte cultures (2-3 x IO6 cells/plate). Cells were har

vested for the measurement of ODC activity 24 h followingexposure to UVB. The results of these experiments are shownin Fig. 1. A significant increase in ODC enzyme activity wasnoted following exposure of keratinocytes to only 1 mJ/cm2,

the lowest dose of UVB tested. Exposure of keratinocytes toUVB doses of 2, 3, and 4 mJ/cm2 resulted in approximately 3-

fold increases in ODC enzyme activity, wherease exposure to 5mJ/cnr produced a 5-fold increase in ODC activity (Fig. 1).Previous studies have shown a good correlation between ODCenzyme activity and the amount of enzyme present, as quantified by radioimmunoassay (19, 20, 27). In view of the knowneffects of serum on the induction of ODC enzyme activity inother cell lines (12), we analyzed the effects of serum on theUVB-induced increase in ODC enzyme activity in rat keratinocyte cultures. The increase in ODC activity following UVBexposure was detected both in cells incubated in medium containing regular serum, as depicted in Fig. 1, and in cells incubated in medium supplemented with low serum (see "Materialsand Methods") concentrations (data not shown).

To determine whether the UVB-induced increase in ODCenzyme activity was associated with an increase in ODC geneexpression, total cellular RNA from irradiated and sham-irradiated keratinocytes was isolated 24 h following exposure todifferent doses of UVB. Northern blot analysis of keratinocyteRNA demonstrated the presence of two distinct ODC mRNAtranscripts, approximatley 2.2 and 2.7 kilobases in size (Fig.2A). Two distinct ODC mRNA transcripts have been demonstrated in a number of different tissues (19, 28, 29) and havebeen shown to differ from each other in the 3'-untranslated

region of the ODC mRNA transcript (30). An increase in thelevels of both ODC mRNA transcripts was seen after exposureto the smallest dose of UVB tested, 1 mJ/cm2. The levels of

ODC mRNA transcripts increased progressively following exposure to increasing doses of UVB. An approximately 7-foldincrease above control was noted with the two highest dosestested, 4 and 5 mJ/cm2 (Fig. 2, A and C). To determine therelative specificity of the UVB-induced increase in ODC geneexpression, we examined the effects of UVB radiation on theexpression of two additional genes. The Northern blot depictedin Fig. 2A was rehybridized sequentially with cDNA probes foractin, a constitutively expressed housekeeping gene, and hex-oxeaminidase A, a gene which encodes a lysosomal enzyme.

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UVB (mJ/cm2)Fig. 1. Effect of increasing doses of UVB on ODC activity in rat keratinocytes.

Keratinocytes in culture were irradiated with UVB (1-5 mJ/cm2) or shamirradiated (0 mJ/cm2). ODC activity was measured 24 h after irradiation and isreported as nmol/CO2/mg protein/h ±SE. Each value represents the mean ofthree separate experiments.

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ULTRAVIOLET B RADIATION INDUCTION OF ODC

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Fig. 2. Northern blot analysis of rat keratinocyte RNA following exposure todifferent doses of UVB irradiation. Total cellular RNA was isolated from ratkeratinocytes 24 h following exposure to 0 (control. C), 1, 2, 3, 4, and 5 mj/cm2

UVB and analyzed by Northern blot analysis. Each lane contains 10 ¡i%of totalcellular RNA. Blots were hybridized with cDNA probes for ODC and actin (A),in A, and hexoseaminidase A (//), in B. The minor transcript variably detectedbelow the 18S RNA band has not been further characterized. Blots were exposedto Kodak X-Omat film for 24 h. ODC mRNA transcript levels were quantitatedby scanning the autoradiograms with a laser densitometer (C).

The effects of UVB exposure on the levels of actin and hexoseaminidase mRNA transcripts are shown in Fig. 2, A and B,respectively. Only a small (< 1.5-fold) increase in actin mRNAtranscripts was detected following UVB exposure. In contrast,the levels of hexoseaminidase A mRNA transcripts remainedconstant, even after exposure of keratinocytes to doses of UVBas high as 5 mJ/cm2. The results of these experiments demonstrated that the UVB-induced increase in ODC gene expressionwas relatively specific and not a generalized cellular responseto UVB radiation.

To delineate the time course of the UVB-induced increase inthe levels of ODC mRNA transcripts, keratinocytes were irradiated with 3 mJ/cm2 and total cellular RNA was isolated at

multiple time points following exposure to UVB (Fig. 3). Totalcellular RNA was also isolated from control sham-irradiated

cells at each time point. An increase in ODC mRNA levelsfollowing UVB exposure was detected at 2 h, the first timepoint studied. Maximum UVB induction of ODC mRNA levelswas seen by 12 h, and the amounts of ODC mRNA transcriptsremained relatively constant through 48 h (48 h time point notshown). In contrast to the rapid and time-dependent increasein the levels of ODC mRNA transcripts following UVB exposure, no significant changes in the levels of either actin (Fig. 3)or hexoseaminidase A (data not shown) mRNA transcripts weredetected under the same experimental conditions.

The results of the experiment shown in Fig. 3 indicated thatthe levels of mRNA transcripts in control cells tended to beconsistently higher immediately after sham irradiation, following which they declined to lower control levels by 12 h. In viewof the well known stimulatory' effect of serum on ODC enzymeactivity and ODC mRNA levels in different cells (12) and sincethe sham-irradiated cells were replenished with fresh mediumcontaining serum, we examined whether serum alone increasedthe expression of the ODC gene in rat keratinocytes. Cells werepreincubated under low serum conditions for 12 h, followingwhich half the cells were then exposed to UVB radiation (3mJ/cm2). After irradiation, half the cells were continued in low

serum, whereas the other half were switched to medium containing regular serum. Sham-irradiated controls were treated inan identical fashion. Sham-irradiated control cells which wereswitched to regular serum had slightly higher (1.3-1.6-fold)levels of ODC mRNA transcripts (Fig. 4; 12 h time pointshown). This effect of serum was also detected in UVB-irradi-ated cells. However, the increase in the levels of ODC mRNAtranscripts following UVB radiation was detected in cells grownin either regular or low serum. These observations demonstrated that the UVB induction of ODC gene expression wasrelatively independent of the serum concentration present inthe medium.

DISCUSSION

UV radiation has been shown to induce ODC enzyme inkeratinocytes, both in vivo and in vitro. Studies in mice haveshown that epidermal ODC activity increases following bothsingle and multiple UVB exposures, in a dose-dependent manner (13). The increase in ODC activity occurred prior to theincrease in DNA synthesis, with a significant increase in ODCenzyme activity noted 2 h after irradiation and a peak at 24-28h (14). The induction of ODC activity was found to be wavelength dependent in the intact mouse, with peak effectivenessoccurring at 290 nm (15, 31). No stimulation of ODC activitywas observed following exposure to 315 nm. These observationssuggested that the interactions between UVR and the subsequent induction of ODC activity might provide a useful modelfor the study of the molecular effects of UV radiation.

The effects of UVR on the regulation of ODC gene expression have not been previously reported. In contrast, a great dealof evidence implicates an important role for a protein kinaseC-dependent pathway in the regulation of ODC gene expression. Incubation of hamster fibroblasts with the tumor promoterTPA leads to an increased accumulation of ODC mRNA transcripts and increased ODC enzyme activity (19). In a murinemodel, TPA-induced cutaneous tumors were found to have

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ULTRAVIOLET B RADIATION INDUCTION OF ODC

6 12 24

Fig. 3. Northern blol analysis of time-dependent increases in ODC mRNA transcriptlevels following DVB radiation. Keratinocytesgrown in low serum medium »ereexposed to 3mj/cm2 UVB or sham-irradiated (C). Total cel

lular RNA was isolated at 2, 6. 12, and 24 h,and 10 jig of RNA were used for Northern blotanalysis. The blot was hybridized with cDNAsfor ODC and actin (A) and exposed for 12 h.

UV UV

iuv C UV

LS RSUV UV

•fFig. 4. Effect of serum on basal (C) and UV-stimulated levels of ODC mRNA

transcripts. Keratinocyte cultures were grown in low serum medium for 12 hprior to irradiation. After irradiation with 3 mJ/cm2 UVB, one group (LS) was

returned to low serum medium. The other group (US) was subsequently grownin medium containing regular serum after irradiation. Twelve h following exposure to UVB, total cellular RNA was isolated and analyzed by Northern blotanalysis with a cDNA probe for ODC.

constitutively elevated levels of ODC activity and mRNA transcripts (18). Compounds which inhibited the induction of ODCactivity by TPA (indomethacin and retinoic acid) were alsofound to inhibit the formation of murine skin tumors (32, 33).Taken together, these observations suggest a possible role forODC in the progression to cutaneous neoplasia.

Additional insights into the control of ODC gene expressionhave come from studies examining the effect of serum andmitogens on the levels of ODC mRNA transcripts. Both serumstimulation and treatment with mitogens and TPA resulted inan increase in the transcription of the ODC gene in mousefibroblasts (4). Withdrawal of serum from BCjH 1 muscle cellsled to a rapid decrease in ODC gene transcription; mitogenicstimulation or protein synthesis inhibition rapidly reinducedODC mRNA levels in these cells, due to an increase in ODCgene transcription. In contrast, different results were obtained

from studies of ODC gene transcription using mouse fibroblasts. Inhibition of protein synthesis strongly inhibited theserum-induced increase in ODC gene transcription, despite theconcomitant superinduction of mRNA transcripts for the proto-oncogenes c-fos and c-myc (4). The UVR induction of theexpression of certain nuclear oncoproteins, including c-myc andc-fos, in human keratinocytes also provides additional evidence

for a close link between the cellular response to UVR and theregulation of cellular proliferation (34).

A number of different studies have examined the effects ofUV radiation on the regulation of gene expression. Irradiationof primary human keratinocyte cultures with UVC (50 J/m2)

produced small but significant qualitative and quantitativechanges in protein synthesis (35). Total mRNA synthesis wasreduced, although Northern blot analysis revealed a highlyselective 2- to 5-fold increase in the level of specific mRNAs(36). Further characterization of the identity of these UVC-induced mRNA species has identified a group of keratins (36)and small proline-rich proteins (35) as members of a UV-inducible gene family. These studies demonstrated that UVC iscapable of inducing protein synthesis and increasing mRNAlevels in a highly selective manner. Among the proteins inducedby UVC, at least two have been shown to be secreted proteinscapable of inducing the synthesis of the UV-induced group ofproteins and mRNAs in previously unexposed cells (37, 38).UVC has also been shown to activate a quiescent gene codingfor metallothionein-1 in murine lymphoma cells, conferringcadmium resistance. The fully expressed gene was found tohave been extensively demethylated after UVC exposure, suggesting that UV-induced gene transcription may be associatedwith altered DNA methylation (39).

Much less is known about the effects of UVA and UVB onthe regulation of gene expression. UVA has been shown toinduce the synthesis of a M, 32,000 protein in normal humanfibroblasts, but the mechanisms responsible for this inductionremain unknown (40, 41). Exposure of human keratinocytes toeither UVA or UVB was much less effective than UVC ininducing the expression of the c-H-ras and c-myc genes inhuman keratinocytes (34). In contrast, human keratinocytes

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ULTRAVIOLET B RADIATION INDUCTION OF ODC

exposed to UVB produced increased amounts of interleukin 1activity, which were associated with increased amounts of interleukin 1 mRNA transcripts (40). The doses of UVB whichwere used in the latter study were much higher than those usedin our experiments and may be due to the irradiation protocol,which involved irradiating the cells through one layer of tissueculture plastic in medium supplemented with 20% serum. Under these conditions, the effective dose of UVB reaching thecells would almost certainly be reduced, necessitating higherdoses. The possibility that human keratinocyte cultures may bemore radioresistant than the newborn rat keratinocyte cellsused in the present study cannot be excluded.

The results of our studies demonstrate that increased expression of the ODC gene accounts, at least in part, for the increasein ODC activity following UVB exposure. Whether UVB radiation results in an increase in ODC gene transcription and/or a change in the stability of ODC mRNA transcripts is notpossible to infer from our current experiments. We proposethat exposure of mammalian cells to UVR may result in theactivation of a subset of cellular genes by stimulating theinteraction of specific /rans-acting factors with as-acting UVresponse elements in the regulatory regions of UV-induciblegenes. Further analysis of the molecular mechanisms whichunderlie the UVB-induced increase in ODC gene expression inrat keratinocytes should increase our understanding of thecellular effects of UVR and may provide a useful model systemfor the study of the molecular effects of UV radiation.

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1990;50:2631-2635. Cancer Res   Cheryl F. Rosen, Dragan Gajic and Daniel J. Drucker  Rat KeratinocytesUltraviolet Radiation Induction of Ornithine Decarboxylase in

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