[CANCER RESEARCH 48, 3045-3049, June l, 1988J

Differential Expression of the Cytochrome P-450c and P-450d Genes inthe Rat Ventral Prostate and Liver1

Peter Söderkvist,2'3Lorenz Poellinger,3 Rune Toftgárd,and Jan-Õke Gustafsson

Department of Medical Nutrition, Karolinska Institute! [P. S., L. P., K. T., J-À.G] and the Center for Biotechnology [R. T.], Huddinge University Hospital F-69,S-I41 86 Huddinge, and Department of Occupational Medicine, Faculty of Health Sciences, S-581 85 LinkópingfP. S.J, Sweden

ABSTRACT

Complementary DNA clones specific for cytochromes P-450c and P-

450d were used to study the expression of corresponding mRNAs in therat ventral prostate. Following treatment with /3-naphthoflavone (BNF),an increase in total cellular levels of cytochrome P-450c mRNA was

observed in this tissue. The induction kinetics were similar in both ratliver and prostate with regard to the expression of the cytochrome P-

450c gene. The mRNA levels reached a maximum at 16 h and decreasedto control levels at about 48 h. In contrast, mRNA specific for cytochromeP-450d was detectable only in the liver following BNF treatment (maximum at 24 h). The tissue-specific expression of cytochromes P-450c andP-450d was further investigated using measurements of nuclear transcription in vitro. RNA transcripts specific for cytochrome P-450c were

detected in nuclei from both liver and prostate following BNF induction.The rate of cytochrome P-450c transcription was maximal at 4 h and 8-

12 h in the liver and prostate, respectively. In the liver, induction by BNFof the rate of transcription of the cytochrome P-450d gene occurred at aslightly later time point as compared to cytochrome P-4SOc gene expression. No elongation of RNA specific for cytochrome P-450d could be

detected in nuclei from the ventral prostate indicating a transcriptionalcontrol of cytochrome IMSOd gene expression in this tissue.

INTRODUCTION

The etiology of prostatic cancer is largely unknown despitethe fact that it is one of the most common cancers in the male.Among other mechanisms, a chemical etiology has been suggested from epidemiological studies (1,2). Chemicals that haveinduced prostatic carcinoma in rats and mice includebenzo(a)pyrene (3), 3-methylcholanthrene (4), and W-nitroso-/V-methylurea (5). Recently, it has been shown that administration of 3,2'-dimethyl-4-aminobiphenyl in the absence or pres

ence of estrogen results in a high incidence of adenocarcinomain rats (6, 7).

We have long been interested in whether the prostatic glandexpresses enzymes necessary for the metabolic activation ofchemicals, i.e., PAH." This activation is generally thought toplay an important role in drug toxicity and chemical carcino-genesis (8). Treatment of rats with BNF or TCDD results in aninduction of the cytochrome P-450-dependent activity aryl hydrocarbon hydroxylase as well as immunodetectable levels ofBNF-inducible cytochrome P-450s (9-11). Moreover, this induction in the prostate has been shown to be accompanied byan increased capability to form mutagenic metabolites from the

Received 7/14/87; revised 1/4/88; accepted 2/8/88.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.

1This work was supported by grants from the Swedish Cancer Society, and

the National Cancer Institute (CA 40037).2To whom requests for reprints should be addressed, at the Department of

Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, F-69, S-141 86 Huddinge, Sweden.

3P. S. is a recipient of a research fellowship from the Swedish Natural ScienceResearch Council; L. P. is a recipient of a research fellowship from the SwedishMedical Research Council.

4The abbreviations used are: PAH, polycyclic aromatic hydrocarbons; TCDD,dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin; BNF, 0-naphthoflavone; PSP, prostatic steroid-binding protein; cDNA, complementary DNA; SSPE, NaCI-sodiumphosphate-EDTA; SSC, NaCI-sodium citrate.

promutagens 2-aminofluorene and benzo(a)pyrene as estimatedby the Ames' Salmonella /microsome assay (12).

PAH, such as benzo(a)pyrene, 3-methylcholanthrene, andTCDD, induce two distinct forms of cytochrome P-450 in ratliver, cytochromes P-450c and P-450d, which have an overallidentity in the coding nucleotide sequence of 75% (13). Theexpression of the cytochrome P-450c and P-450d genes appearsto be mediated by a specific intracellular receptor, the dioxinreceptor, which represents a MT = 100,000 DNA-binding protein similar to steroid hormone receptors (14-17). The ratventral prostate contains both the dioxin receptor and a highlyabundant M, ~ 40,000 protein, PSP (18), which constitutes upto 20% of the total protein content of rat prostatic cytosol andwhich binds TCDD reversibly and with high affinity. In thepresent study, we have investigated the pattern of induction inthe rat prostate of PAH-inducible cytochrome P-450s at themRNA prevalence and transcription level. In addition, we havebeen interested in whether the high concentration of a carcinogen-binding protein such as PSP affects the induction kineticsof cytochrome P-450 gene expression in the prostate as compared to the liver.

MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats weighing 200-300 g were usedthroughout this study. The animals were kept on a standard diet (EwosR3; EWOS, Södertälje,Sweden) ad libitum and had free access towater. For isolation of total RNA or nuclei, the animals received asingle i.p. injection of /3-naphthoflavone (80 mg/kg body weight in cornoil) at different time points prior to sacrifice. Rats were sacrificed bycervical dislocation.

Recombinant Plasmids. Plasmid p210 carries a 758-base pair sequence recognizing the 3'-noncoding region (bases 1822 to 2S80) of

the cytochrome P-450c mRNA; plasmid p72 contains an insert corresponding to a unique noncoding sequence in the 3'-end of the cytochrome P-450d mRNA (bases 1506 to 1771), cloned into pBR322 (19).These probes were kindly provided by Dr. J. Fagan (National CancerInstitute, Bethesda, MD). The cDNA clone pR17 recognizes two phe-nobarbital-induced rat cytochrome P-450s (P-450b and P-450e) andcontains an insert encoding the COOH-terminal 211 amino acids ofcytochrome P-450e, where differences between cytochromes P-450eand P-450b are found (20). A cDNA probe for 0-actin was obtainedfrom Cleveland et al. (21).

Isolation of RNA and Nuclei. Total cellular RNA was isolated byhomogenization of liver or prostatic tissue in 4 M guanidinium thiocy-anate and centrifugation in cesium chloride as described (22). RNAconcentrations were determined from the absorbance at 260 nm. Nucleiwere prepared from the liver and ventral prostate essentially as described (23). Briefly, the tissues were homogenized in 2 volumes of 50HIMTris-HCl-0.25 M sucrose, pH 7.5 (containing 25 mM KC1 and 5mM MgCl2), and mixed with 2 volumes of 50 mM Tris-HCl-2.3 Msucrose, pH 7.5 (containing 25 HIM KC1 and 5 mM MgCl2). Theresulting solution ( -3.5 ml) was then layered onto a I nil cushion of50 mM Tris-HCl-2.3 M sucrose, pH 7.5 (containing 25 mM KC1 and 5mM MgCl2), and centrifuged at -200,000 x g for 1 h. The purifiednuclear pellet was washed twice and resuspended in 50 mM Tris-HCl(pH 8.3)-40% (w/v) glycerol-5 mM MgCl2-0.1 mM EDTA. The hepaticand prostatic nuclei prepared in this fashion were analyzed by electron

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GENE EXPRESSION OF CYTOCHROME P^ÕSOcIN PROSTATE

microscopy and were found to be intact and free of significant contamination with extranuclear cellular components (data not shown). Purified nuclei were quickly frozen in aliquots and stored at -70'C for later

use.RNA Blot Analysis. In RNA gel blot experiments, equal amounts of

heat- and formamide-denatured total cellular RNA were electropho-resed in 1.1% agarose gels containing 25% formaldehyde. The RNAwas transferred by capillary flow to nitrocellulose [Schleicher andSchuell] in 20x SSPE [Ix SSPE is 180 HIM NaCl-10 HIM sodiumphosphate (pH 7.7)-l mM EOT A]. Blots were prehybridized at 42°C

for 16 h in hybridization buffer, i.e., 50% formamide, 5x SSPE, 0.1%sodium dodecyl sulfate, 0.5 mg/ml yeast tRNA, and 2x Denhardt'ssolution [Ix Denhardt's solution is 0.02% (w/v) each of bovine serum

albumin, polyvinylpyrrolidone, and Ficoll). Filter hybridization wasperformed for 16 h at 42'C in hybridization buffer containing 1-2 x10* cpm/ml of radioactive probe. Probes were labeled with [32P]dCTP

(3000 Ci/mmol; Amersham, England) by nick translation (24) to aspecific activity of ~400 x IO6 dpm/ng. The filters were washed at

room temperature for 4 x 15 min twice with 2x SSC (ix SSC is 0.15M NaCl-0.015 M sodium citrate) containing 0.1% sodium dodecylsulfate and twice with O.lx SSC containing 0.1% sodium dodecylsulfate and autoradiographed. For slot blot experiments, RNA wasimmobilized on nitrocellulose with the use of a slot blot apparatus(Schleicher and Schuell), after which the filters were hybridized with"P-labeled probes and washed as above. The signal intensity of theautoradiograms was measured densitometrically with a Shimadzu Dual-Wavelength TLC scanner CS-930 (Kyoto, Japan).

Nuclear Transcription Analysis. Measurements of transcription inisolated nuclei were carried out essentially as described (25). The nucleiwere thawed, and 60 n\ of nuclei corresponding to 100 ^g of DNA weremixed with an equal volume of 10 mM Tris-HCl, pH 8.0 [containing 5mM MgCl2; 0.3 M KC1; 2 units of RNasin/^il (a ribonuclease inhibitorfrom Promega Biotec, Madison, WI); 2 mM dithioerythritol; 0.5 mMATP, CTP, and GTP; 20 ^M UTP; 250 /iCi [a-32P]UTP (3000 Ci/mmol; Amersham, England)], and incubated at 25°Cfor 30 min. A

time course indicated that no further RNA synthesis occurred after 30min. RNA was extracted from the nuclei as described (26). The RNAwas hybridized for 48 h at 42°Cto linearized plasmid DNA (3 ¿ig).TheDNA was denatured in 0.1 M NaOH at 100'C for 5 min, diluted with

1.5 volumes of 20x SSPE, and applied to nitrocellulose using aSchleicher and Schuell slot blot apparatus. The hybridization andwashing conditions were the same as described above. The autoradi-

ographs were analyzed by densitometry as above.

RESULTS

The expression of the genes for cytochromes P-450c and P-450d in the rat ventral prostate was investigated. Rats weregiven 80 mg BNF/kg body weight i.p., after which cellularcytochrome P-450c and P-450d mRNA levels were analyzed byRNA gel blot-hybridization. In nontreated animals, no mRNAcoding for cytochromes P-450c or P-450d could be detected inthe ventral prostate (Fig. 1, Lanes a and d, respectively). Following treatment with BNF for 16 h, a cytochrome P-450cmRNA species of ~2.9 kilobases was induced (Fig. 1, Lane b).In contrast, no cytochrome P-450d mRNA could be detectedin total cellular RNA extracted from the prostate of treatedanimals (Fig. 1, Lane e).

In order to investigate any tissue-specific differences in thetime course of the induction response or in apparent stabilityof the different mR NAs, the induction kinetics for total cellularcytochrome P-450c and P-450d mRNA levels were studied inthe ventral prostate and compared to those of the liver. CellularmRNA levels were analyzed by RNA slot blot hybridization,and the relative amounts of the mRNAs were estimated bydensitometric scanning of the autoradiograms and related tothe cellular levels of /J-actin mRNA at the various time points.Following treatment with BNF, the mRNA coding for cyto-

abc

28S ^

18S »-

d e f

B-ac t i n •Fig. 1. RNA gel blot analysis of cytochromes P-4SOc and P-450d mRNA

levels in total cellular RNA isolated from rat ventral prostate and liver. Totalcellular RNA was isolated both from ventral prostate in control animals (Lanesa and d) and animals treated with 80 mg of BNF/kg for 16 h (Lanes b and c).For comparison, total cellular RNA from BNF-induced rat livers were run in thesame gels (Lanes c and/). Thirty Mg of RNA per lane were separated on 1.1%(w/v) agarose-formaldehyde gels, blotted to nitrocellulose filters, and hybridizedwith a cDNA probe specific for cytochrome P-450c (p210; Lanes a, b, and c) orcytochrome P-450d (p72; Lanes d, e, and /) as described in "Materials andMethods."

12 24 48 96

Time [h]

Fig. 2. Time course for induction of cytochromes P-450c and P-450d mRNAsin total cellular RNA isolated from liver following treatment with BNF. TotalRNA from liver (0.3 fig) was immobilized on slot blots and hybridized with probesspecific for cytochrome P-450c or P-450d mRNA as described in "Materials andMethods." In addition, 1.0 fig of calf thymus DNA was applied to each Tiller to

normalize exposure time to the X-ray film. Slot blots were hybridized andanalyzed as described in "Materials and Methods." Average from the results of

two experiments analyzed by densitometry are presented. The data have beenrelated to the expression of j¡actin but have not been corrected for background.»,cytochrome P-4SOc; O, cytochrome P-4SOd. Deviation from the mean value isless than 15% in any given time point.

chrome P-450c reached maximum levels within 16 h both inliver (Fig. 2) and the prostate (Fig. 3). Cytochrome P-450cmRNA levels then decreased during the following 24 h to reach

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96

Fig. 3. Time course for induction of cytochromes P-450c and P-450d mRNAsin total cellular RNA isolated from the ventral prostate following treatment withBNF. Total cellular RNA was isolated from the ventral prostate at varying timepoints following treatment with BNF and was analyzed for prevalence of cyto-chrome P-45ÛCand P-450d mRNA as described in "Materials and Methods" and

in the legend of Fig. 2. Average from the results of two experiments analyzed bydensitometry and related to the expression of/3-actin are presented. •,cytochromeP-450c; O, cytochrome P-450d. Deviation from the mean value is less than 15%in any given time point.

control levels at 48 h. At 16 h, the magnitude of the inductionresponse was similar in both tissues: ~ 15-20-fold induction ascompared to control levels. At all time points exhibiting inducedlevels of cytochrome P-450c mRNA, the quality of the mRNAwas analyzed by RNA gel blot analysis. In all cases, a 2.9-

kilobase species was observed (data not shown). In the liver,cytochrome P-450d mRNA levels induced by BNF were detectable at later time points than corresponding cytochrome P-

450c mRNA levels, reaching maximal levels at 24 h (Fig. 2).In the prostate, however, it was not possible to detect anymRNA coding for cytochrome P-450d at any time point assayedfollowing administration of BNF (Fig. 3).

In addition to analyses of total cellular mRNA levels, weperformed nuclear runoff transcription assays with highly purified nuclei from both tissues of rats treated for different timeswith BNF to obtain a measure of the rate of transcription atthe time of cell lysis. Transcription of the cytochrome P-450c

gene was found to be maximal in liver nuclei within 4 h andthen decreased down to uninduced (background) levels by 16-

24 h (Fig. 4). In analogy to the kinetics for mRNA prevalence(cf. above), maximal rates of elongation in vitro of the nascentcytochrome P-450d mRNA in liver nuclei tended to occur at aslightly later time point, 12 h, as compared to the rate ofsynthesis of the cytochrome P-450c mRNA (Fig. 4).

In nuclei from the rat ventral prostate, maximal rates oftranscription of the cytochrome P-450c gene were observed ata slightly later time point, 8-12 h, as compared to the liver.Thereafter, the rate of transcription decreased to control levelsby 24 h (Fig. 5). As judged from densitometric scanning of thehybridization signals from several separate experiments, transcription of the cytochrome P-450c gene, as related to theexpression of/8-actin, increased by 8-10-fold in both liver andprostate following administration of BNF. This value is similarto the magnitude of BNF-induced cytochrome P-450 mRNA

accumulation in both tissues (cf. above). Similar observationshave been made regarding the induction of cytochrome P,-450

gene expression by TCDD in mouse hepatoma cells (27). At alltime points tested, transcription of /3-actin remained relatively

constant in both tissues. Analysis of prostatic nuclear transcripts with the p72 probe revealed no hybridization signalsabove the background level observed with cytochrome P-450e

P-450C

P-450d

P-450e

pBR 322B-actin

Time [h] 12 16 24 48 96

3 -

48 96

Time [h]

Fig. 4. Nuclear transcription of cytochromes P-450c and P-450d in BNF-treated rat liver. Nuclei were prepared from the liver of rats treated with BNF forvarying times (0-96 h as indicated). RNA was allowed to elongate in vitro andwas purified as described in "Materials and Methods." The RNA was thenhybridized to filter-immobilized plasmids containing the indicated DNAs. A,autoradiography; B, average from the results of two experiments analyzed bydensitometry. The data were normalized against the expression of .i-actin buthave not been corrected for background. •,cytochrome P-450c; O, cytochromeP-450d. A cDNA clone for cytochrome P-450e, •(which represents a form ofcytochrome P-450 inducible by phénobarbitalbut not PAH), was used in additionto /3-actin to assess control (background) transcription rates. Deviation from themean value is less than 20% in any given time point.

or pBR322 sequences, indicating little or no cytochrome P-450d transcription (Fig. 5).

DISCUSSION

The cytochrome P-450 superfamily of enzymes is character

ized by structural heterogeneity, tissue specificity, and the differential regulation of the various forms of cytochrome P-450.The induction of cytochromes P-450c and P-450d followingadministration of chemicals that interact with the dioxin receptor (e.g., 3-methylcholanthrene, BNF, and TCDD) has beenwell documented in rat liver by characterization of the inducedpolypeptides and the corresponding mRNAs (28-31). The cand d isozymes of cytochrome P-450 show partial immunolog-ical cross-reactivity with each other but exhibit different substrate specificities and NH2-terminal sequences. In the mouse,the equivalent forms of cytochromes P-450c and P-450d, cytochromes Pi-450 and P3-450, respectively, are differentiallyregulated during development, with cytochrome Pj-450 constituting the major induced form in adult liver (32). The inductionmechanisms for cytochromes P-450c and P-450d exhibit similar sensitivities to PAH-like inducers (19, 33), and experimentsperformed with genetic models in mice suggest that the induction of the expression of both genes is regulated by the dioxinreceptor (34). It has also been reported that the cytochrome P3-450 gene is constitutively expressed in mouse liver such that,

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P-450CP-450dP-450e

pBR 322B-aclin

Time h 12 16 24 48 96

Time | h ]

Fig. 5. Nuclear transcription of cytochromes P-450c and P-450d in BNF-treated rat ventral prostate. Nuclei were prepared from the ventral prostate ofrats treated with BNF for varying times (0-96 h as indicated). Nuclear transcription was analyzed as described in "Materials and Methods" and in the legend of

Fig. 4. A, autoradiography; H, average from the results of two experimentsanalyzed by densitometry and related to /3-actin. •,cytochrome P-450c; O,cytochrome P-450d; •.cytochrome P-450e. Deviation from the mean value isless than 20% in any given time point.

in uninduced liver, the concentration of cytochrome 1V450mRNA is substantially higher than that of cytochrome IV450(32). On the basis of these data, it has been speculated that theexpression of cytochrome Pa-450 is regulated by two cellularfactors, one of which determines the PAH-responsiveness (i.e.,the dioxin receptor) and one which controls the "constitutive"

expression (16). In the present study and within our detectionlimits, we observed no "constitutive" expression of cytochromeP-450d in the liver or the prostate of untreated rats. Hybridization signals specific for cytochrome P-450d mRNA weredetectable only in the liver following treatment with BNF.Moreover, little or no expression of the cytochrome P-450dgene was detectable in the rat ventral prostate, as assessed byanalysis of total cellular mRNA levels and nuclear transcriptionrates.

At present, the issue of tissue-specific expression of cytochromes P-450c and P-450d is unclear. Recently, Nebert et al.(32, 35) have reported on expression of cytochrome P3-450 inseveral extrahepatic tissues. In these studies, large tissue-spe

cific discrepancies between transcription and mRNA prevalenceof cytochrome P3-450 were described, indicating transcriptstability to be of importance in the regulation of cellular cytochrome Pn-450 mRNA levels. On the other hand, studies in therabbit have revealed no extrahepatic expression of the equivalent of cytochrome P-450d (36, 37). Cytochrome P-450dexpression is also absent in mouse hepatoma cells (38) or inprimary cultures of adult rat hepatocytes (39). In the presentstudy, low or absent cellular cytochrome P-450d mRNA levelsin the ventral prostate paralleled low or absent nuclear tran

scription rates. Since the rat ventral prostate contains the dioxinreceptor (~1000 sites/cell) (18), the rat ventral prostate mightpresent an attractive model for the study of tissue-specificexpression of cytochrome P-450d. In particular, a comparisonbetween the rat ventral prostate and the rat nasal mucosa,which expresses cytochrome P-450d, but little or no cytochromeP-450C (40), will be interesting.

In addition to the dioxin receptor, the rat ventral prostatecontains a highly abundant M, s 40,000 protein, PSP (18),(~20% of the total protein content of rat prostatic cytosol)which binds TCDD and PAH reversibly and with high affinity.The biological role of PSP and the possible pathophysiologicalsignificance of the PSP-PAH interaction remain unclear. However, it is conceivable that such a high abundance of a PAH-binding protein might be of importance for tissue-specific uptake of procarcinogens in the rat ventral prostate. More importantly, PSP might play a role in the transfer of ligands to thedioxin receptor in vivoin this tissue and thereby elicit a receptor-mediated biochemical and/or toxic response. At present, wehave no experimental data to clarify these issues, but it isinteresting to note that the induction kinetics of cytochrome P-450c in the prostate indicates delayed expression of the gene ata transcriptional level as compared to the liver. Whether thiseffect has a pharmacokinetic background or is due to interactionof the inducer (PAH) with PSP prior to interaction with thedioxin receptor remains to be investigated. In vitro, it is possibleto efficiently label the dioxin receptor in rat liver cytosol byincubation with purified PSP labeled with [3H]TCDD (41).

In conclusion, the study of cytochrome P-4SO gene expressionin the rat ventral prostate might not only be a good model forthe investigation of tissue-specific gene regulation but alsocontributes to the understanding of human prostatic cancer. Itis conceivable that induction of cytochrome P-450c in the ratventral prostate might have implications for the metabolicactivation of procarcinogens in this tissue and therefore haverelevance for the etiology of prostatic cancer.

ACKNOWLEDGMENTS

We thank Dr. Mikael Gillner (Karolinska Institutet, Stockholm,Sweden) for fruitful discussions and Maria Janssen for expert technicalassistance. Dr. J. Fagan (National Cancer Institute, Bethesda, MD)generously provided us with the cytochrome P-450c- and P-450d-speciflc cDNA clones. The cDNA clone for cytochrome P-450e wasprovided by Dr. M. Adesnik (New York University, New York, NY).Dr. Anders Berkenstam (Karolinska Institutet) is gratefully acknowledged for electron microscopic analysis of nuclear preparations.

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