THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 1, Issue of January 10, PP. 160-168, 1970

Printed in U.S.A.

The Role of Ribonucleic Acid and Protein Synthesis in

Microsomal Aryl Hydrocarbon Hydroxylase

Induction in Cell Culture

THE INDEPENDENCE OF TRANSCRIPTION AND TRANSLATION

(Received for publication, September 8, 1969)

D. W. NEBERT* AND H. V. GELBOIN$.

From the Chemistry Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland if0014

SUMMARY

Aryl hydrocarbon hydroxylase is a microsomal mixed function oxygenase induced by benz[a]anthracene and other polycyclic hydrocarbons in mammalian cell culture. Actino- mycin D or Z-mercapto-1-(/3-4-pyridethyl)benzimidazole (inhibitors of RNA synthesis) or cycloheximide or puromycin (inhibitors of protein synthesis), added simultaneously with benz[a]anthracene, completely prevents aryl hydro- carbon hydroxylase induction. During treatment with inducer, the stimulation of hydroxylase activity becomes insensitive to inhibition by actinomycin D but continues to be sensitive to inhibition by cycloheximide.

The phase of induction requiring RNA synthesis is inde- pendent of translation; in cells previously treated with benz[o]anthracene plus cycloheximide, the enzyme activity rises for 8 hours after the cells are placed in fresh control medium. Addition of actinomycin D to these previously treated cells does not inhibit the rise in hydroxylase activity, suggesting that RNA synthesis has occurred in the absence of translation. Furthermore, when actinomycin D is added to cells previously treated with benz[a]anthracene plus Z-mercapto-l-(fl-4-pyridethyl)benzimidazole, the increase in aryl hydrocarbon hydroxylase activity is prevented.

Induction of increased hydroxylase activity seems to require at least two phases. The initial phase appears to involve synthesis of an induction-specific RNA, and this phase is cycloheximide-insensitive and hence translation- independent. The later phase of enzyme induction involves translation related to an induction-specific RNA, and this stage is insensitive to inhibition by actinomycin D and, thus, is transcription-independent. The increased enzyme activity may be due to an increase in the amount of either enzyme protein or other protein involved in enzyme activation.

Microsomal mixed function oxygenases (1) require NADPH and molecular oxygen for the oxidative metabolism of drugs,

* Present address, Section on Developmental Enzymology, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014.

t To whom reprint requests should be addressed.

steroids, and carcinogens (24). The activity of these microso- ma1 enzyme systems in the intact animal varies greatly and is influenced by factors of age, sex, species, strain, nutritional and hormonal. status, and the animal’s exposure to a variety of foreign compounds, such as certain drugs, carcinogens, and in- secticides (2-4). One of these microsomal enzyme systems, aryl hydrocarbon hydroxylase,l is present and inducible in mammalian cells grown in culture (5-7). The assay and properties of the aryl hydrocarbon hydroxylase system were previously described (6). The enzyme system is rapidly induced by the addition of benz[a]anthracene or other polycyclic hydrocarbons to the culture medium of cells derived from different animal species and in various stages of cell growth or subcultures (7). Aryl hydro- carbon hydroxylase induction by BA2 in cell culture is prevented by inhibitors of RNA or protein synthesis, but the induction process becomes insensitive to actinomycin D at later times (7). In this study we have further investigated the requirement for protein and RNA synthesis in the induction process and the relationship between the two processes during enzyme induc- tion.

MATERIALS AND METHODS

Materials

The polycyclic hydrocarbons BA and benzo[a]pyrene were purchased from Eastman and were recrystallized from benzene. Recrystallized 3-hydroxybenzo[a]pyrene was a generous gift of Dr. Hans Falk, National Institutes of Health. Cell culture supplies were obtained and prepared as previously described (6). The standard complete medium consisted of 10% calf serum in Eagle’s No. 2 minimal essential medium, pH 7.1, which contained 100 units of penicillin, 100 pg of streptomycin, and 10 units of Mycostatin per ml; 2 mM glutamine; and the nonessential amino

1 This enzyme system is also called benzpyrene hydroxylase and aryl hydroxylase. We prefer to use aryl hydrocarbon hydroxyl- ase, since the enzyme obtained from rat liver microsomes, or from hamster fetal cells grown in culture, converts a variety of poly- cyclic hydrocarbons to phenolic derivatives and is not specific for benzo[a]pyrene.

2 The abbreviations used are: BA, benz[a]anthracene (1,2- benzanthracene) (the nomenclature is that recommended by the American Chemical Society (8)); MPB, 2-mercapto-1-(P-4-pyrid- ethyl)benzimidazole.

160

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Issue of January 10, 1970 D. W. Nebert and H. V. Gelboin 161

acids. The BA was dissolved in the growth medium, and the concentration of the hydrocarbon was determined spectrophoto- fluorometrically as previously described (6). Control medium without BA was treated identically. An Aminco-Bowman model 4-8202 SPF spectrophotofluorometer was used. NCS solubiliz- ing reagent, uniformly labeled l4C-protein hydrolysate (54 mCi per “milliamino acid”), and generally labeled 3H-uridine (5.0 mCi per 0.093 mg) were purchased from Nuclear-Chicago. Cy- cloheximide and actinomycin D (dactinomycin) were obtained from the Cancer Chemotherapy National Service Center. Puro- mycin was purchased from Nutritional Biochemicals and MPB from the Midland Tar Distillers, Ltd., Birmingham, England. NIH Animal Supply provided us with pregnant hamsters (esti- mated at 10 to 12 days of gestation).

Methods

Cell Culture Techniques-All experiments in this communica- tion were performed on secondary cell cultures (first subcultures) ; the individual cells, derived from whole hamster fetuses estimated at 10 to 14 days of gestational age, were prepared and passaged as previously described (6, 7). The addition of growth medium containing BA, inhibitors of RNA or protein synthesis, or both, or of control medium alone, to the secondary cultures was car- ried out between 32 and 60 hours after the plating of the cells at a density of approximately 0.5 x lo6 per ml; the cells were in optimal logarithmic growth during this time and had not become a confluent monolayer (7). An experiment generally consisted of adding the experimental medium to several dozen dishes, replacing this medium with new experimental medium or fresh control medium alone, and harvesting individual dishes at the indicated times (6, 7). The measurements of 3H-uridine and %-protein hydrolysate incorporation into cellular perchloric acid-precipitable residue and trichloracetic acid-precipitable pro- tein, respectively, were used as parameters of RNA and protein synthesis as previously described (7). There were no apparent irreversible effects of cycloheximide, puromycin, or MPB at the concentrations and exposure times employed. Cells treated with actinomycin D for 4 hours or longer lost enzyme activity rapidly and failed to respond optimally to subsequent inducer treatment. Reduced protein synthesis and slower growth suggested an in- ability to reverse the toxic effect of actinomycin D.

Enzyme Assay-Both the aryl hydrocarbon hydroxylase ac- tivity and the protein concentration were determined in dupli- cate for the cells from an individual loo-mm plastic tissue cul- ture dish, as previously described (6). In the assay for enzyme activity (6), the reaction mixture, in a total volume of 1.00 ml, included 50 pmoles of Tris-chloride buffer, pH 7.5; 0.36 pmole of NADPH; 3 pmoles of MgC12; 0.10 ml of cell homogenate (con- taining 200 to 800 pg of protein); and 80 ml.cmoles of the sub- strate benzo[a]pyrene (added in 40 ~1 of methanol just prior to incubation). Following the 30-min incubation at 37”, the alkali- extractable derivatives were examined spectrophotofluorometri- tally, with activation at 396 rnp and fluorescence at 522 rnp. The enzyme activity is described as the amount of hydroxylated benzo[a]pyrene equivalent to the fluorescence of 3-hydroxybenzo- [ulpyrene (6). One unit of activity equals 1 CcE.cmole equivalent to 3-hydroxybenzo[u]pyrene formed in 30 min.

RESULTS

Kinetics of Aryl Hydrocarbon Hydroxylase Induction by BA- Fig. 1 shows the effect of several concentrations of inducer on

OO3 12 15 HOURS

I FIG. 1. The effect of several concentrations of the polycyclic hvdrocarbon inducer, BA. on arvl hvdrocarbon hvdroxvlase ac- tivity. In this figure and’in all &bskquent figure;, each point is the average of duplicate determinations of both enzyme activity and protein concentration; results of duplicate determinations greater than 10% of each other were excluded from the graphs. The hydroxylase specific activity on the or&in&e in this and sub- sequent figures is expressed in units (micromicromoles of hydrox- ylated benzo[a]pyrene) per mg of protein.

the aryl hydrocarbon hydroxylase activity in hamster fetal cells grown in culture. After a lag period of approximately 35 min, 13 PM BA increased the level of hydroxylase activity linearly for 12 to 16 hours, the activity reaching a peak during the second 24-hour period’(7). With the 2.6 pM and 1.3 pM levels of inducer, the early kinetics of induction observed at 3 and 6 hours was not significantly different from that observed with 13 PM BA. The identical rate of induction indicates that the induction process involves a rate-limiting step which is independent of inducer concentrations above 1.3 PM. This rate-limiting step may be the translation involving an induction-specific RNA, since studies described later suggest an accumulation of such a RNA species.

Although the early kinetics of hydroxylase induction was similar with 1.3 pM, 2.6 pM, and 13 pM BA, the plateau in spe- cific enzyme activity and the time required to attain that plateau were positively correlated with the concentration of BA used. Thus, the peak in the curve of aryl hydrocarbon hydroxylase activity in cells exposed to 2.6 pM BA occurred at approximately 10 hours, and the specific activity was 3 times higher compared to that in cells exposed to 0.65 pM BA, where the plateau in the enzyme activity curve appeared at about 7 hours. The peak in hydroxylase activity in cells exposed to 1.3 PM inducer occurred at approximately 9 hours, and the specific activity attained was twice as high as that in cells exposed to 0.65 PM

inducer. Also, in each case there was a substantial decline in enzymatic activity after the peak activity had been reached. These data suggest that, with low levels of inducer, there may be a depletion of BA to less than saturating amounts. This depletion of inducer is probably caused by the induced enzyme system metabolizing the available BA, since BA is a substrate for the induced aryl hydrocarbon hydroxylase in hamster fetal

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162 RNA, Protein Xynthesis in Microsonaal Hydroxylase Induction Vol. 245, No. 1

cell culture (6) and for the hepatic enzyme system in the rat (9).

Requirements for RNA and Protein Synthesis During Aryl Hydrocarbon Hydroxylase induction-Table I shows the initial requirements for both RNA and protein synthesis for induction of aryl hydrocarbon hydroxylase. When either actinomycin D or MPB (inhibitors of RNA synthesis) or cycloheximide or puromycin (inhibitors of protein synthesis) was added simul- taneously with the inducer, there was a complete block of the BA-induced rise in enzymatic activity. These data do not necessarily signify the production of increased amounts of en- zyme protein, but do indicate that the process resulting in increased hydroxylase activity requires the synthesis of both RNA and protein. This requirement for RNA and protein syn- thesis may be related to the synthesis of a specific protein com- ponent of the aryl hydrocarbon hydroxylase system or, alternatively, may reflect a requirement for the synthesis of a protein necessary for the activation of a pre-existing, inactive enzyme system. It is, of course, possible that actinomycin D and cycloheximide may have effects other than the blockage of RNA and protein synthesis. Actinomycin D, for example, may interfere with the transfer of RNA from nucleus to cytoplasm. We assume, however, that the over-all biological effects of actino- mycin D are due to its interference with RNA synthesis.

Fig. 2 shows the effects of actinomycin D, alone or in combina- tion with BA or with BA plus cycloheximide, on aryl hydrocarbon hydroxylase activity in cells which had been treated previously with inducer for 20 hours. The enzyme activity continued to increase in cells exposed to additional BA. The addition of a high level of actinomycin D, with or without inducer, caused a greater rise in hydroxylase activity during the initial 4 hours than that observed with BA alone. This stimulation was ob- served at concentrations of 0.4 FM to 1.6 PM actinomycin D, but was not found with levels of actinomycin D below 0.2 PM. The insensitivity to actinomycin D for approximately 4 hours sug-

TaBLE 1

Aryl hydrocarbon hydroxylase activity in cells treated simultaneously with BA and several inhibitors of RNA and protein synthesis

The inducer concentration was 13 J.LM BA. At concentrations

of 0.40 pM actinomycin D and 40 PM MPB, these inhibitors each effectively prevented at least 80% of 3H-uridine incorporation into perchloric acid-precipitable residue during the initial 30 min following its addition, and the inhibition was greater than 90% after the initial 30.min period. At levels of 3.5 pM cycloheximide and 60 PM puromycin, these antibiotics each effectively blocked at least 85y0 of W-protein hydrolysate incorporation into trichlora- cetic acid-precipitable protein during the initial 30 min following its addition, and the inhibition was greater than 90% after the in- itial 30-min period.

Cells exposed to

Specific activity

ohr 1 2hr 1 4hr 1 6hr

u?i;~s/mg fwolein

Control medium only. 8 5 14 19 ‘BA only. 40 73 102 BA + actinomycin D.. 13 8 6 BA + MPB 11 7 10 BA + cycloheximide., 8 6 10 BA + puromycin. 10 10 7

gests that during the prior exposure of the cells to BA there occurred an accumulation of an induction-specific RNA. Our data indicate that at least 3 to 4 hours are required for the depletion of this RNA species during the actinomycin D-insensi- tive period. The initial sensitivity to actinomycin D (Table I) and the subsequent insensitivity to high levels of actinomycin D suggest that the cells have passed from a transcription-dependent phase to a transcription-independent stage. The time required to regain actinomycin D sensitivity may reflect the time required for depletion of the induction-specific RNA.

Cycloheximide prevented the stimulation of aryl hydrocarbon hydroxylase activity caused by actinomycin D; this inhibition by cycloheximide was observed whether BA was present or absent. Upon the addition of cycloheximide, the hydroxylase activity decreased; after a 2-hour lag, the rate of decay of induced enzyme activity in cells exposed to BA plus actinomycin D plus cycloheximide was similar to that observed in cells exposed to control medium only. These observations indicate that the stimulation by actinomycin D requires protein synthesis. Since in the presence of cycloheximide and actinomycin D the decay rate is similar to that observed with control medium alone

0’ I I I I 2 4 6 8

HOURS

FIG. 2. The effects of 0.40~~ actinomycin D (AD), fresh inducer (13 )iM BA), control medium alone (CM), and 3.5 PM cycloheximide (CY) on aryl hydrocarbon hydroxylase activity in cells previously exposed to inducer. The cultures had been treated with BA for 20 hours, after which the medium of all dishes was replaced with fresh medium containing the indicated additions. The sharp fall in enzyme activity in cells treated with actinomycin D for more than 4 hours can be explained at least in part by toxic effects on cellular metabolism caused by that inhibitor; prot,ein synthesis was 75% of normal by 4 hours, and less than 25% of normal after 8 hours of treatment with actinomycin D. Such cells exposed to actinomycin D for 8 hours required about 5 days in fresh growth medium before resuming logarithmic growth.

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Issue of January 10, 1970 D. W. Nebert and H. V. Gelboin 163

actinomycin D is not acting by stabilizing the enzyme system and thereby preventing its degradation or inactivation.

Translation-independent Phase of Aryl Hydrocarbon Hydrox- ylase Induction-We asked two questions relating to the accumu- lation of the induction-specific RNA. First, can this RNA accumulate in the absence of translation, and second, can trans- lation then occur in inducer-free medium? Such experiments are depicted in Fig. 3.

Fig. 3A shows that cycloheximide, during a IO-hour period in which the cells were treated with BA, completely prevented the normal rise in aryl hydrocarbon hydroxylase activity. This finding is consistent with our previous reports (5, 6) of a require- ment for protein synthesis during the induction of hydroxylase activity (Table I). Removal of the medium containing BA plus cycloheximide and replacement by medium containing BA alone produced an immediate, linear increase in the microsomal enzyme activity, indicating the effective reversal of the block in protein synthesis. Whether the cycloheximide and BA were replaced with inducer-free medium or with BA-containing me- dium, an identical rise in hydroxylase activity was observed for at least the first 6 hours. The rise in aryl hydrocarbon hydrox- ylase activity was prevented during the second lo-hour period if cycloheximide continued to be present. In cells treated initially

/ BAOnly

L

IO-HR 0 4 8

IC.PRETREATMENT* HOURS

FIG. 3. A, aryl hydrocarbon hydroxylase activity in cells ex- posed to 13 PM inducer (BA) or control medium alone (CM), fol- lowing previous treatment with BA or 3.5 PM cycloheximide (CY) or both. B, aryl hydrocarbon hydroxylase activity in cells ex- posed to 13 PM inducer (BA) or control medium alone (CM), fol- lowing previous treatment with 3.5 pM cycloheximide (CY) and two different concentrations of inducer. The heavy urrow in this

with cycloheximide without BA, the enzyme level did not rise when the cycloheximide-containing medium was replaced with fresh control medium.

Fig. 3B illustrates a similar experiment, where the effects of two different levels of inducer are compared. The addition of inducer-free control medium to cells previously exposed to 13 PM BA plus cycloheximide caused the aryl hydrocarbon hydroxylase activity to reach a plateau after,about 8 hours. In cells treated with one-fifth as much inducer during the IO-hour prior treat- ment period, the addition of control medium caused a similar initial rise in hydroxylase activity, but the peak was attained 4 hours after the control medium had been added. Also, the maximum enzyme activity was less than one-half of that observed in cells previously treated with 13 pM BA and cycloheximide.

These experiments indicate that enzyme activity increases in the presence of control medium alone after the cells are treated with BA during a block of protein synthesis. Two explana- tions are possible. First, RNA synthesis can occur during the cycloheximide block of translation; this transcriptional phase would therefore permit an accumulation of an induction-specific RNA. When translation is restored, the phase of enzyme induc- tion requiring protein synthesis may occur in the absence of inducer. The second possibility is that inducer molecules are inadequately removed from their sites of action, and the removal of cycloheximide permits induction to proceed immediately be- cause of the presence of accumulated inducer molecules at the induction receptor sites. Thus, the effects of the two different levels of BA, as seen in Fig. 3B, may be due to either the ac cumu

E

13,uM BA + CY

CM

IO-HR 0

-PRETREATMENT-

4 8

HOURS

figure and in subsequent figures depicts the time when the pre- liminary treatment medium was removed from all dishes, the surface of the cell layer was washed once with 4 ml of Dulbeccos’ isotonic phosphate buffer, and fresh medium containing the indi- cated additions was added. In those cells previously exposed to cycloheximide, protein synthesis returned to 100% of normal within the first 30-min period following removal of the inhibitor.

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164 RNA, Protein Synthesis in Microsowal Hydroxylase Induction Vol. 245, No. 1

lation and subsequent depletion of different amounts of an induction-specific RNA, or a different number of inducer mole- cules present at induction receptor sites.

We have already shown that the addition of fresh control medium to cells previously exposed to BA alone causes a decrease in enzyme activity after a 2-hour lag (Fig. 2). This finding suggests that the replacement of BA-containing medium with inducer-free control medium is sufficient to remove BA from the inducer receptor sites of the cell. However, aryl hydrocarbon hydroxylase activity has been induced to high levels in these cells, and this high enzyme activity may be an important factor in the rate of removal of inducer molecules from the inducer re- ceptor site. In Fig. 3, the induction of hydroxylase activity in control medium alone, in cells previously treated with BA plus cycloheximide, occurred in cells with low levels of enzyme ac- tivity. We therefore designed an experiment to determine whether this phenomenon occurs in cells with high levels of enzyme activity.

Fig. 4 shows that the hydroxylase activity, in cells previously treated with BA for 14 hours and not allowed to carry out pro- tein synthesis for the last 6 hours, increased when the medium containing BA plus cycloheximide was replaced with inducer-free medium. During the initial 4 hours, the rise in enzyme activity

BA + CY’

I I I I 1 0 8 14 16 18 20 22 24

HOURS

FIG. 4. Aryl hydrocarbon hydroxylase activity in cells exposed to 13 PM inducer (BA) or control medium alone (CM), following previous treatment with inducer or with inducer plus 3.5 pM cyclo- heximide (CY).

in the presence of control medium alone was not significantly different from that in cells exposed to BA, but the enzyme ac- tivity reached a plateau at about 5 hours and declined thereafter. These data indicate that the aryl hydrocarbon hydroxylase in- duction in cells grown in control medium alone, following ex- posure to BA plus cycloheximide, is not due to relatively low levels of enzyme which might prevent effective removal of BA from the relevant cellular binding sites. Therefore, Figs. 3 and 4 show that cells with either low or high levels of hydroxylase activity exhibit a phase of enzyme induction which is independ- ent of translation.

E$ect of Actinomycin D ajter Preuen,tion of Aryl Hydrocarbon Hydroxylase Induction with Cycloheximide-We have already described that, in the presence of inducer, the cells pass from an initial phase of complete sensitivity to actinomycin D (Table I) to a stage of actinomycin D insensitivity (Fig. 2). I f this tran- sition involves the synthesis of an induction-specific RNA, we can use the development of actinomycin D insensitivity as an indication that an induction-specific RNA synthesis has oc- curred. We can thus determine whether RNA synthesis can occur in the absence of translation.

Fig. 5A demonstrates the effect of high levels of actinomycin D on hydroxylase activity in cells previously treated with BA plus cycloheximide. The addition of actinomycin D, with or with- out inducer, to cells previously treated with BA plus cyclohexi- mide stimulated aryl hydrocarbon hydroxylase activity to a greater extent than did BA alone or control medium alone. The continued presence of cycloheximide prevented the actinomycin D effect, indicating that the marked stimulation by actinomycin D requires protein synthesis. Therefore, these results show that, in the presence of inducer and an essentially complete block of protein synthesis, the cells still pass from an actinomycin D sensitivity to an actinomycin D insensitivity. I f this transition represents the synthesis of an induction-specific RNA, we con- clude that the RNA species can be synthesized in the absence of translation. Also, these data suggest that the inducer is operat- ing at a level independent of translation.

Fig. 5B illustrates the effect of different levels of actinomycin D on aryl hydrocarbon hydroxylase activity in cells previously treated with inducer plus cycloheximide. While actinomycin D, at concentrations of 0.40 PM or greater, stimulated the enzyme activity at a faster rate than did BA or control medium alone, lower levels of the antibiotic allowed aryl hydrocarbon hydrox- ylase activity to rise at about the same rate as that in cells exposed to control medium alone. Addition of actinomycin D in concentrations as low as 0.04 PM, simultaneous with the inducer, prevents aryl hydrocarbon hydroxylase induction3 Thus, the data in Fig. 5 indicate that, once the cells have passed from a state of sensitivity to actinomycin D inhibition to a phase of insensitivity to actinomycin D inhibition, microsomal enzyme induction will occur at either high or low levels of the antibi- otic.

Effect of RNA Synthesis Inhibitors on the Translation-inde- pendent Phase of Aryl Hydrocarbon Hydroxylase Induction- Synthesis of an induction-specific RNA seems to occur during the translation-independent, early phase of microsomal enzyme induction. Therefore, if RNA synthesis is blocked during this preliminary treatment period, the subsequent rise in enzyme activity should be inhibited.

3 Unpublished data.

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Issue of January 10, 1970

A

D. W. Nebert and H. V. Gelboin

I / BA+AD BA

BA+CY __------- _----

0’ I I I I I (

Ic- IO-HR 9 4 8

PRETREATMENT HOURS

3

0.40,uWAD /

I I IO-HR 0 4 8

+ PRETREATMENT -+ HOURS

BAtCY .-a- ----__

FIG. 5. A, the effects of 0.40 PM actinomycin D (AD), 13 PM inducer @A), control medium alone (CM), and 3.5 GM cyclo- heximide (CY) on aryl hydrocarbon hydroxylase activity in cells previously treated with inducer plus cycloheximide. B, aryl hydrocarbon hydroxylase activity in cells exposed to 13 by inducer (BA), control medium alone (CM), or three different concentra- tions of actinomycin D (AD), following previous treatment with inducer plus cycloheximide (CY).

Fig. 6 shows the effects of BA or control medium alone on the hydroxylase activity in cells previously exposed to inducer plus cycloheximide, with or without actinomycin D. The addition of BA to cells, previously treated with BA plus cycloheximide plus actinomycin D, produced an increase in aryl hydrocarbon hydroxylase activity at a slower rate than that observed in cells, previously treated with inducer plus cycloheximide. This slower response suggests that actinomycin D, even at 0.04 PM concen- trations, was not effectively removed from the cells, thereby preventing the normal enzyme induction by BA.

The addition of control medium to cells, previously exposed to BA plus cycloheximide plus actinomycin D, caused no rise in hydroxylase activity for 4 hours, after which there was an increase in enzyme activity, but to a lesser extent than when BA had been added to cells receiving similar prior treatment. This temporary and partial inhibition by actinomycin D suggests that at least part of the initial translation-independent phase of microsomal enzyme induction involves RNA synthesis.

MPB is a reversible RNA synthesis inhibitor, which has been shown (10, 11) to block nucleic acid metabolism with little im- mediate effect on protein synthesis. In cell culture, the com- pound inhibits RNA synthesis rapidly, and this inhibition is reversed upon addition of fresh growth medium (12). The mechanism of action of MPB is not thoroughly known, but the compound inhibits nucleoside incorporation into cells (13).

% RNA Synthesis

BA

00 CM

SO

i BA

20 I /

t I I I ,

0 4 8 5-HR

PRETREATMENT HOURS

k >I FIG. 6. Aryl hydrocarbon hydroxylase activity in cells ex-

posed to 13 PM inducer (BA) or control medium alone (CM), fol- lowing previous treatment with inducer plus 3.5 PM cycloheximide (CY), with or without 0.04 PM actinomycin D (AD). During the preliminary treatment period in those cells exposed to actinomy- tin D, the amount of aH-uridine incorporation into perchloric acid-precipitable, residue is depicted in the insert. With higher concentrations of actinomycin D, RNA synthesis was more effectively inhibited during the preliminary treatment period; however, the rate of hydroxylase induction when these cells were exposed to fresh BA was many times slower.

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166 RNA, Protein Synthesis in Microsomal Hydroxylase Induction Vol. 245, No. 1

Other studies (14, 15) have also indicated. that the inhibitor interferes with RNA synthesis. We have found that 40 PM MPB effectively and reversibly prevents 95% of 3H-uridine incorporation into perchloric acid-precipitable residue. Table I shows that MPB completely blocks aryl hydrocarbon hydrox- ylase induction when the inhibitor was added simultaneously with BA. We have found3 by means of fluorescence microscopy and radioisotopic studies that MPB does not interfere with the transport of the polycyclic hydrocarbon inducer into the cells. Also, the addition of this inhibitor to cells previously treated with BA for 20 hours does not stimulate hydroxylase activity,3 as is the case when actinomycin D is added to cells previously exposed to inducer.

Fig. 7 shows the effects of BA, actinomycin D, or control medium alone on aryl hydrocarbon hydroxylase activity in cells previously treated with inducer plus MPB. The addition of fresh medium containing BA produced a linear increase in en- zyme activity, indicating the effective removal of MPB from these cells. Replacement of medium containing inducer plus MPB by fresh control medium alone caused a moderate increase in hydroxylase activity, after a delay of 4 hours. These data are similar to that seen in Fig. 6 with actinomycin D, and suggest that at least part of the early phase of microsomal enzyme induc- tion involves RNA synthesis. The subsequent rise in aryl hydrocarbon hydroxylase activity after 4 hours of growth in fresh inducer-free medium suggests that the initial phase of enzyme induction might also involve a step other than RNA synthesis. Such a step may be the inactivation by BA of a repressor of specific RNA synthesis. This inactivation of repres- sor might also occur in the absence of translation, such as during

FIG. 7. The effects of 13 PM inducer (BA), 0.40 PM actinomycin D (AD), and control medium alone (CM) on aryl hydrocarbon hydroxylase activity in cells previously treated with inducer plus 40 pM MPB. During the preliminary treatment period, RNA syn- thesis was inhibited more than 90%, and the inhibition remained at this effective level in those cells exposed to actinomycin D dur- ing the second period of the experiment. In those cells exposed to fresh BA or control medium alone, however, RNA synthesis re- turned to 100% of normal within the first 30 min following removal of the MPB.

the preliminary treatment of cells with inducer plus cyclohexi- mide. Therefore, the 4-hour lag period observed before signifi- cant hydroxylase activity appears may represent the time re- quired for adequate RNA synthesis to occur, following the removal of a repressor of the induction-specific RNA.

In cells previously exposed to inducer plus MPB, the addition of actinomycin D, with or without BA, did not cause a rise in aryl hydrocarbon hydroxylase activity. Thus, previous treat- ment with MPB in the presence of inducer prevents the cells from passing from a state of sensitivity to inhibition by actino- mycin D to a state of insensitivity to inhibition by actinomycin D. These observations further indicate that the development of insensitivity to actinomycin D inhibition during microsomal enzyme induction is dependent upon synthesis of a RNA species. We have also founda that cells exposed to cycloheximide plus BA plus MPB during the early phase of enzyme induction do not develop an insensitivity to actinomycin D inhibition.

Time Required for Induced Aryl Hydrocarbon Hydroxylase Ac- tivity to Appear after Treatment with BA in Absence of Transla- tion or Transcription-Upon addition of BA-containing medium to hamster fetal cells in culture, there is a 35-min lag time before significant increases in hydroxylase activity are detectable (7). By comparing the lag period in cells exposed to inducer plus MPB with that in cells exposed to BA plus cycloheximide, we hoped to determine whether or not transcription precedes translation during the process of microsomal enzyme induction.

Fig. 8 illustrates aryl hydrocarbon hydroxylase activity in cells, previously treated for 6 hours with inducer and either cycloheximide or MPB, and then exposed to BA alone. In those cells treated with BA plus cycloheximide, significant in- creases in hydroxylase activity occurred during the first 20-min period following removal of the cycloheximide. In those cells treated with inducer and MPB, enzyme activity was not signifi- cantly above basal levels until 40 min after the removal of

0’ 40 80 120 MINUTES

FIG. 8. Increases in aryl hydrocarbon hydroxylase activit.y in cells exposed to 13 PM BA, following previous treatment for 6 hours with either inducer plus 3.5 pM cycloheximide (CU) or inducer plus 40 PM MPB. The graph shows the period after preliminary treatment. Protein and RNA synthesis returned to 100% of normal within the first 30 min after removal of cycloheximide and MPB, respectively.

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Issue of January 10, 1970 D. W. Nebert and H. V. Gelboin 167

MPB. Extrapolation of these curves of enzyme induction to the abscissa produced a lag time of approximately 10 i 6 min for aryl hydrocarbon hydroxylase induction in cells, previously treated with BA and cycloheximide, and a lag period of 26 f 12 min for the induction of the enzyme in cells exposed to inducer plus MPB. These differences were observed in three similar experiments. These results suggest that, during the process of microsomal hydroxylase induction, transcription of RNA is an earlier step than translation into protein. The difference in lag time between the cells previously treated with MPB and those with no prior treatment may reflect the time required for in- ducer molecules to reach the cellular receptor sites and perhaps to inactivate a repressor of specific RNA synthesis. The pos- sibility remains that these data are fortuitous and that the lag periods simply reflect differences in the ability to remove the respective inhibitors from the cells. We do not think this is the explanation, however, since the lag periods in cells, previously treated with BA plus either cycloheximide or MPB, are both shorter than the 35-min lag time found in cells, previously exposed to inducer only (7).

DISCUSSION

The induction of microsomal aryl hydrocarbon hydroxylase activity by BA in hamster fetal cell culture includes an early phase which is translation-independent and a subsequent phase which is insensitive to actinomycin D inhibition. RNA syn- thesis is required only during the early phase. The initial phase of enzyme induction involves in part the synthesis of an induc- tion-specific RNA, and this phase can occur independent of translation. The second stage of microsomal enzyme induction apparently involves translation controlled or directed by the induction-specific RNA. This phase is insensitive to inhibition by actinomycin D and, thus, independent of transcription. The rise in enzyme activity requires that protein synthesis be intact, with one exception: in the case where cycloheximide is added to cells previously exposed to inducer only (Figs. 2 and 4), the hydroxylase activity remained at the same level or increased slightly for 2 or 3 hours before diminishing. This plateau or slight rise in aryl hydrocarbon hydroxylase activity in the ab- sence of protein synthesis also occurs upon addition of fresh control medium alone, and may be related to a step which is independent of amino acid incorporation, such as the assembly of presynthesized polypeptide chains. Although our studies indicate that the synthesis of both RNA and protein is essential for the process of microsomal enzyme induction, our observations do not indicate whether the enzyme protein itself is newly syn- thesized. The requirement for RNA and protein synthesis may reflect the synthesis of a protein moiety of the enzyme system which includes the microsomal electron transport chain (2, 16, 17), or may be necessary for the synthesis of a protein that activates a pre-existing inactive form of the hydroxylase enzyme system.

Our studies involve the induction of a multicomponent, par- ticulate enzyme system by polycyclic hydrocarbons in cell cul- ture. Other studies concerning the mechanism of induction of soluble enzymes in viva (18-20) and in vitro (21-30) by endoge- nous substrates are comparable with our results on aryl hydro- carbon hydroxylase induction. Reel and Kenney (30) reported a stabilizing effect of actinomycin D on tyrosine transaminase. In our studies, the half-life of induced hydroxylase activity is not significantly different in cells grown in fresh control medium

alone, or in cells exposed to actinomycin D plus cycloheximide (Fig. 2). Hence, our data indicate that actinomycin D does not appreciably alter the rate of microsomal oxygenase degradation. Other reports have described the development of insensitivity to inhibition by actinomycin D. The insensitivity to and, at times, stimulation by actinomycin D has been shown for tryp- tophan pyrrolase and tyrosine transaminase induction in rat liver (18)) for alkaline phosphatase in intestinal mucosa (19)) and for dCMP aminohydrolase activity in sea urchin eggs (20). Actinomycin D stimulation was also found during tyrosine transaminase induction by corticosteroids in hepatoma tissue culture cells (23, 24,28,30) and d uring arginase induction follow- ing starvation in Chang liver cells (26).

During the process of aryl hydrocarbon hydroxylase induction by BA, we have found that the initial phase is translation-inde- pendent. Similar observations have been reported (24, 27-29) in studies involving the mechanism of induction of soluble en- zymes by corticosteroids. In hepatoma tissue culture cells previously treated with inducer plus cycloheximide, tyrosine transaminase induction occurs when the block to protein syn- thesis is removed (24, 28). Glutamine synthetase activity is similarly induced in chick embryo retinal organ cultures (27, 29).

The stimulatory effect of actinomycin D has been interpreted (18, 21, 23, 24, 28) as due to the inhibition of synthesis of a labile cytoplasmic translational repressor by relatively high concentrations of the antibiotic. Our data on aryl hydrocarbon hydroxylase induction are not inconsistent ,with the hypothesis (24) that the inducer is acting at the level of tra,nslation. How- ever, we find that, in the absence of translation, the inducer is still effecting a change in RNA synthesis. Thus, we think that the inducer is operating at the level of gene regulation, i.e. RNA synthesis, perhaps by interaction with a repressor protein. The induction-specific RNA may accumulate at a nuclear site bound to DNA, from which it can be displaced by actinomycin D. This would explain the stimulation of induction of actinomycin D. The accumulation of the RNA causes the induction process to pass from a transcription-dependent state to a transcription- independent state. The secondary, translational step involves the synthesis of either enzyme protein or a rapidly synthesized and degraded protein activator of the enzyme complex. This latter stage of induction may involve a process which is inde- pendent of amino acid incorporation and involves polypeptide chain or membrane component assembly. Such a hypothesis accommodates the findings that (a) transcription is initially required and subsequently is not necessary; (b) transcription can occur in the absence of translation, which may indicate that the inducer is not operating at the translation step; (c) a RNA accumulates during the induction process (this is true whether induction proceeds normally or is blocked at the translational level) ; (d) translation proceeds in the absence of RNA synthesis; and (e) protein synthesis is required continuously for increased enzyme activity. The induction-specific RNA involved in microsomal hydroxylase induction may be either messenger RNA or an induction-specific RNA of the transfer RNA or ribosomal RNA type, which may control the translation of pre-existing, stable messenger RNA templates coded for the induction-specific protein.

The similarities between our observations on the induction of a membrane-bound enzyme system by polycyclic hydrocarbons and other studies on the induction of cytoplasmic enzymes by various endogenous substrates suggest that mechanisms regulat-

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168 RNA, Protein Synthesis in Microsomal Hydroxyluse Induction Vol. 245, No. 1

ing enzyme induction and repression in different cellular sites 13. in mammalian cells are similar. Studies on the mechanism of 14* induction of a mixed function oxygenase in cell culture offer 15

’ advantages for the study of factors controlling drug, steroid, and carcinogen metabolism. 16.

AcknmuZedgmentsWe gratefully acknowledge the valuable 17.

technical assistance of Miss Linda Bausserman, Miss M. Pat 18. Fisher, and Mr. Haywood Waters.

19.

REFERENCES 20.

1. 2. 3. 4. 5. 6.

7.

8.

9.

10. 11. 12.

MASON, H. S., Advan. Enzymol., 19, 79 (1957). 21. CONNEY. A. H.. Pharmacol. Rev.. 19. 317 (1967). 22 GILLET&, J. RI, Advan. Pharmacol.; 4, 219 (1967).

--. 23.

GELBOIN, H. V., Advan. Cancer Res., 10, 1 (1967). ALFRED, L. J., AND GELBOIN, H. V., Science, 167, 75 (1967). 24. NEBERT. D. W., AND GELBOIN. H. V.. J. Biol. Chem.. 243,

6242 (i968). ’ ,

NEBERT, D. W., AND GELBOIN, H. V., J. Biol. Chem., 243, 25. 6250 (1968).

PATTERS• N,.A. M., CAPELL, L.T., AND WALKER, D. F. (Ed- 26. itors), The ring index, Ed. 2, American Chemical Society, Washington, D. C., 1960. 27.

NEBERT, D. W., AND GELBOIN, H. V., Arch. Biochem. Biophys., 134, 76 (1969). 28.

BUCKNALL, R. M., J. Gen. Viral., 1, 89 (1967). BUCKNALL, R. M., AND CARTER, S. B., Nature, 213,1099 (1967). 29. SUMMERS, W. P., AND MUELLER, G. C., Biochem. Biophys. 30.

Res. Common., 30, 350 (1968).

SCHOLTISSEK, C., Biochim. Biophys. Acta, 168, 435 (1968). SKEHEL, J. J., HAY, A. J., BURKE, D. C., .QND CARTWRIGHT,

L. N., Biochim. Biophys. Acta, 142, 430 (1967). NAKATA, Y., AND BADER, J. P., Biochim. Biophys. Acta,

190, 250 (1969). OMURA, T., SATO, R., COOPER, D. Y., ROSENTHAL, O., AND

ESTABROOK, R. W., Fed. Proc., 24, 1181 (1965). MASON, H. S.; NORTH, J. C., AND VANNES&,I&, Fed. Proc.,

24, 1172 (1965). GARREN,L: D., 'HOWELL, R.R., TOMICINS,G. M.. ANDCROCCO.

R. M.; Proc.. Nut. Acad. Sci.’ U. 8. A.,‘62, 1121 (1964). . MOOG. F.. Science. 144. 414 (1964). SCARA~O,'E., DE @ETROCEL&, I&, AND AUGUSTI-TOCCO, G.,

Biochim. Biophys. Acta, 87, 174 (1964). MCAUSLAN, B. R., Virology, 21, 383 (1963). KIRK, D. L., Proc. Nat. Acad. Sci. 7J. rS. A., 64, 1346 (1965). THOMPSON, E.B., TOMKINS,G. M., AND CURRAN, J.F.,Proc.

Nat. Acad. Sci. U. S. A., 66, 296 (1966). TOMKINS,G. M., THOMPSON, E.B., HAYASHI,S.,GELEHRTER,

T., GRANNER, D., AND PETERKOFSKY, B.,Cold Spring Har- bor Symp. Qucznt.. Biol., 31, 349 (1966j. . - -

MOSCONA. A. A.. AND PIDDINGTON. R.. Biochim. Biovhus. Acta, lil, 409 (1966).

, , * ”

ELIASSON, E. E., Biochem. Biophys. Res. Commun., 27, 661 (1967).

Mosco~s, A. A., MOSCONA, M. H.. AND SABNZ.N., Proc. Nat. Acad. Sci. U. ‘8. A., 61, i60 (1968). ' '

PETERKOFSKY. B.. AND TOMI~INS. G.M.. Proc.Nat. Acad.Sci. U. S. A., 60, 2i2 (1968). ’ ’

REIF-LEHRER, L., AND AMOS, H., Biochem. J., 106, 425 (1968). REEL, J. R., AND KENNEY, F. T., Proc. Nat. Acad. Sci.

U. S. A., 61, 200 (1968).

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D. W. Nebert and H. V. GelboinOF TRANSCRIPTION AND TRANSLATION

Hydrocarbon Hydroxylase Induction in Cell Culture: THE INDEPENDENCE The Role of Ribonucleic Acid and Protein Synthesis in Microsomal Aryl

1970, 245:160-168.J. Biol. Chem. 

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