ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 190, No. 2, October, pp. 705-711, 1978

Effect of SBromodeoxyuridine on the Induction of Adenosine 3’:5’-Monophosphate Phosphodiesterase in 3T3-L Fibroblasts’

THOMAS MURRAY’ AND THOMAS R. RUSSELL

Departments of Biochemistry and Medicine, University of Miami School of Medicine, Miami, Florida 33152

Received February 22, 1978; revised June 2,1978

Cyclic adenosine 3’:5’-monophosphate (CAMP) phosphodiesterase activity is increased threefold in 3T3-L fibroblasts by l-methyl-3-isobutylxanthine. Actinomycin D (2 pg/ml) and cycloheximide (10 pg/ml) prevent this increase. Cells pretreated with inducer (0.5 mM)

in the presence of cycloheximide for 12 h, rinsed, and then treated with actinomycin D (t = 0) show a nearly twofold increase in enzyme activity in the next 90 min. The results suggest that CAMP phosphodiesterase is an inducible enzyme in 3T3-L cells and that the increase in enzyme activity requires transcription. The induction of CAMP phosphodiester- ase was employed as a model to investigate enzyme induction in cells grown in the presence of the thymidine analog, 5-bromodeoxyuridine. Growth of 3T3-L fibroblasts in 1 PM

bromodeoxyuridine results in an inhibition of the induction of CAMP phosphodiesterase by 1-methyl-3-isobutylxanthine (0.5 mM). This effect increases when bromodeoxyuridine is raised from 0.1 to 10 pM. The basal level of the enzyme is not altered by bromodeoxyuridine. Addition of thymidine (0.1 to 10 pM) to the culture medium prevents the bromodeoxyridine effect, indicating that the analog must be incorporated into DNA to act. An increase in the inducer concentration from 0.1 to 1.0 mM partially overcame the bromodeoxyuridine effect on enzyme induction. These results suggest that the induction of CAMP phosphodiesterase can be inhibited by the incorporation of bromodeoxyuridine into DNA, and that an inducer of the enzyme can override the inhibition but at higher concentrations than are necessary in control cultures.

The thymidine analog, BrdUrd: selec- tively inhibits the expression of cell-specific proteins during cytodifferentiation (l-5), lowers the level of tyrosine aminotrans- ferase in hepatoma cells grown in culture (2, 5), and induces the synthesis of alkaline phosphatase in a number of cells (6) and of prolactin in pituitary tumor cells (7). BrdUrd exerts its effect without altering total DNA, RNA, or protein synthesis (2,3, 8). The mechanism by which BrdUrd func- tions is unknown, but increasing evidence indicates that the analog must be incorpo- rated into DNA to function.

’ This work was supported by the Juvenile Diabetes Research Foundation of Florida and the National In- stitutes of Health (USPHS-AM-21575).

* In partial fulSllment for the requirements for the degree of Doctor of Philosophy.

a Abbreviations used: BrdUrd, 5-bromo-2’-deoxyu- ridine; CAMP, cyclic adenosine 3’:5’-monophosphate; MeiBu-Xan, l-methyl-3-isobutylxanthine.

Lin and Riggs (9) have shown that the lac repressor has a 10 times higher affinity for BrdUrd substituted than unsubstituted DNA. As a result of the increased affinity for BrdUrd-substituted DNA, increasing concentrations of the inducer, isopropyl- thiogalactoside, are required to dissociate the repressor from the DNA (9). Thus, the replacement of the methyl group of thymi- dine by bromine results in tighter binding of a regulatory protein to DNA. However, a sufficient amount of a specific inducer is able to override the increased association of repressor to the BrdUrd-substituted DNA. In eucaryotes, evidence is also accu- mulating which indicates that BrdUrd-sub- stituted chromatin binds both histones and acidic proteins more tightly than unsubsti- tuted chromatin (10, 11). BrdUrd may therefore function in eucaryotes as it does in procaryotes, by increasing the affinity of certain regulatory molecules for DNA.

705

0003-9861/78/1902-0705$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

706 MURRAY AND RUSSELL

Also, as in procaryotes, inducers of enzyme synthesis in eucaryotes may be able to over- come the apparent inhibition caused by BrdUrd.

The enzyme CAMP phosphodiesterase is known to be induced in eucaryotic cells when they are treated with MeiBu-Xan (12). In this paper we report that the addi- tion of BrdUrd to 3T3-L fibroblasts results in an apparent inhibition of the induction of CAMP phosphodiesterase without affect- ing basal enzyme levels, and that increasing concentrations of the enzyme inducer, MeiBu-Xan, are able to override the BrdUrd-dependent inhibition.

MATERIALS AND METHODS

Cell culture. The 3T3-L fibroblasts, a preadipose cell line that differentiates into adipose cells (13, 14), were provided by Dr. Howard Green, Massachusetts Institute of Technology. Cells were plated at a density of lo4 cells/6Omm culture dish and grown in Dul- becco-Vogt’s modified Eagle’s medium (GIBCO) sup- plemented with 10% calf serurn(GIBCO), penicillin (56 microunits/ml), and streptomycin (50 gg/ml). The cells were maintained at 37’C in a humidified 5% COZ atmosphere. Cultures were fed every other day with 5 ml of medium. In experiments involving BrdUrd treat- ment, cells were maintained in the dark and fed under subdued light. All experiments were performed on 3T3-L cells prior to the adipose conversion.

Enzyme induction. Fibroblasts were grown to con- fluency prior to the addition of the inducer MeiBu- Xan. The point of addition was defined as time 0 for the induction studies, except where otherwise stated. The drug was added so as to maintain a total volume of 5 ml of medium in each culture dish. At various times after confluence, total CAMP phosphodiesterase activity was determined. BrdUrd, containing a fourfold excess of deoxycytidine to prevent the development of a deoxycytidineless state (15), was added to cultures shortly after plating and was continually present for the remainder of the culture period. Medium contain- ing BrdUrd was prepared every other week. Thymi- dine solutions were similarly prepared and were added to growing cultures simultaneously with BrdUrd ad- ditions in aliquots comprising 0.4% of the medium volume. When employed, actinomycin D (2 pg/rnl) and cycloheximide (10 pg/ml) were added 20 min prior to the addition of inducer unless otherwise noted.

Phosphadiesterase assay. The CAMP phosphodi- esterase was prepared by washing the monolayer cul- tures three times with cold phosphate-buffered saline (pH 7.2) followed by an additional rinse with cold homogenization buffer (50 rnre T&acetate (pH 6.0)- 3.75 IIIM ,&mercaptoethanol). The cells from each plate were removed with a Teflon scraper and’homog-

enized (0.6 ml of buffer/dish) in a Dounce homogenizer (15 strokes). All steps were carried out at 4°C. Protein values were determined according to the method of Bradford (16) with ovalbumin as standard.

The assay for enzyme activity is a modification of the two-step procedure of Thompson and Appleman (17), which employs QAE-Sephadex to separate the reaction product from CAMP (18). 3H-Labeled CAMP was converted into ‘H-labeled 5’-AMP by cyclic nu- cleotide phosphodiesterase. The AMP was then con- verted into [3H]adenosine by a snake venom nucleo- tidase (Crotalus atrox). In the first stage, the reaction volume of 0.20 ml (pH 7.4) contained 0.05 ml of 4 pM

unlabeled CAMP in 40 mM Tris-Cl (pH 8.0), 0.05 ml of [3H]cAMP (8 pmol, 120,000 cpm) in 40 mM Tris-Cl (pH 8.0) containing 3.75 mM /l-mercaptoethanol and 20 rn~ MgClz, and a total of 0.1 ml of homogenization buffer containing an appropriate amount of enzyme. The reaction was started by adding the ezyme solution to the rest of the mixture that had been equilibrated at 30°C. After a lo-min incubation at 3O”C, the reac- tion was stopped by heating at 100°C for 1 min. The 5’-AMP resulting from this reaction was then con- verted into adenosine by incubation for 10 min at 30°C with 50 pg (0.1 ml) of snake venom. After the snake venom incubation, 0.7 ml of Hz0 was added to each tube to bring the final volume to 1 ml. [3H]Adenosine was then separated from unhydrolyzed [3H]cAMP by passing the reaction mixture over a QAE-Sephadex A- 25 column that had been equilibrated with 20 mM

ammonium formate (pH 7.5) (18). The r3H]adenosine was eluted from the column with an additional 4 ml of 20 rn~ ammonium formate (pH 7.5). Fifteen milliliters of scintillation fluid was added to each eluate, and radioactivity was counted in a Packard Tri-Carb liquid scintillation spectrometer. A unit of enzyme activity is defined as 1 pm01 of CAMP hydrolyzed/min.

Materials. MeiBu-Xan was obtained from Aldrich Chemical Co. and purified by recrystallization from ethanol. [G-3H]cAMP (25 Ci/mmol) was obtained from Schwarz/Mann and purified by chromatography on AG-50-X8. Cycloheximide, thymidine, 5-bromode- oxyuridine, and unlabeled CAMP were purchased from Sigma. Actinomycin D was acquired from Calbiochem.

RESULTS

Induction of CAMP phosphodiesterase. When applied to confluent monolayer cul- tures, MeiBu-Xan raises CAMP phospho- diesterase activity in these cells in a fashion which is dependent upon both the concen- tration of the inducer and the duration of exposure to it. Cells treated with 0.1 mu MeiBu-Xan for 18 h showed an increase in CAMP phosphodiesterase activity of 94 units (Fig. l), which represents the maxi- mum amount of enzyme induced at 18 h as

BROMODEOXYURIDINE AND CAMP PHOSPHODIESTERASE INDUCTION 707

Mei Bu-xm hlM)

FIG. 1. The specific activity of induced CAMP phos- phodiesterase (total specific activity minus specific activity of uninduced control; control specific activity is 100) vs inducer concentration. The cells were treated with inducer for 18 (O- - -0) and 48 (M) h. Specific activity is defined as picomoles of CAMP hydrolyzed per min per mg of protein and was deter- mined at a CAMP concentration of 1.0 pM. Data points represent the average of duplicate determinations, all of which agree within 8%.

no further increase in activity is seen as a function of inducer concentration. When cells were treated with MeiBu-Xan for 48 h, the CAMP phosphodiesterase activity in- creased as a function of inducer concentra- tion (Fig. 1). A maximal induction of more than 2.0 times the basal level occurred at 48 h with 0.5 mru MeiBu-Xan. Inhibitors of the synthesis of RNA and protein have previously been shown to inhibit the induc- tion of CAMP phosphodiesterase (12, 19, 20). The induction of CAMP phosphodies- terase is also blocked by actinomycin D and cycloheximide in 3T3-L fibroblasts (Table I). Thus, transcription seems to be a nec- essary prerequisite for the observed eleva- tion in activity. The long times employed for induction studies of this type (8 and 14 h) were necessitated by the relatively slow response of the 3T3-L cells to the inducer, especially as compared with chicken fibro- blasts (12). The slow response to inducer may be due to the longer half-life (21) (data not shown) of the enzyme in 3T3-L cells compared with chicken embryonic fibro- blasts.

A 12-h treatment of 3T3-L cells with MeiBu-Xan and cycloheximide followed by rinsing and incubation with actinomycin D

(Fig. 2) results in a significant increase in enzyme activity shortly after removal of the inducer. The amount of induction seen at 80 min (Fig. 2) indicates that the prior treatment with inducer caused an increase in the amount of a putative CAMP phos- phodiesterase mRNA, since the same in- crease is not seen for 8 h (Table I) in a standard induction experiment. Compara- bly treated cultures never exposed to the inducer did not show this increase (Fig. 2). Furthermore, superinduction was ruled out, as similar values of specific activity were obtained when the rinse medium did not contain actinomycin D. These results strongly suggest that the increase in CAMP phosphodiesterase caused by MeiBu-Xan involves enzyme induction.

Inhibition of enzyme induction by Brd Urd. The incorporation of BrdUrd into DNA has been shown to alter both induc- ibility and basal levels of some enzymes, while having little or no effect on other enzymes in the same cells (2, 3, 5). The degree to which BrdUrd alters inducibility has also been shown in some cases to vary

TABLE I

EFFECT OF INHIBITORS OF RNA AND PROTEIN SYNTHESIS ON INDUCTION OF CAMP

PHOSPHODIESTERASE”

Effector MeiBu- CAMP (@!/ml) Xan Phosphodiesterase

(1.0 mM) 8h 14 h

None - 1.00* + 0.01 1.00 f 0.06 None + 1.57 + 0.06 1.88 + 0.02 Cycloheximide - 0.82 + 0.10 0.99 + 0.02

(10) Cycloheximide + 0.77 + 0.01 1.20 f 0.01

(10) Actinomycin - 0.82 f 0.02 0.88 f 0.04

DC3 Actinomycin + 0.82 f 0.02 1.10 -t 0.02

DC3 D Medium with MeiBu-Xan and without (control)

was added to the cells. Eight and 14 h later cells Were homogenized, and CAMP phosphodiesterase activity of the homogenate was determined using 1 pM CAMP as substrate. Actinomycin D and cycloheximide were added 20 min prior to the addition of MeiBu-Xan. Data are reported as averages of duplicate determi- nations.

b Data are listed relative to the enzyme activity of untreated cells.

MURRAY AND RUSSELL 708

z a

8 200 m k v

0

e 150 8

/’ F--------------

0 /’ : 100 _-

0’ I I I 1 0 60 120 180 240

Time (min)

FIG. 2. Appearance of increased enzymatic activity after simultaneous exposure to inducer and cyclohex- imide. Twelve hours and 20 min prior to t = 0, cyclo- heximide (10 pg/ml) was added to all cells. Twenty minutes later (12 h before t = O), medium plus Meibu- Xan at 0.1 mM was added (u), while control cultures (C - -0) received no inducer. At t = 0 all cells were rinsed with fresh medium containing acti- nomycin D (2 pg/ml). New medium with actinomycin D was placed in the culture dishes for 10 min at 37°C to rinse the cells. This medium was then replaced by a second addition of fresh medium containing actino- mycin D. Cells were homogenized at the indicated times and CAMP phosphodiesterase activity was de- termined at 1.0 pM CAMP. All duplicates agree within 8%.

with the length of exposure of cells to the inducer (5). A time course (Fig. 3) was employed to examine the effects of BrdUrd on these aspects of CAMP phosphodiester- ase activity and inducibility. Inducer con- centrations of 0.5 mu were used for this study, as this had been shown earlier to elicit maximal induction in a 48-h period (Fig. 1). The time course reveals that basal levels of CAMP phosphodiesterase are not significantly affected by growth in the pres- ence of BrdUrd. However, the drug influ- enced the degree to which the enzyme can be induced during a given period of time. Control cultures exhibit a fairly constant increase in activity, reaching a three- to fourfold increase in 48 h. Cultures exposed to BrdUrd during growth show reduced enzyme induction (Fig. 3).

The response of CAMP phosphodiester- ase activity to MeiBu-Xan is diminished in a concentration-dependent fashion by in- creasing amounts of BrdUrd in the growth

medium (Fig. 4). Cells were grown in the presence of BrdUrd, ranging in concentra- tion from 0.1 to 10 j.rM and treated at con- fluence with MeiBu-Xan for either 18 or 48 h. Under these conditions, there is a sub-

0 12 24 36 48

Time I-CIWS)

FIG. 3. Levels of CAMP phosphodiesterase activity as a function of time in the presence and absence of BrdUrd. Cells treated with 0.5 mM MeiBu-Xan at confluency (t = 0) are indicated by (W), while control dishes are indicated by (-1. Cells were also grown in the presence of 1 PM BrdUrd and then at t = 0 treated with (O- - -0) and without (0- - -0) 0.5 mu MeiBu-Xan. BrdUrd at 1.0 pM was also present during the time course. Activities were measured at 1.0 pM CAMP and are expressed as pico- moles CAMP hydrolyzed per min per mg of protein. All duplicates agree within 12%.

‘z& // 40- xl- ------+

1 1 2 3 ” x rl

FIG. 4. Inhibition of enzyme inducibility after growth in BrdUrd-containing medium. Following plat- ing, cells were continuously exposed to various BrdUrd concentrations. Upon reaching confluency, cells were treated with 0.5 mu MeiBu-Xan. Eighteen h (0) and 48 h (0) later, total CAMP phosphodiesterase activity was determined at 1.0 pM CAMP. Activities in BrdUrd- treated cells are shown as a percentage of total induced activity in cells not exposed to BrdUrd with duplicate determinations within 6%. BrdUrd was also present in the culture medium during the induction period.

BROMODEOXYURIDINE AND CAMP PHOSPHODIESTERASE INDUCTION 709

stantial inhibition of inducibility produced by BrdUrd in the 0.1 to 1 FM range. Further increases in the drug concentration beyond 1 PM are not as effective in reducing the induction of CAMP phosphodiesterase. The pattern of inhibition is similar at both 18 and 48 h, although the absolute value of the reduction is greater at 48 h.

BrdUrd is incorporated into DNA in 3T3-L cells. It has been demonstrated for a number of systems that BrdUrd acts via incorporation into DNA. If this is true it follows that the inclusion of thymidine in the growth medium should competitively block BrdUrd effects. This was confirmed for CAMP phosphodiesterase activity in 3T3-L fibroblasts (Table II). Cells were grown in medium containing 1 pM BrdUrd and varying concentrations of thymidine. Induced specific activities for each condi- tion were determined after a 48-h treatment with 0.5 mM inducer. The degree of enzyme inducibility progressively increases as the concentration of thymidine in the culture medium is raised from 0.1 to 10 PM. A lo- fold excess of thymidine (10 pM) nearly eliminates the inhibitory effects of BrdUrd

TABLE II

EFFECT OF THYMIDINE ON CAMP PHOSPHODIESTERASE INDUCIBILITY IN THE

PRESENCE OF BrdUrd”

Thymidine BrdUrd MeiBu-Xan- Percent (M) bd induced CAMP control

phosphodiesterase activity*

(pmol/min/mg of protein)

0 0 147 + 20 100 W5 0 155 k 9 105

0 lo-” 41+ 11 28 W’ 10-” 62 + 3 42 1o-6 ;;:I 107 f 1 73 1o-” 137 f 3 93

a Thymidine and BrdUrd were added to cultures shortly after plating and were continually present for the remainder of the experiment. At contluency, me- dium with MeiBu-Xan (0.5 mM) or without was added to the cultures. Forty-eight hours later the CAMP phosphodiesterase activity of the homogenate was determined using 1 PM CAMP as substrate. Data are reported as averages of duplicate determinations.

b Basal activity level of 109 pmol/min/mg of protein is subtracted.

’ Induction in the presence of MeiBu-Xan alone is defined as 100%.

on enzyme induction. Parallel studies with [3H]BrdUrd showed that inclusion of thy- midine in the culture medium blocks the incorporation of BrdUrd into DNA (T. R. Russell, manuscript in preparation). Thy- midine has no direct effect on CAMP phos- phodiesterase activity, as 10 PM thymidine added to an assay mixture does not alter the specificity of the enzyme (118 f 6 pmol/min/mg of protein vs. 110 f 5 in control). BrdUrd added to confluent cul- tures only at the time of addition of inducer does not alter enzyme induction (change in specific activity of cells treated with 0.5 mM inducer alone for 18 h is 62 & 6 units; change in specific activity of cells treated for 18 h with 10 PM BrdUrd plus 0.5 mru inducer is 73 + 6 units). Similarly, BrdUrd does not affect the CAMP phosphodiesterase when added directly to the assay. Enzyme activ- ity in assay mixtures containing between 2.5 X 10m4 M and 2.5 X low7 M BrdUrd were all within 9% of control values.

Overcoming Brd Urd inhibition with MeiBu-Xan. The inhibition of CAMP phos- phodiesterase inducibility caused by Brd- Urd incorporation into DNA can be over- riden by increasing the MeiBu-Xan concen- tration (Table III). During an 18-h induc- tion period in control cultures, 0.1 mu MeiBu-Xan causes an increase in specific activity of 94 units/mg of protein. This represents the maximal induction for an 18- h period, as higher MeiBu-Xan concentra- tions elicited the same change in activity (Fig. 1). When cultures are grown in the presence of 1 pM BrdUrd, 0.1 mM inducer can only raise CAMP phosphodiesterase ac- tivity by 30 units/mg of protein or 32% of the inducibility seen in control dishes. As the inducer concentration is raised to 1 m progressive recovery of the degree of induc- tion is obtained, indicating that higher con- centrations of inducer are capable of regain- ing some of the lost response in BrdUrd- substituted DNA.

DISCUSSION

Lin and Riggs (9) have shown that the lac repressor binds more tightly to BrdUrd substituted than to unsubstituted DNA. This increased affinity of the repressor for DNA can be competitively overcome by increasing the concentration of an inducer

710 MURRAY AND RUSSELL

TABLE III EFFECT OF MeiBu-Xan ON THE INDUCTION OF

CAMP PHOSPHODIESTERASE AFTER GROWTH IN BrdUrd”

MeiBu-Xan BrdUrd MeiBu-Xan- Percent Concentration induced CAMP control

(rnM) phosphodiesterase activity6

(pmoW$$x of

0.1 94 f 4 100 0.1 + 30 + 1 32 0.5 + 45 + 9 48 1.0 + 64fl 68

D BrdUrd (1.0 pM) was administered to cultures shortly after plating and was continually present thereafter. When cultures reached confluency, various MeiBu-Xan concentrations were added. Total CAMP phosphodiesterase activity was determined 18 h later at 1 PM CAMP. Data are reported as averages of duplicate determinations.

’ Basal activity level of 87 pmol/min/mg of protein is subtracted.

’ Induction in the presence of MeiBu-Xan alone is defined as 100%.

of the lac operon. The molecular mecha- nism of BrdUrd action in eucaryotes, how- ever, is not as clear. Rutter and others (3, 10,ll) have proposed that an altered inter- action of regulatory proteins with BrdUrd- DNA could be responsible for the altered genetic expression. The results reported here indicate that the inhibitory action of BrdUrd on CAMP phosphodiesterase in- duction in mouse fibroblasts can be com- petitively overcome with an inducer of the enzyme. These findings are consistent with the hypothesis that BrdUrd substitution in eucaryotes may result in an altered inter- action between DNA and molecules that regulate gene expression, although the ac- tual mechanism by which MeiBu-Xan func- tions to compete with BrdUrd is unknown at this time.

For the MeiBu-Xan-dependent increase in CAMP phosphodiesterase to be used as a model to study the effect of BrdUrd on enzyme induction, it is necessary to estab- lish that the increase in CAMP phosphodi- esterase is the result of enzyme induction. Studies with actinomycin D implicate tran- scription as one of the levels where regula- tion of CAMP phosphodiesterase induction occurs in 3T3-L fibroblasts. Actinomycin D blocked the induction of the enzyme when

cells were simultaneously exposed to in- ducer and the drug. Furthermore, treat- ment of cells with inducer in the presence of cycloheximide, followed by removal of both and the addition of actinomycin D, resulted in a greatly accelerated increase in CAMP phosphodiesterase activity over the ensuing 2 h. These results are consistent with the postulate that the inducer stimu- lates the synthesis of an RNA species that cannot be translated into protein due to the presence of cycloheximide in the medium. Upon removal of the cycloheximide, trans- lation of the RNA into new CAMP phos- phodiesterase activity can occur.

Since the induction of the enzyme ap- pears to require transcription of DNA, and BrdUrd is able to block induction, studies were undertaken to determine whether in- corporation of BrdUrd into DNA is neces- sary to block the increase in CAMP phos- phodiesterase. The inhibitory action of BrdUrd is competitively blocked by the inclusion of excess thymidine in the culture medium as is the incorporation of [3H]- BrdUrd into DNA. BrdUrd added to cul- tures that are not actively synthesizing DNA has no effect on induction, nor does it alter the CAMP phosphodiesterase activ- ity when added directly to the assay for the enzyme. Thus, BrdUrd appears to be work- ing here, as in numerous other systems (2, 3), via incorporation into DNA.

Once it was established that CAMP phos- phodiesterase could be induced in a concen- tration-dependent fashion and that BrdUrd incorporation into DNA could inhibit in- duction, it was of interest to determine how changes in MeiBu-Xan and BrdUrd con- centrations would affect enzyme induction when both drugs were present. We find that BrdUrd concentrations between 0.1 and 1 PM are most effective at inhibiting enzyme induction (with 0.5 mM MeiBu-Xan). When the BrdUrd concentration is increased an- other order of magnitude, the inhibition of enzyme induction is proportionately less. A similar pattern was found by Walther et al. (8), who also studied BrdUrd replacement of thymidine in DNA as a function of BrdUrd concentration in the medium. They reported that the decrease in amylase ac- cumulation in pancreatic cultures was most dramatic when BrdUrd concentrations

BROMODEOXYURIDINE AND CAMP

were increased from 0.1 to 1 PM. At 1 PM BrdUrd, less than 10% of the thymidine was replaced (8). Further increases in BrdUrd concentration caused much greater incorporation of BrdUrd into DNA, but were significantly less effective in promot- ing inhibition of amylase accumulation. Thus, it is the initial low level of incorpo- ration which appears to be most effective in the action of BrdUrd on levels of a spe- cific protein.

The ability of a given MeiBu-Xan con- centration to induce the enzyme was found to depend on the length of time cells were exposed to it. Treatment of cultures with 0.1 m inducer is sufficient to elicit maxi- mal induction in an 18-h period, as similar activity is seen at all inducer concentrations between 0.1 and 1.0 ells for this period. Substitution of BrdUrd for thymidine re- duces the effect of a particular MeiBu-Xan concentration on induction of the enzyme. It is possible to examine directly how well a particular BrdUrd concentration can re- duce the efficiency of various concentra- tions of inducer. When such an analysis is performed, the relationship between BrdUrd inhibition and inducer concentra- tions of inducer. When such an analysis is performed, the relationship between BrdUrd inhibition and inducer concentra- nism is such that BrdUrd allows for tighter binding of putative regulatory molecules (histones, acidic proteins, RNA, etc.) to DNA; and (b) the tighter association of the putative regulatory molecules can be over- riden by specific inducers of enzyme syn- thesis. Such a model is attractive since sim- ilar results have been found with the lac operon (9), and since recent results suggest that eucaryotic chromatin proteins do bind more tightly to BrdUrd substituted than unsubstituted DNA (10, 11). However, fur- ther study is required to ascertain the mo- lecular events that occur when MeiBu-Xan competitively overrides BrdUrd-dependent inhibition of CAMP phosphodiesterase in 3T3-L fibroblasts. The mechanism may be CAMP-mediated since MeiBu-Xan in- creases intracellular CAMP levels (22), and the CAMP-dependent protein kinase ap- pears to be necessary for induction of the enzyme (23).

PHOSPHODIESTERASE INDUCTION 711

ACKNOWLEDGMENTS

The authors wish to thank Drs. R. J. Ho, D. H. Mintz, W. A. Scott, and W. J. Whelan for helpful discussions, Mrs. Carol Quigley for technical assist- ance, and Mrs. Sandra Black for technical preparation of the manuscript.

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