A comparison of the uptake, metabolism, and action ot cyclic adenine nucleotides in cultured hepatoma cells

ARCHIVES

OF BIOCkEMlSTBY

AND

BIOPHYSICS

169, 601-615

(1975)

A Comparison of the Uptake, Metabolism, and Action ot Cyclic Adenine Nucleotides in Cultured Hepatoma Cells DARYL

K. GRANNER,’

Departments

of Medicine

LARRY

SELLERS, AGNES LESLIE KUTINA

and Biochemistry, University Administration Hospital, Iowa Received

November

LEE, CAROL

of Iowa College of Medicine City, Iowa 52242

BUTTERS,

and

Veterans

1, 1974

Intracellular radioactivity following incubation of HTC or RLC cells in [3H]cAMP exceeds that following incubation in either [3H]monoor dibutyryl CAMP by 30-fold, yet little [3H]cAMP is found within the cells. Even at early times (30 min) the label derived from [3H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl CAMP (2-10 FM) result from incubation of both cell lines in either N6 mono- or dibutyryl CAMP. A very small percentage of this label is in CAMP, and within 2 h of incubation >65% of the label is again found in ADP or ATP. Liver cytosol contains three major CAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. CAMP is the most effective in vitro activator (lo- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order CAMP > N6 monobutyryl CAMP > > > dibutyryl CAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order CAMP 2 NB monobutyryl CAMP > dibutyryl CAMP. HTC and RLC cell cytosol protein kinase has chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C. The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in uitro binding of [3H]cAMP) was of the order N6 monobutyryl CAMP > dibutyryl CAMP > CAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl CAMP > dibutyryl CAMP > CAMP. This in uivo activation suggests that little additional stimulation would be obtained by adding CAMP to extracts prepared from such cells. This activation can be expressed as the ratio -CAMP: +cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl CAMP > dibutyryl CAMP > CAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells. RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated CAMP derivatives (but not by CAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds. Since HTC and RLC cells accumulate and metabolize CAMP and its derivatives equally, and since they both contain a protein kinase with similar in uivo and in vitro activation properties, it is suggested that the effects of butyrylated CAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase. or by a mechanism independent of protein kinase. ’ To whom

correspondence

should

be addressed. 601

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved

AND

602

GRANNER

3’,5’-adenosine monophosphate ( cAMP2) is generally less effective in cell, organ, or intact animal systems than a variety of its derivatives, most notably dibutyryl CAMP (dbcAMP), yet the latter is less effective in broken cell preparations (1). To account for this it has been stated that, although CAMP is the more active agent intracellularly, dbcAMP is more resistant to hydrolysis by phosphodiesterase and is more readily transported across the plasma membrane than is CAMP (1, 2). Although the resistance of dbcAMP to phosphodiesterase is now well established (3), surprisingly few studies comparing the cellular accumulation of this derivative with CAMP have been done (4-6), and the results are conflicting. Ryan and Durick showed that incubation of Strain L cells in either CAMP or dbcAMP resulted in a higher intracellular concentration of the former and suggested that this was due to active transport of CAMP (4). Kaukel and Hilz, however, demonstrated that HeLa cells exposed to labeled CAMP accumulated labeled products of CAMP metabolism but not CAMP itself and those exposed to labeled dbcAMP accumulate a deacylated derivative of dbcAMP, most probably the N6monobutyryl compound (mbcAMP) (5). It has been postulated that the diverse effects of CAMP are accomplished through activation of protein kinases (7). These enzymes exist in “CAMP-independent” and “CAMP-dependent” forms, the latter consisting of regulatory and catalytic subunits (8, 9). CAMP binds with high affinity to the regulatory protein, causing dissociation of this from the catalytic subunit and thereby activating the latter (8, 9). An active analog of CAMP should therefore compete with CAMP for specific binding sites on the regulatory protein and should coordinately activate protein kinase. Neelon and Birch recently compared the activation of partially purified bovine muscle protein kinase by CAMP and various 2Abbreviations used: CAMP is adenosine 3’5. monophosphate, dbcAMP is the dibutyryl derivative of CAMP, and unless otherwise cited, mbcAMP refers to the Ne-monobutyryl analog of CAMP. TAT is tyrosine aminotransferase.

ET

AL.

butyrylated derivatives (10). They found similar rank orders of effectiveness with respect to displacement of [3H]cAMP binding and protein kinase stimulation, namely CAMP > NGmbcAMP >>> 0”’ mbcAMP > dbcAMP. Studies attempting to demonstrate that such compounds activate protein kinase in intact cells have not been reported. Thus, although dbcAMP is widely used as an analog of CAMP, there has been no comparison of the cellular accumulation and metabolism of CAMP, mbcAMP and dbcAMP with their in vivo and in vitro stimulation of protein kinase, and their effect on differentiated processes, in a single cell system. This paper reports results of such studies in two hepatic-derived permanent cell lines with specific emphasis on tyrosine aminotransferase induction and cell replication. MATERIALS

AND

METHODS

HTC cells, derived from Morris minimal deviation hepatoma 7288~ (ll), and RLC cells, derived from normal liver (12), were used in these studies. Both were grown in suspension culture at cell densities between 4-6 x lo5 cells/ml in a modified Swim’s S77 culture medium containing 50 mM tricine [Ntris(hydroxymethyl)methylglycine] buffer and fetal calf and calf sera at final concentrations of 5% each. Radioactive

Nucleotide

Uptake

Studies

HTC or RLC cells, at 3-5 x 10S/ml, were removed from growth medium by centrifugation at 160g for 5 min. They were resuspended at a concentration of lo6 cells/ml in fresh, 37°C S77 medium containing 1 NCi/ml of the radioactive nucleotide. Unlabeled nucleotide at 1 mM concentration was added where noted. At indicated times triplicate l-ml aliquots were taken. Total cell-associated radioactivity was determined in duplicate aliquots which were washed twice with 10 ml of cold 0.05 M potassium phosphate buffer, pH 7.4, containing 0.15 M KCl. Additional washes remove a very small, constant amount of radioactivity. The cell pellets were resuspended in 100 ~1 H,O, and this and a 100 ~1 wash were added to 0.5 ml Protosol and counted in 10 ml Bray’s solution (13). After washing, the remaining cell aliquot was suspended in 1 ml H,O and disrupted by two 20-s bursts from a Biosonik sonifier. Forty-five microliters of HClO, were added, and precipitated protein was removed by centrifugation. A titrated amount of 7 N KOH was added to the supernatant, and the KClO,

CYCLIC

NUCLEOTIDE

was removed by centrifugation. pernatant was lyophilized prior raphy. Paper

EFFECTS

The protein-free suto paper chromatog-

Chromatography

Lyophilized samples prepared as described above were dissolved in 100 ~1 H,O and spotted on Whatman 3 MM paper. Descending chromatography was performed for 22-24 h in a solvent of absolute ethanol and 0.5 M ammonium acetate (5:2 v/v) at pH 5. Each spot was run with standards consisting of ATP, ADP, 5’ AMP, CAMP, adenosine, N” mbcAMP, and dbcAMP. Spots were located by uv absorbance, cut out, minced, and placed into a scintillation vial containing 2 ml H,O. This was shaken overnight before adding 10 ml Bray’s solution for scintillation counting. This chromatography system allows for complete separation of all the compounds applied (except for occasional overlapping of ATP and ADP). Virtually 100% of the radioactivity applied to the paper is recovered, and the entire procedure starting with HClO, precipitation through the final overnight incubation of the paper does not, of itself, lead to nucleotide degradation. Partial

Purification

of Protein

Protein kinase was from the method of standard incubation rmol sodium acetate, 0.4 pmol theophylline, purified calf thymus acetate and 2 nmol

Kinase

HEPATOMA

Assay

assayed by a procedure derived Kuo and Greengard (14). The volume of 110 ~1 contained 5 pH 6.0, 2 Fmol sodium fluoride, 0.1 gmol dithiothreitol, 50 pg F, histone, 2.2 pmol magnesium ATP containing 2 x lOa cpm

603

CELLS

y-[32P]ATP. Depending on the experiment, either 50 ~1 of column eluate or 40 pg of protein were assayed. Incubation was for 5 min at 37”C, after which CCl,COOH precipitable radioactivity was used to determine kinase activity (14). CAMP

Binding

Assay

CAMP binding activity was assayed by a modification of previously described methods (15, 16). The reaction volume of 250 11 contained 12.5 pmol Tris-HCI, pH 7.4, 3 pmol MgCl,, 0.5 hmol theophylline, 20 pmol [3H]~AMP and 50 ~1 column eluate or 40 pg protein as the source of receptor activity. After a 2 h incubation at 4”C, the [3H]cAMPmprotein complex was collected on Millipore HA cellulose ester filters which then were dried, dissolved, and counted, Tyrosine

Aminotransferase

Assay

TAT (EC 2.6.1.5) was assayed as described previously (17). One unit of enzyme activity represents the formation of 1 rmol of product (phydroxyphenylpyruvate) per min. Protein concentration was determined by the method of Lowry et al. (18), with bovine serum albumin as the standard.

Kinase

Male Sprague-Dawley rats weighing 200 g were killed by decapitation. Livers were rinsed in a buffer consisting of 25 mM Tris-HCl, pH 7.7, 1 mM MgCl,, 1 mM mercaptoethanol, 1 mM EDTA and 10% glycerol (TMG buffer) and homogenized first in a Waring Blendor, then in a Dounce homogenizer. Cell particles were removed by centrifugation at l,OOOg for 15 min, 27,OOOg for 30 min, and 100,OOOg for 90 min, yielding a cytosol fraction to which was added crystalline ammonium sulfate at 0.29 g/liter. The precipitate was dissolved in a small volume of TMG buffer, dialyzed for 18 h against 4 liters of the same buffer and applied to a 2 x 20 cm column of DEAE-Sephadex A-25 equilibrated with TMG buffer. Proteins were eluted with a linear salt gradient established by using 150 ml each of TMG buffer and TMG including 0.4 M KCl. Three milliliter fractions were collected and after protein kinase activity was located, selected fractions were pooled, concentrated by precipitation with 0.5 g/ml ammonium sulfate, and stored at -10°C. HTC and RLC cells were disrupted by sonication, then treated exactly as above. Protein

ON

Materials Culture media was obtained from Grand Island Biological Co. and the sera from the St. Louis Serum Co. Cyclic AMP was purchased from Sigma Chemical Co., the butyrylated derivatives from Boehringer-Mannheim, [3H]cAMP (12.8 Ci/mmol) from Schwarz-Mann, [$H]dbcAMP (8.6 Ci/mmol) from New England Nuclear, and all other compounds from Sigma Chemical Co. The purity of the cyclic nucleotides was established by descending chromatography as described above. CAMP and dbcAMP were devoid of detectable contaminants; mbcAMP contained small amounts of CAMP ( <5%) and trace amounts of dbcAMP (
Cellular

Uptake

of

Cyclic Nucleotides

The uptake of 3H-labeled cyclic nucleotides by HTC and RLC cells was determined in two ways. In the first kind of

604

GRANNER

experiment a tracer amount of labeled compound (1 gCi/ml) was added directly to log phase cells and cell-associated radioactivity was determined as a function of time. Such experiments (Fig. 1) revealed the following: (1) At all times tested the accumulation of radioactivity following addition of [3H]cAMP exceeds by 30-fold that from either mbcAMP or dbcAMP; (2) The uptake of mbcAMP is greater than that of dbcAMP at any time; (3) Accumulation of label from [3H]cAMP increases for about 4 h, then plateaus, whereas that from [3H]mbcAMP or dbcAMP appears to proceed linearly for about 6 h in both HTC and RLC cells, after which it plateaus; and (4) RLC cells appear to accumulate more label from CAMP, mbcAMP, and dbcAMP than HTC cells. RLC cells, however, have a mean cell volume of approximately 5600 pm3 as compared to about 3600 pm3 for HTC cells. Hence, if the data are calculated on the basis of cpm/cell volume, accumulation of any cyclic nucleotide at a given time is about equal in the two cell lines.

ET AL

A second approach consisted of adding larger amounts of label (5-7 pCi/ml) to culture media containing 1 mM concentrations of the corresponding cyclic nucleotide, which is the concentration used for the enzyme induction and growth experiments reported below. Although lower levels of cell-associated radioactivity were obtained and there was more scatter of the data, the findings were qualitatively similar as with the tracer alone. Metabolism of [3H]Cyclic Nucleotides The distribution of the total cellassociated radioactivity following incubation of HTC cells with the various 3Hlabeled nucleotides for 30 or 120 min was determined by descending paper chromatography (Fig. 2). Exactly similar results were obtained using RLC cells so this data will not be shown. At no time after the addition of [3H]cAMP (bottom panel) were amounts of this nucleotide in excess of 2% of the total radioactivity found within the HTC cells. At 30 min most of the label

CAMP mbcAMP dbcAMP

RLC . b .

HTC 0 A 0 I

2 HOURS

4 AFTER

6 ADDITION

FIG. 1. Uptake of 3H-labeled cyclic nucleotides described in Materials and Methods. Data points The left ordinate represents uptake of [SH]mbcAMP represents that of [8H]cAMP (note the difference

6 OF NUCLEOTIDE

by HTC and RLC cells. The procedure used is represent averages of duplicate determinations. and [3H]dbcAMP whereas the right ordinate in scale).

CYCLIC

NUCLEOTIDE

EFFECTS

0.5 HOURS

ON HEPATOMA

2 HOURS

CELLS

INCUBATION

605

TIME

FIG. 2. Metabolism of labeled cyclic nucleotides. HTC cells were incubated for 30 or 120 min in S77 medium containing 3H-labeled CAMP, mbcAMP or dbcAMP. Determination of the intracellular distribution of the radioactivity among the various compounds listed on the abscissa was accomplished by descending paper chromatography as described above. The results are expressed as the 9% of the total counts present in each compound. Very similar results have also been obtained with RLC cells (data not shown). Abbreviations are Ado = adenosine, mbc = N6 monobutyryl cyclic AMP, and dbc = dibutyryl cyclic AMP.

derived from [3H]cAMP was associated with ADP-ATP. Although samples taken at earlier times (15 min or less) occasionally showed larger percentages of CAMP, most of the label was in adenosine,3 suggesting that it enters the cell as such, then is rapidly phosphorylated. At 2 h virtually all the label was in ADP or ATP. Slightly less than 40% of the intracellular radioactivity after a 30 min incubation with [3H]dbcAMP was in the form of dbcAMP, about 20% was mbcAMP, 25% was found as ADP-ATP, and the rest consisted of small amounts of CAMP, adenosine and 5’ AMP. After a 2-h incubation the distribution was similar to that following incubation with [3H]cAMP, i.e., ADP and ATP together 3 In the chromatography system employed adenosine, inosine, and hypoxanthine have the same R,. Further fractionation of this region has not yet been performed.

accounted for about 65% of the radioactivity, with the rest distributed among the other nucleotides; less than 15% remained as the cyclic nucleotides dbcAMP, mbcAMP, or CAMP. The metabolism of [3H]mbcAMP by HTC or RLC cells appears to be similar in some respects to that of both CAMP or dbcAMP. At 30 min, 12% of the radioactivity was in the mbcAMP region of the chromatogram, 32% was in the adenosine region, 12% was 5’ AMP, and the remainder was ADP-ATP. Thus the intracellular metabolism of this compound resembles that of dbcAMP in that significant amounts of cyclic nucleotides, adenosine, and 5’ AMP are present 30 min after initiation of the incubation. A greater percentage of the total intracellular radioactivity, both at 30 and 120 min, is present as ADP-ATP than seen following addition of dbcAMP, thus in this respect the metabolism of mbcAMP resembles that of CAMP.

606

GRANNER

Incubations of mbcAMP and dbcAMP for less than 30 min, which might help identify the compound that enters the cell and its early metabolism, were attempted. The

FRACTION

ET

AL.

results were inconclusive because cellassociated radioactivity was quite low and chromatographic separation resulted in further dilution.

NUMBER

r

RLC

CELLS

FIG. 3. DEAE-Sephadex chromatography of protein Preparation of the cytosol and chromatography was section. Symbols indicate [3H]cAMP binding activity presence (O-----O) or absence of CAMP (O-----O); and bracketed by A, B, C were pooled into three liver corresponding to C, was taken from HTC and RLC cell

.ooo$ 1.

kinase from liver, HTC, and RLC cells. performed as described in the methods (A-----A); protein kinase activity in the conductivity in mmho (----4. Fractions kinase samples and a single sample, fractions.

CYCLIC

DEAE-cellulose

NUCLEOTIDE

Fractionation Kinase

EFFECTS

of Protein

Figure 3 (top panel) shows that liver cytosol CAMP-binding activity can be separated into two major and four minor peaks by DEAE-Sephadex A-25 chromatography. The major peaks and the largest minor peak are associated with peaks of “CAMP-dependent” kinase and were designated as fractions, A, B, and C. Most of the CAMP-dependent protein kinase, peaks A and B, eluted at low salt concentrations. Multiple protein kinases have been found in skeletal muscle (21) and liver cytosol (22), and recent evidence suggests that the regulatory subunit may be responsible for this heterogeneity (23). Previous experiments have shown that HTC and RLC cells lack the high affinity CAMP binding protein found in liver (24). Experiments represented by the middle and bottom panels of Fig. 3 suggest that this binding protein might be associated with BINDING

INHIBITION

ON

60 -

CELLS

Effect of Cyclic Nucleotides on the Protein Kinases Protein kinases A, B, and C from liver, and a pooled fraction (tubes 45-62) from HTC and RLC cells, comparable to fraction C, were all tested for inhibition of KINASE

c-a

STIMULATION

dbcAh4P

602

40-

E

zo-

5

o-

b

100 -

.\”

60.

g

60-

0 5 m

40-

!i 4 ” cy L

’

LO O 100 60. 60-

NUCLEOTIDE

607

protein kinases A and B, as these are lacking in HTC and RLC cells. The CAMPdependent kinases found in RLC and HTC cells elute from DEAE-Sephadex at salt concentrations comparable to that required to elute fraction C of liver (Fig. 3), although elution profiles suggest a degree of heterogeneity not seen in the latter. On exclusion chromatography, however, these three kinase fractions show essentially identical elution profiles (24). The presence of peaks of CAMP-independent kinase in HTC and RLC cell cytosol, not seen in liver, may also reflect the absence of the high affinity CAMP-binding protein in the cytosol of the cultured cells (24).

A

loo-

HEPATOMA

CONCENTRATION

(M)

FIG. 4. Coordinate relationship between (3H]cAMP binding displacement and stimulation of activity of protein kinases A, B, and C from liver. As the concentrations of CAMP (O-----O), mbcAMP (O-----O) or dbcAMP (A-----A) increase there is increased displacement of [3H]cAMP from specific binding sites (left panels) indicated as percent of a control in which only [3H]cAMP was added, and increased stimulation of protein kinase activity (right panels), indicated as the percent increase over a control in which no nucleotide was added. The binding assay was performed as described in Methods except that 2 pmol [3H]cAMP were used.

608

GRANNER

ET

AL.

[3H]cAMP binding and for kinase stimula2.5-fold stimulation of catalytic activity. tion by CAMP, mbcAMP and dbcAMP. Second, within the limits of experimental The results shown in Fig. 4 indicate that, of error, CAMP and mbcAMP appear equally these three nucleotides, CAMP is the most effective in stimulating catalytic activity. effective both in competing with Third, dbcAMP is much more effective in [3H]cAMP for binding to the regulatory competing with [3H]cAMP for binding and protein and in stimulating catalytic activin stimulating fraction C kinase activity ity of protein kinases A, B, and C from than with fractions A and B, with 10 PM liver. concentrations giving virtually maximal Fraction A, which comprises the bulk of stimulation. protein kinase activity in liver, shows a The cytosol protein kinases of cultured 16-fold increase in activity with maximal HTC and RLC cells resembles liver fracstimulation. CAMP is approximately 10 tion C with respect to cyclic nucleotide times more effective than mbcAMP in activation of kinase activity (Fig. 5). Alcompeting for [3H]cAMP binding and is though CAMP is 2-3 times as effective as 4-5 times as effective in stimulating catambcAMP in competing with [3H]cAMP for lytic activity. The dibutyryl derivative is specific binding sites, both are equally essentially ineffective even at 10 kM. effective in stimulating catalytic activity. Small concentrations of CAMP are about With RLC cell cytosol, mbcAMP appears 5 times more effective than those of to give maximal (and greater) activity at a mbcAMP in competing with [3H]cAMP for lower concentration than CAMP. Similar binding or in stimulating catalytic activity results were obtained with HTC cell cytosol. As with liver fraction C, maximal of liver fraction B kinase. Maximally effective concentrations of both result in a stimulation of HTC and RLC kinase is not great (3- to 4-fold), and dbcAMP is effeclo-fold stimulation of catalytic activity. tive, albeit only at high concentrations. Although much less effective, 10 PM concentrations of dbcAMP result in a 50% Tyrosine Aminotransferase Induction inhibition of [3H]cAMP binding and about half maximal stimulation of kinase activHaving demonstrated that butyrylated ity. derivatives of CAMP enter HTC and RLC Fraction C protein kinase is quite differcells and that these cells contain a cyclic ent from the other two. First, there is only a nucleotide dependent protein kinase simiBINDING

2

L s $ g 8 wz 0 z m 2 0 I I!5

INHIBITION

loo so-

KINASE

STIMULATION

HTC CELLS

60-

zo-

40

IOOso60-

4020NUCLEOTIDE

FIG. 5. Coordinate relationship catalytic activity of HTC and RLC same as in Fig. 4.

CONCENTRATION

between [3H]cAMP binding protein kinases. Experimental

(M)

displacement procedures

and stimulation of and symbols are the

CYCLIC

NUCLEOTIDE

EFFECTS

DIBUTYRYL

ON

HEPATOMA

609

CELLS

CAMP

MONOBUTYRYL 200

m bb

0

2

4

HTC CELLS RLC CELLS

6

8 HOURS

n s

M bd

1 22

AFTER

0

2

ADDITITION

I 4

HTC CELLS RLC CELLS ..L

6

1

*

OF MCLEOTIDE

6. Tyrosine aminotransferase induction by butyrylated CAMP analogs in HTC and RLC cells. Exponentially growing cells were placed into fresh S77 medium (at a concentration of lo6 cells/ml) containing 1 mM mbcAMP or dbcAMP where indicated, and lo-ml aliquots were taken at the times indicated. TAT specific activity was determined as percent induced over control to correct for variations in basal activity between experiments. Data points represent means * SE of 5-6 experiments. The left panel indicates experiments with dbcAMP, and the right panel, mbcAMP, with HTC cells (O-----O) and RLC cells (A-----A). FIG.

lar to one from liver, we next tested for an effect of mbcAMP and/or dbcAMP on two processes known to be influenced by these compounds in other cell lines, namely the induction of tyrosine aminotransferase (TAT) (25-27) and cell replication (26-28). Addition of either mbcAMP or dbcAMP to cultures of RLC cells results in a significant induction of TAT activity (Fig. 6). The increased activity is significant by 2 h, is maintained for at least 8 h, and returns to the basal level by 22 h. Although mbcAMP appears to result in greater increases, other experiments with dbcAMP have routinely resulted in a 200-250% increase of TAT activity in RLC cells. Intermediate levels of induction are noted with 0.3 mM concentrations of both nucleotides, and 20-25% increases are seen with 0.1 mM concentrations. Lower concentrations of each are ineffective (data not shown). This suggests that contamination of commercially available dbcAMP with mbcAMP is not the reason for the effectiveness of the former, whereas such could be the case in the binding inhibition-kinase stimulation experiments, as noted above. This does not necessarily imply that dbcAMP is active, for significant cellular accumulation of mbcAMP occurs after incubation of cells with dbcAMP (Fig. 2, Table II). Although TAT induction has routinely been seen in

RLC cells with both mbcAMP and dbcAMP (17 of 18 experiments), these compounds failed to induce HTC cell TAT in 17 consecutive experiments. Cyclic AMP and sodium butyrate are ineffective as inducers in either cell line, and the induction noted with dbcAMP in RLC cells can be blocked completely by 0.1 mM cycloheximide (data not shown). Effect

of Butyrylated Nucleotides Replication

on Cell

Suspension cultures of HTC or RLC cells, maintained in standard S77 medium or medium supplemented with either dbcAMP or mbcAMP in concentrations ranging from 0.25 to 1.0 mM, appear to replicate at the same rate as control cultures. In the representative experiment illustrated in Fig. 7, exponential growth was maintained for 48 h, and at the end of 72 h the cell density was the same in the control and cyclic nucleotide containing cultures. Activation

of Protein Kinase in Intact

Cells

Procedures for determining the state of activation of protein kinases in tissues following hormone treatment have recently been described (29-31) and it has been possible to demonstrate that epinephrine

610

ET AL.

GRANNER DIBUTYRYL

CAMP

MONOBUl-YRYL

IO6

CAMP

IO6 RLC

CELLS

RLC

“F

CELLS

106

HTC CELLS

DAYS

F

AFTER

ADDITION

OF

HTC CELLS

NuCLEOTIDE

FIG. 7. Effect of mbcAMP or dbcAMP on replication of HTC or RLC cells. Cells in exponential growth were placed in fresh S77 * dbcAMP (left panels) or mbcAMP (right panels) at the concentrations indicated. The initial cell concentrations were lo6 cells/ml and the cells were counted daily by hemocytometer.

treatment of isolated fat pads results in the conversion of inactive kinase holoenzyme to the active catalytic subunit (30). Thus, CAMP added to extracts prepared from epinephrine-treated fat pads resulted in little further stimulation of protein kinase activity, and the activity ratio -CAMP: +cAMP approached maximal activation or unity, because of the increase in intracellular CAMP concentration caused by the epinephrine (30). We employed such a technique to determine whether incubation of intact HTC and RLC cells in medium containing cyclic nucleotides results in activation of protein kinase, since the failure of such compounds to induce TAT in HTC cells could be due to insufficient in vivo activation. According to the model of kinase activation (8, 9), incubation of cells in cyclic nucleotides should result in occupation of available sites on the regulatory protein in proportion to the degree of kinase activation. Thus subsequent incubation of the extract in [3H]cAMP would result in less binding than seen in extracts from untreated cells.

Table I, which presents the results as the averages of three experiments, shows that cytosol extracts from RLC cells previously incubated in mbcAMP bind only 30% as much [3H]cAMP as similar extracts from cells cultured in plain S77 medium. Incubation in dbcAMP results in occupation of about 50% of the available sites, and CAMP is least effective. Similar results were obtained with HTC cells. Basal protein kinase activity, expressed as the ratio of cyclic nucleotide treated:control cells, reflects the binding data. The compound most effective in occupying binding sites in vivo, mbcAMP, resulted in the greatest stimulation of basal kinase, i.e., 2.49 times control in RLC cells as compared to 1.6 and 1.3 for dbcAMP and CAMP, respectively. Although the increases were not as striking, particularly for mbcAMP, the same observations were made in HTC cells. The activity ratio ~ CAMP: +cAMP, calculated from extracts prepared from the cells incubated in the presence or absence of cyclic nucleotides, also illustrates in vivo activation of protein kinase in both cell lines.

CYCLIC

NUCLEOTIDE

EFFECTS TABLE

DETERMINATION

Cell type

HTC

RLC

ON

HEPATOMA

611

CELLS

I

OF THE ABILITY OF VARIOUS CYCLIC NUCLEOTIDES, ADDED TO INTACT HTC OCCUPY CAMP BINDING SITES AND TO ACTIVATE PROTEIN KINASE~ Intact cell incubation

[>H] CAMP Binding (% control)

Protein Nucleotide treated:control -

Control CAMP dbcAMP mbcAMP

(100) 71 59 40

1.30 1.46 1.65

Control CAMP dbcAMP mbcAMP

(100) 70 51 30

1.31 1.60 2.49

-

kinase

OR RLC

activity

CELLS,

TO

ratio

-CAMP: +cAMP 0.41 0.52, 0.60 0.68 0.43 0.53 0.69 0.89

u HTC or RLC cells in exponential growth were removed from growth medium and incubated for 30 min at 37°C in fresh S77 medium * 1 mM concentrations of the cyclic nucleotides indicated. The cells were then removed from this medium and washed twice with 0.05 M potassium phosphate buffer, pH 7.6, containing 0.10 M KCl. The cells were disrupted by sonication and the supernatant remaining after centrifugation for 20 min at 35,OOOg was immediately assayed for [3H]~AMP binding and protein kinase activities as described in Methods. The binding data was calculated as the percentage of 13H}cAMP b ound in nucleotide-treated cells as compared to the untreated control; increased occupancy of the sites prior to addition of the [3H]cAMP would result in a lower binding percentage. The activity of protein kinase in the cell extracts was calculated as two ratios. both of which afford an estimation of the degree of stimulation of the enzyme in the intact cells. In the first the basal activity in extracts of cells previously incubated in cyclic nucleotide over that of control cultures was compared. This ratio estimates the degree of endogenous activation by the cyclic nucleotide. In the second, the ability of 1 @M CAMP to stimulate protein kinase in extracts prepared from cells incubated in standard or cyclic nucleotide-containing medium was assessed. A ratio of -CAMP: +cAMP of unity would indicate that the kinase was maximally stimulated by the in viuo incubation conditions.

Again the order of stimulation is mbcAMP > dbcAMP > CAMP, and mbcAMP in particular appears to be more effective in the RLC cells. It must be mentioned that interpretation of this experiment depends upon the assumption that the receptorcatalytic complex is not altered by the isolation procedures, a point discussed in detail by Corbin et al. (31). Intracellular

Concentrations of mbcAMP in HTC and RLC Cells

The protein kinases of intact HTC and RLC cells and of cytosol prepared from such cells are stimulated by N6-mbcAMP (Fig. 5) and the studies in which tracer amounts of this nucleotide were added to cell cultures suggests that each line accumulates the compound equally well (Fig. 1). In view of the lack of effect of mbcAMP on growth in either cell line, and its induction of TAT in RLC but not HTC cells, it became important to estimate the intracel-

lular concentrations of mbcAMP achieved by incubating these cells at 1 mM concentrations of mbcAMP or dbcAMP, those used in the growth and induction experiments.4 The results, shown in Table II, suggest that intracellular mbcAMP concentrations following incubation of cells in ‘It is conceivable that a substantial portion of the cell-associated radioactivity may actually be associated with the plasma membrane and thus not be accessible to cytoplasmic protein kinases. Although it is difficult to absolutely exclude such a possibility, we sought to determine whether enzymes which attack cell surface components resulted in lower uptake values. The experiments were conducted exactly as described in the Methods section, except that serum was omitted from the medium and the cells were incubated in either trypsin (1 mg/ml) or neuraminidase (2 @g/ml) for 15 min at 37°C prior to washing, Such studies gave “uptake” curves exactly like untreated cells. Although limitations are recognized. we thus have assumed that the radioactivity detected is intracellular and have estimated the concentrations of mbcAMP on this basis.

612

GRANNER

either dbcAMP or mbcAMP are in the range of 2-10 PM. This assumes that all of the mbcAMP is in the N6 or active form when N6-mbcAMP is added to the medium. Van Rijn et al. have shown that, following incubation in dbcAMP, about 50% of the intracellular mbcAMP formed is the N6 derivative, the remainder being the 02’ form (27), and studies in our laboratory, using similar procedures, resulted in a similar distribution. Taking this further correction into account, it is apparent that mbcAMP concentrations are achieved, in both cell lines, well above those required for maximal activation of protein kinase (refor maximal activation of protein kinase (refer to Fig. 5). The addition of mbcAMP to the medium results in 3- to 5-fold higher intracellular concentrations of N”mbcAMP than when dbcAMP is employed. DISCUSSION

Rat liver TAT is induced by both glucocorticoid hormones (32) and CAMP (33). Although four rat hepatoma-derived cell lines HTC, RLC, H4-II-E, and MH,C, have been found in which TAT is induced by glucocorticoids as in liver (11, 12, 34, 35), the response of these lines to CAMP or its derivatives has not been uniform either between cell lines or in the same line studied in different laboratories. Several years ago we showed that HTC cells lacked detectable levels of both adenylate cyclase activity and CAMP, and that TAT was not induced by dbcAMP, and suggested separate mechanisms for the action of glucocorticoids and cyclic nucleotides on this induction process (36). Subsequently Stellwagen (37) and van Rijn et al. (27) have demonstrated modest (less than 50%) increases in HTC cell TAT activity using higher concentrations of dbcAMP and monolayer rather than suspension culture conditions. Butcher et al., however, were also unable to induce HTC cell TAT with dbcAMP (25). In our recent series of experiments, now numbering 17, we have never seen induction of HTC cell TAT by either mbcAMP or dbcAMP. Although variations

ET AL.

in culture techniques may be responsible, this difference may be due to the instability of this line with respect to TAT induction, as has been shown by clonal analysis (38). Although the response of the RLC cell line to glucocorticoid hormones is apparently identical to that of HTC cells (39), butyrylated cyclic nucleotides are effective inducers of TAT in RLC cells (Fig. 6). Several studies have been performed in an effort to explain this difference in TAT induction in these two cell lines. Both accumulate and metabolize dbcAMP and mbcAMP (the effective inducers) in a similar manner (Figs. 1 and 2, and Table II). Accepting the postulate that the effects of CAMP in eucaryotic cells are mediated through activation of protein kinase (7), a prime explanation for the difference in the response of these lines to cyclic nucleotides is that RLC cells contain a protein kinase that HTC cells lack. The data of Figs. 3 and 4, and previous work in which the DEAE-Sephadex fractions were further analyzed by Agarose chromatography (24)) do not reveal such a difference, although these analytical procedures would probably not detect a specific kinase present in small amounts, nor would they detect a kinase which had an altered K, for CAMP (or analogs) or one with a defective catalytic subunit. Hence these possibilities cannot be absolutely excluded. Failure of cyclic nucleotides to activate protein kinase(s) in intact HTC cells might also explain the lack of TAT induction. The data presented in Table I tend to exclude this possibility, as all three cyclic nucleotides tested activate HTC and RLC cell kinase in ho, although the extent of activation is greater in RLC cells, particularly those incubated in mbcAMP. It is interesting to note that, although CAMP is thought not to enter cells readily and is ineffective as an inducer of TAT in either cell line, it does result in some activation of protein kinase in intact HTC and RLC cells. It should again be emphasized that such an experiment looks at total protein kinase, and the results do not preclude the possibility that a minor fraction which is

CYCLIC

NUCLEOTIDE

EFFECTS TABLE

DETERMINATION

Compound added

mbcAMP

HEPATOMA

II

Cell type

HTC RLC HTC RLC

Specific

4.5 6.1 4.5 6.1

x x x x

613

CELLS

OF THE MOLARITY OF INTRACELLULAR mbcAMP AFTER INCUBATION OF HTC EITHER mbcAMP OR dbcAMP FOR 30 MIN (SEE FOOTNOTE 3)”

Volume (liters) ( lo6 cells) dbcAMP

ON

10-e 1O-6 10-C 1Om6

CELLS IN

radioactivity

Cells (dpm/lOB cells) 2245 5060 6095 9006

OR RLC

Medium (dpm/nmol)

Molarity (PM)

21,070 21,070 15,020 15,670

2.4 4.1 11 11

D HTC or RLC cells were resuspended in S77 medium warmed to 37°C and containing 1 mM concentrations of mbcAMP (15,020 dpm/nmol for HTC or 15,670 dpm/nmol for RLC) or dbcAMP (21,070 dpm/nmol) at a concentration of lo6 cells/ml. Thirty minutes later cells were harvested as described in Methods. Cell volumes were determined using a Coulter Channelyzer and represent mean values. Final estimation of the intracellular molarity of mbcAMP was based on finding that 20% of the total radioactivity 30 min after addition of dbcAMP to the medium was mbcAMP and 12% of the total 30 min after adding mbcAMP remained as mbcAMP (Fig. 2). The calculations assume that all the mbcAMP is in the NE form when mbcAMP was added to the medium, and that about 50% was the NE derivative when dbcAMP was added to the medium.

critically involved in TAT induction is either absent or not stimulated in HTC cells. Other problems in interpreting this type of experiment have been discussed in detail (31). Cyclic AMP or its butyrylated analogs have been shown to inhibit the replication of a variety of cell lines, including H4-II-E and MH,C, of hepatoma origin (27). Although this effect is also ,allegedly mediated through protein phosphorylation, no direct data support this notion. A positive correlation between the ability of a cyclic nucleotide to inhibit cell replication and to induce TAT has been drawn, however, and both of these have been correlated to protein kinase stimulation (27, 40). The inability of either mbcAMP or dbcAMP to inhibit the replication of HTC or RLC cells, first noted by Van Rijn et al. (27) and confirmed in this paper (Fig. 7), taken with the fact that both nucleotides are excellent inducers of TAT in RLC cells, but not HTC cells, suggests that these two processes are mediated separately. Either each requires a separate, specific protein kinase, or a specific substrate for phosphorylation, or one or both are mediated by a process independent of protein phosphorylation. Opposite effects of CAMP and dbcAMP have been noted on lipolysis (41) and on glycogen content and [3H]thymidine incor-

poration in HeLa cells (42). Several possible explanations of such results are apparent from our study. Since CAMP enters cells poorly, dbcAMP might have an effect whereas exogenous CAMP does not. Also the deacylase required to convert dbcAMP to NE-mbcAMP, which is apparently the active analog (10, 20, Figs. 4 and 5), might not be present in a certain cell type [although these enzymes have been shown to be present in a variety of tissues (43)], thus dbcAMP might be ineffective in mimicking an action of endogenous CAMP. Finally, some of the effects attributed to CAMP may in fact be due to products of its degradation. The extensive intracellular metabolism of cyclic nucleotides is of considerable interest, and is of particular importance in comparing effects of CAMP and butyrylated derivatives, since so much more label from the former enters cells (Ref. 5, Fig. 1). Label from CAMP appears to primarily enter HTC and RLC cells as an unphosphorylated compound, most likely adenosine. It then is rapidly phosphorylated and within 30 min most of the original label is associated with ADP-ATP. Although at least some of the label originally associated with mbcAMP or dbcAMP enters as such, by 2 h it too is primarily in ADP-ATP. Although this observation differs in degree from that of

614

GRANNER

Van Rijn et al. who found that after 1 h of incubation of RLC cells in [3H]dbcAMP approximately 60% of the intracellular radioactivity was still in the form of dbcAMP, it is clear that incubation of these cells (HTC and RLC) in cyclic nucleotides, particularly CAMP, at concentrations commonly used for enzyme induction and growth inhibition studies, must cause tremendous changes in the absolute amounts as well as the ratios of adenine nucleotides. This could affect a variety of processes including the uptake and intracellular pool sizes of amino acids and nucleosides, macromolecular synthesis, any number of enzyme reactions, and finally, complex functions such as cell replication. Such effects should be considered in experiments in which metabolizable derivatives of CAMP are added to cell cultures. ACKNOWLEDGMENTS Supported by Veterans Administration Funds and by USPHS Grant CA12191. Granner is the recipient of an USPHS Career Development Award CA70802.

Research Daryl K. Research

REFERENCES 1. ROBISON, G. A., E. W. (1971) New York. 2. POSTERNAK, T., W. F. (1962) 3. MILLER, J. P., DIMMITT, M. A., ROBINS,

BUTCHER, R. W., AND SUTHERLAND, Cyclic-AMP, Academic Press, SUTHERLAND,

E. W., AND HENION,

Bioehim. Biophys. Acta 65, 558.

SHUMAN, D. A., SCHOLTEN, M. B., K., STEWART, C. M., KHWAJA, T. R. K., AND SIMON, L. N. (1973) Biochemistry 12, 1010. 4. RYAN, W., AND DURICK, M. A. (1972) Science 177, 1002. 5. KALJKEL, E., AND HILZ, H. (1972) Biochem. Eiophys. Res. Commun. 46, 1011. 6. SZABO, M., AND BURKE, G. (1972) Biochim. Bio-

phys. Acta 264, 289. 7. Kuo,

J. F., AND GREENGARD,

P. (1969)

Proc.

Nat.

Acad. Sci. USA 64, 1349. 8. GILL,

G. N., AND GARREN,

L. D. (1970)

Biochem.

Biophys. Res. Commun. 39, 335. 9. REIMANN, E. M., KING, C. A.,

BROSTROM, C. O., CORBIN, J. D., AND KREBS, E. G. (1971) Bio-

them. Biophys. Res. Commun. 42.187. 10. NEELON,

F. A., AND BIRCH,

B. M.

(1973)

J. Biol.

Chem. 248, 8361. 11. THOMPSON,

E. B., TOMKINS,

G. M.,

AND CURRAN, J.

ET

AL.

F. (1966) Proc. Nat. Acad. Sci. USA 56, 296. 12. GERSCHENSON, L. E., ANDERSSON, M., MOLSON, L.. AND OKIGAKI, T. (1970) Science 170, 859. 13. BRAY, G. (1960) Anal. Biochem. 1, 279. 14. Kuo, J. F., AND GREENGARD, P. (1970) J. Biol. Chem. 245, 4067. 15. GILMAN, A. G. (1970) Proc. Nat. Acad. Sci. USA 67, 305. 16. WALTON, G. M., AND GARREN, L. D. (1970) Bio-

chemistry 9, 4223. 17. GRANNER,

D.

K.,

AND TOMKINS,

G.

M.

(1970)

Meth. Enzymol. 17, 633. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 19. POSTERNAK, T. H. (1971) in Cyclic-AMP (Robison, G. A., Butcher, R. W., and Sutherland, E. W., eds.), p. 68, Academic Press, New York. 20. KAUKEL, E., MUNDHENK, K., AND HILZ, H. (1972)

Eur. J. Biochem. 27, 197. 21. REIMANN, E. M., WALSH, D. A., AND KREBS, E. G. (1971) J. Biol. Chem. 246, 1986. 22. CHEN, L. J., AND WALSH, D. A. (1971) Biochemistry 10, 3614. 23. KUMON, A., NISHIYAMA, K., YAMAMURA, H., AND NISHIZUKA, Y. (1972) J. Biol. Chem. 248, 3593, 24. GRANNER, D. K. (1974) Arch. Biochem. Bioph,ys. 165, 359. 25. BUTCHER, F. R., BECKER, J. E., AND POTTER, V. R. (1971) Exp. Cell Res. 66,321. 26. VAN WIJK, R., WICKS, W. D., AND CLAY, K. (1972) Cancer Res. 32, 1905. 27. VAN RIJN, H., BEVERS, M. M., VAN WIJK, R., AND WICKS, W. D. (1974) J. Cell Biol. 60, 181. 28. SHEPPARD, J. R. (1971) Proc. Nat. Acad. Sci. USA 68, 1316. 29. CORBIN, J. D., SODERLING, T. R., AND PARK, C. R. (1973) J. Biol. Chem. 248, 1813. 30. SODERLING, T. R., CORBIN, J. D., AND PARK, C. R. (1973) J. Biol. Chem. 248, 1822. 31. CORBIN, J. D., KEELY, S. L., AND PARK, C. R. (1975) J. Biol. Chem. 250, 218. 32. LIN, E. C. C., AND KNOX, W. E. (1957) Biochim.

Biophys. Acta 26. 86. 33. WICKS, W. D., KENNEY, F. T., AND LEE, K. L. (1969) J. Biol. Chem. 244, 6008. 34. PITOT, H. C., PERAINO, C., MORSE, P. A., AND POTTER, V. R. (1964) Nat. Cancer Int. Monogr. 13, 229. 35. RICHARDSON, U. I., TASHJIAN, A. H., AND LEVINE, L. (1969) J. Cell Biol. 40, 236. 36. GRANNER, D. K., CHASE, L. R., AURBACH, G. D., AND TOMKINS, G. M. (1968) Science 162, 1018. 37. STELLWAGEN, R. H. (1972) Biochem. Biophys. Res. Commun. 47, 1144. 38. LEVISOHN, S. R., AND THOMPSON, E. B. (1972) Nature New Biol. 235, 102.

CYCLIC

NUCLEOTIDE

EFFECTS

39. SELLERS, L. W., AND GRANNER, D. K. (1974) J. Cell Bid. 60, 337. 40. SIMON, C. N., SHUMAN, D. A., AND ROBINS, R. K. (1973) in. Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 3, p. 330, Raven Press, New York.

ON HEPATOMA

CELLS

615

41. SOLOMON, S. S., BRUSH, J. S., AND KITABCHI, A. E. (1970) Science 169, 387. 42. KAUKEL, E., FUHRMANN, U., AND HILZ, H. (1972) Biochem. Biophys. Res. Commun. 48, 1516. 43. BLECHER, M., AND HUNT, N. H. (1972) J. Biol. Chem. 247, 7479.