Inhibition of human prostatic epithelial cell replication by cAMP and selected analogs

Primed in Sweden Copyright @ 1976 by Academic Press. Inc. A// righrs of reproducrion in any form reserved

Experimental Cell Research 102 (1976) 95-103

INHIBITION

OF HUMAN

REPLICATION

PROSTATIC

BY CAMP

R. M. NILES,

AND

EPITHELIAL

SELECTED

CELL

ANALOGS

J. S. MAKARSKI, MARY J. KURTZ and A. M. RUTENEIURG

Department of Surgery, Boston University Medical School, Boston, MA 02118, USA

SUMMARY The inhibition of human prostatic epithelial cell (MA-160) replication by CAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (CAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-CAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the CAMP phosphodiesterases, CAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl CAMP (db-CAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, lmethyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [3H]TdR and [3H]uridine than control cells. Treatment of cells with 0.5 mM CAMP for 24 h, on the other hand, resulted in both substantially increased [3H]TdR uptake and increased [aH]uridine incorporation into RNA. The effects of similar treatment with db-CAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine.

Cell proliferation and DNA synthesis have been shown to be influenced by intracellular cyclic AMP (CAMP) levels [l-5]. Addition of dibutyryl CAMP (db-CAMP) or prostaglandin El (PGE,), which increased cellular CAMP levels by stimulating adenylate cyclase, to the culture medium of transformed cells blocked cell division and restored normal morphology [6-g]. Proliferation of HeLa and L cells has been shown to be inhibited by CAMP by some investigators but not by others [9, lo]. Studies have indicated that CAMP is involved in regulating cellular functions and cellular growth, however, the biochemical mechanism of this effect has not been fully clarified. CAMP inhibited growth of cultured human liver cells and caused a con7-761808

siderable increase in uptake of labelled thymidine and uridine [ll]. CAMP also increased thymidine transport into monkey cells (CV-1) without affecting DNA synthesis [ 121. In contrast, db-CAMP inhibited the transport of thymidine into Chinese Hamster Ovary (CHO) cells [13], while Hilz & Kaukel [14] reported divergent effects between CAMP and db-CAMP on cell proliferation and macromolecular synthesis in HeLa S3 cultures. Plagemann & Sheppard [15] showed that inhibiting phosphodiesterase activity or hormonally stimulating adenylate cyclase activity resulted in inhibition of nucleoside and sugar transport in Novikoff rat hepatoma cells. Subsequent studies [16] with several cultured cell lines, however, did not confirm the correlation Exp

Cell

Res

102 (1976)

96

Nilesetal.

between intracellular CAMP levels and nucleoside and glucose transport. We have previously studied the CAMP system in relation to cell division in WI38 cells, a human diploid tibroblast cell line. Prostaglandin E, and theophylline increased CAMP levels accompanied by an effective halt in DNA synthesis and cell division [ 171. The present communication examines the effect of CAMP and certain analogs on replication and other biochemical parameters in MA-160, an epithelial cell line of human prostatic origin (prostatic adenoma). MATERIALS

AND

METHODS

The cell line used in this study MA-160 (obtained from Microbiological Associates, Bethesda, Md) originated from benign adenoma of human prostate. It may have subsequently undergone transformation in culture such that it now gives rise to tumors in hamster cheek pouches [18] and athymic (nude) mice [ 191. For all experiments herein described cells were seeded in 60 mm plastic tissue culture dishes (Falcon). The seeding medium consisted of Minimal Essential Medium (MEM) with Hanks’ salts, nonessential amino acids. vitamin solution. L-glutamine (2 mM), sodium pyruvate (1 mM), 50’ pgirnl streptomycin sulfate, 50 U/ml oenicillin G. and 10% fetal bovine or heatinactivated fetal bovine (GIBCO) serum, adjusted to a final pH of 7.1. Twenty-four hours after plating, the seeding medium was removed and the cells refed with MEM containing Earle’s salts plus the previously mentioned compounds with or without drugs. The cells were then returned to the 37”C, 95% air, 5% CO, humidified incubator until removal for cell counting or biochemical experiments described below. Cells were counted after removal from the plates by 0.25 % viocase, with the aid of a model B Coulter Counter (Coulter Electronics, Hialeah, Fla). (We are indebted to Dr Arthur Malefont of Instrumentation Labs Inc., Lexington, Mass., for the loan of this equipment.) Culture media and cells were checked for bacterial contamination by inoculation on nutrient agar slants and incubation at 37°C for at least 2 weeks. Possible mycoplasma contamination was checked by the uridineluracil ratio method [20]. Reagents used in this study were obtained from Sigma Chemical Co., except for methyl-thio-CAMP (MES) which was a gift from Squibb Pharmaceuticals and I-methyl-3-isobutylxanthine (MIX) which was purchased from Aldrich Chemical Co., N.J.

Biochemical studies For a given experiment the Falcon culture dishes were seeded at approx. 1-2~ 1t.Pcells/plate. When the cells Exp Cell Res 102 (1976)

reached a density of 5-9~ lo5 cells/plate (34 days later), the medium was aspirated and replaced with Earle’s medium (plus 10% serum) containing 0.5 mM of either methyl-thio-CAMP, CAMP or db-CAMP plus 0.2 mM MIX (a potent phosphodiesterase inhibitor), or 0.03 mM 5’-AMP. Medium alone (with 10% serum) was added to the control plates. After 24 h the cells were incubated for 30 min with either 1 PCi r3H]TdR, 0.5 &i [r4C]tyrosine, or 1 $Zi (New England Nuclear. Boston. Mass.). -13Hluridine Separate plates were used for cell counts at both the time of drug addition and at the time the radioactive tracers were added. After the incubation period the medium was aspirated, the cells washed twice with 3 ml vol of 0.9% NaCl and 1 ml of cold 5% (w/v) trichloroacetic acid (TCA) added. The plates were kept at 4°C for at least 15 min after which the cells were removed with a “rubber policeman”. These 1 ml suspensions of cells and TCA were filtered through Millipore Filters (24 mm diameter; pore size 0.22 pm). One hundred ~1 aliquots of the TCA-soluble filtrates were added to scintillation vials containing 5 ml of

20.017.5 -

15.0 -

12.5 10.0 -

5.0 t & 7.5 -

2.514@4&& 0

12

3

4

5

6

7

Fig. 1. Abscissa: time in culture (days); ordinate: cell number x 105. The effect of CAMP, 5’-AMP, 8-methyl-thio CAMP and uracil on growth of MA-160 cells. Cells were seeded 0.75 x 105/60 mm Falcon dish in Ham’s F-10 plus 10% untreated fetal bovine serum. One day later cells were refed with F-10 media plus 10% serum containing no drug (O-O); 0.25 mM CAMP (O-O); 0.25 mM 5’-AMP (X-X); 8-methyl-thio-CAMP (A-A); and 0.25 mM cAMP+O.S mM uracil (A-A). Media plus additives were replenished at 2 day intervals for the remainder of the experiments. At the indicated days, the cells were removed from the plates with 0.25 % viocase and counted with aid of a Model B Coulter Counter. The data is expressed as the average of triplicate plates.

CAMP and prostatic Aquasol and radioactivity determined in a Packard Tri-Carb liquid scintillation counter. The Millipore filters, containing the TCA-precipitable material, were washed with 5 ml of cold 5% TCA, placed in scintillation vials, and treated with 1 ml of ethylene glycol monoethyl ether and 5 ml of Cellusolve. Radioactivitv in these TCA-insoluble fractions was measured as described above.

cell replication

97

Table 2. The effect of CAMP on MA-160 cell replication in media containing untreated or heat-inactivated fetal bovine serum Cell number x 105

Incubation additives

Untreated fetal calf serum

Heat-inactivated fetal calf serum

ll.Of1.2 1.4f0.2 lO.Of2.0

ll.lk2.3 ll.OkO.8 9.5fl.l

lO.Of0.7

9.1k1.2

RESULTS The initial experiments were carried out in untreated fetal bovine serum containing the various additives shown in fig. 1. A concentration of 0.25 mM CAMP or Y-AMP almost completely inhibited MA- 160 cell replication. The inhibitory effect of S-AMP as well as CAMP on cell division could perhaps have been due to excess adenine nucleotides, acting through a feedback inhibition similar to that observed in the presence of excess TdR. To test this hypothesis, various nucleotide bases were added to the cell cultures in an attempt to reverse the inhibitory effect of CAMP. Of the bases tested only uracil exerted a substantial reversal of the inhibition (fig. 1). The analog 8-methylthio

Table 1. Integrity of CAMP in untreated and heat-inactivated fetal bovine serum Incubation additives No serum Fetal bovine serum Heat-inactivated fetal bovine serum Fetal bovine seru.n+cells Heat-inactivated fetal bovine serum+cells

cpm

[$H]cAMP

%Hydrolysis

14 500 2 610

00.0 82.0

12 905 2 102

12.0 85.5

10 875

25.0

Falcon dishes (60 mm) containing the components listed in the table were incubated with 15000 cpm [S~]~~~~ plus 0.25 mM cold CAMP for 3 days at 3PC. At the end of the incubation period the media were removed and treated with sufficient snake venom (30°C for 30 mitt) to convert any 5’-AMP to adenosine. The treated mixtures were then chromatographed on Dowex 1x2 columns (0.6~4 cm) and the [SH]cAMP eluted with 0.02 N HCI.

None 0.25 mM CAMP 0.25 mM uridine 0.25 mM CAMP+ 0.25 mM uridine

-

Cells were seeded in MEM plus Hanks’ salts with the anorooriate serum (10% v/v) at 1.0~ lOWI mmFalcon diih. ‘one day later the media was changed to MEM plus Earle’s salts with the appropriate serum+the additives listed in the table. After 3 days further incubation, the cells were removed from the plates with 0.25 % viocase and counted with the aid of a Model B Coulter Counter. The data are expressed as means fS.E.M. for quadruplicate plates.

CAMP (MES) was less effective than CAMP in stopping cell replication (fig. 1). Furthermore, MES effects were not reversed by uracil. Since MES is quite resistant to hydrolysis by CAMP phosphodiesterase (PDE) [21], we reasoned that CAMP might be subject to degradation by PDE’s in the serum of the cell growth media, or released by the cells into the media. Table 1 clearly shows that this indeed is the case. After 3 days (the longest time CAMP was present in the medium without replenishment) in media with or without cells, less than 20 % of the CAMP remained intact. To circumvent this problem the fetal bovine serum was heated to 56°C for 30 min, and the experiment repeated. At the end of the 3 day period (table 1) in this serum, media alone had 89% of the CAMP intact, while media with cells had 75 % of the original CAMP. To measure the effect of CAMP on cell replication in media containing heat-infip

Cell

Res 102 (1976)

98

Niles et al.

Table 3. The effect of CAMP and MIX MA-160 cell replication

on

Cell no. X IO” Treatment

Day 3

Day 4

None MIX (0.2 mM) 0.1 mM CAMP 0.1 mM cAMP+MIX (0.2 mM) 0.5 mM CAMP 0.5 mM cAMP+MIX (0.2 mM) 1.O mM CAMP 1.0 mM cAMP+MIX (0.2 mM)

1.43f0.03

2.12+0.10 1.40~0.17

I. 19fO. 13 1.8OkO.08

1.90f0.07

1.24+0.08

1.61kO.20

1.3o+o.I5

1.30f0.12

1.21*0.05

1.65kO.08

0.28f0.04

0.31kO.05

1.02*0.12

1.58f0.11

Cells were seeded at 1.2x105/60 mm Falcon plate in MEM plus Hanks’ salts and 10% heat-inactivated fetal bovine serum. One day later the media was replaced with MEM plus Earle’s salts and 10% serum containing the additives listed in the table. After 3 and 4 days further incubation the cells were removed from the plates by 0.25 % viocase and counted with the aid of a Model B Coulter Counter. The data are expressed as means +S.E.M. of triplicate plates.

activated fetal bovine serum, incubation of cells for 3 days with 0.25 mM CAMP, showed that cell growth in regular fetal bovine serum was markedly repressed (table 2). This inhibition as shown in fig. 1 was almost totally reversed by the addition of 0.25 mM uridine. However, cells incubated with 0.25 mM CAMP in heat-inactivated serum showed no growth inhibition. Heat-inactivation of the serum did not interfere with cytostatic ability of adenosine or 5’-AMP. Other nucleotides, namely GMP, CMP, and UMP did not inhibit cell replication. The ability of CAMP, db-CAMP and MES, to inhibit MA-160 cell growth in the presence or absence of MIX, a potent PDE inhibitor, is illustrated in tables 3-5. All the media used in the remaining studies described in this report was supplemented Exp CellRes

102 (1976)

with heat-inactivated fetal bovine serum. CAMP, at concentrations of 0.5 mM and 1.0 mM significantly inhibited cell replication after 4 days incubation (table 3). This inhibition was negated by including 0.2 mM MIX in the cell growth media. Cell growth inhibition by various concentrations of CAMP was not significantly different (p= >O. 10) than that due to MIX alone. Surprisingly, no concentration of dbCAMP tested was effective in supressing cell growth (table 3). However, when dbCAMP was combined with MIX, inhibition of cell replication was observed which was dependent upon the concentration of dbCAMP and which was significantly different than that found with MIX alone (pcO.02). MES inhibited growth significantly (p< 0.02) even at the lowest concentration tested, 0.1 mM (table 5). After treatment of MA-160 cells for 4 days with 1 mM MES, a

Table 4. The effect of db-CAMP and MIX on MA-160 cell replication Cell no. X 106

Trearment

Day 3

Day 4

None MIX (0.2 mM) 0.1 mM db-CAMP

1.43f0.03 1.19+0.13

2.12f0.10 1.40f0.17

1.51t0.05

1.98kO.08

1.13+0.05

1.52f0.10

“$ r;l~$‘-cAMP+MrX 0.5 mM db-CAMP 0.5 mM d&-AMp+MIX (0.2 mM)

1.60~0.08

2.05f0.08

0.80f0.00

1.13t0.12

1.O mM db-CAMP

1.37f0.07

1.98+0.05

ti~.~~$cAMP+MIX

0.66f0.05

0.8520.05

Cells were seeded at 1.0~ 105 cells/60 mm Falcon dish in MEM plus Hanks’ salts and 10% heat-inactivated fetal bovine serum. After one day the media was replaced with MEM plus Earle’s salts and 10% serum containing the additives listed. After 3 and 4 days, the cells were removed from the plates by 0.25% viocase and counted with the aid of a Model B Coulter Counter. The data are expressed as means f S.E.M. of

triplicateplates.

CAMP and prostatic

Table 5. The effect of MES and MIX MA-160 cell replication

on

Cell no. X lo5 Treatment

Day 3

Day 4

None MIX 0.2 mM 0.1 mM MES 0.1 mM MES+MIX (0.2 mM) 0.5 mM MES 0.5 mM MES+MIX (0.2 mM) I .O mM MES 1.O mM MES+MIX (0.2 mM)

1I .49+0.45 9.28kO.27 7.95kO.21

21.94k1.54 19.17+0.61 18.12+0.70

7.72kO.77 15.35kO.59 4.45kO.3 1 7.86kO.33 5.51f0.20 3.93+0. I6

10.01~0.14 6.45&O. 10

4.59kO.05

8.08?0. I9

Cells were seeded at 1.0~ W/60 mm Falcon dish in MEM plus Hanks’ salts containing 10% heat-inactivated fetal bovine serum. One day later the media was replaced with MEM plus Earle’s salts and 10% serum containing the additives listed in the table. After 3 and 4 days of further incubation, cells were removed from the plates with 0.25 % viocase and counted with the aid of a Model B Coulter Counter. The data are expressed as means tS.E.M. of triplicate plates.

99

demonstrated by radioactivity in the acidinsoluble material (table 7). As described elsewhere, CAMP was 80% degraded after 3 days in normal fetal calf serum (due to PDE activity), but only 25 % degraded after 3 days in heat-inactivated serum containing cells. Using this 25% degradation figure, 0.25 mM CAMP would yield approx. 0.06 mM 5’-AMP (a PDE degradation product of CAMP) after 3 days, or 0.02 mM after 24 h. Since this concentration of 5’-AMP is the maximum amount likely to be formed after

Table 6. Effects of drug treatment on the uptake of labeled TdR, tyrosine and uridine by MA-160 cells cpmlculture Radioactive label [3H]TdR

70% inhibition of cell growth occurred. Addition of 0.2 mM MIX to various concentrations of MES did not lead to greater retardation of cell growth than that observed with MES alone. In other experiments the growth inhibitory properties of CAMP, 5’-AMP, MES, db-CAMP and MIX were correlated with their ability to alter the normal uptake of labeled TdR, tyrosine and uridine, and the incorporation of these precursors into their respective macromolecules. As described in Materials and Methods, all of these experiments were conducted with cells grown in media containing 10% heatinactivated fetal calf serum. Table 6 shows that 24 h treatment with 0.5 mM CAMP resulted in a substantially increased [3H]TdR uptake (174% of control), as well as an increased [3H]uridine incorporation into RNA (163 % of control), as

cell replication

Dw

Control

Drugtreated

% of control

CAMP 5’-AMP MES db-CAMP+ MIX

1 017 610 750

1 770 1 255 353

174b 206” 470

2 078 2 078 229 198 146

1 425 1 535 295 172 179

69’ 74c 129” 87” 123b

216

281 -

1306 -

7069 5 384 6 378

65760 245 1 778

1:; 28O

9 142 9 142

4410 5 037

4gb 55”

MIX [Y]Tyrosine [3H]Tyrosine

CAMP 5’-AMP MES d&b&MP+ MIX

[3H]Uridine

CAMP 5’-AMP MES db-CAMP+ MIX MIX

In these experiments the media of cultures in the log phase of growth was replaced by media containing the drugs indicated. After 24 h the cultures were labeled for 30 min with either r3H]TdR, [“Cl- or [3H]tyrosine, or [3H]uridine. Uptake of thymidine, tyrosine, or uridine was determined by measurements of radioactivity in acid-soluble fractions, as described in Materials. Values are from a representative experiment and are expressed as the mean of 3 replicates. Drug concentrations were as follows; CAMP 0.5 mM, 5’AMP 0.03 mM, MES 0.5 mM, db-cAMP+MIX 0.5 mM and 0.2 mM respectively, and MIX 0.2 mM. a PCO.01. b PCO.05. c PCO.10. Exp CellRes

102 (1976)

100

Nilesetal.

Table 7. Effects of drug treatment corporation of labeled precursors macromolecules by MA-160 cells

on ininto

cpm/culture Fb$oactive Drug [3H]TdR

[“C]Tyrosine [SH]Tyrosine [aH]Uridine

CAMP S-AMP MES db-CAMP+ MIX MIX CAMP 5’-AMP MES db-CAMP+ MIX MIX CAMP 5’-AMP MES db-CAMP+ MIX MIX

Control

Drugtreated

% of control

673 447 839

639 272 375

9.5 61” 45”

1 568 1 568 793 879 672

970 952 1 086 801 537

62a 61a 137 91 80a

176 -

84 -

868 737 1 243

1 414 1041 278

163= 14lb 22”

1 134 1 134

563 685

50a 60”

48

In these experiments the media of cultures in the log phase of growth was replaced by media containing the drugs indicated. After 24 h the cultures were labeled for 30 min with either [3H]TdR, [“Cl or [3H]tyrosine, or [SH]utidine. DNA, protein and RNA synthesis were determined by measuring radioactivity in acidinsoluble fractions, as described in Materials and Methods. Values are from a representative experiment and are expressed as the mean of 3 replicates. Drug concentrations were as follows: CAMP 0.5 mM, 5’AMP 0.03 mM, MES 0.5 mM, db-cAMP+MIX 0.5 mM and MIX 0.2 mM. a P
24 h in heat-inactivated serum with cells, we decided to measure the effects of a slightly higher concentration, i.e., 0.03 mM. Treatment of cells with this concentration of S-AMP yielded exactly the same pattern as CAMP, i.e., an increased TdR uptake (206% of control, table 6) and an increased incorporation of uridine (141% of control, table 7). CAMP increased the uptake of [‘“Cltyrosine (table 6) but did not significantly alter DNA or protein synthesis whereas Exp CellRes

102 (1976)

5’-AMP decreased [14C]tyrosine uptake and DNA synthesis (61% of control, p
DISCUSSION The data presented in this communication illustrate pitfalls involved in treating cells with CAMP and its analogs. The initial experiments which suggested that CAMP inhibited cell division (fig. 1) were negated by the fact that the cyclic nucleotide was almost certainly degraded to S-AMP. We postulate that this event occurred predominantly in the culture medium, since 0.25 mM CAMP added to heatinactivated serum containing media did not affect cell growth, even though intracellular phosphodiesterase activity was not inhibited.

CAMP and prostatic

Our results (fig. 1) would seem to be in agreement with Hilz & Kaukel [14] who found that the actions of exogenous CAMP on HeLa cells were non-specific in that they could be imitated by various adenine nucleotides. They postulate that adenosine is the actual growth-inhibitory compound even though they, as did we, found that exogenously applied adenosine repressed cell growth less than Y-AMP. This discrepancy was explained by noting that adenosine was taken up and metabolized rapidly by the cells while 5’-AMP and CAMP released adenosine slowly and continuously. Slow constant infusion of adenosine to HeLa cells resulted in as great, if not greater, cytostasis than other adenine nucleotides. At high concentrations (0.5-1.0 mM) CAMP caused repression of growth even in heat-inactivated serum (table 3). This was most likely due to its degradation to S-AMP by residual serum or cellular phosphodiesterase, since the addition of MIX, a potent phosphodiesterase inhibitor, reverses this effect. MIX also has a cytostatic effect, probably the result of increasing the intracellular pool of CAMP by negating its hydrolysis and/or by preventing its exodus from the cell in a fashion reported by Kelley & Butcher [22]. Our results with db-CAMP were interesting in that db-CAMP by itself (0.1-1.0 mM) had little effect on cell replication, but when combined with MIX inhibited growth in a concentration dependent manner (table 3). Again Hilz & Kaukel [14] reported that db-CAMP was able to imitate intracellular CAMP only after its conversion of N6-monobutyryl CAMP. It may be that MA-160 cells contain highly active acylases which remove both butyryl groups from db-CAMP forming free CAMP which is in turn rapidly hydrolysed by cellular phosphodiesterase.

cell replication

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Addition of MIX would prevent this degradation of free CAMP and thus markedly raise the intracellular levels of this nucleotide. Alternatively, MIX may in some manner facilitate the transport of db-CAMP into the cell. The addition of MIX to MES did not result in an additive or synergistic repression of cell replication. This would seem to indicate that after a certain level of CAMP is reached within the cell further increases will not cause greater growth inhibition since the system through which CAMP exerts its effect is saturated. Our data suggests that other investigators interested in treating cells with CAMP might consider using MES. In our hands MES was extremely resistant to phosphodiesterase activity and gave a greater inhibition of cell replication than dbCAMP and MIX combined. Insofar as the biochemical mechanism of growth, inhibition is concerned, the agents fall into two major categories when tested in the presence of heat-inactivated fetal bovine serum: (a) those (CAMP, 5’-AMP) which resulted primarily in a substantially increased [3H]TdR uptake and an increased r3H]uridine incorporation into RNA; and (b) those (MES, db-cAMP+MIX, MIX) which resulted in significantly decreased uptake of [3H]TdR and [3H]uridine and the incorporation of these precursors into their respective macromolecules (i.e. apparent DNA and RNA synthesis). Also, no generalizations can be made as to how the first group (CAMP, 5’-AMP) affects tyrosine uptake and protein synthesis, because of variation from experiment to experiment. The second group (MES, db-cAMP?MIX) stimulated tyrosine uptake, while simultaneously decreasing protein synthesis. Eker [ 111 reported marked inhibition of DNA synthesis within 14 h after treatment E.rp Cell

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Niles et al.

of human liver cells with 1 mM CAMP. Our results with CAMP reveal inhibition of DNA synthesis ranging from 5 to 21%; also SAMP significantly inhibited DNA synthesis by 39% (61% of control, table 7). Eker found a considerable increase in the cellular uptake of thymidine and uridine from the medium (within l-4 h after CAMP treatment). Our findings after 24 h treatment with CAMP or 5’-AMP also show increased uptake of TdR; however, results of uridine uptake were variable. In contrast, others have shown that CAMP increases thymidine transport into monkey cells (CV-1) without affecting DNA synthesis [12]. Although Eker reports no significant changes in RNA and protein synthesis, in our experiments CAMP and 5’-AMP both significantly increased RNA synthesis. Hilz & Kaukel [ 141 also reported that CAMP (24 h treatment) results in increased incorporation of r3H]uridine into RNA: however, their results differ from ours in that [3H]TdR incorporation into DNA was elevated by CAMP. It is likely that variability in experimental conditions and differences among cell lines may account for some of these discrepancies. Nonetheless, the fact that both CAMP and 5’-AMP elicit the same general pattern of effects, supports the findings of Hilz & Kaukel [14] that exogenous 5’-AMP produces metabolic alterations similar to CAMP, and their contention that the effect of exogenous CAMP may be due to adenosine produced slowly by the gradual degradation of CAMP and 5’-AMP. Our results with the second group of compounds (MES, db-cAMP+MIX, or MIX) reveal effects that others have reported using db-CAMP on Chinese hamster ovary [13] and HeLa cells [14]; i.e. reduction in the incorporation of [3H]TdR or [3H]uridine and inhibition of [3H]TdR transport. Also, our experiments show excelExp Cell Res 102 (1976)

lent correlation between the degree of uptake inhibition and macromolecular synthesis (incorporation of r3H]TdR and [3H]uridine), suggesting perhaps an effect on cellular transport, as others have reported in the case of db-CAMP. However, in contrast to the inhibition of nucleoside transport and incorporation, our experiments with MES and db-CAMP+ MIX revealed concomitant stimulation of [‘“Cl or [3H]tyrosine uptake, accompanied by an inhibition of tyrosine incorporation (i.e. protein synthesis). This inhibition of protein synthesis may affect the thymidine and uridine transport mechanisms of MA160 cells, since Plagemann et al. [23] have found that the TdR and uridine transport systems of mammalian cells are metabolically unstable and are lost upon inhibition of protein synthesis. Also Hauschka et al. [13] correlated their db-CAMP induced inhibition of thymidine uptake with an observed decrease in thymidine kinase activity. Taken as a whole, our results suggest that investigators treating cells with CAMP and its derivatives should be cautious in ascribing resultant effects to CAMP.

REFERENCES 1. Johnson, G S, Friedman, R M & Pastan, I, Proc natl acad sci US 68 (1971) 425. 2. Burke, R R, Nature 219 (1968) 1272. 3. Hsie, A W & Puck, T T, Proc natl acad sci US 68 (1971) 358. 4. Sheppard, J R, Proc natl acad sci US 68 (1971) 1316. 5. Willingham, M C, Johnson. G S & Pastan. I. Biothem biophys res commun 48 (1972) 743. 6. D’Armiento, M, Johnson, G S & Pastan, I, Proc natl acad sci US 69 (1972) 459. 7. Otten, J, Johnson, G S & Pastan, I, Biochem bionhvs res commun 44 (1972) 1192. 8. Manganiello, V & Vaughn, M, Proc natl acad sci US 69 (1972) 267. 9. Hendrick, M L & Ryan, W L, Cancer res 30 (1970) 376.

CAMP and prostatic 10. Schroder, J & Plagemann, P G W, J natl cancer inst 46 (197 I) 423. 11. Eker, P, J cell sci 16 (1974) 301. 12. Roller, B, Hirai, K & Defendi, V, J cell physiol83 (1974) 163. 13. Hauschka, P V, Everhart, L P & Rubin, R W, Proc natl acad sci US 69 (1972) 3542. 14. Hi;, H &. Kaukel, E, Mol cell biochem I (1973) 15. Plagemann, P G W & Sheppard, J R, Biochem biophys res commun 56 (1974) 869. 16. Sheppard, J R 8t Plagemann, P G W, J cell physiol 85 (1975) 163. 17. Kurtz, M J, Polgar, P, Taylor, L & Rutenburg, A M, Biochem j 142 (1974) 339.

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18. Fralev. E F. Ecker. S & Vincent. M. Science 170 (197Oj540. ’ 19. Niles, R M, Makarski, J S & Rutenburg, A M. Unpublished observations. 20. Schneider, E L, Stanbridge, E J & Epstein, C T, Exp cell res 84 (1974) 311. 21. Biochemical pharmacology Squibb internal reports (1971). 22. Kelley, L A & Butcher, R W, J biol them 249 (1974) 3098. 23. Plagemann, P G W, Richey, D P, Zylka, J M & Erbe, J, J cell biol64 (1975) 29. Received March 1, 1976 Accepted April 23, 1976

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