Loss of adenylate cyclase hormonal sensitivity in chemically transformed epithelial cells

Printed in Sweden Copynght 0 1978 by Academic Pres, Inc. All rights of reproduction in any form reserved 0014-48?7/78/1141-0191%0?.00/0

Experimental Cell Research 114 (1978) 191-195

LOSS OF ADENYLATE

CYCLASE

IN CHEMICALLY

TRANSFORMED

JOSEPH S. MAKARSKI Deparfmenfs

HORMONAL

SENSITIVITY

EPITHELIAL

CELLS

and RICHARD M. NILES

of Surgery and Biochemisfry, Bosfon Universify Boston, MA 02118. USA

School of Medicine,

SUMMARY The hormonal sensitivity of adenylate cyclase from a normal rat liver epithelial cell line (K16) and its chemically transformed derivative (WS) were compared. Intact normal rat liver cells had markedly increased CAMP levels after brief exposure to epinephrine, isoproterenol, norepinephrine or prostaglandin E,. In contrast, the CAMP levels of chemically transformed cells were relatively unaffected by these same compounds even after prolonged incubation. A comparison of broken cell adenylate cyclase activities revealed a decreased basal activity in the chemically transformed cells; the response to NaF was similar in the two cell lines, while the response to catecholamines and prostaglandins paralleled the intact cell studies. These data suggest that one reason for loss of adenylate cyclase hormonal responsiveness in chemically transformed rat liver epithelial cells may be a dysfunction or loss of hormone binding sites.

Specific activities for adenylate cyclase have been examined in a number of control and tumor tissues [I] as well as in normal and transformed cell cultures [2]. No consistent, clear-cut difference prevails when basal and NaF-stimulated adenylate cyclase activities are compared; i.e. increased activity [3], decreased activity [4], and no difference [3] have been observed. A difference that does seem to prevail in malignant cells is the decreased hormonal responsiveness of adenylate cyclase when compared to appropriate normal cells. This difference has been noted both in cultured cells [5], and in vivo [6]. However, the vast majority of studies comparing and contrasting the cyclic nucleotide metabolism of normal and transformed cells have employed cultured fibroblasts. Therefore, we have sought to de13-781813

termine whether similar differences in cyclic nucleotide metabolism might be found in normal and chemically transformed cultured epithelial cells.

MATERIALS

AND METHODS

Normal (K-16) and transformed (W-8) rat liver epithelial cells were kindly provided by Dr I. B. Weinstein of Columbia University, New York. W-8 was derived from K-16 by treatment with the carcinogen N-acetoxy, N-acetylaminofluorene. Radioimmunoassay reagents for determination of CAMP were obtained from Collaborative Research, Waltham, Mass. [“H]ATP, 25-40 Ci/mmole, and r3H]cAMP, 20-30 Cilmmole were purchased from New England Nuclear, Boston, Mass. and purified by Dowex AGlX-2 (CAMP) or Dowex AGSOW-X4 (ATP) chromatography before use. l-Methyl, 3-isobutylxanthine (MIX) was obtained from Aldrich Chemical Co., New Jersey. All other chemicals were purchased from Sigma Chemical co. For measurement of the intact cell response to hormones, cells were seeded at 1.0, 5.0. or 10.0~10~ in 60 mm tissue culture dishes (Falcon) containing 3 ml of Minimal Essential Medium with Earle’s salts (EMEM) Exp Cell Rrs I14 (197b’)

192

Makarski and Niles

Table 1. The effect of catecholamines and prostaglandins on CAMP levels in K-16 and W-8 cells

Table 2. Variation of catecholamine stimulation of CAMP levels with density of K-16 and W-8 cells pmoles CAMP/ 1OBcells

pmoles cAMP/lOG cells Additive None Isoproterenol + MIX Epinephrine + MIX Norepinephrine + MIX PGE, + MIX PGEl + MIX PGF, + MIX

K-16 4.55+0. I5 85.99k6.42 66.69_+1.90 66.98k2.32 29.8350.13 10.87&0.95 13.13k1.23

2.37kO.57 3.61f0.65 3.90_+0.41 2.91f0.35 4.12kO.18 4.99kO.12 2.7850.14

Cells were seeded and prepared for stimulation as described in Materials and Methods. Incubation time was 15 min and all catecholamines were present at concentrations of 1x 1OV M olus 2x lo+ M MIX. while prostaglandins were pre’sent at concentrations of 6x lo-” M plus 2x lo-’ M MIX. CAMP was extracted and quantitated as described in Materials and Methods. The data is represented as the mean +S.E.M. of triplicate plates each assayed in duplicate.

plus 10% fetal bovine serum (Gibco) and antibiotics (50 U/ml penicillin G, 50 &ml streptomycin sulfate). After 24 h the cultures were refed with 2.7 ml of the same media and the following day (48 h after seeding) the experiments were initiated by adding 300 ~1 of the hormone dissolved in EMEM and ethanol (0.5%) where necessary. Control plates received media with or without ethanol. After incubation at 37°C for various time periods. the reaction was terminated bv aspirating tde media, washing the cells twice with saline, and adding 1 ml of 3.5% PCA. The PCA extract was taken fro; the plates, centrifuged to remove cellular debris and the supematant neutralized with 0.3 N KOH. The resultant salt precipitate was removed by centrifugation and the nucleotides in the supernatant acetylated by use of triethylamine and acetic anhydride. CAMP was then quantitated by use of the radioimmunoassay technique [7] as modified by Harper & Brooker [8]. Authenticity of CAMP was tested by digestion of one half of the sample with purified beef heart CAMP phosphodiesterase (Boehringer Mannheim). In all cases assay of the digested sample indicated at least 98 % hydrolysis. At the time of hormonal stimulation, replicate plates were processed for cell counts by removing the cells from the dish with 0.25% viocase and enumerated through the use of a model B Coulter counter. All CAMP values are reuresented as omoles/106 cells. .For adenylate ciclase assay cells were seeded in 90 mm tissue culture dishes (Falcon). All cultures had their media replenished the day befoi-e the experiment. Cells were orocessed for assav bv removing. the media, washing th’e monolayer twice with 0.02 k Tris-HCI, 0.02 M sucrose pH 7.4 buffer, and then scraping the cells from the plate with a “rubber policeman”. This Erp Cdl Res I14 (1978)

Cell line

Density

Control

Epinephrine + MIX

K-16

1.93x 105 5.53x 105 17.59x 105 2.87x lo5 9.71 x 105 22.63x lo5

5.09kO.68 3.25kO.32 2.81kO.52 6.92k2.32 3.55tO.24 2.95kO.21

56.97+ 1.44 60.47k8.39 47.55k3.71 9.33k1.34 12.34kO.86 3.90_+0.41

W-8

W-8

K-16 and W-8 cells were seeded in 3 ml of EMEM + 10% FCS onto 60 mm tissue culture dishes at the initial densities of 1x lo”, 3x lo5 and 9x 105.One day later all dishes were refed with 2.7 ml of EMEM + 10% FCS. Twentyfour hours following refeeding, 300 ~1 of media (control) or epinephrine (1 x lo-* M) + MIX (2x lo-* M) was added and the plates incubated for 1.5 min after which CAMP was extracted and quantitated and replicate plates used to determine cell number as described in Materials and Methods. The data are expressed as the mean +S.E.M. of triplicate plates each assayed in duplicate.

treatment was sufficient to rupture over 90% of the K-16 cells. However, the W-8 cells required sonication (5 set at setting 5 on a Bronson sonifier) in order to disrupt the same percentage of cells. Adenylate cyciase was-measured by the method of Salomon et al. [9] with slight modification. We found that increased separation of r3H]ATP from r3H]cAMP was nossible if Dowex AG50W-X4 columns were washed with 0.01 M Tris-HCl pH 7.4 buffer instead of water after application of the terminated reaction mixture. The CAMP was then eluted with 2 ml of water and passed through an alumina column. The run-off plus 2 ml of 0.01 Tris-HCl pH 7.4 buffer was collected and an aliquot counted in *Aquasol to measure CAMP formed. Recovery was routinely 60-70% and blank r3H]ATP values ranged between 6@100 cpm.

RESULTS There is a marked difference in the ability of various catecholamines and prostaglandins to increase the intracellular levels of CAMP in K-16 cells and W-8 cells (table 1). In preliminary experiments [lo] we observed that the phosphodiesterase inhibitor MIX was a virtual necessity in order to ob-

Adenylate

cyclase hormonal

sensitivity

Table 3. Adenylate and W-8 cells

in epithelial

193

cells

cyclase activity

in K-16

pmoles cAMP/min/mg protein Additive

30

Control NaF 2x 10FzM Isoproterenol 1x 10m4M Epinephrine 1X 10m4M Norepinephrine 1x 10m4M PGE, 5 x lo+ M PGEI 5 x IO+ M PGF*,, 5 x lO-6 M

f II

20

/

K-16

W-8

1.12+_0.01

0.23+0.001

3.36+0.06 2.93rtO.04 2.90+0.20

0.50+_0.017 0.29+0.025 0.31+0.019 0.34+0.003 0.35+0.006

2.05kO.28 1.12+0.02 1.33f0.13 1.31+0.03

0.34+_0.011 0.34+0.002

Fig. 1. Abscissa:

Cells were prepared and assayed as described in the text. The incubations were at 37°C for 20 min, which resulted in linear enzyme kinetics under our conditions. The data are represented as the mean +_S.E.M. where N =3. The entire experiment was repeated twice more with essentially identical results.

serve any stimulation of CAMP levels. Isoproterenol, epinephrine and norepinephrine all markedly stimulated CAMP levels in K-16 cells (15-19-fold). In contrast, CAMP was minimally elevated by catecholamines in W-8 (1.2-1.6-fold). The normal rat epithelial cells are also responsive to prostaglandins with PGE, being most effective (6.6-fold increase in CAMP). The chemically transformed cells are also stimulated by prostaglandins, but to a much lesser extent. An interesting observation is that W-8 cells are more responsive to prostaglandins than to catecholamines, while the opposite is true for K-16 cells. In other experiments (data not shown) we found that glucagon, cortisol or insulin did

not elevate the CAMP levels of either K-16 or W-8. Since the initial hormonal stimulations of K- 16 and W-8 were performed at equivalent cell densities, there existed the possibility that at lower or higher cell densities W-8 might become more hormonally responsive. The results of an experiment designed to investigate this possibility are presented in table 2. As both cell lines increase in cell density there is a decrease in non-stimulated CAMP levels/cell. Stimulation by epinephrine seems to exhibit a biphasic pattern, i.e. highest at an intermediate cell density, somewhat lower at low density, and lowest at high cell densities. However, at all cell densities the hormonal response is much greater in K-16 than in W-8. Another explanation for the lack of responsiveness in W-8 might be that we were looking at the wrong time point, i.e. the response of W-8 to hormones is much slower than K-16. Therefore, we examined the accumulation of CAMP in K-16 and W-8 after incubation with epinephrine for various time periods (fig. 1). The CAMP level

10

i 1;

47

0.5 1.6

5'

I

15

6 30

time (mm); ordinate: pmoles CAMP/ lo6 cells. Time course of epinephrine + MIX stimulation of K-16 and W-8 CAMP levels. K-16 cells (3x105) and W-8 cells (2x 105) were prepared for stimulation as described previously. At the various time periods indicated in the figure, plates were extracted and CAMP levels quantitated as described previously. The concentration of epinephrine was 1X 10m4M, while MIX was 2~ lo+ M. Each time point is the mean of triplicate plates +S.E.M. each assayed in duplicate. O---O, K-16 control; O---O, K-16 stimulated; U-Cl, W-8 control; X-x, W-8 stimulated.

Exp

CPU Rrs

114 (1978)

194

Makarski and Niles

of K-16 rises very rapidly and reaches a peak around 15 min, declining slightly by 30 min. In contrast, there is no significant elevation of CAMP levels in W-8 throughout the 30 min incubation. In other experiments (data not presented) we have incubated W-8 cells for up to 3 h with epinephrine and have observed no significant increase in CAMP. To validate intact cell stimulation experiments, we examined broken cell adenylate cyclase activities (table 3). The basal enzyme activity of K-16 is 6-fold higher than its transformed counterpart. NaF results in a 3-fold and 2.3-fold increase in K-16 and W-8 adenylate cyclase, respectively, suggesting that the catalytic portion of the enzyme from W-8 is not altered in a significant manner. All the catecholamines tested stimulated K-16 adenylate cyclase, while there was little significant stimulation of the enzyme from W-8 cells. The prostaglandins, however, did not activate the K-16 enzyme, while having a small stimulator-y effect on the W-8 enzyme. In general the broken cell adenylate cyclase experiments mimicked the pattern of hormonal response observed in intact cell studies. DISCUSSION The results presented demonstrate that chemically transformed, malignant rat liver epithelial cells become less responsive to hormones which activate adenylate cyclase and increase intracellular CAMP levels in the parental cell. Other studies with viral and chemically transformed fibroblasts [5], and a series of differentially metastasizing mouse melanoma cells [ 111have also shown a loss of hormonal responsiveness in the transformed or more metastatic cells, suggesting that this phenomenon may be a general property in certain classes of malignant cells. &I

Cd RCA 114 (1978~

The component of the CAMP system responsible for the loss of hormonal stimulation in W-8 cells has not yet been identified. In other systems [5] a loss of cell surface receptors has been demonstrated to account for decreased adenylate cyclase stimulation. We are in the process of performing hormone binding studies, but the data presented in this report would suggest that W-8 cells have fewer or altered catecholamine receptors. This conclusion is based on the results of the broken cell adenylate cyclase experiments, where cyclic nucleotide phosphodiesterase activity was negated, and yet the W-8 enzyme was still not stimulated by catecholamines even though NaF activated the enzyme, indicating a presumably normal catalytic subunit. The significance of diminished adenylate cyclase hormonal responsiveness to the abnormal growth properties of transformed cells remains speculative. However, the importance of adenylate cyclase in transmitting an external signal (hormone) into an internal message (CAMP) which in turn regulates many enzyme systems, suggests that alteration in the function of this enzyme may be one way by which cells escape external regulatory restriction upon their growth. We wish to thank Dr I. B. Weinstein of Columbia University for supplying us with cultures of K-16 and W-8 and also MS Joanne Kim for excellent technical assistance. This work has been supported in part by grants CA 10995from the NIH and PDT-16C from the ACS.

REFERENCES 1. Christoffersen, T, Morland, J, Osner, J & Elgio, K, Biochim biophys acta 279 (1%9) 363. 2. Peery, C V, Johnson, G S & Pastan, I, J biol them 246 (1971) 5785. 3. Perkins, J P, MacIntyre, E H, Riley, W D & Clark, R B, Life sci 10 (1971) 1069. 4. Polgar, P, Vera, J C, Kelley, P R & Rutenburg, A M, Biochim biophys acta 297 (1973) 378. 5. Sheppard, J R, Proc natl acad sci 74 (1977) 1091.

Adenylate

cyclase hormonal sensitivity

6. DeRubertis, F R, Chayoth, R & Field, J B, J clin invest 57 (1976) 64 1. 7. Steiner, A L, Pagliara, A S, Chase, L & Kipnis, DM,Jbiolchem247(1972) 1119. 8. Harper, J F & Brooker, G, J cyclic nucleotide res 1 (1975) 207. 9. Salomon, Y, Londos, C & Rodbell, M, Anal biochem 58 (1974) 541.

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cells

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10. Niles, R M, Makarski, J S, Ballinger, N, Kim, H & Rutenberg, A M, In vitro 13 (1977) 467. 11. Niles, R M & Makarski, J S. In preparation.

Received September 23, 1977 Accepted November 21, 1977

Exp Cell Res 114 (1978)