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CYCLIC AMP RESPONSE OF ISOLATED SNELL ADRENOCORTICAL CARCINOMA 494 CELLS TO TROPHIC HORMONES AND OTHER SUBSTANCES
F. G. Peron, D. V. Maudsley and A. Haksar The Worcester Foundation for Experimental Biology Shrewsbury, Mass. 01545
Abstract
Suspensions of viable cells were prepared from solid tumor of the Snell adrenocortical carcinoma 494 without the use of proteo- lytic enzymes. Cyclic AMP formation in these cells was stimulated by ACTH, LH, FSH and TSH but not by prostaglandins (E1, E2, Flg and F2g), insulin and secretin. Glucagon tested at a single dose level of 50 ug increased cyclic AMP to about 65% of the maximum amounts obtained with ACTH. When Ca++ was omitted from the incubation medium, the response to ACTH was considerably reduced while that to LH was essentially unchanged. Low concentrations of EGTA (0.3 mM) abolished the ACTH response almost completely but caused only a partial reduction in the response to LH; as much as 10 mM EGTA was required to obtain complete inhibition of the latter.
Introduction
Suspensions of viable adrenal cortex cells prepared by digestion of adrenal sections with proteolytic enzymes (1-8) have been used by several laboratories to study the steroidogenic action of ACTH as it relates to cyclic 3’5’-AMP (cyclic AMP) production, glycolysis, respiration and the involvement of calcium ions (9-16). While earlier investigations on the Snell adrenocortical carcinoma 494 (17) have been carried out using either slices or tissue homogenates (18-21), preparations of isolated cell sus- pensions derived by trypsin digestion of the solid tumor have been reported (22-24). However, when trypsin and collagenase are used to dissociate this tissue, there is disruption and clumping of most of the cells (unpublished,
Copyright @ 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
this laboratory). Recently, we have reported that intact cell suspensions from the Snell carcinoma can be prepared simply by mechanical agitation of the tissue and gravity settling procedures in a buffer medium containing BSA without the need for enzymatic digestion (25,26). Cell suspensions prepared in this way are relatively free of blood cells and small contam- inating particles. In this communication, we report on cyclic AMP forma- tion and glycolysis in isolated cell suspensions of intact tumor cells subjected to a variety of different stimuli.
Materials and Methods
Cancer Cell Suspensions:
The Snell adrenocortical carcinoma 494 is a solid tumor of rat adrenal origin (17) and grows readily when implanted at subcutaneous sites in young 50 gm male Sprague Dawley rats (Charles River Breeding Laboratories, Inc. ). It was maintained by animal passage as reported previously (24). We have found after about 14 animal passages of the tumor that a three week period of growth yields tissue which is comprised mainly of viable tumor cells with little contaminating necrotic tissue.
Cell suspensions from tumor tissue were prepared by the method described previously (26).
Incubation Procedures:
All incubations were performed in duplicate or triplicate in 10 ml beakers. Each beaker contained 1 ml of cell suspension in Krebs-Ringer phosphate buffer (PH 7.4) containing 0.5% bovine serum albumin (BSA, Pentex, Fraction V, Miles Laboratory, Inc., Kankakee, IL) and 5 mM sodium bicarbonate (KRP-BSA), 8 mM theophylline and test substances dissolved in the buffer to make the final volume 1.5 ml. Incubations were carried out in a Dubnoff metabolic incubator with shaking at 37 ℃ for various periods of time under 02:CO2 (95%:5%) or 100% N2.
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Pyruvate and Lactate Determinations:
After incubation the contents of the beakers were acidified with 15% aqueous HCLO1 and analyses for lactate and pyruvate carried out on the neutralized (pH 7.4) depropteinized incubation medium by methods used previously (28).
Cyclic AMP Determinations:
Incubations were terminated with 3 ml of absolute ethanol. After removal of the precipitated protein by centrifugation, the clear super- natants were evaporated under a stream of N2 at room temperature and the residues reconstituted in assay buffer for cyclic AMP determination as outlined before (27).
Materials:
Purified as-ACTH was obtained from Dr. C. H. Li while LH, FSH, TSH were purified pituitary hormones obtained from NIAMDD, NIH, Bethesda, MD. Glucagon was a generous gift from Eli Lilly Company.
Results and Discussion
Isolated Snell adrenocortical carcinoma 494 cells (hereafter referred to as tumor cells) have a very low steroidogenic activity (24,29) but they do produce substantial amounts of lactic and pyruvic acids which can be accurately and easily measured (24). The glycolytic activity of the tumor cells was, therefore, used as an index of their viability and to standarize the incubation conditions. Preliminary experiments indicated that theophylline (8 mM) was needed in the incubation mixture before increases in cyclic AMP production in response to trophic hormones were detectable. In order to test whether theophylline had effects on other cellular func- tions, pyruvate and lactate production was studied in the presence of this compound. Figure 1 shows that the amount of lactate and pyruvate pro- duced during 120 minutes of incubation carried out without 8 mM
400
120 MIN.
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n moles
200
OR LACTATE
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PYRUVATE
0
5.0
10.0
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25.0
30.0
NUMBER OF CELLS x 10°
theophylline, was related to the number of tumor cells (5 x 105 to 3 x 10°) used. In accompanying incubations where 8 mM theophylline was present, the mean of triplicate pyruvate or lactate values ob- tained with 1.0 to 3.0 x 10° cells were almost superimposable on those of figure 1 obtained without theophylline. Production of these substances with 8 mM theophylline present, was linear up to 120 minutes when 2 x 106 cells were used (figure 2). It is interesting to note that under aerobic conditions, regardless of the incubation time, the lactate concentration was almost twice that of pyruvate.
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n moles/ 10° CELLS
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OR LACTATE
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PYRUVATE
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INCUBATION TIME, MINUTES
FIGURE 2
Since the concentration of glucose used (11 mM) in the incuba- tions was based on that used in the experiments with normal adrenal cells (12), the effect of adding this substance in various amounts was next carried out.
Table I shows that production of pyruvate + lactate reached a maximum at 3-4 mM glucose.
Addition of mitochondrial respiratory chain inhibitors which might be expected to lead to pyruvate and lactate accumulation in incubated tumor cells were also investigated. This was done to further test the viability and “normal” functioning of the tumor cells under our incuba- tion conditions.
| Glucose concentration | Pyruvate | Lactate | Pyruvate + Lactate |
|---|---|---|---|
| 417 UM | 33.4 | 62.8 | 96.2 |
| 837 μΜ | 44.7 | 102.3 | 147.0 |
| 1.67 mM | 58.3 | 114.9 | 163.2 |
| 2.50 mM | 62.9 | 128.5 | 191.4 |
| 3.34 mM | 63.7 | 132.6 | 196.3 |
| 4.17 mM | 66.4 | 146.4 | 212.8 |
| 6.26 mM | 64.9 | 143.7 | 208.6 |
| 8.34 mM | 70.9 | 145.5 | 216.4 |
Incubations carried out for 120 minutes with 2 x 106 cells + 8 mM theophylline. Values are net production of pyruvate or lactate in nmoles/100 cells and are the mean of triplicate incubations.
Both KCN and Antimycin A (Table II) which inhibit the a-glycerol phosphate shuttle in the tumor cells by inhibiting mitochondrial a- glycerol phosphate dehydrogenase (25,26) led to a substantial reduction in pyruvate accumulation but to an increased lactate production. One possible explanation for the latter events can be conjectured if one assumes that because of shuttle inhibition, production of a-glycerol-3- phosphate by the NADH-requiring cytosolic a-glycerol-3-phosphate dehy- drogenase from dihydroxy acetone-3-phosphate became inhibited. This would result in bringing about a larger pyruvate production via glycoly- sis. Since lactic dehydrogenase would not be expected to be inhibited by either KCN or Antimycin A, the “extra” NADH becoming available be- cause of increased glycolysis would be utilized by lactic acid dehy- drogenase for reduction of pyruvate into lactate. Thus, as in our
| Addition | Pyruvate | Lactate | Pyruvate + Lactate |
|---|---|---|---|
| None | 57.9 | 116.9 | 174.8 |
| KCN 3 mM | 6.9 | 276.8 | 283.7 |
| Amytal 3 mM | 27.9 | 151.0 | 178.9 |
| Antimycin A 3 ug | 2.7 | 221.4 | 224.1 |
| Iodoacetate 600 uM | 6.1 | 5.0 | 11.1 |
| N2 gas* | 25.8 | 329.7 | 355.6 |
Incubations carried out for 120 minutes with 2 x 10º cells + 8 mM theophylline. Values are net production of pyruvate or lactate in nmoles/106 cells on triplicate samples.
“Cells were suspended in buffer gassed with N2 before final incubation and aliquoted in appropriate beakers. Final incubation carried out under 100% N2 gas.
experiments, an apparent decrease in pyruvate production would be ob- served because of lactate accumulation.
Amytal, an inhibitor of mitochondrial respiratory flavoprotein- linked NADH-dehydrogenase, an enzyme which has been found to operate efficiently in normal adrenal cortex cells (12), had no effect on pyruvate and lactate production. This is not surprising because the tumor cells do not appear to utilize pyruvate via the Krebs-cycle although normal adrenocortical cells do (25,26). Iodoacetate at levels known to block glycolysis at the glyceraldehyde-3-phosphate level (12), led to an almost complete inhibition of pyruvate and lac- tate production. If incubations were carried out in an anaerobic atmosphere under N2 gas, a condition expected to mimic incubations carried out with KCN, lactate accumulation was again seen to be much greater than that under aerobic conditions. In comparison to KCN, however, anaerobiosis under N2 had a lesser effect upon pyruvate accumulation.
These results together with those published previously (26) indicate that the suspensions prepared in the manner described con- tain intact and viable cells.
An additional procedure for detecting the presence of broken cell fragments is to test for adenyl cyclase in the cell suspension using NaF and ATP (30). As indicated in figure 3 there is only a slight rise in cyclic AMP production in the presence of NaF and ATP indicating that few cell fragments are present in the suspensions. The cyclic AMP responses described below are therefore presumed to be due to stimulation of intact cells.
The cyclic AMP response profile of the tumor cell suspensions is illustrated in figure 3 and is in broad agreement with the results
80
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CAMP pmoles/ 10 CELLS/60MIN.
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20
0
CONTROL
CHOLERA TOXIN
NoF +
ACTH
TSH
FSH
LH
ATP
FIGURE 3
The effect of various agents on cyclic AMP formation by tumor cells (1 x 106). Cells incubated for 60 minutes in the presence of 8 mM theophylline. Doses were as follows: cholera toxin 1 ug; NaF 15 mM + ATP 2 mM; ACTH 10 pmoles; TSH 100 ug; FSH 100 ug and LH 100 µg. Each bar represents the mean of triplicate incubations. The lines represent one standard deviation of the respective means.
obtained by Schorr et al. using tissue homogenates (21). Thus, addi- tion of several hormones e.g. ACTH, LH, FSH and TSH increased cyclic AMP production. The small effect observed with cholera enterotoxin probably reflects the “lag period” which is about 45 minutes in the normal adrenal cell suspensions (14). In some experiments epin-
ephrine produced a slight increase in cyclic AMP levels (<50% above the control values) but this could be due to residual contaminating erythrocytes which reportedly contain a catecholamine-responsive adenyl cyclase (31). In a single experiment glucagon in a dose of 50 µg increased the cyclic AMP levels from 10.5 to 45.2 pmoles/106 cells after a 60 minute incubation. This represented about 65% of the maximum ACTH response observed in the same experiment. A number of other agents were also tested in the tumor cell suspensions. Prostaglandin E1, E2, Fla and F2g were ineffective and so also were insulin and secretin at various concentrations.
A log dose response curve with ACTH is shown in figure 4 and it can be seen that the tumor cells are extremely sensitive to ACTH. Concentrations as low as 0.05 pmoles of ACTH per ml of incubation have produced a statistically significant increase in cyclic AMP levels. This amount of ACTH is ten to twenty times less than that needed to increase cyclic AMP levels in normal adrenal cells (27). On the other hand, the maximum production of cyclic AMP by the tumor cells is only about 5% of that obtained with normal cells (27).
A well established characteristic of ACTH action in normal rat adrenal cells is the obligatory requirement for calcium ions in the stimulation of cyclic AMP and steroidogenesis (10,13). This feature is retained in the Snell carcinoma as shown in table III. Thus, a marked reduction in cyclic AMP levels is obtained when calcium is omitted and EGTA is added to the incubation medium in the presence of maximally stimulating doses of ACTH (2.0 pmoles). Table III also shows that LH may not have as great a requirement for calcium ions since at levels of EGTA (0.30 mM) which completely sup- pressed ACTH-stimulated cyclic AMP production, the response to LH
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100
p moles CAMP/ 10° CELLS/60 MIN.
80
60
40
20
0
.05
15
.25
.50
1.0
2.0
ACTH in PICOMOLES
FIGURE 4
(10 pg) was inhibited by only 25%. Also, ommission of Ca++ from the incubation medium did not seem to affect the response to LH. The response to LH was, however, abolished by 10 mM EGTA. In other experiments not reported here it was found that increasing the con- centration of LH in the presence of 1.0 mM EGTA caused a partial reversal of the EGTA inhibition. On the other hand, large doses of ACTH (50 pmoles) were ineffective in reversing the inhibition by EGTA.
There are now several biochemical characteristics which have been described for the Snell carcinoma 494 which distinguish the tumor from
| Additions | Control | ACTH (2.0 pMoles) | LH (10 µg) | |
|---|---|---|---|---|
| Ca++ | EGTA | |||
| + | - | 15.5 ± 2.0 | 77.7 ± 4.8 | 109.9 ± 11.7 |
| - | - | 15.7 ± 1.8 | 24.9 ± 2.1 | 107.5 ± 4.8 |
| - | 0.3 mM | 17.0 ± 0.2 | 18.9 ± 2.2 | 72.8 ± 1.9 |
| - | 1.0 " | 12.1 ± 1.1 | 12.3 ± 2.0 | 61.4 + 1.3 |
| - | 3.0 " | 10.8 ± 2.3 | 9.2 ± 0.2 | 40.3 ± 3.0 |
| - | 10.0 " | 10.0 ± 0.4 | 8.9 ± 1.7 | 13.6 ± 0.5 |
Incubations carried out with cells (1 x 106) suspended in calcium-free buffer for 60 minutes + 8 mM theophylline. Ca++, 2.5 mM were shown, was added just prior to incubation. EGTA (ethylene glycol-bis (B-aminoethyl ether)-N,N’-tetra- acetic acid) was also added just prior to incubation. The values, expressed as pMoles/100 cells/hour, are mean + S.D. of triplicate incubations.
the normal adrenal gland (26,27,29). In this paper, we have shown principally a lack of hormonal specificity of the tumor cells. This phenomenon is similar, in outline, to that of the isolated fat cell. Perkins (32) has reviewed the explanations for such multivalent regu- lation and discussed the evidence indicating that in the isolated fat cell, separate specific receptors are associated with a single cataly- tic unit. Preliminary evidence suggests that this explanation may also apply to the Snell carcinoma cells since maximum doses of ACTH and LH were not additive when given in combination. Also, the calcium re- quirements for the two hormones were different as was shown in the EGTA experiments and in those where supramaximal amounts of LH were used. We cannot at this time, however, exclude the possibility that we are dealing with a single non-specific receptor.
Finally, the cell suspensions as prepared in our laboratory should serve as a useful experimental model for studies on cyclic AMP metabo- lism. In terms of cell composition they are better defined and more homogeneous than tissue slices or homogenates (19-21). The cell sus- pensions also represent one of the few preparations where enzymatic digestion of the tissue is not necessary and this is particularly advantageous for studying the hormonal regulation of adenyl cyclase.
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ACKNOWLEDGMENTS
Thanks are due to the excellent technical assistance provided by George Gagnon, William F. Robidoux, Jr., and Sara M. Seekings. Supported in part by grant AM-04899 (NIAMDD) and CB-33919.
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