Adenylate Cyclase, Cyclic Adenosine 3 :5 -Monophosphate Phosphodiesterase, and Regression of Walker 256 Mammary Carcinoma1

Home , Cyclic nucleotide

[CANCER RESEARCH 37, 4493-4499. December 1977] Adenylate Cyclase, Cyclic Adenosine 3 :5 -Monophosphate Phosphodiesterase, and Regression of Walker 256 Mammary Carcinoma1

Yoon Sang Cho-Chung2 and Susanne F. Newcomer

Laboratory ol Pathophysiology. National Cancer Institute. NIH. Bethesda. Maryland 20014

SUMMARY nomycin D and cycloheximide. These studies show that adenylate cyclase and cAMP The activities of adenylate cyclase and cyclic adenosine phosphodiesterase from two cell populations of W256 are 3':5'-monophosphate (cAMP) phosphodiesterase have similar in their basal catalytic activities but differ in their been determined in two types of Walker 256 mammary responses to exogenous or endogenous stimulators. carcinoma (W256): one type is responsive and the other is unresponsive to A/6.O2'-dibutyryl cyclic adenosine 3':5'- INTRODUCTION monophosphate treatment in vivo. The basal adenylate cyclase activity in the two cell populations is similar. The Previous studies have suggested an important role for cAMP3 in controlling cell growth. Several investigators (17, enzymes from both cell populations exhibit the apparent KTnfor 5'-adenylylimidodiphosphate and Mg2' of ~0.1 mw 25, 33) have demonstrated that transformed cells main and -2.5 rriM, respectively. The F stimulation of the en tained in vitro contained less cAMP than similar cells that zyme from responsive W256, however, is about 2-fold had not been transformed. Furthermore, addition of cAMP greater than that of the unresponsive W256 enzyme. This or its derivatives restored normal morphological character stimulatory effect of F is due to an increase in Vmaxsince istics to several tumor cell lines and decreased their growth there was no significant effect on the Kmfor substrates 5'- rates (5, 16, 18, 28, 32, 34, 36, 37). adenylylimidodiphosphate or Mg2'. There is also a differ However, the molecular mechanism of cAMP action, ence in the effect of guanosine triphosphate on prosta- especially that involving the control of tumor growth in glandin E, (PGE,) stimulation of adenylate cyclase activity vivo, is poorly understood. We have reported (10) the between responsive and unresponsive W256 enzymes. selection of 2 cell populations from W256; one type is PGE, stimulation of the cyclase activity in crude membrane regressing (DBcAMP responsive), and the other type is preparations from the responsive tumor is dependent on growing (DBcAMP unresponsive) under DBcAMP treat ment. Our recent studies (7-9) have suggested that the the presence of guanosine triphosphate, whereas the en qualitative alteration of cAMP-binding proteins, the regula zyme preparations from the unresponsive tumor show a tory subunit of protein kinase, is probably associated with greatly diminished PGE, stimulation in either the presence "unresponsiveness" to DBcAMP treatment in one type of or absence of guanosine triphosphate. W256. The objective of this study was to assess whether The activities of cAMP phosphodiesterases in the two "responsiveness" to DBcAMP treatment (tumor regression) cell populations are similar. Both cell populations contain at least two different diesterase activities; apparent Kmfor can also be related to cAMP metabolism in the tumor cell. high- and low-affinity enzymes are ~2 ^M and ~20 /*M, The intracellular level of cAMP is determined by the activi respectively. ties of adenylate cyclase and cAMP phosphodiesterase. In Dibutyryl cyclic adenosine 3':5'-monophosphate injec this paper we describe the kinetic properties of adenylate cyclase and phosphodiesterase in DBcAMP-responsive and tions into the host or incubation of tumor slices with PGE, result in a significantly greater increase of Iow-Km cAMP DBcAMP-unresponsive W256 as well as the responses of phosphodiesterase activity in the responsive tumor than in these enzymes to DBcAMP treatment in vivo and PGE, the unresponsive tumor. Adenylate cyclase from only the treatment in vitro. responsive tumor is significantly increased after the incu MATERIALS AND METHODS bation of tumor slices with PGE,. The increase in enzymes is due to an increase in Vmaxwithout affecting the Kmfor Materials. Dowex AG 50W-X8 resin (200 to 400 mesh) in the substrates. The enzyme increases are blocked by acti- a prefilled 2.0-cm-bed-height column and AG 1-X2 resin (200 to 400 mesh) were obtained from Bio-Rad Laborato 1 This is Paper 7 in a series on the role of cAMP in neoplastia cell growth ries, Richmond. Calif. cAMP, GTP, and snake venom and regression, from Laboratory of Pathophysiology. National Cancer Insti tute, NIH, Bethesda. Md. 1 To whom requests for reprints should be addressed, at Laboratory 3 The abbreviations used are: cAMP, cyclic adenosine 3':5'-monophos- of Pathophysiology, Building 10, Room 5B-43, National Cancer Institute, phate; W256, Walker 256 mammary carcinoma; DBcAMP, A/6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate; PGE,, prostaglandin E,; AMP-PNP, NIH, Bethesda, Md. 20014. Received May 16. 1977; accepted September 8. 1977. 5'-adenylyl imidodiphosphate.

DECEMBER 1977 4493

(Ophiophagus hannah, king cobra) were obtained from to the protein concentration up to 2 mg ( 75 /Â¿Iofthe 1:2 Sigma Chemical Co., St. Louis. Mo. [3H]cAMP (27 Ci/ homogenate)/reaction mixture. The NaF-stimulated and the mmole) and theophylline were obtained from Schwarz/ unstimulated enzyme activity showed a broad pH optimum Mann, Orangeburg. N. Y. AMP-PNP and [3H]AMP-PNP (15.8 of 7.0 to 9.0. A unit of enzyme activity was defined as the Ci/mmole) were obtained from ICN Pharmaceuticals, Inc., amount of enzyme that produced 1 pmol cAMP in 20 min at Pikesville, Md. PGE, was a gift from Dr. John Pike, The 30 in the standard assay system. Upjohn Co., Kalamazoo, Mich. Phosphodiesterase Assay. cAMP phosphodiesterase ac Tumor Models. DBcAMP-responsive and DBcAMP-unre- tivity was measured by the 2-step assay system of Thomp sponsive cell populations of W256 were maintained by the son and Appleman (35). The enzyme hydrolyzes cAMP to selective transplantation procedure (10) in female Sprague- 5'-AMP, which is then further converted to adenosine by Dawley rats 3 to 4 months old with an average body weight snake venom 5'-nucleotidase. The reaction mixture of 0.4 of about 200 g. ml contained: [3H]cAMP (200.000 cpm); 1.25 x 10 7 to 1.0 Homogenate and Crude Membrane Preparations. All x 10 4 M cAMP; 5 mM MgCI,: 40 mM Tris-HCI, pH 8.0; and procedures were performed at 0-4 . Tumors from rats killed enzyme [10 tÂ¿\homogenate (1:50) in 10 mw Tris-HCI. pH by decapitation were removed, cleaned of necrotic tissue, 7.5], After 10 min at 30Â°, the reaction was stopped by and homogenized immediately in a Teflon-glass homoge- boiling for 2 min, then 0.1 ml of snake venom (1 mg/ml) nizer with 2 volumes of 10 mM Tris-HCI buffer (pH 7.5). The was added, and the mixture was incubated for another 10 homogenates were filtered through 3 layers of cheesecloth min at 30Â°to convert 5'-AMP to adenosine. This reaction before use. was stopped by the addition of 1.0 ml of a slurry (1:3) of Crude membrane preparations were made as follows. Bio-Rad AG 1-X2 resin; after mixing, the tubes were centri Tumor was homogenized in 5 volumes of 10 mM Tris-HCI fuged at 4000 x g for 10 min, and the radioactivity in the buffer (pH 7.5) and centrifuged at 270 x g for 10 min to supernatant was measured by liquid scintillation. The val separate out the heavy nuclei and unbroken cell fraction. ues of the blanks, prepared either without enzyme or with The supernatant was centrifuged at 10,000 x g for 10 min; heat-inactivated enzyme, were subtracted from the experi the resulting pellet was washed twice by resuspending in mental values. For kinetic analysis the concentration range Tris-HCI buffer to the original volume and was centrifuged is from 1.25 x 10 7to 1 x 10 " M. Activities termed low and at 12,000 x g for 10 min. This pellet was then resuspended high refer to assays at these limits of cAMP concentration. in Tris-HCI buffer to one-half of the original volume and Enzymes are diluted to provide linearity in an assay and, in used for assay. all the assays performed, the rate of [3H]cAMP hydrolysis Adenylate Cyclase Assay. Adenylate cyclase activity was was linear or at least 15 min and directly proportional to assayed by measuring the conversion of [3H]AMP-PNP to the protein concentration over a range of 0.5 to 25 /ng/ [3H]cAMP in crude homogenates or membrane prepara reaction mixture. No more than 15 to 20% of the substrate tions. AMP-PNP, an analog of ATP, was used in kinetic was consumed in each assay. The unit of phosphodiester ase activity is pmoles cAMP hydrolyzed per min at 30Â°per studies of adenylate cyclase since it has been shown to be a substrate for the cyclase but not for the ATPase (29). assay volume (0.4 ml). Protein measurements were made Enzyme activity was measured by the method of Krishna et by the method of Lowry et al. (22). al. (21). The standard incubation medium contained 4 x 10 2 M Tris-HCI buffer (pH 7.5), 5 x 10 3 M MgSO,, IO'2 M theophylline, and 5 x 10 4 M AMP-PNP, together with [3H]- RESULTS AMP-PNP (15.8 Ci/mmole) and enzyme (50 /Â¿Ihomogenate Adenylate Cyclase Activities in DBcAMP-responsive and or crude membrane preparation) Â±NaF (6 mM) in a final DBcAMP-unresponsive W256 volume of 0.5 ml. The reaction was initiated by addition of the enzyme, and this mixture was then incubated at 30 Effect of AMP-PNP Concentration. Adenylate cyclase for 20 min. The reaction was terminated after the addition activity was measured in tumor homogenates in both the of 0.1 ml (0.5 mg) of a solution of carrier cAMP by immer presence and absence of the activator NaF at various sion in a boiling-water bath for 2 min. The tubes were concentrations of AMP-PNP (Chart 1). Normal Michaelis- centrifuged, the supernatant fluids were chromatographed on Dowex columns (see "Materials"), and the barium-zinc Menten kinetics are shown by the enzyme from 2 types of W256 (DBcAMP responsive and DBcAMP unresponsive). precipitation was performed (21). The radioactivity in the Calculations from double-reciprocal plots (data not shown) final supernatant4 (cAMP-rich fraction) was measured by showed essentially the same Km (-0.1 mM) for the enzymes liquid scintillation. The absorption of cAMP at 260 nm was from both W256 in either the presence or absence of NaF. determined by a Gilford spectrophotometer to calculate The affinity of the enzyme for the substrate AMP-PNP the recovery of cAMP; recoveries averaged 30 to 40% in all therefore does not appear to differ between the 2 cell assays. Tubes without homogenates were run through the populations. The V,n;ix of the enzyme reaction in the pres assay as blanks, and the values of these tubes were sub ence of NaF was, however, 2-fold greater in the responsive tracted from each experimental value. The rate of cAMP than in the unresponsive tumor homogenate. The Vmax in production was linear for at least 30 min and proportional the absence of NaF was similar in both tumor homogenates.

4 The purity of the final fraction has been determined by paper Chromato Effect of NaF Concentration. The effect of varying con centrations of F on the adenylate cyclase activity from the graphie technique (21), and 99% of the radioactivity recovered in the assay 2 W256 are shown in Chart 2. At all concentrations of F~, was found to be associated with cAMP.

4494 CANCER RESEARCH VOL. 37

populations in the binding of the Mg-AMP-PNP substrate to the catalytic site on the enzyme. Effect of PGE, and GTP on Crude Membrane Prepara tions. PGE, has been shown to stimulate adenylate cyclase in tissue homogenates (1, 31 ). However, with purified mem brane preparations, a requirement for GTP was observed in the PGE.-dependent activation of adenylate cyclase (20, 29). We therefore tested this effect with crude membrane suspensions of responsive and unresponsive tumors and observed a marked difference in response to PGE, and GTP between the enzymes (Charts 3 and 4). In the absence

0.5 1.0 1.5 2.0 25 O 1.0 1.5 2.0 2.5 of GTP, PGE, at 2 Â¡J.Mdidnot stimulate the basal activity of enzyme from the responsive membranes, whereas the activ AMP PNP ImMI ity was stimulated 40-fold by NaF (Chart 3, left). The PGE, Chart 1. Effect of AMP-PNP concentration on adenylate cyclase activity of DBcAMP-responsive (open symbols) and DBcAMP-unresponsive (closed activation of adenylate cyclase from the responsive mem symbols) W256. Adenylate cyclase was assayed in the presence and absence branes was potentiated by GTP. This stimulation was 100- of 6 mw NaF. Incubations were as described in the text, except for variation fold and 170-fold at 10 5 M and 10 4 M GTP, respectively; in AMP-PNP concentrations as indicated. basal activity was also increased 100-fold by 5 x 10 5 M GTP. The F -stimulated activity was increased 4-fold at 5 x 10 5 M GTP but was markedly .inhibited at higher concentra tions of GTP. Moreover, the stimulating effect of GTP (10s M) was observed at all concentrations of PGE, used in these experiments (Chart 4, left). The adenylate cyclase from unresponsive membranes showed no requirement of GTP for PGE, activation of the enzyme. PGE, activation was inhibited in the presence of 10 5 M GTP (Chart 4, right), and GTP activation was appre ciably lower in the presence of 2 x 10 6 M PGE, (Chart 3, right). The basal activity of the unresponsive membranes was much higher than that of the responsive membranes (30-fold), and stimulation of activity by 2 /U.MPGE, (Chart 4, right) and 5 x 10 5 M GTP (Chart 3, right), were 3-fold and 4-fold, respectively. These experiments showed that stimu Chart 2. Effect of F concentration on adenylate cyclase activity of DBcAMP-responsive (open symbols) and DBcAMP-unresponsive (closed lation of crude preparations from responsive membranes symbols) W256. Incubations were as described in the text, except for by PGE, is completely dependent on the presence of GTP, variations in F concentrations as indicated and Mg2' concentrations of 2.5 whereas unresponsive preparations show a greatly dimin rriM and 10 ITIM ished response to PGE, in either the presence or absence of GTP.5 the stimulation of the activity of responsive tumor enzyme was greater than that of the unresponsive tumor enzyme in the presence of both low and high Mg2' concentrations. cAMP Phosphodiesterase Activities in DBcAMP-respon Furthermore, the shapes of the saturation curves were sive and DBcAMP-unresponsive W256 distinctive for the enzymes from the 2 cell populations. The double-reciprocal plot of the saturation curve from the Effect of cAMP Concentration. The plot of the substrate responsive tumor enzyme was nonlinear, and Hill plots (cAMP concentration) against enzyme activity of tumor (data not shown) showed n = 1.5, indicating a positive homogenates (Chart 5/4) showed that the kinetics of phos- cooperativity (4, 24). phodiesterase activity from both responsive and unrespon Effect of Magnesium Concentration. The relationship sive tumors were similar, with one-half of the Vmaxat about between enzyme activity and Mg2' concentration was stud 20 /UM. Hofstee plots of the same data (Chart 5, B and C), ied (data not shown). The experiments showed that the emphasizing values at lower cAMP concentrations, suggest reaction velocity was markedly increased by the Mg2' con 2 enzyme activities, one with an apparent Km of about 20 x centration, far in excess of AMP-PNP concentration (5 x 10 6 M and the other with higher affinity (apparent Km, ~2.0 10 " M). At pH 7.5, 1 Mg2' is bound to ATP; thus it might be x 10 6 M). High- and low-affinity phosphodiesterase activi expected that the substrate was a Mg-AMP-PNP complex. ties have been described in various other tissues (3, 35), It follows therefore that Mg21 might bind at more than 1 site and serves in a regulatory capacity in addition to its substrate role in association with AMP-PNP. The effect of 5 To rule out the possibility that the diminished stimulatory effect of GTP increased Mg2 ' concentration in the 2 W256 was essentially on unresponsive membrane adenylate cyclase activity was not due to the higher hydrolysis of GTP by the unresponsive membrane preparation, we the same. Both responsive and unresponsive tumors tested various mixtures of responsive and unresponsive membrane prepara showed a similar Km (2.5 x 10 3 M) and Vmax(2 units). Thus, tions; these mixtures showed an additive effect of GTP stimulation, suggest ing that unresponsive membranes do not have a higher hydrolytic activity it appears that no difference exists between the 2 cell toward GTP.

DECEMBER 1977 4495

although their metabolic meaning is unclear. These data show that the kinetics of phosphodiesterase activity are almost identical in both responsive and unresponsive tu mors. PGE, Effect of Exogenous DBcAMP. DBcAMP treatment rap idly increased the level of cAMP in both responsive and unresponsive tumors (Chart 6, left). This increase was accompanied by a sharp increase in the Iow-Km phosphodi

Basai esterase activities in both tumors within 1 hr after treatment (Chart 6, right). The diesterase in the responsive tumor continued to rise for several hr thereafter and maintained 10-5 10" 103 0 105 10" 103 high activity, whereas the unresponsive tumor enzyme decreased sharply to one-half of the peak activity at 3 hr GTP (M) after treatment. At 6 days after treatment, the enzyme levels Chart 3. Effect of GTP concentration on PGE, and F activation of DBcAMP-responsive (open symbols) and DBcAMP-unresponsive (closed in both responsive and unresponsive tumors were 3-fold symbols) W256 membrane adenylate cyclase. Adenylate cyclase was assayed and 2-fold of the basal values, respectively, and the cAMP as described in the text in the presence of various concentrations of GTP content of both tumors had decreased to near basal levels with membrane preparations as the enzyme source. F and PGE, were present at concentrations of 6 mw and 2 x 10 * M, respectively. Basal (Chart 6). Actinomycin D and cycloheximide completely activity in the responsive membrane preparation was 0.02 pmole cAMP (cyclic AMP) per mg protein per 20 min.

E 2.0

+GTP

1.0

o. < y _j u Basal u 10-' 10-6 105 0 10-7 10Â« 106 PGE, (M) Chart 6. cAMP (cyclic AMP) content and phosphodiesterase activity in DBcAMP-responsive ( C) and DBcAMP-unresponsive (â€¢) W256 during Chart 4. Effect of PGE, concentration on DBcAMP-responsive (open sym DBcAMP treatment. DBcAMP (8 mg per 0.1 ml 0.85% NaCI solution per 200 g bols) and DBcAMP-unresponsive (closed symbols) W256 membrane ade rat s.c.) was injected daily as described previously (11). Tumors removed at nylate cyclase. The enzyme was assayed as described in the text in the indicated times were analyzed for cAMP content by the method previously presence of varying concentrations of PGE, and in the absence of F . GTP described (6) and for Iow-Km phosphodiesterase activity as described in the (10 5 M) was added when indicated. Cyclic AMP. cAMP. text. Values represent the mean of 6 tumors Â±S.E.

K,â€ž=20 8,1 M

S (cAMP concentration) (

Chart 5. cAMP phosphodiesterase activity at varying substrate (S) concentrations in DBcAMP-responsive ( O) and DBcAMP-unresponsive (â€¢)W256. The enzyme was assayed as described in the text except for varying cAMP concentrations. A shows enzyme activity (V) plotted against substrate concentration (0.05 to 200 x 10 6 M). B and C are Hofstee plots of the same data: the slope represents the negative value for the apparent Km of the preparation, and the intercept of the ordinate is the apparent Vmax.

4496 CANCER RESEARCH VOL. 37

inhibited the increase of phosphodiesterase activity after -PGE, AT 30Â° DBcAMP treatment (Table 1). These data suggest that the â€¢c 15 increase of diesterase activity after DBcAMP treatment is -A dependent on RNA and protein synthesis and is not due to an activation of the enzyme. 10 Incubation of Tumor Slices in Vitro with PGE, i*

Effect on Adenylate Cyclase Activity. When responsive tumor slices were incubated with PGE, at 30C, within 5 to 10 min the basal and F -stimulated adenylate cyclase activ ities increased markedly (Chart IB). This increase was significantly inhibited by cycloheximide, suggesting that >- O PGE, caused an increase in the synthesis rather than in the _r*s -T1/* â€”â€”â€”â€¢â€¢K*^M [' activation of the enzyme. Further incubation of the slices at 30 for 30 min resulted in the decline of these activities, z whereas incubation at 4Cfor up to 19 hr (Chart 7C) resulted UJ Ã› 0[ in a continuous elevation of both basal and F -stimulated O 10 20 30 O 10 20 30 O 3 activities. Adenylate cyclase activity in unresponsive tumor INCUBATION TIME (min) INCUBATION TIME (hrl slices, however, did not change appreciably after PGE, Chart 7. Adenylate cyclase activity of DBcAMP-responsive (open symbols) incubation either at 30 or 4C(Chart 7, E and F). The kinetic and DBcAMP-unresponsive (closed symbols) W256 exposed to PGE, in vitro. Tumor slices were incubated either at 4 or 30Â°in5 volumes of Tris-HCI analysis of PGE,-stimulated adenylate cyclase activity (10 mM; pH 7.5), theophylline (10 mw), and PGE, (0.1 mM). Cycloheximide showed that the increase in enzyme from responsive tumor (CH) at 10 /Â¿g/mlwas added to the incubation medium when indicated. slices was due to the increase in Vmaxwithout changing the Incubation was stopped by diluting the medium 2.5-fold with cold Tris-HCI at the indicated times. Tumor slices were collected by centrifugation. apparent Km of the enzyme (data not shown). washed twice with cold Tris-HCI, and then homogenized with 5 volumes of Effect on Phosphodiesterase Activity. Chart 8 shows Tris-HCI buffer. The homogenate was used immediately to measure ade that within 5 min of incubation at 30 , PGE, rapidly in nylate cyclase by the method described in the text, in the presence or absence of F . Values are the means Â±range of duplicate incubations. creased cAMP phosphodiesterase activity (low Km) in re sponsive tumor slices to 3-fold that of the control value. -PGE, The enzyme increase was significantly inhibited by cyclo AT 30Â° heximide, suggesting that the increase in activity was due to the increase in enzyme synthesis. No appreciable in crease of phosphodiesterase activity occurred in unrespon 150 sive tumor slices treated with PGE, in the same manner. At 1Ã• 19 hr after incubation at 4 , PGE, stimulated the diesterase 100 activity in responsive tumor slices to 4-fold that of the Q "A control value (zero time), whereas the enzyme activity of unresponsive tumor slices decreased to less than that of II the control value.

Effect on cAMP Content. cAMP content of tumor slices 0 10 20 30 0 10 20 30 03 19 during incubation with PGE, is shown in Chart 9. Within 5 INCUBATION TIME (mini INCUBATION TIME (hrl Chart 8. cAMP phosphodiesterase activity of DBcAMP-responsive ( O) and DBcAMP-unresponsive (â€¢)W256 exposed to PGE, in vitro. Tumor Table 1 slices were incubated as described in the legend to Chart 7. Low-Km Effect of actinomycin D and cycloheximide on DBcAMP-induced phosphodiesterase activity in the homogenates was measured at a concen cAMP phosphodiesterase activities of DBcAMP-responsive and tration of 1 fiM cAMP by the method described in the text. Values are given as the means Â±range of duplicate incubations. DBcAMP-unresponsive W256 DBcAMP was injected for 6 days as previously described (11). min at 30C, cAMP levels in both responsive and unrespon Cycloheximide and actinomycin D at 40 Â¿ig/day/rat s.c. were sive tumor slices increased rapidly to 4-fold those of the injected at the same time as the DBcAMP injection when indicated. The enzyme activity was measured in tumor homogenates as respective control values (-PGE, incubation). After the described in the text, at a concentration of 1 MM cAMP. initial increase the cAMP level in responsive tumor slices cAMP phosphodiesterase continued to rise throughout the incubation period, activity (low Km) whereas the cAMP level in unresponsive tumor slices re mained at a plateau. The initial increase of cAMP content responsive W256 unresponsive W256 in both tumor slices may have been due to the inhibition of TreatmentNone (units/gtumor)900 (units/gtumor)800 cAMP degradation by theophylline, which was added to Â± 80" Â± 70 the incubation medium. However, continued increase of DBcAMP 2600 Â±160 1600 Â±120 cAMP level in only the responsive tumor slices is consistent Actinomycin D + 450 Â± 50 440 Â± 50 with the data on adenylate cyclase; the enzyme activity Cycloheximide +DBcAMPDBcAMPDBcAMP-800 Â± 70DBcAMP- 850 Â±100 increased significantly in responsive but not in unrespon Mean of 6 tumors Â±S.E. sive tumor slices after PGE, incubation (Chart 7).

DECEMBER 1977 4497

sive W256 (12) produced regression of only the responsive tumor. An obligatory requirement for GTP on PGE, stimula tion (Chart 4, left) was observed with the adenylate cyclase preparation of the responsive membrane but not with the enzyme from the unresponsive membrane preparation. The loss of regulation of membrane adenylate cyclase by the GTP-PGE, system has also been found in virus-transformed rat kidney cells (1). Studies on cultured cell lines have suggested that the level of cAMP phosphodiesterase activity is regulated by the intracellular concentration of the substrate cAMP (14, 23, 30). Treatment of tumor slices with PGE, in vitro in the 30 presence of theophylline resulted in the elevation of cAMP in both responsive and unresponsive tumors. Only in the INCUBATION TIME Iminl responsive slices, however, did the significant induction of Chart 9. cAMP content of DBcAMP-responsive (open symbols) and DBcAMP-unresponsive (closed symbols) W256 exposed to PGE, in vitro. phosphodiesterase occur. Moreover, DBcAMP treatment in Circles, incubation in PGE,; triangles, incubation without PGE,. Tumor vivo resulted in a higher elevation of diesterase in the slices were incubated at 30Â°asdescribed in the legend to Chart 7. At the responsive tumor than it did in the unresponsive tumor, times indicated, incubation was stopped by the addition of 5 volumes of cold 10% trichloroacetic acid, and tissues were homogenized. The ether- although the elevation of cAMP was similar in both tumors. extracted trichloroacetic acid supernatants were used to determine cAMP DBcAMP-unresponsive W256, which possesses defective as previously described (6). Values represent the mean Â±range of duplicate cAMP-binding proteins (7-9), is also deficient in the induc incubations. tion of cAMP phosphodiesterase, confirming the observa DISCUSSION tions made on the S49 lymphoma system by Coffino and Yamamoto (13). Thus, phosphodiesterase in the unrespon This study reports that the basal activities of adenylate sive tumor does not respond as well to the cellular con cyclase and cAMP phosphodiesterase are similar for 2 centration of cAMP as it does in the responsive tumor. types of W256: one type is regressing (responsive) under This study shows that adenylate cyclase and cAMP phos phodiesterase in DBcAMP-unresponsive W256 exhibit de DBcAMP treatment and the other type is growing (unre sponsive) under DBcAMP treatment. These data correlate creased or altered responses to both endogenous and with the basal cAMP levels that were also found to be exogenous agents as compared with the enzymes in DBcAMP-responsive W256, despite the fact that their basal similar in these 2 cell populations (6). Adenylate cyclase and phosphodiesterase from respon activities are similar in both types of cell populations. This sive and unresponsive W256 show different responses to finding suggests that the ability of cells to change the stimuli. F stimulation of adenylate cyclase from the re cAMP concentration in response to intracellular or extra cellular stimuli is more closely related to cAMP-mediated sponsive tumor is appreciably higher than that from the growth control than it is to a steady-state level of cellular unresponsive tumor. Activation of adenylate cyclase by F has been classified as a "V" system activation as defined cAMP. The decreased or altered responses of adenylate by Monod et al. (24), since F increases the Vmaii without cyclase and phosphodiesterase might be due to 1 or more affecting the Km for the substrates (15). According to the of the following reasons: alteration of the regulatory subunit model, cooperativity in this system is expected only for the (2, 27) of these enzymes that results in the decreased activator and not for the substrates. In our present study interaction of the subunit with endogenous or exogenous we made the same observations of the responsive tumor stimulators, alteration in the interaction between the regu with adenylate cyclase; cooperativity was observed for F , latory and catalytic subunits of these enzymes, decrease in whereas AMP-PNP and Mg2' obeyed Michaelis-Menten ki the synthesis of the regulatory subunit of these enzymes, netics. Since no cooperativity was observed for F with the and alteration of the tumor membrane that results in the enzyme from the unresponsive tumor, the F stimulation of nonspecific aberration of the regulatory subunit of these this enzyme may be due to an altered mechanism from that enzymes. of the responsive tumor enzyme system. Purification of adenylate cyclase and cAMP phosphodi esterase from DBcAMP-responsive and DBcAMP-unrespon It has been suggested that prostaglandins may play a regulatory role in the activity of adenylate cyclase since sive tumors is now required to examine the above possibil ities and to understand the role of these enzymes in cAMP- PGE, is able to elevate cAMP levels in intact cells (26) as well as to mimic the effects of DBcAMP on cell growth and mediated regulation of tumor growth. morphology (19). In the present study, when tumor slices were incubated with PGE,, adenylate cyclase activity from REFERENCES the responsive tumor increased significantly, whereas the 1. Anderson, W. B., Gallo. M., and Pastan, I. Adenylate Cyclase Activity in cyclase from the unresponsive tumor was not responsive Fibroblasts Transformed by Kirsten or Moloney Sarcoma Viruses. J. to PGE,. This lack of response to PGE, in vitro of adenylate Biol. Chem., 249. 7041-7048, 1974. 2. Appleman, M. M., Thompson. W. J., and Russell, T. R. Cyclic Nucleotide cyclase from the unresponsive tumor correlates with that Phosphodiesterases. Advan. Cyclic Nucleotide Res., 3. 65-98. 1973. observed on tumor growth in vivo. PGE, injected into rats 3. Beavo. J. A., Hardman, J. G.. and Sutherland, E. W. Hydrolysis of bearing both DBcAMP-responsive and DBcAMP-unrespon- Cyclic Guanosine and Adenosine 3'.5'-Monophosphates by Rat and

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DECEMBER 1977 4499

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1977 American Association for Cancer Research. Adenylate Cyclase, Cyclic Adenosine 3′:5′-Monophosphate Phosphodiesterase, and Regression of Walker 256 Mammary Carcinoma

Yoon Sang Cho-Chung and Susanne F. Newcomer

Cancer Res 1977;37:4493-4499.

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