Journal of Neurochemistry, 1977. Vol. 29, pp. 273-218. Pergamon Press. Printed in Great Britain
CYCLIC GMP IN A NEUROBLASTOMA CLONE: POSSIBLE INVOLVEMENT I N MORPHOLOGICAL DIFFERENTIATION INDUCED BY DIBUTYRYL CYCLIC AMP J. ZWILLER,’ C . GO RID IS,^ J. CIESIELSKI-TRESKA and P. MANDEL Centre de Neurochimie du C.N.R.S., 11, rue Humann, 67085 Strasbourg Cedex, France (Received 14 December 1976. Accepted 17 January 1977)
Abstract-DBcAMP induces morphological differentiation of mouse neuroblastoma cells grown in culture. DBcGMP or 8-Br cyclic G M P when added alone also induces a discrete morphological differentiation. When analogues of cyclic GMP were added together with dBcAMP, neurite outgrowth was strikingly enhanced as compared to the effect of dBcAMP alone. Intracellular concentrations of cyclic GMP were increased during dBcAMP treatment and cyclic AMP levels were increased during 8-Br cyclic GMP treatment. Both treatments produced an increased protein kinase activity, supporting the possibility that not only cyclic AMP but also cyclic GMP may be involved in the differentiation process.
C-1300 NEUROBLASTOMA cells grown in uitro retain many of the characteristics of differentiated neurons (SCHUBERT et al., 1969; MANDEL et al., 1973). Process formation by neuroblastoma cells in tissue culture, which can be provoked by a variety of treatments (SEEDSet a/.. 1970; SCHUBERT & JACOB, 1970; FURMANSKI et al., 1971) has been termed morphological or somatic terminal differentiation and may serve as a model for neural maturation. An agent which has been widely used t o elicit morphologic differentiation et al., is dBcAMP (PRASAD& HSIE,1971; FURMANSKI 1971). Cyclic A M P has been implicated in the differentiating response of cultured neuroblastoma cells (PRASAD, 1972; SHEPPARD& PRASAD, 1973) and it has been assumed that dBcAMP acts as a substitute for endogenous cyclic AMP. Neuroblastoma cell differentiation was observed in presence of prostaglandin El or papaverine, both producing a n increase of endogenous cyclic A M P (PRASAD& KUMAR,1975). Neuroblastoma clones contain cyclic G M P in addition t o cyclic A M P (MATSUZAWA& NIRENBERG, 1975). In numerous cases, agents known t o change cyclic A M P levels have been shown to affect also intracellular cyclic GMP (GOLDBERGet al., 1975). Therefore we measured the cyclic GMP content of neuroblastoma cells during morphological differentiation induced by dBcAMP. Since we found that cyclic G M P levels increase in those cells, we investigated also the effects of exogenous cyclic GMP derivatives
on the morphology and cyclic AMP levels of cultured neuroblastoma cells.
MATERIALS AND METHODS The neuroblastoma clone M1, an adrenergic clone, which has been obtained in our laboratory (CIESIELSKITRESKA et al., 1975) was used throughout this study. Cells were grown to confluency in Falcon plastic dishes (75 cm2 for cyclic GMP determinations and 20cm2 for enzyme measurements and morphological observations) in Dutbecco’s modified Eagle’s medium, supplemented with 10% foetal calf serum. Cultures were maintained at 37°C in a humidified atmosphere of 5% COz in air. Confluency was obtained 3 days after subculturing. At this time, the flasks were divided into 2 groups. One group was not treated and served as control. To the other group, dBcAMP, 8-Br cyclic GMP, dBcGMP or combinations of these drugs were added in a final concentration of 1 mM. The medium was replaced every 48 h. Biochemical determinations were performed at different stages of the culture. The medium was rapidly aspirated and the cells washed two times with saline, then, the cells were harvested in 0.4ml 10 mM-Tris-HC1 buffer (pH 7.5) or 1 M-HCIO~, for estimation of enzymatic activity and cyclic GMP content respectively, and homogenized in the same medium. Guanylate cyclase activity was determined on aliquots containing 80-180 pg protein as previously described (GORIDIS & REUTTER, 1975). The MnZ+concentration used (7.7 mM) gave maximum activities. The accumulation of [32P]cyclic GMP was proportional to the amount of protein added and linear with time up to 15 min. Cyclic GMP phosphodiesterase activity was measured as previously described (COQUILe t al., 1975), 1OpM-cyclic GMP was used as substrate. Protein kinase activity was determined by * To whom correspondence should be addressed. Present address: Department of Neuroscience, The the method described by WELLERe t al., 1975. Incubation contained 5 mM-MgZ+, 1 ~ M - Y - ~ ~ P - A T P . Children’s Hospital Medical Center, Boston, MA 02115. mixture 50m~-Tris-HC1(pH 7.9, and 80-12Opg of protein. ReacU.S.A. Ahhreriations used: dBcAMP, N6, 2‘0-dibutyryl cyclic tions were terminated after 4min incubation by the addition of 2 ml of ice-cold 10% trichloroacetic acid. AMP; dBcGMP, N6, 20-dibutyryl cyclic GMP. 273
~~~~
~
274
I.
ZWILLER, C. GORIDIS,J. CIESIELSKI-TRESKA and P. MANDEL t
ea
+:
2
Q
F
OB
\ d
0
E a 0.6 t C
+ al C 0 U
a H a
0.4
.--U U %
V
0.2
Days in the presence of d B c A M P FIG. 1 . Cyclic GMP content and guanylate cyclase activity of neuroblastoma cells during dBcAMP induced morphological differentiation. Confluent cultures of neuroblastoma clone MI were cultivated in the presence or absence of 1 mM-dBcAMP. After various times in the presence of the drug, the cells were harvested for determination of guanylate cyclase activity ( t-. and ) cyclic GMP concentration (A-A). Values for control cultures are given as open symbols and broken lines (@--O and A---A for guanylate cyclase activity and cyclic G M P content, respectively). W designates the cyclic GMP content of cells cultured for the period indicated, where dBcAMP had been omitted from the medium for 24 h, the values for two experiments are shown. Guanylate cyclase activity is expressed as pmol cyclic GMP formed/min/mg protein, cyclic GMP concentrations S.D. is given for 3 cultures. x P < 0.05 x x P < 0.01. as pmol/mg protein. The mean value
Cyclic AMP was estimated using protein binding assay in the presence of dBcAMP (Fig. 1). Whereas cyclic (Amersham kit). Sample purification on neutral aluminium GMP concentrations were quite stable in control culoxide column showed no interference from 8-Br cyclic tures u p t o 10 days after subculturing, the levels in GMP in the cyclic AMP assay. dBcAMP treated cells reached a maximum of more Cyclic GMP was estimated radioimmunologically after than three times the control values 5 days after addisuccinylation following the procedure of CAILLAet a/., tion of the drug and declined thereafter. Higher than 1976. DBcAMP did not interfere with the cyclic GMP control levels were already seen after culturing in the determination as shown by the following criteria: (1) the cyclic GMP-like activity was always proportional presence of dBcAMP for 24 h. The high cyclic GMP to the amount of tissue extract over a 3-fold range of dilu- concentrations were maintained when dBcAMP was omitted from the medium for 24 h. tions. Guanylate cyclase [GTP pyrophosphate-lyase (cyc(2) succinylation of the tissue extract as used in our procedure results in a 50-fold increase of the sensitivity lizing) EC 4.6.1.21, the enzyme which forms cyclic towards native cyclic GMP (CAILLAet al., 1976). Since GMP from GTP, was measured in parallel cultures. dBcAMP cannot be succinylated. immunoreactivity of this The activities increased in control and dBcAMP substance should be indicated by measurement of cyclic treated cultures till day 5 and declined sharply thereGMP-like activity without succinylation. Actually, when after. Guanylate cyclase activity tended to be higher the succinylation step was omitted, no cyclic GMP-like in treated cultures at day 1 and day 5, but this differactivity could be detected. (3) there is less than 1 part in 60,000 cross interference of dBcAMP or N6-monobutyrylcyclic AMP with the cyclic TABLE1. EFFECTOF dBcAMP ON CYCLIC GMP HYDROLYGMP radioimmune assay (GORIDIS, unpublished). AssumSIS BY NEUROBLASTOMA CELLS ing a cellular volume of 11.Opg per mg of cell proteins Phosphodiesterase activity (BORG, in preparation), the cyclic GMP concentrations in (nmol/min/mg protein) control cells are approximately 2.7 x lO-'M. Hence, the Time (day) Control dBcAMP intracellular concentrations of the cyclic AMP derivatives M to result in an equivalent cyclic must attain 1.6 x 0.70 +_ 0.05 0.73 +_ 0.10 3 GMP-like activity, a situation which is highly unlikely. 0.86 & 0.01 0.99 f 0.16 5 (4) suppression of the dBcAMP containing medium for 7 0.86 0.02 0.69 k 0.04 24 h did not result in a change of the cyclic G M P levels. Protein was determined by the method of LOWRYet Confluent cultures of neuroblastoma clone M1 were culal. (1951) using bovine serum albumin as standard. tivated in the presence or absence of l mM-dBcAMP. At various days after addition of the drug, the cells were harRESULTS vested and cyclic GMP phosphodiesterase activity An increase of intracellular cyclic GMP levels was measured with 10 !.IM-CyCk GMP as substrate. Each value represents the mean ( ~ s . D . of measured in the neuroblastoma clone M1 cultivated ) 3 cultures.
215
FIG.2. Morphological appearance of representative cultures of neuroblastoma clone M 1 (a): control culture. (b) and (c): cultivated for 48 h in the presence of 1 mM-dBcAMP (b) or 1 m ~ - 8 - B rcyclic GMP (c) Phase contrast. x 250.
216
Fic3. 3. Morphological appearance of representative cultures of neuroblastoma clone MI, cultiiiated for 48 11 in the presence of both 1 mM-8-Br cyclic G M P and 1 mM-dBcAMP (a) or of 1 mM-dBc(;MP and 1 mM-dBcAMP (b) Phase contrast. x 250.
Neuroblastoma cells/dibutyryl cyclic AMP/cyclic GMP ence did not reach the level of statistical significance (Fig. 1). Similarly, addition of dBcAMP (1 mM) directly to the guanylate cyclase test was without effect (data not shown). It was necessary to test whether dBcAMP could act by inhibiting cyclic G M P hydrolysis. The data of Table 1 show that the activities of cyclic G M P phosphodiesterase (EC 3.1.4.17) in homogenates of neuroblastoma cells were the same in control and treated cultures. Addition of dBcAMP (1 mM) directly to the phosphodiesterase test, however, inhibited the hydrolysis of 10 pM-CyCliC G M P 35%. Apparently, high concentrations of dBcAMP inhibit moderately cyclic G M P phosphodiesterase activity. However such concentrations are not attained in homogenates of treated cells. Our data suggested that cyclic G M P as well as the cyclic AMP-like action of dBcAMP could be involved in the stimulation of neurite outgrowth. Therefore, we studied the morphological appearance of M1 neuroblastoma cells cultivated in the presence of the cyclic G M P derivative 8-Br cyclic GMP. As shown in Fig. 2, dBcAMP and 8-Br cyclic G M P were both capable of inducing process formation. The effect was considerably enhanced when both agents were added together (Fig. 3). The number of cells with processes and the ramification of the neurites were both enhanced as compared to cultures treated with one nucleotide alone. Clumps of undifferentiated cells which still persisted in cultures treated with one of the nucleotides were absent from cultures which had been cultivated for 48 h in the presence of both, dBcAMP and 8-Br cyclic GMP. The same results were obtained when dBcGMP instead of 8-Br cyclic G M P was used (Fig. 3). Adding 2 m of~ one nucleotide alone did not stimulate morphological differentiation more than did 1 mM (not shown). Each of the drugs and drug combinations were tested on 9 cultures with very similar results. In order to decern how cyclic G M P is involved in this process, we examined the cAMP level in cells treated with 8-Br cyclic GMP. Data presented in Table 2 show that cAMP level is increased after this treatment. Moreover, we investigated the long term changes in protein kinase activity in our experimental conditions. There were small increases of endogenous protein kinase activity following treatment with dBcAMP or 8-Br cyclic G M P for 48 h (Table 3), but the effects of the two cyclic nucleotide derivatives were not additive. DISCUSSION
277
TABLE2. EFFECTOF 8-BROMO-CYCLIC GMP ON AMP LEVEL OF M1 NEUROBLASTOMA CELLS
CYCLIC
Cyclic AMP (pmol/mg protein) Control 8-Br cyclic GMP 24 H 48 H
6.2 k 1.3 7.0 i 2.0
14.0 26.6
5.1*
2.87
Cultures were cultivated in the presence or absence of 1 m ~ - l - B rcyclic GMP. Cells were harvested 1 or 2 days later and cyclic AMP level measured. Each value represents the mean ( ~ s . D .of ) 4 cultures. * P < 0.02. t P < 0.01. cyclic G M P levels. This possibility is supported by our observation that cyclic G M P analogues are effective in promoting process outgrowth. Furthermore, addition of 8-Br cyclic G M P produced an increase of cAMP levels which could also be involved in the observed effect. Similar results have been obtained in the brains of 8-Br cyclic G M P treated rats (FERNANDEZ-POL & HAYS,1976). The small increase of protein kinase activity observed after treatment with dBcAMP or 8-BrcGMP suggests the possible involvement of protein kinase in the morphological changes. When both nucleotides were added together, the morphological differentiation was strikingly enhanced as compared t o the effect of one single nucleotide. By contrast, protein kinase activity in neuroblastoma cells treated with both nucleotides was not additive. The same degree of process formation was not obtained by simply adding the double concentration of one nucleotide. The possibility, however, cannot be excluded that 2 m of~ one substance are more toxic to the cells than the presence of both nucleotides in 1 mM concentrations and that the higher toxicity of 2 mM-dBcAMP prevents a further stimulation of neurite outgrowth. In any case, addition of 1 mM-dBcAMP together with 1 m ~ - 8 - B rcyclic G M P is very effective in promoting morphological differentiation of neuroblastoma cells. Work is now in progress to determine whether biochemical correlates of TABLE3. EFFECTOF
CYCLIC NUCLEOTIDE DERIVATIVES ON PROTEIN KINASE ACTIVITY
Protein kinase activity (nmol/4 min/mg protein) Control dBcAMP (1 mM) 8-Br cGMP (1 mM) dBcAMP (1 mM) + 8-Br cGMP (1 mM)
1.84 0.20 2.64 k 0.56* 2.68 & 0.72t 2.36 OA4*
The mode of action of dBcAMP on neuroblastoma cells is unclear at present time. Studies on other systems have shown that dBcAMP and/or N6-butyryl MI neuroblastoma cells were cultivated in the presence cyclic AMP, which can be formed enzymatically from of various cyclic nucleotide derivatives during 48 H. They dBcAMP, are capable of stimulating protein kinase were harvested and protein kinase activity assayed as deactivity, thus substituting for cyclic AMP (KAUKEL scribed in Methods. Each value represents the mean ( ~ s . D . )of 4 cultures. et a!., 1972). Our results indicate that part of the effect * P < 0.05. of dBcAMP could be due to increased intracellular t P = 0.05.
278
J. ZWILLER, C. GORIDIS, J. CIESIELSKI-TRESKA and P. MANDEL
differentiation are also expressed t o a greater extent in cultures treated with a mixture of cyclic AMP and cyclic GMP derivatives. O u r results do not explain how elevated levels of cyclic GMP in differentiated cultures could be brought about. Guanylate cyclase and cyclic G M P phosphodiesterase activities measured in homogenates of M1 cells were not significantly different in d B c A M P treated and control cultures. D B c A M P was, however, a moderately active inhibitor of cyclic GMP hydrolysis when added directly t o the phosphodiesterase test. Such a direct effect of d B c A M P on the degradation of intracellular cyclic GMP is difficult t o reconcile with our observation that cyclic GMP levels remained elevated when dBcAMP had been omitted from the medium for 24 h. Clearly, the elevation of intracellular cyclic GMP levels does not depend on the continuous presence of the drug. Further kinetic work of cyclic GMP synthesis in viuo is required to approach how addition of dBcAMP leads t o increase cyclic GMP concentrations.
FERNANDEZ-POL J. A. & HAYSM. T. (1976) L$e Sci. 19, 3540. FURMANSKI P., SILVERMAN D. J. & LURINM. (1971) Nature, Lond. 233, 413415. GOLDBERC N. D., HADDOXM. K., NICOLS. E., GLASSD. B., SANFORD C. H., KUEHLF. A. & ESTENSEN R. (1975) in Advances in Cyclic Nucleotide Research (DRUMMOND G . J., GREENCARD P. & ROBISON G . A,, eds) Vol. 5, pp. 307-330. Raven Press, New York. GORIDISC. & REUTTERW. (1975) Nature, Lond. 257, 698--700. E., MUNDHENK K. & HILZ H. (1972) Eur. J . BioKAUKEL chem. 27, 197-200. LOWRYO., ROSEBROUCH N. F., FARRA. L. & RANDALL R. J. (1951) J . biol. Chem. 193, 265-275. MANDEL P., CIESIELSKI-TRESKA J., HERMETETJ. c., C. (1973) in Frontiers ZWILLERJ., MACKG. & GORIDIS in Cutecholamine Research, Proc. 3rd Catecholamine Symposium (USDINE. & SNYDERS., eds) pp. 277-284. Pergamon Press, New York. MATSUZAWA H. & NIRENBERC M. (1975) Proc. natn. Acad. Sci., U.S.A. 12, 3472-3476. PRASAD K. N. (1972) Nature N e w Biol. 236, 49-52. PRASADK. N. & HSIE A. W. (1971) Nature, New Biol. Acknowledgements- We are grateful to Mrs M. 0.REVEL 233, 141-142. and G . ULRICHfor their excellent technical assistance. F’RASADK. N. & KUMARS . (1975) Cancer 36, 1338-1343. This work was supported by a Research Grant from SCHUBERT D., HUMPHREYS S., BARONIC. & COHN M. INSERM-ATP 6-74-27. (1969) Proc. natn. Acad. Sci., U.S.A. 64, 316-323. D. & JACOBF. (1970) Proc. natn. Acad. Sci. SCHUBERT REFERENCES U.S.A. 67, 247-254. M. CAILLAH. L., VANNIERC. J. & DELAACE M. A. (1976) SEEDSN. W., GILMANA. G., AMANOT. & NIRENBERC (1970) Proc. natn. Acad. Sci. U S A . 66, 16G167. Analyt. Biochem. 70, 195-202. J. R. & PRASADK. N. (1973) Life Sci. I1 12, J., WARTERS . & MANDELP. (1975) SHEPPARD CIESIELSKI-TRESKA 431439. Neurohiol. 5, 382-392. P. & GORIDIS C. (1975) WELLERM., VIRMAUXN. & MANDELP. (1975) Proc. natn. COQUILJ. F., VIRMAUXN., MANDEL Acad. Sci., U.S.A. 72, 381-385. Biochim. biophys. Actu 403, 425437.
CYCLIC GMP IN A NEUROBLASTOMA CLONE: POSSIBLE INVOLVEMENT I N MORPHOLOGICAL DIFFERENTIATION INDUCED BY DIBUTYRYL CYCLIC AMP J. ZWILLER,’ C . GO RID IS,^ J. CIESIELSKI-TRESKA and P. MANDEL Centre de Neurochimie du C.N.R.S., 11, rue Humann, 67085 Strasbourg Cedex, France (Received 14 December 1976. Accepted 17 January 1977)
Abstract-DBcAMP induces morphological differentiation of mouse neuroblastoma cells grown in culture. DBcGMP or 8-Br cyclic G M P when added alone also induces a discrete morphological differentiation. When analogues of cyclic GMP were added together with dBcAMP, neurite outgrowth was strikingly enhanced as compared to the effect of dBcAMP alone. Intracellular concentrations of cyclic GMP were increased during dBcAMP treatment and cyclic AMP levels were increased during 8-Br cyclic GMP treatment. Both treatments produced an increased protein kinase activity, supporting the possibility that not only cyclic AMP but also cyclic GMP may be involved in the differentiation process.
C-1300 NEUROBLASTOMA cells grown in uitro retain many of the characteristics of differentiated neurons (SCHUBERT et al., 1969; MANDEL et al., 1973). Process formation by neuroblastoma cells in tissue culture, which can be provoked by a variety of treatments (SEEDSet a/.. 1970; SCHUBERT & JACOB, 1970; FURMANSKI et al., 1971) has been termed morphological or somatic terminal differentiation and may serve as a model for neural maturation. An agent which has been widely used t o elicit morphologic differentiation et al., is dBcAMP (PRASAD& HSIE,1971; FURMANSKI 1971). Cyclic A M P has been implicated in the differentiating response of cultured neuroblastoma cells (PRASAD, 1972; SHEPPARD& PRASAD, 1973) and it has been assumed that dBcAMP acts as a substitute for endogenous cyclic AMP. Neuroblastoma cell differentiation was observed in presence of prostaglandin El or papaverine, both producing a n increase of endogenous cyclic A M P (PRASAD& KUMAR,1975). Neuroblastoma clones contain cyclic G M P in addition t o cyclic A M P (MATSUZAWA& NIRENBERG, 1975). In numerous cases, agents known t o change cyclic A M P levels have been shown to affect also intracellular cyclic GMP (GOLDBERGet al., 1975). Therefore we measured the cyclic GMP content of neuroblastoma cells during morphological differentiation induced by dBcAMP. Since we found that cyclic G M P levels increase in those cells, we investigated also the effects of exogenous cyclic GMP derivatives
on the morphology and cyclic AMP levels of cultured neuroblastoma cells.
MATERIALS AND METHODS The neuroblastoma clone M1, an adrenergic clone, which has been obtained in our laboratory (CIESIELSKITRESKA et al., 1975) was used throughout this study. Cells were grown to confluency in Falcon plastic dishes (75 cm2 for cyclic GMP determinations and 20cm2 for enzyme measurements and morphological observations) in Dutbecco’s modified Eagle’s medium, supplemented with 10% foetal calf serum. Cultures were maintained at 37°C in a humidified atmosphere of 5% COz in air. Confluency was obtained 3 days after subculturing. At this time, the flasks were divided into 2 groups. One group was not treated and served as control. To the other group, dBcAMP, 8-Br cyclic GMP, dBcGMP or combinations of these drugs were added in a final concentration of 1 mM. The medium was replaced every 48 h. Biochemical determinations were performed at different stages of the culture. The medium was rapidly aspirated and the cells washed two times with saline, then, the cells were harvested in 0.4ml 10 mM-Tris-HC1 buffer (pH 7.5) or 1 M-HCIO~, for estimation of enzymatic activity and cyclic GMP content respectively, and homogenized in the same medium. Guanylate cyclase activity was determined on aliquots containing 80-180 pg protein as previously described (GORIDIS & REUTTER, 1975). The MnZ+concentration used (7.7 mM) gave maximum activities. The accumulation of [32P]cyclic GMP was proportional to the amount of protein added and linear with time up to 15 min. Cyclic GMP phosphodiesterase activity was measured as previously described (COQUILe t al., 1975), 1OpM-cyclic GMP was used as substrate. Protein kinase activity was determined by * To whom correspondence should be addressed. Present address: Department of Neuroscience, The the method described by WELLERe t al., 1975. Incubation contained 5 mM-MgZ+, 1 ~ M - Y - ~ ~ P - A T P . Children’s Hospital Medical Center, Boston, MA 02115. mixture 50m~-Tris-HC1(pH 7.9, and 80-12Opg of protein. ReacU.S.A. Ahhreriations used: dBcAMP, N6, 2‘0-dibutyryl cyclic tions were terminated after 4min incubation by the addition of 2 ml of ice-cold 10% trichloroacetic acid. AMP; dBcGMP, N6, 20-dibutyryl cyclic GMP. 273
~~~~
~
274
I.
ZWILLER, C. GORIDIS,J. CIESIELSKI-TRESKA and P. MANDEL t
ea
+:
2
Q
F
OB
\ d
0
E a 0.6 t C
+ al C 0 U
a H a
0.4
.--U U %
V
0.2
Days in the presence of d B c A M P FIG. 1 . Cyclic GMP content and guanylate cyclase activity of neuroblastoma cells during dBcAMP induced morphological differentiation. Confluent cultures of neuroblastoma clone MI were cultivated in the presence or absence of 1 mM-dBcAMP. After various times in the presence of the drug, the cells were harvested for determination of guanylate cyclase activity ( t-. and ) cyclic GMP concentration (A-A). Values for control cultures are given as open symbols and broken lines (@--O and A---A for guanylate cyclase activity and cyclic G M P content, respectively). W designates the cyclic GMP content of cells cultured for the period indicated, where dBcAMP had been omitted from the medium for 24 h, the values for two experiments are shown. Guanylate cyclase activity is expressed as pmol cyclic GMP formed/min/mg protein, cyclic GMP concentrations S.D. is given for 3 cultures. x P < 0.05 x x P < 0.01. as pmol/mg protein. The mean value
Cyclic AMP was estimated using protein binding assay in the presence of dBcAMP (Fig. 1). Whereas cyclic (Amersham kit). Sample purification on neutral aluminium GMP concentrations were quite stable in control culoxide column showed no interference from 8-Br cyclic tures u p t o 10 days after subculturing, the levels in GMP in the cyclic AMP assay. dBcAMP treated cells reached a maximum of more Cyclic GMP was estimated radioimmunologically after than three times the control values 5 days after addisuccinylation following the procedure of CAILLAet a/., tion of the drug and declined thereafter. Higher than 1976. DBcAMP did not interfere with the cyclic GMP control levels were already seen after culturing in the determination as shown by the following criteria: (1) the cyclic GMP-like activity was always proportional presence of dBcAMP for 24 h. The high cyclic GMP to the amount of tissue extract over a 3-fold range of dilu- concentrations were maintained when dBcAMP was omitted from the medium for 24 h. tions. Guanylate cyclase [GTP pyrophosphate-lyase (cyc(2) succinylation of the tissue extract as used in our procedure results in a 50-fold increase of the sensitivity lizing) EC 4.6.1.21, the enzyme which forms cyclic towards native cyclic GMP (CAILLAet al., 1976). Since GMP from GTP, was measured in parallel cultures. dBcAMP cannot be succinylated. immunoreactivity of this The activities increased in control and dBcAMP substance should be indicated by measurement of cyclic treated cultures till day 5 and declined sharply thereGMP-like activity without succinylation. Actually, when after. Guanylate cyclase activity tended to be higher the succinylation step was omitted, no cyclic GMP-like in treated cultures at day 1 and day 5, but this differactivity could be detected. (3) there is less than 1 part in 60,000 cross interference of dBcAMP or N6-monobutyrylcyclic AMP with the cyclic TABLE1. EFFECTOF dBcAMP ON CYCLIC GMP HYDROLYGMP radioimmune assay (GORIDIS, unpublished). AssumSIS BY NEUROBLASTOMA CELLS ing a cellular volume of 11.Opg per mg of cell proteins Phosphodiesterase activity (BORG, in preparation), the cyclic GMP concentrations in (nmol/min/mg protein) control cells are approximately 2.7 x lO-'M. Hence, the Time (day) Control dBcAMP intracellular concentrations of the cyclic AMP derivatives M to result in an equivalent cyclic must attain 1.6 x 0.70 +_ 0.05 0.73 +_ 0.10 3 GMP-like activity, a situation which is highly unlikely. 0.86 & 0.01 0.99 f 0.16 5 (4) suppression of the dBcAMP containing medium for 7 0.86 0.02 0.69 k 0.04 24 h did not result in a change of the cyclic G M P levels. Protein was determined by the method of LOWRYet Confluent cultures of neuroblastoma clone M1 were culal. (1951) using bovine serum albumin as standard. tivated in the presence or absence of l mM-dBcAMP. At various days after addition of the drug, the cells were harRESULTS vested and cyclic GMP phosphodiesterase activity An increase of intracellular cyclic GMP levels was measured with 10 !.IM-CyCk GMP as substrate. Each value represents the mean ( ~ s . D . of measured in the neuroblastoma clone M1 cultivated ) 3 cultures.
215
FIG.2. Morphological appearance of representative cultures of neuroblastoma clone M 1 (a): control culture. (b) and (c): cultivated for 48 h in the presence of 1 mM-dBcAMP (b) or 1 m ~ - 8 - B rcyclic GMP (c) Phase contrast. x 250.
216
Fic3. 3. Morphological appearance of representative cultures of neuroblastoma clone MI, cultiiiated for 48 11 in the presence of both 1 mM-8-Br cyclic G M P and 1 mM-dBcAMP (a) or of 1 mM-dBc(;MP and 1 mM-dBcAMP (b) Phase contrast. x 250.
Neuroblastoma cells/dibutyryl cyclic AMP/cyclic GMP ence did not reach the level of statistical significance (Fig. 1). Similarly, addition of dBcAMP (1 mM) directly to the guanylate cyclase test was without effect (data not shown). It was necessary to test whether dBcAMP could act by inhibiting cyclic G M P hydrolysis. The data of Table 1 show that the activities of cyclic G M P phosphodiesterase (EC 3.1.4.17) in homogenates of neuroblastoma cells were the same in control and treated cultures. Addition of dBcAMP (1 mM) directly to the phosphodiesterase test, however, inhibited the hydrolysis of 10 pM-CyCliC G M P 35%. Apparently, high concentrations of dBcAMP inhibit moderately cyclic G M P phosphodiesterase activity. However such concentrations are not attained in homogenates of treated cells. Our data suggested that cyclic G M P as well as the cyclic AMP-like action of dBcAMP could be involved in the stimulation of neurite outgrowth. Therefore, we studied the morphological appearance of M1 neuroblastoma cells cultivated in the presence of the cyclic G M P derivative 8-Br cyclic GMP. As shown in Fig. 2, dBcAMP and 8-Br cyclic G M P were both capable of inducing process formation. The effect was considerably enhanced when both agents were added together (Fig. 3). The number of cells with processes and the ramification of the neurites were both enhanced as compared to cultures treated with one nucleotide alone. Clumps of undifferentiated cells which still persisted in cultures treated with one of the nucleotides were absent from cultures which had been cultivated for 48 h in the presence of both, dBcAMP and 8-Br cyclic GMP. The same results were obtained when dBcGMP instead of 8-Br cyclic G M P was used (Fig. 3). Adding 2 m of~ one nucleotide alone did not stimulate morphological differentiation more than did 1 mM (not shown). Each of the drugs and drug combinations were tested on 9 cultures with very similar results. In order to decern how cyclic G M P is involved in this process, we examined the cAMP level in cells treated with 8-Br cyclic GMP. Data presented in Table 2 show that cAMP level is increased after this treatment. Moreover, we investigated the long term changes in protein kinase activity in our experimental conditions. There were small increases of endogenous protein kinase activity following treatment with dBcAMP or 8-Br cyclic G M P for 48 h (Table 3), but the effects of the two cyclic nucleotide derivatives were not additive. DISCUSSION
277
TABLE2. EFFECTOF 8-BROMO-CYCLIC GMP ON AMP LEVEL OF M1 NEUROBLASTOMA CELLS
CYCLIC
Cyclic AMP (pmol/mg protein) Control 8-Br cyclic GMP 24 H 48 H
6.2 k 1.3 7.0 i 2.0
14.0 26.6
5.1*
2.87
Cultures were cultivated in the presence or absence of 1 m ~ - l - B rcyclic GMP. Cells were harvested 1 or 2 days later and cyclic AMP level measured. Each value represents the mean ( ~ s . D .of ) 4 cultures. * P < 0.02. t P < 0.01. cyclic G M P levels. This possibility is supported by our observation that cyclic G M P analogues are effective in promoting process outgrowth. Furthermore, addition of 8-Br cyclic G M P produced an increase of cAMP levels which could also be involved in the observed effect. Similar results have been obtained in the brains of 8-Br cyclic G M P treated rats (FERNANDEZ-POL & HAYS,1976). The small increase of protein kinase activity observed after treatment with dBcAMP or 8-BrcGMP suggests the possible involvement of protein kinase in the morphological changes. When both nucleotides were added together, the morphological differentiation was strikingly enhanced as compared t o the effect of one single nucleotide. By contrast, protein kinase activity in neuroblastoma cells treated with both nucleotides was not additive. The same degree of process formation was not obtained by simply adding the double concentration of one nucleotide. The possibility, however, cannot be excluded that 2 m of~ one substance are more toxic to the cells than the presence of both nucleotides in 1 mM concentrations and that the higher toxicity of 2 mM-dBcAMP prevents a further stimulation of neurite outgrowth. In any case, addition of 1 mM-dBcAMP together with 1 m ~ - 8 - B rcyclic G M P is very effective in promoting morphological differentiation of neuroblastoma cells. Work is now in progress to determine whether biochemical correlates of TABLE3. EFFECTOF
CYCLIC NUCLEOTIDE DERIVATIVES ON PROTEIN KINASE ACTIVITY
Protein kinase activity (nmol/4 min/mg protein) Control dBcAMP (1 mM) 8-Br cGMP (1 mM) dBcAMP (1 mM) + 8-Br cGMP (1 mM)
1.84 0.20 2.64 k 0.56* 2.68 & 0.72t 2.36 OA4*
The mode of action of dBcAMP on neuroblastoma cells is unclear at present time. Studies on other systems have shown that dBcAMP and/or N6-butyryl MI neuroblastoma cells were cultivated in the presence cyclic AMP, which can be formed enzymatically from of various cyclic nucleotide derivatives during 48 H. They dBcAMP, are capable of stimulating protein kinase were harvested and protein kinase activity assayed as deactivity, thus substituting for cyclic AMP (KAUKEL scribed in Methods. Each value represents the mean ( ~ s . D . )of 4 cultures. et a!., 1972). Our results indicate that part of the effect * P < 0.05. of dBcAMP could be due to increased intracellular t P = 0.05.
278
J. ZWILLER, C. GORIDIS, J. CIESIELSKI-TRESKA and P. MANDEL
differentiation are also expressed t o a greater extent in cultures treated with a mixture of cyclic AMP and cyclic GMP derivatives. O u r results do not explain how elevated levels of cyclic GMP in differentiated cultures could be brought about. Guanylate cyclase and cyclic G M P phosphodiesterase activities measured in homogenates of M1 cells were not significantly different in d B c A M P treated and control cultures. D B c A M P was, however, a moderately active inhibitor of cyclic GMP hydrolysis when added directly t o the phosphodiesterase test. Such a direct effect of d B c A M P on the degradation of intracellular cyclic GMP is difficult t o reconcile with our observation that cyclic GMP levels remained elevated when dBcAMP had been omitted from the medium for 24 h. Clearly, the elevation of intracellular cyclic GMP levels does not depend on the continuous presence of the drug. Further kinetic work of cyclic GMP synthesis in viuo is required to approach how addition of dBcAMP leads t o increase cyclic GMP concentrations.
FERNANDEZ-POL J. A. & HAYSM. T. (1976) L$e Sci. 19, 3540. FURMANSKI P., SILVERMAN D. J. & LURINM. (1971) Nature, Lond. 233, 413415. GOLDBERC N. D., HADDOXM. K., NICOLS. E., GLASSD. B., SANFORD C. H., KUEHLF. A. & ESTENSEN R. (1975) in Advances in Cyclic Nucleotide Research (DRUMMOND G . J., GREENCARD P. & ROBISON G . A,, eds) Vol. 5, pp. 307-330. Raven Press, New York. GORIDISC. & REUTTERW. (1975) Nature, Lond. 257, 698--700. E., MUNDHENK K. & HILZ H. (1972) Eur. J . BioKAUKEL chem. 27, 197-200. LOWRYO., ROSEBROUCH N. F., FARRA. L. & RANDALL R. J. (1951) J . biol. Chem. 193, 265-275. MANDEL P., CIESIELSKI-TRESKA J., HERMETETJ. c., C. (1973) in Frontiers ZWILLERJ., MACKG. & GORIDIS in Cutecholamine Research, Proc. 3rd Catecholamine Symposium (USDINE. & SNYDERS., eds) pp. 277-284. Pergamon Press, New York. MATSUZAWA H. & NIRENBERC M. (1975) Proc. natn. Acad. Sci., U.S.A. 12, 3472-3476. PRASAD K. N. (1972) Nature N e w Biol. 236, 49-52. PRASADK. N. & HSIE A. W. (1971) Nature, New Biol. Acknowledgements- We are grateful to Mrs M. 0.REVEL 233, 141-142. and G . ULRICHfor their excellent technical assistance. F’RASADK. N. & KUMARS . (1975) Cancer 36, 1338-1343. This work was supported by a Research Grant from SCHUBERT D., HUMPHREYS S., BARONIC. & COHN M. INSERM-ATP 6-74-27. (1969) Proc. natn. Acad. Sci., U.S.A. 64, 316-323. D. & JACOBF. (1970) Proc. natn. Acad. Sci. SCHUBERT REFERENCES U.S.A. 67, 247-254. M. CAILLAH. L., VANNIERC. J. & DELAACE M. A. (1976) SEEDSN. W., GILMANA. G., AMANOT. & NIRENBERC (1970) Proc. natn. Acad. Sci. U S A . 66, 16G167. Analyt. Biochem. 70, 195-202. J. R. & PRASADK. N. (1973) Life Sci. I1 12, J., WARTERS . & MANDELP. (1975) SHEPPARD CIESIELSKI-TRESKA 431439. Neurohiol. 5, 382-392. P. & GORIDIS C. (1975) WELLERM., VIRMAUXN. & MANDELP. (1975) Proc. natn. COQUILJ. F., VIRMAUXN., MANDEL Acad. Sci., U.S.A. 72, 381-385. Biochim. biophys. Actu 403, 425437.
