Proceedings of the National Academy of Sciences Vol. 67, No. 2, pp. 786-792, October 1970 Regulation of Acetylcholinesterase in Neuroblastoma Cells A. Blume}, F. Gilbert, S. Wilson, J. Farber, R. Rosenberg, and M. Nirenberg LABORATORY OF BIOCHEMICAL GENETICS, NATIONAL HEART AND LUNG INSTITUTE, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND 20014 Communicated July 31, 1970 Abstract. The specific activity of mouse neuroblastoma acetylcholinesterase (EC 3.1.1.7) increased 25-fold when the rate of cell division was restricted. The results show that acetylcholinesterase activity is regulated in neuroblastoma cells and that the regulatory mechanism is inversely related to the rate of cell division. Under the same conditions the specific activity of catechol-O-methy] transferase (EC 2.1.1.6) did not change significantly. Mouse neuroblastoma C-1300 has been transplanted from animal to animal approximately 1000 times over a period of 30 years; nevertheless, cells, after 5000-8000 cell generations, continue to follow a program of neuron differentia- tion. Cloned cells contain acetylcholinesterase (AChE), choline acetyltrans- ferase (EC 2.3.1.6), and enzymes for norepinephrine synthesis.’? Cells also are capable of extending processes, several millimeters in length, that contain microtubules,** neurofilaments, and dense core vesicles? and possess membranes capable of generating action potentials in response to electrical stimulation’ * or acetylcholine. Burkhalter e al.2 and Goodwin and Sizer" have shown that, the acetylcholines- terase activity of cultured chick embryo intestine and skeletal muscle can be regulated. In this report the activities of mouse neuroblastoma acetylcholines- terase and catechol-O-methyl transferase were investigated.11 We wish to report that neuroblastoma acetylcholinesterase activity is regulated and that the regulatory process is coupled to the rate of cell division. Materials and Methods. Cells: Experiments were performed with mouse neuroblastoma C-1300, clone N-18,° passage 43-60. Cells were grown in Dulbecco’s modi- fication of Eagle’s medium supplemented with: 50 units of penicillin G and 10 yg of streptomycin sulfate/ml, 10% fetal calf serum, and 10% horse serum (except where noted) in 10% CO.-90% air. Cells were kept in logarithmic growth in monolayer cul- ture (ie., cells grown attached to the surface of a Petridish) for 7~10 generations before each experiment. For experiments, cells were grown in 150-mm Petri dishes (Falcon Co.) containing 20 ml of growth medium. The medium was changed every other day. Cells in logarithmic phase growth were dissociated by incubation without Ca?+ or Mg?t for 10-15 min in saline D,,!* supplemented with 5.6 mM glucose and 59 mM sucrose (modi- fied D,). Cells in stationary phase growth were firmly attached to the growth surface, and their subculture required an additional incubation with 0.05% “trypsin” (Difco 1:250) in modified D, for 10-15 min at 37°C. Homogenates: The medium from each dish was transferred to a centrifuge tube and the cell monolayer was washed 3 times with modified D,. Washes and growth medium were 786 Vou. 67, 1970 NEUROBLASTOMA ACETYLCHOLINESTERASE 787 pooled and centrifuged (250 X g, 10 min, 3°C); the cell pellet was suspended in modi- fied D,, then washed twice more in the same manner. Dishes were drained, and cell monolayers recovered by scraping. Dishes were then washed twice with small amounts of a solution containing 50 mM potassium phosphate buffer, pH 6.8, and 1 mM EDTA. The washes, scraped cells, and cell pellet were combined and sonicated. Homogenates were stored in small portions at —196°C. No effect of storage on AChE or catechol-O- methyl transferase activities was detected. Assays: Cell concentrations were determined in duplicate with a hemocytometer, cell viability was determined by the nigrosin method," and protein was determined by a modification of the method of Lowry ef al.\4 Catechol-O-methyl transferase activity was determined by a modification of the method of Nikodejevic, Senoh, Daly, and Creveling."* Each reaction contained the fol- lowing, in a final volume of 0.05 ml: 5 mM potassium phosphate buffer, pH 6.8; 0.8 mM EDTA; 5 mM MgCl; 1 mM dihydroxybenzoic acid; 1.1 mM [!C]methyl-S- adenosyl-L-methionine iodide (4.6 »Ci/umol); and homogenate protein. Reactions were incubated at 37°C for 20 min. The rate of reaction was proportional to enzyme concentrations. Each reaction mixture was acidified and then extracted with toluene. AChE was measured by a modification of the method of Reed et al.,!” to be described in detail elsewhere (S. Wilson, ef al., in preparation). In brief, 1-[}4C]acetylcholine is incubated with homogenate; then 1-[!*C]acetate is separated from the substrate and counted. Each reaction contained the following components, in a final volume of 0.05 ml, except where noted: 0.05 M potassium phosphate buffer, pH 6.8; 0.2 M NaCl; 1 mM EDTA; 0.5% Triton X-100 (Packard Instrument Co.); 3.3 mM 1-[4Clacetyl- choline iodide (0.39 »Ci/umol); and 0-0.05 mg homogenate protein. Reactions were incubated for 10 min at 37°C except where noted, and terminated by the addition of 1.5 ml of a solution, at 3°C, containing 2 »M 1,5-bis-(4-allyldimethylammoniumphenyl) pentane-1,3-dibromide (BW284C51, Burroughs-Wellcome Co.), an inhibitor of AChE." In all cases, the rate of reaction was proportional to homogenate concentration. The diluted reaction mixture, and a subsequent 1.5 ml wash containing the AChE inhibitor, were passed through a disposable 0.5 X 5 cm column of BioRad AG50X8 (Nat-form, 100-200 mesh) washed with water. The eluates were collected in a scintillation vial, 10 ml scintillation fluid (1000 g Triton X-100-2000 ml toluene-165 ml Liquifluor [New England Nuclear]) was added and the radioactivity was determined. Most values re- ported represent the average of triplicate homogenates; each homogenate was assayed at four concentrations. The reproducibility of AChE and catechol-O-methyl trans- ferase specific activity determinations is +15%. One unit of AChE or catechol-O-methy] transferase activity is defined as 1.0 nmol [44C]-product formed per minute. Specific activity of the enzymes is expressed as units of enzyme per mg protein. Enzyme activity per dish corresponds to units of enzyme per dish. Results. Enzyme activity as a function of cell division: The relation be- tween AChE and catechol-O-methy] transferase activities and the rate of multi- plication of neuroblastoma cells is illustrated in Fig. 1. Each Petri dish was inoculated with 2 X 10° cells. After a slight lag, cells multiplied rapidly with a generation time of 24 hr. The maximum cell concentration (28 X 108 cells) was attained on approximately the 6th day of incubation. The specific activity of AChE did not change appreciably during the period of rapid cell division. However, during the stationary phase of growth, the 6th through the 17th day of incubation, the specific activity of AChE increased 25-fold. To investigate the effect of logarithmic growth upon AChE specific activity, stationary phase cells were subcultured at lower cell concentrations on the 13th day of incubation and then maintained in logarithmic growth. A 50% decrease 788 BIOCHEMISTRY: BLUME ET AL. Proc. N. A. S. 40 [a” WEES, Praren T 5 25 a 40 & ~ ~ "5 20 /s SUBCULTURE i 30 Zz % 15st fe * o 5 o wolff || IP & aa a t a a a 5t te oh iy 1 '0 2 Fig. 1. Neuroblastoma cells in logarithmic oe ae 0 growth were subcultured without trypsin. On soe ACE TYLCHOLINESTERASE 16000 the 13th and 17th days of incubation, some of 2 Perry the cells were dissociated with trypsin (indicated wi IS) petiwry\, v4 9000 | by the vertical dotted lines). Closed and open 2 oor 14000 2 — symbols represent the values obtained before and es 75 ji \ | 43000 3 after subculture, respectively. In panel A, cells/ = sof | J2000 = dish (@) and mg protein/dish (4) are shown. Zz sen} : a More than 85% of the cells were viable throughout 2 25 DISH + loo0 & . : < vA 2 the experiment. In panel B, AChE specific wy of ia Oo S activity (@) and AChE activity/dish (A) are & O18 [C caNsreRAse, | © )6©6 shown. In panel C, catechol-O-methyl trans- * Qg . - woe . © O15 Tf speemic 15 2 ferase specific activity (@) and activity/dish (a) PER wetnt 8 o.l2 ren 40 4 © are presented. 2 0.09} Te 13 9 pos, fk fy 2 3 8 0.03 + x pa | | c < 4 Q , 9 36 9 i215 182i 7 DAYS in AChE specific activity was observed within 40 hr; after 6 days the specific activity returned to the basal level. The amount of protein per average cell doubled, from 0.6 to 1.2 ng after cells shifted from logarithmic to stationary growth. Over the same period, AChE activity per dish increased greatly, resulting in a 50-fold rise in AChE/cell. When stationary phase cells were shifted to logarithmic growth, corresponding decreases in protein and AChE per cell were found. No significant change in the specific activity of catechol-O-methyl transferase was noted throughout the course of the experiment. The possibility that subculturing procedures affected AChE activity was also investigated. Incubation of stationary phase cells with trypsin resulted in a 30% decrease in cell protein and in AChE activity; thus the specific activity of AChE remained constant. In other experiments not shown here, stationary phase cells were dissociated with trypsin and then plated at the original cell con- centration and incubated for 2 days. No change in AChE specific activity was detected throughout the period of incubation. In addition, two cell populations were maintained separately in logarithmic growth by subculturing every other day for 10 days. One population was subcultured by the trypsin procedure, the ‘other with modified D,. Cells then were incubated for 17 days. Values ob- tained with cells treated with trypsin did not differ significantly from those obtained with cells passaged with modified D, shown in Fig. 1. The results suggests that AChE, but not catechol-O-methyl-transferase, responds to a regula- tory mechanism that is coupled to the rate of cell division. Neuroblastoma cells stop dividing in the absence of serum but continue to Vou. 67, 1970 NEUROBLASTOMA ACETYLCHOLINESTERASE 789 synthesize protein.’ If AChE activity and cell division are inversely related, then removal of serum from the growth medium should result in an increase in AChE activity. The effect of serum concentration on AChE activity and cell growth is shown in ]‘ig. 2. In the presence of serum, cell growth was logarithmic and AChE specific activity remained constant (Fig. 2A). In the absence of serum, the cell population quadrupled within 2 days and then remained constant. When cell division was restricted, a marked increase in AChE specific activity was observed (Fig. 2B). In this experiment, the cell concentration at the time that AChE specific activity increased was 31,000 cells/cm? compared with 200,000 in the experiment illustrated in Fig. 1. Thus regulation of AChE activity is not a function of cell concentration under these conditions. 2 io T T T T T T T T T t r T T T T xr 5 A SERUM B NO SERUM C SERUM ON 3rd DAY ai e t 30 + 600 & 100440 r a } AChE AChE > SP. ACT. = S eot32 = - 13 PROTEIN Z 941 4802 g ce « 2 a = a a ceLts w N ae = g 8 iI & 18 +3600 W 60724 4 + ia 9 i ~ oO tu o a PROTEIN © iG 4 a 4 + 124+240£ # 407 16 a CELLS ACHE Q us AChE g ° 1 S 20+ 8 ACHE 6+ 120M a AChE ra wn 4 —@SP ACT cELLs SP ACT. a a yg O",__PROTEWN | o 2 2 , es 4 5°70 1 @¢3 4 5 °°3 456 7 c DAYS Fic. 2. The rate of cell division was regulated by adjusting the serum concentration. Neuroblastoma cells were incubated for 24 hr prior to sero time in 150-mm dishes containing the medium described, except that horse serum was omitted. At zero time the medium was removed and cells were washed once with growth medium; then fresh medium with or without 10% fetal calf serum was added as specified in panel A and B, respectively. Some cells were incubated for 3 days without serum, as shown in panel B; nondividing cells were then shifted up to the rapidly dividing state by the addition of 10% fetal calf serum (panel C). The me- dium was changed and fresh medium containing 10% fetal calf serum was added at 4 and 5.5 days. Symbols correspond to the following: @, AChE specific activity; O, AChE ac- tivity/dish; A, cells (X10-*)/dish; 0, mg protein/dish. Cells were incubated without serum for 3 days (Fig. 2B); serum was added on the 8rd day to shift cells from stationary to logarithmic growth without subculturing them (Fig. 2C). A decrease in AChE specific activity resulted. The results again show that regulation of AChE activity is coupled to cell division and is not a consequence of subculturing procedures. Cell division was also restricted by incubating cells with 2 mM thymidine in the presence of serum and growth medium. An increase in AChE specific activity was found (data not shown). The possibility that the changes in AChE activity were due to activation or inhibition of the enzyme was investigated by mixing homogenates prepared from cells in the logarithmic and stationary phase of growth. Acetylcholinesterase activity was additive; thus no enzyme effector was detected. The relation between protein synthesis and AChE regulation was studied by determining the effect of cycloheximide upon AChE activity, cell viability, and 790 BIOCHEMISTRY: BLUME ET AL. Proc. N. A. S. Z T T T T ka 5 sofA ACh & q!20 2 a 5 fi0oo § = = ra 480 wi < ‘= 3 4 60 3 Fie. 3. Neuroblastoma cells were incubated — —. for 2 days without serum, as described in the +? 440 +° legend to Fig. 2, to restrict cell division and in- ars “z erease AChE activity. Symbols denote the Ww j20 “ following: Open symbols, no cycloheximide; 2 S closed symbols, 3.5 x 10-§ M cycloheximide © oh} +-++4+ o * added at zero time (48 hr after removal of B CELL VIABILITY serum). In panel A, AChE specific activity and AChE: activity/dish are shown both in the presence and absence of cycloheximide. In panel B the percent of cells that were viable and 1 mg of protein/dish are shown, both in the presence and absence of cycloheximide. Each dish contained 4.0 « 108 cells, denoted on the ordinate as 100%. The number of cells/dish did not change significantly during the course of the experiment. %NIABLE CELLS C PROTEIN 2 -CH 4 +CH MG PROTEIN/DISH Oo 6 (2 18 24 HOURS protein (Fig. 34, B, and C respectively). Cells were incubated without serum for 48 hr before cycloheximide was added. In the absence of cycloheximide, AChE specific activity and units/dish increased 2.5-fold within 24 hr; in the presence of the drug, AChE specific activity and units decreased. The amount of protein and number of viable cells per dish also declined in the presence of cycloheximide. The results suggest that the increase in AChE activity is dependent upon protein synthesis but further data are needed to substantiate this possibility. Properties of neuroblastoma AChE: A number of enzymes catalyze acetyl- choline hydrolysis, including AChE, cholinesterase (EC 3.1.1.8), and acetyl- esterase (EC 3.1.1.6). The enzymes differ from one another in relative activity towards various substrates and in sensitivity to inhibitors. The effects of selec- tive esterase inhibitors on AChE from stationary phase cells are shown in Fig. 4. Acetylcholine hydrolysis was inhibited 50% in the presence of the following compounds: 1 X 10-8 M diisopropyl fluorophosphate, an inhibitor of AChE, cholinesterase, and carboxylesterase (EC 3.1.1.1), but not arylesterase (EC 3.1.1.2); 6 X 10-§ M 1,5-bis-(4-allyidimethylammoniumpheny]) pentane-1,3- dibromide (BW284C51), a more potent inhibitor of AChE than cholinesterase; 7 X 10-7 M neostigmine sulfate, a more potent inhibitor of AChE or cholines- terase than arylesterase or carboxylesterase; and 4 X 10~‘ M tetramonoisopropy] Vou. 67, 1970 NEUROBLASTOMA ACETYLCHOLINESTERASE 791 Fic. 4. Effects of esterase inhibitors on AChE 1 Ne from stationary phase neuroblastoma cells. The INHIBITION OF AChE dotted lines correspond to reactions with a final volume of 0.1 ml, containing the components de- scribed and 12.3 pg homogenate protein/reaction, that were incubated at 37°C for 15 min. Abbrevia- tions are as follows: DFP, diisopropylfluorophos- phate; BW, = 1,5-bis(4-allyldimethylammonium- pheny!)pentane-!,3-dibromide; NS, neostigmine sulfate; DPDA, tetramoncisopropyl pyrophosphor- tetramide; NEM, N-ethylmaleimide. The solid lines correspond to reactions incubated 180 150 120 30 60 430 % MAXIMAL VELOCITY ae 6 in two stages. Stage 1 reactions contained the OW" FS dae . : : owoKn eo ow oe components described in Materials and Methods MOLARITY NMOLES ACETATE FORMED/MIN/MG PROTEIN minus [4C]acetylcholine, the esterase inhibitors indicated at 5 times the molarity shown on the abscissa, and 12.3 ug of homogenate protein in a final volume of 20 ul. Stage 1 reactions were incubated for 10 min at 37°C, then placed in an ice bath. Stage 2 reactions were prepared by the addition of 4.13 mM ["C]acetylcholine iodide and other reaction components de- scribed so that the final volume of each reaction was 0.1 ml. The final molarity of esterase inhibitors corresponded to that shown on the abscissa. Stage 2 reactions were incubated 15 min at 37°C, pyrophosphortetramide, a more potent inhibitor of cholinesterase than AChE. N-Ethylmaleimide had relatively little effect on the hydrolysis of acetylcholine even at 1 X 10-2 M. In addition, the relative rates of hydrolysis of [*C]- acetylcholine and [!C]butyrylcholine were determined; butyrylcholine hydroly- sis was 1% that of acetylcholine. Although definitive characterization awaits the analysis of purified enzyme fractions, the neuroblastoma enzyme appears to be AChE (EC 3.1.1.7). Discussion. The results show that neuroblastoma acetylcholinesterase ac- tivity is regulated and that the regulatory mechanism is inversely related to the rate of cell division. Acetylcholinesterase specific activity increased about 25- fold when the rate of cell division was restricted. Under the same conditions, the specific activity of catechol-O-methyl transferase remained relatively con- stant. Acetylcholinesterase activity in stationary phase neuroblastoma cells was approximately twice that of mammalian brain or erythrocytes, both known to contain relatively high levels of the enzyme. Studies with neuroblastoma cells revealed that the shift from the dividing to the nondividing state influences the expression of a set of characteristics required for neuron differentiation. Thus far, five properties have been found to increase upon restriction of neuroblastoma cell division: acetylcholinesterase activity, protein per cell, axon-dendrite development, formation of electrically active membranes, and synthesis of acetylcholine receptors. Neuroblastoma cells are still committed to a program of neuron differentiation even though thousands of cell generations have elapsed since the tumor orig- inated. Commitment to the program may be thought of as the initiating event in the process of differentiation. Once initiated, the progeny apparently do not terminate such a program. Although the mechanism of initiation is not known, the stability of the phe- nomenon suggests that it is not fully reversible, It should then be relatively 792 BIOCHEMISTRY: BLUME ET AL. Proc. N. A. S. easy to explore the possibility that normal sympathetic neuroblasts are also irreversibly committed to a pattern of neuronal differentiation. We acknowledge the invaluable contributions of Drs. A. Gilman and T. Amano and the help of Mrs. H. Carpenter in the preparation of this manuscript. Abbreviation: AChE, acetylcholinesterase. + NIH Postdoctoral fellow (HE 29,138-02). 1 Augusti-Tocco, G., and G. Sato, Proc. Nat. Acad. Sci. USA, 64, 311 (1969). 2 Schubert, D., 8S. Humphreys, C. Baroni, and M. Cohn, Proc. Nat. Acad, Set. USA, 64, 31 (1969). 3 Olmsted, J. B., K. Carlson, R. Klebe, F. Ruddle, and J. Rosenbaum, Proc. Nat. Acad. Sct. USA, 65, 129 (1970). ‘Nelson, P., W. Ruffner, and M. Nirenberg, Proc. Nat. Acad. Sci. USA, 64, 1004 (1969). 5 Seeds, N. W., A. G. Gilman, T. Amano and M. W. Nirenberg, Proc. Nat. Acad. Sct. USA, 66, 160 (1970). ‘ Harris, A. J.. and M. J. Dennis, Science, 167, 1253 (1970). 7 Nelson, P. G., J. H. Peacock, T. Amano, and J. Minna, in preparation. 8 Nelson, P., J. H. Peacock, and T. Amano, in preparation. ® Burkhalter, A., R. M. Featherstone, F. W. Schueler, and M. Jones, J. Pharmacol., 120, 285 (1958). Goodwin, B. C., and I. W. Sizer, Develop. Biol., 11, 136 (1965). 11 A preliminary report of these findings has been published. Blume, A. J., and F. Gilbert, Fed. Proc., 29, 861 (1970). 2 Ham, G. R., and T. T. Puck, in Methods in Enzymology, eds. 8. P. Colowick and N. O. Kaplan (New York: Academic Press, 1962), vol. 5, pp. 90-119. 13 Phillips, H. J., and J. E. Terryberry, Hap. Cell Res., 13, 341 (1957). “Lowry, O. H., N. F. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 6 Nikodejevic, B., S. Senoh, J. W. Daly, and C. R. Creveling, J. Pharmacol., in press. 6 Austin, L., and W. K. Berry, Biochem. J., 54, 695 (1953). ” Reed, D. J., K. Goto, and C. H. Wang, Anal. Biochem., 16, 59 (1966).