Neurochemical Research, Vol. 4, No. 6, 1979
PHOSPHORUS-31 NMR OF NEUROBLASTOMA CLONAL LINES Effect of Cell Confluency State and Dibutyryl Cyclic AMP JAY W. PETTEGREW, t THOMAS GLONEK, 2 FRED BASKIN, 3 AND ROGER N . ROSENBERG 3 1Department of Neurology and Pediatrics University of Texas Health Science Center, Dallas, Texas, 75235 2Research Resources Center, University of Illinois Medical Center, Chicago, Illinois, 60612 3Department of Neurology University of Texas Health Science Center, Dallas, Texas, 75235
Accepted May 7, 1979
Phosphorus nuclear magnetic resonance is used to study changes in the levels of the major phosphate-containing intermediary metabolites concomitant with induced cellular differentiation in the N-18 and C-46 neuroblastoma clonal lines. The study reveals differences between the 31p-NMR profiles of the two clonal lines and also striking differences attendant to dibutyryI cAMP-mediated morphological differentiation in the N-18 clone. Phosphorus-31 NMR would appear to provide a new technique with which to study genetic differentiation.
INTRODUCTION The genetically determined program of neuronal differentiation remains incompletely defined. In an attempt to gain further understanding of this process, the major phosphorus containing metabolites of murine neuroblastoma clonal lines N-18 and C-46 were simultaneously analyzed and quantitated by the technique of phosphorus-31 nuclear magnetic resonance (31p-NMR). The N-18 clonal line has been shown previously, in 795 0364-3190/79/1200-0795503.00/0 9 1979 Plenum Publishing Corporation
796
PETTEGREW ET AL.
this and other laboratories, to differentiate, as evidenced by neurite formation, when grown in the presence of dibutyryl cyclic AMP (db-cAMP) (I, 2), and observed in the more slowly dividing confluent state (3, 4). That is, there is an increase (from 20 to 80%) in the fraction of cells with greater than 30 p~m long processes. In contrast, the C-46 clonal line does not morphologically change to any significant degree under either of these conditions. This study investigates the 31p-NMR profiles of cytoplasmic metabolites of these two neuroblastoma clonal lines as a function of the cell confluency stage and the presence or absence of the differentiating agent db-cAMP. The analytical method employed (31p-NMR) yields spectroscopic profiles of the principal intracellular phosphate metabolic pools which in turn reflect the cellular metabolic state (for reviews see references 5 and 6). To date, various phosphorus-containing inorganic and organic molecules have been measured in cellular organelles, whole cells, tissues, and perfused organs. In addition it has been unequivocably demonstrated that metabolites quantified by 3~p-NMR agree with conventional analytical methods (7, 8). Marked alterations in the 3Jp-NMR profiles attendant to morphological differentiation are documented in the present study providing a new, sensitive, and more comprehensive approach to cellular differentiation.
EXPERIMENTAL PROCEDURE Mouse neuroblastoma clones N-18 and C-46 were grown in monolayer culture as previously described (1, 9). Confluent cells were trypsinized and 6 x 106 cells were replated in 150-mm Falcon tissue culture dishes and grown in Dulbecco's modified Eagle's medium H21 (Grand Island Biological Co.) with 10% fetal calf serum, 200 p~g/ml kanamycin, 125 mg/ ml Spectinomycin at 37~ with 90% air, 10% CO2, and 100% humidity. In the db-cAMPtreated cells, 1.0 mM db-cAMP was included in the medium at the time the cells were plated, and medium and db-cAMP were changed every day. The logarithmic growth phase was defined as a phase of approximately 30-50% cellular confluency (18-24 x 106 cells/150-mm plate) and active cellular division as determined by phase microscopy. The confluent cells were harvested at 90-100% confluency (>60 • 106 cells/150-mm plate) as determined by phase microscopy. Neurite formation, one marker of morphological differentiation, was only observed in the N-18 cells at 90-100% confluency when grown in the presence of 1.0 mM db-cAMP (1-4). To eliminate catabolism of metabolites during extraction from control and experimental cells, 20 confluent plates of cells, first rinsed twice at 4~ with 10.0 ml of 0.85% sodium chloride, were gently scraped directly into 0.1 volume of 60-62% perchloric acid at 4~ As one control, cells from additional 150-mm dishes grown with or without dibutyryl cAMP and harvested as described above, were resuspended in culture media and reincubated. After 48 hr, phase microscopy, accompanied by a quantitation of unattached cells with a Coulter ZB~ cell counter, revealed healthy attached cells which were approximately
PHOSPHORUS-31 NMR OF NEUROBLASTOMA CLONAL LINES
797
75% confluent. The absence of lysed or floating cells in this culture medium corresponds to a plating efficiency greater than 98%. This served as a check on the physiological integrity of the rinsed cells at the stage of the perchloric acid extraction procedure. The perchloric acid'extract was centrifuged at 17,300 g for l0 rain at - 4~ The supernatant was alkalinized to pH 10.0-11.0 with 10 N potassium hydroxide with care taken to avoid warming. The potassium perchlorate was removed by centrifugation at 17,300 g for l0 rain at -4~ The supernatant was lyophilized at -60~ to dryness. The resulting light brown powder was solubilized with the minimal amount of 4~ distilled deionized water (3.0 ml) and placed drop wise on a 1 x 5-cm Na-chelex-100 column. The eluent and 10.0 ml of distilled deionized water washings, pH 10.5, were lyophilized to dryness at - 60~ and stored at -70~ until the NMR spectra were run. The experiments were repeated 8 separate times. The reproducibility of all peaks was _+5% and the error in the peak aeras -+ 1.5%.
R E S U L T S AND DISCUSSION Both confluency and growth in the presence of db-cAMP have been previously shown to additively increase intracellular cAMP levels and the percentage of N-18 cells bearing long neurites (1-4). We contrast here the two extremes in differentiated state: cells subject to neither differentiation-promoting condition with cells subject to both conditions. The results are shown in Figure 1 and pertinent phosphorus NMR parameters are tabulated in Table I. Three points deserve particular attention. The first is the 3~p-NMR profile of the N-18 clone which greatly varies as a function of cellular differentiation as shown by comparing cells grown without added db-cAMP and harvested in the log phase to cells grown in the presence of db-cAMP and harvested in the confluent phase. The second point is that similar changes are not observed for the C-46 clone which does not morphologically differentiate under the conditions of this study. The third point is that the 3~P-NMR profile of C-46 differs from that of N-18 under both culture conditions. More specifically for the N-18 clone, the process of differentiation is associated with a decrease in another nucleotide monophosphate (NMP) from 17.5% to undetectable levels, a 17.4% decrease in phosphodiester (PDE), and smaller decreases in the percentages of phosphorylcholine (PC), glycerol 3-phosphorylethanolamine (GPE), and glycerol 3-phosphorylcholine (GPC) (2.1%, 0.9%, and 0.7%, respectively). Similarly there is a 10.4% increase in di(pyro)phosphodiester (DPDE), a 6.2% increase in Pyro, and a 2.2% increase in anomeric sugar phosphate (ASP) (Table I). Conversely the C-46 clone which does not morphologically differentiate under the conditions of this study demonstrates only trace amounts of NMP but large amounts (23.5-28.6%) of DPDE under both conditions. Comparing cells cultured without added db-cAMP and harvested in the
798
P E T T E G R E W ET AL.
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,
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I
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799
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PETTEGREW ET AL.
A
B
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FIG. 1. (A) N-18 clone cultured without added db-cAMP and harvested in log phase. (B) N-18 clone cultured with 1,0 mM db-cAMP and harvested in confluent phase, (C) C-46 clone cultured without added db-cAMP and harvested in log phase. (D) C-46 clone cultured with 1.0 mM db-cAMP and harvested in confluent phase. The NMR spectrometer employed was a Bruker HFX-5 with 2D stabilization operating at 36.43 MHz for 3~p (21 kG magnetic field, ~H frequency 90.00 MHz). Time constant used was - 3 which equals 1.2 Hz broadening/ Dine; sweep width = 2500 Hz with 4096 data points in the FID; all spectra are proton broadband decoupled. Transients: spectrum A, (30,615); B, (71,535); C, (120,878); D, (44,644). Na~EDTA (approx. 25 mg/l.0 ml sample) was added.
PHOSPHORUS-31 NMR OF NEUROBLASTOMA CLONAL LINES
801
log phase to cells grown in the presence of db-cAMP and harvested in the confluent phase, there is a 1.7% decrease in PC, a 0.5% decrease in PDE, and increases of 1.1% and 1.5% in ASP and Pyro, respectively. Comparing the N-18 and C-46 clonal lines, the most striking differences are the presence of changes in the N-18 31p-NMR profiles as a function of differentiation, and the absence of similar changes in the C-46 clone. Also prominent is a large percent of NMP and PDE in the N-18 cells cultured without added db-cAMP and harvested in the log phase and only trace amounts of these peaks in the C-46 cells cultured and harvested under both conditions. Other significant differences include the higher percentage of DPDE in the C-46 cells cultured and harvested under both conditions, and the higher percentage of Pyro in the N-18 cells in the presence of db-cAMP and harvested in the confluent stage. In summary, the 3~P-NMR analysis of two neuroblastoma clonal lines (N-18 and C-46) which differ in their ability to morphologically differentiate have been analyzed by 3~p-NMR. The study reveals differences in the 3Jp-NMR profiles of the two clonal lines and also striking differences attendant to differentiation in the N-18 clone. The C-46 clone which does not morphologically differentiate under the conditions of this study did not reveal similar 3~p-NMR changes. Preliminary experiments analyzing the separate contributions of confluency and the addition of 1.0 mM dbcAMP to N-18 cells suggest that only confluent cultures grown with dbcAMP display these significant shifts in their 3Jp metabolite profile.
REFERENCES 1. PRASHAD, N., WISCHMEYER, B., EVETTS, C., BASKIN, F., and ROSENBERG, R. N. 1977. Identification and quantification of proteins synthesized by neuroblastoma cells during differentiation. Cell Diff. 6:147-157. 2. PRASHAD, K. N., and KUMAR, S. from "Control of Proliferation in Animal Cells" pp. 581-594, 1974, Cold Spring Harbor. 3. SEEDS, N. W., GILMAN, A. G., AMANO, T., and NIRENBERG, M. W. 1970. Regulation ~ of axon formation by clonal lines of a neural tumor. Proc. Natl. Acad. Sci. U.S.A. 66:160. 4. SEEDS, N. W., and MACCIONI, R. B. 1978. Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization. J. Cell. Biol. 76:547-555. 5. BURT, C. T., GLONEK, T., and BARANY, M. 1977. Analysis of living tissue by phosphorus-31 magnetic resonance. Science 195:145-149. 6. DWEK, R. A., CAMPBELL, I. D. RtCHARDS, R. E., and WmLIAMS, R. J. P. (eds.). 1977. NMR in Biology, 372 pp., Academic Press, New York. 7. HOULT, D. F., BUSBY, S. J. W., GADIAN, D. G., RADDA, G. K., RICHARDS, R. E., and SEELEY, P. J. 1974. Observations of tissue metabolites using 31p nuclear magnetic resonance. Nature 252:285-287.
802
PETTEGREW ET AL.
8. BURT, C., GLONEK, T., and BARANY, M. 1976. Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. J. Biol. Chem. 251:2584-2591. 9. BASKIN, F., CARLIN, S. C., KRAUS, P., FRIEDKIN, M., and ROSENBERG; R. N. 1975. Experimental chemotherapy of neuroblastoma, lI. Increased thymidylate synthetase activity in a 5-fluorodeoxyuridine resistant variant of mouse neuroblastoma. Mol. Pharmacol. 11:105-117.
PHOSPHORUS-31 NMR OF NEUROBLASTOMA CLONAL LINES Effect of Cell Confluency State and Dibutyryl Cyclic AMP JAY W. PETTEGREW, t THOMAS GLONEK, 2 FRED BASKIN, 3 AND ROGER N . ROSENBERG 3 1Department of Neurology and Pediatrics University of Texas Health Science Center, Dallas, Texas, 75235 2Research Resources Center, University of Illinois Medical Center, Chicago, Illinois, 60612 3Department of Neurology University of Texas Health Science Center, Dallas, Texas, 75235
Accepted May 7, 1979
Phosphorus nuclear magnetic resonance is used to study changes in the levels of the major phosphate-containing intermediary metabolites concomitant with induced cellular differentiation in the N-18 and C-46 neuroblastoma clonal lines. The study reveals differences between the 31p-NMR profiles of the two clonal lines and also striking differences attendant to dibutyryI cAMP-mediated morphological differentiation in the N-18 clone. Phosphorus-31 NMR would appear to provide a new technique with which to study genetic differentiation.
INTRODUCTION The genetically determined program of neuronal differentiation remains incompletely defined. In an attempt to gain further understanding of this process, the major phosphorus containing metabolites of murine neuroblastoma clonal lines N-18 and C-46 were simultaneously analyzed and quantitated by the technique of phosphorus-31 nuclear magnetic resonance (31p-NMR). The N-18 clonal line has been shown previously, in 795 0364-3190/79/1200-0795503.00/0 9 1979 Plenum Publishing Corporation
796
PETTEGREW ET AL.
this and other laboratories, to differentiate, as evidenced by neurite formation, when grown in the presence of dibutyryl cyclic AMP (db-cAMP) (I, 2), and observed in the more slowly dividing confluent state (3, 4). That is, there is an increase (from 20 to 80%) in the fraction of cells with greater than 30 p~m long processes. In contrast, the C-46 clonal line does not morphologically change to any significant degree under either of these conditions. This study investigates the 31p-NMR profiles of cytoplasmic metabolites of these two neuroblastoma clonal lines as a function of the cell confluency stage and the presence or absence of the differentiating agent db-cAMP. The analytical method employed (31p-NMR) yields spectroscopic profiles of the principal intracellular phosphate metabolic pools which in turn reflect the cellular metabolic state (for reviews see references 5 and 6). To date, various phosphorus-containing inorganic and organic molecules have been measured in cellular organelles, whole cells, tissues, and perfused organs. In addition it has been unequivocably demonstrated that metabolites quantified by 3~p-NMR agree with conventional analytical methods (7, 8). Marked alterations in the 3Jp-NMR profiles attendant to morphological differentiation are documented in the present study providing a new, sensitive, and more comprehensive approach to cellular differentiation.
EXPERIMENTAL PROCEDURE Mouse neuroblastoma clones N-18 and C-46 were grown in monolayer culture as previously described (1, 9). Confluent cells were trypsinized and 6 x 106 cells were replated in 150-mm Falcon tissue culture dishes and grown in Dulbecco's modified Eagle's medium H21 (Grand Island Biological Co.) with 10% fetal calf serum, 200 p~g/ml kanamycin, 125 mg/ ml Spectinomycin at 37~ with 90% air, 10% CO2, and 100% humidity. In the db-cAMPtreated cells, 1.0 mM db-cAMP was included in the medium at the time the cells were plated, and medium and db-cAMP were changed every day. The logarithmic growth phase was defined as a phase of approximately 30-50% cellular confluency (18-24 x 106 cells/150-mm plate) and active cellular division as determined by phase microscopy. The confluent cells were harvested at 90-100% confluency (>60 • 106 cells/150-mm plate) as determined by phase microscopy. Neurite formation, one marker of morphological differentiation, was only observed in the N-18 cells at 90-100% confluency when grown in the presence of 1.0 mM db-cAMP (1-4). To eliminate catabolism of metabolites during extraction from control and experimental cells, 20 confluent plates of cells, first rinsed twice at 4~ with 10.0 ml of 0.85% sodium chloride, were gently scraped directly into 0.1 volume of 60-62% perchloric acid at 4~ As one control, cells from additional 150-mm dishes grown with or without dibutyryl cAMP and harvested as described above, were resuspended in culture media and reincubated. After 48 hr, phase microscopy, accompanied by a quantitation of unattached cells with a Coulter ZB~ cell counter, revealed healthy attached cells which were approximately
PHOSPHORUS-31 NMR OF NEUROBLASTOMA CLONAL LINES
797
75% confluent. The absence of lysed or floating cells in this culture medium corresponds to a plating efficiency greater than 98%. This served as a check on the physiological integrity of the rinsed cells at the stage of the perchloric acid extraction procedure. The perchloric acid'extract was centrifuged at 17,300 g for l0 rain at - 4~ The supernatant was alkalinized to pH 10.0-11.0 with 10 N potassium hydroxide with care taken to avoid warming. The potassium perchlorate was removed by centrifugation at 17,300 g for l0 rain at -4~ The supernatant was lyophilized at -60~ to dryness. The resulting light brown powder was solubilized with the minimal amount of 4~ distilled deionized water (3.0 ml) and placed drop wise on a 1 x 5-cm Na-chelex-100 column. The eluent and 10.0 ml of distilled deionized water washings, pH 10.5, were lyophilized to dryness at - 60~ and stored at -70~ until the NMR spectra were run. The experiments were repeated 8 separate times. The reproducibility of all peaks was _+5% and the error in the peak aeras -+ 1.5%.
R E S U L T S AND DISCUSSION Both confluency and growth in the presence of db-cAMP have been previously shown to additively increase intracellular cAMP levels and the percentage of N-18 cells bearing long neurites (1-4). We contrast here the two extremes in differentiated state: cells subject to neither differentiation-promoting condition with cells subject to both conditions. The results are shown in Figure 1 and pertinent phosphorus NMR parameters are tabulated in Table I. Three points deserve particular attention. The first is the 3~p-NMR profile of the N-18 clone which greatly varies as a function of cellular differentiation as shown by comparing cells grown without added db-cAMP and harvested in the log phase to cells grown in the presence of db-cAMP and harvested in the confluent phase. The second point is that similar changes are not observed for the C-46 clone which does not morphologically differentiate under the conditions of this study. The third point is that the 3~P-NMR profile of C-46 differs from that of N-18 under both culture conditions. More specifically for the N-18 clone, the process of differentiation is associated with a decrease in another nucleotide monophosphate (NMP) from 17.5% to undetectable levels, a 17.4% decrease in phosphodiester (PDE), and smaller decreases in the percentages of phosphorylcholine (PC), glycerol 3-phosphorylethanolamine (GPE), and glycerol 3-phosphorylcholine (GPC) (2.1%, 0.9%, and 0.7%, respectively). Similarly there is a 10.4% increase in di(pyro)phosphodiester (DPDE), a 6.2% increase in Pyro, and a 2.2% increase in anomeric sugar phosphate (ASP) (Table I). Conversely the C-46 clone which does not morphologically differentiate under the conditions of this study demonstrates only trace amounts of NMP but large amounts (23.5-28.6%) of DPDE under both conditions. Comparing cells cultured without added db-cAMP and harvested in the
798
P E T T E G R E W ET AL.
ll?Ti
,
z
44~
I
,t
~
o
~
O ~,..O ~ ~ IZl .
,.
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CLONAL LINES
799
800
PETTEGREW ET AL.
A
B
i I 15.0 10,0
i 5,0
i I 0,0 -5,0 PPM
i ,,I I -10.0-'15.0-20.0-25,0
i
FIG. 1. (A) N-18 clone cultured without added db-cAMP and harvested in log phase. (B) N-18 clone cultured with 1,0 mM db-cAMP and harvested in confluent phase, (C) C-46 clone cultured without added db-cAMP and harvested in log phase. (D) C-46 clone cultured with 1.0 mM db-cAMP and harvested in confluent phase. The NMR spectrometer employed was a Bruker HFX-5 with 2D stabilization operating at 36.43 MHz for 3~p (21 kG magnetic field, ~H frequency 90.00 MHz). Time constant used was - 3 which equals 1.2 Hz broadening/ Dine; sweep width = 2500 Hz with 4096 data points in the FID; all spectra are proton broadband decoupled. Transients: spectrum A, (30,615); B, (71,535); C, (120,878); D, (44,644). Na~EDTA (approx. 25 mg/l.0 ml sample) was added.
PHOSPHORUS-31 NMR OF NEUROBLASTOMA CLONAL LINES
801
log phase to cells grown in the presence of db-cAMP and harvested in the confluent phase, there is a 1.7% decrease in PC, a 0.5% decrease in PDE, and increases of 1.1% and 1.5% in ASP and Pyro, respectively. Comparing the N-18 and C-46 clonal lines, the most striking differences are the presence of changes in the N-18 31p-NMR profiles as a function of differentiation, and the absence of similar changes in the C-46 clone. Also prominent is a large percent of NMP and PDE in the N-18 cells cultured without added db-cAMP and harvested in the log phase and only trace amounts of these peaks in the C-46 cells cultured and harvested under both conditions. Other significant differences include the higher percentage of DPDE in the C-46 cells cultured and harvested under both conditions, and the higher percentage of Pyro in the N-18 cells in the presence of db-cAMP and harvested in the confluent stage. In summary, the 3~P-NMR analysis of two neuroblastoma clonal lines (N-18 and C-46) which differ in their ability to morphologically differentiate have been analyzed by 3~p-NMR. The study reveals differences in the 3Jp-NMR profiles of the two clonal lines and also striking differences attendant to differentiation in the N-18 clone. The C-46 clone which does not morphologically differentiate under the conditions of this study did not reveal similar 3~p-NMR changes. Preliminary experiments analyzing the separate contributions of confluency and the addition of 1.0 mM dbcAMP to N-18 cells suggest that only confluent cultures grown with dbcAMP display these significant shifts in their 3Jp metabolite profile.
REFERENCES 1. PRASHAD, N., WISCHMEYER, B., EVETTS, C., BASKIN, F., and ROSENBERG, R. N. 1977. Identification and quantification of proteins synthesized by neuroblastoma cells during differentiation. Cell Diff. 6:147-157. 2. PRASHAD, K. N., and KUMAR, S. from "Control of Proliferation in Animal Cells" pp. 581-594, 1974, Cold Spring Harbor. 3. SEEDS, N. W., GILMAN, A. G., AMANO, T., and NIRENBERG, M. W. 1970. Regulation ~ of axon formation by clonal lines of a neural tumor. Proc. Natl. Acad. Sci. U.S.A. 66:160. 4. SEEDS, N. W., and MACCIONI, R. B. 1978. Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization. J. Cell. Biol. 76:547-555. 5. BURT, C. T., GLONEK, T., and BARANY, M. 1977. Analysis of living tissue by phosphorus-31 magnetic resonance. Science 195:145-149. 6. DWEK, R. A., CAMPBELL, I. D. RtCHARDS, R. E., and WmLIAMS, R. J. P. (eds.). 1977. NMR in Biology, 372 pp., Academic Press, New York. 7. HOULT, D. F., BUSBY, S. J. W., GADIAN, D. G., RADDA, G. K., RICHARDS, R. E., and SEELEY, P. J. 1974. Observations of tissue metabolites using 31p nuclear magnetic resonance. Nature 252:285-287.
802
PETTEGREW ET AL.
8. BURT, C., GLONEK, T., and BARANY, M. 1976. Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. J. Biol. Chem. 251:2584-2591. 9. BASKIN, F., CARLIN, S. C., KRAUS, P., FRIEDKIN, M., and ROSENBERG; R. N. 1975. Experimental chemotherapy of neuroblastoma, lI. Increased thymidylate synthetase activity in a 5-fluorodeoxyuridine resistant variant of mouse neuroblastoma. Mol. Pharmacol. 11:105-117.
