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Differences between murine C1300 neuroblastoma clones detected by rosette formation with nerve growth factor-coated sheep red blood cells
Brain Research, 118 (1976) 453-459
453
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
D I F F E R E N C E S B E T W E E N M U R I N E C1300 N E U R O B L A S T O M A C L O N E S D E T E C T E D BY R O S E T T E F O R M A T I O N W I T H N E R V E G R O W T H F A C T O R - C O A T E D S H E E P R E D B L O O D CELLS
LEILA DIAMOND*, ROBERTO REVOLTELLA and LUISA BERTOLINI C.N.R. Laboratory of Cell Biology, Rome (Italy)
(Accepted April 20th, 1976)
SUMMARY The expression of receptors for nerve growth factor ( N G F ) on the cell surface was assayed by rosette formation with ligand-coated sheep red blood cells (SRBC). Cell clones derived from the murine C1300 neuroblastoma and from hybrids between a neuroblastoma clone and L cell clones showed a wide variation in the capacity to form rosettes with N G F - c o a t e d SRBC. All the neuroblastoma, L cell and hybrid clones formed rosettes with phytohemagglutinin-coated SRBC and none formed rosettes with cytochrome c- or ferritin-coated SRBC or with SRBC not coated with ligand.
INTRODUCTION Nerve growth factor ( N G F ) is a protein that enhances the growth and differentiation of sympathetic nerve cells in vivo and in vitro 8. Cell stimulation during N G F treatment of newborn mice is characterized by a marked increase in metabolic processes, including a selective induction of some of the key enzymes in the catecholamine biosynthetic pathway9,14. Recently, we demonstrated by rosette formation and by radioimmunoassay that cells of the murine C1300 neuroblastoma, a spontaneous tumor of sympathetic neurons of strain A mice 4, bind the N G F molecule 11-13. Rosettes are formed when N G F coated sheep red blood cells (SRBC) react at 2 °C with receptors exposed on the cell surface membrane and adhere to the tumor cells11,13. This phenomenon does not occur with normal strain A fibroblasts or with other tumor cells such as L cells and
* Permanent address: The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. 19104 (U.S.A.)
454 $37 sarcoma cells. We now report a segregation of this cell surface marker in several clones derived from the C1300 neuroblastoma and in clones derived from hybrids between a neuroblastoma clone positive for rosette formation and L cell clones. The study demonstrates the great variation among clones in the expression of membrane receptors for NGF. MATERIALS AND METHODS
Celh" A cell line (NB) derived from the murine C1300 neuroblastoma was obtained from Dr. F. Jacob, Institut Pasteur, and several clones (NB/R series) were isolated from it. The characterization of these clones which differ from each other in growth properties, morphology and the expression of neuron-specific properties will be reported elsewhere 1. Clone NA, a hypoxanthine-guanine phosphoribosyl-transferasedeficient subclone isolated by R. J. Klebe from clone Neuro-2a of C1300, was kindly supplied by Dr. F. Arthur McMorris, Wistar Institute. The 3 fibroblast clones studied were: L cells (NCTC clone 929), derived from subcutaneous tissue of adult C3H mice, and two L cell clones deficient in thymidine kinase, L M ( T K - ) 5 and L M ( T K - ) l D 3. The three hybrid [NA × LM(TK-)] clones of the NL-I series were supplied by Dr. McMorris; their expression of some neuronal phenotypes has been described 6,7,a0. Hybrid [NA × L M ( T K - ) I D ] clones of the 54-31 series were isolated and supplied by Dr. Carlo Croce, Wistar Institute. The hybrid nature of these clones was confirmed by karyotype and isozyme (glucese phosphate isomerase) analysis; these and other properties of the clones will be described elsewhere a. NB and NB/R cells were maintained as suspension cultures. They were grown in bacteriological Petri plates in Dulbecco's modified Eagle's medium (EDM) supplemented with 2 0 ~ fetal bovine serum (FBS) in an atmosphere of 12~o CO2 in air. When transferred to tissue culture or glass Petri plates, these cells attach to the surface and extend cytoplasmic processes of varying lengths. All other clones were maintained as monolayer cultures. They were grown in Eagle's minimum essential medium supplemented with 1 0 ~ FBS in an atmosphere of 5 ~ CO2 in air. NA cells had a 'neuronal' morphology with many cells showing cytoplasmic processes. Two of the hybrid clones (NL-I-7A and NL-]-8) had a similar morphology while all the others (NL-I-18 and the 54-31 series) had a non-neuronal morphology and resembled the L cell parents1, v. Coating of NGF on SRBC Mouse salivary gland N G F was isolated as a 27,500 mol.wt, dimer from SwissWebster mice by the method of Bocchini and Angeletti 2. When assayed by the method of Levi-Montalcini 8, 10 ng of this N G F induced the formation, within 12 h, of a halo of nerve fibers from chick embryonic sensory and sympathetic ganglia. The procedure for coating N G F on SRBC has been described t1,13. Briefly, SRBC in Alsever's solution were washed 3 times in 0.05 M potassium phosphate-0.15 M NaC1, pH 7.4 (PBS) and fixed for 5 min at room temperature in 1 ~ glutaralde-
455 hyde in 0.1 M phosphate buffer, pH 7.4. The SRBC were then washed exhaustively a n d 0.5 ml o f p a c k e d cells was i n c u b a t e d in 2 ml o f p h o s p h a t e buffer c o n t a i n i n g 5 m g purified N G F a n d 10 m g 1 - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) - c a r b o d i i m i d e h y d r o c h l o r i d e ( O t t C h e m i c a l Co., M u s k e g o n , Mich.). A f t e r 60 rain at r o o m t e m p e r a t u r e , the cells were w a s h e d in PBS until no p r o t e i n was detectable in the s u p e r n a t a n t by a b s o r b a n c e readings at 280 rim. N G F - c o a t e d S R B C elicited the f o r m a t i o n o f a fibrillar h a l o when i n c u b a t e d in vitro with 13-day chick e m b r y o s y m p a t h e t i c ganglia, i n d i c a t i n g t h a t the biological activity o f N G F was r e t a i n e d after the c a r b o d i i m i d e reaction a n d cell a t t a c h m e n t . As controls, S R B C were c o a t e d with u n r e l a t e d p r o t e i n s such as insulin, c y t o c h r o m e c, horse spleen ferritin ( W o r t h i n g t o n Biochemical Co.) a n d p h y t o h e m a g g l u t i n i n - W ( P H A ) ( W e l l c o m e :Research L a b o r a t o r i e s ) or were t r e a t e d similarly b u t w i t h o u t an a d d e d protein. The c o a t e d S R B C did n o t agglutinate s p o n t a n e o u s l y a n d were stable for at least one week when tested for passive h e m a g g l u t i n a t i o n with s t a n d a r d p r e p a r a t i o n s o f specific h y p e r i m m u n e r a b b i t antibodies. Test f o r rosette f o r m a t i o n
F o r assessment o f rosette f o r m a t i o n , cells were seeded in 50 m m Petri plates c o n t a i n i n g glass coverslips; the cells o f each clone a d h e r e d to the glass surface. A f t e r
TABLE I Rosette formation by CI300 neuroblastoma and L cell clones with ligand-coated sheep red blood cells (SRBC) Cell clones
Rosette formation* NGF-coated SRBC
PHA-coated SRBC
++ +q 4 ! + -! +-+ -± t
++-{-+ -~+~ + +q q q +q ++ -} +-I + + q +q4-+4 +
----
q + ++ ++
Cytochrome cor ferritincoated S R B C
Uncoated S R B C
C1300 neuroblastoma
NB NB1R NB6R NB7R NB9R NBIOR NA
m
m
L cells
L (NCTC clone 929) LM (TK-) L M ( T K - ) 1D
m
* A rosette was considered to be a single cell with 5 or more adherent erythrocytes. Rosette formation was evaluated on an arbitrary scale ranging from q (25 ~ rosette-forming cells) to q- + + / (100 ~ rosette-forming cells). A score of + + + + + was used when 100 ~ of the cells formed rosettes with an average of 20 or more adherent SRBC/celI.
456
Fig. 1. Rosette formation by cells of C1300 neuroblastoma clone, NB1R, exposed to sheep red blood cells coated with nerve growth factor. Nomarski. × 400.
incubation at 37 °C for 48 h, the coverslips were rinsed with E D M without serum, drained, placed in 100 m m plates and transferred to 4 °C. Two ml o f a 2 ~o suspension of SRBC (approximately 5 x 107 cells) in E D M without serum were overlaid on the coverslips. After incubation at 4 °C for 30 rain, the coverslips were rinsed gently with 15 ml cold E D M and the cells were examined microscopically for rosette formation ; a rosette was considered to be a cell with at least 5 adherent erythrocytes. Cell viability during the test remained close to 100 ~ as monitored by the capacity of the cells to remain adherent to the coverslips and to exclude the dye, erythrosine B. RESULTS AND DISCUSSION The ability of neuroblastoma and L cell clones to form rosettes when exposed to SRBC coated with N G F or other proteins is summarized in Table I. Some of the neuroblastoma clones (NB, NB 1R and NB7R) exhibited high NGF-binding capacity; the coated SRBC were firmly attached to the membrane of the cell body and along the network of axon-like cytoplasmic processes (Fig. 1). Repeated rinsing of the coverslips did not reduce the number of erythrocytes attached to the cells. Two neuroblast o m a clones (NB6R and NA) bound N G F to a slight extent. Other clones (NB9R, NB10R and the three L cell clones) showed essentially no rosette formation with N G F - c o a t e d SRBC; the erythrocytes bound to the few rosette-forming cells in this
457 T A B L E II
Rosette formation by neuroblastoma × L cell hybrid clones HybrM cell clones
Rosette formation* NGF-coated SRBC
PHA-coated SRBC
-± F ± kt ÷ + + + +(--)**
+ + ~ + +++% ++++ q +++ -+ + + + + F+ +
- 8
-F + + ( - - ) * *
+4+~
-18
~-q + + +
+-r-F+
NA × L M ( T K - ) I D 54-31-27 -29 -35 -36 -43 -46 NA × L M ( T K ) N L - I - 7a
Cytochrome eor ferritincoated S R B C
Uncoated S R B C
m
m
m
m
++-F+
* See legend o f Table I. ** S o m e areas on the coverslips h a d rosette-forming cells; others did not.
group were easily detached by further rinsing of the coverslips. The ability of hybrid clones to form rosettes is summarized in Table II. The clones showed a wide variation in the capacity to form rosettes with NGF-coated SRBC. In some cases (54-31-43 and NL-I-18), 1 0 0 ~ of the cells formed rosettes. In other cases (54-31-46 and NL-I-8), there were areas on the coverslips in which each cell formed a rosette with 10 or more firmly bound erythrocytes but other areas of the same coverslip had only a few rosette-forming cells with various numbers of attached erythrocytes. These differences in rosette-forming capacity among cells of the same or different clones cannot be explained by cell death occurring during the course of the test since cell viability remained essentially unchanged. The expression of N G F - b i n d i n g capacity in these clones appeared to be a stable characteristic; for example, clones NL-I-18 and 54-31-43 (both highly positive for rosette formation) and clone 54-31-27 (negative for rosette formation) maintained these characteristics over the course of 20 passages in culture. Similarly, the high capacity for rosette formation of the non-hybrid clone NB1R did not change over an even longer period of observation. Tests for rosette formation with erythrocytes coated with other proteins showed that all the neuroblastoma, L cell and hybrid clones formed rosettes with PHA-coated SRBC and none formed rosettes with cytochrome c- or ferritin-coated SRBC or with SRBC not coated with ligand (Tables I and II). These data show that rosette formation is a rapid and sensitive procedure for comparing the expression of membrane receptors for N G F in individual cells and cell clones. Only the neuroblastoma parent of the hybrid clones was able to form rosettes
458 with N G F - c o a t e d SRBC, and this only to a slight extent, while the hybrid clones showed wide differences ranging from negative to + ÷ + + - ~ in the ability to form rosettes. As described previously la, rosette formation is a specific reaction dependent in part on the n u m b e r o f N G F - c o a t e d S R B C added to the cell monolayers and the n u m b e r o f N G F molecules coated on each erythrocyte. The preparation o f N G F coated S R B C used in the present experiments was sufficiently labeled to allow saturation o f rosette formation by responding cell clones, within the standard incubation time, while under the same conditions, the n u m b e r o f rosette-forming cells a m o n g negative clones was less than 3 ~ . The exposure o f distinct receptor sites for P H A did not differ a m o n g the various clones tested and there are several possible interpretations for this difference in the behavior o f receptors for P H A and N G F on the m e m b r a n e surface o f these cells. One is that the clones have heritable quantitative differences in the expression o f receptors for N G F , but not for P H A . A n o t h e r possibility is that the clones have similar numbers o f receptor molecules for N G F but they are masked to different degreees by an as yet undetermined mechanism. The isolation and purification o f NGF-specific membrane receptors(s) f r o m different clones m a y help to explain the observed differences in rosette-forming capacity. ACKNOWLEDGEMENTS This research was supported by funds f r o m the Consiglio Nazionale delle Ricerche and N A T O G r a n t No. 864. L.D. was supported, in part, by Grants C A 08936 and H D 06323 from the U.S.P.H.S. REFERENCES 1 Bertolini, L., Diamond, L., Vigneti, E., Rizzoni, M., McMorris, F. A. and Revoltella, R., in preparation. 2 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000-molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787-794. 3 Dubbs, D. R. and Kit, S., Effect of halogenated pyrimidines and thymidine on growth of L-cells and a subline lacking thymidine kinase, Exp. Cell Res., 33 0964) 19-28. 4 Dunham, L. J. and Stewart, H. L., A survey of transplantable and transmissible animal tumors, J. nat. Cancer Inst., 13 (1952-1953) 1299-1377. 5 Kit, S., Dubbs, D. R., Piekarski, L. J. and Hsu, T. C., Deletion of thymidine kinase activity from L-cellsresistant to bromodeoxyuridine, Exp. Cell Res., 31 (1963) 297 312. 6 McMorris, F. A., Kolber, A. R., Moore, B. W. and Perumal, A. S., Expression of the neuronspecific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids, J. Cell PhysioL, 84 (1974) 473-480. 7 McMorris, F. A. and Ruddle, F. H., Expression of neuronal phenotypes in neuroblastoma cell hybrids, Develop. Biol., 39 (1974) 226-246. 8 Levi-Montalcini, R., The nerve growth factor: its modes of action on sensory and sympathetic nerve ceils, Harvey Lect., 60 (1966) 217-259. 9 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534 569. 10 Revoltella, R., Bertolini, L., Diamond, L., Vigneti, E. and Grasso, A., A radioimmunoassay for measuring the 14-3-2 protein in cell extracts, J. Neurochem., 26 (1976) 831-834. 11 Revoltella, R., Bertolini, L. and Pediconi, M., Unmasking of nerve growth factor membranespecific binding sites in synchronized murine C 1300 neuroblastoma cells, Exp. Cell Res., 85 (1974) 89-94.
459 12 Revoltella, R., Bertolini, L. Pediconi, M. and Vigneti, E., Specific binding of nerve growth factor (NGF) by murine C1300 neuroblastoma cells, J. exp. Med., 140 (1974) 437451. 13 Revoltella, R., Bosman, C. and Bertolini, L., Detection of nerve growth factor binding sites on neuroblastoma cells by rosette formation, Cancer Res., 35 (1975) 890-895. 14 Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. and Kettler, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-fl-hydroxylase in the rat superior cervical ganglia, Proc. nat. Acad, Sci. (Wash.), 68 (1971) 1598-1602.
453
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
D I F F E R E N C E S B E T W E E N M U R I N E C1300 N E U R O B L A S T O M A C L O N E S D E T E C T E D BY R O S E T T E F O R M A T I O N W I T H N E R V E G R O W T H F A C T O R - C O A T E D S H E E P R E D B L O O D CELLS
LEILA DIAMOND*, ROBERTO REVOLTELLA and LUISA BERTOLINI C.N.R. Laboratory of Cell Biology, Rome (Italy)
(Accepted April 20th, 1976)
SUMMARY The expression of receptors for nerve growth factor ( N G F ) on the cell surface was assayed by rosette formation with ligand-coated sheep red blood cells (SRBC). Cell clones derived from the murine C1300 neuroblastoma and from hybrids between a neuroblastoma clone and L cell clones showed a wide variation in the capacity to form rosettes with N G F - c o a t e d SRBC. All the neuroblastoma, L cell and hybrid clones formed rosettes with phytohemagglutinin-coated SRBC and none formed rosettes with cytochrome c- or ferritin-coated SRBC or with SRBC not coated with ligand.
INTRODUCTION Nerve growth factor ( N G F ) is a protein that enhances the growth and differentiation of sympathetic nerve cells in vivo and in vitro 8. Cell stimulation during N G F treatment of newborn mice is characterized by a marked increase in metabolic processes, including a selective induction of some of the key enzymes in the catecholamine biosynthetic pathway9,14. Recently, we demonstrated by rosette formation and by radioimmunoassay that cells of the murine C1300 neuroblastoma, a spontaneous tumor of sympathetic neurons of strain A mice 4, bind the N G F molecule 11-13. Rosettes are formed when N G F coated sheep red blood cells (SRBC) react at 2 °C with receptors exposed on the cell surface membrane and adhere to the tumor cells11,13. This phenomenon does not occur with normal strain A fibroblasts or with other tumor cells such as L cells and
* Permanent address: The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pa. 19104 (U.S.A.)
454 $37 sarcoma cells. We now report a segregation of this cell surface marker in several clones derived from the C1300 neuroblastoma and in clones derived from hybrids between a neuroblastoma clone positive for rosette formation and L cell clones. The study demonstrates the great variation among clones in the expression of membrane receptors for NGF. MATERIALS AND METHODS
Celh" A cell line (NB) derived from the murine C1300 neuroblastoma was obtained from Dr. F. Jacob, Institut Pasteur, and several clones (NB/R series) were isolated from it. The characterization of these clones which differ from each other in growth properties, morphology and the expression of neuron-specific properties will be reported elsewhere 1. Clone NA, a hypoxanthine-guanine phosphoribosyl-transferasedeficient subclone isolated by R. J. Klebe from clone Neuro-2a of C1300, was kindly supplied by Dr. F. Arthur McMorris, Wistar Institute. The 3 fibroblast clones studied were: L cells (NCTC clone 929), derived from subcutaneous tissue of adult C3H mice, and two L cell clones deficient in thymidine kinase, L M ( T K - ) 5 and L M ( T K - ) l D 3. The three hybrid [NA × LM(TK-)] clones of the NL-I series were supplied by Dr. McMorris; their expression of some neuronal phenotypes has been described 6,7,a0. Hybrid [NA × L M ( T K - ) I D ] clones of the 54-31 series were isolated and supplied by Dr. Carlo Croce, Wistar Institute. The hybrid nature of these clones was confirmed by karyotype and isozyme (glucese phosphate isomerase) analysis; these and other properties of the clones will be described elsewhere a. NB and NB/R cells were maintained as suspension cultures. They were grown in bacteriological Petri plates in Dulbecco's modified Eagle's medium (EDM) supplemented with 2 0 ~ fetal bovine serum (FBS) in an atmosphere of 12~o CO2 in air. When transferred to tissue culture or glass Petri plates, these cells attach to the surface and extend cytoplasmic processes of varying lengths. All other clones were maintained as monolayer cultures. They were grown in Eagle's minimum essential medium supplemented with 1 0 ~ FBS in an atmosphere of 5 ~ CO2 in air. NA cells had a 'neuronal' morphology with many cells showing cytoplasmic processes. Two of the hybrid clones (NL-I-7A and NL-]-8) had a similar morphology while all the others (NL-I-18 and the 54-31 series) had a non-neuronal morphology and resembled the L cell parents1, v. Coating of NGF on SRBC Mouse salivary gland N G F was isolated as a 27,500 mol.wt, dimer from SwissWebster mice by the method of Bocchini and Angeletti 2. When assayed by the method of Levi-Montalcini 8, 10 ng of this N G F induced the formation, within 12 h, of a halo of nerve fibers from chick embryonic sensory and sympathetic ganglia. The procedure for coating N G F on SRBC has been described t1,13. Briefly, SRBC in Alsever's solution were washed 3 times in 0.05 M potassium phosphate-0.15 M NaC1, pH 7.4 (PBS) and fixed for 5 min at room temperature in 1 ~ glutaralde-
455 hyde in 0.1 M phosphate buffer, pH 7.4. The SRBC were then washed exhaustively a n d 0.5 ml o f p a c k e d cells was i n c u b a t e d in 2 ml o f p h o s p h a t e buffer c o n t a i n i n g 5 m g purified N G F a n d 10 m g 1 - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) - c a r b o d i i m i d e h y d r o c h l o r i d e ( O t t C h e m i c a l Co., M u s k e g o n , Mich.). A f t e r 60 rain at r o o m t e m p e r a t u r e , the cells were w a s h e d in PBS until no p r o t e i n was detectable in the s u p e r n a t a n t by a b s o r b a n c e readings at 280 rim. N G F - c o a t e d S R B C elicited the f o r m a t i o n o f a fibrillar h a l o when i n c u b a t e d in vitro with 13-day chick e m b r y o s y m p a t h e t i c ganglia, i n d i c a t i n g t h a t the biological activity o f N G F was r e t a i n e d after the c a r b o d i i m i d e reaction a n d cell a t t a c h m e n t . As controls, S R B C were c o a t e d with u n r e l a t e d p r o t e i n s such as insulin, c y t o c h r o m e c, horse spleen ferritin ( W o r t h i n g t o n Biochemical Co.) a n d p h y t o h e m a g g l u t i n i n - W ( P H A ) ( W e l l c o m e :Research L a b o r a t o r i e s ) or were t r e a t e d similarly b u t w i t h o u t an a d d e d protein. The c o a t e d S R B C did n o t agglutinate s p o n t a n e o u s l y a n d were stable for at least one week when tested for passive h e m a g g l u t i n a t i o n with s t a n d a r d p r e p a r a t i o n s o f specific h y p e r i m m u n e r a b b i t antibodies. Test f o r rosette f o r m a t i o n
F o r assessment o f rosette f o r m a t i o n , cells were seeded in 50 m m Petri plates c o n t a i n i n g glass coverslips; the cells o f each clone a d h e r e d to the glass surface. A f t e r
TABLE I Rosette formation by CI300 neuroblastoma and L cell clones with ligand-coated sheep red blood cells (SRBC) Cell clones
Rosette formation* NGF-coated SRBC
PHA-coated SRBC
++ +q 4 ! + -! +-+ -± t
++-{-+ -~+~ + +q q q +q ++ -} +-I + + q +q4-+4 +
----
q + ++ ++
Cytochrome cor ferritincoated S R B C
Uncoated S R B C
C1300 neuroblastoma
NB NB1R NB6R NB7R NB9R NBIOR NA
m
m
L cells
L (NCTC clone 929) LM (TK-) L M ( T K - ) 1D
m
* A rosette was considered to be a single cell with 5 or more adherent erythrocytes. Rosette formation was evaluated on an arbitrary scale ranging from q (25 ~ rosette-forming cells) to q- + + / (100 ~ rosette-forming cells). A score of + + + + + was used when 100 ~ of the cells formed rosettes with an average of 20 or more adherent SRBC/celI.
456
Fig. 1. Rosette formation by cells of C1300 neuroblastoma clone, NB1R, exposed to sheep red blood cells coated with nerve growth factor. Nomarski. × 400.
incubation at 37 °C for 48 h, the coverslips were rinsed with E D M without serum, drained, placed in 100 m m plates and transferred to 4 °C. Two ml o f a 2 ~o suspension of SRBC (approximately 5 x 107 cells) in E D M without serum were overlaid on the coverslips. After incubation at 4 °C for 30 rain, the coverslips were rinsed gently with 15 ml cold E D M and the cells were examined microscopically for rosette formation ; a rosette was considered to be a cell with at least 5 adherent erythrocytes. Cell viability during the test remained close to 100 ~ as monitored by the capacity of the cells to remain adherent to the coverslips and to exclude the dye, erythrosine B. RESULTS AND DISCUSSION The ability of neuroblastoma and L cell clones to form rosettes when exposed to SRBC coated with N G F or other proteins is summarized in Table I. Some of the neuroblastoma clones (NB, NB 1R and NB7R) exhibited high NGF-binding capacity; the coated SRBC were firmly attached to the membrane of the cell body and along the network of axon-like cytoplasmic processes (Fig. 1). Repeated rinsing of the coverslips did not reduce the number of erythrocytes attached to the cells. Two neuroblast o m a clones (NB6R and NA) bound N G F to a slight extent. Other clones (NB9R, NB10R and the three L cell clones) showed essentially no rosette formation with N G F - c o a t e d SRBC; the erythrocytes bound to the few rosette-forming cells in this
457 T A B L E II
Rosette formation by neuroblastoma × L cell hybrid clones HybrM cell clones
Rosette formation* NGF-coated SRBC
PHA-coated SRBC
-± F ± kt ÷ + + + +(--)**
+ + ~ + +++% ++++ q +++ -+ + + + + F+ +
- 8
-F + + ( - - ) * *
+4+~
-18
~-q + + +
+-r-F+
NA × L M ( T K - ) I D 54-31-27 -29 -35 -36 -43 -46 NA × L M ( T K ) N L - I - 7a
Cytochrome eor ferritincoated S R B C
Uncoated S R B C
m
m
m
m
++-F+
* See legend o f Table I. ** S o m e areas on the coverslips h a d rosette-forming cells; others did not.
group were easily detached by further rinsing of the coverslips. The ability of hybrid clones to form rosettes is summarized in Table II. The clones showed a wide variation in the capacity to form rosettes with NGF-coated SRBC. In some cases (54-31-43 and NL-I-18), 1 0 0 ~ of the cells formed rosettes. In other cases (54-31-46 and NL-I-8), there were areas on the coverslips in which each cell formed a rosette with 10 or more firmly bound erythrocytes but other areas of the same coverslip had only a few rosette-forming cells with various numbers of attached erythrocytes. These differences in rosette-forming capacity among cells of the same or different clones cannot be explained by cell death occurring during the course of the test since cell viability remained essentially unchanged. The expression of N G F - b i n d i n g capacity in these clones appeared to be a stable characteristic; for example, clones NL-I-18 and 54-31-43 (both highly positive for rosette formation) and clone 54-31-27 (negative for rosette formation) maintained these characteristics over the course of 20 passages in culture. Similarly, the high capacity for rosette formation of the non-hybrid clone NB1R did not change over an even longer period of observation. Tests for rosette formation with erythrocytes coated with other proteins showed that all the neuroblastoma, L cell and hybrid clones formed rosettes with PHA-coated SRBC and none formed rosettes with cytochrome c- or ferritin-coated SRBC or with SRBC not coated with ligand (Tables I and II). These data show that rosette formation is a rapid and sensitive procedure for comparing the expression of membrane receptors for N G F in individual cells and cell clones. Only the neuroblastoma parent of the hybrid clones was able to form rosettes
458 with N G F - c o a t e d SRBC, and this only to a slight extent, while the hybrid clones showed wide differences ranging from negative to + ÷ + + - ~ in the ability to form rosettes. As described previously la, rosette formation is a specific reaction dependent in part on the n u m b e r o f N G F - c o a t e d S R B C added to the cell monolayers and the n u m b e r o f N G F molecules coated on each erythrocyte. The preparation o f N G F coated S R B C used in the present experiments was sufficiently labeled to allow saturation o f rosette formation by responding cell clones, within the standard incubation time, while under the same conditions, the n u m b e r o f rosette-forming cells a m o n g negative clones was less than 3 ~ . The exposure o f distinct receptor sites for P H A did not differ a m o n g the various clones tested and there are several possible interpretations for this difference in the behavior o f receptors for P H A and N G F on the m e m b r a n e surface o f these cells. One is that the clones have heritable quantitative differences in the expression o f receptors for N G F , but not for P H A . A n o t h e r possibility is that the clones have similar numbers o f receptor molecules for N G F but they are masked to different degreees by an as yet undetermined mechanism. The isolation and purification o f NGF-specific membrane receptors(s) f r o m different clones m a y help to explain the observed differences in rosette-forming capacity. ACKNOWLEDGEMENTS This research was supported by funds f r o m the Consiglio Nazionale delle Ricerche and N A T O G r a n t No. 864. L.D. was supported, in part, by Grants C A 08936 and H D 06323 from the U.S.P.H.S. REFERENCES 1 Bertolini, L., Diamond, L., Vigneti, E., Rizzoni, M., McMorris, F. A. and Revoltella, R., in preparation. 2 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000-molecular weight protein, Proc. nat. Acad. Sci. (Wash.), 64 (1969) 787-794. 3 Dubbs, D. R. and Kit, S., Effect of halogenated pyrimidines and thymidine on growth of L-cells and a subline lacking thymidine kinase, Exp. Cell Res., 33 0964) 19-28. 4 Dunham, L. J. and Stewart, H. L., A survey of transplantable and transmissible animal tumors, J. nat. Cancer Inst., 13 (1952-1953) 1299-1377. 5 Kit, S., Dubbs, D. R., Piekarski, L. J. and Hsu, T. C., Deletion of thymidine kinase activity from L-cellsresistant to bromodeoxyuridine, Exp. Cell Res., 31 (1963) 297 312. 6 McMorris, F. A., Kolber, A. R., Moore, B. W. and Perumal, A. S., Expression of the neuronspecific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids, J. Cell PhysioL, 84 (1974) 473-480. 7 McMorris, F. A. and Ruddle, F. H., Expression of neuronal phenotypes in neuroblastoma cell hybrids, Develop. Biol., 39 (1974) 226-246. 8 Levi-Montalcini, R., The nerve growth factor: its modes of action on sensory and sympathetic nerve ceils, Harvey Lect., 60 (1966) 217-259. 9 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534 569. 10 Revoltella, R., Bertolini, L., Diamond, L., Vigneti, E. and Grasso, A., A radioimmunoassay for measuring the 14-3-2 protein in cell extracts, J. Neurochem., 26 (1976) 831-834. 11 Revoltella, R., Bertolini, L. and Pediconi, M., Unmasking of nerve growth factor membranespecific binding sites in synchronized murine C 1300 neuroblastoma cells, Exp. Cell Res., 85 (1974) 89-94.
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