Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2562-2566, April 1984 Neurobiology and insulin-like II permit nerve growth factor binding and the formation response in cultured human neuroblastoma cells (/differentiation/nerve /pheochromocytoma PC12) ESPERANZA RECIO-PINTO, FREDERICK F. LANG, AND DOUGLAS N. ISHII* Department of Pharmacology and Cancer Research Center, College of Physicians and Surgeons of Columbia University, New York, NY 10032 Communicated by I. S. Edelman, January 3, 1984

ABSTRACT In serum-free medium, SH-SY5Y human mouse (16); purity was confirmed by the single band in neuroblastoma cells specifically and reversibly lost the capaci- polyacrylamide gels subjected to isoelectric focusing (pH ty to bind 1251-labeled nerve growth factor (NGF) to the high- 3.5-10), or NaDodSO4 gel electrophoresis and by bioassay affinity sites (slow sites) and to respond by neurite outgrowth, (17). Anti-insulin antiserum was from Cappel Laboratories unless physiological concentrations of insulin or insulin-like (Cochranville, PA). The cloned cell line SH-SY5Y (7) was a growth factor II were present. In serum-containing medium, kind gift from June L. Biedler and Barbara A. Spengler. anti-insulin antiserum decreased the neurite formation re- Cells between passage numbers 9 and 29 were studied. The sponse to NGF, and insulin supplementation increased the cloned PC12 cell line (18) was the kind gift of Lloyd A. number of available NGF slow sites. The low-affinity NGF fast Greene and was subcloned prior to use. sites are absent from SH-SY5Y cells and did not emerge on Cell Culture. Cells were maintained in the logarithmic treatment with insulin. Insulin potentiated the induction of phase of growth in Roswell Park Memorial Institute medium by NGF in rat pheochromocytoma PC12 cells also. 1640 (RPMI 1640) supplemented with 12% fetal calf serum, These results implicate a wider role for insulin and its homo- sodium penicillin G at 50 units/ml, and streptomycin sulfate logs in the . at 25 ,ug/ml at 370C in humidified 5% C02/95% air (4, 5). Neurite Outgrowth. For studies in serum-containing medi- Nerve growth factor (NGF) is important to the development um (SCM), cells were seeded onto microwell plates in the of the sympathetic and sensory of vertebrates and above growth medium and permitted to attach for 3 days, can cause outgrowth of axons in vitro and in vivo (1-3). We and then fresh warm RPMI 1640 medium with 12% fetal calf have used the human neuroblastoma SH-SY5Y cell as a serum and various test solutions was added. For studies in model in which to study the mechanism of neurite formation SFM, after attachment, cells were washed three times with (4-6). Cloned SH-SY5Y cells (7) respond to NGF with in- warm RPMI 1640 medium, then warm RPMI 1640 medium creased neurite outgrowth (4, 8) and veratridine-dependent containing various test solutions was added. The percentage Na' uptake (8). The neurites show ultrastructural maturation of cells bearing neurites was determined by replicate counts in cells treated with NGF (8) and end in typical neuronal on more than 100 cells in several randomly chosen fields un- growth cones (9). der low-power modulation-contrast microscopy as described High-affinity sites (slow sites) and low-affinity sites (fast (4, 5). sites) that bind 1251-labeled NGF are present in sensory (10, Binding Assay. Purified NGF was iodinated (0.1-0.2 mol 11), sympathetic (12), and pheochromocytoma PC12 (13, 14) of 125i per mol of NGF) with lactoperoxidase (4). Cells were cells. Both sites are also present in some human neuroblasto- detached from nonconfluent monolayer cultures in Hanks' ma cell lines, but only the slow type site is present in SH- salts solution with 1 mM EDTA. Cells (2-4 x 106 per ml) SYSY cells (4), suggesting that binding to the fast sites is not were resuspended in RPMI 1640 medium containing bovine required for neurite outgrowth. The hypothesis that the slow serum albumin at 5 mg/ml and incubated for 1 hr at 37°C. In sites are the receptors for neurite outgrowth would be con- the absence of this incubation, down modulation of binding siderably more attractive if it were possible to activate and can often, but not always, be observed (4). Then, 0.1 nM (2.5 inactivate the capacity of these sites to bind NGF and to ng/ml) 125I-labeled NGF was added. Other conditions are show corresponding activation and inactivation of the capac- described in the legends. In cell lines without fast sites, non- ity to respond by neurite outgrowth. specific binding was assayed in parallel incubations addition We show that SH-SY5Y cells reversibly lose virtually all ally containing 100-fold excess NGF at the onset, and this capacity to bind NGF and respond by neurite outgrowth in binding was subtracted from other values. However, a 1000- serum-free media (SFM). Moreover, both capacities are re- fold excess of NGF is required to displace all specific bind tained in media containing insulin or insulin-like growth fac- ing in cell lines with lower affinity fast sites. Binding to fast tor II (IGF-II). sites is lost and binding to slow sites is quantitatively re tained after 10 min on ice with 1000-fold excess NGF, as in MATERIALS AND METHODS PC12 cells (13). Samples were centrifuged in needlenosed Materials. Pork pancreatic insulin (24 units/mg) and phor- 400-,ul capacity tubes (W. Sarstedt, Princeton, NJ) for 30 bol 12,13-dibutyrate (PBt2) were from Sigma. Insulin was sec. Tubes were quickly frozen in an acetone/dry ice bath, dissolved in 0.01 M HCl and stored at -20°C. Rat cell the tips were severed (tip volume was about 2 ,ul), and radio multiplication-stimulating activity, which is the same as activities were measured (4). IGF-II (15), of about 85% purity, was from Collaborative Re- Statistics. Values are means and SEM; n values are given search. The ,3 subunit of NGF was prepared from male Abbreviations: IGF-II, insulin-like growth factor II (rat liver multi- The publication costs of this article were defrayed in part by page charge plication-stimulating activity); NGF, nerve growth factor; PBt2, payment. This article must therefore be hereby marked "advertisement" phorbol 12,13-dibutyrate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

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in legends. Analysis of variance and the Bonferroni multiple NGF plus 0.1 nM IGF-II, 51.9 ± 5.5%. The values are means t test (19) were used to compare experimental groups. Data ± range (n = 2 replicate cultures in which 400 cells were expressed as percentages were transformed to arc sin values scored). IGF-II alone directly enhanced neurite outgrowth; a prior to statistical analysis. suboptimal concentration for direct enhancement of neurite formation was studied. NGF alone had no significant activi- RESULTS ty even at a very high concentration. When NGF and IGF-II Effect of Insulin on SH-SY5Y Cell Neurite Formation in were added in combination, the neurite outgrowth response SFM. To avoid the variable amounts of serum components to NGF was potentiated. Thus, IGF-II, like insulin, had per- that may affect neurite formation, some tests were conduct- missive as well as direct effects on neurite outgrowth. ed in SFM, in which SH-SY5Y cells can survive at least a Effect of Anti-Insulin Antiserum on the SH-SY5Y Cell week without loss in number (4). Growth resumes on the Neurite Formation Response to Insulin, NGF, and PBt2. reintroduction of serum. The maximum neurite outgrowth Neurite outgrowth was increased in insulin-supplemented response to insulin is reached in 3 days in SFM. SCM (Table 1). Anti-insulin antiserum completely blocked In SFM, up to 0.75 ,uM NGF alone did not enhance neurite this increase. NGF increased the proportion of cells with outgrowth (Fig. 1A), showing that other serum factors are neurites from 27% to 70%. The anti-insulin antiserum, when required for the response. With 1 nM insulin alone, neurite added together with NGF, inhibited 77% of this increase, outgrowth was increased to about 13%. This will be referred showing that the low level of insulin normally present in se- to as the "direct" effect, since other serum factors were not rum was required for expression of a major fraction of the required. When 1 nM insulin was present, the simultaneous NGF activity. The anti-insulin antiserum also decreased the addition of various concentrations of NGF caused a syner- spontaneous neurite outgrowth to less than half the level in gistic, dose-dependent increase in neurite outgrowth. This untreated cultures; the residual spontaneous neurite out- shall be referred to as the "permissive" effect of insulin. The growth may be due to the activity of other serum factors. true sensitivity to NGF is obscured in Fig. LA because of its The antiserum does not, however, nonspecifically inhibit strong tendency to adsorb to culture vessel walls (20). The neurite outgrowth. Tumor promoters can reversibly increase inclusion of bovine serum albumin to reduce nonspecific ad- neurite formation in SH-SY5Y cells (5, 24). The promoter sorption resulted in a sharp shift in the NGF response curve PBt2 increased neurite outgrowth in a manner that was not to the left, such that the half-maximal response was at about inhibited by the anti-insulin antiserum (Table 1). 0.2-0.4 nM (Fig. 1B). The small increase in response to NGF These results suggest that the capacity of SH-SY5Y cells seen even in cultures untreated with insulin was probably to respond to NGF depends on the activity of serum insulin due to the activity of impurities in the albumin preparation. and its homologs. The permissive effect is best shown in The permissive effect was seen at insulin concentrations SFM. In SCM the serum insulin, and probably IGF-II, al- as low as 10 pM; at that concentration insulin had no detecti- ready exert a high background of permissive activity (Table ble direct activity on neurite outgrowth after 3 days (untreat- 1). ed, 2.6 ± 1.0%; 10 pM insulin, 3.3 ± 1.3%; 8 nM NGF, 2.8 ± Effect of Culture in Insulin-Supplemented SCM on the Time 0.5%; 8 nM NGF plus 10 pM insulin, 10.0 ± 0.7%; means ± Course of 125I-Labeled NGF Binding and Dissociation in SH- range; n = 2). Both the direct and permissive effects were SY5Y Cells. Cells were cultured in SCM, with and without reversible. insulin supplementation, and the time course of 125I-labeled Effect of IGF-II on SH-SY5Y Cell Neurite Formation in NGF binding was studied (Fig. 2). Specific binding, defined SFM. Insulin and IGF-II are closely related in structure, and as the difference between total and noncompetable binding, cross-occupy one another's receptors (21-23). Cells were reached a steady state within 40 min. Insulin increased spe- cultured for 3 days in SFM with NGF, IGF-II, and the com- cific binding by 80% (Fig. 2A). The noncompetable binding bination of the factors, and neurite formation was scored. increased at a constant rate, causing total binding to rise The results were as follows: untreated, 16.5 ± 1.0%; 0.1 nM IGF-II, 32.2 ± 7.5%; 400 nM NGF, 20.3 ± 1.7%; and 400 nM Table 1. Effect of anti-insulin antiserum on neurite formation in SH-SY5Y cells in response to insulin, NGF, and PBt2

A Neurite 50 Conditions Serum outgrowth, % Untreated + 27.0 ± 3.2 Insulin antiserum, 20 pl/ml + 12.1 ± 1.5* Insulin, 100 nM + 42.7 ± 3.8tt ,30 Insulin, 100 nM, plus antiserum, 20 pl/ml + 9.2±1.2t± NGF, 8 nM + 69.9 ± 7.4t C0 NGF, 8 nM, plus antiserum, 20 dul/ml + 37.0 ± 1.4tt z 0 10 Untreated - 8.1 ± 1.8 Insulin, 100 nM - 81.2 ± 5.9t I I Insulin antiserum, 10 dul/ml - 1.9 ± 0.9 0 109 i68 167 16 0 1610 1i9 1 8 1o7 Insulin, 100 nM, plus antiserum, NGF (M) NGF (M) 10 Al/ml - 11.8 ± 2.8f PBt2, 200 nM - 70.8 ± 1.3t FIG. 1. Dependence of neurite outgrowth on NGF concentration in the presence and absence of insulin in SFM in human neuroblas- PBt2, 200 nM, plus antiserum, 20 gl/ml - 68.6 ± 1.2t toma SH-SY5Y cells. (A) Cells were cultured in SFM for 3 days with Cells were cultured in RPMI 1640 medium containing the indicat- the indicated concentrations of NGF either without (o) or with (0) 1 ed compounds either with 12% fetal calf serum or without serum and nM insulin. Neurite outgrowth was scored. Values are means ± neurite outgrowth was scored. The values are means ± SEM (n = 4 range (n = 2 replicate cultures, 400 cells scored per culture). (B) As replicate cultures). in A except all cultures contained in addition bovine serum albumin *P < 0.05 between treated and untreated cultures. at 0.2 mg/ml. Values are means + SEM (n = 3 replicate cultures, tp < 0.01 between treated and untreated cultures. 100 cells scored per culture). tP < 0.01 between group and group immediately preceding. Downloaded by guest on September 27, 2021 2564 Neurobiology: Recio-Pinto et al. Proc. NatL Acad Sci. USA 81 (1984)

that the half-times of loss of binding were 25 and 22 min for untreated and insulin-treated cultures, respectively. Thus, the 1.8-fold increase in specific binding was not due to a de- creased 125I-labeled NGF dissociation rate in insulin-treated cultures. Insulin did not cause X20~~~~~~~~~~~~~~~~. emergence of the low-affinity NGF fast sites. All of the specific binding was to the slow 0 20 40 sites. FIG. 2.Tie ous o l5IlaeedNG bndn isocaminad Insulin increased the content of cells cultured for 2 10~~~~~~~ days in SCM by about 30%, but a simple difference in pro- tein content between treated and untreated cells could not LL r;_ U explain the increased specific 1251-labeled NGF binding ca- z 0.520- pacity (untreated, 588 + 51; 0.1 /iM insulin, 1314 ± 134 cpm per ,ug of protein; means ± SEM, n = 3 cultures). Effect of Insulin and IGF-II on SH-SY5Y Cell 125j-Labeled Lo0 11 NGF Binding Capacity in SFM. About 87% of the total spe- cific binding capacity was lost after culture for 2 days in SFM, and all detectable binding capacity after 5 days (Fig. 3). When serum or insulin was added to cultures grown in SFM for 2 days, the binding capacity reemerged. All of the specific binding was to slow sites. Insulin treatment for 3 days in SFM increased the cell protein content (untreated, tion in SH-SY5Y cells cultured in SCM with and without insulin 119 ± 0.3; 0.1 1tM insulin, 129 ± 8 ,ug per 106 cells; means ± supplementation. Cultures were grown in SCM for 2 days with (A) SEM, n = 3 cultures). The increased protein content in and without (B) 0.1 1tcM insulin. The cells were harvested and incu- SFM, as in SCM, did not account for the insulin-increased bated at 370C with 0.1 nM (59 cpm/pg) '251-labeled NGF alone and 125I-labeled NGF binding capacity (untreated, 2.8 + 0.4; 0.1 with 1000-fold excess NGF: o, total; z, noncompetable;e*, 1000-fold ,uM insulin, 19.8 ± 1.2 cpm/Ag of protein; means ± SEM, n excess NGF added at 30 mmn (Inset) Specific dissociation curves = from A and B are plotted by the method of least squares. The non- 3). competable binding has been subtracted: 0, untreated cultures, r = IGF-I also maintained specific 125I-labeled NGF binding 0.98 and the half-time of loss = 25 min;e*, insulin-treated cultures, r capacity in cells grown for 2 days in SFM and assayed as in = 0.96 and the half-time of loss = 22 min. Fig. 3 (12% serum, 1955 ± 163; serum-free, 265 + 135; se- rum-free plus 10 nM IGF-II, 1167 ± 140 cpm per 106 cells; means ± SEM, n = 3). steadily with time. Insulin increased the rate of noncompeta- ble binding, suggesting it is under metabolic regulation. The dissociation rate was measured by adding excess NGF at 30 min. After displacement of all specific binding, the dissocia- tion curves began to rise steadily at 2 hr due to the influence E30 of the noncompetable binding. The noncompetable binding c~~ was subtracted and the resultant specific dissociation curves are shown in Fig. 2B Inset. The least-squares plot showed

- 0

100~~ us 042.60. 0. x BON7(ml/0 cls E 0.6 [ o3

zi ,-! '0 0.4 u2 E -6 10 E z-,2 LO 3 0.2 z

us V - 0 j 2 4 0 0.2 0.4 0.6 0.8 1.0 TIME (days) FREE (nM)

FIG. 3. Loss of '25I-labeled NGF binding capacity in SH-SY5Y FIG. 4. Effect of culture in insulin-supplemented SCM on the cells grown in SFM, and recovery after treatment with insulin or 125I-labeled NGF binding isotherm in SH-SY5Y cells. Cultures were serum. Monolayer cultures were grown for 2 days in RPMI 1640 grown for 2 days in SCM without (o) and with (0) 100 nM insulin. medium with (e) or without (o) 12% serum. Cultures were assayed The cells were harvested and incubated with various concentrations for capacity to bind 0.1 nM (190 cpm/pg) 1251-labeled NGF after 1 hr of 125I-labeled NGF at 4°C for 7 hr. Noncompetable binding was at 37°C. Some of the cultures initially grown without serum were determined in parallel incubations containing NGF at 0.25 ,ug/ml. cultured for 3 more days without serum (o), with 200 nM insulin (A), Only specific binding is shown. (Inset) Scatchard plot of the data. or with 12% serum (A), and assayed for the binding capacity as be- The lines were fit by the method of least squares. Untreated, r = fore. Noncompetable binding was subtracted. Only specific binding 0.97, Kd = 280 pM, maximal binding Bmax = 0.448 fmol per 106 cells; to the slow sites is shown. Fast sites were not detected. Values are insulin-treated, r = 0.96, Kd = 270 pM, Bmax = 0.828 fmol per 106 means ± SEM (n = 3 replicate cultures). cells. Downloaded by guest on September 27, 2021 Neurobiology: Recio-Pinto et aL Proc. NatL. Acad. Sci. USA 81 (1984) 2565 Effect of Culture in Insulin-Supplemented SCM on the 1251_ High affinity, saturability, and inability of other Labeled NGF Binding Isotherm in SH-SY5Y Cells. The cells to compete for binding are properties consistent with recep- were cultured for 2 days in SCM with and without insulin tors, but they do not exclude the possibility that sites are supplementation. To reduce the likelihood of internalization, nonspecific (27) or, instead, are specific transport or degra- binding was studied at 40C, at which the maximal binding is dative sites. NGF does undergo retrograde transport (28) reached after 5-6 hr (4). The least-squares Scatchard plots and is degraded (10). NGF modified by exposure to bromo- were linear, consistent with the presence of a single type of succinimide (29, 30), or complexed to its antibodies (31) site (Fig. 4 Inset). The number of available binding sites in shows a reduced binding to the high- and low-affinity sites insulin-treated cells was increased 1.8-fold, consistent with and a diminished activity for neurite outgrowth. However, the amount of increased binding earlier observed in Fig. 2. the possibility that the true NGF receptor is neither of these The same qualitative results were obtained at 370C; the affin- sites is not excluded, because modified or complexed NGF ity of binding was higher, but the same, for insulin-treated may fail to bind to the other types of sites as well as to the and untreated cultures. receptors. The curves for the dose-dependent reversible in- Effect of Insulin on NGF-Induced Neurite Formation in hibition of neurite outgrowth and the dose-dependent revers- PC12 Cells. In contrast to its direct effect on neurite forma- ible inhibition of NGF binding caused by the tumor promot- tion in SH-SY5Y cells (Table 1), insulin at concentrations as ers saccharin and cyclamate were closely parallel (32, 33), high as 10 AM did not directly induce neurite formation in suggesting by the pharmacological antagonism that one of PC12 cells cultured in SCM. But, insulin at 1 nM did potenti- the two sites is the receptor. Inhibition of binding to the oth- ate the neurite outgrowth response to NGF (untreated, 0.0 ± er types of sites should not cause antagonism of neurite out- 0.0%; 1 nM insulin, 0.0 + 0.0%; 30 pM NGF, 7.2 + 1.4%; 30 growth, but the possibility of a fortuitous parallelism must at pM NGF plus 1 nM insulin, 26.1 ± 2.8%; means ± SEM, n = least be considered. Since binding to both high- and low-af- 4). At 10 nM insulin there was a decrease in the magnitude of finity sites was inhibited, none of the above studies reveal potentiation (not shown). A suboptimal NGF concentration which site is the receptor. The dose-response curve for was studied to demonstrate clearly the synergistic nature of neurite outgrowth, and the dose-total binding curve, are not insulin's potentiating effect. NGF did induce neurites in closely parallel. The NGF concentration causing the half- SFM, despite the absence of other serum factors. maximal neurite outgrowth response in sensory neurons (34) The neurite formation response in PC12 cells "primed" by has about the same value as the Kd of the high-affinity sites prior exposure to NGF is transcription independent but de- (10, 11), but a significant number of low-affinity sites can be pendent in unprimed cells (25, 26). Priming with NGF for 9 occupied even at low NGF concentrations because there are days did not confer a state in which insulin could directly at least 10-fold more low- than high-affinity sites (10, 35). If a enhance neurite outgrowth. The ability of insulin to potenti- spare receptor system (36) were involved, a maximal re- ate the response to NGF is not confined to human neuroblas- sponse would be attainable with occupancy of only a fraction toma cells. However, whether the same mechanism is in- of the low-affinity sites. Thus, there is meaningful strong but volved has not been studied. not incontrovertible evidence that the sites are receptors, and the relative importance of the two sites to neurite out- DISCUSSION growth is not fully established for sensory and sympathetic neurons. Insulin and IGF-II are permissive for NGF-directed neurite The trypsin-sensitive and kinetic binding properties of fast formation and can increase NGF binding activity. The re- and slow sites in human neuroblastoma cells (unpublished sults show that the slow binding sites are in fact NGF recep- data) are very similar to those in the PC12 cell (13). The Kd tors for neurite outgrowth in SH-SY5Y cells. of fast sites at 40C is about 3 nM, and that of slow sites is In SFM, the cells reversibly lost the capacity to form neur- about 0.2-0.3 nM (Fig. 4) (4). Binding to slow sites can be ites in response to NGF (Fig. 1). The loss of NGF respon- detected in intact SH-SY5Y cells at 40C (4) (Fig. 4) and in siveness is not part of a generalized inability to form neurites enriched membrane fractions (unpublished data). Hence, the in SFM, since insulin, IGF-II, and tumor promoters (24) (Ta- slow sites are not due to internalized NGF. Fast sites are ble 1) retain their capacity to directly enhance neurite out- absent from SH-SY5Y and LA-N-5 cells (4), suggesting by growth. The presence of nanomolar amounts, or less, of in- elimination that the slow sites are the receptors for neurite sulin and IGF-1I prevented the loss of responsiveness to outgrowth and other demonstrated effects. The possibility NGF in SFM (Fig. 1). This was termed the permissive effect. that the slow sites represent other than receptors is not en- The anti-insulin antiserum blocked serum insulin's permis- tirely excluded by this observation alone. Our finding that sive effect, thereby diminishing SH-SY5Y cell responsive- inactivation and activation of NGF binding to slow sites is ness to NGF (Table 1). IGF-II is likely to be among the other correlated with activation and inactivation of the neurite for- serum factors that support NGF activity. mation response is potent evidence that these sites are in- Insulin's permissiveness for NGF-directed neurite out- deed the receptors. growth correlates with its effect on the NGF binding capaci- Insulin and IGF-II, in addition, have direct effects on ty of SH-SY5Y cells. That capacity is virtually completely neurite outgrowth whose expression does not require the and specifically lost after culture in SFM (Fig. 3). The capac- presence of other serum factors. Insulin's direct effect is not ity to bind insulin, IGF-II, and tumor promoters is not lost, inhibited by anti-NGF antiserum, and highly purified human as is clear from the intact responses (Table 1). The loss of insulin is as potent as porcine insulin (6). Both the direct and NGF binding capacity can be prevented by insulin and IGF- permissive effects are produced well within the range of the II and can be reversed by insulin. Supplementation with in- circulating concentrations of these factors. sulin increased the NGF binding capacity in SCM (Fig. 2), The response to insulin and NGF differs in PC12 cells in due to an increase in the number of slow sites (Fig. 4). The several respects. In SFM, neurite formation was directly en- affinity of existing sites (Fig. 4) and the dissociation rate hanced by insulin in SH-SY5Y, but not in PC12 cells. How- (Fig. 2 Inset) was unchanged. LA-N-5 is another human neu- ever, insulin did potentiate the response to NGF in both roblastoma cell line responsive by neurite outgrowth to NGF PC12 and SH-SY5Y cells. NGF, but not insulin, inhibits the (4) in which insulin can increase binding to slow sites. Thus, growth rate, is required for survival, and can induce neurite the modulation of NGF binding by insulin is not restricted to outgrowth in PC12 cells in SFM (18, 37). the SH-SY5Y cell. Insulin did not cause emergence of NGF Supraphysiological insulin concentrations can enhance fast sites in either cell line. neurite outgrowth (38, 39) and support survival (38, 40) of Downloaded by guest on September 27, 2021 2566 Neurobiology: Recio-Pinto et al. Proc. NatL Acad ScL USA 81 (1984)

cultured embryonic sensory neurons; IGF-II can enhance 15. Acquaviva, A. M., Bruni, C. B., Nissley, S. P. & Rechler, neurite outgrowth half-maximally at around 4 nM (39). These M. M. (1982) 31, 656-658. observations do not reveal whether insulin and its homologs 16. Burton, L. E., Wilson, W. H. & Shooter, E. M. (1978) J. Biol. are survival factors and neurite outgrowth is spontaneous, Chem. 253, 7807-7812. 17. Ishii, D. N. & Shooter, E. M. (1975) J. Neurochem. 25, 843- whether they are true agonists for neurite formation, or both. 851. Also, insulin might be acting indirectly through the remain- 18. Greene, L. A. & Tischler, A. S. (1976) Proc. Natl. Acad. Sci. ing nonneuronal cells. Our results, in cloned cells that re- USA 73, 2424-2428. quire neither insulin nor NGF for survival, suggest that insu- 19. Dunn, 0. J. & Clark, V. A. (1974) Applied Statistics: Analysis lin and its homologs directly enhance neurite formation. of Variance and Regression (Wiley, New York). 20. Pearce, F. L. (1972) in Nerve Growth Factor and Its Antise- rum, eds. Zaimis, E. & Knight, J. (Athlone Press, London), We thank Mary J. Savage for helpful technical assistance in some pp. 253-261. of these studies. This work was supported in part by Grant R01 NS 21. Rinderknecht, E. & Humbel, R. (1978) Fed. Eur. Biochem. 14218 from the National Institute of Neurological and Communica- Soc. Lett. 89, 283-286. tive Disorders and Stroke and Grant R01 AM32841 from the Nation- 22. Rinderknecht, E. & Humbel, R. (1978) J. Biol. Chem. 253, al Institute of Arthritis, Diabetes, and Digestive and Dis- 2769-2774. eases. D.N.I. is a recipient of U.S. Public Health Service Research 23. Zapf, J., Schoenle, E. & Froesch, E. R. (1978) Eur. J. Bio- Career Development Award 1 K04 NS 00375. chem. 87, 285-296. 24. Spinelli, W. & Ishii, D. N. (1983) Cancer Res. 43, 4119-4125. 1. Mobley, W. C., Server, A. C., Ishii, D. N., Riopelle, R. J. & 25. Burstein, D. E. & Greene, L. A. (1978) Proc. Natl. Acad. Sci. Shooter, E. M. (1977) N. Engl. J. Med. 297, 1096-1104 (Part USA 75, 6059-6063. I), 1149-1158 (Part II), and 1211-1218 (Part III). 26. Greene, L. A., Burstein, D. E. & Black, M. M. (1980) in Tis- 2. Harper, G. P. & Thoenen, H. (1980) J. Neurochem. 34, 5-16. sue Culture in Neurobiology, eds. Giacobini, G., Vernadakis, 3. Vinores, S. & Guroff, G. (1980) Annu. Rev. Biophys. Bioeng. A. & Shahar, A. (Raven Press, New York), pp. 313-319. 9, 223-257. 27. Cuatrecasas, P., Hollenberg, M. D., Chang, K. J. & Bennett, 4. Sonnenfeld, K. H. & Ishii, D. N. (1982) J. Neurosci. Res. 8, V. (1975) Recent Progr. Horm. Res. 31, 37-94. 375-391. 28. Hendry, I. A., Stockel, K., Thoenen, H. & Iversen, L. L. 5. Spinelli, W., Sonnenfeld, K. H. & Ishii, D. N. (1982) Cancer (1974) Brain Res. 68, 103-121. Res. 42, 5067-5073. 29. Baierjee, S. P., Snyder, S. H., Cuatrecasas, P. & Greene, 6. Recio-Pinto, E. & Ishii, D. N. (1983) Brain Res., in press. L. A. (1973) Proc. NatI. Acad. Sci. USA 70, 2519-2523. 7. Biedler, J. L., Roffler-Tarlov, S., Schachner, M. & Freedman, 30. Cohen, P., Sutter, A., Landreth, G., Zimmermann, A. & L. S. (1978) Cancer Res. 38, 3751-3757. Shooter, E. M. (1980) J. Biol. Chem. 255, 2949-2954. 8. Perez-Polo, J. R., Werrbach-Perez, K. & Tiffany-Castiglioni, 31. Zimmermann, A., Sutter, A. & Shooter, E. M. (1981) Proc. E. (1979) Dev. Biol. 71, 341-355. Natl. Acad. Sci. USA 78, 4611-4615. 9. Burmeister, D. W. & Lyser, K. (1982) Diss. Abstr. Int. 43, 32. Ishii, D. N. (1982) Cancer Res. 42, 429-432. 1334B. 33. Ishii, D. N. (1982) J. Natl. Cancer Inst. 68, 299-303. 10. Sutter, A., Riopelle, R. J., Harris-Warrick, R. M. & Shooter, 34. Ishii, D. N. (1978) Cancer Res. 38, 3886-3893. E. M. (1979) J. Biol. Chem. 254, 4972-4982. 35. Claude, P., Hawrot, E., Dunis, D. A. & Campenot, R. B. 11. Olender, E. J., Wagner, B. J. & Stach, R. W. (1981) J. Neuro- (1982) J. Neurosci. 2, 431-442. chem. 37, 436-442. 36. Stephenson, R. P. (1956) Brit. J. Pharmacol. 11, 379-393. 12. Olender, E. J. & Stach, R. W. (1980) J. Biol. Chem. 255, 9338- 37. Greene, L. A. (1978) J. Cell Biol. 78, 747-755. 9343. 38. Snyder, E. Y. & Kim, S. U. (1980) Brain Res. 196, 565-571. 13. Schechter, A. L. & Bothwell, M. A. (1981) Cell 24, 867-874. 39. Bothwell, M. (1982) J. Neurosci. Res. 8, 225-231. 14. Landreth, G. E. & Shooter, E. M. (1980) Proc. Nati. Acad. 40. Bottenstein, J. E., Skaper, S. D., Varon, S. S. & Sato, G. H. Sci. USA 77, 4751-4755. (1980) Expt. Cell Res. 125, 183-190. Downloaded by guest on September 27, 2021