Vol. 7, 775-785, June 1996 Cell Growth & Differentiation 775

Protein Kinase C-#{128}Is Implicated in Neurite Outgrowth inDifferentiating Human Neuroblastoma Cells’

Sofia Fagerstrom,2 Sven �Carolina Gesthlom,2 and Eewa N#{226}nbergDepartment of Pathology, University Hospital, 5-751 85 Uppsala,

Sweden

Abstract

A combination of basic fibroblast growth factor (bFG9and insulin-like growth factor-I (IGF-l) or 16 n�.i 12-0-tetradecanoylphorbol-13-acetate (TPA) and seruminduces human SH-SY5Y neuroblastoma cells toundergo differentiation and acquire a neuronalphenotype. Nerve growth factor (NGF) added to SH-5Y5Y cells stably transfected with the NGF-receptorTRK-A (SH-SY5YItrk) induces a similar differentiatedphenotype. SH-SY5Y cells express protein kinase C(PKC)-a, PKC-�3l, PKC-e, and PKC-C protein, andphorbol ester- or growth factor-induced differentiationresults in a sustained activation of PKC. The specificPKC inhibitor GF 109203X blocked TPA- and bFGF-IGF-I-induced neurite outgrowth in wild-type SH-SY5Y cellsand NGF-induced neurfte outgrowth in SH-SY5Y/trkcells. When added to differentiated cells, GF 109203Xcaused rapid retraction of growth cone filopodia. InTPA- and bFGF-IGF-l-treated cells, addition of GF109203X also blocked induced expression of growthassociated protein-43 and neuropeptide tyrosine whilethe increase in expression of these two genes was onlyslightly affected by the inhibitor in NGF-treatedSH-SY5Y/trk cells. Thus, a portion of the NGF-inducedphenotypic changes appears not to be mediated viaPKC-dependent signaling. A high concentration of TPA(1.6 g.u�,) down regulated PKC-a and PKC-�3l almostcompletely and PKC-#{128}partially in wild-type SH-SY5Yand SH-SY5Y/trk cells. Cells with down-regulated PKC-a and PKC-�l after I .6 � TPA treatment stilldifferentiated with growth factors. In these cells, thePKC-#{128}level was restored, and the PKC-E protein wasenriched in the growth cones. The I .6 ,.t� TPA-induceddown-regulation of PKC-e was counteracted by bFGF

Received 1 1/27/95; revised 3/8/96; accepted 3/20/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 Supported by grants from the Swedish Cancer Society(S. P., E. N.), TheChildren’s Cancer Foundation of Sweden, HKH Kronprinsessan Lovisasforening fOr bamasjukv#{226}rd, Hans von Kantzows och Ollie och Elof Erics-sons stiftelser (S. P.), and GOran Gustavssons and Magnus BergvallsStiftelser (E. N.).2 Present address: Department of Medicine, Lund University, MalmO Uni-versity Hospital, 5-205 02 MalmO, Sweden.3 To whom requests for reprints should be addressed. Phone: +46-40337403; Fax: +46-40337322; E-mail: sven.pahlman@medforsk.mas.lu.se.

and NGF but not by platelet-derived growth factor orIGF-l. These data indicate that PKC activity is vital forneurite formation, and that the cells can differentiateunder conditions when PKC-a and PKC-�3l areextensively down regulated. The close correlationbetween differentiation and presence of PKC-#{128}proteinsuggests an important function for this isoform duringthis process.

IntroductionSH-SY5Y neuroblastoma cells develop a neuronal sympa-thetic phenotype when grown in serum-containing mediumsupplemented with low concentrations (1 6 nM) of TPA4 (1 , 2).16 nr,i WA is not sufficient for inducing differentiation underserum-free conditions, but do so if combined with one ofseveral defined peptide growth factors e.g., members of thePDGF, IGF, and fibroblast growth factor families (3-5). Acombination of bFGF and IGF-l in the absence of WA alsoinduces differentiation under both serum-free and serum-containing conditions (3). NGF, on the other hand, almostcompletely lacks trophic, mitogenic, and differentiating ef-fects on wild-type SH-SY5Y (SH-SY5Y/wt) cells. However,cells stably transfected with the NGF receptor gene, TRK-A,

(SH-SY5Y/trk) differentiate in response to NGF treatment (6,7). Differentiated SH-SY5Y cells show increased expressionof markers such as GAP-43 and NPY and extend neuriteswith varicosities and growth cones. The growth cones, whichcan be isolated by subcellular fractionation (8, 9), are char-acterized by enrichment of GAP-43, the synaptic vesicleprotein synaptophysmn, and the tyrosin kinases pp60c-�,pp60��, pp5gfYn, and pp62�Yee.

PKC is a family of serine-threonine kinases that consists ofat least 10 members. The isoforms are divided into threegroups: the classical (a, �l, p11, and ‘y)that are Ca2� depend-ent; the novel Ca2�-insensitive forms (6, #{128},0, and i); and theatypical, Ca2�- and phospholipid-insensitive isoforms (� andA) (reviewed in Refs. 10-1 2). PKC has been suggested tohave important functions for growth factor-dependentevents such as gene regulation, growth control, and differ-entiation. The classical and novel PKC isoforms can be ac-tivated by diacylglycerol and phorbol esters. Activation of,e.g., receptor protein tyrosine-kinases, triggers phospho-lipase-dependent hydrolysis of phospholipids, mainly phos-phatidylinositol-4,5-bisphosphate and phosphatidylcholine,which generates diacylglycerol (1 0, 1 1). Receptor-dependentactivation of phosphoinositide- and phosphatidylcholine-

4 The abbreviations used are: WA, 12-O-tetradecanoylphorbol-13-ace-tate; PDGF, platelet-derived growth factor; IGF, insulin-like growth factor;bFGF, basic fibroblast growth factor; NGF, nerve growth factor; wt, wildtype; GAP-43, growth-associated protemn-43; NPY, neuropeptide tyro-sine; PKC, protein kinase C; MARCKS, myristoylated alanine-rich C ki-nase substrate; FBS, fetal bovine serum.

776 PKC-E and Neurite Outgrowth

specific lipases may lead to activation and translocation of

individual PKC isoforms because of generation of diacylg-

lycerol with varying fatty acid composition in the absence orpresence of Ca2� mobilization (13). In addition to differentsensitivity to activating signals, PKC isoforms show varia-tions in susceptibility to down-regulation, in tissue-specificdistribution, and in substrate specificity. It is therefore likelythat they have specific cellular functions. However, little isknown about this issue, mainly because of a lack of identifiedisoform-specific activators, inhibitors, or substrates (re-

viewed in Ref. 14).It has been shown previously that SH-SY5Y cells express

PKC-a, PKC-#{128},and PKC-� protein and that treatment with 16nM TPA and serum for 96 h slightly down regulates PKC-aand PKC-#{128},while PKC-C remains unaffected (15). During thedifferentiation process, a sustained PKC activity (measuredas phosphorylation of MARCKS) is seen (1 6). Treatment with1 .6 p.M TPA in the presence of serum down regulates PKC-atotally and PKC-#{128}partially. These cells still proliferate andshow a poor differentiation response. Taken together, previ-ous data have suggested a role primarily for PKC-a, but alsofor PKC-#{128}during differentiation (1 5). Cells differentiated with

a combination of bFGF-IGF-I also show an elevated phos-phorylation of MARCKS over several days, but this effect isless pronounced than after treatment with 16 nt�i TPA (3).PKC-a and PKC-#{128}are enriched in growth cones preparedfrom SH-SY5Y cells treated either with 16 n� TPA and serumor bFGF-IGF-l. This growth cone enrichment of PKC-a andPKC-#{128}suggests a functional role for these isoforms in growthcone activity (1 5). At least two identified PKC substrates,GAP-43 and pp6Oc�c, are also enriched in growth cones ofdifferentiating SH-SY5Y cells (8), and it is conceivable that

PKC acts via phosphorylation of these proteins.To investigate further the role of PKC-a and PKC-#{128}in

SH-SY5Y/wt and SH-SY5Y/trk cells during growth factor-induced differentiation, defined as neurite outgrowth andinduction of NPY and GAP-43 expression, we have studieddifferentiating cells in which PKC-a, PKC-pl, and PKC-#{128}activities were inhibited with PKC inhibitors or the proteinswere down regulated by treatment with 1 .6 �M TPA.

ResultsPKC Inhibitors Impair Neurite Formation. The hypothesisthat PKC is important for sympathetic neuronal differentia-tion of neuroblastoma cells was addressed by studying theeffect of specific PKC inhibitors on neurite formation andgene expression. Used at 2 p.t�i, a concentration that blocksPKC-dependent phosphorylation of MARCKS (data notshown), the inhibitor GF 109203X (1 7) completely preventedneurite outgrowth induced by 1 6 nM TPA and serum,

whereas bFGF-IGF-l-induced neurite outgrowth in serumwas partially inhibited (Table 1). After 96 h in the absence ofinhibitor, the TPA-treated cells showed a differentiated mor-phology with extended neurites with varicosities and growthcones. These morphological changes were prevented bytreatment with GF 109203X prior to addition of TPA (Fig. 1A).bFGF-IGF-l-treated cells showed extended neurites that

were longer than after TPA treatment, but with fewer van-cosities and growth cones, whereas cells treated with the

Table 1 Effect of GF 109203X on TPA- and growth factor-inducedneurite formation

SH-SY5Y/wt and SH-SY5Y/trk cells were grown and treated as de-scribed in Figs. 1A and 2A before being fixed. For each condition, 300cells from each of triplicate plates were scored. Cells with growth cone-terminated neurites longer than one cell body diameter, or neurites longerthan two cell body diameters, were scored as positive. The values rep-resent means from triplicate plates ± SD.

. . % cells with neuritesAdditions

(Mean ± SD)

SH-SY5Y/wt

Control 19.9 ± 2.3GF 109203X 17.4 ± 0.5916nMTPA 54.3±1.5a

16 n�i WA + GF 109203X 14.3 ± 2.4k’bFGF/1GF-l 64.5 ± 1 .2�

bFGF/IGF-l + GF 109203X 30.6 ± 4.2a, bSH-SY5Y/ta*

Control 18.3 ± 2.5GF 109203X 7.19 ± 2#{149}6bNGF 62.6 ± 448NGF + GF 109203X 25.3 ± 2o�L�

a Statistically different from control cells (P < 0.001).b Statistically different (P < 0.001) from treatment with an identical corn-

pound in the absence of GF 109203X. Data were analyzed by the x� test.

same growth factors and GF 109203X only displayed shortand delicate processes (Fig. 1A). NGF-induced neurite out-growth was also almost completely blocked when the inhib-itor was added to SH-SY5Y/trk cells treated subsequentlywith NGF (Fig. 2A; Table 1). Similar effects of GF 109203X onneunite outgrowth was observed under serum-free condi-tions (data not shown), but cell attachment was affectedseverely, and thus the morphology was poorly developed.Another widely used PKC inhibitor, calphostin C, had aneffect similar to that of GF 109203X with respect to neuniteformation, but in addition led to partial cell detachment evenin serum-containing medium (data not shown) and wastherefore used only in a limited number of experiments. Theinhibition of neurite formation was not a toxic effect of GF109203X, because the inhibitor-treated cultures continued to

proliferate, measured as cell number and thymidine incorpo-ration (data not shown). GF 109203X also had no generaleffect on transcription (see below).

Effect of GF 109203X on Transcriptional Activity.Based on early findings that 16 n� TPA in FBS inducesneurite outgrowth and induction of specific marker genes,e.g., NPY and GAP-43, we investigated the effect of GF109203X on TPA- and growth factor-induced NPY andGAP-43 transcripts detected by in situ hybridization. SixteennM TPA and bFGF-IGF-l strongly increased the amount of

NPY transcript after 24 h. This effect was blocked almostcompletely by pretreatment with GF 109203X for 30 mmbefore addition of TPA or growth factors (Fig. 1B; Table 2).GAP-43 mRNA levels showed the same pattern (data notshown). In SH-SY5Y/trk cells, on the contrary, GF 109203Xonly reduced NGF-induced NPY-expression by approxi-mately 30% (Fig. 2B; Table 2) and with similar effects onGAP-43 expression (data not shown).

Effect of GF 109203X on Growth Cone Structure. Toinvestigate further the importance of PKC for the mainte-

GF 109203X

�-‘ �.

‘\ .�

GF 109203X

,�

b FBS . . a b

d 16nMTPA,.

�..

*�ji. .�w,

‘

. ...

� .... C d

bFGFIIGF.1 -� -�

,,

L�-T�-�-�

:� �‘ � � � - �‘S. � e f”�””�\ f bFGF/IGF.1

4�$�fS e #{149}� � � � .

A SH-SY5Y/wt B

Cell Growth & Differentiation 777

-j� � . i

:�FBS

.“� �. .�: � �‘�‘#{149}

,� �8 � � � ‘-��-.:I6nMTPA � :“ , c

�.‘:i�’\ 4”

.,�. . 4 .‘.-�. \

Fig. 1 . A, phase contrast microscopy of SH-SY5Y/wt cells grown in serum-containing medium. Cells were pretreated for 30 mm with 2 j.LM GE 109203X(b, d, I) before supplementation with: no addition (a, b); 16 nM TPA (C, a); or 3 nM bFGF and 5 n�i IGE-l (e, I); and the incubation was continued for 96 h.B, cells treated as described in A were fixed after 24 h in 4% paraformaldehyde, and NPY in situ hybridization was performed.

nance of already-formed neurites and growth cones, SH-

SY5Y/wt cells differentiated with bFGF-IGF-l under serum-free conditions for 96 h were treated with GF 109203X orvehicle. Within 20 s after addition ofthe PKC inhibitor, growth

cones started to change their structure. Over an 8-mm pe-

nod, the growth cones retracted their filopodia and obtained

a more condensed and clublike shape (Fig. 3) that wasconserved for several days. No neurite retraction or cell

detachment was seen.

Growth Factor-induced Differentiation in PKC Down-regulated Cells. We have reported previously that a highconcentration of TPA (1 .6 �M) only induces poor neuronal

differentiation of SH-SY5Y cells and down regulates PKC-a

completely (1 5, 1 6) and PKC-e to a large extent (1 5). To

investigate further the importance of PKC-a protein on

growth factor-dependent neurite outgrowth and gene regu-

lation, SH-SY5Y cells were pretreated for 24 h with 1 .6 �MTPA before changing to serum-free medium with bFGF-IGF-Iand fresh TPA. These cells differentiated as judged by mor-

phology, and after 96 h of exposure to growth factors, ap-proximately 50% of the cells showed extended long neurites

with varicosities and growth cones and resembled cells dif-

ferentiated with bFGF-IGF-l in the absence of TPA. In con-

trast, control cells and cells treated with 1 .6 p.M TPA in

serum-free or serum-containing medium only displayed

short processes, and those cells were in minority (1 0% and

6%, respectively; Fig. 4A; Table 3; Ref. 1 5). SH-SY5Y/trk

cells were also treated with a high concentration of TPA (1.6

JLM)for 24 h before changing to serum-free medium with NGF

in combination with fresh TPA. These cells showed a differ-

entiated morphology similar to that of NGF-treated SH-

SY5Y/trk cells in the absence ofTPA, whereas, again, control

cells and cells grown in the presence of 1 .6 j�M TPA failed to

form neunites (Fig. 4B; Table 3). In agreement with these

morphological effects, SH-SY5Y/wt cells treated with bFGF-

IGF-l and 1 .6 �M TPA and SH-SY5Y/trk cells treated with

NGF and 1 .6 j.LM TPA for 96 h expressed high levels of NPY

transcript compared to control cells and cells treated with 1.6

�M TPA only. TPA worked synergistically with bFGF-IGF-l

and NGF to affect NPY transcript level (Fig. 5). SH-SY5Y/wt

cells treated with bFGF-IGF-l and 1 .6 �M TPA and SH-SY5Y/

trk cells treated with NGF and TPA also expressed high

amounts of GAP-43 mRNA compared to control cells and

cells treated with 1 .6 j.tM TPA only (Fig. 5). Thus, neurite

formation was accompanied by increased expression of neu-

ronal marker genes.

Restored PKC-#{128}Protein in Growth Factor-differenti-ated Cells. SH-SY5Y/wt cells treated with 1 .6 �M TPAand/or bFGF and IGF-l in serum-free medium for 24 and 96

h as described above were analyzed for their PKC-a, PKC-f31, and PKC-#{128}protein content with isoform specific antibod-

ies. After 24 h, control cells and cells differentiated with

bFGF-IGF-l expressed equally high levels of PKC-a, whereasin cells treated with 1 .6 jiM TPA alone or in combination withbFGF-IGF-l, PKC-a was completely down regulated (Fig. 6A,upper left). After 96 h, control and bFGF-IGF-l-treated cells

still showed intact levels of PKC-a, whereas the protein

remained down regulated in cells treated with 1 .6 �M TPA

alone or in combination with growth factors (Fig. 6A, upper

right). We have not been able to detect PKC-f3 on transcript

A ________ Table 2 Effect of GE 109203X on TPA- and growth factor-inducedNPY expression

FBS

NGF

GF 109203X

. .�. ‘,

\ �: �

, , .� � �

� �“�Z�-.:>�’ .

� �‘i.�

CF 109203X

SH-SY5Y/wt and SH-SY5Y/tr* cells were grown and treated as de-scribed in Figs. 1 and 2 and were fixed after 24 h of treatment. NPYin situhybridization was performed as described in “Materials and Methods.”Five to eight randomly selected microscopic fields from each treatmentwere analyzed by a color-based analysis system. Data in the table rep-resent percentage positive signal area of total cell area ± SD.

. . % signal area of total cell areaAdditions

(Mean ± SD)

SH-SY5Y/wtControl 18.9 ± 6.0GE 109203X 13.7 ± 3.216nMTPA 42.3±9.8”16 n� TPA + GE 109203X 20.0 ± 11b

bEGE/IGE-l 42.6 ± 19”bEGE/IGE-l + GE 109203X 18.2 ± �

SH-SY5Y/trk

Control 16.1 ± 6.2GE 109203X 10.9 ± 5.9

NGF 64.0 ± 3.5”NGF + GE 109203X 42.6 ± 6.4”’�’

Ba Statistically different from control cells (P < 0.001).

b Statistically different (P < 0.001) from treatment with identical corn-

pound in the absence of GE 109203X. Data were analyzed by Fisher’sexact test.

�. : �

;�‘ C

778 PKC-#{128}and Neurite Outgrowth

�i#{149}

. C

FBS #{149} . S � a

NGF

Fig. 2. SH-SY5Y/tr* cells were preplated for 24 h in medium containing10% FBS prior to additions. The cells on the right (b, d) were treated withGE 109203X for 30 mm before supplementation with no additions (a, b) orwith 1 00 ng/ml NGF (c, d). A, phase contrast microscopy 96 h afteradditions. B, NPY in situ hybridization on cells that were fixed 24 h afteradditions.

level in SH-SY5Y cells (1 6). However, a PKC-f3l-specific an-

tiserum recognized a doublet at Mr 80,000 and 82,000, re-

spectively, which was blocked by the corresponding anti-

genic peptide. The bands are very close to PKC-a (Mr

84,000) but were not recognized by PKC-a antiserum after

immunoprecipitation with PKC-�3l antiserum (data not

shown). In the experiment described above, PKC-�l showed

the same expression pattern as PKC-a (data not shown).

Thus, we conclude that SH-SY5Y cells are able to differen-

tiate in the absence of detectable PKC-cx and PKC-131 pro-

teins. As shown in Fig. 6A, lowerleft, control and bFGF-IGF-

I-induced SH-SY5Y/wt cells contained equal amounts of

PKC-#{128}protein. Treatment with 1 .6 �M TPA for 24 h underserum-free conditions down regulated PKC-#{128}to a large ex-

tent, but not completely. In contrast, addition of bFGF-IGF-l

and TPA for 24 h after pretreatment with TPA partially re-

stored the PKC-#{128}protein (Fig. 6A, lowerleft). The effect of thegrowth factors was even more pronounced after 96 h; i.e., in

cells exposed to 1 .6 .LM TPA, PKC-#{128}remained almost corn-

pletely down regulated, whereas cells treated with growth

factor and phorbol ester contained amounts of PKC-#{128}protein

comparable to that of control cells (Fig. 6A, lower right). The

lower band in the blots corresponds to PKC-a, as demon-

strated by sequential immunoprecipitations (data notshown).

d Similarly to SH-SY5Y/wt, the SH-SY5Y/trk cells expressed

PKC-a, -al, -#{128}, and -� protein (data not shown). NGF-differ-

entiated cells expressed slightly higher levels of PKC-a than

control cells after 24 h, whereas treatment with 1 .6 �M TPA

for 24 h under serum-free conditions completely down reg-

ulated PKC-a. PKC-a was also totally down regulated in cells

pretreated with 1 .6 �M TPA prior to subsequent addition of

NGF for 24 h (Fig. 6B, upperleft). This effect of TPA remained

after 96 h (Fig. 6B, upper right), and a similar pattern was

identified for PKC-f31 (data not shown). Control and NGF-

treated cells expressed equal amounts of PKC-#{128}protein,

whereas cells treated for 24 h with TPA alone or in combi-

nation with NGF were almost totally depleted of PKC-#{128}(Fig.6B, lower left). However, after 96 h of treatment with the

combination ofTPA and NGF, PKC-#{128}protein was restored to

the same level as in NGF-treated cells (Fig. 6B, lower right).

These results indicate that addition of differentiating growth

factors counteract TPA-dependent down-regulation of

PKC-#{128}.To investigate further the potential of different growth fac-

tors to prevent TPA-induced down-regulation of PKC-#{128},SH-SY5Y/wt cells were treated with PDGF-BB, bFGF, IGF-l, or

the combination of bFGF-IGF-l in addition to 1 .6 �.tM TPA for

96 h after 24 h of pretreatment with TPA. Western blot

I

‘*��

�

:0

0

�-

�3

0

-�----‘�.

Cell Growth & Differentiation 779

�t

N

�

�-S�

Fig. 3. Time-lapse photographyof SH-SY5Y/wt cells treated with2 jw GE 109203X after treatmentfor 96 h with bEGE-IGE-l. Notethe time-dependent changes instructure in the growth cones (ar-rows).

analysis revealed that PKC-a was completely down regu-

lated with all TPA-growth factor combinations (Fig. 7). The

amount of PKC-#{128}protein, on the other hand, was closely

correlated to the degree of induced morphological differen-

tiation. Second to treatment with bFGF-IGF-l and TPA, bFGF

and TPA induced the longest, although fewer, neurites (Table

3; data not shown). Under these two conditions, PKC-e was

increased compared to cells treated with TPA alone (Fig. 7).

IGF-l and TPA was as efficient as bFGF and TPA to induceneurite formation, but these neunites were shorter (Table 3;

data not shown). The amount of PKC-#{128}in these cells equaledthat in cells treated with TPA only. Finally, PDGF-BB and TPA

gave rise to cells with only a few short processes and poorattachment (Table 3). In these cells, the amount of PKC-#{128}protein was even lower than in cells treated with TPA alone

(Fig. 7).

A ________ Table 3 Effect of 1 .6 �tM TPA and growth factors on neurite formationSH-SY5Y/wt and SH-SY5Y/trk cells were grown for 24 h in serum-

containing medium with or without 1 .6 �M TPA before changing to serum-free medium with additions as indicated. After additional 96 h the cellswere fixed and cells were counted and scored for neurites as described intable 1.

.d�

�

4.�0�� �

. . -�. ‘�Ti�� ‘

SHTE � :. . . � �

.*,�-‘_T.S � %“�

C:. � �

.� �# S

FGF/IGF � -� �.-�-_f .G

1.6�tMTPA

5”

WS � _

1� � �

�. .55 - 5. 5

,T�

/�:: � � . �

� .�

1.6�tMTPA

B SH-SY5Y/trk

#{149}“;c #{149} � � � ‘ � ��. � �

I #{149}���:: �q�43� �.� S � �‘ j � �‘ � .4 ‘,�.‘ .. Sg” ( �. I �l’ � #{149}“ � .5 1-.‘: � � 5- � S �

. a #{149}:. S.

a Statistically different from control cells (P < 0.001).

b Statistically different from cells treated with 1 .6 �M TPA (P < 0.001). Data

were analyzed by the � test.

SHTE

NGF

Fig. 4. A, phase contrast microscopy of SH-SY5Y/wt cells cultured for96 h under serum-free conditions. Cells were preplated for 24 h in serum-containing medium, with addition of 1 .6 p�i TPA (b, d), before changing toserum-free medium. After changing medium, cells were treated with: noaddition (a); fresh TPA (1 .6 �.u�i; b); 3 n� bEGE and 5 ni�i IGE-l (C); and 1.6p.M TPA in combination with bEGE and IGE-l (d) for 96 h. B, phase contrastmicroscopy of SH-SY5Y/trk cells preplated in serum-containing mediumand pretreated with 1 .6 j.�M TPA (b, d) as described for SH-SY5Y/wt cellsabove. After changing to serum-free conditions, the medium was supple-mented with: no additions (a); 1 .6 �.tM TPA (b); 1 00 ng/rnl NGE (C); and TPAand NGE (d) for 96 h.

7� PKC-#{128}and Neurite Outgrowth

b

� � ----� �� S

-�--�----c.:::3ic�� 55

‘ � ) � � � ) :‘

PKC-#{128}Is Enriched in Growth Cones in Cells Differen-tiated by the Combination of Growth Factors and I .6 g�MTPA. In a previous work (1 5), we have shown that growthcones isolated from SH-SY5Y cells differentiated with 1 6 nM

TPA in serum or bFGF-IGF-l in serum-free medium, are

enriched in PKC-a and PKC-#{128},as compared to cell body

contents. Here, we demonstrate that growth cones prepared

from SH-SY5Y/trk cells, induced by NGF, showed similar

growth cone enrichment of PKC-a and PKC-#{128}(Fig. 8).

Growth cones were also isolated from SH-SY5Y/wt cells

differentiated with bFGF-IGF-l and 1 .6 �.tM TPA and SH-SY5Y/trk cells treated with NGF and 1 .6 .tM TPA for 96 h,respectively. Western blot analysis revealed that cell bodies

and growth cones from both preparations were virtually de-

.Additions

% cells with neurites(Mean ± SD)

SH-SY5Y/wt

Control 9.64 ± 1.11.6 �.tM TPA 6.19 ± 0.65bEGE/IGE-l 56.4 ± 4.3” b1.6 �M TPA + bEGE/IGE-l 50.6 ± 0.51” b1 .6 �.tM TPA + PDGE-BB 8.00 ± 2.1

1.6p.�iTPA+bEGE 216#{247}59”.b

1.6 �M TPA + IGE-l 19.5 ± 1.3� bSH-SY5Y/trk

Control 12.1 ± 0.26

1.6 .w TPA 9.32 ± 2.3NGE 70.0 ± 4.1� b

1.6 �t�i TPA + NGE 60.5 ± 2.4” b

void of PKC-a (Fig. 8). However, both cell types contained

PKC-#{128}(as shown in Fig. 6), and the protein was enriched ingrowth cones (Fig. 8). Thus, the growth factor-induced res-

toration of PKC-#{128}protein in differentiated cells described

above correlates with a relative enrichment of PKC-#{128}in the

growth cones.

DiscussionIn previous reports, conflicting conclusions have been pre-

sented as to whether SH-SY5Y differentiation is caused byinhibition (18, 19) or by activation (16, 20) of PKC. Here, wereport data that further support the latter hypothesis. First,PKC activity was required for neurite outgrowth and main-tenance of growth cone structure in the defined differentia-

tion protocols used. Secondly, TPA- and bFGF-IGF-l-in-

duced development of the differentiated phenotype as

defined by expression of the differentiation marker genes

NPY and GAP-43, was blocked by PKC inhibitors. In addi-

tion, our data suggest a specific role for PKC-#{128}in growth

cone function.To study the importance of PKC for morphological and

biochemical differentiation, two approaches have been used,one employing specific PKC inhibitors and another usinghigh TPA concentrations to down regulate primarily classical

PKC isoforms. The PKC inhibitor GF 109203X blocked TPA-and bFGF-IGF-l-induced NPY and GAP-43 expression, mdi-cating a PKC-dependent transcriptional control of these

genes. Our results also demonstrate that NGF-evoked ex-pression of these two genes was to a much lesser extent

achieved via activation of PKC. This in turn means that

NGF-TRK-A signaling uses an alternative signal transductionpathway different from that of TPA and bFGF-IGF-l. ThatNGF can regulate NPY expression independently of PKC inSH-SY5Y/trk cells is supported further by an observation that

activation of a NPY-promotor-chloramphenicol acetyltrans-

GAP-43 -

NPY -

GAPDH -

S

�- -

,��� #{231}�c�

����I) - .0 .0+ (11

,����

�. �

,-� zz#{247}

Fig. 5. GAP-43 and NPY rnRNA levels in differentiating SH-SY5Y cells.Cells were preplated for 24 h in serum-containing medium and then grownunder serum-free conditions with additions as indicated in the figure. After96 h, cells were harvested, and total RNA was separated on a formalde-hyde agarose gel and transferred to Hybond C filters by the method ofNorthern blotting before hybridizing with probes for GAP-43 and NPY,respectively. Glyceraldehyde-3-phosphate dehydrogenase expressionwas used as reference for loaded RNA.

5 I. Johansson, V. Larsson, C. Minth-Worby, S. P#{226}hlman, and G.Andersson. Transcription of the human NPY gene in differentiatingSH-SY5Y neuroblastorna cells is dependent on AP-1 and NGE-respon-sive elements, manuscript in preparation.

Cell Growth & Differentiation 781

SH-SY5Y/wt SH-SY5Y/trk�

.:S�M

#{149}..,

ferase reporter construct is inhibited totally by GF 109203X in

TPA- but only partially in NGF-treated SH-SY5Y/trk cells.5

Present and previously published findings that PKC-a and

PKC-e, but not PKC-�, are enriched in growth cones isolatedfrom differentiated SH-SY5Y cells make these two PKC iso-forms interesting candidates for involvement in the induction

of neunite outgrowth and growth cone function. We now

demonstrate that PKC activation as such is essential formorphological differentiation of SH-SY5Y neuroblastoma

cells, because TPA-, bFGF-IGF-l- and NGF-induced neurite

formation was blocked by treatment with PKC inhibitors. This

is in agreement with a previous report on the effect of GF

1 09203X on TPA-induced differentiation (20). Once the neu-

rites and growth cones are formed, sustained PKC activity

appears to be essential for growth cone functionality also

later during differentiation, as illustrated by the retraction of

filopodia in differentiated cells treated with PKC inhibitors.

The major cytoskeletal component of the filopodia are actin

filaments (reviewed in Ref. 21). The PKC inhibitor-inducedretraction ofthe filopodia suggests that active PKC is needed

for actin polymerization and formation of filopodia. This con-

clusion is in agreement with several reports describing a role

for PKC in actin polymerization and reorganization in non-

neuronal cell systems (22-24). However, the inhibitors used

in this study are not PKC isoform-specific, and no such

inhibitors or activators are available. To demonstrate a po-

tential role for specific PKCs, we have used instead the

strategy to down regulate susceptible PKCs (mainly PKC-a

and PKC-j31) by pretreatment with a high concentration of

TPA. Activated PKC is cleaved in the hinge region into theregulatory and catalytic subunits that are further proteolyti-

cally degraded (reviewed in Ref. 25). Previous reports have

also demonstrated that mutated, catalytically inactive PKC-a

undergoes proteolytic degradation upon binding of phorbol

ester (26). Thus, prolonged treatment of cells with high con-

centrations of phorbol esters leads presumably to both ac-

tivation-dependent and -independent down-regulation of

PKC protein, with the exception of the phorbol ester-insen-sitive isoforms such as PKC-�. This has been used exten-

sively as a method to study PKC dependency but is princi-

pally different from the use of specific inhibitors, inasmuch as

it allows activation of newly translated protein. In the SH-

SY5Y cells, 2-5 days of treatment with 1 .6 j.�M TPA downregulated the classical PKCs PKC-a and PKC-131 almost

completely, whereas PKC-#{128}was down regulated to a much

lesser degree. Under these conditions, bFGF-IGF-l and NGF

were still able to induce neurite outgrowth. In fact, these cells

showed an even more differentiated phenotype compared

with cells treated with growth factors alone, as revealed by

GAP-43 and NPY mRNA measurements. A role for PKC-aand PKC-�l during initiation of differentiation cannot be ruled

out, because the down-regulation protocol allows a small

residual PKC activation, but the results show that differenti-

ation can continue for several days in almost complete ab-

sence of these PKC isozymes.The finding that preformed growth cones retracted their

filopodia upon treatment with PKC inhibitors indicates that

normal growth cone function cannot be maintained without

active PKC. As already mentioned in the “Introduction,” 5ev-

eral PKC substrates are found in neuronal growth cones, and

among these are pp6Oc&c and GAP-43 (8, 27, 28). Recent

reports using transgenic GAP-43-depleted mice (29) andanimals with deregulated postnatal expression of GAP-43

(30) have demonstrated the importance of this protein during

neuronal sprouting and outgrowth. Interestingly, expression

of a PKC phosphorylation-site mutant (31) failed to promote

neuronal sprouting (30). Because growth cone integrity was

maintained with PKC-a and PKC-�l being virtually absent,

PKC-#{128},due to its expression pattern, remains an attractivecandidate for providing the PKC activity needed for growthcone function.

PKC-E was partially down regulated by 1 .6 �M TPA, butafter 96 h of differentiation induced by bFGF-IGF-l or NGF in

the presence of this high TPA concentration, the amount of

PKC-#{128}protein was restored to almost the same level as incells differentiated by growth factors alone. The TPA-

96h

782 PKC-#{128}and Neurite Outgrowth

A24h

SH-SY5YIwt B

24h

SH�SY5Y/frk

96h

PKC-a--- � PKC-a--

.-� p � ,�

PKC-c :: � � � �

- -.S. �

- PKC-c =� - �. .

‘d� ��“ �4 �4

� c� c��� �z � �� �. c� #{231}�o� �,a � �(l� .-� ,0 .0+ cl�

.,� -� �4

�- ��� �S4

�. c�� �4

- .0 .0+

..� � 4��l � �4�- � �-

� �. �4 �o � �. �� � �.� �q � � � �q #{231}�c1� ‘- z z+ Cl) ,-� Z Z+

Fig. 6. A, PKC-cr and PKC-#{128}proteins in SH-SY5Y cells. Total lysates were prepared from cells cultured under serum-free conditions, with addition of 1.6�.tM TPA and/or 3 nM bEGE and 5 n� GE-I for 24 and 96 h, respectively. One hundred �tg protein/sample were separated by SDS-PAGE and transferredto nitrocellulose filters. The irnrnunoreactivities of PKC-a and PKC-#{128}were detected by isoform-specific antisera. Bars to the right indicate the positions ofMr markers of 98,000 and 69,000, respectively. B, total cell lysates from SH-SY5Y/trk cells grown in serum-free medium supplemented with 1 .6 �M WAand/or 100 ng/rnl NGE for 24 and 96 h, respectively, were analyzed for PKC-cr and PKC-#{128}protein as described in A.

dependent decrease of total PKC-#{128}protein was prevented by

NGF, bFGF-IGF-l, or bFGF alone, but not by IGF-l or PDGF,indicating that NGF and bFGF specifically are involved in the

regulation of PKC-#{128}protein. Whether the higher PKC-#{128}pro-

tein level in differentiated cells is a consequence of de-

creased down-regulation of protein, transcriptional regula-

tion and/or increased mRNA stability remains to be

investigated. There are a fair number of reports in the liter-

ature that describes an altered expression pattern of PKC

isozymes during differentiation of various cell lines, although

only a few of these reports have demonstrated an isoform-

specific induction of differentiation. One such example isTPA-induced macrophage differentiation of HL-60 cells,

which requires and is driven by activated PKC-�l (32, 33).

PKC-� has been reported to drive maturation of amphibianoocytes (34) and proliferation of murine fibroblasts (35),

whereas stable overexpression of PKC-� in the human

monocytic cell line U937 induced an increased AP-1 activity

concomitant with a longer doubling time and a more differ-

entiated phenotype (36). Also, PKC-a seems to have distinct

cell type-specific functions. Liao and Jaken (37) have re-

ported PKC-a to be localized in focal adhesion sites and

possibly involved in cell attachment and migration of REF-52

cells. Interestingly, during progressive SV4O-driven transfor-

mation of REF-52 cells focal adhesion association of PKC-a

was lost with increasing anchorage-independent growth.The total amount of PKC-a remained constant, whereas

PKC-� was up regulated (38). On the other hand, expressionof an antisense PKC-a construct in U-87 glioma cells in-

duced longer doubling time and prevented tumor growth inxeno-transplanted nude mice (39).

To address the question whether the induction of PKC-#{128}in

differentiated cells promotes or is a consequence of differ-

entiation, SH-SY5Y/wt cells were transfected transiently with

a constitutively active PKC-e (PKC-#{128}E159) that has beenpoint mutated in the pseudosubstrate region (40). PKC-

#{128}E159-transfected SH-SY5Y/wt cells grown in serum or

treated with bFGF, IGF-l, or 1 6 nM TPA under serum-free

conditions, did not show any morphological changes or

tendency to form neurite-like processes. Neither did PKC-

#{128}E159-transfection potentiate bFGF-IGF-l-induced neurite

outgrowth in SH-SY5Y cells or NGF-induced neurite out-

growth in SH-SY5Y/trk cells (data not shown). Thus, PKC-#{128}

activity alone as produced in these transfection experiments

did not seem to be sufficient to initiate neunite outgrowth or

to bypass or potentiate the differentiation pathways used by

growth factors. We therefore conclude that PKC-#{128}protein is

specifically up regulated during and probably as a conse-

PKC-oc--- � S..

PKC-a- �- - � S.

PKC-E = r� � � 1�

Cell Growth & Differentiation 783

SHSY5Y/wt SH-SYSY/wt SH-SY5Y/trk

.�..;

:‘� ��

PKC-c :: � �J � �

TPA - + + #{247} + #{247}

��

U I �‘4 � �

Fig. 7. PKC-a and PKC-#{128}proteins in total cell lysates from SH-SY5Y/wtcells treated for 96 h with: no addition; 1 .6 �M TPA; or 1 .6 �M TPA incombination with either PDGE-BB, bEGE, IGE-l, or bEGE-IGE-l underserum-free conditions. TPA cells (1 .6 �M) were pretreated for 24 h beforeadditions of growth factors and fresh TPA.

quence of differentiation, and that this process can be in-

duced by specific growth factors. Furthermore, the data

point to a vital role of PKC-e in neurite extension and main-

tenance, two processes for which a functional growth coneis essential.

Materials and MethodsCell Culture. The human neuroblastorna cell lines SH-SY5Y (41, 42) andSH-SY5Y/trk (6) were cultured in Eagle’s MEM supplemented with 10%

EBS (Life Technologies, Inc.), 100 lU/mI penicillin, and 50 j�g/rnl strepto-mycin in an atmosphere of 5% CO2 at 37#{176}C.For all differentiation proto-

cols, 1 06 cells/i 0-cm or 50,000 cells/3.5-crn culture dish were plated in

serum-containing Eagle’s MEM. GE 109203X (Calbiochern) was dissolvedin DMSO. Cells treated with GE i09203X were preplated for 24 h before

additions. GE 109203X was added 30 mm before growth factors or TPA.For differentiation induced under serum-free conditions, the cells werewashed twice with RPMI 1640 before changing to SHTE medium (RPMI

1 640 containing 30 n� selenium, 1 0 n�,i hydrocortisone, 30 �g/rnl trans-femn, 10 nM f3-estradiol). At the same time, 3 np�i bEGE (Promega) and 5

nM GE-I (Kabi-Pharmacia) (3), 100 ng/rnl NGE (Prornega) or 10 ng/rnlPDGE-BB (provided generously by Dr. C-H. Heldin) were added (6).

Quantitatlon of Neurites. Cells were grown and treated as describedabovefor 96 h before being fixed in 4% paraformaldehyde for 1 mm. Threehundred cells from each of triplicate plates were scored in two separateexperiments. Cells with at least one growth cone-terminated neurite Ion-ger than one cell body diameter, or with neurites longer than two cell bodydiameters, were scored as positive.

In Situ Hybridization and Northern Blot Hybridization. Antisense35S-Iabeled riboprobes used for in situ hybridization were transcribedfrom NPY and GAP-43 cDNA sequences cloned into a pBlue SK+ vector

and a pGern-3 vector, respectively. Sense RNA was employed as negative

controls. Cells were seeded, stimulated, and fixed as described above.The cells were then hybridized with 1 .8 x 1 06 cprn of antisense or sense

RNA for 4 h at 50#{176}C,followed by RNase treatment, as described (43). Thecells were exposed to Kodak NTB2 emulsion for 10-1 2 days before

development and counterstaining. The intensity of the signal was ana-

CB GC CB GC CB GC CB GC

!� �I �

Fig. 8. Subcellular distribution of PKC isoforms in SH-SY5Y/wt and SH-SY5Y/tr* cells. Left, SH-SY5Y cells differentiated for 96 h in serum freemedium by bEGE-IGE-I or 1 .6 p�i TPA and bEGF-IGE-I after pretreatmentwith WA were fractionated into cell bodies and growth cones. Onehundred �Lg protein from each fraction were resolved by SDS-PAGE andtransferred to nitrocellulose filters. The PKC isoforms were detected byPKC-a- and PKC-#{128}-specific antisera. Right, SH-SY5Y/trk cells were in-duced to differentiate in serum-free medium by NGF alone or by thecombination of 1 .6 �M TPA and NGE after pretreatment with WA for 24 hin serum-containing medium. After 96 h, cell body and growth conefractions were prepared, and PKC-a and PKC-#{128}protein was detected asdescribed above.

lyzed by a color-based analysis system developed at the Centre for Image

Analysis, Uppsala University (44), and the signal was expressed as per-

centage signal area of total cell area. Total RNA was extracted by theguanidin-isothiocyanate-phenol-chloroform method (45). Ten �g RNA’

sample were separated on a formaldehyde agarose gel and transferred to

Hybond C Extra filters (Arnersharn) by the method of Northern blotting

(46). The filters were hybridized in 50% formamide for 20 h at 42”C with

probes for either NPY (47), GAP-43 (48), or glyceraldehyde-3-phosphatedehydrogenase(49)Iabeled with 32P-deoxyribonucleotides by the methodof random priming (Amersham). The filters were washed in 2 x SSC (1 5 m�i

sodium citrate, 300 mM sodium chloride) and 0.5% SDS for 30 mm at

55”C.

Immunodetection of PKC. Total cell homogenates were prepared by

lysing cells in radioirnmunoprecipitation assay buffer [10 rn�i Tris-HCI (pH

7.2), 160 mM NaCI, 1 % Triton X-i00, 1% sodium deoxycholate, 0.1%

SDS, 1 nM EGTA, 1 n�i EDTA, 10 �g/rnl aprotinin, 10 �g/rnl leupeptin, and1 mM phenylrnethylsulfonyl fluoride] followed by sonication for 1 0 s and

centrifugation. One hundred �g of proteiMane [determined by the methodof Lowry et aL (50)] were separated by 7.5% SDS-PAGE (51), followed by

blotting to Hybond C Extra filters (52). Filters were incubated with 2 �g/rnl

polyclonal PKC-a antiserum (Life Technologies, Inc.), 0.1 pg/mI polyclonalPKC-/3l antiserum (Santa Cruz Biotech), or 2 �g/rnl polyclonal PKC-#{128}

antiserum (Santa Cruz Biotech), respectively, as described (1 5). The im-

munoreactivity was detected by the enhanced chernilurninescence

method.Subcellular Fractionation. Growth cones and cell bodies from differ-

entiated SH-SY5Y/wt and SH-SY5Y/trk cells were isolated by subcellular

fractionation as described previously (8). Briefly, after harvesting, cells

were homogenized in a Teflon/glass homogenizer in ice-cold EDTA buffer[0.54 m�i EDTA, 137 mM NaCI, 10 rn�i NaHPO4, 2.7 rn�i KCI, 0.15 mp�i

KH2PO4, (pH 7.4)] and layered onto a 20% sucrose cushion and centri-

fuged for 4 mm at 500 x g. Growth cones were recovered in the EDTAbuffer supernatant and cell bodies in the pellet. Each fraction was cen-

trifuged at 20,000 x g for 20 mm. After lysis in ice-cold RIPA, 100 �g of

each fraction was separated by SDS-PAGE followed by imrnunoblotting

as described above.

784 PKC-E and Neurite Outgrowth

AcknowledgmentsWe thank Dr. Peter Parker for providing the PKC-#{128}E159construct and Dr.

June Biedler for providing the SH-SY5Y cells.

References1 . PAhlman, S., Odelstad, L, Larsson, E., Grotte, G., and Nilsson, K.

Phenotypic changes of human neuroblastoma cells in culture induced by12-tetradecanoyl-phorbol-13-acetate. Int. J. Cancer, 28: 583-589, 1981.

2. P#{226}hlman,S., Ruusala, A-I., Abrahamsson, L, Odelstad, L, and Nils-son, K. Kinetics and concentration effects of TPA-induced differentiation

of cultured human neuroblastoma cells. Cell Differ., 12: 165-170, 1983.

3. Lavenius, E., Parrow, V., N#{226}nberg,E., and P#{227}hlman,S. Basic EGF andIGF-1 promote differentiation of human SH-SY5Y neuroblastoma cells inculture. Growth Factors, 10: 29-39, 1994.

4. P#{227}hlman,S., Meyerson, G., Undgren, E., Schalling, M., and Johans-

son, I. Insulin-like growth factor-i shifts from promoting cell division topotentiating maturation during neuronal differentiation. Proc. NatI. Aced.Sci. USA, 88: 9994-9998, 1991.

5. P#{227}hlman,S., Johansson, I., Westermark, B., and Nist#{233}r,M. Platelet-

derived growth factor potentiates phorbol ester-induced neuronal differ-entiation of human neuroblastorna cells. Cell Growth & Differ., 3:783-790,

1992.

6. Lavenius, E., Gestblom, C., Johansson, I., N#{225}nberg,E., and P#{226}hlman,

S. Transfection of TRK-A into human neuroblastoma cells restores theirability to differentiate in response to nerve growth factor. Cell Growth &

Differ., 6: 727-736, i995.

7. Poluha, W., Poluha, D. K., and Ross, A. H. TrkA neurogenic receptorregulates differentiation of neuroblastoma cells. Oncogene, 10: 18�-189,1995.

8. Meyerson, G., Pfenninger, K. H., and P#{226}hlman,S. Acomplex consistingof pp�0c&C/pp�oCS�CN and a 38-kDa protein is highly enriched in growthcones from differentiated SH-SY5Y human neuroblastoma cells. J. CellSci., 103: 233-243, 1992.

9. Meyerson, G., and P#{226}hlman,S. pp59� and pp62cY�� are enriched inSH-SY5Y neuroblastorna growth cones but do not associate to the 38-kDa protein which complexes with pp60cs�� and pp60csrcN. FEBS Left.,332: 27-30, 1993.

10. Nakamura, S., and Nishizuka, Y. Lipid mediators and protein kinase C

activation for the intracellular signaling network. J. Biochem. (Tokyo), 115:1029-1034, 1994.

1 1 . Nishizuka, Y. Intracellular signaling by hydrolysis of phospholipidsand activation of protein kinase C. Science (Washington DC), 258: 607-614, 1992.

12. Stabel, S., and Parker, P. J. Protein kinase C. Pharmacol. & Ther., 51:71-95, 1991.

13. Ha, K-S., and Exton, J. H. Differential translocation of protein kinase

C isozymes by thrombin and platelet-derived growth factor. J. Biol.Chern., 268: 10534-10539, 1993.

14. Dekker, L V., and Parker, P. J. Protein kinase C: a question ofspecificity. Trends Biochem. Sci., 19: 73-77, 1994.

15. Parrow, V., Fagerstrom, S., Meyerson, G., N#{225}nberg,E., and P#{227}hlman,S. Protein kinase C-a and -#{128}are enriched in growth cones of differenti-ating SH-SY5Y human neuroblastoma cells. J. Neurosci. Res., 41: 782-791, 1995.

16. Parrow, V., N#{225}nberg,E., Helkkilft, J., Hammerling, U., and P#{226}hlman,S.

Protein kinase C remains functionally active during WA induced neuronal

differentiation of SH-SY5Y human neuroblastoma cells. J. Cell. Physiol.,

152: 536-544, 1992.

17. Toullec, D., Pianefti, P., Coste, H., Bellevergue, P., Grand-Perret, T.,

Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L,

Charon, D., and Kirilovsky, J. The bisindolylmaleimide GE 109203X is apotent and selective inhibitor of protein kinase C. J. Biol. Chem., 266:15771-15781, 1991.

18. Leli, U., Parker, P. J., and Shea, T. B. Intracellular delivery of protein

kinase C-a or -#{128} isoform-specific antibodies promotes acquisition of amorphologically differentiated phenotype in neuroblastoma cells. FEBSLeft., 297: 91-94, 1992.

19. Shea, T. B., Beermann, M. L, Leli, U., and Nixon, R. A. Opposinginfluences of protein kinase activities on neurite outgrowth in humanneuroblastorna cells: initiation by kinase A and restriction by kinase C. J.Neurosci. Res., 33: 398-407, 1992.

20. Heikkil#{228},J., Jalava, A., and Eriksson, K. The selective protein kinaseC inhibitor GE 109203X inhibits phorbol ester-induced morphological andfunctional differentiation of SH-SY5Y human neuroblastoma cells. Bio-chem. Biophys. Res. Commun., 197: 1 185-1 193, 1993.

21 . Smith, S. J. Neuronal cytomechanics: the actin-based motility of

growth cones. Science (Washington DC), 242: 708-715, 1988.

22. Apgar, J. R. Regulation of the antigen-induced F-actin response in ratbasophilic leukemia cells by proteln kinase C. J. Cell Biol., 112: 1157-1163,1991.

23. Downey, G. P., Chan, C. K., Lea, P., Takai, A., and Grinstein, S.Phorbol ester-induced actin assembly in neutrophils: role of protein kinase

C. J. Biol. Chem., 116: 695-706, 1992.

24. Trachsel, S., and Keller, H. U. Selective responses (actin polymeriza-tion, shape changes locomotion, pmnocytosis) to the PKC inhibitor Ro31-8220 suggest that PKC discriminately regulates function of humanblood lymphocytes. J. Leukocyte Biol., 57: 587-591 , 1995.25. Hug, H., and Sarre, T. F. Protein kinase C isoenzymes: divergence insignal transduction? Biochern. J., 291: 329-343, 1993.

26. Pears, C., and Parker, P. J. Down-regulation of a kinase defectivePKC-a. FEBS Left., 284: 120-122, 1991.

27. Helmke, S., and Pfenninger, K. H. Growth cone enrichment andcytoskeletal association of non-receptor tyrosmne kinases. Cell Motil. Cy-toskeleton, 30: 194-207, 1995.

28. Maness, P. F., Aubry, M., Shores, C. G., Frame, L, and Pfenninger, K.H. c-Sit gene product in developing rat brain is enriched in growth conemembranes. Proc. NatI. Aced. Sci. USA, 85: 5001-5005, 1988.

29. Striftmafter, S. M., Eankhauser, C., Huang, P. L, Mashimo, H., andFishman, M. C. Neuronal pathfinding is abnormal in mice lacking theneuronal growth cone protein GAP-43. Cell, 80: 445-452, 1995.

30. �4Jgner, L, Arber, S., Kapfhammer, J. P., Laux, T., Schneider, C.,Botteri, F., Brenner, H-R., and Caroni, P. Overexpression of the neuronalgrowth-associated protein GAP-43 induces nerve sprouting in the adultnervous system of transgenic mice. Cell, 83: 269-278, 1995.

31 . Widmer, F., and Caroni, P. Phosphorylation-site mutagenesis of the

growth-associated protein GAP-43 modulates its effects on cell spreadingand morphology. J. Cell Biol., 120: 503-512, 1993.

32. Tonefti, D. A., Henning-Chubb, C., Yamanishi, D. T., and Huberman,E. Protein kinase C-f3 is required for macrophage differentiation of humanHL-60 leukemia cells. J. Biol. Chem., 269: 23230-23235, 1994.

33. Macfarlane, D. E., and Manzel, L Activation of p-isozyme of proteinkinase C (PKC 13) Is necessary and sufficient for phorbol ester-induceddifferentiation of HL-60 prornyelocytes. Studies with PKC �3-defective PETmutant. J. Biol. Chem., 269: 4327-4331 , 1994.34. Cornet, M. E., Sanz, L, and Moscat, J. Evidence for a role of proteinkinase C � subspecies in maturation of Xenopus Iaevis oocytes. Mol. Cell.Biol., 12: 3776-3783, 1992.

35. Berra, E, Diaz-Meco, M. T., Dominguez, I., Municio, M. M., Sanz, L,Lozano, J., Chapkin, R. S., and Moscat, J. Protein kinase C � isoform iscritical for mitogenic signal transduction. Cell, 74: 555-563, 1993.

36. Ways, D. K., Posekany, K., deVente, J., Garris, T., Chen, J., Hooker,J., Qin, W., Cook, P., Fletcher, D., and Parker, P. Overexpression ofprotein kinase C-C stimulates leukemic cell differentiation. Cell Growth &Differ., 5: 1 195-1203, 1994.

37. Liao, L, and Jaken, S. Effect of a-protein kinase C neutralizing anti-bodies and the pseudosubstrate peptide on phosphorylation, migration,

and growth of REF52 cells. Cell Growth & Differ., 4: 309-316, 1993.

38. Uao, L, Ramsey, K., Jaken, S. Protein kinase C isozymes In progres-sively transformed rat embryo fibroblasts. Cell Growth & Differ., 5: 1185-1194, 1994.

39. Ahmad, S., Mineta, T., Martuza, R. L, Glazer, R. I. Antisense expres-sion of protein kinase C-a inhibits the growth and tumorigenicity of human

glioblastoma cells. Neurosurgery (Baltimore), 35: 904-909, 1994.

Cell Growth & Differentiation 785

chem., 162: 156-159, 1987.

40. Genot, E. M., Parker, P. J., and Cantrell, D. A. Analysis of the role ofprotein kinase C-a-#{128}and -� in T cell activation. J. Biol. Chem., 270:9833-9839, 1995.

41 . Biedler, J., Helson, L, and Spengler, B. Morphology and growth,tumorigenicity and cytogenetics of human neuroblastoma cells in contin-

uous culture. Cancer Res., 33: 2643-2652, 1973.

42. Biedler, J., Roffler-Tarlov, S., Schachner, M., and Freedman, L Mul-tiple neurotransmitter synthesis by human neuroblastoma cell lines and

clones. Cancer Res., 38: 3751-3757, 1978.43. Meyerson, G., Parrow, V., Gestblom, C., Johansson, I., and P#{226}hlman,

S. Protein synthesis and mRNA in isolated growth cones from differenti-ating SH-SY5Y neuroblastoma cells. J. Neurosci. Res., 37: 303-312,1994.

44. Ranefall, P., Nordin, B., and Bengtsson, E. A new metric in colourspace used for supervised and unsupervised image segmentation. In: K.Malmquist (ed.), Proceedings of Swedish Society for Automated ImageAnalysis: Symposium on Image Analysis, pp. 77-80. HOgskolanHalmstad, Halmstad, Sweden, 1994.

45. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolationby acid auanidinium thiocvanate-nhenol-chloroform extraction. Anal. Rio-

46. Sambrook, J., Fritsch, E., and Maniatis, T. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Labo-ratory, 1989.

47. Minth, C., Andrews, P., and Dixon, J. Characterization, sequence, andexpression ofthecloned human neuropeptide Y gene. J. Biol. Chem., 261:11974-11979, 1986.

48. Ortoft, E., P#{226}hlman,S., Andersson, G., Parrow, V., Betsholtz, C., andHammerling, U. Human GAP-43 gene expression. Multiple start sites forinitiation of transcription in differentiating human neuroblastoma cells.

Mol. Cell. Neurosci., 4: 549-561 , 1993.49. Tso, J., Sun, X-H., Kao, T-H., Reese, K., and Wu, A. Isolation and

characterization of rat and human glyceraldehyde-3-phosphate dehydro-genase cDNAs: genomic complexity and molecular evolution of the gene.Nucleic Acids Res., 13: 2485-2502, 1985.

50. Lowry, 0., Rosebrough, N., Farr, A., and Randall, R. Proteln mess-urement with thefolin phenol reagent. J. Biol. Chem., 193: 265-275, 1951.

51 . Laemmli, U. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.

52. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer ofproteins from polyacrylamide to nitrocellulose sheets: procedures andsome applications. Proc. NatI. Acad. Sd. USA, 76: 4350-4354, 1979.