Vol. 8, 861-869, August 1997 Cell Growth & Differentiation 861

Characterization of Platelet-derived Growth Factor (PDGF)Action on a Mouse Neuroblastoma Cell Line, NB4I , byIntroduction of an Antisense PDGF p3-Receptor RNA1

Keiko Funa2 and Aive AhgrenLudwig Institute for Cancer Research, Biomedical Center, 5-751 24

Uppsala, Sweden

Abstract

We have shown previously that platelet-derived growthfactor (PDGF) has trophic effects on dopaminergicneurons in vitro. We now examined a mouseneuroblastoma cell line, NB41 , for its response toPDGF and studied their phenotypic characteristicsfollowing introduction of an antisense PDGF 13-receptorRNA. NB41 cells produce both PDGF-AA and -BB;however, they carry only PDGF 13-receptors,responding to BB but not to AA. Culturing the cellswith PDGF-BB induced mRNA for c-fos and PDGF-13receptor as well as that of neuron-specific enzyme,tyrosine hydroxylase. In contrast, mRNA ofchromogranin A, which is produced by chromaffincells, decreased. Introduction of an antisense PDGFfl-receptor RNA in NB41 cells completely suppressedneurite extension and cell growth. We compared thePDGF-13 receptor sense and antisense clones for theirsurvival. Following serum withdrawal, NB4I cellsshowed a DNA ladder, which by an addition of theneurotoxin, 6-hydroxy dopamine (6-OHDA), resufted ina further enhancement of the DNA ladder. The additionof PDGF-BB prior to 6-OHDA rescued cells fromundergoing apoptosis, seen as a reduction of the DNAladder. The antisense clone, regardless of the presenceof PDGF-BB in the culture, showed a pronounced DNAladder after serum withdrawal, which was furtherenhanced by the addition of 6-OHDA.

IntroductionPDGF,3 having been characterized primarily as a potent mi-togen for mesenchyme-denved cells (1 , 2), has recently been

Recelved 8/i 3/96; revised 4/30/97; accepted 5/i 5/97.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 This work was partly supported by the Fredrik and Ingrid Thuring Foun-dation and grants from the the Swedish Lundbeck Fund and the WibergFoundation.2 To whom requests for reprints should be sent, at present address:Department of Anatomy and Cell Biology, University of GOteborg, Medici-naregatan 3-5, 5-413 90 Gothenburg, Sweden. Phone: 46 31 773-3300;Fax: 46 31 773-3330.3 The abbreviations used are: PDGF, platelet-derived growth factor; CNS,central nervous system; TH, tyrosine hydroxylase; NPY, neuropeptide Y;ChA, chromogranin A; IGF, insulin-like growth factor; 6-OHDA, 6-hydroxydopamine.

shown to be an important factor also for neuroepithelial cells(3, 4). PDGF is a disulfide-bonded dimer of PDGF A- and

B-chains, occurring in three isoforms, PDGF-AA, PDGF-BB,and PDGF-AB. These isoforms bind with different affinities to

two distinct receptors, a- and p-receptors (1 , 2, 5.-B). The

a-receptor binds both A- and B-chains with high affinity,

whereas the p-receptor shows high affinity only for the B-

chain. Ligand binding induces receptor dimerization and

subsequent activation of the tyrosine kinase domains, lead-

ing to transduction of intracellular signals into cell nuclei (1,

2). PDGF and its receptors are widely distributed throughoutthe developing and mature CNS (3, 9, 1 0). Both PDGF chains

are expressed by CNS neurons (9, 10).

GIial precursors, known as O-2A cells, possess PDGF

a-receptor and respond to PDGF with proliferation and a

timed differentiation to oligodendrocytes and type II astro-cytes (1 1 , 12). In contrast, the CNS neurons express PDGF

/3-receptor and respond to PDGF-BB by increased survival

and neurite outgrowth (3, 4, 13-1 5). In primary cultures,

PDGF-BB induced differentiation and prolonged survival ofGABAergic, dopaminergic, and striatal neurons (3, 4, 13-15).

In the embryonic mesencephalic cells, PDGF-BB also in-

duced mRNA of TH. Furthermore, in intraocular transplanta-

tion of rat embryonic ventral mesencephalon, PDGF-AA pro-

moted dopaminergic fiber outgrowth from the grafts,whereas PDGF-BB stimulated survival of the TH-positive

cells in the grafts (1 6). Recently, there are several reports

demonstrating the association of PDGF a-receptor expres-

sion with neuronal differentiation on a rat pheochromocy-

toma cell line, PC12 (17, 18).

Several factors have been shown to act on neuroblastomacells. Some of them stimulate the growth of the tumor cells,

and others promote differentiation of the cells (19-21). A

majority of established human neuroblastoma cell lines pos-

sess PDGF receptors and respond to PDGF by cell prolifer-ation, chemotaxis, and neurite extension (22, 23). We havestudied the action of PDGF on a mouse neuroblastoma cell

line, NB41 , which carries only the 13-subunit of PDGF recep-tors. Furthermore, the importance of PDGF in survival of the

cells was demonstrated by suppression of the receptor ex-

pression through an introduction of antisense PDGF p-re-

ceptor RNA.

ResultsSynthesis of PDGF by the NB4I Cell Line and Its Sub-clones with PDGF 13-Receptor Sense, Antisense, andVector RNA. We performed ELISA assays to determine the

PDGF production in NB41 cells and its derivatives, sepa-rately. We calculated the means of PDGF concentration inthe NB41 parental cells and those cells with sense, anti-

sense, and vector RNA, because there were no significant

862 Antisense PDGF fJ-Receptor in Murine NB41 Neuroblastoma

200 kDa-.

97 kDa-

PDGF-BB PDGF-AA

+ - +

Fig. 1. Ligand-stimulated kinase activity of PDGF receptors on theparental NB41 cells. The cells were incubated with or without 30 ng/ml ofPDGF-A.A or PDGF-BB, and the cells were immunoprecipitated withPDGF-R7 (anti-PDGF �-receptor) or PDGF-R3 (anti-PDGF f3-receptor). AMr 180,000 phosphorylated band corresponding to the mature PDGFp-receptor was detected.

differences between NB41 parental cells and other transfec-

tants. We could detect that these cell lysates contained

0.50 ± 0.08 ng of PDGF-AA and 1 .65 ± 0.29 ng of PDGF-BB

per flask, which were seeded in 1 x 1 06 cells 3 days before

harvesting the cells. The conditioned medium contained a

detectable but much lower level of PDGF-AA and -BB.

Expression of PDGF Receptors on the NB4I Cell Line.To examine whether the functional receptors were present,

we performed an in vitro kinase assay before and after the

addition of PDGF-AA or PDGF-BB, which revealed the pres-

ence of the PDGF n-receptor (Fig. 1) but not the a-receptor.

We have also done receptor binding assays with 125I-labeled

PDGF ligands. PDGF-AA binding was not detectable. How-

ever, PDGF-BB bound to the a-receptor, and the binding

was blocked by increasing amounts of unlabeled ligands

(data not shown).

Induction of c-fos, PDGF 13-Receptor, TH mRNA, andDown-Regulation of ChA mRNA in NB4I Cells. After stim-

ulation of the NB41 cells with PDGF-BB, levels of mRNA for

c-fos, PDGF p-receptor, TH, and NPY as well as ChA were

determined by reverse transcription-PCR. The PDGF a-re-

ceptor mRNA increased gradually (Fig. 2A). The c-fos mRNA

increased after 0.5 h, thereafter decreasing to the initial level,

whereas the level of TH mRNA increased later, reaching its

maximal level at 4 h (Fig. 2B). The level of ChA mRNA

decreased successively, whereas the level of NPY mRNA

remained at the same level during 48 h (Fig. 2C). Culturing

NB41 cells with PDGF-BB, however, did not cause any ev-

dent changes in the cell morphology.

Expression of PDGF 13-Receptor in Antisense NB4ICells. Southern blot hybridization of genomic DNA from

sense clones, antisense clones, and NB41 cells confirmed

the integration of the PDGF a-receptor DNA in genomic DNA.

We have altogether screened six antisense clones, three

sense clones, and one vector clone. Hybridization with thereceptor probe after restriction with BamHl/Hindlll revealed

the presence of randomly integrated DNA, shown as various

sizes and numbers of PDGF p-receptor DNA when com-

pared with that of the parental NB41 cells (Fig. 3). Eleven

antisense and 3 sense clones were examined for the pres-

ence of the functional receptor by in vitro kinase assay.

Clones expressing antisense PDGF p-receptor RNA showed

varying but clearly lower levels of phosphorylated receptors

when compared with parental NB41 cells (Fig. 4) and cells

expressing sense PDGF a-receptor or vector RNA (data not

shown). Western blot analysis on the extracted protein from

an antisense clone lacked a band corresponding to the p-re-

ceptor, whereas a sense and a vector clone as well as the

parental NB41 cell line showed a clear PDGF p-receptor

band of Mr 1 80,000 (Fig. 5). The following experiments were

carried out on the antisense clones with greatly reduced

levels of PDGF p-receptor expression (AS1 , AS12, and

AS1 5).Morphological and Growth Characteristics of Anti-

sense and Sense PDGF 13-Receptor and Parental NB4IClones. Parental NB41 cells grow in heterogeneous mor-

phology with various lengths of neurites and perikarya sizes

(Fig. 6A). After selection of clones by the puromycin resist-

ance method, clones expressing PDGF a-receptor vector,

sense, and antisense RNA demonstrated more homogene-

ous morphology (Fig. 6, B-D). Some of those clones grew in

a flattened monolayer (data not shown). A majority of clones

with the p-receptor antisense RNA showed immature mor-phology with shorter neurite extension and round cell bodies,

and the cells attached less well to the plastic surface (Fig.

6D).When these cells were cultured in 24-well plates and cul-

tured in serum-free media, these clones showed consider-

able differences in their growth rates (Fig. 7). The growth of

sense and vector clones was still seen for 4 days after serum

withdrawal, whereas most of the antisense cells died within

3 days. When these cells were cultured in the N2 serum-free

medium supplemented with progesterone, putrescine, sele-

nium, and transferrin (24), these antisense cells survived

longer than those cultured in the serum-free medium (Fig.8A). When PDGF-BB was added in the N2 medium, the

growth of the sense clone was stimulated, whereas the re-

sponse of the antisense clone was rapidly saturated (Fig. 8B).

In contrast, the growth-stimulatory effect of IGF-l was still

maintained in the antisense clone but clearly at a lower level.

The same antisense clone of different passages (p7 and p18after being stored frozen), examined in the same experiment,

showed similar growth responses (Fig. 8).

Apoptosis of PDGF 13-Receptor Antisense, Sense, andParental NB41 Cells following Serum Withdrawal or Ad-dition of 6-OHDA. By the second day following serum dep-rivation from the parental NB41 cells, we could see a con-

siderable number of cells beginning to detach from the flask.They showed cell shrinkage and chromatin condensation in

accordance with morphological features of apoptosis. Toinduce clear apoptosis in the cells, we added a neurotoxin

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Fig. 3. Southern blot analysis of NB41 cells and two antisense and onesense PDGF j3-receptor clones. Genomic DNA was digested with Hindlll/BamHl. The filter was hybridized with a human PDGF (3-receptor cDNA.Lane 1, NB41 ; Lane 2, sense 13; Lane 3, antisense 1 ; Lane 4, antisense i 5.A series of analyses performed on various clones is shown.

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Cell Growth & Differentiation 863

specific for dopaminergic cells, 6-OHDA, in the NB41 cell

culture. This treatment induced very rapid apoptosis in the

cells, which was easily confirmed when the genomic DNA

was extracted and run on an agarose gel (Fig. 9). The cells

detached from the plastic surface showed a severe nucleo-

somal DNA ladder, and the severity of the ladder was de-

pendent on the concentration of 6-OHDA. In contrast, the

adherent cells contained intact DNA. Next, we compared an

antisense clone with no receptor expression with a senseclone in the same manner. The antisense clone began to die

rapidly after serum withdrawal, and we harvested the cells

when there were still remaining adherent cells in the flask.

The detached fraction of the antisense clone demonstrated a_______ severe DNA ladder when compared with the sense clone

treated in the same fashion (Fig. 10). The adherent cellscontained intact DNA (data not shown). The DNA ladder offragmented DNA was evident for the antisense clone after

serum removal, which was more pronounced after the addi-

tion of 6-OHDA (Fig. 10). The much less pronounced DNA

ladder was seen for the sense clone even after treatment with

6-OHDA. The extent of PDGF-BB effects in reducing apop-

tosis was compared in the sense, antisense, and control

NB41 cells. PDGF-BB showed a clear protective effect on

the parental NB41 cells as well as on the sense cells follow-ing serum withdrawal and 6-OHDA treatment (Figs. 9 and

1 0). A weak protective effect of PDGF-BB was also detected

on the antisense clone (Fig. 1 0). In addition, the antisense

cells were examined for their response to GE-I following

Fig. 2. A, PDGF p-receptor mRNA expression in NB41 cells, determined The levels were expressed as means of percentage changes of the initialby a comparative PCR method. The PDGF (3-receptor (PDGFRb) rnRNA level, which was set to 100%; bars, SE. B, the induction of c-fos (C-FOS)content was increased after stimulation of the cells by PDGF-BB. Each and TH mRNA in NB41 cells after PDGF-BB stimulation determined asvalue was predicted from the linear regression curves (r > 0.6) based on described above; bars, SE. C, the induction of NPY and ChA mRNA inthe four hybridization signals obtained from the dilution series of mRNA. NB41 cells after PDGF-BB stimulation determined as above; bars, SE.

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864 Antisense PDGF (3-Receptor in Murine NB41 Neuroblastoma

which has been shown to be capable of expressing dopam-

NB41 AS1 A52 AS5 AS6 AS7 AS15 M

PDGF-BB - + - + - + - + - + - + - +

200 kDa

S AS VE NB41

Fig. 5. lmmunoblot analysis on an antisense (AS) clone compared with asense (5) or a vector (VE) RNA expressing clone as well as the parentalNB41 cells (NB41). The PDGF (3-receptor was detected by the PDGF-R3antibody.

6-OHDA treatment. To see the effect, we harvested the cells

before the serum-free control cells started to die. The result

demonstrated a clear protective effect of IGF-l on the cells

treated with 6-OHDA (Fig. 1 1).

DiscussionIn our previous studies, we have characterized PDGF-BB as

a potent survival factor with certain differentiation effects on

primary cultures of various types of neurons (3, 4, 13-15).

The embryonic mesencephalic dopaminergic neuron was the

most thoroughly examined cell type in our studies. Herein we

extended our study by using an antisense strategy to block

PDGF p-receptor expression, thereby confirming the func-

tional role of the PDGF p-receptor in neuronal cells. We have,

therefore, selected a mouse neuroblastoma cell line, NB41,

Fig. 4. Ligand-stimulated ki-nase activity of PDGF receptorson the subclones expressing an-tisense PDGF (3-receptor RNAcompared with that of the paren-tal NB41 cells. The cells were in-cubated with or without 30 ng/mlof PDGF-BB, and the cellswere immunoprecipitated withPDGF-R3 (anti-PDGF (3-recep-tors). All antisense clones (AS)exhibited variable but lower 1ev-els of the receptor expressionthan NB41 cells. Results ob-tamed from two of three assayson various clones are shown. M,molecular weight marker.

ne receptors (25). This cell line expresses only the p-recep-

tor, which is an advantage in this study because the a-re-

ceptor binds all three dimers. There are several intracellular

signaling pathways common to both types of receptors (26).

We have examined the function of PDGF on the parental

NB41 cells. We could show that this cell line responded toPDGF-BB by induction of c-fos as well as PDGF p-receptormRNA. The up-regulation of PDGF p-receptor upon stimu-

lation of PDGF itself has been observed previously (27). We

are currently studying the transcription mechanism of PDGF

p-receptor to clarify this mechanism.4 We could also dem-

onstrate that PDGF-BB transiently induced the neuron-spe-

cific enzyme TH mRNA and prevented differentiation of the

cells to a chromaffin phenotype by suppressing the expres-

sion of ChA. It has been reported previously that pheochro-

mocytoma PCi 2 cells can differentiate to a sympathetic

phenotype upon NGF stimulation, whereas hydrocortisone

stimulated their differentiation into chromaffin cells by induc-

tion of ChA (28).

However, we could not see any evident phenotypical

changes upon culturing the cells in the presence of

PDGF-BB for a longer period. Probably, this cell line is not an

optimal cell line to monitor phenotypical differentiation ef-

fects. In fact, this cell line contains several morphologically

different cells, and some of them exhibit rather differentiated

phenotype with long neurites. This notion was confirmed by

4A. Ishisaki, A. E. Ballagi, T. Murayama, and K. Funa. NF-Y controls thebasal transcription activity of the mouse PDGF (3-receptor gene, Eur. J.Biochem., in press.

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Fig. 6. The morphological charac-teristics of various clones. A, NB41parental cells. B, a clone expressingvector RNA. C, a clone expressingsense ANA. D, an antisense clone.The same clones examined in Fig. 5.Note the absence of neunte out-growth in the antisense clone.

Cell Growth & Differentiation 865

the observation that the cells changed its phenotype and

began to exhibit homogeneous culture following selection of

the individual clones after introduction of the plasmids. It is

also possible that this cell line uses an autocrine stimulation

via PDGF p-receptor because they produce PDGF-BB by

themselves. Nevertheless, the total amount of PDGF de-

tected in the cells was smaller than that which is necessary

for exogenously affecting the cells. It might be sufficient forthe autocrine stimulation. This may have rendered it difficult

to see the phenotypical changes by an exogenous addition

of PDGF in the culture. In fact, when comparing several

antisense clones with various degrees of PDGF p-receptor

expression, we were confident that the antisense PDGF p-re-

ceptor RNA truly blocked the neurite extension. The clones

with shorter neurites grew much slower. Moreover, they

tended to detach from the plastic surface.

NB41 cells and sense clones showed clear DNA ladders

following serum deprivation or by an addition of a neurotoxin,6-OHDA. This DNA fragmentation was reduced when the

cells were cultured with PDGF-BB prior to the challenges, in

concordance with our previous study on embryonic mesen-

cephalic cell culture (29).

In the serum-free medium without supplements, the anti-

sense clone did not grow at all but gradually underwent

apoptosis, which was verified by DNA fragmentation shown

as a DNA ladder. The weak rescuing effect by PDGF-BB was

detected also in an antisense clone, although not as clearly

as that seen in the parental NB41 or sense cells. It is possible

that an up-regulation of the endogenous receptor might oc-

cur at the time of injury. We have indeed reported that this

occurred in the neuronal cells following facial nerve axotomy

(30). In contrast to the response to PDGF, the antisense cells

still maintained the intact response to IGF-l in respect to its

antiapoptotic effect, suggesting that a common downstream

signal, which is still present in the cells, is responsible for theeffect.

In the N2 defined serum-free medium containing trans-

ferrin, selenium, progesterone, and putrescine but without

insulin, a slow increase in cell number was still observed

even for antisense clones. In this experiment, we have corn-

8 ___________________

7

6

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Day

Fig. 7. Cell numbers counted after serum withdrawal of a sense, vectorRNA clone compared with an antisense clone; the same clones as exam-med in Fig. 5. Each value represents the mean of triplicate wells; bars, SE.

pared the same antisense clone of different passages show-ing the same pattern of growth response to different stimuli.

This confirms the stability and uniformity of the clone follow-

ing several passages. The growth response of the antisense

clone to PDGF-BB seems to become saturated as judged by

a rapid flattening of the growth curve, in contrast to itsresponse toward IGF-l, which remained intact. However, thegrowth response to IGF-I was clearly retarded when corn-pared to that of the sense clone, suggesting the presence ofan autocrine stimulation via PDGF 13-receptor, which is

blocked in the antisense clone, accounting for the differenceseen after IGF-l stimulation. Furthermore, PDGF-BB pro-duced in the cells that carry PDGF 13-receptor could up-

regulate IGF-l receptor expression (31 , 32), resulting in anincreased response to IGF-l.

Our results agree with a previous study in PCi 2 cells withtransfected PDGF wild-type receptors, which rescued thecells from apoptosis induced by serum withdrawal (33). In

our study, we have confirmed this function of PDGF p-re-

ceptor by a different approach, where the expression of thePDGF 13-receptor was more natural for the cells. The formerapproach might fail to show the true effect of the receptorwhen the intracellular substrates necessary for the signalingare not available in the examined cells.

In conclusion, we demonstrated, on a neuroblastoma cell

line, that deprivation of PDGF p-receptor converted the cells

to morphologically immature neuroblasts and induced their

apoptosis. Our results imply that PDGF-BB not only acts asan autocrine growth factor but also as a differentiation and

Antisense PDGF /3-Receptor in Murine NB41 Neuroblastoma

C AS, serum--.- s, serum--� . vecter, serum-

survival factor in neuroblastoma.

Materials and MethodsCell Culture and Transfection. A mouse neuroblastoma cell line, NB41,was obtained from American Type Culture Collection (Rockvllle, MD) andmaintained in Ham’s F-b medium supplemented with 2.5% of FCS (Life

Technologies, Inc.), 15% horse serum, 100 lU/mI penicillin, and 100 �g/mlstreptomycin. An extracellular domain of 1-460 bp of mouse PDGF (3-re-

captor cDNA (7) was inserted either in a sense or an antisense orientationin a pBabe retroviral vector containing Moloney mouse Ieukaemia viruslong terminal repeat and puromycin-selectable marker (a kind gift of Dr.Hartmut Land, Imperial Cancer Research Fund, London, United Kingdom;Ref. 34). Constructs were transferred into cells via Upofectin (Life Tech-nologies, Inc., Renfrewshire, ScOtland, United Kingdom) according to thevendors’ manual. The titration of purornycin was done on the parentalNB41 cell line, and colonies were selected with 2 �g/ml puromycin.Several clones were selected, and integration of the DNA into the genomicDNA was estimated by Southern blot analysis.

Southern Blot Analysis. Preparation of genomic DNA and Southernblot analysis were done essentially according to Sambrook etei. (35). Theextracted DNA from all antisense clones as well as several sense andvector plasrnid clones was digested with Hindlll/BamHl and run, 20 �.&g ineach lane, on 0.7% agarose gel. After electrophoresis, DNA was trans-ferred to a nitrocellulose filter and dried. Hybridization was performedusing a 32P-Iabeled extracellular domain of human PDGF (3-receptorcDNA (8).

ELISA. Cells were seeded at a density of 1 x 10�/ml in 10 ml of theserum containing F-b medium, and on the second day, cells werewashed twice with PBS, and the medium was changed to serum-freemedium. After 2 days of culture, the cells were trypsinized and oounted.The cell pellet was homogenized in 2 ml of 10 m� Tris (pH 8), 0.5%

deoxycholate, 0.05% Tween 20 containing 1 m� EDTA, 1 m� o-phenan-throline, 1 pM pepstatin, 20 p.� leupeptmn, and 50 kalllkrein inhibitor unitsper ml of aprotinin and incubated end over end for 2 h at room temper-ature, diluted to 12 ml in the same buffer without deoxycholate and Tween20, and centrifuged at 31 .000 x g for 10 h. All of the chemicals werepurchased from Sigma Chemical Co. The supernatant was washed andconcentrated in a Centriprep concentrator (Amicon, W. A. Grace, Dan-vera, MA). EUSA assay was performed in 96-well, flat-bottomed micro-

plates(Nunc)as described as before(36). Recombinant PDGF-M or-BB(37) was used as the standard. For PDGF-AA, the plate was costed witha mouse monoclonal anti-PDGF-AA, 3E205 (a kind gift of Amgen, Oak-wood, CA) at the dilution of 5 hg/mI overnight at room temperature.Biotinylated IgG fraction of a rabbit polyclonal anti-PDGF-M (38) wasused for the detection with streptoavidin-peroxidase complex (Dako-paffs). For PDGF-BB, the lgG fraction ofa rabbit polyclonal anti-PDGF-BB(38), as well as a biotinylated mouse monoclonal anti-PDGF-BB, 4E4(Amgen), was used. The level of bound antibody was determined by theaddition of reaction buffer (0.1 M sodium citrate, pH 5, containing 0.5ng/mlo-phenylen diamine and 0.5 p.1/mI of 30% H2O�). The color reactionwas monitored by a spectrophotometer set at 450 nm.

RNA Preparation and Reverse Transcription. Total RNA was pro-pared from NB41 cells. Briefly, the cells were grown on six-well plates(Becton Dickinson, Lincoln Park, NJ), and guanidmniurn thiocyanate solu-tion [containing 4 PA guanidinlum thiocyanate, 0.1 pA Tris (pH 7.5), and 0.1M (3-mercaptoethanol] was added directly on the PBS-washed cells. Afterphenol extraction and precipitation, the RNA content of the differentlytreated samples were diluted 5-fold in three steps, and all dilution serieswere reverse transcribed into cDNA as described (4) using the Moloneymurine leukemia virus reverse transcriptase (Life Technologies, Inc.,Gaithersburg, MD).

ComparatIve PCR. We performed a comparative estimation of mRNAlevels by a quantitative PCR method described earlier (39). We used thelevel of glyceroaldehyde 3-phosphatedecarboxylase mRNA, a house-

keeping gene, in each ANA sample as a standard. The reactions wereperformed as described earlier (4). The primers and probes for glyceroal-dehyde3-phosphatedecarboxylase and PDGF (3-receptor were describedearlier(39), as well asforc-fos and TH (14). The primers for ChA were AGGTG.A TGA AGT GCG TCC T and CCC UG TCA GM TCC 1fl C, and theprobe sequence was CAT CCT CM AGC TGC TGT TGT GT. The primersfor NPY were TAC CCC TCC MG CCG GAC M and GAT GAG Gil’ GAT

GTA GTG TCG, and the probe sequence was GTA GTA TCT GGC CATGTC CT.

In Vitro Kinase Assay. Cells were cultured in a Petri dish as a sub-confluent monolayer and kept serum-free overnight. After treatment of thecellsfor 10 mm at 37#{176}Cwith 30 ng/mI PDGF-AA, -BB, orvehicle alone, thecells were nnsed twice with cold washing buffer containing 20 m� Tris/HCI(pH 7.5), 150 mM NaCI, and 50 �tt�i Na3VO4 and lysed in 1 ml of lysis bufferconsisting of 0.5% Triton X-iOO, 20 mp,i Tris/HCI (pH 7.5), 150 m� NaCI,10% glycerol, 2.5 mM EDTA (pH 7.5), 100 lU/mI Trasylol, 1 m� phenyl-

6�

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Cell Growth & Differentiation 867

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Fig. 8. Growth-response curves of the antisense and the sense clones shown in Fig. 5. In A, the cells were cultured in serum-free N2 defined medium.B, in the same medium supplemented with 30 ng/ml PDGF-BB. C, in the medium supplemented with 30 ng/mI IGF-l. Each value represents the mean oftriplicate wells; bars, SE.

Fig. 9. Agarose gel electrophoresis of DNA extracted from NB41 paren-teral cells after serum withdrawal. Lanes 1-6, DNA taken from the floatingcells detached from the plastic surface; Lanes 7-12, DNA from the ad-herent cells in the same cell cultures shown in Lanes 1-6. Lane 1, noaddition; Lane 2, with 30 ng/ml PDGF-BB; Lane 3, DNAfrom cells culturedin the presence of 25 pi�i (Lane 3) or 37.5 .tM (Lane 5) 6-OHDA; Lanes 4 and6, corresponding cultures to those in Lanes 3 and 5, but PDGF-BB wasadded prior to the addition of 6-OHDA. A representative experimentperformed four times is shown. M, molecular weight marker.

methylsulfonyl fluoride, 50 ,.�M Na3VO4, and i m� DII. The lysates were

centrifuged, and the supematants were incubated with an anti-PDGF(3-receptor antiserum, R3 (4), or an anti-PDGF a-receptor antiserum, R7

(5). Immunoprecipitation was carried out by protein A-Sepharose, and theimmunoprecipitated samples were subjected to kinase assays in 25 p3 of20 m� Hepes (pH 7.5), 10 mr�i MnCI2, 0.5 m� DTT, 100 m�i NaCI, and

0.05% Triton x-ioo containing iO xCi of [-y-32P]ATPfor 10 mm on ice. Thereaction was stopped by adding 25 �d of buffer containing 4% SDS, 0.2

M TriS/HCI (pH 8.8), 0.5 M sucrose, 5 mM EDTA, 0.01 % bromphenol blue,and 2% 2-mercaptoethanol and boiled for 3 mm. The samples wereanalyzed by SDS-gel electrophoresis. The gels were treated with 1 M KOHfor 30 mm at 55AC, destained, and dried before autoradiography.

Immunoblotting. Cells having been cultured in a Petri dish as a sub-confluent monolayer were lysed in lysis buffer consisting of 0.5% Triton

1 2 3 4 5 67 8M

Fig. 10. Agarose gel electrophoresis of DNA extracted frorn floatingdetached cells of a sense clone (Lanes 1-4) and of an antisense clone(Lanes 5-8). The treatment was exactly the same as in Lanes 1-4 of Fig.9. The antisense clone demonstrating severe DNA ladders when corn-pared with the sense clone treated in the same manner is shown. Thesense and antisense clones correspond to Si 3 and AS1 , respectively, inFigs. 3 and 4. M, molecular weight marker.

x-ioo, 0.5% deoxycholate, 0.1% SDS, 20 mr�i Tris-HCI (pH 7.5), 150 m�NaCI, 1 0 mM EDTA, 1 % Trasylol, and 1 mr�i phenylmethylsulfonyl fluoride.After incubation on ice for 30 mm, the lysates were centrifuged, and thesupernatants were incubated with wheat germ lectin-Sepharose 6 MB(Pharmacia) for 2 h at 4”C The precipitates were separated by SDS-gelelectrophoresis, and the proteins of the gel were transferred onto nitro-cellulose membranes (Hybond-C extra; Amersham Corp.). Blots wereblocked and Incubated with the R3 antiserum at a dilution of i :500. Theblots were washed and incubated with the peroxidase-conjugated swineanti-rabbit immunoglobulmns at 1 :3000 dilution. After washing, antibody

binding was visualized by ECL Western blotting detection system (Amer-

sham).Counting of Cell Numbers. Cells were seeded into 24-well tissue

culture plates (Costar, Cambridge, MA) at a density of 1 x 1 05/ml inserum-free Ham’s F-b medium or the N2 defined serum-free mediumconsisting of DMEM/Ham’s F-i2 (Life Technologies, Inc.; 1:1) mixture

supplemented with 100 lU/mI penicillin, 100 �g/ml streptomycin, 2 m�L-glutamlne, 1 00 �.tg/ml transferrmn, 1 00 mr�i putrescine, 20 n�i progester-

868 Antisense PDGF (3-Receptor in Murine NB41 Neuroblastoma

6-OHDAIGF-l

Floating

M 1 2 3 4 5 6 7

Adherent

8 9 �O 11 12

Fig. 1 1 . Agarose gel electrophoresis of DNA extracted from an antisenseclone (AS 1 5 in Fig. 4). Cells were harvested before the control cells (Lane1) began to die. Lanes 1-6, DNA taken from the detached cells from theplastic surface. Lanes 7-12, DNA from the adherent cells in the same cellcultures shown in Lanes 1-6. Lane 1, no addition; Lane 2, with 30 ng/mlIGF-l; Lane 3, DNA from cells cultured in the presence of 25 tM (Lane 3)or 37.5 �tM (Lane 5) 6-OHDA. Lanes 4 and 6, corresponding cultures tothose in Lanes 3 and 5, but IGF-l was added prior to the addition of6-OHDA. M, molecular weight marker.

one, and 30 nM selenium, but insulin was omitted (24). On the second day,

the medium was changed to serum-free medium, and growth factor (30ng/ml PDGF-BB or 30 ng/ml IGF-l) or vehicle was added. During thesuccessive 5 or 6 days, cells in each well were trypsinized, and the viablecells that excluded trypan blue dye were counted in triplicate under amicroscope. Then one-half of the medium was changed on the fifth day.

Neurotoxin-induced Cell Death in N841 Cells. The NB4i cell line

and its subclones with antisense, sense PDGF (3-receptor DNA and vectorplasmids were cultured in 75-cm2 flasks at a total number of 1 x i06

cells/flask. On the second day, the cells were rinsed three times withserum-free medium, and the cells were resuspended in serum-free me-

dium with or without 30 ng/ml PDGF-BB or 30 ng/ml IGF-l (Sigma), two orthree flasks each. On the third day, 25 MM or 37.5 �tM 6-OHDA, dissolved

3 Mg1p1 in 0.2 mg/mI ascorbate/saline, were added in one of the flasks withonly serum-free medium and one of the flasks with PDGF-BB or IGF-l

containing medium. In the PDGF-BB- or IGF-l-containing flasks, a finalconcentration of 30 ng/mI PDGF-BB was added again. On the next day,floating cells together with the cells collected after a gentle rinse with

serum-free medium were pooled in a tube separately from the cells

attached on the bottom for each flask. Genomic DNA was extractedaccording to Walkinshaw and Waters (40). Cells were centrifuged andincubated in a buffer containing 5 m� Tris, 20 mr�i EDTA, and 0.5% Triton

x-ioo for 20 mm at 4CC. High molecular weight DNA was removed bycentrifugation at high speed for 1 5 mm at 4CC, and the samples were then

centrifuged for 10 mm in 0.1 % SDS. Samples were sequentially extractedwith equal volumes of phenol:phenol-chloroform:isoamyl alcohol (25:24:1), and the DNA was suspended in 10 mr�i Tris, pH 8, and 1 mr�i EDTA and

loaded on a 1 .2% agarose gel containing 0.1 �tgJml ethidium bromide. The

samples were then subjected to electrophoresis at 40 V in TBE runningbuffer containing 0.045 M Tris-borate and 0.001 M EDTA. Following elec-trophoresis, the gel was incubated for 3 h with TBE running buffer con-

taming 0.1 mg/mI RNase A.

AcknowledgmentsWe thank Dr. Carl-Henrik Heldin for his continuous support, Dr. Hartmut

Land, Imperial Cancer Research Fund, London, United Kingdom, for the

pBabe vector plasmid, and Amgen, Inc. for the monoclonal antibodies forPDGF receptors.

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