Human Neuroblastoma Cell Lines

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Induction of Transforming Growth Factor β1 and Its Receptors during all- trans-Retinoic Acid (RA) Treatment of RA-responsive Human Neuroblastoma Cell Lines Pamela S. Cohen, John J. Letterio, Carlo Gaetano, et al. Cancer Res 1995;55:2380-2386. Published online June 1, 1995.

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Downloaded from cancerres.aacrjournals.org on July 17, 2011 Copyright © 1995 American Association for Cancer Research

[CANCER RESEARCH 55, 2380-2386,

June 1, 1995]

Induction of Transforming Growth Factor ßland Its Receptors during all-irans-Retinole Acid (RA) Treatment of RA-responsive Human Neuroblastoma Cell Lines Pamela S. Cohen,1 John J. Letterio, Carlo Gaetano, Jane Chan, Kazue Matsumoto, Michael B. Sporn, and Carol J. Thiele Department of Pediatrics. Division of Pediatrie Hematology-Oncoloxy. Harbor-University of California at Los Angeles Medical Center and the Research and Education Institute, Harbor-Universitv of California at Los Angeles Medical Center. Torrance. California 90509 ¡P.S. C., J. CJ, and Laboratory of Chemopreveniion ¡J.J. L, M. B. S.I and the Cell and Molecular Biology Section ¡C.G.. K. M.. C. J. T.¡,Pediatrie Branch. National Cancer Institute. NIH. Belhesda, Maryland 20892

ABSTRACT Recent work on a variety of normal and malignant cell lines has shown that induction and secretion of biologically active TGF-ßmay occur after exposure to all-frans-retinoic acid (RA), coincident with decreased growth rate and/or differentiation. This study evaluates the expression and reg ulation of transforming growth factor ß(TGF-ß)and its receptors during RA-induced cell growth arrest and induction of differentiation in the RA-sensitive human neuroblastoma cell line SMS-KCNR and the RAresistant neuroblastoma cell line SK-N-AS. RA treatment of SMS-KCNR cells results in a 40-fold increase in TGF-ßl iiiRN A after 4 days of RA, a dose-dependent increase in TGF-ßl secretion, an increase in types I (TBR,) and III (TBRIM) TGF-ßreceptor proteins, and an increase in type II TGF-ßreceptor (TBR,,) niRN.V coincident with RA-responsiveness of the cells. However, in the RA-resistant line SK-N-AS, TGF-ßl is constitutively secreted at levels that are unchanged after RA treatment, and although TBR, and TBR,,, mRNA is expressed in untreated SK-N-AS cells, levels of TBR, and TBR,,, protein and TBR,, mRNA decrease after RA treatment. Thus, in RA-sensitive neuroblastoma cells, RA treatment may result in the induction of a negative autocrine TGF-ßl growth regulatory loop. These results suggest the hypothesis that: (a) a TGF-ßl negative autocrine growth loop may be a necessary for RA-responsiveness of neuroblastoma cells in vivo; and (b) to induce or maintain this TGF-ßl negative autocrine growth a mechanism of RA resistance in neuroblastoma.

induction of component the inability loop may be

creased growth rate and/or differentiation. Cells that respond in such a manner include normal keratinocytes (14), the human promyelocytic leukemia cell line HL-60 (15), and mouse and human embryonal carcinoma (EC) cell lines (16, 17). This study evaluates the expression and regulation of TGF-ßand its receptors during RA-induced cell growth arrest and induction of differentiation in human neuroblastoma cell lines.

MATERIALS

AND METHODS

Cell Lines and Cell Culture. The cell lines derived from human neuro blastoma tumors used for analysis included SMS-KCNR (provided by Dr. C. P. Reynolds, Children's Hospital of Los Angeles, Los Angeles, CA), and SKN-AS (Dr. L. Helson, New York Medical College, Valhalla, NY). The cell line CLL64 is derived from mink lung epithelial cells (American Type Culture Collection). All cell lines were cultured in RPMI 1640 containing 10% PCS, glutamine, penicillin, and streptomycin. For all RNA isolations, 3 X IO6 cells were plated onto 150-mm tissue culture plates and incubated at 37°Cand 5% CO,. In experiments using RA, media was removed from cells, adjusted to 5 JIM RA (Sigma Chemical Co.), and added back to these cells. Control cells were treated similarly with equal volumes of ethanol solvent. Cells were subsequently fed every 2 days with media containing 5 JAMRA or solvent control and harvested at various times up to 8 days of culture. To test for TGF-ßresponsiveness, 1 X IO4 cells/well were plated in media containing 10% PCS (as above) in quadruplicate in %-well plates with varying concentrations of porcine platelet-derived TGF-ßl (courtesy of Dr. Larry

INTRODUCTION

Ellingsworth. Celltrex Labs, Palo Alto, CA), which shares complete amino acid homology with human TGF-ß(18). Cells were refed every 3 days and

TGF-ß2is a multifunctional regulatory peptide that has been shown to have pleiomorphic effects in both normal and malignant cells (1-4). Among its most striking effects is the ability to inhibit prolif eration of both tumor and normal cells, sometimes in association with the induction of differentiation. Particularly responsive in this manner are epithelial cells, including cells derived from intestine, skin, liver, and bronchus, and endothelial and hematopoietic cells (2, 3, 5-8). The ability of TGF-ßto inhibit the growth of neural tube-derived fetal retinal cells suggests that TGF-ßmay be involved in regulating the growth of cells of neuroectodermal origin (9). The in vitro treatment of neuroblastoma cell lines with the retinoid RA has provided a useful model for the study of neuroblastoma growth and differentiation (10-12). After 6-8 days, cell growth is markedly inhibited, and induction of neuronal differentiation occurs (12, 13). Recent work on a variety of normal and malignant cell lines in vitro and in vivo has shown that induction and secretion of biolog ically active TGF-ßoccurs after RA treatment, coincident with deReceived 11/11/93; accepled 3/31/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed, at Picower Institute, 350 Community Drive, Manhasset, NY 11030. 2 The abbreviations used are: TGF-ß,transforming growth factor ß;RA, M-transretinoic acid; RS-PCR, RNA template-specific PCR; RT-PCR. reverse transcription PCR; GAPDH. glyceraldehyde-3-phosphate dehydrogenase; SELISA, sandwich ELISA.

harvested at various times up to 8 days of culture. Wells were observed for effects on growth assessed by ['HJthymidine incorporation. For this, wells were pulsed with 1 ¿¿Ci of ['HJthymidine for the last 24 h of culture and then harvested using a PHD cell harvester (Cambridge Tech, Inc., Watertown, MA). For actinomycin D experiments, SMS-KCNR cells treated for 2 days with either RA or solvent control were incubated with 5 fxg/ml of actinomycin D for time points up to 24 h. Total nRNA was isolated by the method of Chomczynski and Sacchi (20) and evaluated by quantitative RS-PCR analysis as described below using 12P-labeled TGF-ßl oligo nos. 2 or 5 for determi nation of TGF-ßl and ß2-mexpression, respectively. The mRNA half-life was determined by linear regression analysis of the RS-PCR product, as measured by quantitative densitometric scanning. Isolation and Analysis of RNA. Total cellular RNA where used was isolated from tissue culture cells by either the hot phenol method (19) or the method of Chomczynski and Sacchi (20) as indicated. Poly(A)+ RNA was prepared using the Fast Track kit (Invitrogen, San Diego, CA). Northern blot hybridizations were performed by electrophoresis of 3 ^tg poly(A)+ RNA in a 1.2% formaldehyde-agarose gel and blotted on Nytran membranes (Schleicher and Schuell, Keene, NH) by capillary blotting on 10X SSC (IX SSC = 0.15 M NaCl plus 0.015 M sodium citrate) buffer. Blots were hybridized to random primer-labeled [3-P]dCTP DNA at 42°Cfor 16 h. Then membranes were washed as described previously (21) and washed in a final wash of 0.1X SSC-1% SDS at 65°Cfor 30 min. Membranes were exposed to XAR5 film (Kodak) using an intensifying screen at -70°C. Prior to rehybridization, Nytran membranes were treated with 50% formamide-O.lX 1 h at 75°C.Quantitative densitometric scanning of appropriately

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SSC for exposed

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Table 1 Oligonucleolide primers used for RS-PCR GeneTGF-J31

size326 5 ' -AACTATTGCTTCAGCTCC- 3 ' 2 5 ' -TGACAAGCTTAGGTATCGATA- 3 'S'-TGACAAGCTTAGGTATCGATACAATCCAAATGCGGCATGTT-S' 345Sequence5'-TGACAAGCTTAGGTATCGATAGATCATGTTGGACAGCTG-3'

ß-mOligo1

1978-1996 bp 1691-1709 sequencebp Unique 35 13-3532 bp 1544-1563Product

5 ' -ACCCCCACTGAAAAAGATGA- 3 'bp

bp

130 bpRef.(23)

(31)

autoradiograms was used to assess the relative amounts of mRNA detected using a Bio-Rad 620 densitometer.

varying amounts of time. Densitometric analysis was plotted on a log-log scale against nanograms of RNA3 used in PCR. Using RS-PCR on total RNA from

The DNA fragment or plasmid probes used in this study include pNbl (N-myc; Ref. 22), Aßc(TGF-J31; Ref. 23), pGIG2 (TGF-ß2;Ref. 24), type II TGF- ßreceptor (25), and GAPDH (26). In Vitro Transcription. Approximately IO8 cells were treated for 7 days in

control and RA-treated SMS-KCNR cells, a linear range of RNA expression was found at 125 ng RNA template for TGF-ßl and 32 ng RNA template for ß2-mexpression in these cells. After this linear range template concentration

the presence of RA or control solvent as described above. The nuclear run-on procedure was performed as described previously (27) using 2 x IO6 intact cell nuclei. Briefly, [32P]UTP-labeled RNA was hybridized for 48 h at 42°Cto Nytran membranes containing 2 (¿geach of TGF-ßl, NMYC, GAPDH, and pGEM plasmid (Promega, Madison, WI). The filters were washed at a final wash of 0.5X SSC-0.1% SDS at 55°C.Densitometric analysis was performed using a Bio-Rad 620 densitometer. Quantitative RS-PCR. Total RNA from RA-treated and control SMSKCNR cells was prepared by the Chomczynski and Sacchi method (20) and was subjected to RS-PCR (28, 29), a modification of the RT-PCR method designed to be RNA template specific. Quantitation of gene expression was performed using the method of Murphy et al. (30). This quantitative method uses a serial dilution of cDNA template prior to PCR amplification. The results of each reaction are analyzed by gel electrophoresis to determine an amount of template that lies within a linear range of signal intensity when measured densitometrically or by radioactivity. By performing an RT-PCR analysis using a starting template concentration that lies within relative levels of steady-state mRNA between samples can Oligonucleotide primers for the RS-PCR reaction were published sequences for the human TGF-ßl and ß2-mgenes

this linear range, be compared. synthesized using (Table 1; Refs. 23

and 31). Reverse transcription of both genes was performed using a unique 21-mer sequence (notated in bold in Table 1) attached to the 3' end of the

was determined, it was used to compare gene expression from cells treated for various durations of time with actinomycin D (from 0 to 24 h). Measurement of K.I -ßl and TGF-02 in Conditioned Media. Cells were grown to near confluence in RPMI 1640 with 10% FCS. The cells were then washed and changed to medium containing 1% dialyzed charcoal-stripped serum and allowed to condition media for 36 h in the presence of either 5 nM or 5 (¿MRA or vehicle control. Conditioned media were centrifuged, and protease inhibitors (leupeptin, pepstatin, and aprotinin at 2 (xg/ml; phenylmethylsulfonyl fluoride, 120 fig/ml) were added prior to storage at —70°C. Samples were concentrated by trichloroacetic acid precipitation and processed for assay of TGF-ßl and TGF-ß2by SELISA as described previously (32). For quantitation of unknown concentrations of TGF-ßl and TGF-ß2, data points were fit to a quadratic or cubic regression equation with Dynatech's Immunosoft program. Chemical Cross-Linking

of TGF-ß Receptors.

Cross-linking studies

were performed as described previously (33). Cells were harvested at the end of a 48-h exposure to RA. They were washed twice with serum-free RPMI medium, once with binding buffer (RPMI with l mg/ml BSA), and resuspended in binding buffer at 5 x IO6 cells/ml. Cells were aliquoted into siliconized (Sigmacote; Sigma) Eppendorf tubes. The cells were incubated with 125I-labeled TGF-ßl in the presence (+) or absence (-) of unlabeled competitor TGF-ßl for 2 h at 4°C.Cell pellets were washed and resuspended

gene-specific priming oligomer. This unique 21-mer was subsequently used alone as the downstream 3' oligomer in combination with an upstream 5',

in cross-linking buffer (RPMI without BSA containing 100 /¿g/mlof disuccinimidyl suberate; Pierce Chemical, Rockford, IL). After a 30-min incubation at 4°C,cells were washed three times and lysed. Cell lysates were subjected

gene-specific oligomer for the subsequent PCR amplification step. The addi tion of a unique 21-mer sequence is designed to eliminate false positive RT-PCR results due to contaminating DNA. Thus, RS-PCR of TGF-ßl used

to electrophoresis under reducing conditions using a 10% linear gel. Autora diograms were obtained after a 7-day exposure to Kodak X-OMAT film at -70°C.

oligo no. 1 for the priming reaction, and oligo nos. 2 and 3 for the amplification reaction, yielding a 326-bp product. RS-PCR of ß2-mused oligo no. 4 for the priming reaction and oligo nos. 3 and 5 for the amplification reaction, yielding a 130-bp product. The method used for RS-PCR was as follows. Reverse transcription of 1 /j.g of total RNA was performed for 30 min at 42°Cusing 2.5 units/jj.1 Moloney

RESULTS

Expression of TGF-ßl mRNA in Neuroblastoma Cell Lines in Response to RA. When treated for 8 days with 5 /XMRA, SMSKCNR cells morphologically differentiate along a neuronal phenomurine leukemia virus reverse transcriptase in IX RT-PCR buffer [10 mM type, and growth is decreased substantially, as measured by [3H]thyTris-HCl (pH 8.3), 50 mM KC1, 5 mM MgCl2, and 1 mM each dATP, DGTP, midine uptake (10-12, 34). In contrast, SK-N-AS cells do not dCTP, dTTP) containing 1 unit//xl RNasin (Promega) and 0.75 /J.Mof down stream gene-specific primer to which a unique tag sequence was added at the morphologically differentiate, and growth is only moderately inhib 3' end (oligo 1 for TGF-ßl and oligo 4 for ß2-m).In each experiment, water ited when treated with RA (34). Thus, SMS-KCNR cells are consid ered RA sensitive and SK-N-AS cells are considered RA resistant was substituted for mRNA as a negative control for contamination. The samples were heated for 5 min at 95°Cto inactivate the reverse transcriptase. (34). Fig. 1 shows the steady-state mRNA levels of TGF-ßl and The resulting cDNA was then serially diluted in IX RT-PCR buffer. Samples TGF-ß2mRNA in RA-treated SMS-KCNR and SK-N-AS cells. In for PCR contained 10 fil serially diluted cDNA, 1.25 units AmpliTaq DNA the RA-sensitive cell line SMS-KCNR, TGF-ßl mRNA increases polymerase, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2 mM MgCl2, 1 unit/ju.1 6-fold after 2 days of RA, peaks to a 40-fold increase after 4 days of RNasin (Promega), 1 mM each of dATP, dGTP, dCTP, and dTTP, 0.15 H.M RA, and decreases thereafter. TGF-ß2mRNA is constitutively ex oligo nos. 2 and 3 (for TGF-ßl) or oligo nos. 5 and 3 (for ß2-m),and IO6 pressed but is unchanged after RA treatment. The cell line SK-N-AS cpm/reaction of 5' primer (oligo nos. 2 and 5 for TGF-ßl and ß2-m,respec tively) 32P-labeled by T4 polynucleotide kinase (specific activity, IO9 cpm/ maintains a higher basal level of TGF-ßl mRNA than is detected in /ig). The reaction mixture final volume was 50 /j.1and was overlaid with 25 ¡u SMS-KCNR, but in contrast, TGF-ßl and TGF-ß2mRNA are con mineral oil. Samples were amplified for 1 cycle at 94°Cfor 5 min and 60°Cfor stitutively expressed in SK-N-AS, and levels do not change during RA 2 min and then amplified for 38 sequential cycles at 94°Cfor 1 min and 60°C treatment. for 2 min. The final extension cycle was 94°Cfor 1 min and 60°Cfor 10 min.

Ten fil of each PCR reaction from serially diluted cDNA was electrophoresed on gels made of 1% agarose/2% NuSieve GTG (FMC Byproducts, Rockland, ME). Gels were dried and exposed to XAR5 film (Kodak) at -70°C for

3 Any amount of RNA indicated in this study as being used as a template for PCR refers to the amount of reverse transcribed cDNA generated from the stated amount of RNA.

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TGF-B INDUCTION

KCNR I 1 23456

A.

I

SK-N-AS I 7 8 9101112 I

IN RA-TREATED

Probe

28S — TGF0-1

TGFß-2

NEUROBLASTOMA

Secretion of TGF-ßl by SMS-KCNR Cells in Response to RA. The increase in steady-state levels of TGF-ßl mRNA following treatment of SMS-KCNR with RA is associated with enhanced secre tion of TGF-ßl (Table 2). Steady-state TGF-ßl levels were only one-tenth those produced by SK-N-AS cells, but there was a 4-fold increase at 5 JU.MRA (3 to 14 pM) and a 2-fold induction at 5 nM (4.5 to 8 pM). There was no detectable TGF-ß2produced by either cell line, before or after RA treatment, implicating posttranscriptional control over the expression of this isoform as well. Regulation of TGF-ßReceptor Expression in Neuroblastoma Cells by RA. To determine if exogenous TGF-ßlcould affect SMSKCNR cell growth, cells were cultured with various concentrations of TGF-ßl, and cell growth was assessed. At concentrations ranging

5 hrs 18hrs 40 hrs C RA C RA C RA GAPDH

f

Fig. 1. Northern blot hybridization analysis of TGF/3-1 (A), TGFJ3-2 (fi). GAPDH (C) mRNA expression in neuroblastoma cell lines SMS-KCNR (Lanes 1-6) and SK-N-AS (Lanca 7-12) after treatment with control solvent (odd-numbered lanes) or 5 /AM RA (even-numbered lanes) for 2 days (Lanes I. 2. 7, and 8), 4 days (Lanes 3, 4, 9, and 10), or 8 days (Lanes 5, 6. II, and 12).

Evaluation of TGF-ßl mRNA expression in RA-treated SMSKCNR cells indicated that significant changes were not detected after 5 h of RA treatment, yet levels were markedly increased by 18 h (Fig. 2). Among the earliest changes noted in neuroblastoma cells induced to differentiate by RA is a dramatic decrease in N-mvc mRNA steady-state levels. Analysis indicated that the increases in TGF-ßl mRNA levels occurred when N-mvc levels were decreased (Fig. 2). The Control of TGF-ßl mRNA Expression Occurs at a Posttranscriptional Level. In order to ascertain the mechanisms contrib uting to the RA-induced increase in TGF-ßl mRNA expression, a nuclear run-on assay was performed (Fig. 3). After 7 days of treatment with 5 JIM RA, in vitro transcription of TGF-ßlmRNA was decreased when compared to control cells. In this experiment, in vitro transcrip tion of the gene N-mvc was decreased as described previously (13), and in vitro transcription of the GAPDH gene remained the same. Thus, although steady-state levels of TGF-ßl mRNA increase, TGF-ßl transcription decreases after RA treatment. To evaluate TGF-ßlmRNA stability, RS-PCR was used to analyze TGF-ßlmRNA levels in actinomycin D-treated cultures. Control and 2-day RA-treated SMS-KCNR cells were treated with actinomycin D for 0, 8, 16, or 24 h. The resultant RNA half-life was measured using quantitative RS-PCR after various durations of actinomycin D expo sure. Standard curves generated to ascertain the PCR product inten sities of both TGF-ßl and ß2-min control and RA-treated SMSKCNR cells at varying starting RNA concentrations are shown in Fig. 4, A and B. From this analysis, a starting concentration of 125 and 32 ng for TGF-ßl and ß2-m,respectively, was chosen to measure gene expression by RS-PCR in the cells treated with actinomycin D. The half-life of TGF-ßlmRNA in untreated SMS-KCNR cells was found to be approximately 17 h, whereas the half-life of the RA-treated cells was substantially greater than the 24-h observation period (Fig. 4, C and E). For comparison, the half-life of ß2-mexpression in untreated cells appears to be the same or slightly longer than that of the RA-treated cells, both being substantially greater than 24 h (Fig. 4, D and E). These experiments indicate that the stability of TGF-ßl mRNA after RA treatment may contribute to the increase in steadystate mRNA levels.

TGFß,

NMYC Lane

123456

«-28S

Fig. 2. Northern blot hybridization analysis of mRNA expression in SMS-KCNR cells after treatment for 5 h (Lanes I and 2), 18 h (Lanes 3 and 4), or 40 h (Lanes 5 and 6) with solvent control (Lane\ I. 3. and 5), or 5 /AMRA (Lanes 2, 4, and 6) probed with either TGF-ßl (upper pane!) or N-mvc (middle panel). Lower panel, an ethidium bromide staining of the original get.

7DRA

NMYC GAPDH pGEM

r KCNR

Fig. 3. In vitro transcription of TGF-ßl and N-mvc during 7 days treatment of SMS-KCNR cells with control solvent or 5 fiw RA. In vitro transcription of a positive control (GAPDH) and negative control (pGEM) are also shown.

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A.

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Standard

NEUROHLASTOMA

Curve RT-PCR TGF-Bl

B.

Standard Curve RT-PCR

I Q 0

Il

ni of RNAtcnpUte Fig. 4. Evaluation of TGF-ßRNA message sta bility using RS-PCR analysis. A and B. standard curve of signal intensity using serial dilutions of cDNA tempiale prior to RS-PCR analysis of TGF-ßl (A) and ß2-mRNA (B) expression in SMS-KCNR cells with and without treatment with RA. C and D. the results of RS-PCR analysis of TGF-ßl (C) and ß2-m(D) expression in control (•)and RA-treated (•)SMS-KCNR cells after actinomycin D exposure. One hundred twenty-five ng of starling cDNA template and 32 ng of cDNA template were used for TGF-ßl and ß2-mexpres sion, respectively. Ten ^.1 of each RS-PCR reaction were loaded on a gel, electrophoresis was performed (see "Materials and Methods"), and autoradiograms

c.

TGF-ßlmRNA half life

of the dried gel were generated and evaluated by densitometry. The autoradiograms from which C and D were generated are shown in K.

Hours of Actiaomjcfn

1M

ng of RNA template

D.

D

IÌ2-MmRNA

half life

Hours of Ac ti nom yci n D

E.

from 0.1-10 ng/ml, the addition of TGF-ßl had no effect on cell growth (data not shown). Lack of a direct effect of TGF-ßl on SMS-KCNR cells suggested that TGF-ßreceptors were either not expressed or expressed in insufficient numbers prior to RA treatment. To determine whether TGF-ßreceptors were expressed, SMSKCNR and SK-N-AS cells were evaluated for TGF-ßreceptor ex pression after 3 days of either 5 nM or 5 JAMRA treatment by affinity labeling and cross-linking (Fig. 5). Only TBR, (Mr 70,000-80,000) is detected in untreated SMS-KCNR control cells, and the levels of

Table 2 TGF-ßlevels ip\t) in conditioned media an assayed bv SELISA Subconfluent cultures were treated for 36 h in RPMI 1640, and supernatant was processed as described in "Materials and Methods." TGF-ßland TGF-ß2concentrations were calculated using cubic and quadratic regression equations with Dynatech's immunosoft program. Condition cells5 and

Treated32.3

UM RA AS KCNR5

+ 3.1 ±1.74.5 3.1 + 1.3

nw RA KCNRTGF-ßlControl+ 1.2

DISCUSSION

Treated0 24.0 ±2.4 13.8

0 à 0 œ0

8.0 ±1.7TGF-ß2Control

TBR, and TBRm (M, 300,000) receptors increase after RA treatment at both RA doses (Fig. 5A). In contrast, in the the RA-resistant line SK-N-AS, TBR, and TBR,,, are detectable, but both receptors de crease after exposure to RA (Fig. 5ß).We were unable to demonstrate TBR,, protein by affinity labeling in either cell line; therefore, we looked at TBR,, mRNA expression by Northern blot in both SMSKCNR cells and SK-N-AS cells treated with RA (Fig. 6). In SMSKCNR cells that are RA-responsive, TBR,, mRNA is expressed at low levels constitutively, increases markedly after 2 days of 5 /J.M RA treatment, and decreases thereafter at 4 and 8 days of RA (Fig. 6/4, Lanes 1-6). In SK-N-AS cells that are RA resistant, exposure to RA at 2 days results in a slight decrease in TBRH expression (Fig. 65, Lanes 7 and 8). Thus, the data obtained by Northern blot for TBR,, expression after RA treatment of these two neuroblastoma cell lines is similar to that obtained for TBR, and TBR,,, by affinity labeling.

0

The cascade of events by which RA mediates its dramatic effects on neuroblastoma growth and differentiation in vitro are not completely understood. Changes associated with RA treatment of neuroblastoma include decreases in the steady-state mRNA expression of the proto2383

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KCNR Control

Fig. 5. Regulation of TGF-ß receptor expression in neuroblastoma cells by RA. Affinity labeling and cross-linking of TGF-ßreceptors in SMS-KCNR (KCNR) (A) or SK-N-AS (AS) (B) neuroblastoma cell lines treated with RA or solvent control for 3 days. Whole cells were grown in media containing either ethanol solvent (Control) or 5 HMor 5 JAMRA and subsequently incubated with l25I-TGF-ßlin the presence (+) or absence (—)of unlabeled TGF-ßlcompetitor, as described in "Materials and Methods." A similar analysis performed on the pos itive control cell line CCL64 is shown in Fig. 6ß. The migration of standard protein size markers (in kilodaltons) are at the left of both panels.

SuM RA

5nM RA

B

CCL64

AS Control 5>lM Control RA

MW x10-3

200-

f1'"

97.56946-

:• "

MW x103

20097.56946-

5nM RA

30-

3014.3-

2114.4-

—. •»•

(ColdTGF-P,)

(Cold TGF-ß,)

-+-

+

-

+

A. C

RA C

RA C RA

1 23456 TGF-ß type II R

GAPDH

Fig. 6. Regulation of TBRU mRNA expression in neuroblastoma cells by RA. Northern blot analysis of SMS-KCNR cells (A) or SK-N-AS cells (B) treated with solvent control (C) or RA for 2 days (Lanes 1,2,7, and 8), 4 days (Lanes 3 and 4), or 8 days (Lanes 5 and 6). The blot was stripped and rehybridized with GAPDH to control for equal RNA loading in each lane.

ff •••

oncogenes N-myc, c-myb, and c-Ha-ras, increases in steady-state mRNA expression of the proto-oncogenes c-ets-l, c-fos, and c-erb-B, dephosphorylation of the tumor suppressor gene product RB, and a marked decrease in the expression of p34cdc2, a protein kinase that plays an important role in controlling cell cycle progression (12, 35, 36). We have shown that TGF-ßlmRNA and TGF-ßlprotein secre tion is increased, and types I, II, and III TGF-ßreceptor protein or mRNA are induced when the RA-sensitive SMS-KCNR neuroblas toma cell line is treated with 5 /AMRA (Figs. 1, 5A, and 6A; Table 2). These results are similar to those of Falk et al. (15), who demonstrated enhanced secretion of bioactive TGF-ß2and increased expression of all three TGF-ßreceptors on the surface of HL-60 cells treated with RA. In contrast, after RA treatment of the RA-resistant neuroblastoma cell line SK-N-AS, although TGF-ßl is secreted at 10-fold higher levels than SMS-KCNR cells and TGF-ßl secretion remains at ap proximately this level after RA treatment, types I, II, and III TGF-ß receptors are detectable at the protein or mRNA level, but expression of TBR, and TBRIH protein and TBR,, mRNA decreases after expo sure to RA (Figs. 5B and 6B). Thus, these data suggest that induction and maintenance of a TGF-ßlnegative autocrine growth loop may be a necessary component for RA responsiveness of neuroblastoma cells in vitro.

TBRn protein could not be demonstrated by affinity labeling and cross-linking in both cell lines, before or after RA treatment, despite repeated attempts. This may be due to low numbers of TBR,, recep tors expressed or some alteration of the receptor that alters the ability to cross-link it with ligand. The TBR, receptor binds TGF-ßonly in the presence of TBR,, (37, 38). Thus, one may infer that the increase in TBR, visualized by cross-linking analysis is most likely accompa nied by an increase in type II receptor as well. This conclusion is supported by the mRNA expression of TBR,, seen in Fig. 6. Further more, we have been able to use antibodies to the cytoplasmic portion of the type II receptor to immunoprecipitate the type I receptor cross-linked to labeled ligand in RA-treated KCNR cells.4 This further suggests that TBR,, protein is present and that it does complex to the type I receptor, but that it is not readily detectable by cross-linking analysis. No change was seen in the growth of SMS-KCNR cells treated directly with TGF-ßl (data not shown). This may be due to the fact that untreated SMS-KCNR cells expressed insufficient amounts of TBR,,, which may be below the threshold for TGF-ßresponsiveness. 4 J. Letterio, unpublished data.

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Type II receptors are responsible for TGF-ß-mediated growth inhibi tion and hypophosphorylation of the retinoblastpma gene product, pRB, possibly in conjunction with type I receptors (39). In SMSKCNR cells, TBR,, mRNA increased markedly after 2 days exposure to both 5 nin and 5 JU.MRA (Fig. 6/4). RA treatment may have increased the number of TBR,, receptors above a threshold level, which when taken along with an increased amount of secreted TGFßl,may explain the RA responsiveness of SMS-KCNR cells. Con versely, the relative RA resistance of SK-N-AS cells may be ex plained by the decrease of TBR,, expression (Fig. 6ß). Regulation of TGF-ßlgene expression is known to be complex. In these experiments, both transcriptional and posttranscriptional regu lation was observed, with a paradoxical decrease in transcriptional rate, along with an increase in message stability. The TGF-ßlhalf-life of 17 h, which we observed in untreated cells, correlates well with the 15-h half-life reported in untreated AKR-2B cells (40). In addition, we observed that RA increased TGF-ßlmessage stability to much greater than 30 h. An increase in steady-state TGF-ßmRNA due to an increase in mRNA stability has also been observed in AKR-2B cells after autoinduction with TGF-ßl and in colon carcinoma cells after serum starvation (40, 41). Recent work of Kim et al. (42) have demonstrated a stem-loop structure in the 5' untranslated region of the

differentiation induced by RA in NB cells is also mediated via induction of trkB in cells constitutively producing the trkB ligand brain-derived neurotrophic factor (48, 49). Growth inhibitory activi ties have not been reported to be induced by activation of the brainderived neurotrophic factor/trkB signal transduction pathway in NB cells (49).s Our evaluation of the regulation and expression of TGF-ß and its receptor in NB cells is consistent with the hypothesis that TGF-ßl stimulates growth inhibitory as well as differentiation re sponses in RA-sensitive NB cells. ACKNOWLEDGMENTS We thank Dr. Frank Ruscelli for the gift of TGF-ßl protein and Sharon Sickafuse for her expert technical support and data analysis.

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TGF-ßlgene that negatively regulates gene expression posttranscriptionally via interference with translation. This mechanism may also be responsible for the increased secretion of TGF-ßl protein observed after RA in these experiments but remains to be evaluated. The TGF-ßl secretion detected by SELISA (Table 2) could repre sent either active or latent TGF-ßl, because this cannot be distin guished by SELISA. Latency of the produced protein could not be evaluated by the CCL64 growth inhibition assay, because the presence of RA in conditioned media would also inhibit these epithelial cells. However, as recognized in other systems, retinoids and other steroid compounds that act to induce TGF-ßexpression typically result in the secretion of TGF-ßin its biologically active form (43). Pietenpol et al. (44) have shown that c-myc expression is negatively regulated by TGF-ßvia dephosphorylation of pRB. We found that there is an inverse correlation between TGF-ßlexpression and N-myc gene expression. Since N-myc and c-myc share sequence homology, these data suggest the possibility that TGF-ßmay also regulate N-myc expression, perhaps by a mechanism similar to that of the TGF-ß/cmyc interaction. Further studies are needed to clarify the nature of this interaction, if any, and to evaluate whether such an interaction occurs in other situations in which N-myc is expressed, such as small cell lung carcinoma and some Wilm's tumors. These results also suggest that in neuroblastoma cell lines, an increase in TGF-ßl secretion and TGF-ß receptor expression is associated with the induction of neuronal differentiation. At a dose of 5 nM RA, both TGF-ßl secretion and expression of type II receptors is increased, and we observed a detectable morphological evidence of neuronal differentiation in the absence of growth inhibition (data not shown). An association between TGF-ßl expression and neuronal differentiation has also been reported by McCune et al. (45) in a histochemical analysis of neuroblastoma tumors. TGF-ßl expression was found in 1 of 9 poorly differentiated neuroblastoma tumors but was strongly expressed in the differentiating neuronal cells of 3 of 3 ganglioneuroblastomas. The induction of a neural differentiation path way has been associated with the RA-mediated induction of TGF-ß secretion in at least one other situation. In the human embryonal carcinoma cell lines, Tera-2 clone 13 and NTera-2 clone Dl, neuroectodermal derivatives form in association with increased biologically active TGF-ß2secretion (46, 47) following RA treatment. RA induces arrest of cell growth and induction of differentiation in neuroblastoma cells. Recent studies indicate that the morphological

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