control of the expression of c-sis mrna in human ...increase in c-sis mrna even at 1.0 fim (data not...

[CANCER RESEARCH 49, 2914-2920, June 1, 1989]

Control of the Expression of c-sis mRNA in Human Glioblastoma Cells by PhorbolEster and Transforming Growth Factor ÃŸ1

Richard D. Press, Anita Misra, Glenda Gillaspy, David Samols, and David A. Goldthwait2

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT

The regulation of c-vi.v oncogene expression in human glioblastomacell line Al 72 has been investigated using a sensitive RNA-RNA solutionhybridization method. Enhanced expression of c-sis mRNA was induced

by phorbol ester (PMA) and diacylglycerol, each of which activatesprotein kinase C. c-.vi'.vmRNA was also induced by transforming growth

factor ÃŸ(TGF-,8). The response to PMA and TGF-/3 was transient, andin each case the decrease in t-sis mRNA level following maximumstimulation occurred with a half-life similar to the mRNA half-life

previously determined. Cycloheximide had no significant effect on theinduction of c-sis mRNA by either PMA or TGF-/3. The increases in c-sis mRNA following addition of either PMA or TGF-/3 correlated wellwith increases in c-sis transcription as observed by the nuclear run-ontechnique. In cells in which protein kinase C had been down-regulated,there was no inhibition of the c-sis mRNA response to TGF-/3. Furthermore in cells pretreated with TGF-/3, induction by PMA was unaffected.Thus the TGF-/S signal pathway does not involve activation of protein

kinase C, and at least two initially distinct intracellular signaling routeslead to activation of c-sis gene expression in this glioblastoma cell line.

The protein kinase inhibitor H7 abolished the ability of not only PMAbut also of TGF-/S to induce c-sis mRNA. The ability of H7 to inhibit theTGF-/Ã• stimulation suggests that a protein kinase other than proteinkinase C is involved in the signal transduction by TGF-/3.

INTRODUCTION

The observation that the B chain of PDGF3 is encoded bythe c-sis protooncogene (1, 2) provided the first direct linkbetween the product of a transforming gene and a factor regulating cell growth. Cellular transformation induced by the sis/PDGF-B gene product (3, 4) may be the result of excessive orinappropriate PDGF-mediated stimulation of growth inPDGF-responsive cell types such as fibroblasts, glial cells, andsmooth muscle cells (5). PDGF-mediated autocrine growthstimulation (6) may therefore be an in vivo mechanism by whichsarcomas and glioblastomas are initiated or maintained. Theexpression of c-sis mRNA in human sarcoma and glioblastomacell lines compared to its absence in normal fibroblast or glialcells (7-10) seems to support this autocrine hypothesis oftumorigenesis. The expression of c-sis mRNA has been observed in a series of normal human cells which includes endo-thelial cells (11, 12), placen tal cells (13), and activated mono-cyte/macrophages (14, 15). c-sis mRNA is also expressed in avariety of transformed cell types such as glioblastomas (7-10),fibrosarcomas (8), osteosarcomas (16, 17), breast carcinomas(18), and melanomas (19).

The focus of this laboratory has been on the regulation of c-

Received 6/13/88; revised 11/17/88, 3/1/89; accepted 3/7/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by Grants NIH 5-R01-42121, NIH 5-K06-GM 21444, NIH P30CA 43703, and DOE-DE-FG02-87ER60587.

2To whom requests for reprints should be addressed.3The abbreviations used are: PDGF, platelet-derived growth factor; TGF-0,

transforming growth factor ft ATCC, American Type Culture Collection;DMEM, Dulbecco's modified Eagle's medium; PCS, fetal calf serum; DTT,dithiothreitol; PMA, phorbol-12-myristate-13-acetate; DiC8, S,n-1,2-dioctanoyIglycerol; DMSO, dimethyl sulfoxide; PDB, phorbol-12,13-dibutyrate; BSA, bovine serum albumin; GAPDH, glyceraldehyde phosphate dehydrogenase.

sis gene expression in human glioblastoma cells. Previous studies have demonstrated expression oÃc-sis mRNA in the majorityof glioblastoma cell lines and a c-sis mRNA half-life in glioblastoma cell lines A172 and A2781 of 3.4 and 2.6 h, respectively, only slightly longer than the 1.6-h half-life observed innormal human umbilical vein endothelial cells (10). This suggested that, in the steady state, the posttranscriptional controlof sis gene expression is similar in tumor cells versus normalcells.

In the studies reported here, we have used a sensitive RNA-RNA solution hybridization procedure to examine quantitatively the kinetics of c-sis mRNA expression in glioblastomaA172 cells exposed to phorbol ester and TGF-/3. We havedemonstrated a transient c-sis mRNA response to these agents,and we provide evidence that this response is primarily on genetranscription. In addition, we show that, when TGF-0 stimulates synthesis of c-sis mRNA, the signals do not involve theprotein kinase C system.

MATERIALS AND METHODS

Cell Culture. Human glioblastoma cell lines A2781, A172, andA1235 were provided by Dr. Stuart Aaronson (NIH). Cell linesU373MG and U87MG were obtained from the ATCC. Normal glialcells were obtained from Dr. Steven D'Ambrosio (Ohio State Univer

sity) (20). The glioblastoma cells obtained from Dr. Aaronson weregrown in DMEM (Gibco) with 10% PCS (Gibco) plus 3.4 g/liter ofglucose. Those obtained from the ATCC were grown in 10%FCS:DMEM with nonessential amino acids. The normal glial cellswere grown in supplemented DMEM (20). For drug treatments, cellswere incubated in FCS-containing medium with the appropriate dilution of drug at 37Â°Cfor the specified length of time. When cells received

two sequential treatments, the monolayer was rinsed at least twice indrug-free medium before the second incubation.

Plasmid Constructs, Antisense RNA Synthesis, Solution Hybridization, and Nuclear Run-On Experiments. Analysis of the levels of c-sis,a-tubulin, and GAPDH mRNA was by RNA-RNA hybridization usinglabeled antisense RNA probes driven from the T-7 promoter. Thesource of the genes used, the construction of the plasmids, and thepreparation of the radioactive RNA probes have been described elsewhere (10) as have the isolation of RNA, the hybridization conditions,and the gel electrophoresis and autoradiography (10).

For nuclear run-on experiments, the nuclei were isolated as describedby others (21), and nuclear transcription reactions were performed withminor modifications. Each reaction consisted of 90 p\ of nuclei (10 x10' nuclei) and 70 ^1 of reaction mixture 135 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 400 mM KC1, 13.5 mM NaF, 80 MMEDTA, 32% glycerol, 9.5 HIM MgCl2, 297 Mg/ml of creatine kinase,5.9 mg/ml of creatine phosphate, 10.8 mM DTT, 1.35 mM each of CTPand GTP, 2.7 mM ATP, and 20 Mlof [a-32P]UTP (200 ^Ci, 800 Ci/

mmol). Incorporation of label was approximately 15%. Purified RNA(21), 35 to 40 x IO6 cpm, was added to a final volume of 1 ml ofhybridization buffer. Nitrocellulose filters with 5 ÃŸgof slot-blottedpBR322, pSM-1, and a-tubulin (10) were used for overnight hybridization and were exposed to Kodak XAR film in cassettes with intensifying screens at â€”70Â°Cfor 2 days. Quantitation of hybridization was

by densitometric scanning with an LKB Ultrascan XL apparatus.PMA, PDB, vasopressin, bradykinin, and cycloheximide were ob

tained from Sigma. TGF-/3 was obtained from R & D Systems, diacyl-

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on July 21, 2021. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

c-Ã®ismRNA IN HUMAN GLIOBLASTOMA CELLS

glycerol (DiCg) from Avanti Polar Lipids, and protein kinase inhibitorH7 from Seikagaku America.

RESULTS

Induction of c-.v/.vmRNA by Phorbol Ester and Diacylglycerol.To assess the effect of protein kinase C activation on c-sis geneexpression, growing cultures of glioblastoma A172 were treatedwith the tumor-promoting phorbol ester PMA (100 HM) forvariable lengths of time before cell lysis, preparation of totalcellular RNA, and quantitation of c-sis mRNA by RNA-RNAsolution hybridization analysis. As seen in Fig. 1, PMA treatment led to the accumulation of significantly higher levels of c-

sis RNA beginning approximately 2 h after stimulation andreaching a peak at 3 h. A return to the basal level was observedat approximately 14 h. The 153 nucleotide protected fragmentis from exon 6, and the 357 nucleotide protected fragment isfrom exon 7. In this experiment, no response was observedwhen the vehicle 0.002% DMSO alone was tested. Hybridization with probes for a-tubulin and GAPDH was done as acontrol for the amount of RNA added. The mRNA levels ofboth of these genes showed no significant change after PMAaddition (Fig. 1). Quantitative c-sis mRNA levels in this andsubsequent experiments are therefore presented as a ratio ofthe ,y/.ysignal to the tubulin signal.

Quantitative analysis of the time course of c-sis mRNAinduction by PMA in glioblastoma A172 cells is shown in Fig.2. The c-sis mRNA level was increased approximately 20-foldafter 3 h of drug treatment and then declined to a steady-statebasal value in 14 h. Since the rate at which an induciblesubstance approaches its postinduction steady-state level isdirectly related to its intrinsic stability (22), the half-life of thec-sis mRNA can be estimated from the data in Fig. 2. A semilogplot (Fig. 2, inset) of the differences between the c-sis mRNAlevels during the decay phase and the c-sis mRNA steady-statelevel as a function of time of PMA exposure reveals a straightline (correlation coefficient = â€”0.992)with a half-life of 2.3 h(22). This estimate of the c-sis mRNA half-life in the PMA-stimulated glioblastoma A172 cells is similar to the 3.4-h half-life previously reported using the transcription inhibitor acti-nomycin D in the absence of message induction (10).

The induction of c-sis mRNA has been determined in A172glioblastoma cells after a 3-h exposure to PMA concentrations

ranging from 2 to 400 nM. All PMA concentrations in excessof 25 nM were able to stimulate c-sis mRNA to near-maximal

levels (data not shown). A concentration of 100 nM PMA wastherefore used in subsequent experiments. Additional experiments with PMA stimulation utilized 4 h as the time foranalysis, as this reflected more accurately the peak c-sis mRNAlevel.

PDB (0.1 JIM), another active tumor promoter, induced anincrease in glioblastoma A172 cell c-sis mRNA similar to thatinduced by PMA, while phorbol itself, the biologically inactiveparent alcohol of both PMA and PDB (23), produced noincrease in c-sis mRNA even at 1.0 fiM (data not shown). Thesynthetic diacylglycerol DiCg (24), which also binds and activates protein kinase C, at 0.2 HIMinduced a transient 10-foldincrease of c-sis mRNA maximal after 2 to 3 h (data not shown).a-Tubulin mRNA levels were not significantly affected by thesetreatments (data not shown). Vasopressin (25), bradykinin (26),and phospholipase C (27), agents also known to signal throughthe protein kinase C system, elevated c-sis mRNA levels inthese cells (data not shown). Thus, a series of agents known toactivate protein kinase C can stimulate c-sis mRNA.

Induction of c-sis mRNA following TGF-/3 Addition. The effectof TGF-/3 on glioblastoma A172 cell c-sis mRNA expressionhas been investigated. Maximal c-sis mRNA induction wasobserved at approximately 2 h after TGF-/3 addition (Fig. 3)followed by a rapid decline to the baseline. The a-tubulin andGAPDH probes revealed no significant change in these mRNAsafter TGF-/3 addition. In later experiments, the peak of c-sismRNA following TGF-ÃŸaddition was generally at 2 to 3 h andwas 1 h earlier than the peak following PMA addition. Thehalf-life of the c-sis mRNA as it approached its new steady stateafter the peak of TGF-/3 induction (Fig. 3, inset) was approximately 3.3 h, a value similar to both the half-life seen afterPMA stimulation (2.3 h; Fig. 2), and the half-life measured inthe presence of actinomycin D (3.4 h) (10). The relationship ofthe concentration of TGF-/8 to the induction of c-sis mRNA inglioblastoma A172 cells was also examined. Concentrations inexcess of approximately 0.1 ng/ml were required for maximalincreases of c-sis mRNA (data not shown).

Fig. 1. Induction of c-sis mRNA by PMA.Near-confluent cultures of glioblastoma A172cells were incubated in medium containing 0.1MMPMA plus 0.002% DMSO. Time pointswere taken after 1, 2, 3, 5, 8, 10, 12, 14, 16,19, and 24 h of PMA exposure. Equal amounts(10 jig) of each RNA sample were simultaneously assayed for c-sis and tubulin mRNAexpression by RNA-RNA solution hybridization. Aliquots of the same RNA samples (10iig) were then assayed for GAPDH expression.VS represents cells treated with only 0.002%DMSO vehicle for 8 h. ND (no drug) represents untreated control cells. The predicted c-vf.vproic.-ctc.-ilfragments are 357 and 1S3 bases.The predicted a-tubulin- and GAPDH-pro-tected fragments are 136 and 110 bases, respectively. No RNA was available for analysisof GAPDH transcripts in the sample treatedwith PMA for 2 h.

357 SIS

153 SIS

TUBULIN

ND V8 1 23 5 8 10 12 14 16 19 24

tÂ«t iiftftfÂ«* GAPDH

2915



c-sis mRNA IN HUMAN GLIOBLASTOMA CELLS

240

200

m 16030

Â§5Â«K 120

UJC

80

40

O 4 8 12 16 20 24

TIME OF PMA EXPOSURE (.1uM) (HR)

Fig. 2. Induction of c-sis mRNA by PMA as a function of time. The autora-diograms from Fig. 1 were quantitated densitometrically, and the data wereplotted as a relative ratio of sis mRNA to tubulin mRNA versus time of exposureto PMA (O). The 0-h time point represents untreated control cells. The RNAratio in cells exposed to 0.002% DMSO for 8 h is also shown (H). The inset is asemilog plot of the same data showing the approach of c-sis mRNA to a newsteady state. This is a plot of the difference between the ii'j/tubulin ratio at each

descending time point minus the s/i/tubulin ratio at 14 h of PMA treatmentversus the time of PMA exposure.

Prolonged Exposure to Phorbol Ester Eliminates the c-sismRNA Induction following Subsequent Phorbol Ester Addition.Although glioblastotna A172 cell c-sis mRNA can be induced5- to 50-fold by a short 3- to 5-h exposure to PMA or PDB,

SIS

TUB

GAPDH

B sis

10

3CD=>O

W <

Â£ OC

UJDC

8 12 16 20 24

TIME OF TGF-B EXPOSURE (HR)

Fig. 3. Induction of c-i/j mRNA by TGF-0 as a function of time. Near-confluent cultures of glioblastoma A172 cells were exposed to media containing3 ng/ml of TGF-/3 for 0.4, 1.0, 2.0, 3.5, 7.0, 12, or 24 h (0). Control culturesreceived an identical dilution of vehicle (4 MMHCI, 1.5 Mg/m' of bovine serumalbumin) for either 5 or 25 h (Q). The inset shows a semi-log plot of the differencebetween the .m,'tubulin ratios at 3.5, 7, and 12 h and the .v/.v/inbiilinratio at 24 has a function of time of exposure to TGF-/J.

PMA and TGF-/8 Both Increase Nuclear Transcription. PMAand TGF-0 both are able to induce transient responses in thelevel of the c-sis mRNA. To determine whether this responsewas due to an alteration in the rate of transcription of the c-sisgene, nuclear run-on experiments were performed. For theseexperiments, glioblastoma A172 cells were exposed to eitherPMA for 2.5 h or TGF-/3 for 1.5 h prior to determination oflevels of both c-sis nuclear RNA transcription rate and c-sismRNA. Figs. 4 and 5 show that, with both PMA and TGF-/3,the increase in c-sis mRNA was similar to the increase in thec-sis nuclear RNA transcription rate. Times were chosen inorder to examine synthesis prior to any peak of activity, andalso in recognition of the l h difference in the time of maximummRNA level after PMA versus TGF-0 addition. We concludefrom these data that an increase in transcription of c-sis nuclearRNA is responsible for the increase in c-sis mRNA.

2916

SIS

TUB

Fig. 4. Comparison of levels of c-sis transcription rate and messenger RNAbefore and after addition of PMA or TGF-0. Glioblastoma A172 cells wereincubated with 0.1 Â¡iMPMA for 2.5 h or 3 ng/ml of TGF-/3 for 1.5 h. A, nuclearrun-on technique. The 32P-labeled RNAs were hybridized with 5 ^g each of c-sis(SIS), a-tubulin (TUB), and GAPDH DNA. B, RNA-RNA hybridization with c-

sis probe as in Fig. 1.

UJ

6.0-

5.0-

4.0-

Nucleartranscription rate

mRNA

Fig. 5. Quantitation of c-sis transcription rate and messenger RNA before andafter addition of PMA or TGF-/3. Data were obtained from densitometer tracingsof autoradiograms shown in Fig. 4 and are expressed as a ratio of the sis signalto that of a-tubulin. C, control cells.



C-SIJmRNA IN HUMAN GLIOBLASTOMA CELLS

chronic exposure to a phorbol ester (17 h) yielded basal values(Fig. 2, Table 1). In addition, acute stimulation of these chronically pretreated cells with fresh PMA yielded no observable c-

sis mRNA induction. In Table 1, cells pretreated with PDB for72 h or cells pretreated with PMA for 17 h were each nonresponsive to addition of fresh PMA. This inhibition of phorbolester-stimulatory activity by prior chronic exposure to phorbolester reflects protein kinase C down-regulation. In this condition, cells become nonresponsive to phorbol ester, and there isan alteration in the intracellular form of protein kinase C (28-31).

Medium harvested from cells incubated with PMA for 17 h,when applied to fresh cells, stimulated c-sis mRNA expressionat least as well as fresh PMA (Table 1, Experiment 2). Thissuggests that active PMA remained in the medium during thetime when the cells became refractory to additional phorbolester stimulation. The low level of c-sis mRNA in these chronically treated cells was therefore not due to degradation of thephorbol ester.

Prolonged Exposure to TGF-ÃŸReduces the c-sis mRNA Increase following Subsequent TGF-/3 Addition. When glioblas-toma A172 cells were exposed to 3 ng/ml of TGF-ÃŸfor 24 hand then washed and reexposed to a second addition of TGF-ÃŸ,the subsequent increase in c-sis mRNA was only 30% of theincrease in control cells without TGF-0 preincubation (Table2, Experiment 1). This could be due to a partial down-regulationof TGF-ÃŸreceptors during the long preincubation or to otherintracellular responses. Biologically active TGF-ÃŸis consideredto be present in the medium during the preincubation, as thismedium retains 73% of the capacity of the original TGF-/3 tostimulate c-sis mRNA in fresh cells (Table 2, Experiment 2).

TGF-/ÃŽDoes Not Stimulate c-sis mRNA through Protein Kinase C. Since either PMA or PDB pretreatment of cells caneffectively down-regulate protein kinase C so that subsequentexposure to fresh phorbol ester fails to stimulate c-sis mRNA(Table 1), this procedure can be used to determine whetherTGF-/3 signaling operates via protein kinase C activation. If thesignal pathway from the TGF-ÃŸreceptor to c-sis regulatorysequences utilizes protein kinase C, then down-regulation ofprotein kinase C should block the response to TGF-/3. Glio-blastoma A172 cells were therefore preincubated with PDB for28 h and were then challenged with either PMA or controlDMSO (Table 3, Experiment 1). No significant c-sis mRNAresponse was observed after addition of PMA compared to

Table 1 Effect of down-regulation of protein kinase C on the level of c-sis mRNANear-confluent A172 cell cultures were treated for the indicated times with

PMA (0.1 MMwith 0.002% DMSO), PDB (0.1 MMwith 0.002% DMSO), orcontrol DMSO (0.002%). In Experiment 2, medium from cells incubated for 17h with either PMA (0.1 MMPMA with 0.002% DMSO) or DMSO (0.002%) washarvested and added to previously untreated cells for 3 h.

Incubation IncubationFirst addition (h) Second addition(h)Experiment

1DMSOPMAPDBPDBPDBPDBPDBExperiment

2DMSOPMAPMA5517244772723317NoneNoneNoneNoneNoneNonePMA

6NoneNonePMA

3Medium"-DMSO3MediumÂ°-PMA

3WA

lubulinRNAratio1.04.90.60.60.50.80.91.06.20.711.38.7

Table 2 Effect of prolonged exposure to TGF-ÃŸon the ability of cells torespond to TGF-ÃŸ

Near-confluent cultures of glioblastoma A172 cells in Experiment 1 wereexposed for 24 h to 1.5 Mg/ml of BSA (control) or 3 ng/ml of TGF-0 plus 1.5Mg/ml of BSA. The cells were rinsed, and TGF-ÃŸat the same concentration wasthen added for the incubation. In Experiment 2, cells were exposed to similarconcentrations of BSA or TGF-ÃŸplus BSA for 17 h. Medium from these cells orfresh K.I.) was then added to fresh cells for the incubation.

PreincubationAdditionExperiment

1BSABSATGF-ÃŸTGF-ÃŸExperiment

2NoneNoneNoneNoneh24242424IncubationAdditionNoneTGF-ÃŸNoneTGF-ÃŸNoneTGF-/3MediumÂ°-BSAMedium"-TGF-/3h33333Relative

j/s/tubulinRNAratio1.07.00.82.11.03.70.92.7

" Medium from cells exposed for 17 h to either BSA or TGF-ÃŸplus BSA.

Table 3 PMA and TGF-ÃŸstimulation of c-sis mRNA levels through independentpathways

Near-confluent cultures of glioblastoma A172 cells were preincubated for 28h in Experiment 1 with 0.1 MMPDB or 0.002% DMSO prior to addition of either0.1 MMPMA, 2 ng/ml of TGF-ÃŸ,control DMSO (0.002%), control BSA (1 Mg/ml), or nothing. Cells in Experiment 2 were exposed to I Mg/ml of BSA controlor 2 ng/ml of TGF-/3 for 25 h prior to addition of 0.1 MMPMA, 2 ng/ml of TGF-ÃŸ,or nothing. Vehicles for PMA and TGF-ÃŸwere DMSO and BSA, respectively.

PreincubationConditionExperiment

1DMSODMSODMSOPDBPDBPDBPDBPDBExperiment

2BSABSABSATGF-ÃŸTGF-ÃŸTGF-ÃŸh2828282828282828252525252525IncubationConditionNonePMATGF-ÃŸNoneDMSOPMABSATGF-0NoneTGF-ÃŸPMANoneTGF-ÃŸPMAh43443334345i5/tubulin

RNAratio1.035113.85.86.37.4191.014202.89.226

" Medium from cells incubated for 17 h with either PMA or DMSO.

DMSO (6.3 with 5.8), implying efficient down-regulation ofprotein kinase C. In contrast, preincubation with controlDMSO failed to inhibit the subsequent stimulation of c-sismRNA by PMA addition. When the same down-regulated cellswere washed and restimulated with TGF-ÃŸ,c-sis mRNA wasincreased to 19, compared to 11 in cells without prior proteinkinase C down-regulation. The difference probably does notrepresent a superinduction of c-sis mRNA by phorbol esterpretreatment as the basal level of c-sis mRNA was higher in thePDB-pretreated cells than in the original control cells (3.8versus 1.0). If the relative sis level after 28 h of PDB and 3 h ofbovine serum albumin (7.4) is subtracted from 19, the increasedue to TGF-ÃŸis still 11.6, a value comparable to the responseof 11 in control-pretreated cells. The different incubation timeswith PMA (4 h) and TGF-/3 (3 h) were selected because of thedifferent times at which the peak of the c-sis mRNA responseto these agents occurred. Thus, cells no longer responsive toPMA stimulation because of protein kinase C down-regulationwere still responsive to TGF-ÃŸ.The TGF-ÃŸsignaling pathwayfor c-sis gene expression therefore does not utilize proteinkinase C.

A similar type of experiment was done which involved prein-

2917



c-Ã/smRNA IN HUMAN GLIOBLASTOMA CELLS

cubation of glioblastoma A172 cells with TGF-0 (Table 3,Experiment 2). When cells, preincubated with TGF-ÃŸfor 25 h,were washed and reincubated with TGF-0 for an additional 3h, the c-sis mRNA level increased, but to only 66% of the levelattained when TGF-ÃŸwas added to cells without prior TGF-0preincubation (Table 3, Experiment 2). However, after the samepreincubation with TGF-0, PMA increased the c-sis mRNArelative level from 9.2 to 26. By subtraction of the 2.8 base linevalue, the specific PMA stimulation was 23.2, a value similarto the PMA stimulation of 20 in control-pretreated cells. Thus,there is no impairment of the c-sis mRNA response to PMAby preincubation with TGF-0 consistent with the hypothesisthat the initial signal pathway of TGF-ÃŸinduced c-sis geneexpression does not involve protein kinase C.

H7 Inhibition of Stimulation by Either PMA or TGF-ÃŸ.Thesynthetic protein kinase inhibitor H7 [l-(5-isoquinolinyl sulfo-nyl)-2-methylpiperazine] inhibits a number of different intra-cellular protein kinases, including protein kinase C, via a directinteraction with the catalytic sites of the enzymes (32). Theresponse of the TGF-0 signaling pathway to H7 could not bepredicted as the involvement of protein kinases in the TGF-0pathway has not been described. Cells were preincubated withor without 50 ^M H7 before the addition of either TGF-0 orPMA (Table 4). As expected, the preincubation with H7 effectively inhibited the c-sis mRNA expression induced by PMA.The H7 also inhibited c-sis mRNA expression induced by TGF-ÃŸ.There was no morphological change in cells exposed to H7.Since data presented here indicate that the signaling from TGF-ÃŸto the c-sis gene does not involve protein kinase C, the resultswith the protein kinase inhibitor imply that another H7-sensi-tive protein kinase is involved in the TGF-0 pathway.

Protein Synthesis Not Required for the Induction of c-sismRNA by Phorbol Esters or TGF-ÃŸ.When glioblastoma Al 72cells were preincubated with or without cycloheximide prior toaddition of PMA or TGF-0, there was no significant effect ofthe cycloheximide on the c-sis mRNA response (data notshown). It can be concluded that the immediate induction of c-sis mRNA following TGF-0 or PMA addition is not dependentupon de novo protein synthesis.

Response of c-sis mRNA to PMA and TGF-ÃŸin a Series ofGlioblastoma Cell Lines. All of the work so far presented wasdone with the glioblastoma cell line Al72. A survey was therefore done of a number of other cell lines to determine whetherthe c-sis gene responded to addition of either PMA or TGF-0.

The results are shown in Table 5. In glioblastoma A2781 withthe highest relative sw/tubulin ratio, a response to PMA but no

Table 4 Effect of protein kinase inhibitor H7 on the induction of c-sis mRNA byPMA and TGF-ÃŸ

Near-confluent glioblastoma A172 cells were either not preincubated or preincubated for 40 min with either protein kinase inhibitor H7 (50 MM)or a controldilution of water. The cells were then treated for the indicated times with eitherPMA (0.1 MM with 0.002% DMSO), TGF-0 (3 ng/ml), or control DMSO(0.002%). Cultures initially preincubated with H7 were maintained in H7-con-taining medium throughout the subsequent treatments. Control cultures weretreated only with H7 (50 MM,55 min) or were untreated.

Table 5 c-sis mRNA levels in human cells without and with added PMAor TGF-ÃŸ

Experiments with PMA and TGF-/3 were each done at different times. ForPMA experiments, 0.002% DMSO or 0.1 to 0.2 MMPMA with 0.002% DMSOwas added for 3 to 5 h. For the TGF-0 experiment, l Mg/""' of BSA or 2 ng/mlof TGF-0 in the BSA were added for 3 h.

PreincubationAddition

minNoneH7NoneNoneH2OH7NoneH20H75540404040IncubationAdditionNoneNoneDMSOPMAPMAPMATGF-ÃŸTGF-0TGF-0h3.63.73.73.72.62.62.6.vi.v/lubiilinRNAratio1.00.63.014320.412160.9

No PMA orTGF-ÃŸ(relative

s/i/tubulinCells RNAratio)Glioblastoma

A2781Glioblastoma A 172Glioblastoma U373 MGGlioblastoma A 1235Glioblastoma U87 MGGlioblastoma U 138 MGNormal glial100

4592000Added

PMA(foldincreased".siv/tubulin

RNA ratio)3

4-49250

0eAdded

TGF-ÃŸ(fold increased"

j/j/tubulinRNA ratio)0

3.7-18370

ND*

ND" Increase over control values determined in that experiment.* ND, not determined.c When normal human glial cells were stimulated with PMA, c-sis mRNA

increased from an undetectable level to a level approximately 5% of the basal c-s/s mRNA level of A172 cells.

response to TGF-0 was observed. In glioblastomas U373MGand A1235 which had relatively low i/s/tubulin ratios, therewas a moderate response to each agent. In glioblastoma U87MG, in which there was no sis expression, PMA or TGF-ÃŸdidnot alter this. In normal untransformed human glial cells withno detectable basal expression of c-sis mRNA, PMA induced avery low level of c-sis mRNA roughly equivalent to 5% ofthatexpressed in the unstimulated A172 cells.

DISCUSSION

These studies have demonstrated that, in the glioblastomaA172 human cell line, the level of c-sis mRNA is increasedafter addition of either phorbol ester or TGF-ÃŸ.Furthermore,experiments have demonstrated that the increase in cytoplasmicc-sis RNA following stimulation by either of these agents correlates with a comparable increase in transcription in the nucleus as measured by the nuclear run-on technique. This supports the concept that the c-sis mRNA response followingaddition of PMA or TGF-ÃŸis due to an increase in transcription. The increase in c-sis mRNA observed after either PMAor TGF-|0 exposure is transient. Following addition of PMA orTGF-ÃŸto the glioblastoma A172 cells, the c-sis mRNA levelrises to a peak in approximately 2 to 4 h and then falls rapidly.In each case, the decrease in mRNA level is exponential suchthat its half-life can be calculated. After PMA addition, thehalf-life of c-sis mRNA is approximately 2.3 h, while afterTGF-jS addition it is approximately 3.3 h. The steady-state half-life for c-sis mRNA in these cells has been determined and is3.4 h (10). Minor differences in half-lives between those determined after drug stimulation and those determined after acti-nomycin D (10) are probably not significant. The data suggestan increase of transcription which lasts for a short period oftime, following which there is a rapid readjustment in the rateof transcription either to zero or to a low value, while theelevated mRNA level falls exponentially due to its normaldegradation system. This implies that the signals which increasethe transcription are transient and are eliminated by a secondsystem which becomes active. This sort of "cross-talk" has been

suggested for the protein kinase C system (33, 34) and is understudy.

The rapid induction of glioblastoma cell c-sis mRNA by PMAand TGF-0 is in contrast to the slow induction in some othersystems. Induction of c-sis mRNA by TGF-0 in human endo-thelial cells occurs in 30 h (35), and induction in mouse AKR-2B fibroblasts occurs in 12 to 24 h (36). The human leukemia

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C-iÂ«mRNA IN HUMAN GLIOBLASTOMA CELLS

cell lines K562, U937, and HL60, each of which responds toPMA treatment with a slow induction of c-sis gene expression(37-40), do not produce increased levels of c-sis mRNA following TGF-0 stimulation (39). These data suggest that both theability of TGF-/3 to induce c-sis gene expression and the kineticsof the induction are specific for the cell type.

The induction of glioblastoma cell c-sis mRNA by eitherprotein kinase C activation or TGF-/3 suggests that the elevatedlevels of c-sis mRNA in transformed versus normal glial cells

(10) could be the result of an inappropriate activation of eitherof these primary pathways. The expression in human glioblastoma cells (including A172) of TGF-/3 mRNA (41; Footnote 4)might then imply that an initial autocrine stimulation by TGF-ÃŸresults in expression of the c-sis (PDGF-/3) gene product

which causes inappropriate growth stimulation. Because mostcells secrete TGF-/3 in a biologically inactive form requiringsubsequent activation by target cell-associated factors (42), glioblastoma cells would have to possess such an activity in orderto respond to autocrine TGF-/3 stimulation.

Down-regulation of protein kinase C by phorbol ester hasbeen studied extensively in many systems. On addition ofphorbol ester, a rapid decrease in the free cytosolic enzyme isobserved associated with an increase in the membrane-boundactivity (28). This membrane-bound activity is converted to anenzymatically inactive form (29), and there is evidence that aproteolytic reaction cleaves the membrane-bound form to aphospholipid and Ca2+-independent catalytic unit and a regu

latory unit (30, 31). The response of c-sis mRNA to phorbolester was shown to be eliminated in the glioblastoma A172 cellspretreated with phorbol ester, and under these same conditions,the response of c-sis mRNA TGF-/3 was not impaired. Weconclude that two separate pathways exist for signals thatstimulate the transcription of c-sis mRNA. The conclusionregarding two separate pathways is supported by data from cellsexposed for a prolonged period to TGF-0 which then showedan increased response of c-sis mRNA to phorbol ester but adecreased response to TGF-/3. In endothelial cells, stimulationof c-sis mRNA by PMA and TGF-/3 is also thought to occurthrough separate signal transduction pathways (35).

The inhibition of the response of c-sis mRNA to both phorbolester and TGF-/3 by the inhibitor H7 is of interest. H7 has beenshown to inhibit a series of protein kinases which includeprotein kinase C and the cyclic AMP and cyclic GMP-depend-ent protein kinases but not the calcium-calmodulin-dependentprotein kinase (32). One would predict that the phorbol esterresponse should be inhibited by H7 as it was. However, theinhibition of the TGF-/3 response suggests that another as yetunidentified protein kinase may be active in the TGF-0 signaling pathway. It is possible that such a protein kinase is alsoinvolved in the more terminal steps of signaling through proteinkinase C.

Induction of c-sis gene transcription by the separate PMA-and TGF-0-mediated signal pathways suggests that separateenhancer sequences related to these agents may be present inthe 5'-flanking region of the c-sis gene. The AP-1 enhancer

sequence, known to be involved in the transcriptional responseof cells after the addition of phorbol ester (43, 44), isTGAGTCA (43, 44). It is to this sequence that the fos-junprotein complex binds to initiate transcription (45-48). In the5'-flanking DNA of the human c-sis gene (49, 50), this sequence

is present 2948 bases from the transcription start site. Othercontrol elements have been found at similar distances. The

4 Unpublished results.

glucocorticoid response element is 2.5 kilobases upstream ofthe tyrosine aminotransferase gene (51 ), and elements controlling the a-fetoprotein gene are over 3 kilobases upstream (52).Several AP-1-like sequences with one base altered are alsopresent in the c-sis 5'-flanking DNA.

The NF-1 site has been shown to be involved in the transcriptional stimulation of the type 1 collagen gene by TGF-0 (53).The sequence of this NF-1 site is TCGN5GCCAA (53). It hasnot been shown that the 5'-portion of this sequence is needed

for response to TGF-/3. A consensus sequence derived from aseries of transcription factor sites is GCCAA, and this isthought to be the binding sequence of the NF-1 protein (54).In the 5'-flanking sequence of the human c-sis gene, theGCCAA sequence is present in three copies at nucleotides -284(complementary strand), â€”620,and â€”3918 (complementarystrand) before the transcription start site. In several otherpromoters with the GCCAA consensus sequence, there is noadditional homology with the TCGN5GCCAA NF-1 binding

sequence of the type 1 collagen gene (54). There is also nosequence 5' to any of the three c-sis GCCAA sequences that is

similar to the NF-1 type 1 collagen sequence (53).The responsiveness of c-sis mRNA to PMA and TGF-/3 was

tested in a small series of glioblastoma cell lines. In 4 of 6 celllines, c-sis mRNA was expressed. Clearly the expression of thisgrowth factor gene is not necessary in all glioblastomas. Inthose cell lines in which it is expressed, phorbol ester increasedc-sis expression to a variable extent. However, in the two celllines that showed no basal level of c-sis mRNA, there was noresponse to PMA, suggesting that enhancer sequences may notbe readily available in genes that are transcriptionally silenced.Of interest is the lack of response of glioblastoma A2781 toaddition of TGF-ÃŸ.This cell line has the highest steady-statelevel of c-sis mRNA expression, and these data raise the question whether the c-sis gene is already maximally stimulated byTGF-/3. Normal glial cells, which do not express c-sis mRNA,responded only minimally to addition of PMA.

In conclusion, we have demonstrated that, in a glioblastomacell line, the steady-state level of c-sis mRNA is transientlyincreased on addition of phorbol ester or TGF-/3. This increasecorresponds to a brief stimulation of transcription. There is nosignificant change in the half-life of the c-sis mRNA after PMAor TGF-/3 stimulation compared to the half-life of the steady-state mRNA, and this is consistent with a rapid cessation oftranscription at the time of maximum stimulation. The signaling pathway from TGF-/3 does not utilize the phorbol esterprotein kinase C pathway, and the presence of separate enhancer elements related to phorbol ester and TGF-0 is consistentwith these separate pathways.

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1989;49:2914-2920. Cancer Res Richar D. Press, Anita Misra, Glenda Gillaspy, et al.

βFactor Glioblastoma Cells by Phorbol Ester and Transforming Growth

mRNA in HumansisControl of the Expression of c-

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control of the expression of c-sis mrna in human ...increase in c-sis mrna even at 1.0 fim (data not...

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