Insulin-like Growth Factor I and Transforming Growth Factor a as

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  • [CANCER RESEARCH 50. 103-107. January I. I990|

    Insulin-like Growth Factor I and Transforming Growth Factor a as AutocrineGrowth Factors in Human Pancreatic Cancer Cell Growth1

    Eiji 0.16 nisi), whereas cell growth of human fibroblasts wasstimulated by it. 12-0-Tetradecanoyl-phorbol-13-acetate also reducedthe binding of 12*I-TGF-a, but not 12'I-IGF-I, to PC cells. Decrease inTGF-a binding was mainly due to the reduced affinity of receptors to theligand.

    These results suggest that IGF-I and TGF-a are involved in PC cellproliferation as autocrinc factors. Further, the inhibition of PC cellgrowth by phorbol ester could be, at least partly, due to the decreasedbinding of TGF-a to the cells.


    Because of a diminished, or complete lack of, requirementfor serum or specific growth factors, neoplastic cells in cultureare presumed to have autonomous activating pathways in theirgrowth system, namely: (a) autonomous production of growthfactors; (b) synthesis of an altered receptor for growth factors;and (c) activation of postreceptor pathway (1-3).

    Many types of tumor cells grown in culture secrete a varietyof peptide growth factors into their culture medium, and thesame cells usually have functional receptors for such growthfactors (2, 3). For example, a recent study has shown that mostmalignant cells derived from solid tumors synthesize TGF-2and all such cells express mRNA for EGF/TGF- receptor (4).

    Received 6/13/89; revised 9/26/89; accepted 10/3/89.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.

    1The present work was supported by grants from the Ministry of Education,

    the Ministry of Health and Welfare, and the Foundation for Growth Science.2The abbreviations used are: TGF, transforming growth factor; EGF, epider

    mal growth factor; IGF, insulin-like growth factor; PC cell. MIA-PaCa 2 humanpancreatic carcinoma cell; DM EM. Dulbecco's modified essential medium; FCS,fetal calf serum; BSA, bovine serum albumin; TPA, 12-O-tetradecanoy Iphorbol-13-acetate: CM, conditioned medium; IR, immunoreactive; PK-C protein kinase C.

    In addition, growth factors such as IGF-I, TGF-, TGF-/3,platelet-derived growth factor, and bombesin have been reported to act on cell growth in an autocrine manner in neoplastic and nonneoplastic cells (2, 3, 5-11). Furthermore, a specificmonoclonal antibody against a receptor for a growth factor cansuppress the cell growth, which is consistent with the autocrinetheory in tumor growth (12, 13).

    The role of growth factors in the development of pancreaticcancer has been reported by several investigators (14-17). Chester et al. (14) have shown that the incidence of pancreatic cancerinduced by A'-nitrosobis(2-oxopropyl)amine was increased by

    EGF in Syrian hamsters. EGF has been shown to promotephosphorylation in cultured pancreatic cancer cells, MIA PaCa-2 (15). Moreover, over-expression of EGF receptor has beenfound in pancreatic cancer cells ( 16). Recently, Smith etal.(\l)revealed that mRNA of TGF-a, which is thought to act throughthe EGF receptor, is expressed in several kinds of pancreaticcancer cells.

    In the present study, we demonstrate that IGF-I and TGF-stimulate pancreatic cancer cell growth, presumably in an autocrine manner. In addition, we show that phorbol ester, aprotein kinase C activator (18), has a potent inhibitory activityon the cell growth. The mechanism(s) of this inhibitory actionin terms of the above two growth factors will be discussed.


    Cell Culture. MIA-PaCa 2 human pancreatic carcinoma cells wereobtained from the American Type Culture Collection (Rockville, MD)and maintained by once weekly passage in DM EM (Gibco, GrandIsland, NY) containing 4% FCS (Filtron, Australia) and antibiotics(Gibco). Human fibroblast cultures were established from the forearmskin of normal adults and were grown in RPMI (Gibco) supplementedwith 10% FCS and antibiotics. The cells were cultured at 37Cin 5%

    CO2/95% humidified air atmosphere.Proliferation Study. PC cells or human fibroblasts were seeded into

    12-well plates (Costar, MA) at the density of 1.5-2.0 x IO4cells/well.

    Cultured cells were usually used after growing for 3 days in 4% FCS/DMEM. The cells were washed with serum-free DMEM and proliferation studies were performed in 0.3% BSA/DMEM. The test materials,dissolved with 0.3% BSA/DMEM, were added at the initiation of theexperiment. Media and materials were changed every 2-3 days. At theend of the experiments, cells isolated by trypsinizing were countedusing a Coulter Counter (Coulter Electronics, Inc.) Recombinant IGF-I and TCP- were kindly supplied by Fujisawa Pharmaceutical Co.(Osaka, Japan) and Earth Chemical Co. (Hyogo, Japan). TPA wasobtained from Sigma Chemical Co. (St. Louis, MO).

    The mean SD was calculated for each group and significance wasdetermined by the Student's / test.

    Binding Studies. Confluent cells cultured in nmlii\\cll (12 wells)plates were used for the binding studies. The cells were washed oncewith ice-cold 0.3% BSA/DMEM supplemented with 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid (pH 7.4) and then incubated at 4Cfor 24 h in the same medium with or without peptides.

    Labeled ligands (40,000-50,000 cpm/well) were added and incubatedat 4Cfor 20 h. Reaction was stopped by removing the medium and

    washing the cells twice with the ice-cold fresh medium. PC cells were


    Research. on February 5, 2018. 1990 American Association for Downloaded from


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    Fig. I. A, PC cell growth in two different concentrations (2% and 10%) ofFCS. B, PC cell growth in serum-free medium. Initially, cells were cultured in4% FCS/DMEM; then medium was changed to 0.1% or 0.5% BSA/DMEM.Medium was changed every 2 days. Values are means SD (bars) of threedeterminations.

    then solubili/ed with 0.5 ml l N NaOH and transferred to tubes forcounting radioactivity.

    Collection and Extraction of Conditioned Medium. PC cells weregrown in flasks to confluence in 4% FCS/DMEM. Cells were washedonce with serum-free DMEM. Following 48 h of culture of these cellsin 0.3% BSA/DMEM, CM was collected and centrifuged at 500 x gfor 5 min. The supernatant was dialyzed (molecular weight cutoff,3500) against distilled water for 48 h at 4C.Freeze-dried CM prepa

    rations were treated with I Macetic acid for 20 h at 4 ( and centrifugedat 2000 x g for IO min. The supernatant was neutralized when necessaryand analyzed for the growth factors of interest.

    Radioimmunoassay for IGF-I or IGF-a. Antibodies against synthetichuman TGF- (Sumitomo Pharmaceutical Co., Osaka, Japan) andrecombinant IGF-I (Fujisawa) were obtained by immunizing rabbitswith peptides conjugated with thyroglobulin. Antibodies to IGF-I andTGF-a were used at final dilution of 1:50,000. Human IGF-I and ratTGF-a (Peninsula, CA) labeled with Na'"I (NEN, Boston, MA) by thechloramine-T method were used as tracers. The sensitivity in the IGF-I assay system was 20 pg/tube and the 50% inhibitory dose was almost0.2 ng/tube. IGF-II cross-reacted with this antibody at approximately0.1%. In the TGF-a radioimmunoassay system, the sensitivity was 0.3ng/tube, and 50% inhibitory dose was about 3 ng/tube. Cross-reactionof EGF in this assay system was less than 0.01%.


    Growth of PC Cells in Medium Supplemented with FCS orBSA. Fig. \A shows the growth of PC cells in 2% and 10%FCS/DMEM. The doubling time was about 24 h at the twoconcentrations. When FCS/DMEM was changed to 0.1% or0.3% BSA/DMEM on day 5, the growth rate of PC cells becameslower, but the final cell density on day 13 was comparable withthat obtained in the presence of 5% FCS (Fig. \B).

    Effect of Dialyzed CM on PC Cell Growth. To determinewhether PC cells produce factor(s) which modulate their owncell growth, we investigated the effect of CM from PC cells ontheir own cell growth. Culture medium (0.3% BSA/DMEM)conditioned by confluent PC cells for 48 h was dialyzed againstDMEM for 72 h at 4C(molecular weight cutoff, 3500). Then,

    the CM was filtered with a Millipore filter (0.22 ^m) and usedas a test material for PC cell growth. As a control material,0.3% BSA/DMEM not exposed to cells was processed in thesame way. As shown in Fig. 2, PC cell growth was stimulatedby the medium exposed to the cells. Maximum stimulatingactivity (1.8-fold above control value) was observed at a concentration of 50% of the test material. The control medium hadno stimulatory activity on the cell growth. In the same condi-


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    C 25'/. 50* 100*


    Fig. 2. Effect of CM from PC cells cultured in 0.3% BSA/DMEM on theproliferation of PC cells (13). Nonconditioned medium (0.3% BSA/DMEM) wasused for control materials (D). CM and noncondilioned medium were dialyzedagainst DMEM for 72 h at 4Cand then filtered and used as test materials.

    Growth study was performed using PC cells cultured in 0.3% BSA/DMEM. Thecell number was counted 3 days after the addition of samples (see "Materials andMethods"). Values are means SD (bars) of three determinations (**. P< 0.01.

    compared with control).




    1 TT.B5-lri00" C!IGF-KNG/ML)TGF**L.111



    Fig. 3. Effects of IGF-I (A) and TGF-(B) on PC cell growth. Cells grown in4% FCS/DMEM were washed with DMEM and then cultured in 0.3% BSA/DMEM with or without test materials. Media and test materials w