(CANCER RESEARCH 50, 4979-4983. August 15. 1990]

Paracrine Growth Stimulation of Androgen-responsive Shionogi Carcinoma 115 byIts Autonomous Subline (Chiba Subline 2)1

Yuzo Furtiva. Naohide Sato, Koichiro Akakiira, Tomohiko Ichikawa, Noriaki Suzuki, Ryoko Sato,and Jun Shima/akrDepartment of Urology, School of Medicine [Y. F., N, S., K. A., T. /., R. S., J. S.], and Laboratory of Biophysical Chemistry, Faculty of Pharmaceutical Sciences[N. S.J, Chiba University, Chiba-shi, 280 (School of Medicine) and 260 (Faculty of Pharmaceutical Sciences), Japan

ABSTRACT

Shionogi Carcinoma 115 cells (SC 115 cells) and Chiba Subline 2 cells(CS 2 cells) are clones of an androgen-responsive mouse tumor cell lineand its autonomous subline, respectively. Since it was reported that thegrowth of cells from SC 115 was regulated by an androgen-inducedfibroblast growth factor (FGF)-like peptide (1), the present study wasaimed at examining whether a similar growth factor was secreted by CS2 cells in the absence of testosterone. Although SC 115 cells did not growin the serum-free medium without androgens, SC 115 cells could proliferate in mixed culture with CS 2 cells, suggesting stimulation of SC 115cells by CS 2 cells. It was shown that CS 2 cells secreted a growth factorwithout the influence of testosterone, and this factor promoted the growthof SC 115 and CS 2 cells, as well as that of BALB/3T3 cells. The factorwas partially purified from serum-free conditioned medium obtained fromcultures of CS 2 cells.

It showed an affinity for heparin, stability to heat and acid treatments,a decrease in activity when anti-basic FGF antibody was added to cultures,and an estimated molecular weight of approximately 50,000. Therefore,the factor seemed to have the nature of an FGF-like peptide. It wasconcluded that, in the absence of testosterone, CS 2 cells produced anFGF-like growth factor which controlled the growth of both CS 2 cellsand parent SC 115 cells, in autocrine and paracrine manners, respectively.

INTRODUCTION

Since it has been reported that some hormone-responsivetumors are controlled by hormone-induced growth factors (1-4), the significance of autocrine effects on the actions of steroidhormones has become an important issue. SC 1153 is an androgen-responsive mouse tumor (5). Recently, it was reported thata clone from SC 115 secreted an FGF-like peptide when cultured in serum-free medium in the presence of androgens andthat the growth of SC 115 was regulated exclusively by theFGF-like peptide in an autocrine manner (1, 6-8). An andro-gen-unresponsive subline, CS 2, was derived from SC 115 (9-11), and a clone from CS 2 (CS 2 cells) has subsequently beenmaintained in our laboratory. The present study was undertakento examine growth factor secretion by CS 2 cells without theinfluence of androgens.

MATERIALS AND METHODS

Chemicals. Acidic and basic FGFs were purchased from R & DSystems (Minneapolis, MM). PDGF and TGF-fi were obtained from

Received 9/15/89; revised 4/27/90.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.

1Supported in part by a Grant-in-Aid for Cancer Research from the Ministry

of Education, Science and Culture (01010011): from the Ministry of Health andWelfare (1-42); and for Scientific Research from the Ministry of Education,Science and Culture (62480333), Japan.

2To whom requests for reprints should be addressed.'The abbreviations used are: SC 115, Shionogi Carcinoma 115; TGF-n and

-ß,transforming growth factor «and ß;IGF-I and -II, insulin-like growth factor-I and -II; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor;FBS, fetal bovine serum; CS 2, Chiba Subline 2; CM, conditioned mediumobtained from CS 2 cells: CS 2-GF, growth factor produced by CS 2 cells; SDS,sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate.

Oxford Biomedicai Research (Oxford, MI) and Biomedicai Technologies Inc. (Stoughton, MA), respectively. IGF-I, IGF-II, and TGF-awere purchased from Bachern Inc. (Torrance, CA). Epidermal growthfactor was obtained from Sigma Chemical Co. (St. Louis, MO). Anti-recombinant human basic FGF antiserum (rabbit, polyclonal) was agift from Takeda Chemical Industries (Osaka, Japan). The IgG fractionof the antiserum was obtained by using Protein A-Sepharose affinitychromatography, according to the method of Coding (12). '"I-BasicFGF assay system and ['Hjthymidine (40 Ci/mmol) were purchasedfrom Amersham International Pic (Buckinghamshire, UK). 125I-Protein

A (80 uCi/ug) was obtained from New England Nuclear (Boston, MA).Heparin-Ultrogel was obtained from IBF Biotechnics (Villeneuve-la-Garenne, France), and the molecular weight markers were purchasedfrom Bio-Rad (Richmond, CA) and BRL (Gaithersburg, MD).

Cells. Cloning of SC 115 and CS 2 cells was described previously(13). SC 115 and CS 2 cells were maintained in minimum essentialmedium/Ham's F-12 (1/1, v/v) containing 10% FBS (0.05% dextran-

coated, 0.5% charcoal-treated, designated as maintenance medium).For cultures of SC 115 cells, the medium contained 10~"Mtestosterone

(in ethanol, with a final concentration of 0.01%). BALB/3T3 cells weredonated by the Japanese Cancer Research Resources Bank (Tokyo,Japan) and were maintained in minimum essential medium containing10% FBS.

Growth of Cells. To determine cell growth, 5 x 10" cells/dish wereplated onto 35-mm dishes containing 2 ml of maintenance mediumsupplemented with 10~8 M testosterone. After 48 h, the medium wasreplaced by minimum essential medium/Ham's F-12 (1/1, v/v; serum-

free medium) with or without testosterone. The medium was changedevery other day. Viable cells were counted by trypan blue exclusion,using a hemocytometer. Separate counting of the cell numbers for SC115 and CS 2 cells in mixed culture was performed by morphologicaldifferences and chromosomal analysis (13).

Determination of Growth-promoting Activity by Thymidine Incorporation. SC 115 cells (1 x IO4 cells/well) were plated in 24-well plateswith 500 u\ of maintenance medium supplemented with IO"8 M testos

terone. After 48 h, the medium was changed to the serum-free mediumwithout testosterone except when examining the effects of androgens.Ninety-six h after changing to the serum-free medium, samples to betested (5-10 u\) were added to the wells and 24 h later [-'Hjthymidine

(0.5 uCi/5 fíl/well)was added to the medium. Culture was continuedfor 3 h and the radioactivity incorporated into DNA was measured(14). Effects of testosterone were examined in cultures containingtestosterone during the entire culture period. When CS 2 or BALB/3T3 cells were used, the following modifications were performed (15):2 x IO4cells/well were plated, and 6 h later the media were changed tothe serum-free (CS 2 cells) or 0.5% FBS-containing ones. Then, testsamples were added either immediately (CS 2 cells) or 48 h later(BALB/3T3 cells). The cells were cultured for 48 h (CS 2 cells) or 16h (BALB/3T3 cells), and thymidine incorporation was measured.Growth-promoting activity was expressed either as dpm/well or aspercentage of the control value obtained by incubation without any testsamples.

Preparation of CM. CS 2 cells (5 x IO5cells/dish) were plated onto100-mm dishes containing 8 ml of maintenance medium. Two dayslater, the medium was replaced by the serum-free medium withouttestosterone, and the medium was changed every other day. The medium obtained between the third and seventh days after changing to theserum-free medium was combined and filtered through a nylon membrane (pore size, 0.22 um; Costar, Cambridge, MA). The filtrate wasconcentrated 50-fold by ultrafiltration with M, 10,000 cut-off mem-

4979

on March 19, 2020. © 1990 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

GROWTH FACTOR OF ANDROGEN-RESPONS1VE TUMOR

brane discs (PM 10; Amicon Corp., Danvers, MA) and was mixed withCHAPS (Sigma Chemical Co.) to make the final concentration 0.1%.The mixture was dialyzed against 0.01 M Tris-HCl buffer (pH 7.5)containing 0.1% CHAPS.

Heparin Affinity Chromatograph). Concentrated and dialyzed CMwas applied to a heparin-Ultrogel column. The column was eluted witha 0.1-3.0 M NaCl gradient in 0.01 M Tris-HCl buffer (pH 7.5) containing 0.1% CHAPS, according to the method of Shing er a/. (16).

Size Exclusion Chromatograph}'. Aliquots of the samples were applied

to a TSKG 3000 SWXL high performance liquid chromatographycolumn (Tosoh Corp., Tokyo, Japan). The column was eluted with 0.1Mphosphate buffer (pH 7.0) containing 0.3 M NaCl and 0.1 % CHAPS.

SDS-PAGE. Aliquots of the samples were denatured and subjectedto electrophoresis on slab gels by the method of Laemmli (17). Thegels were stained using a Bio-Rad silver nitrate stain kit. To elute thesamples from the gels, electroelution was performed in 0.025 M Tris/0.192 M glycine buffer (pH 8.3) supplemented with 0.1% SDS, using aMaxyield GP (Atto Corp., Tokyo, Japan).

Western Blot Analysis. Aliquots of the samples were separated bySDS-PAGE and electrotransferred onto Clear Blot Membrane-P (AttoCorp.). The membrane was washed, incubated with anti-basic FGFantibody, and treated with '"I-Protein A, according to the method of

Towbin et al, ( 18).Protein Determination. Protein was measured with a Bio-Rad protein

assay kit or by UV absorption with bovine serum albumin as a calibration standard.

RESULTS

Growth of SC 115 and CS 2 Cells in Serum-free Culture. SC115 cells showed exponential growth in the presence of testosterone, while removal of testosterone caused a decline in cellnumbers after a transient initial increase, showing the androgenresponsiveness of the growth (Fig. 1). The maximal effects oftestosterone were observed at 10~8 M, between 10"'°and 10~6

M. Proliferation of CS 2 cells was almost identical irrespectiveof the presence or absence of testosterone. Previously it wasreported that the growth rate of SC 115 cells was higher thanthat of CS 2 cells in fetal calf serum-containing medium (13),

1x10"-

1x10'

1X10'

•¿�II.T(-)

0 4 8 12Days after Serum-free Culture

Fig. 1. Growth of SC 115 and CS 2 cells cultured separately or in combination(Mix.) in serum-free medium. Cells were cultured in maintenance medium in thepresence of testosterone (SC 115, combination) for 2 days and transferred to theserum-free medium with \T (+)\ or without [T f—)\10~*M testosterone. In mixed

culture, the ratio of SC 115 and CS 2 cells was 1:1 at the start of culture. Eachpoint was calculated from three dishes and SE was not depicted due to its verylow level. Percentage of the number of SC 115 cells in the combination was 45 ±4% (with testosterone) and 47 ±3% (without testosterone) at 4 days after serum-free culture. 47 ±3% (with testosterone) and 47 ±5% (without testosterone) at8 days, and 48 ±5% (with testosterone) and 49 ±K (without testosterone) at12 days.

but in the serum-free medium supplemented with testosteronethe growth rate of SC 115 cells was reduced to become identicalto that of CS 2 cells. This can be explained by the high growthdependency on fetal calf serum of SC 115 cells. When equalnumbers of SC 115 and CS 2 cells were mixed and culturedsimultaneously in the presence or absence of testosterone, thetotal number of cells increased markedly, due to the equalproliferation of both cells. The rate of increase of respectivecells in mixed culture was greater than that obtained by cultur-ing them separately (P < 0.001), and unknown cooperativeeffects may be considered during mixed growth of the two cells.Because SC 115 cells could not grow alone without testosterone,the proliferation of SC 115 cells in the mixed culture withouttestosterone might be supported by CS 2 cells, probably due tothe secretion of growth-promoting substance(s) by CS 2 cells.Therefore, CM was prepared in the absence of testosterone andexamined for its growth-promoting activity.

Effects of Testosterone, CM, and Growth Factors on Thymi-dine Incorporation of SC 115, CS 2, and BALB/3T3 Cells. Theeffects of IO"8 M testosterone on thymidine incorporation of

SC 115 cells were evident, but testosterone did not influenceCS 2 and BALB/3T3 cells (Table 1). Addition of CM stimulatedthymidine incorporation of SC 115 cells in the serum-freemedium in a dose-dependent manner. CM also increased DNAsynthesis of CS 2 cells in the serum-free medium but the rateof increase was less than that observed for SC 115 cells. Thismay be explained by autocrine control by growth factor(s) fromCS 2 cells during culture. Addition of CM stimulated thegrowth of 3T3 cells up to 10 /¿g/ml,and then the activitydiminished.

The effects of various growth factors on DNA synthesis ofSC 115 and CS 2 cells were examined. Acidic FGF stimulatedDNA synthesis of SC 115 cells, and much greater effects wereobserved with basic FGF, the activity of which was similar tothat of CM. In the case of CS 2 cells, only a slight increase ofDNA synthesis was observed with both acidic and basic FGFs,

Table 1 Effects of testosterone, conditioned medium from CS 2 cells, and growthfactors on f'HJthymidine incorporation into DNA ofSC 115, CS 2, and

BALB/3T3 cellsEffects on DNA synthesis were measured in the serum-free medium (SC 115

and CS 2 cells) or 0.5% FBS-containing medium (BALB/3T3 cells), as describedin "Materials and Methods." Values were obtained from at least three independent

experiments and are shown as mean ±SE from percentage of control (noadditives). Counts of control were: 473 ±30 dpm/well (SC 115 cells). 2485 ±72(CS 2 cells), and 7990 ±120 (BALB/3T3 cells).

Additives

[JH]Thymidine incorporation (%)

SC 115 cells CS 2 cells BALB/3T3 cellsTestosterone. 10"* M 700 ±42" 100 ±1 100 ±2

CM0.1°

0.31.03.0

10.030.0Acidic

FGF,1.0*Basic

FGF,0.1'TGF-0,

1.0*PDGF,

10.0'100

±3136±\'255

±17"521 ±32°

1150 ±54°1822 +89°1203

±39°1694

±55°146

±5¿199

±4°100

±5109 ±11126 ±6'153 ±13C166 ±19C220 ±\f,"150

±5"166

±5^101

±2100±3117±

12361 ±22°506 ±32°672 ±39°718 ±40°420±51°%g

of protein/ml.' P < 0.05.

'Maximum effective concentration examined (0.001-10.0 ng/ml for acidicand basic FGFs and TGF-f); 0.01-10.0 unit/ml for PDGF).

4980

on March 19, 2020. © 1990 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

GROWTH FACTOR OF ANDROGEN-RESPONSIVE TUMOR

probably due to the presence of autocrine growth factor(s).TGF-0 and PDGF showed weak stimulatory effects on SC

115 cells but other growth factors such as TGF-«, epidermalgrowth factor, IGF-I, or IGF-II (10 pg to 100 ng/ml) did nothave any influence on thymidine incorporation of SC 115 orCS 2 cells.

Fractionation of CM by Heparin Affinity Chromatography.The elution pattern of CM revealed three peaks showinggrowth-promoting activity: flow through and 0.7 M and 1.0 MNaCl fractions (Fig. 2). Since the 1.0 M NaCl fraction exhibitedthe greatest stimulating activity, this fraction was used in thesubsequent experiments as partially purified CM. Specificgrowth-promoting activity of the 1.0 M NaCl fraction wasapproximately 100 times that of the original CM.

Characteristics of Growth Factor from CS 2 Cells. The effectsof various treatments on the growth-promoting activity of partially purified CM were examined (Table 2). The activity wasresistant to heat and acidic treatments. Trypsin treatment completely abolished the activity, showing that the growth-promoting activity was associated with a protein-like factor.

Since basic FGF exhibited a potent growth-promoting activity on SC 115 cells, the effects of anti-basic FGF antibody onDNA synthesis stimulated by partially purified CM were ex-

Fraction Number

Fig. 2. Heparin affinity Chromatograph} of conditioned medium from CS 2cells. CM (27 mg of protein) was applied to a heparin-Ultrogel column (1.0 x 5.4cm) and eluted with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.1% CHAPSand a gradient of NaCl, at a flow rate of 23.4 ml/h. Each 2-ml fraction wascollected. Protein was measured by UV absorption at 280 nm. Ten /J of eachfraction were used for the determination of the growth-promoting activity to SC115 cells in serum-free medium. Subtraction of the control value (no additives,305 ±20 dpm/well) was omitted.

Table 2 Stability of growth-promoting activity in conditioned mediumfrom CS 2 cells

The 1.0 M NaCl fraction from heparin affinity Chromatography of concentratedand dialyzed CM was used. Treatment with HC1 was performed by mixing thefraction with HC1 and the mixture was kept at 4'C overnight, neutralized withNaOH. and dialyzed against 0.01 M Tris-HCl buffer containing 0.1% CHAPS.Exposure to dithiothreitol was performed for 2 h at room temperature, followedby dialysis. Trypsin treatment was performed at 37°Cfor 2 h. Remaining activitywas determined by [3H]thymidine incorporation into DNA of SC 115 cells. Valueswere obtained from at least three independent experiments and are shown asmean ±SE from percentage of untreated CM. Counts in the presence and absenceof the untreated CM were 1443 ±85 and 382 ±37 dpm/well, respectively.

TreatmentHeat

70°C,5 min100°C,5minHC1,

1NDithiothreitol.

0.05MTrypsin,

Kl ni: mlRemaining

activity(%)91

±730 ±364

±564

±60

amined (Fig. 3). In this experiment, the amount of partiallypurified CM was reduced in an attempt to show clear effects oflimited amounts of anti-basic FGF antibody. Neither anti-basic

FGF antibody nor preimmune rabbit IgG had any effects onDNA synthesis of SC 115 cells. Growth-promoting activity ofbasic FGF was completely blocked by the antibody, which wasconfirmed to have no effects on acidic FGF. The activity ofpartially purified CM was reduced to approximately half byaddition of anti-basic FGF antibody. The inhibition rate by theantibody was constant during purification steps for CS 2-GF,

but the inhibition was not caused by the presence of basic FGF,since the 125I-basic FGF assay system did not detect it in

partially purified CM (data not shown).Partially purified CM was rechromatographed on a heparin

affinity column and the fraction eluted with 1.0 M NaCl wasobtained. The specific activity of the fraction was increased toapproximately 500 times that of the original CM. The fractionthus obtained was dialyzed against distilled water overnight at4°C,lyophilized, and dissolved in 0.1 M phosphate buffer con

taining 0.1 % CHAPS and 0.3 M NaCl. An aliquot of the samplewas applied to a TSKG 3000 SWXL high performance liquidChromatography column and fractionated, followed by measurement of the growth-promoting activity (Fig. 4). The activity

was localized in a single peak with a molecular weight ofapproximately 50,000.

An aliquot of the peak fraction on a TSKG 3000 SWXLcolumn was subjected to immunoblotting with anti-basic FGF

antibody (Fig. 5). A main band and an additional band werefaintly detected at M, 50,000 and 57,000, respectively. Althoughthe amount applied to respective lanes was similar on the basisof the growth-promoting activity, the binding of the antibodywith basic FGF was greater than that with CS 2-GF. The peakfraction was run on SDS-PAGE and the gels were stained (Fig.

6). Main and additional bands were observed at the same siteswith immunoblotting. Electroelution of the gel containing thesetwo bands revealed that the growth-promoting activity wasassociated with this region. These results suggested that CS 2-

GF was related to but not identical to basic FGF and the

MPPCM PPCM PPCM bFGF bFGF

Prefflwume Anti-bfGf PretmmuneIjG «itiboii isG

»nli-tiFSFintibodr

Fig. 3. Effect of anti-basic FGF antibody on the growth-promoting activity ofthe growth factor from CS 2 cells. The 1.0 M NaCl eluate of heparin affinityChromatography was used as partially purified conditioned medium (PPCM).Forty ng of protein/ml partially purified CM, 100 Mg/ml preimmune rabbit IgG,100 //g/ml anti-basic FGF antibody, or 0.1 ng/ml basic FGF (bFGF) were addedto respective wells. The growth-promoting activity was measured with SC 115cells cultured in serum-free medium. Each column represents mean ±SE obtainedfrom at least three determinations. The control value (no additives, 531 ±50dpm/well) was subtracted in all instances. *,P< 0.01 from partially purified CMand preimmune IgG; **. />< 0.001 from basic FGF and preimmune IgG.

4981

on March 19, 2020. © 1990 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

158K 44K UK 1 35K

GROWTH FACTOR OF ANDROGEN-RESPONS1VE TUMOR

1 2

5 10 15 20 25 30 35 40

Fraction Number

Fig. 4. Size exclusion Chromatograph) of growth factor from CS 2 cells.Thirty-five jig of protein of the 1.0 M NaCI fraction of CM from two cycles ofheparin affinity chromatography were applied to a TSKG 3000 SWXL highperformance liquid chromatography column, at a flow rate of 1.0 ml/min. Each0.25-ml fraction was collected and 5 fi\ of each fraction were measured for thegrowth-promoting activity to SC 115 cells. Subtraction of the control value (noadditives, 337 ±16 dpm/well) was not performed. In a parallel run. the elutionsites of molecular markers were checked: bovine -y-globulin (M, 158.000). oval-bumin (M, 44,000). myoglobin (A/, 17,000), and vitamin B,2 (M, 1.350).

1 2Top97.4K68K

43 K

29K

18.4K

FrontFig. 5. Western blot analysis of growth factor from CS 2 cells and basic FGF.

Ten fjg of protein of the peak fraction from size exclusion chromatography, asshown in Fig. 4 (lane I), and 100 ng of basic FGF (lane 2) were subjected toSDS-PAGE and to immunoblotting with anti-basic FGF antibody. Black arrow,a site of M, 50,000. Molecular weight markers used were phosphory läseB (M,97,400). bovine serum albumin (M, 68,000). ovalbumin (M, 43,000). carbonicanhydrase (M, 29,000). and /i-lactoglobulin (A/, 18.400).

molecular weight of this FGF-like peptide was assumed tentatively to be approximately 50,000.

DISCUSSION

There have been some reports concerning growth-promotingsubstances produced by SC 115 and its related tumors. An

Top

92.5K66.2K

45K

31K

21.5K

14.4K

FrontFig. 6. SDS-PAGE of growth factor from CS 2 cells. One |jg of protein of the

peak fraction from size exclusion chromatography, as shown in Fig. 4 (lane I),was subjected to electrophoresis. Molecular weight markers used (lane 2) werephosphorylase B (M, 92.500), bovine serum albumin (M, 66,200), ovalbumin (M,45.000). carbonic anhydrase (M, 31.000). soybean trypsin inhibitor (M, 21.500).and lysozyme (M, 14,400).

autonomous subline derived from SC 115 produced a tumor-stimulating factor which stimulated the growth of the originaltumor, but this factor did not exert any influence on its owngrowth (19). In this regard, the factor reported by these authorsseemed to be different from CS 2-GF. Significant mitogeniceffects of the conditioned medium from androgen-treated SC115 cells for mouse fibroblasts were observed when cells werecultured in serum-supplemented medium (20). This indicatedthat a growth-promoting substance was secreted by SC 115cells under the influence of androgens. It is well known thatserum contains many growth-promoting substances; therefore,to analyze the factor(s) secreted by tumor cells, it is necessaryto develop a well defined culture system. Recently, culturing ofSC 115 cells in serum-free medium has been successfully studiedand the production of an autocrine growth factor in the presenceof androgens was discovered (21). It was reported that, amongmany growth factors, only FGF stimulated the growth of cellsfrom SC 115 in serum-free culture. However, anti-basic FGFantibody did not block the growth-promoting activity of theconditioned medium in culturing cells from SC 115 completely;therefore, it was assumed that the growth factor from SC 115was an FGF-like peptide (1, 7, 8). The present experimentsshowed that CS 2 cells also secreted a growth factor whichresembled FGF.

There are many similarities between the growth factor derivedfrom SC 115 ( 1,6-8) and CS 2-GF: affinity to heparin, stability,and inhibition of activity by anti-basic FGF antibody. Althoughthere is a slight difference in the molecular weight, it seemsprobable that these two growth factors are very similar to eachother, if not identical.

CS 2-GF shows similarities with authentic FGF, but thereare some differences between the two factors. The molecular

4982

on March 19, 2020. © 1990 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

GROWTH FACTOR OF ANDROGEN-RESPONSIVE TUMOR

weight of CS 2-GF is greater than that of authentic FGF (22,23). CS 2-GF is resistant to heat and acid treatments, to whichFGF is susceptible (24, 25). FGF does not contain a signalpeptide (26), but CS 2-GF may be secreted into medium. CS 2-

GF is eluted with 1.0 M NaCl from a heparin affinity columnand the elution profile is similar to that of acidic FGF (27) butnot to that of basic FGF, although it has been reported thatacidic FGF is labile to heat and acid treatments (25). In thepresent study, the antibody, which does not inhibit the activityof acidic FGF, reduced the effects of CS 2-GF. These resultsstrongly suggest that CS 2-GF is not acidic or basic FGF itselfbut contains some structures homologous to FGF, presumablyin the active domain. Alternatively, it may be FGF in association with some proteins making a large molecule (28), but thisseems unlikely, since CS 2-GF appears to be a large moleculewhen subjected to electrophoresis under denaturing conditions.

There have been previous reports concerning growth factorsthat resembled FGF in some respects. FGF-like substance(s)was found in some tumors, and autocrine control of the tumorsby this substance(s) was suggested (29). A high molecular weightform of basic FGF, estimated to be M, 25,000, was isolatedfrom porcine brain and its biological activity was blocked completely by anti-basic FGF antibody against authentic M, 18,000FGF (30). It was reported that bovine pituitary follicular cellssecreted a M, 45,000 heparin-binding endothelial cell growthfactor which was a cationic heat- and acid-stable protein beinga dimer of 23,000 (31, 32). CS 2-GF does not seem to beidentical to these substances but is one of the FGF-like peptideswhich controls its own growth and that of its parent tumors inautocrine and paracrine manners, respectively.

ACKNOWLEDGMENTS

The authors thank Dr. Toshio Kuroki, Institute of Medical Science,University of Tokyo, and Dr. Katsuichi Sudo, Takeda Chemical Industries, for providing BALB/3T3 cells and human recombinant anti-basicFGF antibody, respectively.

REFERENCES

1. Nonomura, V, Nakamura, N.. Sudo, K., Sato, B., and Matsumoto. K.Proliferation of Shionogi carcinoma cells caused by androgen-induced fibro-blast growth factor (FGF)-like peptide in an autocrine mechanism. In: M.Serio (ed.). Perspectives in Andrology, pp. 431-438. New York: Raven Press,1989.

2. Huff, K. K., Kaufman, D., Gabbay, K. H.. Spencer, E. M., Lippman, M. E.,and Dickson, R. B. Secretion of an insulin-like growth factor-I-related proteinby human breast cancer cells. Cancer Res., 46: 4613-4619, 1986.

3. Dickson, R. B., Bates, S. E., McManaway, M. E., and Lippman, M. E.Characterization of estrogen responsive transforming activity in humanbreast cancer cell lines. Cancer Res.. 46: 1707-1713, 1986.

4. Manni, A., Wright, C., Feil, P., Baranao, L., Demers, L., Garcia, M., andRochefort, H. Autocrine stimulation by estradiol-regulated growth factors ofrat hormone-responsive mammary cancer: interaction with the polyaminepathway. Cancer Res., 46: 1594-1598. 1986.

5. Minesita, T., and Yamaguchi. K. An androgen-dependent mouse mammarytumor. Cancer Res., 25: 1168-1175. 1965.

6. Nonomura, N., Nakamura, N., Uchida, N., Noguchi, S., Sato, B., Sonoda,T., and Matsumoto, K. Growth-stimulatory effect of androgen-induced au-tocrine growth factor)s) secreted from Shionogi carcinoma 115 cells onandrogen-unresponsive cancer cells in a paracrine mechanism. Cancer Res.,48: 4904-4908, 1988.

7. Sato, B., Nakamura, N., Noguchi, S., Uchida, N., and Matsumoto, K.Characterization of androgen-dependent autocrine growth factor secretedfrom mouse mammary' carcinoma (Shionogi carcinoma 115). In: H. Imura,K. Shizume, and S. Yoshida (eds.). Progress in Endocrinology 1988, Vol. 1,pp. 99-104. Amsterdam: Elsevier Science Publishers, 1988.

8. Lu, J., Nishizawa. Y., Tanaka, A., Nonomura, N., Yamanishi, H., Uchida,

N.. Sato, B., and Matsumoto, K. Inhibitory effect of antibody against basicfibroblast growth factor on androgen- or glucocorticoid-induced growth ofShionogi carcinoma 115 cells in serum-free culture. Cancer Res., 49: 4963-4967, 1989.

9. Fuse, H., Akimoto, S., Sato, R., Miyauchi, T., Wakisaka, M., Hosoya, T.,and Shimazaki, J. Changes in cytosolic androgen receptor after administration of testosterone of androgen-dependent mouse mammary tumor (Shionogi carcinoma) and its sublines of altered androgen dependency. Endocrino!.Jpn.. 30: 189-197, 1983.

10. Suzuki. N., Urata, M., Miyauchi, T., Wakisaka, M., Shimazaki. J., andHosoya. T. In vivo effect of androgen on RNA synthesis in nuclei fromandrogen-independent subline of Shionogi carcinoma (CS 2). Endocrino!.Jpn., 50:657-661, 1983.

11. /ama. S., Akimoto, S., Yazawa, S., Ichikawa, T., Hayata, I., Petrow, V.,Shimazaki, J. Androgen receptor, testosterone uptake and karyotype inandrogen-dependent mouse tumor (SC 115) and its androgen-independentsubline (CS 2). Endocrino!. Jpn., 34: 279-289, 1987.

12. Coding, J. W. Conjugation of antibodies with fluorochromes: modificationsto the standard methods. J. Immunol. Methods, 13: 215-226, 1976.

13. Ichikawa, T., Akimoto, S.. Hayata, I., and Shimazaki, J. Progression andselection in heterogeneous tumor composed of androgen-responsive Shionogicarcinoma 115 and its autonomous subline (Chiba Subline 2). Cancer Res.,49:367-371, 1989.

14. Nishikawa, K. Intracellular DNA synthesis factor from rat rhodamine fibrosarcoma. Biochem. Int., 4: 169-175. 1982.

15. Klagsbrun, M., Langer, R., Levenson, R., Smith, S., and Lillehei, C. Thestimulation of DNA synthesis and cell division in chondrocytes and 3T3 cellsby a growth factor isolated from cartilage. Exp. Cell Res., 105:99-108,1977.

16. Shing, Y., Folkman, J., Sullivan, R., Butterfield, C, Murray, J., and Klagsbrun, M. Heparin aliimi): purification of a tumor-derived capillary endothelial cell growth factor. Science (Wash. DC), 223: 1296-1299, 1984.

17. Laemmli, U. K. Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature (Lond.), 227: 680-685. 1970.

18. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer of proteinsfrom polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nati. Acad. Sci. USA, 76: 4350-4354, 1979.

19. Noguchi, S., Uchida, N., Sato, B., Koyama, H., and Matsumoto, K. Growthcontrol of androgen-dependent Shionogi carcinoma cells by growth factor(s)produced from androgen-independent Shionogi carcinoma cells in a paracrinemachanism. J. Steroid Biochem., 32: 479-483. 1989.

20. Jung-Testas, I., (lase. J. M.. and Baulieu, E. E. Effects of androgen andantiandrogen on growth, morphology and synthesis of specific proteins inmouse mammary carcinoma cells. J. Steroid Biochem., JO: 353-358, 1988.

21. Nakamura, N., Nishizawa, Y., Noguchi, S., Uchida, N., Sato, B., and Matsumoto, K. Action mechanisms of physiological doses of androgen or pharmacological doses of estrogen in growth stimulation of Shionogi carcinoma115 in mice. J. Steroid Biochem., 27:459-464, 1987.

22. Gospodarowicz. D., Cheng, J., Lui, G. M., Baird, A., and Bohlent, P.Isolation of brain fibroblast growth factor by heparin-Sepharose affinitychromatography: identity with pituitary fibroblast growth factor. Proc. Nati.Acad. Sci. USA, 81: 6963-6967, 1984.

23. Esch, F., Ueno, N., Baird, A., Hill, F., Denoroy, L., Ling, N., Gospodarowicz,D.. and Gillemin. R. Primary structure of bovine brain acidic fibroblastgrowth factor (FGF). Biochem. Biophys. Res. Commun., 133: 554-562,1985.

24. Gospodarowicz, D., Massoglia, S., Cheng, J., Lui, G. M., and Bohlen, P.Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization. J. Cell.Physiol../22: 323-332, 1985.

25. Gospodarowicz, D., and Cheng, J. Heparin protects basic and acidic FGFfrom inactivation. J. Cell. Physiol., ¡28:475-484, 1986.

26. Rogelj, S.. Weinberg, R. A., Fanning, P., and Klagsbrun, M. Basic fibroblastgrowth factor fused to a signal peptide transforms cells. Nature (Lond.), 331:173-175, 1988.

27. Klagsbrun. M., and Shing, Y. Heparin affinity of anionic and cationiccapillary endothelial cell growth factors: analysis of hypothalamus-derivedgrowth factors and fibroblast growth factors. Proc. Nati. Acad. Sci. USA,82:805-809, 1985.

28. Nishi, N., Matsuo, Y., and Wada, F. Partial purification of a major type ofrat prostatic growth factor: characterization as an epidermal growth factor-related mitogen. Prostate, 13: 209-220, 1988.

29. Moscatelli, D., Presta, M., Joseph-Silverstein. J., and Rifkin. D. B. Bothnormal and tumor cells produce basic fibroblast growth factor. J. Cell.Physiol., 129:273-216, 1986.

30. Moscatelli. D., Joseph-Silverstein, J., Manejias, R., and Rifkin, D. B. M,25,000 heparin-binding protein from guinea pig brain is a high molecularweight form of basic fibroblast growth factor. Proc. Nati. Acad. Sci. USA,84: 5778-5782, 1987.

31. Ferrara, N., and Henzel, W. J. Pituitary follicular cells secrete a novelheparin-binding growth factor specific for vascular endothelial cells.Biochem. Biophys. Res. Commun., 161: 851-858, 1989.

32. Gospodarowicz, D., Abraham. J. A., and Schilling, J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derivedloll in ilo stellate cells. Proc. Nati. Acad. Sci. USA, 86: 7311-7315, 1989.

4983

on March 19, 2020. © 1990 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1990;50:4979-4983. Cancer Res   Yuzo Furuya, Naohide Sato, Koichiro Akakura, et al.   Carcinoma 115 by Its Autonomous Subline (Chiba Subline 2)Paracrine Growth Stimulation of Androgen-responsive Shionogi

  Updated version

  http://cancerres.aacrjournals.org/content/50/16/4979

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/50/16/4979To request permission to re-use all or part of this article, use this link

on March 19, 2020. © 1990 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from