Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptorsin Human Prostate Cancer

By Coralia I. Rivas, Juan Carlos Vera, Fernando Delgado-Lopez, Mark L. Heaney, Victor H. Guaiquil,

Rong H. Zhang, Howard I. Scher, Ilona I. Concha, Francisco Nualart, Carlos Cordon-Cardo, and David W. Golde

We studied the expression and function of the granulocyte-

macrophage colony-stimulating factor (GM-CSF) receptor in

the human prostate carcinoma cell line LNCaP and looked for

its presence in normal and neoplastic human prostatic

tissue. The GM-CSF receptor is composed of two subunits, aand b. While the isolated a subunit binds GM-CSF at

low-affinity, the isolated b subunit does not bind GM-CSF by

itself; but complexes with the a subunit to form a high-

affinity receptor. Quantitative reverse transcriptase-polymer-

ase chain reaction (RT-PCR) showed expression of mRNAs

encoding the a and b subunits of the GM-CSF receptor in

LNCaP cells, and the presence of the a and b proteins was

confirmed by immunolocalization with anti-a and anti-bantibodies. Receptor binding studies using radiolabeled GM-

CSF showed that LNCaP cells have about 150 high-affinity

sites with a kd of 40 pmol/L and approximately 750 low-

affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a

time- and dose-dependent manner, for protein tyrosine

phosphorylation and induced the proliferation of the LNCaP

cells. Immunolocalization studies showed low level expres-

sion of GM-CSF a and b subunits in normal prostate tissue,

with substantial expression in benign prostatic hyperplasia

and prominent expression in neoplastic prostate tissue.

Maximal expression of both subunits was observed in pros-

tatic carcinomas metastatic to lymph node and bone. Tumor

cells that stained positively with anti-a subunit antibodies

were also reactive with anti-b subunit antibodies, indicating

that they express high-affinity GM-CSF receptors. Our data

show that the LNCaP cells express functional GM-CSF recep-

tors and that prostatic carcinomas have prominent GM-CSF

receptor expression. These findings imply that both hyper-

plastic and neoplastic prostatic tissues may be responsive to

GM-CSF.

r 1998 by The American Society of Hematology.

GRANULOCYTE-MACROPHAGE colony-stimulating fac-tor (GM-CSF) is an important regulator of the prolifera-

tion and differentiation of myeloid precursor cells and enhancesthe function of mature granulocytes and mononuclear phago-cytes.1 Receptors for GM-CSF are expressed on myeloidprogenitors and mature mononuclear phagocytes, monocytes,eosinophils and neutrophils.1-4 The GM-CSF receptor is com-posed of two subunits,a andb.5,6 The isolateda subunit bindsGM-CSF at low affinity (kd, 1 to 7 nmol/L). The isolatedbsubunit does not bind GM-CSF by itself; however, in a complexwith thea subunit forms a high-affinity receptor (kd, 20 to 100pmol/L). The binding of GM-CSF to its cognate receptortriggers a number of cellular responses.7-12 In human neutro-phils, GM-CSF induces increased protein tyrosine phosphoryla-tion through the activation of specific protein tyrosine cas-cades9,13-15and transcriptional activation of early genes such asc-fos, c-jun, and c-myc.16 Signaling through the GM-CSFreceptor may occur via participation of botha and b sub-units5,10,17,18and also through the isolateda subunit.19,20

Receptors for GM-CSF are also present in nonhematopoieticcells such as placental trophoblasts, endothelial cells, andoligodendrocytes in the central nervous system.21-24 Someprimary neoplasms and tumor cell lines such as melanoma,small cell lung cancer, colon, pancreatic, renal, and ovariancarcinomas have also been reported to express GM-CSFreceptors.25-29

Although there is evidence indicating that GM-CSF maystimulate the growth of some nonhematopoietic tumor celllines,29-34 the physiologic role of the GM-CSF receptors ex-pressed in normal or neoplastic nonhematopoietic tissue isunknown. In these studies, we provide evidence that humanLNCaP prostate cancer cells express functional high-affinityGM-CSF receptors that transduce signals involving proteintyrosine phosphorylation and cell proliferation. We also showthat thea andb subunits of the GM-CSF receptor are expressedat low level in normal human prostatic tissue, with substantialexpression in benign prostatic hyperplasia and prominentexpression in prostatic carcinoma.

MATERIALS AND METHODS

Cell culture. The human prostatic cell line LNCaP35 was culturedin RPMI-1640 supplemented with 6% heat-inactivated fetal bovineserum, 1% L-glutamine, and antibiotics. The human leukemia cell lineHL-6036 was maintained in Iscove’s modified Dulbecco’s medium(IMDM) supplemented with 10% heat-inactivated fetal bovine serum,1% L-glutamine, and antibiotics.

Proliferation assay. Cell proliferation was assessed by [3H]-thymidine37 and [125I]-deoxyuridine incorporation. Briefly, 40,000 cellsper well were plated in sextuplicate in 6-well plates (Costar, Cambridge,MA), and bacterial recombinant human GM-CSF (0.01 to 100 mmol/L)(a gift from Amgen, Thousand Oaks, CA) was added at the beginning ofthe culture. Every day for 6 days of culture, 2 µCi of [3H]-thymidine(DuPont NEN, Boston, MA) or 0.45 µCi of [125I]-dUridine (DuPontNEN) was added to each well. The cells were harvested after 20 hoursusing a cell harvester (Skatron, Sterling, VA) or extracted with 1 NNaOH. The radioactivity incorporated into DNA was quantitated byliquid scintillation org spectrometry.

From the Program in Molecular Pharmacology and Therapeutics,Departments of Medicine and Pathology, Memorial Sloan-KetteringCancer Center, New York, NY; Instituto de Bioquı´mica, Facultad deCiencias, Universidad Austral de Chile, Valdivia, Chile; and Departa-mento de Histologı´a y Embriologı´a, Facultad de Ciencias Biolo´gicas,Universidad de Concepcio´n, Concepcio´n, Chile.

Submitted June 30, 1997; accepted October 2, 1997.Supported in part by Grants No. R01 CA30388, R01 HL42107,

CA-DK-47650, and P30 CA08748 from the National Institutes ofHealth, Bethesda, MD; by Memorial Sloan-Kettering Institutionalfunds; by the Schultz Foundation, Verona, NJ; by the PepsiCo Founda-tion, Purchase, NY; and by the David H. Koch Charitable Foundation,Wichita, KS.

Address reprint requests to David W. Golde, MD, Program inMolecular Pharmacology and Therapeutics, Box 451, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021.

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked ‘‘adver-tisement’’ in accordance with 18 U.S.C. section 1734 solely to indicatethis fact.

r 1998 by The American Society of Hematology.0006-4971/98/9103-0034$3.00/0

Blood, Vol 91, No 3 (February 1), 1998: pp 1037-1043 1037

Reverse transcription-polymerase chain reaction (RT-PCR).TotalRNA was isolated from LNCaP and HL-60 cells using guanidiniumthiocyanate (RNAzol B, Cinna/Biotecx Laboratories, Houston, TX).Single-stranded cDNA synthesis and quantitative PCR were performedas previously described.38 The primers used were:a-subunit primer:58:AGCCCGAGCAAAACACA, position 1009-1026 and 38:CCATG-CCA TTCCTACACCCT, position 1360-1379;b-subunit primer:58:CTACAAGCCCAGCCC AGATGC, position 859-879 and 38:AC-CCGTAGATGCCACAGAAGC, position 1390-1410. The PCR condi-tions were 94°C for 1 minute and 65°C for 2 minutes for 35 cycles. Forquantitation of theb subunit, the GM-CSF receptorb cDNA subclonedinto pBluescript (Stratagene, La Jolla, CA) was transcribed in vitro(Megascript, Ambion, Austin, TX) and the RNA was digested withDNase and quantitated spectrophotometrically and by the addition oftrace levels of [a32P] uridine triphosphate (UTP) (DuPont NEN). RT-PCR ofserial dilutions was performed in the presence of 1 µg of RNA derivedfrom HeLa cells, a cell line that does not express theb subunit.

Binding assays. For binding assays, 73 106 cells were suspendedin RPMI-1640 containing 0.2% bovine serum albumin (BSA) andincreasing concentrations of125I-labeled human GM-CSF (DuPontNEN) with or without excess unlabeled human GM-CSF. Afterincubation for 20 hours at 4°C, the cells were centrifuged for 5 minutesat 4°C through a cushion of fetal bovine serum and the cell pellets werewashed with cold phosphate-buffered saline (PBS) pH 7.4. BoundGM-CSF was quantitated byg spectrometry.

Immunoblotting. Cells were serum starved in RPMI-1640 contain-ing 0.2% BSA for 18 hours, washed, resuspended at 13 107 cells/mLand incubated in the absence or in the presence of increasing concentra-tions of GM-CSF (0.01 nmol/L to 1 µmol/L) for different periods oftime (2 seconds to 20 minutes) at 37°C. The cells were washed with coldPBS and the cell pellets were resuspended in 100 µL of lysis buffer14 anddisrupted by sonication. The soluble proteins were resolved by SDS-PAGE(100 µg of cell lysate per lane) in a 10% polyacrylamide gel andtransferredto immobilon (Millipore, Bedford, MA). Proteins phosphorylated ontyrosine residues were detected using an antiphosphotyrosine antibody(UBI, Lake Placid, NY). Phosphorylated mitogen-activated protein(MAP) kinase was localized using an antiphosphoMAP kinase antibody(New England Biolabs,Beverly, MA).Anti-JAK2 antibody was purchasedfrom UBI. The antibody blots were developed by chemiluminescence(New England Biolabs).

Immunocytochemistry.For immunoperoxidase localization,39

LNCaPcells cultured in 6-chamber microscopic slides were fixed in bufferedparaformaldehyde-acetone, treated with 0.3% H2O2 for 5 minutes andincubated for 30 minutes at room temperature in 4% BSA-PBS pH 7.8,followed by incubation overnight at 4°C in 1% BSA-PBS pH 7.8 andanti-a or anti-b GM-CSF receptor subunit antibodies (1:500) (AlphaDiagnostics, San Antonio, TX). Cells were washed and incubated withantirabbit IgG-horseradish peroxidase (1:100) (Amersham, ArlingtonHeights, IL) for 2.5 hours at room temperature. Immunostaining wasdeveloped using 0.05% diaminobenzidine and 0.03% H2O2. As con-trols, cells were incubated with antibodies preabsorbed with therespective peptide used to generate the antibodies. Cells were counter-stained with hematoxylin.

Archived, formalin fixed, and paraffin embedded human prostatetissues were obtained from the Department of Pathology at MemorialSloan-Kettering Cancer Center. For immunostaining,39 tissue sectionswere rehydrated, treated with 3% hydrogen peroxide for 15 minutes atroom temperature, and blocked with 4% BSA-PBS pH 7.8, followed byincubation in a humid chamber overnight at 4°C with anti-a or anti-bsubunit GM-CSF receptor antibodies (1:500) in 1% BSA-PBS pH 7.8.After extensive washing, sections were incubated for 2.5 hours at roomtemperature with antirabbit IgG-horseradish peroxidase (1:100, Amer-sham). The peroxidase activity was developed with 0.05% diaminoben-zidine and 0.03% H2O2. Tissues were counterstained with hematoxylin.

RESULTS

LNCaP cells express GM-CSF receptors.Aband of approxi-mately 370 nucleotides, the expected size of the amplificationproduct for the membrane-bound form of thea-subunit mRNA,was amplified by RT-PCR from LNCaP cells RNA (Fig 1A).Primers specific for glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) were used as an internal standard for theefficacy of the RT-PCR procedure. A similar approach usingprimers complementary to theb subunit of the GM-CSFreceptor showed the expected amplification product of approxi-mately 570 nucleotides (Fig 1A). The size of the amplified

Fig 1. Expression of GM-CSF receptors in LNCaP cells. (A) RT-PCR

analysis. Total RNA was isolated from LNCaP (lane 1) or HL-60 (lane 2)

cells and subjected to RT-PCR using radiolabeled primers specific for

the a (left panel) or the b (right panel) subunits of the GM-CSF

receptor. PCR products were size-fractionated on 5% acrylamide gels

and autoradiographed. Shown are the PCR products corresponding

to the a (370 bp) and the b (570 bp) subunits of the GM-CSF receptor

obtained in the presence (1) or in the absence (-) of reverse transcrip-

tase (RT). (B) Binding analysis. Cells were incubated with radiolabeled

GM-CSF at concentrations that ranged from 5 pmol/L to 10 nmol/L.

GM-CSF binding was dose dependent and saturable approximately at

10 nmol/L. (C) Scatchard analysis of data from (B) showing the

presence of two classes of binding sites in the LNCaP cells. (D through

G) Immunostaining with antihuman GM-CSF receptor antibodies.

Cells were cultured, fixed, and incubated with anti-a (D and F) or

anti-b subunit (E and G) antibodies in the absence (D and E) or the

presence (F and G) of the peptides used to elicit them (original

magnification 3 160).

1038 RIVAS ET AL

bands corresponded exactly to the size of the respective bandsamplified from RNA obtained from HL-60 cells, which expressabundant mRNA for thea and theb subunits of the GM-CSFreceptor (Fig 1A).38 No amplification products were observed inparallel reactions in which the reverse transcriptase was omitted(Fig 1A), confirming the absence of contaminating DNA in theRNA preparations. The content ofa andb subunit mRNAs inLNCaP cells was quantitated by interpolation on a standardcurve generated by performing RT-PCR with known amounts ofeithera or b subunit RNA in parallel with LNCaP cell-derivedRNA. LNCaP cells expressed 0.02 to 0.33 fg ofa subunitmRNA per 105 cells (0.24 to 4 copies per 103 cells), a value thatis 60 to 200 times lower than thea subunit mRNA levelsexpressed in HL-60 cells (Fig 1A). LNCaP cells expressed 12 to83 fg ofb subunit mRNA per 105 cells (80 to 550 copies per 103

cells), which is 30 to 200 times lower than theb subunit mRNAlevels expressed in HL-60 cells (Fig 1A). LNCaP cells alsoexpressed low levels of mRNA encoding the soluble isoform ofthea subunit (data not shown).

Radiolabeled GM-CSF bound to LNCaP cells in a dose-dependent and saturable manner (Fig 1B). Scatchard analysis ofthe binding data (Fig 1C) showed that the LNCaP cellsexpressed approximately 150 high-affinity binding sites forGM-CSF with a kd of 40 pmol/L, and 750 low-affinity bindingsites. The presence of thea and b subunits of the GM-CSFreceptor in the LNCaP cells was confirmed by immunolocaliza-tion with antibodies specific for each subunit. The LNCaP cellswere immunoreactive with both antibodies, with intense immu-nostaining in both the cytoplasm and the plasma membrane (Fig1D and F). The LNCaP cells consistently showed enhancedimmunoreactivity with the anti-a antibody compared with theanti-b antibody. No immunoreactivity was observed when theprimary antibodies were preabsorbed with the peptides used togenerate them (Fig 1E and G).

GM-CSF signaling in LNCaP cells.Proliferation assays,measuring the incorporation of [3H]-thymidine or [125I]-deoxyuridine in DNA, showed that GM-CSF stimulated prolif-eration of the LNCaP cells in a dose- and time-dependentmanner (Fig 2B and C; only [3H]-thymidine incorporation is

shown). An increase in [3H]-thymidine incorporation of 20% to40% was observed after 3 or 4 days of culture in the presence of0.3 or 100 nmol/L GM-CSF (Fig 2B). The effect of GM-CSF on[3H]-thymidine incorporation was evident during the exponen-tial phase of cell growth and decreased at latter stages (Fig 2Aand B). Dose-response studies indicated a biphasic effect ofGM-CSF on [3H]-thymidine incorporation (Fig 2C). GM-CSFinduced a measurable increase in [3H]-thymidine at a concentra-tion of 0.03 nmol/L, an effect that reached saturation at 1nmol/L GM-CSF, with no further increase observed at concen-trations of GM-CSF from 1 to 30 nmol/L. However, 100 nmol/LGM-CSF induced an additional increase in [3H]-thymidineincorporation, which was also evident in the time-courseexperiments (Fig 2B and C).

We next analyzed whether GM-CSF induced protein tyrosinephosphorylation in the LNCaP cells. A transient increase intyrosine phosphorylation was observed in several proteins whenLNCaP cells were treated with GM-CSF (Fig 3A). Proteins withapparent molecular weights of 160, 130, 75 to 80, 68 to 70, 60,55, 47, and 40 kD (black arrowheads, Fig 3A) showed maximalphosphorylation during the first 30 seconds of treatment with 1nmol/L GM-CSF and remained phosphorylated for 20 minutes,with phosphorylation returning to basal levels after 1 to 2 hours(Fig 3A). Phosphorylation of these proteins was induced atconcentrations of GM-CSF ranging from 0.03 nmol/L to 1µmol/L (Fig 3A). Interestingly, we observed the rapid dephos-phorylation of proteins with an apparent molecular weight of 45and 110 kD after incubating the cells with 1 nmol/L GM-CSF(white arrowheads, Fig 3A). These proteins were dephosphory-lated after treating the cells with GM-CSF at concentrationsfrom 0.03 nmol/L to 1 µmol/L and remained dephosphorylatedfor at least 18 hours (Fig 3A).

We did not detect tyrosine phosphorylation of MAP kinase inLNCaP cells treated with GM-CSF using antiphosphotyrosineantibodies (Fig 3A). The p42 MAP kinase was identified byreprobing the membrane with an anti-MAP kinase antibody andwas found to be present at a constant level at the expectedposition in the blots (Fig 3B). Phosphorylation of MAP kinase,however, was evident when using a monoclonal antibody

Fig 2. GM-CSF signals for proliferation in LNCaP cells. (A) Growth curve. LNCaP cells were maintained in continuous culture with no

stimulation and the cell number was determined by counting the cells every day for 6 days and the cell viability was assessed by exclusion of

trypan blue. (B) Time course. Cells were incubated with 0.3 (W) or 100 nmol/L (X) GM-CSF for 1 to 6 days and parallel cultures were pulse-labeled

with [3H]-thymidine for 20 hours every day. (C) Dose response. Cells were incubated with increased amounts of GM-CSF (0.01 to 100 nmol/L) for 3

(W) or 4 days (X) and pulse-labeled with [3H]-thymidine for the last 20 hours.

GM-CSF RECEPTOR IN PROSTATE CANCER 1039

specific for tyrosine phosphorylated MAP kinase (Fig 3B).Phosphorylation of MAP kinase occurred only at concentrationsof GM-CSF of 3 nmol/L or higher and reached a maximal levelat 100 nmol/L GM-CSF. Maximal phosphorylation was ob-served at 30 seconds, with phosphorylation returning to basallevels after 20 minutes (Fig 3B). No phosphorylation of JAK2was evident in cells treated with GM-CSF.

Increased expression of GM-CSF receptors in human pros-tate tumors. We examined whether thea andb subunits of theGM-CSF receptor were expressed in normal prostate, benignprostatic hyperplasia, and prostatic carcinomas, including pri-mary tumors and metastatic lesions to lymph node and bone(Fig 4 and Table 1). Very weak to undetectable immunostainingwas observed in the luminal epithelial cells of the acini of thenormal prostate, although weak to moderate reactivity wasevident in basal cells (Fig 4). Ducts exhibited a more intenseimmunoreactivity than acini for both antibodies. Moderateimmunostaining was also found in the epithelial cells of most ofthe samples of benign prostatic hyperplasia analyzed with bothantibodies, although moderate to strong reactivity was observedin two specimens (Fig 4). In addition, basal cell hyperplasia, ahistologically different form of hyperplasia, was seen in onesample and displayed strong reactivity with both antibodies. Allprimary tumors analyzed, which exhibited Gleason scores from5 to 9, were positive for both antibodies, with homogeneous andheterogeneous staining patterns of moderate to strong intensity(Fig 4 and Table 1). The metastatic tumors displayed homogene-ity, as well as heterogeneity in immunostaining profiles, withmoderate to strong reactivity to both antibodies and hadincreased staining compared with the primary tumors (Fig 4).One sample from a bone metastasis was negative for bothantibodies. Tumor cells that were immunopositive for the anti-aantibody were also positive for the anti-b antibody, as immuno-histochemistry was conducted in consecutive sections andnumerous lesions showed homogeneous staining. In addition,most of the cases studied showed a more intense anti-aimmunoreactivity than the anti-b immunoreaction.

DISCUSSION

GM-CSF is a hematopoietic growth factor and a host defenseregulator used clinically to stimulate hematopoietic cell prolif-eration after chemotherapy, as well as autologous or allogeneicbone marrow transplantation.40-42 The physiologic role ofGM-CSF receptors in nonhematopoietic tissue is unknown.Even more problematic are the implications and consequencesof GM-CSF receptor expression on malignant, nonhematopoi-etic tissue. Distinct types of neoplastic cells have been shown tohave functional GM-CSF receptors.25,30,31 Whether or nottherapeutically administered hematopoietic growth factors canstimulate nonhematopoietic cell growth, including solid tumorcells, has remained a controversial issue.

We report here a detailed study addressing the issue ofGM-CSF receptor expression in human prostate cancer. Thepresence of GM-CSF receptors in the LNCaP cells was definedby quantitative RT-PCR, ligand binding, immunolocalization,and functional assays. Quantitative RT-PCR showed that theLNCaP cells expressed mRNAs for thea andb subunits of theGM-CSF receptor. The immunolocalization experiments con-firmed the presence of thea andb proteins in the LNCaP cells

Fig 3. GM-CSF signals for protein tyrosine phosphorylation in

LNCaP cells. (A) Protein tyrosine phosphorylation. Cells were incu-

bated in the absence (2) or in the presence (1) of 1 nmol/L GM-CSF

from 2 seconds to 20 minutes at 37°C (upper panel), or with 0.01

nmol/L to 1 mmol/L GM-CSF for 2 minutes at 37°C (bottom panel).

Tyrosine phosphoproteins were identified by immunoblotting with

antiphosphotyrosine antibodies. Black and white arrowheads indi-

cate proteins phosphorylated and dephosphorylated in response to

GM-CSF, respectively. (B) Phosphorylation of MAP kinase. Cells were

incubated with 10 pmol/L to 0.3 mmol/L GM-CSF for 1 minute at 37°C,

or were incubated in the absence (2) or in the presence (1) of 100

nmol/L GM-CSF from 2 seconds to 20 minutes at 37°C. Phosphoryla-

tion of MAP kinase was assessed by immunoblotting with an

antiphospho MAP kinase antibody. MAP kinase was identified with

an anti-MAP kinase antibody. The arrowheads indicate the positions

of the p42 and p44 MAP kinases.

1040 RIVAS ET AL

and the ligand-binding studies showed that LNCaP expressedboth high- and low-affinity GM-CSF receptors. The number ofhigh-affinity receptors present in the LNCaP cells was similar tothe number present in cells of hematopoietic origin in whichGM-CSF induces proliferation and differentiation43 and innonhematopoietic cells such as mouse fibroblasts expressing thehuman high-affinity receptors, which respond to GM-CSF withcell proliferation and protein phosphorylation.10,44The identifi-cation of approximately 750 low-affinity GM-CSF receptors inthe LNCaP cells, compared with about 150 high-affinity sites,indicates the presence of an excess ofa as compared withbsubunit in these cells. Consistent with these findings, theimmunolocalization experiments showed greater immunoreac-tivity with the anti-a subunit antibodies than with the anti-bsubunit antibodies in the LNCaP cells. The RT-PCR experi-ments, however, indicated that LNCaP cells express a highernumber of mRNA molecules per cell encoding theb than theasubunit of the GM-CSF receptor. These data suggest that, in the

LNCaP cells, the expression of thea andb subunit proteins isregulated at the level of translation or protein stability.

Biologic response analyses confirmed the presence of func-tionally active GM-CSF receptors in the LNCaP cells. Aprevious study34 reported a 2.8-fold increase in LNCaP cellproliferation in the presence of suprapharmacologic concentra-tions of GM-CSF (.1 µmol/L). These concentrations ofGM-CSF are at least four orders of magnitude higher than theconcentrations we used here (<100 pmol/L) and are notcompatible with the expression of high-affinity GM-CSF recep-tors in the LNCaP cells. On the other hand, although our dataindicated that GM-CSF increased LNCaP cell proliferation atconcentrations consistent with the presence of high-affinityreceptors in these cells, these concentrations of GM-CSF (<100pmol/L) were at least one order of magnitude higher than thatnecessary in cells such as HL-60 (<10 pmol/L), which expressa similar number of high-affinity GM-CSF receptors. The originof these discrepancies is not evident from these studies;

Fig 4. Immunolocalization of GM-CSF receptors in benign and malignant human prostate tissues. Consecutive tissue sections were

incubated with anti-a (left panel) or anti-b–subunit (right panel) antibodies. (A) Low to undetectable levels in the epithelial cells of the normal

gland. (B) Moderate levels of expression in the epithelial cells of benign prostatic hyperplasia. (C) Increased immunoreactive pattern in a primary

tumor. (D) High levels of expression in a lymph node and a bone (E) metastases. Stars in bone metastases images indicate bone location. Two

different magnifications of each immunostained tissue section are shown (1603 and 4003).

GM-CSF RECEPTOR IN PROSTATE CANCER 1041

however, the data suggest the existence of cell-specific effectsthat modulate the functional activity of the GM-CSF receptor.

Tyrosine phosphorylation of p42 and p44 MAP kinases is anearly step in GM-CSF signal transduction.9,10,13-15,45We foundthat GM-CSF induced time- and dose-dependent tyrosinephosphorylation of several proteins in the LNCaP cells andinduced tyrosine phosphorylation of the p42 and p44 MAPkinases. Phosphorylation of proteins other than MAP kinasewas observed at GM-CSF concentrations of 0.1 nmol/L or less,which is consistent with the presence of high-affinity GM-CSFreceptors in the LNCaP cells. On the other hand, phosphoryla-tion of MAP kinase was triggered only at GM-CSF concentra-tions of at least 3 nmol/L. Furthermore, we failed to observeJAK2 phosphorylation in these cells. Because the LNCaP cellsexpress about 750 low-affinity sites that likely correspond toexcess ofa subunits (in addition to approximately 150 high-affinity sites), the data raise the intriguing possibility that excessof a-subunit expression may modulate signaling through thehigh-affinity receptor.

The immunohistochemical analysis of human prostatic tissueusing anti-a and -b antibodies confirmed the presence of theaand theb subunits of the GM-CSF receptor in prostate tumors.The lack of immunoreactivity or weak pattern of stainingobserved in normal epithelial cells, contrasts with the substan-tial immunoreactivity with anti-a and -b antibodies observed inall cases of benign prostatic hyperplasia. Although primarytumors showed higher levels of expression compared withbenign prostatic hyperplasia, we observed a further increase inthe immunoreactivity of metastases to lymph node and bonecompared with that of primary tumors. These data are compat-ible with increased expression of the GM-CSF receptor as thedisease progresses from localized tumors to the development ofmetastatic disease.

Although the action of growth factors and their receptors innormal prostate physiology and progression of prostate cancerare not well understood, our results suggest that GM-CSF mayhave a role in maintenance of function in the normal prostate, aswell as in prostate cancer progression. The presence of bothsubunits of the GM-CSF receptor in the tumor cells indicate thatthey express functional high-affinity GM-CSF receptors andtherefore this hematopoietic growth factor may have an effect

on prostate carcinoma cells, which have a proclivity to metasta-size to bone. The increased expression of GM-CSF receptors inprostatic hypertrophy and neoplastic prostate ephitelium sug-gest a relationship between prostatic epithelial cell growth andGM-CSF.

REFERENCES

1. Gasson JC: Molecular physiology of granulocyte-macrophagecolony-stimulating factor. Blood 77:1131, 1991

2. Gasson JC, Kaufman SE, Weisbart RH, Tomonaga M, Golde DW:High-affinity binding of granulocyte-macrophage colony-stimulatingfactor to normal and leukemic human myeloid cells. Proc Natl Acad SciUSA 83:669, 1986

3. Park LS, Friend D, Gillis S, Urdal DL: Characterization of the cellsurface receptor for human granulocyte/macrophage colony-stimulatingfactor. J Exp Med 164:251, 1986

4. Cannistra SA, Koenigsmann M, DiCarlo J, Groshek P, Griffin JD:Differentiation-associated expression of two functionally distinct classesof granulocyte-macrophage colony-stimulating factor receptors byhuman myeloid cells. J Biol Chem 265:12656, 1990

5. Hayashida K, Kitamura T, Gorman DM, Arai K, Yokota T,Miyajima A: Molecular cloning of a second subunit of the receptor forhuman granulocyte-macrophage colony-stimulating factor (GM-CSF):Reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad SciUSA 87:9655, 1990

6. Kitamura T, Sato N, Arai K, Miyajima A: Expression cloning ofthe human IL-3 receptor cDNA reveals a shared beta subunit for thehuman IL-3 and GM-CSF receptors. Cell 66:1165, 1991

7. Isfort RJ, Ihle JN: Multiple hematopoietic growth factors signalthrough tyrosine phosphorylation. Growth Factors 2:213, 1990

8. Kanakura Y, Druker B, Cannistra SA, Furukawa Y, Torimoto Y,Griffin JD: Signal transduction of the human granulocyte-macrophagecolony-stimulating factor and interleukin-3 receptors involves tyrosinephosphorylation of a common set of cytoplasmic proteins. Blood76:706, 1990

9. Okuda K, Sanghera JS, Pelech SL, Kanakura Y, Hallek M, GriffinJD, Druker BJ: Granulocyte-macrophage colony-stimulating factor,interleukin-3, and steel factor induce rapid tyrosine phosphorylation ofp42 and p44 MAP kinase. Blood 79:2880, 1992

10. Eder M, Griffin JD, Ernst TJ: The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiatingsignal transduction in NIH3T3 cells. EMBO J 12:1647, 1993

11. Hanazono Y, Chiba S, Sasaki K, Mano H, Miyajima A, Arai K,Yazaki Y, Hirai H: c-fps/fes protein-tyrosine kinase is implicated in asignaling pathway triggered by granulocyte-macrophage colony-stimulating factor and interleukin-3. EMBO J 12:1641, 1993

12. Ihle JN: Cytokine receptor signalling. Nature 377:591, 199513. Gomez-Cambronero J, Huang CK, Gomez-Cambronero TM,

Waterman WH, Becker EL, Sha’afi RI: Granulocyte-macrophagecolony-stimulating factor-induced protein tyrosine phosphorylation ofmicrotubule-associated protein kinase in human neutrophils. Proc NatlAcad Sci USA 89:7551, 1992

14. Raines MA, Golde DW, Daeipour M, Nel AE: Granulocyte-macrophage colony-stimulating factor activates microtubule-associatedprotein 2 kinase in neutrophils via a tyrosine kinase-dependent pathway.Blood 79:3350, 1992

15. Welham MJ, Duronio V, Sanghera JS, Pelech SL, Schrader JW:Multiple hemopoietic growth factors stimulate activation of mitogen-activated protein kinase family members. J Immunol 149:1683, 1992

16. Watanabe S, Muto A, Yokota T, Miyajima A, Arai K: Differentialregulation of early response genes and cell proliferation through thehuman granulocyte macrophage colony-stimulating factor receptor:Selective activation of the c-fos promoter by genistein. Mol Biol Cell4:983, 1993

Table 1. Expression of GM-CSF Receptors in Human Prostatic Tissue

GMRa GMRb

Positive

Cases* Intensity†

Positive

Cases Intensity

Normal prostate 4/7‡ 6/1 4/7 6/1

Benign hyperplasia 7/7‡ 1/11 7/7 1/11

Prostatic carcinoma

Grade (Gleason score)

5 3/3 11 3/3 11

7 3/3 11/111 3/3 11/111

8 4/4 11/111 4/4 11/111

9 1/1 111 1/1 111

Metastases

Bone 4/5 111 4/5 11/111

Lymph node 4/4 11/111 4/4 111

*No. of positives cases/total no. of cases studied.

† 6 , very weak; 1, weak; 11, moderate; 111, strong.

‡Epithelial cells.

1042 RIVAS ET AL

17. Lopez AF, Shannon MF, Hercus T, Nicola NA, Cambareri B,Dottore M, Layton MJ, Eglinton L, Vadas MA: Residue 21 of humangranulocyte-macrophage colony-stimulating factor is critical for biologi-cal activity and for high but not low affinity binding. EMBO J 11:909,1992

18. Sakamaki K, Miyajima I, Kitamura T, Miyajima A: Criticalcytoplasmic domains of the common beta subunit of the humanGM-CSF, IL-3 and IL-5 receptors for growth signal transduction andtyrosine phosphorylation. EMBO J 11:3541, 1992

19. Ding DX, Rivas CI, Heaney ML, Raines MA, Vera JC, GoldeDW: The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphor-ylation-independent pathway. Proc Natl Acad Sci USA 91:2537, 1994

20. Spielholz C, Heaney ML, Morrison ME, Houghton AN, Vera JC,Golde DW: Granulocyte-macrophage colony-stimulating factor signalsfor increased glucose uptake in human melanoma cells. Blood 85:973,1995

21. Bussolino F, Wang JM, Defilippi P, Turrini F, Sanavio F, EdgellCJ, Aglietta M, Arese P, Mantovani A: Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cellsto migrate and proliferate. Nature 337:471, 1989

22. Gearing DP, King JA, Gough NM, Nicola NA: Expressioncloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J 8:3667, 1989

23. Baldwin GC, Benveniste EN, Chung GY, Gasson JC, Golde DW:Identification and characterization of a high-affinity granulocyte-macrophage colony-stimulating factor receptor on primary rat oligoden-drocytes. Blood 82:3279, 1993

24. Brosnan CF, Shafit-Zagardo B, Aquino DA, Berman JW: Expres-sion of monocyte/macrophage growth factors and receptors in thecentral nervous system. Adv Neurol 59:349, 1993

25. Baldwin GC, Gasson JC, Kaufman SE, Quan SG, Williams RE,Avalos BR, Gazdar AF, Golde DW, DiPersio JF: Nonhematopoietictumor cells express functional GM-CSF receptors. Blood 73:1033, 1989

26. Metcalf D, Nicola NA, Gearing DP, Gough NM: Low-affinityplacenta-derived receptors for human granulocyte-macrophage colony-stimulating factor can deliver a proliferative signal to murine hemopoi-etic cells. Proc Natl Acad Sci USA 87:4670, 1990

27. Miyagawa K, Chiba S, Shibuya K, Piao YF, Matsuki S, Yokota J,Terada M, Miyazono K, Takaku F: Frequent expression of receptors forgranulocyte-macrophage colony-stimulating factor on human nonhema-topoietic tumor cell lines. J Cell Physiol 143:483, 1990

28. Baldwin GC, Golde DW, Widhopf GF, Economou J, Gasson JC:Identification and characterization of a low-affinity granulocyte-macrophage colony-stimulating factor receptor on primary and culturedhuman melanoma cells. Blood 78:609, 1991

29. Guillaume T, Sekhavat M, Rubinstein DB, Hamdan O, SymannML: Transcription of genes encoding granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 6 receptors and lack ofproliferative response to exogenous cytokines in nonhematopoietichuman malignant cell lines. Cancer Res 53:3139, 1993

30. Dedhar S, Gaboury L, Galloway P, Eaves C: Human granulocyte-macrophage colony-stimulating factor is a growth factor active on avariety of cell types of nonhemopoietic origin. Proc Natl Acad Sci USA85:9253, 1988

31. Berdel WE, Danhauser-Riedl S, Steinhauser G, Winton EF:Various human hematopoietic growth factors (interleukin-3, GM-CSF,G-CSF) stimulate clonal growth of nonhematopoietic tumor cells.Blood 73:80, 1989

32. Joraschkewitz M, Depenbrock H, Freund M, Erdmann G, MeyerHJ, De Riese W, Neukam D, Hanauske U, Krumwieh M, Poliwoda H,Hanauske A-R: Effects of cytokines on in vitro colony formation ofprimary human tumour specimens. Eur J Cancer 26:1070, 1990

33. Nachbaur D, Denz H, Zwierzina H, Schmalzl F, Huber H:Stimulation of colony formation of various human carcinoma cell linesby rhGM-CSF and rhIL-3. Cancer Lett 50:197, 1990

34. Lang SH, Miller WR, Duncan W, Habib FK: Production andresponse of human prostate cancer cell lines to granulocyte macrophage-colony stimulating factor. Int J Cancer 59:235, 1994

35. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H,Chu TM, Mirand EA, Murphy GP: LNCaP model of human prostaticcarcinoma. Cancer Res 43:1809, 1983

36. Tomonaga M, Gasson JC, Quan SG, Golde DW: Establishmentof eosinophilic sublines from human promyelocytic leukemia (HL-60)cells: Demonstration of multipotentiality and single-lineage commit-ment of HL-60 stem cells. Blood 67:1433, 1986

37. Sherwood ER, Ford TR, Lee C, Kozlowski JM: Therapeuticefficacy of recombinant tumor necrosis factor alpha in an experimentalmodel of human prostatic carcinoma. J Biol Res Mod 9:44, 1990

38. Heaney ML, Vera JC, Raines MA, Golde DW: Membrane-associated and soluble granulocyte/macrophage-colony-stimulating fac-tor receptor alpha subunits are independently regulated in HL-60 cells.Proc Natl Acad Sci USA 92:2365, 1995

39. Nualart F, Hein S, Rodriguez EM, Oksche A: Identification andpartial characterization of the secretory glycoproteins of the bovinesubcommissural organ-Reissner’s fiber complex. Evidence for theexistence of two precursor forms. Brain Res Mol Brain Res 11:227,1991

40. Lieschke GJ, Burgess AW: Granulocyte colony-stimulatingfactor and granulocyte-macrophage colony-stimulating factor (1) and(2). N Engl J Med 327:28 and 99, 1992

41. Appelbaum FR: The use of colony stimulating factors in marrowtransplantation. Cancer 72:3387, 1993

42. Vose JM, Armitage JO: Clinical applications of hematopoieticgrowth factors. J Clin Oncol 13:1023, 1995

43. Chiba S, Tojo A, Kitamura T, Urabe A, Miyazono K, Takaku F:Characterization and molecular features of the cell surface receptor forhuman granulocyte-macrophage colony-stimulating factor. Leukemia4:29, 1990

44. Watanabe S, Mui AL, Muto A, Chen JX, Hayashida K, Yokota T,Miyajima A, Arai K: Reconstituted human granulocyte-macrophagecolony-stimulating factor receptor transduces growth-promoting signalsin mouse NIH 3T3 cells: Comparison with signalling in BA/F3 pro-Bcells. Mol Cell Biol 13:1440, 1993

45. Polotskaya A, Zhao Y, Lilly ML, Kraft AS: A critical role for thecytoplasmic domain of the granulocyte-macrophage colony-stimulatingfactor alpha receptor in mediating cell growth. Cell Growth Differ4:523, 1993

GM-CSF RECEPTOR IN PROSTATE CANCER 1043