if2 receptors as potential biomarkers of proliferation in...

[CANCERRESEARCH57, 156-161.January1. 19971

ABSTRACT

E7j and a2 receptors have been shown to exist in a number of rodent and

human tumor cell lines. Although their expression is heterogeneous andtheir function is unknown, u receptors have been proposed as potentialtargets for diagnostic tumor.imaging agents. In this study, the density ofU2 receptors in proliferative (P) and quiescent (Q) cells of the mouse

mammary adenocarcinoma, line 66, was examined. Scatchard analyses ofU2 receptors were performed on membrane preparations of 66 P cells

from 3-day cultures and 66 Q cells from 7., 10-, and 12-day cultures. TheScatchard studies revealed that 66 P cells had â€”10times more u@ receptars/cell than the 66 Q cells from 10-day cultures. Although >97% of thecells were quiescent after 7 days in culture, the maximum differential inthe 02 expression between 66 P and 66 Q cells was not attained until thesecells had been in culture for 10 days. These data suggest that ligandslabeled with positron-emitting or single photon-emitting radlonudlides,which selectively bind u2 receptors, have the potential to noninvasivelyassess the proliferative status of human breast tumors.

INTRODUCTION

The diagnosis and subsequent treatment of breast cancer has histoncally relied on a number of anatomical determinants such as tumorsize and axillary lymph node involvement. Tumors less than 1 cm indiameter usually have a good prognosis, with conventional treatmentsresulting in a 10-year survival rate exceeding 90% (1). Because thereis a strong correlation between axillary lymph node involvement andthe metastatic potential of the primary tumor, the presence of axillarylymph node involvement is a major prognostic variable for selectingbreast cancer therapies and predicting their outcome (1). However, therelatively high recurrence rate (25â€”30%)for patients initially diagnosed as node negative (2) suggests that future improvements in theclinical management of breast cancer could be made if other prognostic variables were available to help select breast tumor therapiesand accurately predict their outcome.

From both experimental and modeling data, it is known that theproliferative status of a breast tumor is a strong prognostic variable ofits response to various chemotherapy and radiotherapy regimens (3â€”7). In 1992,a DNA CytometryConsensusConferencereviewedtheclinical utility of cytometric DNA content measurements (ploidy andS-phase fraction) in selecting or predicting the outcome of therapiesfor several different tumor types (8). A strong association was foundbetween a high S-phase fraction (a measure of cell proliferation) andincreased risk of recurrence and mortality for patients with bothnode-negative and node-positive breast cancer (5). Unfortunately,30â€”40% of the cytometric samples were unevaluable in many studies

due to technical problems. Thus, many patients were unable to benefitfrom these cytometric DNA content measurements. The technicalproblems were related to: (a) sampling and dissociating the tumor intosingle cells that are representative of the proliferative status of the

Received 8/13/96; accepted 11/15/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by NIH Grants NS-31907 (to R. H. M.) and CA-45156 (to

K. T. W.).2 To whom requests for reprints should be addressed, at Department of Radiation

Oncology, Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC27157.

whole tumor; (b) staining the tumor and host cells uniformly for thevarious markers; and (c) analyzing DNA histograms that contain largeamounts of debris. These technical difficulties all contributed tosamples being either unevaluable or highly variable. Many of theseproblems might be overcome by the development of a noninvasiveimaging procedure that can assess the proliferative status of an entirebreast tumor.

A number of recent studies have reported an overexpression of o@receptors in a variety of human and murine tumor cell lines (9, 10). o@receptors represent a class of proteins that were originally, and falsely,classified as a subtype of the opiate receptors. Subsequent studiesrevealed that o@binding sites represented a distinct class of receptorsthat are located in the central nervous system as well as in a variety oftissues and organs (11). Radioligand binding studies and biochemicalanalyses have shown that there are two types of o@receptors, termed0@1 and Â°@2(11). Â°@lreceptors have a molecular weight of â€”25,000,

whereas Â°@2receptors have a molecular weight ranging from 18,000â€”21,500 (1 1, 12). The radioligand [3H](+)-pentazocine has a high (-â€”3nM) affinity for the o-@receptor and a low (>1000 nM) affinity for the

Â°@2 receptor, whereas [3H]DTG3 is equipotent at both o@ and o@2

receptors (11, 12). The role of u receptors as potential biomarkers forbreast cancer became apparent when it was reported that the radiotracer [â€˜251]N-(N-benzylpiperidin-4-yl)-4-iodobenzamide, which possesses a high affinity for o@and @2receptors, labeled MCF-7 humanbreast cancer cells in vitro (13, 14). A subsequent study revealed thatMCF-7 cells possessed a high density of o@2receptors, as measured by[3HJDTG in the presence of dextrallorphan to mask if1 sites (10).There was no detectable binding of the o@,radiotracer, [3H](+)-pentazocine, to these MCF-7 cells (10). Although these results suggested that the Â°@2receptor may be a potential target for imagingstudies of breast cancer, they provided no information on the relationship between the density of if2 receptors and the proliferativestatus of breast cancer cells.

The goal of our present investigation was to test the hypothesis thatthe density of Â°@2receptors varies with the proliferative status of breastcancer cells. This investigation was carried out with the well-characterized line 66 from a heterogeneous mouse mammary adenocarci

noma cell model, where pure (97%) populations of proliferative (P)and quiescent (Q) cells could be obtained in tissue culture (15, 16).These 66 P cells have a cell cycle distribution and a tritiated thymidinelabeling index characteristic of a mammalian cell line with a 13.8-hdoubling time (15). The 66 Q cells have predominantly a G1 DNAcontent (90%), a reduced cell volume (@50%), a mitotic and tritiated thymidine labeling index of virtually zero, and a reduced RNAcontent (40â€”50%).All of these Q cells are viable (98% tryptan blueexcluding) and can be recruited back into the P cell compartment (16).Consequently, these 66 mouse breast tumor cells have all of thecharacteristics required to test the hypothesis stated above.

3 The abbreviations used are: [3H]DTG, [3H]l,3,-di-o-tolylguanidine; PET, positron

emission tomography; [â€˜tF]FDG,[â€˜8F]2-fluoro-2-deoxyglucose;SPECT, single-photonemission computed tomography

156

if2 Receptors as Potential Biomarkers of Proliferation in Breast Cancer1

Robert H. Mach, Cynthia R. Smith, Isaf Al-Nabulsi, Brian R. Whirrett, Steven R. Childers, and Kenneth T. Whee1er@

Departments of Radiology fR. H. M., C. R. S., B. R. W.J, Radiation Oncology (I. A., K. T. WI, and Physiology and Pharmacology [R. H. M., S. R. C.], Bowman Gray School ofMedicine, Winston-Salem, North Carolina 27157

on May 27, 2018. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

BIOMARKERSOF TUMORCELLPROLIFERATION

MATERIALS AND METHODS

Drugs. DTG and (+)-pentazocine were purchased from Research Biochemicals International (Natick, MA). [3H}DTGwas purchased from DupontNEN (Bilerica, MA).

Cell Culture Procedures. The 66 cells used in these experimentswerederived from a mouse mammary adenocarcinoma. Cells were rejuvenated fromfrozen stock and tested for Mycoplasma at 3â€”6-month intervals. Unlike mostexperimental tumor cell models, these tumor cells enter a true quiescent stateunder nutrient-deprived conditions. The growth conditions required to establish these pure (>97%) P- and Q-cell populations have been described in detailpreviously (15, 16).

Briefly, exponentially growing 66 cells were trypsinized and seeded at: (a)1â€”2X l0@ cells per 25-cm2 flask in 5 ml of Waymouth's medium supplemented with 3% fetal calf serum, 6% newborn calf serum, 6% horse serum, 1%glutamine, 80.5 mg/mi streptomycin, and 80.5 units/ml of penicillin for thegrowth curve experiments; and (b) 1â€”2X 106cells per 175-cm2flask in 30 mlof Waymouth's medium for the if2 receptor experiments. In the growth curveexperiments, three flasks were trypsinized at various times after seeding, thecells were counted and sized on a Coulter counter/channelizer system, and themean Â±1 SD of the three cell counts was plofted as a function of the incubationtime. The doubling time was determined from a regression analysis of the day1â€”4data.

In the Â°â€˜2receptor experiments, 17 flasks of 66 cells were set 3 days priorto harvesting the 12 flasks of Q cells so that a 3-day P-cell population could beanalyzed simultaneously with each 7-, 10-, or 12-day Q-cell population. Toharvest the P and Q cells, the flasks were placed on ice, the medium wasremoved, and the flasks were rinsed with 15 ml of ice-cold Waymouth's salts.After rinsing, 15 ml of ice-cold Waymouth's salts were added, and the cellswere scraped. These cells were then transferred to 50-mi conical tubes andstored on ice. In each experiment, the cells in three flasks were trypsinized andcounted, and their protein content was determined by the method of Bradford(17). For the various calculations, it was assumed that the number of scrapedcells recovered per flask and their protein content were identical to thoseobtained by trypsinizing the cells.

Membrane Preparation. The ice-cold scraped cells were centrifuged for10 rain at 1000 rpm. After decanting the supernatant, the cell pellet wasresuspended in ice-cold 10 nmuTris-HCI/0.32 M sucrose (pH 7.4) to a concentration of 5.0 x 106_1.5X l0@cells/mi (volume A). Homogenization wascarried out at 4Â°Cusing a Potter-Elvejhem tissue grinder, and the ice-coldhomogenate was centrifuged for 10 mm at 1000 X g. The supernatant wassaved on ice, and the pellet was resuspended to 20% of volume A in ice-cold10 mMTris-HCI/0.32 Msucrose (pH 7.4) by vortexing. After centrifuging at1000 X g for 10 mm at 4Â°C,the pellet was discarded, and the ice-coldsupernatants were combined and centrifuged for 15 rain at 31,000 X g. Thepellet was resuspended to 30% of volume A in 10 nmi Tris-HC1(pH 7.4), byvortexing, and the suspension was incubated at 25Â°Cfor 15 mm. Following anice-cold centrifugation at 31,000 X g for 15 mm, the final pellet was gentlyresuspended in 10 mM Tris-HCI (pH 7.4) to a protein concentration of 1 mg/mi.Aliquots were stored at â€”80Â°Cuntil used. The protein concentration was alsodetermined on homogenized whole cells to obtain the milligrams of protein/cell. All protein assays were conducted by the method of Bradford (17).

Radioligand Binding Assays. Saturation binding was carried out on bothP- andQ-cell membranepreparationsof the 66 cell line. u2 bindingsites werelabeled by incubating cell membranes (30â€”60,.tg of protein) with 4 revs[3HIDTG (38.3 Ci/mmol) and varying amounts of unlabeled DTG (0.1â€”1000nM) in the presence of (+)-pentazocine (100 revs) to mask o@ sites. Incubations

were carried out in 50 mMTris-HC1 (pH 8.0) for 40 mm at 25Â°Cin a finalvolume of 0.5 ml. Nonspecific binding was determined in the presence of 5 @.tMDTG. Assays were terminated by the addition of ice-cold 10 mrvsTris-HC1(pH8.0), followed by rapid filtration through a Brandel cell harvester (Gaithersburg, MD). Filters were then washed twice with 5 ml of ice-cold buffer. Liquidscintillation counting was carried out in Ecolite (+) (ICN Radiochemicals,Costa Mesa, CA) using a Beckman LS 60001C spectrometer with acounting efficiency of 50%.

Association binding kinetics were carried out by incubating 3 nM [3H]DTGwith cell membranes (30â€”60,.tg of protein) for different lengths of time (305 to 120 miss). Nonspecific binding was determined at each time point in the

presence of 5 ,.tM DTG; (+)-pentazocine (100 nM) was used to mask o@ sites.

Dissociation binding was determined by the addition of cold DTG (5 @.tM)atdifferent time points following a 40-mm incubation of 3 flM [3H]DTG with cellmembranes (30â€”60,.@gof protein). Nonspecific binding was determined in thepresence of 5 @.tMDTG; (+)-pentazocine (100 nM) was used to mask [email protected] conditions and filtration procedures were the same as describedabove.

Data Analysis. The association kinetics of [3H]DTG to both P- and Q-cellmembranes were obtained by plotting the amount of bound [3H]DTG as afunction of time. The dissociation kinetics was obtained by plotting the amountof bound [3H]DTG as a function of time following the addition of unlabeledDTG. Saturation binding data were analyzed with the Scatchard programEBDA (Biosoft, Milltown, NJ) using the COLD option to calculate the KDandBm,,,@ values. To calculate the number of if2 receptors/cell, the number of

receptors/fmol (6.02 X 108) was multiplied by both the mg of protein/celldetermined by the Bradford assay and the fmoles of receptor/mg of proteinobtained from the Scatchard analysis.

RESULTS

In these experiments, the 66 cells grew exponentially for 4 dayswith a doubling time of 16.0 Â±1 h (Fig. 1). This doubling time issimilar to the 13.8-h doubling time reported by Wallen et a!. (15) forthis cell line. A plateau of â€”8X 106 cells/25-cm2 flask was reachedafter 5â€”6days in culture, which is identical to that reported by Wallenet a!. (15). However, once the plateau was attained, no decrease in the

cell number was observed over the next 7 days as reported by Walleneta!. (15).

The association experiments demonstrated that the binding of[3HIDTG to both P and Q cells was complete within 30 mm andremained stable for at least 2 h (Fig. 2, top). Dissociation of the[3HIDTG from if2 sites was complete within 10 mm; therefore, allassays had to be performed rapidly (Fig. 2, bottom). The associationand dissociation kinetics for [3H]DTG were identical for both P and Qcells, indicating that the same site, if2 receptors, was labeled in the twoproliferative states. The higher level of binding of [3H]DTG in P cells(Fig. 2, top) indicates that the P cells have a higher density of if2receptors than the Q cells.

Representative data from Scatchard studies of 3-day P and 10-dayQcellsareshowninFig.3.ThePcells(top)havea Bmaxof5,250fmol/mg of protein and a KD of 62.2 nM. The Q cells (Fig. 3, bottom)have a Bmax of 2462 fmol/mg of protein and a KD of 34.8 flM. Theaverage values of Bmaxand KD from the three experiments are shownin Table 1. The@ receptors/cell were calculated from the Scatchard

. V10@

106

10@

106

Cl)

LI.

w

Dz-J

@1wC)

/=16.0Â±1.Oh V

V

0 2 4 6 8 10 12 14 16

TIME (DAY)

Fig. 1. Growth curve for mouse mammary adenocarcinoma cells, line 66, seeded in25-cm2 flasks. Each point represents the mean of the cell counts from two to threeindependent experiments; bars, SD. If not shown, error bars lie within the point.

157



0 10 20 30 40 50 60

aProliferative.Quiescent

Table 1 Summary ofthe @2receptor analyses for 3-day 66 P10-day66 Q cellsâ€•cells

and either 7-dayorBm,,,,

(fmol/mg protein) KD(nM)Receptors/cell3-day

P cells7-day Q cellsP:Qratio6,573

Â±541 56.3 Â±7.34,543 Â±523 41.6 Â±4.3

1.4510,000

Â±50,000180,000 Â±60,000

2.83-day

P cells10-day Q cellsP:Q ratio5,351

Â±436 43.8 Â±8.12.271 Â±214 34.0 Â±7.2

2.4840,000

Â±110,00088,000 Â±10,000

9.5

BIOMARKERS OF TUMOR CELL PROLIFERATION

the mortality rate from breast cancer has recently declined for the firsttime in centuries (18). In spite of this promising trend, there continuesto be an increase in the number of new cases of breast cancerdiagnosed each year, and approximately one in eight women inWestern culture will be diagnosed with this disease in her lifetime (2).

It is known from previous studies that the proliferative status of abreast tumor is an excellent predictor of its response to therapy (3â€”7).Flow cytometric DNA content measurements of fine needle aspirates

Association Kinetics

3 Day Proliferative Cells

Cu

E0

Cu

Cuas6

Minutes

Fig. 2. E3HIDTG association (top) and dissociation (bottom) curves for 3-day 66 P (0)and 10-day 66 Q (â€¢)cells incubated at 25'C.

I I I I

0 30 60 90 120

Minutes

Dissociation Kinetics

@-â€˜ 3000

.@ 2000Cu0

@ 1000

F-

â€˜Cu0Cu0

B (fmol/mg)

10 Day Quiescent Cells

data as described in â€œMaterialsand Methods.â€•The Bmas P:Q ratioexpressed per cell was greater than that expressed per mg of protein,because the P cells had a higher protein content than the Q cells. TheKD5 obtained in these experiments were similar to the KD5 reportedfor rat liver and other munne tumors (10â€”14).Note that the P:Q ratiofor the number of if2 receptors/cell increased from 3 to -â€”10 betweenday 7 and day 10 in culture, because the number of @y2receptors/Q celldecreased substantially during that period. No further change in thenumber of O@2receptors/cell was observed after 12 days in culture(data not shown). These results indicate that a prolonged quiescentperiod is required to maximize the loss of cr2 receptors from these 66cells.

DISCUSSION

Breast cancer is the second leading cause of cancer deaths inwomen in the United States. In 1994, over 182,000 women werediagnosed with this disease. Increased public awareness and thewidespread use of mammography have resulted in the successful, andoften early, diagnosis of breast cancer. Because of an early diagnosis,

B (fmol/mg)

Fig. 3. Representative Scatchard plots for 3-day P and 10-day Q cells. For the P-cellexperiment (top), the B,,.@ was 5250 fmol/mg of protein, and the KD was 62.2 nsi. For theQ-cell experiment (bottom). the Bmaxwas 2462 fmol/mg of protein, and the KD was34.8 nM.

a All valuesare the means Â±1 SE from at leastthree independentexperiments.TheP-cell experiments were run simultaneously with the corresponding Q-cell experiments.

158




or tissue biopsies have been used extensively to measure the proliferative status of breast tumors. However, this procedure is severelylimited by difficulties in obtaining tissue samples that are representative of the entire tumor and technical problems that make manysamples unevaluable. Therefore, the development of a noninvasiveprocedure to assess the proliferative status of an entire breast tumorcould overcome many of these problems and significantly improve theclinical management of breast cancer by allowing a patient' s therapyto be individualized using the proliferative status of her own tumor asone of the prognostic variables.

One technique that has shown promise as a noninvasive procedurefor identifying tumor cell properties in situ is PET. PET is an imagingtechnique that measures the spatial and temporal distribution of aradiotracer in a living subject. Most of the radiotracers used in PETstudies of cancer to date are radiolabeled analogues that target a

variety of metabolic processes that are enhanced in comparison to that

of normal tissue. Examples include: (a) [18F]FDG, which measuresglucose utilization; (b) [1â€˜C]methionine,which measures the rate ofprotein synthesis; and (C) [â€˜â€˜C]thymidine,which measures the DNAsynthesis in proliferating cells. Early studies suggested that it may bepossible to differentiate malignant from benign breast cancer with[â€˜tFJFDG(19, 20). Other imaging studies in malignant melanoma andhead and neck tumors have shown a moderate to high correlationbetween [â€˜tF]FDGuptake and a number of cell proliferation markerssuch as Ki-67, S-phase fraction, and thymidine labeling index (21,22). However, subsequent studies utilizing ovarian and melanoma celllines in vitro have suggested that [3H]FDG uptake does not necessarily correlate with the proliferative activity of the cancer cells but maybe related to the number of viable cells (23) or their oxygenationstatus (24). A recent study demonstrated that the uptake of [18F]FDGin FM3A mouse mammary tumors was higher in cells from the G0-G1and G2 phases of the cell cycle than it was in cells from the S and Mphases of the cell cycle (25). These data question the suitability ofFIX; as a radiotracer for assessing the proliferative status of breasttumors.

S-phase fraction is measured in vitro by either [3H]thymidineincorporation and subsequent autoradiography or bromodeoxyuridinelabeling followed by immunohistochemical or flow cytometric identification of the bromodeoxyuridine-labeled cells (26). These procedures led to the development of [â€˜â€˜C]thymidineas a potential radiotracer for assessing S-phase fraction with PET (27). Although[1 â€˜C]thymidine is incorporated into tumor cells, the overall utility of

this PET radiotracer is limited by its rapid metabolic degradation in

vivo (27). Perhaps the most promising of the metabolic PET radio

tracers for measuring tumor cell proliferation is [t â€˜C]methionine.Initial studies have shown a high correlation (r = 0.6â€”0.8)between[1 â€˜C]methionine uptake and the S-phase fraction of both non-small

cell lung carcinoma (28) and breast cancer (29, 30). However, additional validation studies must be conducted before [1â€˜C]methionineuptake is accepted as a true measure of the proliferative status oftumor cells.

An alternative approach to metabolic imaging studies is the use ofradiotracers that possess a high affinity for a protein that is overexpressed in tumor cells. The most prominent example of this approachis the use of radiolabeled monoclonal antibodies possessing a highaffinity for tumor-associated antigens. Although some success hasbeen obtained in this area, a number of complications, including theheterogeneity of antigen-containing tumor cells, low tumor uptake,nonspecific radiotracer uptake in adjacent or other nontumor tissues,the presence of circulating antigens that compete with tumor cells forantibody, and the potential immunogenicity of the monoclonal antibody, have limited the general utility of this approach in cancer celldetection (31). Furthermore, the use of radiolabeled monoclonal an

tibodies in breast cancer imaging has been limited to the staging ofprimary breast cancer based on the presence or absence of axillarylymph node metastases (32). An alternative approach to monoclonalantibodies, which usually have molecular weights greater than100,000, is the use of radiolabeled small molecules that possess a highaffinity for receptors that are overexpressed in tumor cells. Examplesof this approach in breast cancer imaging include the use of radiolabeled estrogen and somatostatin analogues to determine the estrogenand somatostatin receptor status of a tumor (33â€”35).The goal of theseimaging studies is to predict the success of antiestrogens or somatostatin agonists in the treatment of breast cancer. The relationship

between the density of estrogen and somatostatin receptors and theproliferative status of breast tumors is not known at this time.

Recently, Vilner et a!. (10) reported that MCF-7 breast cancer cellspossessed a high density of if2 receptors and displayed no detectablebinding of the radioligand [3H](+)-pentazocine, indicating that cr1receptors were absent (10). These data suggest that cr2 receptors are apotential biomarker of breast tumors, and that high affinity u2 ligandspossessing a y-emitting radionuclide have the potential to visualizeprimary breast tumors by using the imaging techniques, PET andSPECT. For example, the radiotracer [â€˜251]N-(N-benzylpiperidin-4-yl)-4-iodobenzamide has been shown to label MCF-7 cells in vitro(13, 14) and suggests that the corresponding â€˜23I-labeledanaloguemay be a potential SPECT imaging radiotracer for breast tumors.

The goal of the current study was to determine if cr2receptors havethe potential to be biomarkers of tumor cell proliferation in breastcancer. A series of radioligand binding experiments were conductedon membrane homogenates obtained from cell culture samples of themouse mammary adenocarcinoma, line 66. Previous studies haveshown that 66 cells display exponential growth kinetics for a period of4 days,followedby aplateauphasethatremainsstableupto day14(15). In vitro studies also revealed that the exponentially growing 66P cells have â€”50%of the population in G1 and a 24-h [3H]thymidinelabeling index of >90%, whereas plateau phase 66 Q cells have>97% of the population in G1 and a 24-h [3H]thymidine labeling

index of â€”2% (15). Furthermore, subsequent studies revealed that100% of the 66 Q cells can be recruited into the P-cell compartment(16). The induction of a pure (>97%) quiescent tumor cell populationfrom cells that were initially in exponential growth and the completerecruitment of 66 Q cells into the P-cell compartment indicates thatthis cell line is an appropriate in vitro model system for studyingbiomarkers of cell proliferation.

In the current study, the cr2receptor density was measured in 3-day66 P cells and in 7-, 10-, and 12-day 66 Q cells. Since the transitionfrom a pure 66 P-cell population to a pure 66 Q-cell population is notcomplete until day 6â€”7in this in vitro model, these Q-cell populationscorrespond to about 1, 3, and 5 days after quiescence. All bindingstudies with the 66 Q cells were paired with a corresponding bindingstudy of 3-day 66 P cells in an attempt to minimize the amount ofexperimental error that might result from making comparisons withcell cultures harvested on different days. The results of Scatchardstudies with 7-day 66 Q cells revealed a@ receptor density of â€”4500fmol/mg of protein (Table 1). The corresponding 3-day P cells had aO@2receptor density of â€”-6,500 fmol/mg of protein, resulting in a P:Q

ratio of 1.4: 1. However, comparing the ratio of cr2receptor densitiesbetween 66 P and 66 Q cells on a fmol/mg of protein basis is notentirely valid because 66 P cells have a significantly higher amount ofprotein per cell than 66 Q cells (15). Conversion of the@ receptordensity to a receptors/cell basis resulted in 180,000 O@2receptors/cellin 7-day 66 Q cells and 510,000 if2 receptors/cell in the corresponding3-day 66 P cells to give a P:Q ratio of â€”2.8:1.The if2 receptor densityin 10-day 66 Q cells was â€”2,300 fmol/mg of protein or 88,000receptors/cell (Table 1). The u2 receptor density in the corresponding

159




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160

3-day 66 P cells was â€”-5,400fmol/mg of protein or 840,000 receptors/ REFERENCEScell, resulting in a P:Q ratio of â€”9.5:1. However, there was no furtherchange in the cr2 receptor density of these 66 Q cells after 12 days inculture (data not shown). These data suggest that a prolonged periodin quiescence is required to maximize the loss of if2 receptors from 66cells.

Although the number of @2receptors in the 3-day 66 P cells ona receptors/cell basis appears to be exceptionally high (5 10,000â€”840,000 receptors/cell), this number is consistent with the numberof if2 receptors/cell reported in other tumor cell lines (10). Thedensity of if2 receptors in the 66 P cells is also much higher thanthat observed in other receptor systems expressed in tumor cells.For example, the density of somatostatin receptors in MCF-7 cellshas been reported to be â€”250receptors/cell (36), and the densityof estrogen receptors in estrogen receptor-positive breast tumorbiopsies has been reported to be <200 fmol/mg of protein (34),which is substantially lower than the density of if2 receptorsobserved in breast tumor biopsies (37).

Recent advances in molecular biology have resulted in the investigation of a variety of biomarkers that are overexpressed in tumorcells. Examples of these include epidermal growth factor receptor,transforming growth factor (3mRNA, insulin-like growth factors I andII, somatostatin receptors, and the oncogene proteins, HER/neu andc-myc. However, the expression of these markers in breast tumors ishighly variable and their relationship to other indices of cellularproliferation, such as S-phase fraction, is not known (38). The resultsof the current study indicate that cr2 receptors can be added to the listof proteins that are overexpressed in breast tumors. However, our dataalso indicate that the density of if2 receptors is dependent on theproliferative status of the 66 cells. This suggests that u2 receptorligands labeled with a radionuclide that can be used in conjunctionwith the in vivo imaging techniques, PET or SPECT, have the potential to assess the proliferative status of primary breast tumors. However, additional studies are clearly needed to determine if the higherdensity of if2 receptors in 66 P cells is caused by a nonspecificup-regulation of a variety of proteins, or if if2 receptors play anintegral role in the cell proliferation process. A recent study demonstrated that g ligands inhibit cell proliferation in mammary carcinoma

cell lines in vitro by inducing a sequence of morphological changesresembling those that occur during apoptosis (39). Furthermore, ifnonselective ligands were more potent than ifI-selective ligands ininhibiting tumor cell growth, which suggests that if2 receptors mayplay an important role in cell growth and/or proliferation (39). Therelative importance of u2 receptors as a potential biomarker of theproliferative status of breast cancer will not be clear until the biochemical function of @2receptors in vivo is completely understood.

In conclusion, the results of the current study confirm the previousreport indicating that@ receptors are expressed in high density inbreast tumor cell cultures (10). Furthermore, the @y2receptor densitywas higher in 66 P cells than in 66 Q cells. These data suggest that if2receptors may be a biomarker for noninvasively assessing the proliferative status of human breast tumors, thereby allowing a patient's

therapy to be individualized using the proliferative status of her owntumor as one of the prognostic variables.

ACKNOWLEDGMENTS

Tissue culture medium and Mycoplasma testing were provided by theTissue Culture Core Laboratory of the Comprehensive Cancer Center of Wake

Forest University, which is supported in part by NIH Grant CA 12197.




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A. H., and Katzenellenbogen,J. A. Breast cancer: PET imaging of estrogen receptors.Radiology. 169: 45â€”48,1988.

35. van Eijck, C. H., Krenning. E. P., Bootsma, A., Oei, H. Y., van PcI, R., Lindemans,J., Jeekel, J., Reubi, J. C., and Lamberts, S. W. J. Somatostatin-receptor scintigraphyin primary breast cancer. Lancet, 343: 640â€”643, 1994.

36. Setyono-Han, B., Henkelman, M. S., Foekens, J. A., and Klijn, J. G. M. Directinhibitory effects of somatostatin (analogues) on the growth of human breast cancercells. Cancer Ret., 47: 1566â€”1570, 1987.

37. John. C. S., Vilner, B. J., Schwartz, A. M., and Bowen, W. D. Characterization ofsigma receptor binding sites in human biopsied solid breast tumors. J. NucI. Med., 37:267P, 1996.

38. Klijn, J. G. M., Berns, P. M. J. J., van Putten, W. L. J., Bontenbal, M., AlexievaFigusch, J., and Foekens, J. A. Critical review of growth factors as clinical tools inprimary and metastatic breast cancer. Recent Results Cancer Ret., 127: 77â€”88,1993.

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1997;57:156-161. Cancer Res Robert H. Mach, Cynthia R. Smith, Isaf Al-Nabulsi, et al. Cancer

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