[CANCER RESEARCH 52. 1598-1605, March 15, 1992]

Serum Prostate Specific Antigen Levels in Mice Bearing Human Prostate LNCaPTumors Are Determined by Tumor Volume and Endocrine and Growth Factors1

Martin E. Cleave,2 Jer-Tsong Hsieh, Hsi-Chin Wu, Andrew C. von Eschenbach, and Leland W. K. Chung

Urology Research Laboratory, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT

The ability of prostate-specific antigen (PSA) to predict tumor volumeand stage in patients with prostate cancer would be improved if factorsregulating its production and clearance were better defined. A thoroughunderstanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA has been precluded, however, by the absenceof an in vivo animal model. The purposes of this study are to develop amurine model for evaluating PSA pharmacokinetics in vivo and to assessfactors that influence PSA production in vitro. The human prostatecancer cell line, LNCaP, was chosen because it is androgen sensitive andPSA positive. Although LNCaP cells are usually nontumorigenic wheninoculated s.c. in athymic mice, coinoculation of 1 x IO6 LNCaP cellswith 1 x 10' human bone fibroblasts reliably produces PSA-secreting

carcinomas. This LNCaP model provides accurate correlation betweentumor volume and serum PSA levels (r = 0.94) and demonstrates thattumor volume and androgens are codeterminants of circulating PSAlevels. Following castration, serum PSA levels decrease rapidly up to 8-fold and increase up to 20-fold following androgen supplementation,without detectable castration-induced tumor cell death or concomitantchanges in tumor volume. Serum PSA levels increase 0.24 ng/ml/mm3 oftumor, which is approximately 5-fold less than that estimated for humans.Most likely this reduced PSA index (PSAitumor volume ratio) resultsfrom a 7-fold faster clearance of PSA in athymic mice than in humans;other than this shorter half-life, PSA elimination in the murine modelappears similar to that in humans, with both following first-order kineticscharacteristic of a two-compartment model. Interestingly, following prolonged growth (>21 days) in castrate hosts, LNCaP tumors are capableof adapting to an androgen-deprived environment whereby LNCaP tumors regain the ablility to secrete PSA in amounts similar to theprecastrate state.

In LNCaP cells, androgens increase PSA mRNA levels 4-fold in vivoand in vitro. PSA mRNA expression is also altered by various growthfactors. Changes in PSA production induced by androgens and growthfactors do not always parallel changes in LNCaP cell growth rate inducedby these factors, suggesting that PSA production occurs independentlyof cell growth rate and may be influenced by various interrelated factors,including hormonal and stromal milieu. Observations from this murinemodel suggest that androgens and tumor volume are independent determinants of serum PSA levels and imply that decreases in circulating PSAfollowing antiandrogen therapy may not always reflect a correspondingreduction in tumor volume.

INTRODUCTION

PSA3 is a M, 34,000 species- and tissue-specific glycoprotein

produced only by human prostatic epithelial cells. Since itsisolation by Wang et al. (1) in 1979, clinical experience withPSA has helped to define its utility and limitations in thediagnosis and management of patients with prostate cancer.The role of PSA in following patients with prostate cancer after

Received 7/24/91; accepted 1/6/92.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.

1This work was supported by Grants DK-38649 and CA 56307.2To whom requests for reprints should be addressed.'The abbreviations used are: PSA, prostate-specific antigen; BPH, benign

prostatic hyperplasia; DHT, dihydrotestosterone; FBS, fetal bovine serum; bFGF,basic fibroblast growth factor, GF, growth factor, TCM, defined media supplement; TGF, transforming growth faeton TP, testosterone propionate; TRPM-2,testosterone-repressed prostate-specific message.

therapy (2, 3) and in the immunohistochemical confirmation ofthe prostatic origin of a poorly differentiated adenocarcinoma(4) is undisputed. However, its role in staging and screening ofprostate cancer remains controversial (5, 6); clinical investigations indicate that serum PSA levels are roughly proportionalto tumor volume and stage (7, 8) but wide variations exist inmany patients with either localized disease or advanced mot-astatic disease (7, 9). Thus, except within broad ranges, serumPSA levels poorly predict total tumor volume in any givenpatient, presumably due to tumor heterogeneity with development of subpopulations of cells that variably produce themarker. Alternatively, variable PSA levels may be related tochanges in tumor cell microenvironment or hormonal, GF, orextracellular matrix milieu. With the exception of tumor volume, little is known regarding factors that influence serum PSAlevels; regulators of PSA production at the cellular and molecular level and routes of metabolism and excretion remain poorlydefined.

Recent studies suggest that in vitro PSA production by thehuman prostate cancer cell line, LNCaP, may be regulated byandrogens and various GFs (10, 11). Furthermore, serum PSAlevels in patients with BPH are reversibly decreased by antiandrogen therapy (12) and increased by androgen supplementation (13). However, controlled investigation of the regulationof PSA production and its pharmacokinetic parameters in vivohas been precluded because there are relatively few animalmodels available for study. Of the available human prostatecancer cell lines, including PC-3 (14), DU-145 (15), PC-82 (16),LNCaP (17), and HONDA (18), only the LNCaP cell line isandrogen responsive, PSA secreting, and immortalized in vitro(17, 19). However, with few exceptions, the LNCaP cell line isgenerally considered nontumorigenic when inoculated s.c. inathymic mice (20).

Recently we have reliably induced LNCaP tumor growth invivo by coinoculating LNCaP cells with nontumorigenic humanbone fibroblasts (20). The tumors are histologically poorlydifferentiated carcinomas that secrete PSA. The s.c. location ofthese tumors permits rapid, accurate, and sequential tumorvolume measurements. This model allows for the study of PSAproduction both in vitro and in vivo and provides a means todefine the pharmacokinetic profile (regulation of production,metabolism, and excretion) of PSA and its relationship totumor volume, serum androgen levels, and tumor cell microenvironment or metastatic site.

In this communication, we evaluated the relationship betweentumor volume and PSA and whether this relationship is alteredby androgen ablation or supplementation. This LNCaP tumormodel provides accurate correlation between tumor volume andserum PSA levels (r = 0.94) and demonstrates that tumorvolume and androgen are codeterminants of serum PSA levels.Serum PSA levels decrease up to 8-fold following castrationand increase up to 20-fold following androgen supplementationwithout detectable cell death or concomitant changes in tumorvolume. Interestingly, we observed that LNCaP tumors arecapable of adapting to an androgen-deprived environment; following a prolonged period of growth (>21 days) in castrated

1598

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

DETERMINANTS OF SERUM PROSTATE SPECIFIC ANTIGEN LEVELS

hosts, LNCaP tumors regain their ability to secrete PSA inamounts similar to their precastrate state without androgensupplementation. Differential changes in PSA mRNA expression in vitro induced by androgens and various GFs suggestthat LNCaP cell growth rate and PSA production are notcorrelated. These results suggest that serum PSA levels arelikely influenced by numerous factors in addition to tumorvolume, including tumor cell hormonal and straniai milieu.

MATERIALS AND METHODS

Cell Lines and Establishment of LNCaP Tumors. LNCaP cells, passage 29 of the original line developed by Horoszewicz et al. (17), werekindly supplied by Dr. Gary Miller (University of Colorado, Denver,CO) and grown in RPMI 1640 (Irvine Scientific, Santa Ana, CA) with5% FBS. A human bone fibroblast cell line, MS, derived from a patientwith an osteogenic sarcoma, was established by Dr. A. Y. Wang (TheUniversity of Texas M. D. Anderson Cancer Center). MS cells weremaintained in T-medium (20) with 5% FBS and passages 35-40 were

used.Six- to 8-week-old male nude mice (BALB/c strain; Charles Rivers

Laboratories, Wilmington, MA) were coinoculated s.c. with 1 x IO6LNCaP and 1 x IO6 MS bone fibroblasts. Up to 5 x IO6 LNCaP and2 x IO6 MS cells are nontumorigenic when inoculated s.c. alone. Cells

were suspended in RPMI 1640 with 5% FBS prior to injection and 0.1ml was inoculated using a 0.27-gauge needle. Tumors were measuredtwice weekly and their volumes were calculated using the formula L xWx H x 0.5236 (21).

To determine whether PSA is regulated by androgen in vivo, 10animals bearing LNCaP/MS chimeric tumors were castrated and theirserum PSA level and tumor volume were followed for 4 weeks (seebelow). Castration was performed transabdominally under methoxy-fluorane anesthesia (Pitman-Moore, Mundelein, IL). Five of the 10

castrated mice were treated with 3 mg/kg/day of TP (Sigma ChemicalCo., St. Louis, MO) injected s.c. in 0.1 ml peanut oil for 10 consecutivedays. TP therapy was started 8 days after castration in 2 mice and 26days after castration in 3 mice. Another cohort of 3 intact male animalswas treated by s.c. injection daily for 7 days with 0.3 mg/kg 17/3-estradiol dipropionate suspended in 0.1 ml peanut oil. Serum PSA andtumor volume were measured over a 4-week observation period.

At sacrifice, sternotomy was performed to obtain serum for PSAanalysis. Tumor specimens were subjected to various morphologicaland biochemical analyses (see below). Selected tumors were cut into 1-2-mm cubes and transplanted in male recipient mice.

Determination of Serum PSA Values. Once tumors became measurable blood samples for sequential PSA measurements were obtained.The dorsal tail +vein was incised and a sample was collected using a75-nim microhematocrit capillary tube. Usually, 60-100 ¿ilof blood

were obtained with each measurement. The samples were allowed toclot and centrifuged, and the sera were stored at —¿�20°Cuntil assayed.

PSA values were determined using a dual-site reactive enzymatic im-munoassay kit (Hybritech, Inc., San Diego, CA). With a 100-Mlserumsample, the lower limit of sensitivity of the assay is 0.4 ng/ml. Seracollected from 4 control male mice bearing human transitional cellcarcinomas had undetectable PSA levels, confirming that no murineserum protein cross-reacts with the 2 PSA antibodies used in theHybritech assay. The PSA index is defined as the increase in serumPSA in ng/ml/mm3 of tumor volume.

PSA Half-Life Determination. To establish the metabolic clearancerate of PSA in this murine model, the half-life of serum PSA was

determined. Following excision of LNCaP tumors in 3 mice, PSAvalues were obtained at 2, 6, 12, 24, 36, 48, and 60 h postexcision. Alog-linear regression model was used to evaluate the clearance rate andhalf-life of serum PSA. For each mouse, the natural log of the PSA

level (ng/ml) at time (f) divided by the PSA value at time zero wascalculated and plotted against time.

Histology and Immunohistochemistry. For routine histology, specimens were fixed in 10% neutral buffered formalin and embedded in

paraffin. Fixed sections were cut and stained with hematoxylin andeosin. For immunohistochemical studies, sections from intact andcastrate hosts were deparaffinized with xylene, rehydrated with 70%ethanol, and treated with 0.1% trypsin for 10 min at 37°C.Sections

were then incubated with monoclonal rabbit anti-PSA antibodies orrabbit serum as negative controls (Biogenex, Dublin, CA). The avidin-biotin complex method was used with all specimens using alkalinephosphatase/fast red as the chromogen (Biogenex). Slides were coun-terstained with aqueous hematoxylin and mounted with glycerol forvisual inspection and photography.

Determination of PSA Production in Vitro. For in vitro assays, LNCaPcells were weaned gradually from serum using a serum-free definedmedia supplement (T-medium plus 2% TCM; Celox Co., Minnetonka,MN). The effects of androgens (testosterone, DHT; Sigma), antiandro-gens (4-hydroxyflutamide; Schering Corp., Bloomfield, NJ), and GFs(TGFa, TGF/3, bFGF; Upstate Biotechnology, Inc., Lake Placid, NY)on LNCaP cell PSA mRNA expression and growth rate were assessed.LNCaP cell growth rate was determined using a 96-well plate (Falcon)

assay based on the uptake and elution of crystal violet (20, 22). Thecells were fixed with 1% glutaraldehyde (Sigma) and stained with 0.5%crystal violet (Sigma). Plates were washed with water and air-dried, andthe dye was eluted with 100 ¿tlof Sorensen's solution (9 mg of trisodium

citrate in 305 ml of distilled H20, 195 ml of 0.1 N HC1, and 500 ml of90% ethanol). The absorbance of each well was measured by a TitertekMultiscan TCC/340 (Flow Laboratories, McLean, VA) at 560 nm.Control experiments demonstrated that absorbente is directly proportional to the number of cells in each well.

Northern analysis was used to determine the effects of androgensand GFs on PSA mRNA levels by LNCaP cells. LNCaP cells wereplated in 150-mm tissue culture dishes (Falcon) in T-medium, 2%TCM, and 1% charcoal-stripped calf serum. When the cells were 70-80% confluent, the media were changed, cells were washed with PBS,and media were replaced with serum free T-medium with 2% TCM andvarious concentrations of androgens or GFs, or 100 ng/ml of 4-hydroxyflutamide (see above). Medium was changed on day 2, and 2days later cells were harvested and their RNA was isolated and subjectedto Northern blot analysis as described below.

RNA Isolation and Northern Blot Analysis. Total cellular RNA wasprepared from cultured LNCaP cells or LNCaP/MS chimeric tumorsusing the 4 M guanidinium thiocyanate extraction method (23). Typicalyields of total cellular RNA were about 300 fig/200 mg tissue asquantified spectrophotometrically using 40 ^g RNA//<260 unit. RNAwas denatured in 50% formamide/18% formaldehyde at 55"C and

fractionated by electrophoresis in a 0.9% denaturing formaldehydeagarose gel. Samples were transferred onto a Zetaprobe membrane(Bio-Rad) by a capillary method, and the membrane was then bakedfor 2 h at HOT".Following this, the membrane was prehybridized in the

presence of 1 M NaCl, 10% dextran sulfate, 1% sodium dodecyl sulfate,and 200 /¿g/mlsalmon sperm DNA for at least 2 h at 65°C.Hybridization was carried out at 65°Covernight with a random-primer-labeled

probe. The complementary DNA probe for PSA was a kind gift fromDr. D. Tindall (Mayo Clinic, Rochester, MN) (24) and a complementary DNA probe for TRPM-2 was kindly donated by Dr. R. Buttyan(Columbia University, New York, NY) (25). Finally, the membranewas washed under high stringency conditions (0.5 x standard saline-citrate-1 % sodium dodecyl sulfate at 65°C).Autoradiograms were prepared by exposing Kodak X-Omat AR film to the membrane at —¿�80"C

with intensifying screens.

RESULTS

Relationship between Tumor Volume and Serum PSA. Coin-oculation of LNCaP and MS cells s.c. in athymic mice inducedtumor formation at approximately 60% of inoculation sites.The tumors first appeared 6-8 weeks postinoculation. Morerecently, we have been successful in transplanting establishedLNCaP/MS chimeric tumors from host to host, with an approximate 60% take rate. LNCaP/MS tumors do not form infemale hosts or male hosts castrated at the time of inoculation.

1599

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

DETERMINANTS OF SERUM PROSTATE SPECIFIC ANTIGEN LEVELS

Fig. 1 illustrates the relationship between serum PSA andtumor volume. Serum PSA levels increase proportionally toincreases in tumor volume with a high degree of correlation (/•

= 0.94; Fig. 1). Serum PSA values ranged from 2.1 ng/ml in amouse with a tumor volume of 14 mm3 to 672 ng/ml in amouse with a tumor volume of 2855 mm3. In this model, serumPSA increases 1 ng/ml for every 4.2-mm3 tumor. Conversely,

the PSA index, or the change in serum PSA per unit change intumor volume, is 0.24 ng/ml/mm3.

Effect of Castration on Serum PSA. Castration of male micebearing LNCaP tumors produced a rapid fall in serum PSAindependent of changes in tumor volume (Fig. la). A similarbut less dramatic and more variable effect was observed usings.c. 17/i-estradiol dipropionate administration (data not shown).This reduction in the PSA index began 12 h following castrationand continued for 2 weeks postcastration after which PSAvalues stabilized (Fig. 2a). The PSA index in 3 mice graduallyincreased 21 days after castration without exogenous androgenadministration.

During a 4-week postcastration period, no reduction in tumorvolume occurred; several tumors remained unchanged in size,but most continued to grow slowly. Therefore, it appears thatalthough initiation of LNCaP tumor growth by coinoculationwith MS bone fibroblasts is androgen dependent (becauseLNCaP/MS tumors do not form in female or castrated malehosts), growth of established tumors occurs in an androgen-independent fashion. This observation corroborates an earlierreport by Horoszewicz et ai. (17). The decline in serum PSAfollowing castration occurs independent of changes in tumorvolume and results in a 3.4-fold reduction in PSA index from0.24 ng/ml/mm3 tumor to 0.07 ng/ml/mm3 tumor (range,0.013 to 0.09 ng/ml/mm3) (Fig. 2b).

Effect of Testosterone Administration on Serum PSA in Castrate Hosts. Administration of 3 mg/kg TP daily in castratemale mice resulted in a marked and rapid rise in serum PSAlevels (Fig. 3). Within 24 h of the first injection, PSA levelsrose independent of tumor volume and increased up to 20-fold(0.8 ng/ml to 16 ng/ml) above castrate levels. Stimulation ofPSA production by exogenous TP returned the PSA index to

•¿�oo

400.

200.

y = 78516 •¿�0.22029X FT2 = 0.938

r=0.94

PSA Index =0.24 ng/ml/mm3

1000 2000 3000 4000

Tumor Volume (mm3)

Fig. 1. Serum PSA increases proportionally to tumor volume with a highdegree of correlation (r = 0.94). Serum PSA ranged from 2.1 ng/ml in a mousewith a tumor volume of 14 mm3 to 672 ng/ml in a mouse with a tumor volumeof 2855 mm3. The PSA index, or the increase in serum PSA for every 1 mm3 oftumor volume, is 0.24 ng/ml/mm3.

levels equal to or higher than the precastrate state (range, 0.24to 0.82 ng/ml/mm3, n = 5).

Determination of PSA Half-Life. PSA clearance was evaluated in 3 mice to calculate its serum half-life in this model.Using a log-linear regression model, the half-life of serum PSAwas determined to be 11.7 ±0.3 h (Fig 4). In all cases, the datafit a two-compartment model of first-order elimination kinetics,in which the plot of the natural log of PSA(f)/PSA(0) againsttime produced a straight line (Fig. 4b). PSA elimination ischaracteristic of a two-compartment model in which the initial,more rapid elimination (a) phase has a half-life of 6.4 h andthe second, more gradual (ft) phase has a half-life of 12.8 h.

Histology and Immunohistochemistry of LNCaP Tumors. Tumors from intact and castrate hosts were sectioned and examined for changes in histomorphology and PSA staining. Nohistomorphological differences were observed in tumors fromintact and castrate hosts (Fig. 5, A and B). Specifically, minimalevidence of necrosis or apoptosis was observed in tumors fromcastrated males. To confirm that castration-related tumor celldeath was not responsible for the decrease in serum PSA levels,Northern analysis for TRPM-2 expression was performed. Tumors from intact hosts or from castrated hosts removed ondays 3, 4, and 8 following castration did not express TRPM-2,suggesting that cell death resulting from androgen ablation didnot occur (data not shown). Immunohistochemical staining forPSA revealed reduced staining for PSA in tumors from castrated hosts compared to tumors from intact hosts (Fig. 5, Cand D). Furthermore, tumors from castrate hosts that eitherreceived TP or were followed for longer than 21 days alsostained strongly for PSA. Although immunohistochemical studies are not quantitative, these observations agree with the resultsof Northern blot analysis assessing PSA mRNA expression (seebelow).

Effects of Androgens on PSA mRNA Expression by LNCaPTumors in Vivo. Tumors from intact and castrate hosts werebiochemically analyzed to evaluate changes in PSA mRNAexpression associated with castration. Total cellular RNA fromLNCaP tumors was isolated (see above) and subjected to Northern blot analysis. PSA mRNA expression decreases 4-fold 3and 8 days following castration and levels are restored toprecastrate levels with exogenous TP administration (Fig. 6).However, 21 days following castration, PSA mRNA expressionin some tumors returned to precastrate levels without exogenous TP, suggesting that escape from androgen-dependent PSAproduction has occurred. These data correlate with the gradualincrease in serum PSA levels observed in castrate animalsbearing LNCaP tumors when these tumors were maintained incastrated hosts for a prolonged period (Fig. 2a).

Effect of Androgens and Growth Factors on PSA Productionin Vitro. Northern analysis was performed to assess the effecton PSA mRNA levels by androgens and GFs involved inprostate cancer growth and progression. As determined bydensitometry, DHT increased PSA mRNA expression 3- to4.4-fold in a concentration-dependent manner (Fig. la). Testosterone resulted in similar increased expression (data notshown). Surprisingly, the antiandrogen 4-hydroxyflutamideproduced similar increases in PSA expression and did not blocktestosterone- or DHT-induced increases in PSA expression.

We observed that changes in PSA mRNA expression inLNCaP cells exposed to androgens or GFs are not alwaysaccompanied by corresponding changes in growth rate ofLNCaP cells; androgens increase LNCaP cell growth up to180% (20) and increase PSA mRNA expression 4-fold, while

1600

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

3 0.4

DETERMINANTS OF SERUM PROSTATE SPECIFIC ANTIGEN LEVELS

800

Days Post-Castration

y = 5.9628+ 7.3672e-2xR«2«0.922y =7.8516+ 0.22029XR»2- 0.938

IntactPSA Index =

0.24 ng/ml/mm3

CastratePSA Index =

0.07 ng/ml/mm3

1000 2000 3000 4000

Tumor Volume (mm3)

Fig. 2. (a) Castration of S male mice bearing LNCaP tumors resulted in a rapid decrease in sreum PSA levels independent of changes in tumor volume, beginning12h postcastration and continuing for 2 weeks, (b) Castration reduces the PSA index 3.4-fold from 0.24 ng/ml/mm3 to 0.07 ng/ml/mm3. Data based on serum PSA

measurements drawn 7 to 10 days postcastration.

bFGF stimulates LNCaP cell growth 180% but decreases PSAmRNA expression by 50% (Fig. Ib). TGF/3 inhibits LNCaPcell growth by 70% and yet increases PSA mRNA expression180%. Taken together, these data suggest that PSA mRNAexpression and growth are independent of one another andinfluenced by various factors such as androgens and GFs.

DISCUSSION

PSA is a serine protease produced exclusively by humanprostatic epithelial, but not stromal, cells (19). This M, 34,000glycoprotein is a single polypeptide chain of 240 amino acidsthat shows strong homology with human tissue kallikreins (26,27). Normally PSA is secreted in high concentrations into theseminal fluids where it functions to liquefy the seminal coagulimi. In patients who have prostate cancer, PSA has beenstudied extensively as a tissue-specific tumor marker, where itsdiscovery has raised hope for early detection of prostate cancerat a potentially curable stage. Serial serum PSA measurementsare the most specific and reliable indicator to monitor responseto therapy, and to signal residual and recurrent disease (2, 3,27). Preoperative PSA levelscorrelate statistically with capsularpenetration, seminal vesicle invasion, and lymph node metastasis but are not sufficiently reliable to predict clinical stage(27). Because of considerable overlap of PSA concentrations inpatients with early stage prostate cancer and BPH, it is difficultto select a cutoff value that would enable earlier detection ofcancer. Furthermore, the wide range of PSA values in patientswith the same clinical stage suggests that factors other thantumor volume alone determine final PSA levels.

An improved understanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA wouldhelp clinicians better to interpret PSA levels in individualpatients. Circulating PSA levels represent a steady state between total PSA production and its rate of metabolism andexcretion. Routes of excretion of PSA are unknown, and theconsequences of liver and renal dysfunction on serum PSAlevels remain undefined. However, factors that determine totalPSA production are becoming better defined. For instance,prostate cancer volume is one of the most important tumor

factors affecting the amount of PSA produced (7, 8, 28). However, prostatic adenocarcinomas are heterogeneous tumors withvariable PSA production throughout the tumor mass dependingon vascular supply, hormonal milieu, and grade. Variable PSAlevels may result partially from decreased cellular PSA production by poorly differentiated tumors compared to normal, hy-perplastic, or well differentiated tumors (29, 30). However,serum PSA levels may be higher on a g-for-g basis for cancerthan BPH, despite lower cellular production, because a higherpercentage of cancer cell PSA enters the circulation as a resultof more distorted architecture and obstructed ducts. Furthermore, because it is difficult to accurately measure tumor volume, interpretation of the PSA index in patients with prostatecancer is impaired.

Several host factors independent of prostate cancer, including

30 40

T TDays Post-Castration

Fig. 3. Administration of testosterone propionate (T) to castrate mice produceda marked and rapid rise in serum PSA levels. Up to 20-fold increase in the PSAindex was observed, returning the PSA index back to levels equal to or higherthan the precastrate state (0.24-0.82 ng/ml/mm3).

1601

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

DETERMINANTS OF SERUM PROSTATE SPECIFIC ANTIGEN LEVELS

a

f»&

¡U0>en

500

400

300 -

200-

100

y = 121.06 ' 10*(-2.5867e-2x) R«2»0.995

y = 370.65 •¿�10A(-2.6040e-2x) R"2 . 0.976

y = 385.31 ' 10"(-2.3926e-2x) R"2 - 0.973

= ll.7W-0.3h

-2-

-3-

y = - 0.22238 - 5.9963e-2x R"2 = 0.976

t1/2= 11.7+/-0.3hrs

10 20 30 40 50 60

Time (hours) Time (hours)Fig. 4. To establish the metabolic clearance rate of PSA in this murine model, LNCaP tumors were completely excised in 3 mice and serum PSA levels were

sequentially measured, (a) Serum PSA decays exponentially with a half-life of 11.7 ±0.3 h. (A) A log-linear regression model demonstrates that PSA clearancefollows a 2-compartment model of first order elimination kinetics.

volume and histology of BPH and circulating androgen levels,also influence the total amount of PSA produced. Because mostpatients with prostate cancer also have BPH, serum PSA isvariably elevated by the glandular component of BPH. Furthermore, circulating androgen will also affect PSA production.PSA levels decrease dramatically in patients with BPH (12) andprostate cancer (31, 32) shortly following androgen ablationtherapy. Age-related measurements have determined that PSAlevelsare testosterone dependent at all ages (33); hence, changesin serum PSA levels associated with antiandrogen therapy mayresult from death of androgen-dependent prostatic cells withsubsequent reduction in prostatic epithelial cell volume, or froma reduction in the androgen-regulated production of PSA.

The purpose of this study was to develop a PSA-producingprostate cancer model that would allow us to evaluate factorsinfluencing PSA levels in vivoand to evaluate whether changesin PSA levels following androgen ablation or administrationare related to changes in tumor volume or to changes in androgen-regulated PSA synthesis and secretion. The s.c. location ofthe LNCaP cell and bone fibroblast chimeric tumors enablessimple and accurate measuremaents of tumor volume andserum PSA levels. In intact male hosts, serum PSA is directlyproportional to tumor volume, and the PSA index is 0.24 ng/ml/mm3. Because the average athymic mice weighs 35 g (I/2000 of average 70-kg male), this measurement represents aPSA index of 0.12 ng/ml/cm3 of cancer in a human, which isless than the 0.30 ng/ml/cm3 estimated for BPH (2) and the3.5 ng/ml/cm3 for cancer (34) using the Yang assay (0.16 and1.9 ng/ml/cm3 respectively, using the Hybritech assay).

The PSA index in this model is less than that calculated byStamey et al. (34) in patients with prostate cancer because of afaster PSA clearance in the murine model, where the PSA half-life in the latter (11.6 h) is 7-fold less than that in humans [2.2or 3.2 days using Yang (2) or Hybritech (5) assays, respectively]. Other than a shorter half-life, PSA elimination in the murinemodel appears similar to that in humans, with both followingfirst-order kinetics characteristic of a two-compartment model.If corrected for differences in half-life, the PSA index in theLNCaP tumor model approaches that measured in patientswith prostate cancer (34).

Previous reports have documented that PSA expression invitro is stimulated by androgéns (10, 11). However, this is thefirst report to document a similar increase in PSA mRNAexpression and circulating protein levels in vivoindependent ofchanges in tumor volume. Similarly, androgen ablation resultsin rapid and dramatic decreases in serum PSA levels independent of tumor volume. No evidence of cell death, as assessed bythe absence of post-castration tumor volume reduction, histo-morphological evidence of cell necrosis, and TRPM-2 expression on Northern analysis, was observed to account for the fallin serum PSA. The PSA index decreased 3.4-fold followingcastration from 0.24 ng/ml of PSA/mm3 of tumor to 0.07 ng/ml/mm3 tumor. The observation that the antiandrogen, 4-hydroxyflutamide, increases PSA expression in vitro similar tothat produced by androgens contrasts with findings observedby Bilhartz et al. (10). LNCaP cells are known to have astructurally aberrant androgen receptor with a broad bindingspecificitywhich may result in abnormal responses to androgenreceptor antagonists (35).

The absence of gross, histológica!,or biochemical evidenceof castration-induced LNCaP cell death suggests that the initialdecrease in PSA results from reduction in androgen-regulatedPSA production. The gradual return to a normal PSA index incastrated mice suggests that a subpopulation of LNCaP cellsadapts to an androgen-deprived environment, a phenomenonconsistent with a process of progressive state selection (36)wherebydifferent subpopulations within a heterogeneous tumordifferentially respond when confronted by environmentalchange. Following castration, some LNCaP cells continue without change to produce PSA, while others variably decrease PSAproduction, following which, some are capable of regainingnormal PSA synthesis in the absence of testicular androgens.

In addition to studying control of PSA by endocrine factors,we also investigated whether autocrine or paracrine GFs, implicated as possible mediators of androgen-induced growth andprostate cancer progression (37-40), affect PSA production.PSA mRNA expression was increased by TGF/3and decreasedby bFGF. Changes in PSA expression did not parallel changesin LNCaP cell growth rate produced by these GFs, demonstrating that changes in PSA production do not simply reflect

1602

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

DETERMINANTS OF SERUM PROSTATE SPECIFIC ANTIGEN LEVELS

¿.yi '.;

/ .

- g. , M-;' ;-!-'->>'"«;V*."-

<c f*. ¡f--

*é^f vi »-•

i•¿�

<».-•«-„¿�•»: . ;/ »•*?•¿�-jL¡;& * * •¿�'' + t ' * •¿�)^N > /ï fr ''q '

Fig. S. LNCaP tumor PSA staining is reversibly androgen sensitive. No histomorphological differences were visible in tumors from intact (A) and castrate (B)hosts. Specifically, areas of necrosis were similarly distributed in tumors from intact and castrate hosts. Immunohistological staining using anti-PSA antibodiesrevealed increased staining for PSA in tumors removed from intact hosts (C) compared to those from third day postcastrate hosts (/'). Furthermore, tumors fromcastrate hosts treated with TP (lì)and tumors from mice followed for more than 21 days postcastration (I i also stained strongly for PSA. H & E.

changes in growth rate. Because we have not observed changesin the expression of TGF/3, TGFa, or bFGF in response to invitro androgen stimulation," these observations suggest thatPSA production and LNCaP cell growth rate are independentand that GFs do not mediate androgen-induced changes in PSAexpression. Furthermore, differential PSA production in re-sponse to androgens GFs, and extracellular matrix (10, 11)suggests that serum PSA levelsare influenced by various factors

4j. T.Hsieh,unpublishedobservations.

such as hormonal and stromal milieu and imply that tumor cellPSA production may vary depending on its metastatic site.

Finally, we should address how the findings in this study can^ extrapolated to the human situation. The major limitationof this study ¡sthe use of the imrnortaiize(j cell line (LNCaP)to produce a relatively homogeneous and uniform PSA-produc-. carcinoma. This contrasts with the clinical situation where

.„ . r „¿�.i,prostate cancers are heterogeneous, composed of cells with aspectrum of grade, hormonal responsiveness, and PSA produc-

1603

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

DETERMINANTS OF SERUM PROSTATE SPECIFIC ANTIGEN LEVELS

Controll 2 3

Day 3 T.P.E^ Day 2145678 9-i»-28S

-18S

Fig. 6. Total LNCaP tumor mRNA was isolated and subjected to Northernanalysis to determine whether decreases in PSA protein production were accompanied by concomitant changes in PSA mRNA expression. PSA mRNA expression decreases 4-fold 3 days following castration (Lanes 4 and 5), or 17/3-estradioldipropionate (/>/>;) (Lane 7) and is restored to precastate levels with exogenousTP (Lane 6). By 3 weeks postcastration, however, PSA mRNA expression returnsto precastrate levels without TP (Lanes S and 9), suggesting that an escape fromandrogen-dependent PSA production has occurred.

a

50

[DHT] nM10 1 0.1

bFGF TGFcx(ng/ml)50 10 1 10 1

TGFßl 0.1

oU

-28S

tion within any given tumor. These factors, along with variablehost hormonal status and BPH volume, will continue to makeinterpretation of PSA levels difficult for any given individual.However, our results demonstrate that androgens and tumorvolume are codeterminants of serum PSA levels and suggestthat the decrease in circulating PSA that follows antiandrogentherapy may not always reflect a corresponding reduction intumor volume.

ACKNOWLEDGMENTS

We acknowledge and thank C. Davis and D. Evans for their excellentsecretarial and editorial assistance, respectively.

REFERENCES

1. Wang, M. C., Valenzuela, L. A., Murphy, G. P., and Chu, T. M. Purificationof a human prostate specific antigen. Invest. Urol., 17: 159-163, 1979.

9.

10.

11.

12.

13.

14.

15.

16.

17.

20.Fig. 7. (a) DHT increases LNCaP cell PSA mRNA expression 4-fold in a

concentration-dependent manner in vitro, (b) LNCaP cell PSA mRNA expressionincreases 180% with TGF/3, decreases 50% in a concentration-dependent manner 21.with bFGF, and is unchanged by TGF».

22.

23.

24.

25.

26.

27.

28.

29.

30.

Stamey, T. A., Yang, N., Hay, A. R., McNeal, J. E., Freiha, F. S., andRedwine, E. Prostate-specific antigen as a serum marker for adenocarcinomaof the prostate. N. Engl. J. Med., 317:909-916, 1987.Lange, P. H., Èrcole, C. J., Lightner, D. J., Fraley, E. E., and Vessella, R.The value of serum prostate specific antigen determinations before and afterradical prostatectomy. J. Urol., 141: 873-879,1989.Ford, T. F., Butcher, D. N., Masters, J. R. W., and Parkinsson, M. C.Immunocytochemical localization of prostate-specific antigen: specificity andapplication to clinical practice. Br. J. Urol., 57: 50-55, 1985.Oesterling, J. E., Chan, D. W., Epstein, J. L, Kimball, A. W., Jr., Bruzek,D. J., Rock, R. C., Brendler, C. B., and Walsh, P. C. Prostate specific antigenin the preoperative and postoperative evaluation of localized prostatic cancertreated with radical prostatectomy. J. Urol., 139: 766-772, 1988.Cooner, W. H., Mosley, B. R., Rutherford, C. L., Beard, J. H., Pond, H. S.,Terry, W. J., Igel, T. G., and Kidd, D. D. Prostate cancer detection in aclinical urologie practice by ultrasonography, digital rectal examination andprostate specific antigen. J. Urol., 143: 1146-1154, 1990.Brawer, M. K., and Lange, P. H. Prostate-specific antigen: its role in earlydetection, staging, and monitoring of prostatic carcinoma. J. Endourol., 3:227-236, 1989.Stamey, T. A., and Kabalin, J. N. Prostate specific antigen in the diagnosisand treatment of adenocarcinoma of the prostate. I. Untreated patients. J.Urol., ¡41:1070-1075, 1989.Scardino, P. T. Early detection of prostate cancer. Urol. Clin. North Am.,76:635-655, 1990.Bilhartz, D. L., Young, C. Y. F., Flanagan, W. F., He, W., and Tindall, D.J. Androgen regulation of prostate specific antigen (PSA) in human prostatecarcinoma. J. Urol., 143: 203A, 1990.Sherwood, E., Kozlowski, J., and Lee, C. Effect of androgens and prostaticstroma on growth and PSA/PAP secretion by the human prostate carcinomaline LNCaP. J. Urol., 143: 203A, 1990.Weber, J. P., Oesterling, J. E., Peters, C. A., Partin, A. W., Chan, D. W.,and Walsh, P. C. The influence of reversible androgen deprivation on serumprostate-specific antigen levels in men with benign prostatic hyperplasia. J.Urol., 141: 987-992, 1989.Jackson, J. A., Waxman, J., and Spiekerman, A. M. Prostatic complicationsof testosterone replacement therapy. Arch. Intern. Med., 149: 2365-2366,1989.Kaighn, M., Shakar Narayan, K., Ohnuki, Y., Lechner, J., and Jones, L.Establishment and characterization of a human prostatic carcinoma cell line(PC-3). Invest. Urol., 17: 16-23, 1979.Mickey, D., Stone, K., Wunderli, H., Mickey, G., Vollmer, R., and Paulson,D. Heterotransplantation of a human prostatic adenocarcinoma cell line innude mice. Cancer Res., 37:4049-4058, 1977.Hoehn, W., Schroeder, F., Riemann, J., Joebsis, A., and Hermanek, P.Human prostatic adenocarcinoma. Some characteristics of a serially trans-plantable line in nude mice (PC-82). Prostate, /: 95-99, 1980.Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., Rosenthal, H.,Chu, T. M., Mirane!, E. A., and Murphy, G. P. LNCaP model of humanprostatic carcinoma. Cancer Res., 43: 1809-1818, 1983.Ito, Y., and Nakazato, Y. A new serially transplantable human prostaticcancer (Honda) in nude mice. J. Urol., 132: 384-387, 1984.Papsidero, L. D., Kuriyama, M., Wang, M. L., Horoszewicz, J. S., Leong,S. S., Valenzuela, L., Murphy, G. P., and Chu, T. M. Prostate antigen: amarker for human prostate epithelial cells. J. Nati. Cancer Inst., 66: 37-42,1981.Cleave, M. E., Hsieh J. T., Gao, C., von Eschenbach A. C., and Chung, L.W. K. Acceleration of human prostate cancer growth in vivo by factorsproduced by prostate and bone fibroblast. Cancer Res., 51:3753-3761,1991.Janek, P., Briand, P., and IIan man. N. R. The effect of estrone-progesteronetreatment on cell proliferation kinetics of hormone-dependent GR mousemammary tumors. Cancer Res., 35: 3698-3704, 1975.Gillies, R. J., Didier, N., and Dentón, M. Determination of cell number inmonolayer cultures. Anal. Biochem., 159:109-113, 1986.Chomcjymski, P., and Sacelli. N. Single step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. Anal.Biochem., 162: 156-159, 1986.Lundwall, A., and Lilja, H. Molecular cloning of human prostate specificantigen cDNA. FEBS Lett., 214:317-322, 1987.Buttyan, R., Olson, C. A., Pintar, J., Chang, C. Bandyk, M., Ng, P. Y., andSawezuk, I. S. Induction of the TRPM-2 gene in cells undergoing programmed death. Mol. Cell. Biol. 9: 3473-3481, 1989.Lilja, H. A kallikrein-like serine protease in prostatic fluid is the predominantseminal vesicle protein. J. Clin. Invest., 76:1899-1903, 1985.Oesterling, J. E. Prostate specific antigen: a critical assessment of the mostuseful tumor marker for adenocarcinoma of the prostate. J. Urol., 145: 907-923, 1991.Chybowski, F. M., Larson Keller, J. J., Bergstralh, E. J., and Oesterling, J.E. Predicting radionuclide bone scan findings in patients with newly diagnosed, untreated prostate cancer: prostate specific antigen is superior to allother clinical parameters. J. Urol., 145: 313-318, 1991.Svanholm, H., and Horder, M. Clinical application of prostatic markers. I.Classification of prostatic tumors using immunohistochemical techniques.Scand. J. Urol. Nephrol., Suppl. 107, 65-70, 1988.Fiener, H. D., and Gonzalez, R. Carcinoma of the prostate with atypicalimmunohistogical features. Clinical and histologie correlates. Am. J. Surg.Pathol., 10: 765-770, 1986.

1604

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

DETERMINANTS OF SERUM PROSTATE SPECIFIC ANTIGEN LEVELS

31. Killian, C. S., Yang, N., Enrich, L. J., Vargas, F. P., Kuriyama, M., Wang,M. C., Slack, N. H., Papsidero, L. D., Murphy, G. P., Chu, T. M., andInvestigators of the National Prostatic Cancer Project. Prognostic importance of prostate-specific antigen for monitoring patients with stages B2 toDl prostate cancer. Cancer Res., 45: 886-891, 1985.

32. Stamey, T. A., Kabalin, J. N., Ferrari, M., and Yang, N. Prostate specificantigen in the diagnosis and treatment of adenocarcinoma of the prostate.IV. Anti-androgen treated patients. J. Urol., 141: 1088-1090, 1989.

33. Goldfarb, D. A., Stein, B. S., Shamozadeh, M., and Petersen, R. O. Age-related changes in tissue levels of prostatic acid phosphatase and prostate-specific antigen. J. Urol., 136:1266-1269, 1986.

34. Stamey, T. A., Kabalin, J. N., McNeal, J. E., Johnstone, I. M., Freiha, F.,Redwine, E. A., and Yang, N. Prostate specific antigen in the diagnosis andtreatment of adenocarcinoma of the prostate. II. Radical prostatectomytreated patients. J. Urol., 141: 1076-1083,1989.

35. Veldscholt, J., Voorhorst-Ogink, M. M., BoIt-deVries, J., vanRooij, H. C.J., Trapman, J., and Mulder, E. Unusual specificity of the androgen receptorin the human prostate tumor line LNCaP: high affinity for progesterone and

estrogenic steroids. Biodi im. Biophys. Acta, 1052: 187-194, 1990.36. Rubin, A. L., Arnstein, P., and Rubin, H. Physiologic induction and reversal

of focus formation and tumorigenicity in NIH 3T3 cells. Proc. Nati. Acad.Sci. USA, 87:10005-10009, 1990.

37. Wilding, G., Valverius, E., Knabbe, C., and Gelmann, E. P. Role of transforming growth factor-a in human prostate cancer cell growth. Prostate, IS:1-12, 1989.

38. Schuurmans, A. L. G., Bolt, J., and Mulder, E. Androgen receptor-mediatedgrowth and epidermal growth factor receptor induction in human prostatecell line LNCaP. Urol. Int., 44:71-76, 1989.

39. Lu, J., Nishizawa, V., Tamaka, 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.

40. Mydlo, J. H., Michaeli, J., Heston, W. D. W., and Fair, W. R. Expressionof basic fibroblast growth factors mRNA in benign prostatic hyperplasia andprostatic carcinoma. Prostate, 13: 241-247, 1988.

1605

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

1992;52:1598-1605. Cancer Res   Martin E. Gleave, Jer-Tsong Hsieh, Hsi-Chin Wu, et al.   Endocrine and Growth FactorsProstate LNCaP Tumors Are Determined by Tumor Volume and Serum Prostate Specific Antigen Levels in Mice Bearing Human

  Updated version

  http://cancerres.aacrjournals.org/content/52/6/1598

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/52/6/1598To request permission to re-use all or part of this article, use this link

Research. on September 8, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from