bone and kidney adenylate cyclase-stimulating activity ... · was stimulated by the tumor extract...

[CANCER RESEARCH 47, 690-695, February 1, 1987]

Bone and Kidney Adenylate Cyclase-stimulating Activity Produced by a

HypercalcÃ©mieCanine Adenocarcinoma Line (CAC-8) Maintained inNude Mice1

Thomas J. Rosol,2 Charles C. Capen, and Charles L. Brooks

Department of Veterinary Pathobiology, The Ohio State University, Columbus, Ohio 43210

ABSTRACT

The tumor line, CAC-8, is a serially transplantable adenocarcinoma

maintained in nude mice which originated from a hypercalcÃ©mie dog.Nude mice with CAC-8 developed a syndrome of humoral hypercalcemiaof malignancy. CAC-8 contained a protein factor which stimulated ade-

nylate cyclase of bone and kidney cells in vitro. The adenylate cyclase(AC) of rat osteosarcoma cell lines, ROS 17/2.8 (ROS) and UMR-106,

was stimulated by the tumor extract and potentiated by forskolin (0.1Mivi). The ROS cells responded to the lowest concentration of CAC-8

extract, but UMR cells responded with a greater increase in AC activitycompared to controls following exposure to CAC-8 extract. Pretreatmentof ROS 17/2.8 cells with dexamethasone enhanced the response to CAC-

8 extract. The opossum kidney cell line (OK) was less sensitive to theAC-stimulating activity of CAC-8 extract, but AC stimulation was increased in the presence of forskolin. Bovine (1-34) parathyroid hormone

(Brill) (10 HM) stimulated AC equally in ROS, UMR, and OK cells.Isoproterenol (1.0 MM) stimulated AC activity in ROS and UMR cellsbut not in OK cells. The AC-stimulating activity of CAC-8 appeared to

bind to the parathyroid hormone receptor of ROS, UMR, and OK cellssince addition of the parathyroid hormone receptor antagonist,["'norleucine, "tyrosineJBPTH (3-34) amide, inhibited CAC-8-mediatedcyclic adenosine S'-monophosphate production and alone did not stimu

late AC activity. The AC-stimulating activity of CAC-8 was acid and heatstable. Trypsin digestion reduced HI*111 and CAC-8 stimulation of AC

to near basal levels and treatment of CAC-8 extract with dithiothreitol

reduced AC stimulation in UMR cells by approximately 50%.Extracts of the hypercalcÃ©mie tumor line (CAC-8) contained bone and

kidney AC-stimulating activity which was enhanced by forskolin anddexamethasone, inhibited by |*-'"Mi-, "TyrjBPTH (3-34) amide, heat

stable, trypsin sensitive, inactivated by reduction, and had a relativemolecular weight of 34,000 by gel exclusion chromatography. Isolationand characterization of the factors) produced by CAC-8 that stimulate

AC activity will be useful in further understanding the pathogenesis ofhumoral hypercalemia of malignancy in animal and human patients.

INTRODUCTION

A serially transplantable tumor line, designated CAC3-8, has

been developed from a canine hypercalcÃ©mieadenocarcinoma( 1) in nude mice that develop an identical clinical syndrome ofHHM as reported in human patients (2). Nude mice with CAC-8 developed hypercalcemia, hypophosphatemia, increased

Received 6/26/86; revised 9/15/86; accepted 10/21/86.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 from The Ohio Canine Research Fund(611216) and the Morris Animal Foundation (20322-55-00). Nude mouse facilities were provided by the Comprehensive Cancer Center of The Ohio StateUniversity (USPHS Grant CAI6058-12). T. J. R. was supported by a Schering-Plough Corporation fellowship and a National Research Service Award (l F32CA07818-01A1) from the National Cancer Institute.

2To whom requests for reprints should be addressed, at The Ohio StateUniversity, Department of Veterinary Pathobiology, 1925 Coffey Road, Columbus, OH 43210.

3The abbreviations used are: CAC, canine adenocarcinoma; AC, adenylatecyclase; HHM, humoral hypercalcemia of malignancy; cAMP, cyclic AMP; PTH,parathyroid hormone; HP 111. bovine parathyroid hormone (1-34); Nie, norleu-cine; Tyr, tyrosine; KTK, i (tosylamiiiu 2-phenyl)ethylchloro methyl ketone;FBS, fetal bovine serum; ROS, ROS 17/2.8 (a rat osteosarcoma cell line); UMR,UMR-106 (a rat osteosarcoma cell line); TGF, transforming growth factor.

serum 1,25-dihydroxycholecalciferol, increased urinary excre

tion of cAMP, and increased rates of bone formation andrÃ©sorptionwithout bone metastasis (2). Tumor extracts andconditioned tissue culture medium contained a factor whichincreased in vitro bone resorbing activity but was not suppressedby indomethacin, stimulated osteoclast hyperplasia and hypertrophy in neonatal mouse calvarÃa, and had transforminggrowth factor activity that was not dependent on the presenceof epidermal growth factor (3, 4).

Humoral hypercalcemia of malignancy is an important clinical syndrome resulting from the secretion of unidentified substances by tumor cells which results in increased osteoclasticbone rÃ©sorption, altered renal excretion of phosphorus andcalcium, and increased nephrogenous cAMP (5). In HHM ofhuman beings and laboratory animals there is increased totaland fractional excretion of phosphorus and increased total anddecreased fractional excretion of calcium (6, 7). These featuresalso are a characteristic response to PTH excess (8). The effectson bone and kidney may result from one or more humoralfactors released by tumor cells. Prostaglandins, PTH, TGFs,and PTH-like peptides have been suggested as the factors

produced by tumor cells that result in the development of HHM(9). Most forms of HHM have been shown not to be the resultof excessive secretion of PTH or prostaglandins (10). It hasbeen hypothesized that HHM is due to a combined secretionof TGF and PTH-like peptides by the neoplastic cells (9).Transforming growth factors stimulate in vitro bone rÃ©sorptionand the TGF activity of the rat Leydig cell tumor and theWalker carcinosarcoma coelute on chromatography with boneresorbing activity (11, 12). Tumors associated with HHM inhuman beings and animals have been reported to contain factorswhich bind to PTH receptors in target cells and stimulateadenylate cyclase (13-15). It is not known whether the bone-resorbing activity and the adenylate cyclase-stimulating activityof tumors associated with HHM are the same or differentproteins (15).

The tumor line CAC-8 contains in vitro bone-resorbing activ

ity (3), TGF activity (4), and activity that stimulates adenylatecyclase in both bone and kidney. This triad of biological activities is typical of the few well-described laboratory animalmodels of HHM (8). PTH-like activity from a collection ofhuman tumors associated with hypercalcemia correlated wellwith occurrence of HHM and could be used to classify thepatients into "HHM" and "non-HHM" categories (15). In

addition, the tumor line CAC-8 is a well-differentiated epithelialmalignancy generally similar in type to the forms of cancer thatproduce HHM in human patients. The tumor doubling rate ofCAC-8 in nude mice is approximately 14 days which allows

time for morphological changes consistent with HHM to develop in nude mice.

The objectives of this investigation were to (a) determinewhether hypercalcÃ©mieand normocalcemic canine adenocarci-nomas contained activity capable of stimulating adenylate cyclase in ROS 17/2.8 (rat osteosarcoma) cells, UMR-106 (rat

690

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CARCINOMA STIMULATION OF AC

osteosarcoma) cells, and OK (opossum kidney) cells; (Â¿>)evaluate the effects of forskolin and dexamethasone on adenylatecyclase stimulation; (c) determine whether adenylate cyclasestimulation could be inhibited by the PTH antagonist, [8-'8Nle,34Tyr]BPTH (3-34); (d) compare the adenylate cyclase stimulation of CAC-8 extract to BPTH (1-34); and (e) determine thechemical stability and approximate molecular weight of theadenylate cyclase-stimulating activity in this animal model ofHHM.

MATERIALS AND METHODS

Materials. Bovine PTH (1-34), forskolin, isoproterenol, dexamethasone, isobut ylniet hylxiint hiÃ±e,phenylmethylsulfonyl fluoride, pepstatinA, ATP, ADP, AMP, cAMP, adenine, adenosine, dithiothreitol, soybean trypsin inhibitor, and calcium- and magnesium-free Hanks' bal

anced salt solution were obtained from Sigma Chemical Co. (St. Louis,MO); ["-'"Nie, "TryJBPTH (3-34) amide from Bachern, Inc. (Torrence,CA); trypsin treated with TCPK from Cooper Biomedicai, Inc. (Mal-vern, PA); 12.X-'IIjuduninc from New England Nuclear (Boston, MA);basal medium Eagle's and F12 medium from Grand Island BiologicalCo. (Grand Island, NY); and Fractogel TSK HW-50s from Pierce

Chemical Co. (Rockford, IL).Cell Culture. The ROS 17/2.8 cells were kindly provided by Dr. R.

Majeska (University of Connecticut, Farmington, CT) and maintainedin F12 medium with 5% FBS as previously described (16). The UMR-106 and OK cell lines were kindly provided by Dr. G. Strewler (University of California, San Francisco, CA) and maintained in basalmedium Eagle's supplemented with 10% FBS, 2 HIM L-glutamine,

penicillin (SO units/ml), streptomycin (SO Mg/ml), and neomycin (100Mg/ml). The ROS cells are a cloned cell line from a rat osteosarcomaselected for an optimum response of adenylate cyclase to PTH andisoproterenol (16). The UMR cells are a cloned cell line from a ratosteosarcoma with a high level of AC responsiveness to PTH andprostaglandins (17). The ROS and UMR cells may represent differentphases in the life cycle or different subpopulations of osteoblasts (18).The OK cells are an uncloned cell line derived from opossum kidneythat have PTH-responsive adenylate cyclase (19).

Preparation of Tumor Extract. CAC-8 tumor tissue (0.5-2.0 g/mouse)was removed from hypercalcÃ©mienude mice (Life Sciences, St. Petersburg, FL) and frozen at â€”70"C(13). Positive and negative control

tissues consisted of an anal sac adenocarcinoma removed from a nor-mocalcemic dog (negative control, CAC-23) and an anal sac adenocarcinoma removed from a hypercalcÃ©miedog (positive control, CAC-22).Tumor tissue was frozen in liquid nitrogen and shattered by compression. The tissue was thawed in a solution (4.0 ml/g of tissue) consistingof 375 ml 95% (vol/vol) ethanol, 7.5 ml concentrated HC1, 33 mgphenylmethylsulfonyl fluoride, and 1.9 mg pepstatin A. Distilled water(2 ml/g of tissue) was added, and the tissue was homogenized in an icewater bath using a Polytron homogenizer (Brinkman Instruments, Inc.,Westbury, NY) (three 15-s bursts). The mixture was extracted overnightat 4Â°C,centrifugea at 15,000 x g for 30 min, and the residue was

reextracted for 2 h with 80 ml of a solution consisting of 375 ml 95%ethanol, 105 ml distilled water, and 7.5 ml concentrated HC1. Thesupernatants were pooled, and the pH was adjusted to 5.2 with concentrated ammonium hydroxide followed by the addition of 1 ml of 2 Mammonium acetate buffer, pH 5.3/85 ml of extract. Two volumes ofice-cold absolute ethanol and 4 volumes of ice-cold anhydrous etherwere immediately added. The mixture was cooled to â€”20Â°Cfor 48 h.

The resulting precipitate was collected by rapid filtration on WhatmanNo. 1 paper, redissolved in l M acetic acid (4 ml/g tissue), andcentrifuged to remove nonsoluble precipitate. The solution was dialyzed(1:100) against 0.17 M acetic acid for 48 h at 4*C with 4 changes of

dialysate (Spectropor tubing, M, 3500 cut-off; American ScientificProducts, Columbus, OH). The samples were lyophilized and stored at-20Â°C (20). One batch of tumor extract (CAC-8 extract) was used in

all the experiments described in this investigation, except in the initialexperiment in which all tumor extracts (CAC-8 extracts 1 and 2, CAC-22, and CAC-23) were compared in a single AC-stimulating assay using

ROS cells. The protein of tumor extracts was determined using theBradford assay with bovine serum albumin as a standard (21).

Adenylate Cyclase-stimulating Assay. The AC-stimulating assay wasperformed as described (13) with minor modifications. Briefly, cellswere plated at 10,000 cells/cm5 and grown to confluence in either 4cmVwell or 2 cm2/well plates (Costar, Cambridge, MA). The experi

ments which compared the 4 tumor extracts and determined the effectof pretreatment with BPTH (1-34) utilized 4 cm2/well plates while all

other experiments utilized 2 air/well plates. The ROS cells required 8days and the UMR-106 and OK cells required 4 days to reach confluence in the 2 cm2/well plates. The cells were incubated with 1 ml ofF12 medium with 5% FBS containing 1 Â¿tCi[3H]adenine (100 pCi/ml)for 2 h at 37*C. The wells were washed twice with 1 ml of Hanks'

balanced salt solution and incubated with 0.5 ml of F12 medium with2% FBS and 1 mM isobutylmethylxanthine for 10 min at 37Â°C.Positive

control and test substances were added to result in specified concentrations to the wells for a treatment period of 5 min, and the reaction wasstopped with the addition of 50 ni of 1.2 M trichloroacetic acid. Themedium was transferred to plastic tubes and 50 v\ of a carrier solutionwas added that contained 5 mM of ATP, ADP, AMP, cAMP, adenine,and adenosine. The samples were frozen at â€”20Â°Covernight, thawed,

and centrifuged for 10 min at 1200 x g. The supernatant was neutralizedwith 4 N KOH (25 M'), the cAMP was separated by the methods ofSalomon (22) using serial Dowex X50 (Bio-Rad Laboratories, Richmond, CA) and alumina column chromatography, and the radiationwas determined by scintillation spectrophotometry.

The effect of 4 canine tumor extracts (extracts 1 and 2 of CAC-8,CAC-22, and CAC-23) was determined on the AC activity of ROScells. Bovine PTH (5-20 nM) was the positive control in each experiment. The effect of 2 h pretreatment of BPTH was evaluated on CAC-8- and BPTH-stimulated AC of ROS cells. A time response curve (1-20 min) was developed on the AC stimulation of UMR cells by CAC-8 extract. The dose response of AC stimulation by CAC-8 extract wasdetermined using UMR and ROS cells. The effect of BPTH, isoproterenol (1 MM)(/3-adrenergic agonist), and CAC-8 extract (0.5 mg/ml) wascompared on the AC stimulation of ROS, UMR, and OK cells. Forskolin (0.1 MM)was added directly to test substances or the 3 cell lineswere incubated with medium supplemented with 30 nM dexamethasonefor 48 h prior to the AC assay in order to determine the effects offorskolin or dexamethasone on the stimulation of AC in ROS, UMR,and OK cells by BPTH and CAC-8 extract.

The BPTH receptor antagonist, ["â€¢"Nie,"TyrJBPTH (3-34) amidewas added to ROS, UMR, and OK cells in addition to BPTH (1-34) orCAC-8 extract to determine the dependence of AC stimulation onbinding to the PTH receptor. Bovine PTH and CAC-8 extract wereexposed to 60Â°Cfor 1 h, 100Â°Cfor 3 min, or trypsin-TCPK (50 Mg/ml)for 1 h at 37Â°Cto determine the effect of heat treatment and protease

digestion on AC stimulation of UMR cells. The trypsin digestion wasstopped by the addition of soybean trypsin inhibitor (100 Mg/ml). Thetrypsin control solution contained both trypsin-TCPK and trypsininhibitor at the same concentrations. CAC-8 extract was treated withdithiothreitol (0.062 M) for 1 h at 25Â°Cand then extensively dialyzed

against 0.17 M acetic acid. A paired batch of CAC-8 extract was treatedidentically without exposure to dithiothreitol.

Gel Chromatography. CAC-8 tumor extract (20 mg; l ml of sample)was dissolved in l N acetic acid, applied to a column (2.5 x 80 cm) ofFractogel TSK HW-50s (25-40 Mm),and eluted with l N acetic acid ata flow rate of 2 ml/h. Fractions (1 ml) were collected, evaluated forabsorbante of light at 280 nm, lyophilized, and stored at -20V untilassayed for AC-stimulating activity on UMR cells in the presence offorskolin (0.1 MM).Chromatographie molecular weight markers included bovine serum albumin (M, 67,000), chymotrypsinogen (M,25,000), and insulin (M, 6,000).

Statistical Analysis. The AC stimulation assays were performed intriplicate. The data were expressed as cpm/well Â±SE. Data consistingof 3 or more groups were analyzed by analysis of variance and the NewMultiple Range test (23). Data with 2 groups were evaluated withStudent's i test. A significant difference was considered to exist when

P < 0.05.

691




RESULTS

Three canine tumor extracts produced significant stimulationof AC in ROS cells (Table 1). The 2 batches of CAC-8 extractwere not significantly different and produced approximately47% the magnitude of stimulation of AC activity compared toBPTH. The extract from CAC-22 (a second anal sac adenocar-cinoma from a hypercalcÃ©miedog) significantly stimulated ACbut was less active than the extracts from CAC-8 obtained bytumors grown in nude mice. Tumor extract designated CAC-23 (adenocarcinoma of anal sac from a normocalcemic dog) didnot significantly stimulate AC activity as compared to CAC-8,CAC-22, and controls despite numerical doubling of AC activity(one-way analysis of variance). The protein content of CAC-8extracts 1 and 2, CAC-22, and CAC-23 was 0.28, 0.29, 0.20,and 0.27 mg protein/mg dry extract, respectively. Pretreatmentof ROS cells with BPTH for 2 h significantly reduced theresponse of AC to stimulation by both BPTH and CAC-8extract by 45% (Table 2).

The time response curve (Fig. 1) of AC stimulation by CAC-8 extract demonstrates that cAMP accumulates in UMR cellsin a linear pattern during the initial 10 min of incubation andthen plateaus from 10-20 min. The point of median stimulationoccurs at approximately 5 min. The dose response curve of ACcyclase stimulation in ROS cells (Fig. 2) by CAC-8 demonstrates a sigmoidal pattern with significant stimulation of ACat 0.01 mg of extract/well. In comparison, the UMR cellsrespond to CAC-8 extract with significant stimulation of ACat 0.035 mg CAC-8 extract/well (Fig. 3).

In Table 3 results are compared of AC activity in ROS,UMR, and OK cells after stimulation by BPTH, isoproterenol,and CAC-8 extract. The concentrations used for the 3 agonistswere maximal for AC stimulation of the cell lines and werekept constant to permit comparisons between cell lines. Although the AC of ROS cells was stimulated by BPTH, isopro-

Table I Effect of canine adenocarcinoma extracts and BPTH on the stimulationofadenylate cyclase in KOS 17/2.8 (bone) cells

The 4 tumor extracts (1.5 mg/well) and BPTH (20 n\i) were added to 4-cm-wells of confluent ROS 17/2.8 cells. The A<'-si imulatinÂ«assay was performed asdescribed in "Materials and Methods." The data were evaluated with one-way

analysis of variance and New Multiple Range test.

Treatment cpm/well

ControlBPTH (20 nivi)CAC-8 (1.5 mg, extract 1)CAC-8 (1.5 mg, extract 2)CAC-22 (1.5 mg)CAC-23 (1.5mg)

486 Â±33"23,040 Â±1,682*10,895 Â±691Â»10,931 Â±594*6,554 Â±470*1,011 Â±85'

" Mean Â±SE from triplicate wells of a representative experiment.* Different from control, at P< 0.001.' Different from control, at P < 0.01.'' Not significantly different from control.

Table 2 Effect of pretreatment with BPTH on the stimulation ofadenylatecyclase in ROS 17/2.8 (bone) cells by BPTH and CAC-8 extract

Bovine l'Ili (20 DM)or a control solution without BPTH was added to 4-crn2

wells of confluent ROS 17/2.8 cells in F12 medium containing 2% fetal calfserum for 2 h. The wells were washed twice with Hanks' balanced salt solutionand the AC-stimulating assay was performed with BPTH (20 IIM) and CAC-8extract (0.6 mg/well) as described in "Materials and Methods." The data fromthe pretreatment and no pretreatment groups were compared using Student's t

test.

TreatmentControl

BPTH (20 nM)CAC-8 (0.6 mg)No

pretreatment(cpm/well)524

Â±54"

29,732 Â±4529,313 Â±654Pretreatment

withBPTH(cpm/well)1,670

Â±58*16,4 14 Â±834*5,150 Â±351*

* Mean Â±SE from triplicate wells.* Different from corresponding no pretreatment group, at P < 0.01.

6000-,

5000

- 4000

I 3000-OQ.

u 2000

1000

0 1 10

MINUTES

20

Fig. I. Time response curve from 0-20 min of AC stimulation of UMR-106(bone) cells by CAC-8 extract. Points, mean of triplicate wells Â±SE (bars).

JL

n_CONTROL

0.0

CAC-8 EXTRACT (mg/WELL)

0.01 0035 0075

â€¢= D<0 OS ,,= |><0.01

Fig. 2. Dose response curve of AC stimulation of ROS 17/2.8 (bone) cells byCAC-8 extract. Ãœ,mean of triplicate wells Â±SE (bars) of a representativeexperiment. Data were evaluated using one-way analysis of variance and the NewMultiple Range test.

I

2 3000

riCONTROL CAC-8 EXTRACT (mg/WELL)

0.500.0 0.001 0.01 0035 0.079 0.20

â€¢*P<O.OS **=p<0.01

Fig. 3. Dose response curve of AC stimulation of UMR-106 (bone) cells byCAC-8 extract. D, mean of triplicate wells Â±SE (Aur.v)of a representativeexperiment. Data were evaluated using one-way analysis of variance and the NewMultiple Range test.

692




Table 3 Effect ofBPTH, Â¡soproterenol,and CAC-8 extract on the stimulation ofadenylate cyclase in KOS 17/2.8, UMR-106, and OK cell lines

Bovine PTH (20 DM), isoproterenol (1 MM),or CAC-8 extract (0.5 mg/well)were added to 2-cm2 wells of confluent ROS, UMR, or OK cells. The AC-stimulating assay was performed as described in "Materials and Methods." Thedata from each cell line were compared using one-way analysis of variance andNew Multiple Range test.

Cell line(cpm/well)TreatmentControl

BPTH (20 nM)Isoproterenol (1 I/M)CAC-8 (0.5 mg)ROS

17/2.8(bone)237

Â±37Â«7219 Â±849*8942 Â±1261*2639 Â±103"UMR-106

(bone)230Â±

115906 Â±263*1394Â±212CJ*5086 Â±507*OK

(kidney)538

Â±686593 Â±210*

485 Â±50771Â±72

" Mean * SE from triplicate wells of a representative experiment.* Different from control, at P < 0.01.c Different from control, at P < 0.05.* Different from BPTH and CAC-8, at P < 0.01.' Different from BPTH and isoproterenol, at P< 0.01.

Table 4 Effect of forskolin and dexamethasone on the stimulation of adenylatecyclase in KOS 17/2.8, UMR-106, and OK cells by BPTH and CAC-8 extractBovine PTH (10 nM), CAC-8 extract (specified amount per well), or control

solutions were added to 2-cm2 wells of confluent ROS, UMR, or OK cells. Inseparate wells forskolin (0.1 MM)was added directly to the test substance, or the3 cell lines were incubated with medium supplemented with 30 nM dexamethasonefor 2 days prior to assay. The AC-stimulating assay was performed as describedin "Materials and Methods." The data from each cell line were compared to

control values and the treatment data were compared to no treatment data usingthe analysis of variance and New Multiple Range test.

TreatmentROS

I7/2.8 (bone) cellsControlCAC-8 (0.05 mg)BPTH (10nM)UMR-106

(bone) cellsControlCAC-8 (0.1 25 mg)BPTH (10nM)OK

(kidney) cellsControlCAC-8 (0.5 mg)BPTH (10 nM)No

treatment238

Â±20Â°797 Â±56*

3,721 Â±344C295

Â±353,074 Â±160e5,91 8 Â±664e449

Â±51881 Â±93

3,672 Â±215'cpm/wellForskolin(0.1

MM)249

Â±151,349 Â±73tJ/5,774 Â±91'*310

Â±274,063 Â±91"7,947 Â±780"275

Â±271,412 Â±126*'9,957 Â±868^Dexamethasone(30

DM)199

Â±131,690 Â±85'-''

10,595 Â±684''479

Â±492,168 Â±214"6,567 Â±635C497

Â±69712Â± 112

3,130Â±265C

" Mean Â±SE from triplicate wells of a representative experiment.* Different from control, at P < 0.05.e Different from control, at P < 0.01.* Different from no treatment group, at P < 0.01.' Different from no treatment group, at P < 0.05.

production of OK cells with or without forskolin, but the ACstimulation was increased significantly compared to controlsonly in the presence of forskolin. Forskolin, at 0.1 /Â¿M,did notstimulate the AC activity of the cell lines in the absence ofanother agonist. Preincubation of the cell lines for 2 days inmedium with 30 HM dexamethasone significantly increasedcAMP production by ROS cells in response to BPTH andCAC-8 extract. The AC stimulation of UMR and OK cells byBPTH was not potentiated by dexamethasone pretreatment,and cAMP production of UMR cells induced by CAC-8 extractwas significantly reduced compared to control cells. Dexamethasone pretreatment of the cell lines did not alter cAMP production in the absence of an AC agonist.

The PTH receptor competitive antagonist [8-18Nle,"Tyr]-BPTH (3-34) amide significantly reduced AC stimulation ofROS, UMR, and OK cells by BPTH (1-34) and CAC-8 extract(Table 5). The ["-'"Nie, 34Tyr]BPTH (3-34) amide did not stim

ulate AC activity of the 3 cell lines.Heat treatment of CAC-8 extract (Table 6) demonstrated

that the AC-stimulating activity of CAC-8 or BPTH in UMR

Table 5 Effect ofl*-"Nle, "TyrJBPTH (3-34) amide on stimulation of adenylatecyclase in KOS 17/2.8, UMR-106, and OK cell lines by CAC-8 extract

The BPTH receptor antagonist [*-"Nle, ^TyrJBPTH (3-34) amide (1 MM)wasadded to 2-cm2 wells of confluent ROS, UMR, or OK cells alone, in addition toBPTH (1-34) (5 nM) or with CAC-8 extract (specified amount per well). The AC-stimulating assay was performed as described in "Materials and Methods." The

data from each cell line were compared using analysis of variance and NewMultiple Range test.

Cell line(cpm/well)TreatmentControl

3-34 BPTH (1MM)BPTH

(5 HM)BPTH + 3-34 BPTH(1MM)CAC-8

(0.05, 0.2, 0.5 mg)CAC-8 + 3-34 BPTH (I

MM)ROS

17/2.8(bone)238

Â±20*

205 Â±233721

Â±344*441 Â±51'-*797

Â±56*186 Â±35'UMR-106

(bone)222

Â±20385Â±97867

Â±867*4050 Â±282M4522

Â±587*846 Â±54'OK(kidney)449

Â±51190 Â±293672

Â±215*1680 Â±237M881

Â±93'230 Â±16'

* Mean Â±SE of triplicate wells of a representative experiment.* Different from control, at P < 0.01.' Different from control, at P < 0.05.d Different from BPTH, at P < 0.01.' Different from CAC-8, at P < 0.01.

terenol, and CAC-8 extract, the magnitude of AC stimulationby CAC-8 was significantly less than with BPTH or isoproterenol. Bovine PTH, isoproterenol, and CAC-8 extract stimulatedthe AC activity of UMR cells compared to controls, but thecells were significantly less responsive to isoproterenol. The ACactivity of OK cells was stimulated by BPTH and not byisoproterenol. There was a numerical increase in cAMP production by OK cells in response to CAC-8, but when comparedwith BPTH (analysis of variance) it was not significantly different from controls. The UMR cells produced the greatest increase in AC activity compared to controls in response to CAC-8 extract (0.5 mg/ml) (Table 3). All 3 cell lines respond to asimilar degree of BPTH, but there are significant differences inAC stimulation by CAC-8 extract and isoproterenol.

The effects of forskolin and dexamethasone on AC stimulation of ROS, UMR, and OK cells are presented in Table 4. Theconcentration of CAC-8 extract was specified for each cell lineto produce a significant yet submaximal stimulation of ACactivity. Forskolin at 0.1 /Â¿Msignificantly increased the ACstimulation of ROS, UMR, and OK cells by BPTH and CAC-8 extract. CAC-8 extract numerically increased the cAMP

Table 6 Effect of heat, trypsin, and dithiothreitol on the stimulation of adenylatecyclase in UMR-106 (bone) cells by CAC-8 extract and BPTH

Bovine PTH (5 nM) and CAC-8 extract (0.2 mg/ml) were dissolved in serum-free F12 medium and exposed to 60'C for 1 h, 100'C for 3 min, or trypsin-TCPKfor l h at 37'C. The trypsin digestion was stopped by the addition of soybean-

trypsin inhibitor (100 Mg/ml). The trypsin control solution contained both trypsin-TCPK and trypsin inhibitors at the same concentrations as treatment samples.CAC-8 extract was treated with dithiothreitol (0.062 M) for l h at 25'C and thenduly ml extensively against 0.17 M acetic acid. The CAC-8 extract and BPTHsamples were added to 2-cm2 wells of confluent UMR cells. The AC-stimulatingassay was performed as described in "Materials and Methods." The data from

heat and trypsin treatments were compared to controls using analysis of varianceand New Multiple Range test. The dithiothreitol data were compared to CAC-8control using Student's t test.

TreatmentControl

60 C, 1 h100 C, 3 minTrypsin (50Mg/ml)Control

DithiothreitolNo

agent451Â±46Â°538

Â±76cpm/wellCAC-8

(0.2mg)2175

Â±1102144 Â±692771 Â±27*

569 Â±68*2389

Â±3401444 Â±192'BPTH

(5nM)5319

Â±4495694 Â±2034269 Â±439

841 Â±96*

* Mean Â±SE from triplicate wells.* Different from control, at P < 0.01.' Different from CAC-8 control, at P < 0.01.

693




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nla 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

FRACTION NUMBER

Fig. 4. Gel exclusion chromatography of AC-stimulating activity in CAC-8extract in UMR-106 (bone) cells. An acid-ethanol extract of adenocarcinoma(CAC-8) (20 mg, dissolved in 1 ml of 1 N acetic acid) was applied to a column ofFractogel INK HW-50s (2.5 x 80 cm) and eluted with l N acetic acid at a flowrate of 2 ml/h. Fractions (1 ml) were collected, evaluated for absorbance of lightat 280 nm, and assayed for AC-stimulating activity in the presence of forskolin(O.I Â»IM),//.vi. bovine serum albumin (M, 67,000); C, chymotrypsinogen (M,25,000); /, insulin (M, 6,000).

cells was not reduced by 60Â°Cor 100Â°C.The AC-stimulating

activity of CAC-8 extract was significantly increased with treatment at 100'C. Trypsin digestion significantly reduced AC

stimulation of UMR cells by both BPTH and CAC-8 extractwhile the mixture of trypsin-TCPK and soybean-trypsin inhibitor did not significantly affect AC activity. Reduction of CAC-8 extract by dithiothreitol decreased AC stimulation of UMRcells (P < 0.01 ) when compared to control CAC-8 extract (Table

6).Specific fractions of CAC-8 extract obtained by gel exclusion

chromatography demonstrated a single peak of AC-stimulatingactivity using UMR cells. The approximate relative molecularweight was 34,000 (Fig. 4).

DISCUSSION

The tumor line, CAC-8, which had been shown to producethe syndrome of HHM in nude mice as well as in the dogcontained a protein factor which stimulated the AC of boneand kidney cells. The presence of AC-stimulating activity incanine adenocarcinomas correlated well with the developmentof clinical hypercalcemia. An anal sac adenocarcinoma whichwas taken from a dog with hypercalcemia contained AC-stimulating activity, but AC-stimulating activity was not detectablein an adenocarcinoma from a normocalcemic dog.

The cell lines utilized in this study, ROS 17/2.8, UMR-106,and OK cells, responded similarly to BPTH (1-34) but had amore varied response to CAC-8 tumor extract. The ROS cellsresponded to the lowest concentration of CAC-8 extract, butthe UMR cells with equal concentrations of CAC-8 extract hadthe greatest increase in AC activity compared to controls. Inresponse to equal concentrations of CAC-8 extract the cell linesproduced approximately 50% (ROS cells), 80% (UMR cells),and 15% (OK cells) stimulation of AC activity compared toBPTH (1-34). This may indicate that the tumor-related factorwhich stimulated AC was not capable of interacting with different PTH receptors to the same degree as with native PTH. Thisobservation is consistent with the hypothesis that more thanone class of PTH receptors exist (9). The PTH-like factor(s)released by some tumors associated with HHM may interactwith certain PTH receptors to a greater degree than with others.The bone and kidney alterations seen in human patients withhyperparathyroidism that are not present in patients with HHMinclude renal bicarbonate wasting, increased serum 1,25-dihy-

droxycholecalciferol, and increased bone formation (9). However, the syndrome of HHM which develops in nude mice withtransplanted CAC-8 does include increased serum 1,25-dihy-droxycholecalciferol and an increased bone formation rate (2).The reason for this difference compared to human patients maybe due to an increased severity of HHM in the nude mousemodel induced by a large tumor volume to body size withresultant PTH-like effects in tissues which are less responsiveto the tumor products. The cell lines also varied in their ACresponse to the adrenergic agonist, isoproterenol, but in adifferent pattern compared to CAC-8 extract. ROS cells werethe most responsive to isoproterenol, UMR cells were muchless responsive, and OK cells did not respond to isoproterenol.

Although CAC-8 extract did not stimulate the AC complexof the 3 cell lines to the same degree as did BPTH, when thetumor extract was compared to BPTH by other physical andchemical means they resulted in parallel AC stimulation. Pretreatment of ROS cells with BPTH for 2 h prior to exposureto CAC-8 extract or BPTH did result in desensitization of theAC stimulation similar to cultured chick kidney cells whichundergo homologous desensitization to PTH after l h of prein-cubation with PTH (24). AC stimulation in response to CAC-8 extract and BPTH was enhanced in all 3 cell lines by thepresence of forskolin (0.1 /Â¿M).This low concentration of forskolin alone did not stimulate cAMP production. Forskolin isa potent activator of the PTH-responsive adenylate cyclasesystem in a number of cell lines and can directly stimulate ACactivity in cells at concentrations as low as 1.0 //M (25). Thesite of action of forskolin is unknown but may be the nucleosideregulatory unit of the AC catalytic system (25, 26). Preculturewith dexamethasone enhanced AC stimulation by either CAC-8 extract or BPTH only in ROS cells (27). The mechanism ofdexamethasone enhancement of AC in ROS 17/2.8 cells isunknown, but it has been reported that dexamethasone increased the guanine nucleotide regulatory protein of the ACcatalytic system (28). The antagonist of the PTH receptor,[".'"Nie, 34TyrjBpTH (3.34) amide significant^ reduced ACstimulation induced by CAC-8 extract or BPTH in ROS, UMR,and OK cells. The PTH receptor antagonist did not stimulateAC alone. We concluded that CAC-8 contains a protein factorwhich stimulated the AC of bone and kidney cells by bindingto the PTH receptor, since the response to CAC-8 extractparalleled the response to BPTH in AC desensitization, potentiation of AC with forskolin or dexamethasone, and inhibitionof AC by the PTH receptor-antagonist.

Heat treatment of CAC-8 extract and BPTH at 60"C orl(10Â°C did not reduce AC-stimulating activity. The cause of the

small increase in AC-stimulating activity of CAC-8 extract afterheating at 100Â°Cis not known but may represent a direct

enhancement of the AC-stimulating activity or removal of aninhibitory factor by denaturation. Bovine PTH and CAC-8 AC-stimulating activity were deactivated by exposure to trypsinindicating that both substances are proteins that were susceptible to trypsin-induced proteolysis. The AC-stimulating activity of CAC-8 extract was decreased significantly by reductionwith dithiothreitol, suggesting that disulfide bonds are important structural components of the AC-stimulating protein.

Gel chromatography of CAC-8 extract demonstrated peakAC-stimulating activity at a relative molecular weight of 34,000.This is similar in size to AC-stimulating activities present in ahuman renal cell carcinoma, the rat Leydig cell tumor, andconditioned medium from human keratinocytes (29, 30). ThePTH-like activity expressed by human keratinocytes may represent a similar factor that is released by squamous cell carci-

694




nomas associated with hypercalcemia. PTH-like peptides havebeen purified from a human squamous cell carcinoma and therat Leydig tumor (H-500) (31). Peptides which were active inA( '-stimulÃ¢tionassays had molecular weights of 9000 and 9500

by amino acid analysis. In addition, the rat Leydig cell tumorcontained an active peptide of M, 28,000. These findings suggest that the approximate molecular weights of AC-stimulatingactivities from gel exclusion chromatography experiments maybe overestimated due to possible protein glycosylation and thelack of denaturing conditions. Two classes of AC-stimulatingactivity have been isolated from a murine squamous cell carcinoma model of HHM (32). Class I consisted of 3 closely relatedpeaks observed in reverse phase high-performance liquid chromatography fractions that stimulated AC in disrupted andintact cells and were inhibited by [Â»-"Nie,34Tyr]BPTH (3-34)

amide. Class II was a single peak which stimulated AC only indisrupted cellular membranes and was not inhibited by [8J8Nle,34Tyr]BPTH (3-34) amide. It was postulated that Class II AC-

stimulating activity acts by a postreceptor mechanism andpotentiates the activity of the Class I factor. The CAC-8 tumor-related peptide described in our study appears to more closelyresemble Class I activity of the murine squamous cell carcinoma.

In summary the tumor line CAC-8 contains a protein fac-tor(s) which stimulates the AC of bone and kidney cells bybinding to the PTH receptor. The tumor-related activity wasenhanced by forskolin and dexamethasone, was heat and acidstable, was inactivated by reduction, and had a relative molecular weight of approximately 34,000. Future purification andcharacterization of the AC-stimulating activity in CAC-8 willbe useful in understanding the pathogenesis of HHM.

ACKNOWLEDGMENTS

We are grateful to Sevgi B. Rodan, Ph.D., for her helpful instructionin development of the adenylate cyclase stimulating assay.

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1987;47:690-695. Cancer Res Thomas J. Rosol, Charles C. Capen and Charles L. Brooks (CAC-8) Maintained in Nude MiceProduced by a Hypercalcemic Canine Adenocarcinoma Line Bone and Kidney Adenylate Cyclase-stimulating Activity

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