Proc. Nati Acad. Sci. USAVol. 80, pp. 1454-1458, March 1983Medical Sciences

Identification. of adenylate 'cyclase-stimulating activity andcytochemical glucose-6-phosphate dehydrogenase-stimulating activity in extracts of tumors from,patients with humoral hypercalcemia,of malignancy

(pseudohyperparathyroidism/ectopic hyperparathyroidism/calcium)

ANDREW F. STEWART*t, KARL L. INSOGNAt, DAVID GOLTZMANt, AND ARTHUR E. BROADUSt

*Department of Medicine, West Haven Veterans Administration Medical Center, West Haven, Connecticut 06516; tDepartment of Medicine, Yale University Schoolof Medicine, 333 Cedar Street,.New Haven, Connecticut 06511; and tDepartment of Medicine, McGill University and Royal Victoria Hospital,Montreal, Quebec, Canada H3A LAL

Communicated by Alfred Gilman, November 19, 1982

ABSTRACT Humoral hypercalcemia of malignancy (HHM)most commonly results from secretion by tumors of an unidenti-fied circulating calcemic factor that appears in clinical studies tostimulate both a parathyroid hormone (PTH)-sensitive proximaltubular adenylate cyclase and a distinct PTH-sensitive renal tu-bular glucose-6-phosphate dehydrogenase complex. In the presentstudy, 8 M urea extracts of tumors from patients with HHM.havebeen shown to contain both in vitro adenylate cyclase-stimulatingactivity and glucose-6-phosphate dehydrogenase-stimulating ac-tivity as detected in a sensitive cytochemical bioassay. Both theadenylate cyclase-stimulating activity and cytochemical bioactivityare due to specific binding of a substance in the tumor extracts torenal PTH receptors, as demonstrated by competitive inhibitionstudies using the bovine PTH fragment analogue [Nle8"8,Tyr4]bPTH-(3-34) amides Preincubation with an antiserum to PTHresults in no loss of activity in the tumor extract, and the activityappears both on gel filtration and ultrafiltration to be far largerthan PTH (estimated Mr 70,000). These studies demonstrate thatextracts of tumors from patients with HHM contain a substancethat binids to the PTH receptors in the nephron responsible for ac-tivation of both the PTH-sensitive glucose-6-phosphate dehydro-genase and the PTH-sensitive adenylate cyclase. This substance ischromatographically and immunologically distinct from PTH. Itsrole in the genesis of HHM requires further study.

Humoral hypercalcemia of malignancy (HHM) results from thesecretion by tumors remote from bone of a circulating, calcemicfactor (or factors) that stimulates bone resorption (1, 2). The na-ture of the factor or factors responsible for this humorally me-diated hypercalcemia remains elusive. Although vitamin D me-tabolites, prostaglandins of the E series, and parathyroid hormone(PTH) have in the past been suggested as causative agents (1-6), the available evidence suggests that these agents can accountfor at best a minority of instances of HHM (1, 2, 7-9). Thus, anas yet unidentified humoral substance appears to be'responsiblefor the majority of instances of this syndrome (10).

Of the calcemic hormones listed above, the humoral factorresponsible for HHM most closely resembles PTH. Both pa-tients with HHM and those with primary hyperparathyroidism,the prototype of humorally mediated hypercalcemia, display(i) increased osteoclastic activity in bone (11), (ii) increasednephrogenous cAMP excretion (2, 12, 13), and (iii) increasedplasma cytochemical bioactivity in a PTH-sensitive cytochem-

ical bioassay (14). However, there are also a number of distinc-tive points of pathophysiological difference between these twohumoral syndromes, in that, unlike primary hyperparathroid-ism, HHM is associated with: (i) markedly increased fractionalcalcium-excretion (2, 3), (ii) strikingly decreased plasma con-centrations of 1,25-dihydroxyvitamin.D (2), (iii) decreased toabsent osteoblastic activity in bone (11), (iv) low to undetectablecirculating levels of immunoreactive PTH (2, 5-7), and (v) anelution profile ofplasma cytochemical bioactivity which differsfrom that encountered in hyperparathroid plasma (14).

The observations that nephrogenous cAMP and plasma cy-tochemical bioactivity are increased in patients with HHM sug-gest that the humoral substance responsible for HHM binds toand stimulates both the proximal tubular PTH-sensitive ade-nylate cyclase complex and the renal tubular glucose-6-phos-phate dehydrogenase system responsible for activity in the cy-tochemical bioassay. The present study was designed to examinethese possibilities.'

METHODSPatient and Tissue Samples. Tumor tissue was obtained at

autopsy from five patients with HHM. Humoral hypercalcemiawas defined by using a previously described (2, 11) constellationof biochemical findings, including hypercalcemia (mean serumcalcium, 13.7 mg/dl; range, 12.4-15.2), a reduced renal phos-phorus threshold (mean, 1.5 mg/dl; range, 0.7-2.7), a de-creased plasma 1,25-dihydroxyvitamin D concentration (mean,17 pg/ml; range, 9-35), increased nephrogenous cAMP excre-tion (mean, 5.5 nmol/dl of glomerular filtrate; range, 3.11-12.49), and normal or low values for immunoreactive PTH. Threeof the five patients had no evidence of bone metastases; onepatient had a single bone metastasis as determined by bone scan,and the fifth had skeletal involvement of uncertain extent dis-covered at autopsy.

Control tumor tissue was obtained at surgery or autopsy fromnine normocalcemic patients. In this group there were threebreast carcinomas, two ovarian carcinomas, one renal carci-noma, one squamouscarcinoma of the uterine cervix, one coloncarcinoma, and one anaplastic pulmonary carcinoma. A tenthtumor sample was obtained at autopsy from a patient with breastcarcinoma, severe hypercalcemia, and extensive skeletal met-

Abbreviations: HHM, humoral hypercalcemia of malignancy; Nle, nor-leucine; PTH, parathyroid hormone; bPTH, bovine PTH. PTH frag-ments are indicated as, for example, bPTH-(1-34) for the fragment con-sisting of amino acid residues 1-34.

1454

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

27, 2

020

Proc. Natl. Acad. Sci. USA 80 (1983) 1455

astatic disease. In this patient hypercalcemia appeared to be dueto bone metastases rather than humoral mechanisms, and thistumor tissue served as a "nonhumoral hypercalcemic control."A parathyroid adenoma was obtained at surgery from a patientwith primary hyperparathyroidism. This adenoma was ex-tracted in the same fashion (see below) as the tumor samples andserved as a "positive" control. All samples were promptly trans-ported to the laboratory on ice, dissected free of adjacent nor-mal tissue, and frozen at -700C until extraction.

Extraction Procedure. All procedures were performed at 40C.Tissue was homogenized in a Waring blender in 3:1 (ml/g) 0.1M Tris HCI, pH 7.4. The homogenate was centrifuged for 15min at 27,000 X g. The pellet was then extracted in 2:1 (ml/goriginal wt) 8 M urea/0.2 M HCI/0. 1 M cysteine. At the endof 1 hr, the extract was centrifuged at 27,000 X g for 15min. The resultant supernatant was dialyzed against 12 liters ofwater over 24 hr, using Spectrapor-3 dialysis tubing (SpectrumMedical Industries, Los Angeles, CA). This dialysate was eitherassayed directly or was divided into aliquots and lyophilized forfuture use. The crude extracts prepared in this manner are sub-sequently referred to as "humoral hypercalcemia extract,""control extract," or "parathyroid extract." No loss of activitywas observed after lyophilization. The protein concentration inthe crude extracts was determined by the Lowry method (15)and ranged between 3.7 and 39.5 mg/ml.

Adenylate Cyclase Assay. Purified canine renal corticalmembranes were prepared as previously described (16). Thefragment of bovine parathyroid hormone consisting of aminoacid residues 1-34 [bPTH-(1-34) Beckman Bioproducts, PaloAlto, CA] was used as standard. Each sample was assayed intriplicate in tubes containing 100,000 cpm of [a-32P]ATP(Amersham), 50 mM Tris HCl at pH 7.4, 5.0 mM MgCl2, 10mM KC1, 1.0 mM EGTA, bovine serum albumin (Sigma) at 1mg/ml, creatine kinase (Sigma) at 0.11 unit/ml, 5 mM creatinephosphate, 0.125 mM ATP (Sigma), 1.0 mM cAMP (Sigma), 2.0mM dithiothreitol (Sigma), and 10 ,tM 5'-guanylyl imidodi-phosphate (Sigma). Unknown samples and dilutions of PTHstandard were added in 10-,ul volumes, yielding a total reactionvolume of 70 ,ul. The reaction was begun by the addition ofmembrane preparation (60 ,ug of membrane protein per tube),and tubes were incubated for 30 min at 30°C. The reaction wasterminated by the addition of 100 ,u1 of"stopping solution" con-taining 2.0 mM ATP, 0.5 mM cAMP, and 5,000-10,000 cpmof [3H]cAMP as a recovery standard. Reaction tubes were thenboiled for 3 min at 1000C, brought up to 1.0 ml with water, andapplied sequentially to Dowex AG 5OW-X4 (200-400 mesh,hydrogen form, Bio-Rad) and alumina (Sigma) columns (17).Triplicates were usually within 5-15%, and recovery of[3H]cAMP ranged between 70% and 95%. Results are ex-pressed as pmol ofcAMP formed per mg of membrane proteinper 30-min incubation.

Samples containing as little as 100 pg of bPTH-(1-34) per ml(1 pg per tube) can clearly be distinguished from basal activity,and 100 ng of bPTH-(1-34) per ml (1 ng per tube) produces max-imal stimulation. Dose-response curves for parathyroid extractand bPTH-(1-34) are superimposable, with 1,000 ,ug of proteinper ml of extract yielding maximal stimulation and 10 ,ug of pro-tein per ml of extract yielding approximately half-maximal ac-tivity.

Corticotropin (ACTH; porcine, grade II) and bovine calci-tonin were obtained from Sigma. Arginine vasopressin was ob-tained from Parke-Davis (Morris Plains, NJ). Prostaglandin E2was obtained from Upjohn.

Cytochemical Bioassay. The cytochemical bioassay was per-formed as described (14, 18). Guinea pig renal cortical segmentswere cultured with or without graded doses of hormone stan-

dard (50 fg/ml to 50 pg/ml) or with dilutions of tumor extract.Hormone standard was human PTH (National Institute of Bi-ological Standard and Control, Holly Hill, England), Code79/500, containing 0.1 international unit (or estimated 100 ng)of human PTH-(1-84) per ampoule. Lyophilized tumor extractswere resuspended in 0.01 M acetic acid containing bovine serumalbumin at 1 mg/ml and were then further diluted 1:100 and1:1,000 in Trowell's T8 medium (GIBCO) containing 1% (vol/vol)hypoparathyroid plasma. All samples were bioassayed in culturemedium containing a final concentration of 1% (vol/vol) of hy-poparathyroid plasma. Results are expressed as glucose-6-phos-phate dehydrogenase activity (relative absorbance) or as pgequivalents of human PTH standard.

Competitive Inhibition and Antiserum Preincubation Stud-ies. [Nle8'18,Tyrm]bPTH-(3-34) amide (Nle, norleucine) waspurchased from Bachem Fine Chemicals (Torrance, CA). Thisinhibitor was added to appropriate samples immediately priorto assay in concentrations given in Figs. 2 and 3 and in the text.

Antiserum preincubation studies were performed with anti-serum G-5, a goat anti-human PTH antiserum that reacts withboth amino- and carboxyl-terminal portions of the PTH se-quence (19). This antiserum was generously supplied by L. Mal-lette (Houston, TX). Samples were incubated at 4°C with an ap-propriate amount of antiserum for 48 hr prior to assay. Incubationof parathyroid extract and humoral hypercalcemia extract in theabsence of antiserum resulted in no loss of activity.

Gel Filtration and Ultrafiltration Studies. Gel filtration wasperformed using a 0.9 x 50 cm column of Sephadex G-100(Pharmacia) in 0.1 M ammonium acetate, pH 5.0. Fractions (2ml) were collected, dialyzed against 4 liters of water for 24 hrin Spectrapor-3 dialysis tubing, lyophilized, and reconstitutedin 0.01 M acetic acid at 1/10th of their prelyophilization vol-ume. The column was calibrated by using ferritin (Mr, 440,000;Sigma), tetracycline (Mr, 250; Pfizer), bovine serum albumin(Mr, 67,000, Sigma), and l25I-labeled bPTH-(1-84). Ultrafiltra-tion was performed by using an Amicon stirred cell. Lyophilizedtumor extract was suspended in 0.01 M acetic acid and filteredsequentially through an XM-100A membrane (nominal Mr cut-off, 100,000), an XM-50 membrane (nominal Mr cutoff, 50,000),and a UM02 membrane (nominal Mr cutoff, 500). The retentatefrom each membrane was assayed, or saved and lyophilized forfuture assay.

RESULTSAdenylate Cyclase Assay. The results of adenylate cyclase

assay of the 10 control extracts and the 5 tumor extracts frompatients with humoral hypercalcemia are shown in Fig. 1. Sevenof the 10 control extracts displayed nondetectable activity whereas3 (1 renal, 1 squamous carcinoma of the cervix, 1 anaplastic pul-monary carcinoma) contained small amounts of adenylate cy-clase-stimulating activity that displayed a poor dose-responserelationship. In contrast, four of the five humoral hypercal-cemia extracts contained adenylate cyclase activity, with threeof these tumors displaying striking dose-related adenylate cy-clase stimulation. The activity in the humoral hypercalcemia ex-tracts from patients 1 and 2 produced approximately half-max-imal stimulation in the assay, equivalent to parathyroid extractat 10 ,ug/ml.

Cytochemical Bioassay. The four humoral hypercalcemia ex-tracts that contained adenylate cyclase-stimulating activity (pa-tients 1-4, Fig. 1) and five control extracts were examined in thecytochemical bioassay (Table 1). Potent bioactivity was presentin all four humoral hypercalcemia extracts, ranging between 164pg equivalents/ml and 900 ng equivalents/ml (4.8-60.8 pgequivalents/mg of protein). These values are similar quanti-tatively to those obtained by using the adenylate cyclase assay

Medical Sciences: Stewart et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

27, 2

020

1456 Medical Sciences: Stewart et al.

600

0.,.I

o500

0

Q 400d to

¢t

U"4 300

Control

15 25 50 100 200 400 15 25 50 100 200 400Protein, gg per tube

(Fig. 1 legend). Dilutions of humoral hypercalcemia extractelicited enzyme stimulation parallel to that generated by gradeddoses of PTH. In contrast to the humoral hypercalcemia ex-tracts, activity was undetectable (less than 50 fg equivalents/ml)in all five control extracts.

Specificity Studies. Vasopressin (0.1-10 PM), corticotropin(0.01 and 0.1 A.M), calcitonin (0.1 and 1 AM), and prostaglandinE2 (0.05-5 AM) displayed no activity in the adenylate cyclaseassay. To further assess the specificity of the response to theparathyroid extract and the humoral hypercalcemia extract, thesynthetic analogue and inhibitor of PTH [Nle8 18,Tyrm]bPTH-(3-34) amide (20) was employed. Fig. 2A shows the peptide'sdose-related inhibition of the adenylate cyclase assay in re-sponse to 10 Atg of protein per ml of parathyroid extract, a con-centration that closely approximates the activity in the humoralhypercalcemia extracts from patients 1 and 2. The adenylate cy-clase-stimulating activity in the humoral hypercalcemia extractfrom patient 1 (Fig. 2B) was also abolished by increasing con-centrations of the inhibitor, with activity being totally inhibitedby 250 ,uM inhibitor. Similarly, the activity in the extracts frompatients 2 and 4 was completely inhibited by 250 AM inhibitor.

As with the adenylate cyclase studies, increasing concentra-tions of inhibitor progressively diminished the cytochemicalbioactivity produced by both human PTH-(1-84) and humoralhypercalcemia extract from patient 1, with 1 ,M inhibitor pro-ducing near-maximal inhibition (Fig. 3).

Table 1. Bioactivity of humoral hypercalcemia extracts in thecytochemical bioassay

Bioactivitypg equivalents/ pg equivalents/

Patient ml mg protein1 900 60.82 440 31.43 171 15.24 164 4.8Controls (n = 5)* <0.05 <0.070

*Control extracts assayed included those from four normocalcemicpatients (two breast carcinomas, one ovarian carcinoma, one coloncarcinoma) and from the patient with breast carcinoma and hyper-calcemia.

FIG. 1. Adenylate cyclase-stimulat-ing activity in the tumor extracts. Thenumbers next to the curves of the hu-moral hypercalcemia extracts refer to thepatients with humoral hypercalcemia.Patient 1 had a squamous carcinoma ofthe skin, patient 2 a squamous carcinomaof unknown primary site, patient 3 a renalcarcinoma, patient 4 a squamous carci-noma of the bladder, and patient 5 an oatcell carcinoma of the pancreas. The ex-tracts from patients 1-4 contained 18.9,30.0, 8.0, and 3.5 pg equivalents of bPTH-(1-34) per mg of extract protein, respec-tively. The extract of the hypercalcemiapatient with breast carcinoma and wide-spread skeletal metastases is indicated bytriangles among the controls.

Fig. 4 illustrates the effects of parathyroid antiserum prein-cubation on the adenylate cyclase-stimulating activity in theparathyroid extract and in the humoral hypercalcemia extractfrom patient 1. Extract from patient 1 was selected for this andsubsequent experiments, because large amounts of tumor wereavailable for extraction, in contrast to the limited amounts oftumor available from patients 2-4. Fig. 4A displays the pro-gressive disappearance of adenylate cyclase-stimulating activityobserved after incubation of parathyroid extract with increasingamounts of antiserum. Activity was completely extinguished byusing an antiserum dilution of 1:160. In contrast, preincubationof humoral hypercalcemia extract with an antibody dilution of1:160 resulted in no loss in adenylate cyclase-stimulating activ-ity (Fig. 4B).

Stability and Physical Characteristics. In an attempt to de-fine the stability of the adenylate cyclase-stimulating activityfrom patient 1, the extract was: (i) frozen at -20°C and thawed,(ii) snap-frozen in acetone/dry ice and thawed, (iii) lyophilized,and (iv) boiled for 3, 5, 10, or 15 min at 100°C. None of these

A. Parathyroid extract

100-~ Extract alone.,-

C)

cu Q

"0 L

.a-.

.1-4*o >-

75

50

25

| Parathyroid extract

"/inhibitor

-

10-8 10-7 10-6 10-5 10-4

B. Humoral hypercalcemiaextract

100

75

50

25

k Extract alone

Tumor extract~~X ~+

innhibitor

10-8 10-7 10-6 10-5 10-4

Inhibitor concentration, M x 0.4

FIG. 2. Competitive inhibition of adenylate cyclase stimulation with[Nle,8"18,Tyr34]bPTH-(3-34) amide. Activity is expressed asthe percentof activity of the parathyroid extract or humoral hypercalcemia ex-tract in the absence of inhibitor. Bars indicate the range of triplicatesat each concentration. Progressively increasing concentrations of in-hibitor extinguished the activity both in the parathyroid extract (10,g/ml) (A) and in the humoral hypercalcemia extract (B). SyntheticbPTH-(1-34), at 1.0 ng/ml, which produced half-maximal stimulationin the assay, was extinguished by 2.5 x 10-5 M inhibitor (not shown).

Proc. Nad Acad. Sci. USA 80 (1983)

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

27, 2

020

Proc. Natl. Acad. Sci. USA 80 (1983) 1457

00 10-8 10-7 10-6

Inhibitor concentration, M

FIG. 3. Influence of the inhibitor [Nle8 '8,Tyr`]bPTH-(3-34) amideon the cytochemical glucose-6-phosphate dehydrogenase (G6PD) pro-duced by human PTH at 5 fg/ml (o) and on the activity of humoral hy-percalcemia extractfrom patient 1, 3 pg equivalents/ml (A). Each pointrepresents the mean ± SEM of 10 measurements. The broken line rep-resents the detection limit in the assay.

maneuvers resulted in loss of activity.This extract has been chromatographed on three occasions.

On each occasion, the adenylate cyclase-stimulating activity ap-peared in the column fractions migrating with albumin, well be-fore either the 1"I-labeled bPTH-(1-84) marker or the adenyl-ate cyclase-stimulating activity in the parathyroid extract. Re-coveries from the Sephadex column were extremely low. Theactivity in the humoral hypercalcemia extract from patient 1partitioned equally across an XM-100A membrane, but no ac-tivity was detected in the filtrate of an XM-50 membrane. Incontrast, the adenylate cyclase activity in the parathyroid ex-tract passed readily through the XM-50 membrane. Under theconditions of study, both of these observations are compatiblewith an initial estimate of molecular weight of between 50,000and 100,000.

DISCUSSIONMalignancy-associated hypercalcemia is a common clinical syn-drome. In some instances hypercalcemia occurs as the result ofskeletal metastases, but in many patients, typically those withsquamous carcinomas, renal carcinomas, and urothelial carci-nomas, hypercalcemia results from tumor elaboration of a sys-temic, humoral factor (or factors) that stimulates bone resorp-

A

500

250 k

Parathyroid extract

Parathyroid extractIBlvd + anti-PTH.

Basal

1:1,280 1:640 1:320 1:160

Parathyroid antiserum dilution

100 W

50

tion (1-3, 7, 21). This humoral factor (or factors) has been thesubject of intense interest since Albright postulated its exis-tence in 1948 (22), yet its nature remains undefined (1, 7-10).Recent clinical studies have identified three characteristics ofthis substance that might provide the basis for the developmentof in vitro detection systems: (i) the material stimulates osteo-clasts (11), (ii) the material is active in a cytochemical bioassayfor PTH-like activity (14), and (iii) the material stimulates thePTH-sensitive adenylate cyclase in the proximal nephron (2, 12,13). With these characteristics in mind, we have sought to fur-ther define the factor responsible for HHM by using extracts oftumors obtained from well-characterized patients with this syn-drome.

In the present report, we describe the occurrence of adenyl-ate cyclase-stimulating activity in extracts of 4 of 5 tumorsfrom patients with HHM. Activity was undetectable in 7 andpresent in small amounts in 3 of 10 control tumors. It was ofinterest that these 3 tumors included a renal carcinoma, a pul-monary carcinoma, and a squamous carcinoma of the cervix; tu-mors which are commonly associated with the humoral syn-drome (1, 2, 21). Adenylate cyclase-stimulating activity was notdetectable in the extract of a tumor from a hypercalcemic pa-tient with breast cancer and widespread skeletal metastases, asituation in which humorally mediated hypercalcemia would notbe expected to occur. That the adenylate cyclase-stimulating ac-tivity in the humoral hypercalcemia extracts represents a spe-cific interaction with the proximal tubular PTH receptor is sup-ported (i) by the finding that hormones other than PTH, bothin our hands and others' (16), do not stimulate the activity and(ii) by the finding that the adenylate cyclase-stimulating activityencountered in tumor extracts could be competitively inhibitedby the specific PTH inhibitor [Nle8'18,Tyrm]bPTH-(3-34) amide.

All four humoral hypercalcemia extracts that contained ade-nylate cyclase-stimulating activity also displayed marked stim-ulation in the cytochemical bioassay, and the stimulation of thisactivity was quantitatively similar to that of the activity en-countered in the adenylate cyclase assay. None of the five con-trol extracts assayed, including that from the patient with breastcancer and hypercalcemia, contained cytochemical bioactivity.[Nle8"18,Tyrm]bPTH-(3-34) amide competitively inhibited thecytochemical bioactivity associated with both bPTH-(1-84) andhumoral hypercalcemia extract. Existing evidence indicates thatthe major site of action of this PTH analogue in inhibiting theaction of PTH agonists in the kidney is at the level of the PTHreceptor (23). The findings in the present study therefore stronglysuggest that the cytochemical bioactivity in the humoral hy-

B

PTH ]

FIG. 4. (A) Adenylate cyclase ac-tivity after incubation of parathyroidextract (10 pg/ml) with G-5, an anti-serum to parathyroid hormone, at in-creasing concentrations. (B) Adenyl-ate cyclase activity after incubation ofparathyroid extract (10 jLg/ml) andpatient 1 HHM extract alone or with

PTH HHM HHM G-5 antiserum (AS) at a dilution of- + 1:160. Bars indicate the range of tri-AS AS plicates for each experiment.

110

"- 1000U,~Qw 90

4> 80

> 70._,coM 60Co0c

0

cc 0

C.)

*-4

CQ)

00

0..¢

co

Medical Sciences: Stewart et al.

:t.lz.Q

C. CZ

.E WEn

U Cdi. -,u0 >-.

t.- uC a)

4-.

-E.:r.C.)-0Cd

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

27, 2

020

1458 Medical Sciences: Stewart et al.

percalcemia extracts, like the adenylate cyclase-stimulating ac-tivity in the same extracts, is due to a specific interaction witha renal tubular PTH receptor.

Although these data suggest that the humoral material inquestion interacts with PTH receptors, the material does notappear in other ways to resemble native parathyroid hormone.The most telling observations in this regard were (i) the anti-serum preincubation studies, which failed to influence theadenylate cyclase-stimulating activity in the humoral hypercal-cemia extract, and (ii) the gel filtration and ultrafiltration stud-ies, which yielded an estimate of molecular weight far in excessof that of PTH or its precursors. These molecular weight es-timates were obtained under low ionic strength conditions andshould be considered preliminary. Nevertheless, the findingsresemble the results of gel filtration studies on plasma from pa-tients with HHM, performed by Goltzman et al using the cy-tochemical bioassay (14).

Hirshorn et al (24) described the presence of adenylate cy-clase-stimulating activity in an extract of a breast carcinoma froma patient who appeared to have humorally mediated hypercal-cemia. This extract also appeared to have osteoclast-stimulatingactivity in a fetal bone resorption assay. The adenylate cyclase-stimulating activity migrated on gel filtration near the positionof PTH-(1-84). We have not been able to identify adenylate cy-clase-stimulating activity by using the extraction procedure de-scribed by Hirshorn et aL and cannot account for the differencesin the material described by these authors or our own. Recently,Minkin et al. (25) have demonstrated the presence of bone-re-sorbing activity in tumor homogenates from patients with HHM.Although these authors did not attempt to characterize this bone-resorbing activity, it seems likely that the activity described byMinkin et aL is distinct from that described in the present studybecause similarly prepared aqueous extracts of our tumors con-tained no adenylate cyclase-stimulating activity.The major unanswered question in the present study regards

the relationship between the observed adenylate cyclase-stim-ulating activity and cytochemical bioactivity in the tumor ex-tracts and the role of this activity in the genesis of hypercal-cemia. Because the amount of activity identified in tumor extractsby using the adenylate cyclase assay and cytochemical bioassaysis far below the amount required for detection in standard fetalbone resorption systems or in vivo hypercalcemia bioassays, anexperimental approach to this question must await the devel-opment of methods to purify and concentrate the activity.

In summary, we have identified in extracts of tumors frompatients with HHM a material that resembles PTH in its abilityto stimulate activity in both a canine renal adenylate cyclase as-say and a sensitive cytochemical bioassay. This material specif-ically interacts with PTH receptors in the renal tubule but dif-fers from parathyroid hormone in that (i) the activity cannot beblocked by using an antiserum to PTH, and (ii) the material hasan apparent molecular weight far larger than that of PTH. A re-lationship of this material to HHM seems probable, but thedemonstration of a direct causal role will require further in-vestigation.

The authors thank Dr. Howard Rasmussen for his constant supportduring these studies. We thank Drs. Victoria Altmeyer and Daniel Ben-ninghoff at Greenwich Hospital (Greenwich, CT) for providing us withtissue from patients 2 and 4. We thank members of the Pathology De-partments at the Yale-New Haven Hospital and the West Haven Vet-erans Administration Medical Center for providing the remaining tu-mor samples. We thank Ms. Robin Jones and Dr. Mary Ann Mitnick forsuperb technical assistance and Ms. Mary Murray and Ms. DeborahBeauvais for outstanding manuscript preparation. This work was sup-ported by the Veterans Administration (West Haven, CT), National In-stitutes of Health Grants RR 125 and AM 30102, the General ClinicalResearch Center of the Yale-New Haven Hospital, and by grants toD.G. from the Fonds de la Recherche en Sante du Quebec, the MedicalResearch Council, and the National Cancer Institute of Canada.

1. Rodman, J. S. & Sherwood, L. M. (1978) in Metabolic Bone Dis-ease, eds. Avioli, L. V. & Krane, S. M. (Academic, New York),Vol. 2, pp. 555-631.

2. Stewart, A. F., Horst, R., Deftos, L. J., Cadman, E. C., Lang,R. & Broadus, A. E. (1980) N. Engl. J. Med. 303, 1377-1383.

3. Seyberth, H. W., Segre, G. V., Morgan, J. L., Sweetman, B. J.,Potts, J. T. & Oates, J. A. (1975) N. Engl. J. Med. 293, 1278-1283.

4. Robertson, R. P., Baylink, D. J., Metz, S. A. & Cummings, K.B. (1976)J. Clin. Endocrinol. Metab. 43, 1330-1335.

5. Benson, R. C., Riggs, B. L., Pickard, B. M. & Arnaud, C. D. (1974)Am. J. Med. 56, 821-826.

6. Benson, R. C., Riggs, B. L., Pickard, B. M. & Arnaud, C. D. (1974)J. Clin. Invest. 54, 175-181.

7. Powell, D., Singer, F. R., Murray, T. M., Minkin, C. & Potts, J.T. (1973) N. Engl J. Med. 289, 176-181.

8. Brenner, D. E., Harvey, H. A., Lipton, A. & Demers, L. A. (1982)Cancer 49, 556-561.

9. Metz, S. A., McRae, J. R. & Robertson, R. P. (1981) Metabolism30, 299-316.

10. Sherwood, L. M. (1980) N. Engl. J. Med. 303, 1412-1413.11. Stewart, A. F., Vignery, A., Silverglate, A., Ravin, N. D., Li-

Volsi, V., Broadus, A. E. & Baron, R. (1982)J. Clin. Endocrinol.Metab. 55, 219-227.

12. Rude, R. K., Sharp, C. F., Fredericks, R. S., Oldham, S. B., El-baum, N., Link, J., Irwin, L. & Singer, F. R. (1981)J. Clin. En-docrinol Metab. 52, 765-771.

13. Kukreja, S. C., Shemerdiak, W. P., Lad, T. E. & Johnson, P. A.(1980)J. Clin. Endocrinol Metab. 51, 167-169.

14. Goltzman, D., Stewart, A. F. & Broadus, A. E. (1981)J. Clin. En-docrinol. Metab. 53, 899-904.

15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.(1951)J. Biol. Chem. 193, 265-275.

16. Nissenson, R. A., Abbott, S. R., Teitelbaum, A. P., Clark, 0. H.& Arnaud, C. D. (1981)J. Clin. Endocrinol. Metab. 52, 840-846.

17. Solomon, Y., Londos, C. & Rodbell, M. (1974) Anal Biochem. 58,541-548.

18. Goltzman, D., Henderson, B. & Loveridge, N. (1980)J. Clin. In-vest. 65, 1309-1317.

19. Mallette, L. E. & Bradley, W A. (1981)J. Lab. Clin. Med. 98, 886-895.

20. Rosenblatt, M., Callahan, E. N., Mahaffey, J. E., Pont, A. & Potts,J. T. (1977)J. Biol. Chem. 252, 5847-5851.

21. Lafferty, F. W. (1966) Medicine (Baltimore) 45, 247-260.22. Albright, F. (1941) N. Engl J. Med. 225, 789-791.23. Goltzman, D., Peytremann, A., Callaghan, E., Tregear, G. W.

& Potts, J. T. (1975)J. Biol. Chem. 250, 3199-3203.24. Hirshorn, J. E., Vrhovsek, E. & Posen, S. (1979)J. Clin. Endo-

crinol Metab. 48, 217-221.25. Minkin, C., Fredericks, R. S., Pokress, S., Rude, R. K., Sharp,

C. F., Tong, M. & Singer, F. R. (1981)J. Clin. Endocrinol. Metab.53, 941-947.

Proc. Nad Acad. Sci. USA 80 (1983)

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

27, 2

020