Vol. 4, 9 1 1 -920, November 1993 Cell Growth & Differentiation 911

Biochemical Characterization of Peptide a-AmidationEnzyme Activities of Human NeuroendocrineLung Cancer Cell Lines’

Anthony M. Treston, Frank M. Scott, Michele Vos,Naomichi lwai, Richard E. Mains, Betty A. Eipper,Frank Cuttitta, and James I.. Mulshine2

Intervention Section, Biomarkers and Prevention Research Branch,National Cancer Institute Division of Cancer Prevention and Control,

Rockville, Maryland 20850 IA. M. I., F. M. S., M. V., N. I., F. C.,

I. L. Ml, and Department of Neuroscience, Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland 21205 ER. E. M., B. A. E.l

AbstractPeptide a-amidation is a posttranslational modificationof approximately half of all endocrine andneuroendocrine peptide hormones, including severalhormones with mitogenic effeds for tumor cells, and istypically essential for complete hormonal bioadivity.a-Amidated peptide hormones have been reported to beautocrine growth fadors for small cell lung cancercells. We report here that a variety of human lungtumor cell lines express both enzymes required for thetwo-step conversion of inadive glycine-extendedpeptides into their adive COOH-terminal a-amideanalogues. Human tumor cell peptidylglycinea-amidation enzymes are present in multiple molecularforms. Both proteins are metalloenzymes which arepresent at highest concentrations in secretory granulesin neuroendocrine cell lines. The expression of theseenzymes is positively correlated with expression of othermarkers of the neuroendocrine phenotype, such asDOPA decarboxylase. Peptidylglycine a-amidatingenzyme-specific adivities are approximately 50-foldhigher in extracts of endocrine cell lines (lung small celland carcinoid) than of nonendocrine lines. Biochemicalcharaderization of the peptidylglycine a-amidatingenzymes will enable development of tools for detedionof endocrine processes in the early stages of neoplasiaand for interruption of autocrine stimulation pathwaysin tumor cells.

Introduction

Peptide hormones are synthesized as inactive prohormonesand require posttranslational processing to produce theactive form of the hormone. The most common posttrans-lational modification of peptide hormones is COOH-

Received 7/14/93; revised 8/25/93; accepted 8/31/93.

1 The opinions and assertions contained herein are the views of the authorsand are not to be construed as official or as reflecting the views of the De-partment of Health and Human Services. Portions of this work are funded by

a generous grant from the G. Harold and Leila Y. Mathers Charitable Trust andby grants from the National Institute on Drug Abuse (DA-00266, DA-00097,

and DA-00098).2 To whom requests for reprints should be addressed, at Intervention Section,Biomarkers and Prevention Research Branch, National Cancer Institute Di-vision of Cancer Prevention and Control, Suite 300, 9610 Medical CenterDrive, Rockville, MD 20850.

terminal a-amidation, which involves conversion of aglycine-extended prohormone into a peptide with an a-car-boxyamido amino acid at the COOH terminus (Fig. 1 a) (1).This modification is found in approximately 50% of knownneuroendocrine and gastrointestinal peptide hormones, in-cluding several tumor cell growth factors and autocrinegrowth factors (2).GRP,3 an a-amidated peptide hormone, isan example of an autocrine factor for small cell lung cancercells (3). The cardinal requirements ofthe autocnine growthhypothesis (growth factor synthesis, secretion, binding toreceptors, and growth response in a homogeneous cellpopulation) have been demonstrated for a large number ofpeptide hormones (2, 4). Autocrine growth-stimulatoryloops have been proposed to play a role both in preneo-plastic cell hyperplasia (5) and in nonregulated cancer cellproliferation (6).

However, some specific aspects of autocrine tumor biol-ogy have not previously been tested, particularly the pres-ence in tumor cells ofenzymes needed for production of theposttranslationally processed, bioactive form of proposedgrowth-stimulating factor(s). Tumor cells purportedly pro-ducing an a-amidated autocnine growth factor-for ex-ample, GRP-must express enzymatic machinery capable ofCOOH-terminal a-amidation of the glycmne-extended pro-hormone. In all species studied thus far, only one activitywhich catalyzes the conversion of a glycmne-extended pep-tide to an a-amidated peptide has been identified (1).

Peptidylglycine a-amidation is carried out in two sequen-tial enzymatic steps (Fig. 1a). The first reaction is the mo-nooxidation of the a-pro-S hydrogen of a glycine-extendedpeptide to its a-hydroxyglycine analogue (7-1 1). At acidicpH in vitro, and therefore presumably in the moderately

acidic environment of a mature secretory granule, a secondenzyme is required to catalyze lysis of this intermediateand formation of the bioactive a-carboxyamido-peptideproduct. A variety of names have been proposed for theseenzymes (8-1 0, 1 2, 1 3), but in this article, these enzymeswill be referred to as PHM and PAL (F#{232}ptidylgly-cine a-Hydroxylating Monooxygenase and F�ptidyI-a-hydroxyglycine a-Amidating Lyase), respectively (1 3). Theoriginal name given to the monooxygenase, PAM (F�pti-dylglycine a-Amidating Monooxygenase), is reserved for theoverall two-step amidation reaction and for the bifunctionalgene product described by Eipper etal. (1 4). In several mam-malian species, both PHM and PAL are encoded by the PAMgene, are coexpressed on a single mRNA, and are initiallypart ofa single proenzyme (1 3, 1 5). Alternative splicing gen-erates at least seven forms of PAM mRNA in the rat (1 6). Thestructures of the proenzymes encoded by the two charac-

3 The abbreviations used are: GRP, gastrin-releasing peptide; cDNA, comple-

mentary DNA; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic

acid; acYFG, N-acetyl-Iyr-Phe-Gly; acYFamide, a-N-acetyl-Iyr-Phe-amide;acYF(aHO)G, N-acetyl-Iyr-Phe-a-hydroxygh ne.

a

Glycine-extendedSubstrate

molecular oxygen

ascotbate

copper (Cu2’)

b

hPAM-A

PHM domain

rPAL domain

A

r�4 I Ill

Signal

hPAM-B1/�’ TMD Cyto

�A I III

‘5

amiio acidsI I I I I I I I I

0 200 400 600 800

I bPHM.B I

I bPHM-A 1

I bPAL I

912 Peptide a-Amidating Enzymes in Lung Cancer Cells

H 0 H

I II IPeptide-C-C-NH-C-COcy

A H

PHM ==monooxygenase

H,

H 0 0

I II IPeptide - C - C- NH - C- C00

A , H a-hydroxylatedintermediate

H0 or PAL Peptidyl-a-hydroxygtycinea.amidating lyase _____________________

H 0 0

I II IIPeptide-C-C-_NH2 HC-C00

A Peptide Glyoxalatea-amide

Fig. 1. a, reactions catalyzed by peptidylg)ycine a-amidation enzyme activities. a-Amidated peptides are formed from glycine-extended precursors by a two-stepreaction involving sequential monooxidation by PHM and lysis by PAL. b, structure of human PAM proenzymes. Approximate sizes and positions of PHM andPAL enzyme domains within the common PAM precursor. The prohormones depicted are those designated human PAM-A and human PAM-B by Glauder et a!.(1 7) and correspond to rat PAM-i and -2 described by Stoffers et a!. (46). The identities proposed for the PHM and PAL enzymes purified from bovine neuro-intermediate pituitary tissues are shown: bPHM-A, bPHM-B, and bPAL could all arise via endoproteolytic cleavage at pairs ofbasic amino acids (1 3, 14). Human

PAM-B lacks the 1 07-amino acid region shown lightly shaded in PAM-A, which includes the paired basic site Lys432-Lys433 (A) cleaved to separate bovine PAL

and PHM-A enzymes: an alternative potential cleavage site exists in human PAM-A and PAM-B at Lys378-Arg379 (Lx). Signal, signal sequence; TMD, transmembranedomain; Cyto, cytoplasmic domain; V, potential N-g)ycosylation site; I , paired basic amino acid site.

tenized human PAM cDNAs are shown in Fig. 1 b (1 7). BothcDNAs encode PHM and PAL enzymes, but human tumorcell PAL has not been characterized. A report of peptidyl-glycine a-amidation enzyme activity in the plasma of pa-tients with endocrine tumors involved a method now knownto assay only PHM activity (18).

Autocnine activity in cancer cells may be responsible forthe rapid proliferation of these cells and their consequenthighly malignant phenotype. The identification of autocninegrowth stimulation in tumor cells has already provided av-enues for novel antitumor therapies (3, 19). Small cell lungcancer and other well-differentiated lung tumor cell lines,particularly the subset classified as “classic” small celllung cancer, are defined by their frequent expression ofneuroendocnine markers (20), including GRP (21), and sowere expected to contain peptidylglycine a-amidation en-zymes. We therefore chose to investigate the levels ofthese enzymes in neuroendocnine cell lines and to charac-tenize the enzymes biochemically. Characterization of thehuman tumor cell peptidylglycine a-amidation enzymeswill enable rational design of tools for detection of theseactivities and for potential therapeutic interruption of thefunction of these enzymes in the biosynthesis of tumorautocnine growth factors.

Results

Previous studies on human peptidylglycine a-amidation en-zymes preceded elucidation of the two-step nature of theamidation reaction (1 7, 1 8, 22). The cDNAs encoding hu-

man PAM closely resemble those encoding rat and bovinePAM; therefore, human tumor PHM was expected to cata-lyze the conversion of a COOH-terminal glycine-extended

peptide into its a-hydroxylated analogue. The PAL enzymewould complete the transformation into an a-amidated pep-tide at acid pH. Also, human tumor cell PHM should be a

Cu2�- and ascorbate-dependent monooxygenase. BothPHM and PAL should be colocalized to secretory granules.We have identified and characterized PAM, PHM, and PALactivities in human tumor cell lines using variations of aradiochemical assay based on an N-acetylated tnipeptidesubstrate (23). Reverse-phase high-performance liquid chro-

matography and thermospray mass spectrometry were usedto confirm the identity ofthe product ofthe PAM reaction asauthentic a-carboxyamidated peptide (data not shown). The

biochemistry of these enzymes, in particular the specificmetal ion dependence and substrate selectivities, has en-abled us to identify several in vitro enzyme inhibitors whichmay be important as potential in vivo therapeutic and/orintervention agents.

We first confirmed that the sequential enzymatic activityofPHM followed by PAL enzyme is essential for a-amidationof the peptidylglycine substrate. Conversion of the substrateto amidated product was seen only in samples in which thePHM reaction was carried out before the PAL reaction. Thereaction conditions for the a-amidation assays can be ma-n ipulated to enable measu rement ofeach enzyme separatelyin complex mixtures. PHM at pH 5.5 converts the pepti-dylglycine substrate into an intermediate which isnot meas-

2300

300

200

a:

>.-

�(00

Ici.

b:

100

1000

800�

600’s

400

200’-

Fig. 2. Metal ion requirements of PHM and PAL enzymes. ,s, PHM assayswere carried out on an extract of NCI-H720 cells as described in “MatErials

and Methods,” except that 5 psi diethyldithiocarbamic acid was added to

chelate endogenous copper, and 1 5 � of eah metal ion shown was added.

PHM activity in the absence of added metal ions was not significantly differentfrom the blank (no enzyme) control. All activities except that in the presence

of adder) Cu2 � were not significantly different from the blank control. Similarresults were obtained with 50 psi ofeach metal ion. h, PAL assays were carrier)out on an extract of NCI-H720 cells as describer) in “Materials and Methods,”

except that 1 00 is, EDTA was added to complex endogenous metal ions, and

1 10 psi of each metal ion shown was added. Bars, SD.

Cell Growth & Differentiation 913

unable under the conditions of the ethyl acetate extraction,but which can be measured after base treatment of the ne-action mix, as previously described for an ct-hydnoxyglycinepeptide (8, 9). The dependence of this base-catalyzed re-action rate on pH demonstrates that it is a nonenzymaticprocess. Adjustment of pH to approximately 1 3 therefore

enables determination of PHM activity independent of thepresence of PAL. For analysis of PAL activity, preformed

a-hydroxyglycine peptide is enzymatically converted toa-amidated product by PAL at acid pH (pH 5.5) wherehydroxide-catalyzed conversion rates are negligible. ThesepH manipulations were used for separate analyses of PHMand PAL activities in cell extracts for all results shown below.

Human PHM and PAL Are Metalloenzymes. HumanPHM from all cell lines tested shows dependence on andspecificity for Cu2 � ions (results shown are for NCI-H 720extract; Fig. 2a). Each of oven 30 small cell and non-smalllung cancer cell lines tested to date requires Cu2� for maxi-nial PHM activity in the cell extract. This dependence on

exogenous Cu2 � for maximal activity under in vitro assayconditions is seen with both crude cell extracts and partiallypurified PHM preparations. Concentrated secretory granuleextracts have measurable but not maximal PHM activitywithout added Cu2 � , but more dilute samples, whole cellextracts, and partially purified PHM enzyme need exog-enous Cu2r for measurable activity. PHM is selective forCu� � in that only Cu2 can restore the activity of PHM in-hibited by metal ion chelation (Fig. 2a). Maximal PHM ac-tivity is also dependent on the presence of ascorbate and

catalase (data not shown). In contrast, human PAL is a nela-tively nonselective metalloenzyme. PAL activity is de-creased by metal ion chelation, and a number of metal ionscan restore the activity to varying degrees (Fig. 2b). Maximalrestoration ofactivity was observed in the presence of Zn24,although significant increases in activity were also seen withother metal ions. This nonselectivity ofthe human tumor PALmetal ion requirement is consistent with literature reports ofbovine PAL metal ion dependency (1 3).

In Vitro Inhibition of PHM and PAL Activities. We haveattempted to identify specific inhibitors of PHM and PALthrough characterization of their biochemical properties.As the second (PAL-catalyzed) step in peptidylglycinea-amidation can be replaced by base-catalyzed lysis and hasa slow but measurable rate at physiological pH and tem-perature, inhibition of PHM may be of more utility in a po-tential clinical setting.

Fig. 3, a-d, shows results for inhibition of PHM and PALactivities by metal ion chelatons and substrate analogues (ne-suIts shown are for partially purified enzymes from NCI-H345 cells; similar results were seen with a crude NCI-H720extract). The stnuctunes ofthe substrate analogues are shownin Fig. 3ewith the COOH-tenminal structure ofthe PHM andPAL substrate peptides. PHM and PAL activity can be in-hibited by nonselective chelatons such as EDTA and the nela-tively specific Cu2� chelaton diethyldithiocanbamate (Fig. 3,a and c). PAL is less sensitive to low amounts of the copperchelator (Fig. 3a), as expected from its relative nonselectivityfor divalent metal ions (Fig. 2). PHM is inhibited efficientlyby 4-phenyl-3-butenoate and less efficiently by 4’-amino-cinnamate (Fig. 3d), both ofwhich fitthe criteria determinedby Bradbuny et a!. (1 2) for covalent PHM inhibitors. PAL isnot inhibited by either of these compounds but is inhibitedby a-hydnoxy-hippunate (Fig. 3b), which is a substrate forPAL (9). Similar degrees of inhibition of both PHM and PALwere seen with three other chelatons, and somewhat poorer

:: c o (I) � cu C 82 0 � C

�OO�Q�uOZ0N�

Metal ion

�

g � � c o U) � � � �.- � � a)a) w �

:� Treatment

inhibition of PHM activity was achieved with four alterna-tive substrate analogues.

Expression of Peptidylglycine Amidation Activities Cor-relates with Expression of a Panel of Markers of the Neu-roendocrine Phenotype. Table 1 shows data for PAM,PHM, and PAL enzymatic activities in whole cell extractsof several cell lines with a wide range of expression of neu-noendocnine markers. The data on expression of neunoen-docnine markers are summarized from several published ne-ports from our group (24-26). The markers measured were:dense core secretory granules by electron microscopy; syn-aptophysin, chromognanin A, and Leu-7 antigens by immu-nohistochemistny; and neuron-specific enolase, BB isoen-zyme of creatine kinase, DOPA decarboxylase, and

[Inhibitor] (jiM)

a

>-.,

(00

a-

C

.�

coo

Ia-

e

b

�.tuuu

>,-.

cu2__J

a-

d

.�

coo

Ia-

N-acctyl-TyrPhe-(a-Hydmxy)-Gly(PAL assay substrate)

[Inhibitor] (jiM) [Inhibitor] (jiM)

N-acetyl-TyrPheGly(PHM assay substrate)

R-CH-C -NH-CH-000

�2

4-Pbenyl-3-Butenoate(PHM inhibitor)

c:yr__.CH CH-CH

4’-Amino-Cinnamate(PHM inhibitor)

H2N_Tj__.CH=C__C00

a-Hydroxy-Hippurate

(PAL inhibitor) 0 OH

�_C�-�H�LcOO

914 Peptide a-Amidating Enzymes in Lung Cancer Cells

a(H0)Hipp

0 .1 1 10 100 1000

[Inhibitor] (jiM)

0 OH

R-CHCNHCCOO

�2 H

Fig. 3. Inhibition of PHM andPAL activities in vitro. Assays for

a, b, and d were carried out usinga whole cell extract of NCI-H345

cells for 1 h; for c, an extract of anNCI-H345 secretory granule sub-

cellular fraction was assayed for 3h, as whole cell extract has very

little PHM activity in the absenceof added Cu2�. The standard re-action conditions were changed

to allow assay of endogenous

PHM activity in c (no Cu2� wasadded); for d, 10 �M Cu2� wasadded to provide maximal activ-ity; for a and b, 4 �M Cu2’ waspresent from the preparation ofthe PAL substrate. Metal ion chela-

tors tested were: ddc, diethyl-

dithiocarbamate ( #{149}, r� ); Phen,i,10-phenanthroline fY, V); andEDIA (�r, *). Substrate analogues

tested were: PhBu, 4-phenyl-3-butenoate (#{149},0); AmCinn, 4’-amino-cinnamate (A, es); anda(HO)Hipp, a-hydroxyhippurate

(U, 0). In e, N-acetyl-tyrosyl por-

tion of PHM and PAL substrates isrepresented by R-.

bombesin-like immunoreactivity by enzyme assay and ra-dioimmunoassay. Results were not available for all markerson every cell line. For the results shown in Table 1 , all lungtumor lines and MCF-7 were grown and maintained inserum-free culture medium, as we have found that the fetalbovine serum used for cell culture contains significant levelsof both PHM and PAL activities.

Every tumor cell line assayed to date expresses at least alow level of peptidylglycine a-amidation enzymes, evenlines such as MCF-7 (breast adenocarcinoma) and AS49(bronchioalveolar carcinoma), which are considered non-endocrine and express no neuroendocrine markers (Table1 ). For most cell lines, similar levels of expression are seenin cells grown in medium supplemented with fetal bovine

serum. The hemopoietic lines assayed for peptidylglycinea-amidation enzyme activities (BL21 26 and BL21 07,B-Iymphoblastoid; H929, B-myeloma; U937, macrophage;Molt 4, T-cell) will not grow in serum-free medium; there-fore, the possibility exists that the low activities in these linesmay reflect residual contamination of the cells by serum

enzymes. As expected from their coexpression as productsof a single gene, PHM and PAL levels vary approximately inparallel.

PAM activity was statistically compared to percentage ofpositive neuroendocnine markers in a set of eight cell lines(NCI-H34S, H510, H11SS, H720, N4i7, A549, SNU-C5,and MCF-7). For these lines, there is a statistically significantcorrelation between expression of PAM, PHM, or PAL ac-

28S-�

4.O-� �

b

28S-�

18S#{248}�

Fig. 4. Northern blot analysis of total RNA from human tumor cell lines. a,total RNA from human tumor cell lines probed for expression of PAM gene.Results are representative of analyses of at least two separate extracts of each

cell line. Faint hands can be seen for Molt 4 and MCF-7 upon longer exposure

of the Northern blots. b, the same gel was photographer) under UV light to

demonstrate integrity and equivalent loading of RNA.

Cell Growth & Differentiation 915

Table 1 Expression of markers of the neuroendocrine phenotype, levels

of PAM enzyme activities, and PAM mRNA levels in human tumor lines

Cell lines used are: carc, lung carcinoid; SCLCc, classic small cell lung

cancer; NSC-ha, non-small cell hronchioalveolar carcinoma; NSC-ad, non-small cell lung adenocarcinoma; SCLCv, variant small cell lung cancer; and

breast adenocarcinoma; the five hemopoietic cell lines assayed are listed inthe text. Expression of neuroendocrine markers was summarized from severalpreviously published reports from our group (details in “Materials and Meth-

ods”). ND, not done. Amidation enzyme assays of whole cell extracts of celllines were carried out as described in “Materials and Methods.” Values are

averages of 2-5 determinations )SEM, 1 0-40”/,,), except for H727 and the fivehemopoietic lines, which are single determinations. Relative mRNA levels are

estimated from Fig. 4. <, below sensitivity of PAM assay (0.5-1 .0 pmol/h/mg).

Neuroendocrine Enzymatic activity

Cell line (tumor) markers, )pmol/h/mg) mRNA/,, positive -

(no. tested) PAM PHM PAL

Lung

H720)carc) 100)3) 140 870 410 +++

H727 )carc) 100)3) 360 950 490 +++

H345)SCLCc) 100)7) 220 610 320 ++++

H510(SCLCc) 100)4) 64 607 314 ++++

H820)NSC-ba) ND 34 200 130 ++++

H1155 )NSC-ad( 75 (4) 32 290 87 ++

N417)SCLCv) 43)7) 6 94 65 ++

A549 )NSC-ha) 0 (5) 1 14 6 ++

Non-lung

MCF-7 (breast) 0 (3) 2 68 1 6 +

Hemopoietic )n = �5) ND < 1-17 1-12 +

tivities and expression of neuroendocnine markers. The pen-centage of positive neunoendocnine markers was treatedas a linear variable, yielding correlation coefficients of 0.95-0.98, and P-values less than 0.001.

Localization of PAM Enzymes to Secretory Granules. Inseveral species, subcellulan fractionation has demonstratedthat peptidylglycine a-amidation enzymes in endocrine tis-sues are colocalized to secretory granules, where processingof propeptides to bioactive hormones occurs (27). Colocal-ization of immunoneactive peptidylglycine a-amidation en-zymes and amidated peptide hormones has recently beenshown by immunoelectnon microscopy in human pancreaticislets (28). As shown in Table 2 for a typical cell line, humanPAM, PHM, and PAL activities are concentrated in a sub-cellular fraction containing secretory granules and other

membrane-bound components. The increased specific ac-tivity of PHM in secretory granules was supported by West-em blot analysis.

PHM and PAL activities are released from cultured tumorcells into the culture medium. Release does not have anabsolute dependence on the presence of secretory granules.After 3 days in culture, spent medium from a group of bothendocrine and non-endocrine tumor cell types containedPAM activities above the level in nonconditioned medium(1 2 of 14 had PHM and 1 4 of 1 4 had PAL activities greatenthan 75% of total cell-derived activities).

Molecular Analysis of Human PAM Gene Expression.Northern blot analysis of mRNAs from several tumor celllines is shown in Fig. 4. Previous reports have shown that thelevels of PAM mRNA in a variety of tissues do not correlatedirectly with the level of PAM enzyme activities in extractsfrom those tissues (29). In the tumor lines examined here, thelevels of PAM mRNAs detected by Northern blotting ap-proximately correlate with expression of enzymatic activity(Table 1 ). In each cell line examined, the human PAM cDNA

Table 2 Suhcellular fractionation of PAM, PHM, and PAL activities in

H720 (lung carcinoid) cell line

Specific activities )pmol/h/mg) were calculated from enzyme activities andprotein concentrations determined as describer) in “Materials and Methods.”Fold concentrations are relative to the specific activity in the whole cell

extract.

Subcellularfraction

Specific

PAM

activity (fold conce

PHM �

ntration)

PAL

Whole cell 82 79)) 250

Nuclei 81 (1 .0) 760 (1 .0) 200 0.8)

Cytosol 7 (0.1) 140)0.2) 70)0.3)

Granules 720 (8.8) 2400 (3.0) 720 ) 3.0)

0 .� �C) 0

e -i C.) 0_i Eto C) (I) #{248}o! �

C.) Cl) Z zcnm III � 1 II II

� � .� Se�

a

,w � w ‘� w

�i.�II..M�Ui�”-

probe visualized a broad band approximately 4.0 kilobasesin size. The width of the band in each lane suggests thepresence of multiple mRNA species in these cell lines. Pre-liminary polymenase chain reaction analysis of the mRNAfrom several cell lines confirms the presence of multiple al-ternatively spliced PAM mRNA forms in most of the celllines.

The characterized full-length human PAM mRNAs wouldencode a 974-amino acid hPAM-A pnoenzyme and an 867-amino acid hPAM-B pnoenzyme (1 7). Both include a trans-membrane domain, and so would be synthesized as bifunc-tional integral membrane proteins. In the rat, alternativesplicing can lead to the production of proteins lacking thetnansmembnane domain, which are predicted to be solubleenzymes (16, 30). Most of the purified peptidylglycinea-amidation enzymes from a variety of sources have beenshown to be soluble monofunctional proteins (1 ). To deter-mine whether the peptidylglycine a-amidation enzyme ac-

SCLCc

H187 H847b H446 H345 N417 H820 A549 H1155

(121) (596) (3724) (7058) (1610) (436) (520) (1850)

NSC-ba NSC-adSCLCv

916 Peptide a-Amidating EnzymesinLung Cancer Cells

Cl)

a)

>

C-)

-J

Q�co

Cu-

I0�ti)

0(I)

Fig. 5. Soluble PHM and PAL ac-tivities in human tumor cell line

extracts. Proportion of PHM andPAL activities extractable by soni-

cation of cells in water compared

to total activity released by themannitolfrriton x-ioo method de-

scribed in uMaterials and Meth-ods.” Abbreviations of cell linetype are defined in Table 1 . Total

PAM activity of each cell line inpmol/h protein is shown in paren-

theses below the cell line name.

tivities in lung tumor cell lines are soluble or membranebound, cells were divided into two batches and extracted byeither the distilled water or Triton X-i 00 method describedin “Materials and Methods.” Extraction with Triton X-1 00solubilizes membrane-bound proteins, whereas the waterextract would release only proteins which are already in asolubleform. Resultsareshown in Fig. S. lnthisgroupof lungtumor cell lines with total PAM activity ranging from i21(NCI-Hi 87)to 7058 (NCI-H354) pmol/h, approximately 20-60% of both PHM and PAL activities are present in solubleform. This range is similar to that reported PAM activity ex-tracted from a number of rat tissues (29). There is no obviousvariation in proportion ofsolubleto membrane-bound formswith tumor cell type.

Several lung tumor cell extracts were further characterizedby fractionation using hydrophobic interaction (phenyl-Sepharose) chromatography, to determine whether the ac-tive enzymes in tumor cells are present as a single bifunc-tional PAM enzyme or as separate PHM and PAL enzymes.Cells were extracted in the presence of protease inhibitors tominimize artifactual separation of PAM into PHM and PAL.Fig. 6 shows the resolution of soluble PHM and PAL activi-ties present in a neuroendocnine lung carcinoid cell line,NCI-H720. Similar results were obtained with extracts of thesmall cell lung cancer cell line NCI-H34S, and with enzymesin serum-free media which had been conditioned by NCI-H345, NCI-H820 (non-small cell with endocrine features),and A549 cell lines. The major portion of soluble PAM ac-tivity in the NCI-H720 extract is present as separate PHMand PAL activities. PAL activity is eluted from the hydro-phobic interaction column under low ionic strength condi-tions, whereas elution of PHM activity requires addition ofa mobile phase modifier such as ethylene glycol. Proteinseluting at intermediate polarities, at about fraction i 3-14and 18-i 9 (92 and i i 7 mm, respectively), catalyze bothPHM and PAL reactions and possibly represent bifunctionalPAM enzymes.

Discussion

Elucidation ofthe central mechanisms oftumor cell biologyis essential in developing rational approaches to more ef-

fectively diagnose and control the progression of cancers.We have previously demonstrated that the peptide hormonemediators ofautocnine growth stimulation can provide noveltargets for therapy of tumor cells. A monoclonal antibodywhich neutralizes the autocrine growth-stimulating activi-ties ofGRP by binding to its a-amidated terminus is currentlybeing used in a Phase II clinical trial in small cell lung cancerpatients, with promising results in late-stage patients (31). Areport of increased immunoreactivity of a bombesin-like na-

ture in asymptomatic smokers (32) has led us to suggest thatautocrine processes may also be relevant in the early stagesof tumor promotion, and so may provide targets for earlydetection and intervention approaches (33, 34). We havesuggested that interruption of autocrine growth stimulationmay be more central to early stages of tumor development,when rapidly proliferating preneoplastic cells are often con-fined to the surface of an epithelium. Although bombesin/GRP is a useful model of an autocnine growth factor, manypeptide hormones have been reported to mediate autocrinegrowth stimulation in a variety of tumor types (35); thus,focused approaches to therapy using agents directed againstsingle autocrine factors may not be optimal. Greater benefitmay be gained by identifying control points in the pathwaysof synthesis of multiple autocrine growth factors, and usingthose points as targets for interrupting many autocrinegrowth loops simultaneously. We have therefore investi-

gated the biochemistry of a major pathway for peptide hor-mone processing which is common to the production ofdiverse autocrine factors.

Since all peptide hormones must be activated by process-ing of the precursor prohormone, several posttranslationalprocessing activities must be present in cell lines whichare dependent on autocnine loops involving peptide hor-mones. For an a-amidated hormone such as GRP, theseactivities presumably include a subtilisin-like prohormoneendoprotease, carboxypeptidase H, and peptidylglycinea-amidation activities (1 ). We have suggested that inter-ruption of tumor autocrine growth loops may be achiev-able via inhibition of the specific posttranslational process-ing enzymes which are necessary for biosynthesis of auto-crine hormones (36). These enzymes would be found at

[Ammonium Sulfate](M)

PAM Enzyme Activities

PAL frnoVh

0

0

PHM fmoVh

xx

Fraction Number

Cell Growth & Differentiation 917

Absorbance at 210 nm

[Ethylene Glycol]

(%)

Fig. 6. Resolution of PAM, PHM, and PAL enzymatic activities. The column (10 ml phenyl-Sepharose), loaded with an extract of approximately 5 X 10’ cellsof the carcinoid cell line NCI-H720, in buffer A (20 msi TES, pH 7.4, with 0.5 si ammonium sulfate), was washed with 1 5 ml buffer A to remove loosely boundproteins and eluted successively with 5 ml 20% buffer A-80% 20 msi IES, pH 7.4 (buffer B), a 5-mI linear gradient to buffer B, 1 5-mI buffer B, a 30-mI linear

gradient to 80% buffer B-20% ethylene glycol, and then washed with 50% buffer B-50% ethylene glycol. After an initial fraction of 20 ml (40 mm), fractions were

collected every 5 mm (2.5 ml). PHM and PAL activities in each fraction were assayed by the procedures described in “Materials and Methods.”

high levels in tumor cell types known to express markersof an endocrine or neuroendocnine phenotype. However,cell types which are generally considered to be nonendo-cnine have also been shown to express posttranslationalprocessing enzymes, suggesting that interruption of

peptide processing may be possible in many tumor celltypes (37).

In order to develop strategies for inhibiting the function ofposttranslational processing enzymes in tumor cells, wehave focused on the best studied of these activities, the pep-tidylglycine a-amidation enzymes. Several well-differen-tiated lung tumor cell lines, particularly the subsets classifiedas classic small cell and carcinoid, are defined by their fre-quent expression of neuroendocnine markers, including pep-tide hormone production (20, 2i ), and so were expected tocontain active PHM and PAL enzymes. PHM has been char-acterized in secretory granules of a tumor cell line originat-ing from the mouse anterior pituitary gland, AtT-20 (38), andin the rat medullary thyroid carcinoma line CA-77 (i 2, 39).The sequence of human PAM cDNA was determined fromclones isolated from a human thyroid carcinoma library (1 7).Furthermore, there has been a report of elevated PHM ac-tivity in the plasma of patients with endocrine tumors, al-

though a tumor site of origin for the PHM activity was notascertained, and plasma of normal controls also had PHM

activity (1 8). We therefore chose to investigate the levels of

these a-amidation enzymes in neuroendocnine lung cancer

cell lines and to characterize the enzymes biochemically to

develop procedures for inhibiti ng posttranslational process-ing leading to autocrine hormone production.

The results reported here confirm our hypothesis that

human tumor cell lines express the enzyme activities re-

sponsible for glycine-directed peptidyl a-amidation. As hasbeen described for PAM and PHM enzymes from other spe-

cies, human tumor PHM is a secretory granule-associatedCu2 � and ascorbate-dependent monooxygenase, capableof converting a COOH-terminal glycine-extended peptide

into an intermediate which has the characteristics of ana-hydroxyglycine analogue (Fig. i ). The PAL enzyme thenconverts this intermediate into an a-carboxyamidated pep-tide. The maintenance of expression of both enzyme activi-ties in long-term tumor cell culture suggests that productionof a-amidated peptide hormones may be an essential factorin the proliferation of the cell lines, and therefore of the

tumors from which these lines were developed.

918 Peptide cu-Amidating Enzymes in Lung Cancer Cells

Several aspects ofthe biochemistry ofthe peptidylglycinea-amidation enzymes described in this paper suggest ap-proaches which could be taken to inhibit production of ami-dated peptide hormones in neuroendocnine tumor cells. Theabsolute requirement of PHM for Cu2�, and the ability ofchelating agents like diethyldithiocarbamate to inhibit PHMin vitro(Figs. 2 and 3), suggest such a biochemical approach.Although endocrine tissues appear to have mechanisms tomaintain levels of Cu2� in spite of copper deficiency (40),it may be possible to deplete Cu2� in neoplastic and pre-neoplastic cells and so inhibit PHM activity and peptidea-amidation. An alternative approach would be to use non-peptide substrate analogues of PHM and PAL, several ofwhich have been recently described in the biochemical lit-erature (1 2, 41 , 42). Bradbury et a!. (1 2) have described thechemical characteristics of a family of PHM covalent in-hibitors. One of these inhibitors, 4-phenyl-3-butenoate, ef-

ficiently inhibits human tumor PHM in biochemical assays(Fig. 3d). We have elsewhere presented preliminary resultswhich show that both copper chelators and the covalentPHM inhibitor 4-phenyl-3-butenoate can inhibit in vitrogrowth of several lung cancer cell lines (36, 43).

Further biochemical characterization of tumor cell pep-tidylglycine a-amidation enzymes will be necessary to en-able rational design of inhibitors for therapeutic studies. Forexample, although the quantitatively major forms of activeamidation enzymes in the neuroendocnine cell lines studiedare the separate PHM and PAL enzymes (Fig. 6), all linesappear to produce active bifunctional PAM enzymes.Whether these are incompletely processed intermediates ofthe ultimate relevant forms of the enzyme, or whether theyhave some independent functional or regulatory signifi-cance, requires further study. It is possible that the presenceof multiple enzymatic forms is related to alternative splicingat the mRNA level. In the rat, alternative splicing of exonsin the region of the transmembrane domain results in asoluble form of PAM (30) which can be secreted withoutfurther processing. Release of soluble forms of PAM wouldbe expected to occur rapidly, with secretory granules pro-viding a site for storage. However, release of PHM and PALfrom cells expressing integral membrane forms of PAM re-quires endoproteolytic cleavage close to the transmembranedomain. Our results show the presence of both soluble andmembrane-bound forms of both PHM and PAL in a varietyof tumor cell lines (Fig. 5). The two forms of PAM cDNAdescribed by Glauder et a!. (1 7) differ by a i 07-amino acidsegment (shown in Fig. 1 ) which encodes a paired basicamino acid site that is the cleavage site for bovine PHM-Aand PAL enzymes (1 3, 14). The broad band seen on North-em blot for each of the human cell lines suggests the pres-ence of multiple mRNA species in these lines (Fig. 4). Pre-liminary polymerase chain reaction analysis of the mRNAfrom several cell lines confirms the presence of multiple al-ternatively spliced PAM mRNA forms (data not shown).Studies are under way to identify any correlation betweenenzyme activity levels or enzyme biochemical characteris-tics and particular splice variants in human tumor cells. Dif-ferences in the molecular forms of PAM, PHM, and PALbetween tumor and nonneoplastic cells may be crucial todesigning inhibitors selective for tumor cell-associated pep-

tide amidation.Our data show a parallel between cellular levels of pep-

tidylglycine a-amidation enzyme activities, PAM mRNA ex-pression, and average expression of the panel of neuroen-docnine markers (Table i). The high levels of expression of

peptidylglycine a-amidation enzyme activities by the en-docnine tumor cell lines could therefore be viewed as asimple corollary to the presence of secretory granules inthese tumor cells. However, the small but measurable levelsofPAM enzymes in extracts and culture medium ofcells withno granules (for example, A549) demonstrates expression ofPAM by a non-granule-associated pathway. Eipper eta!. (37)have recently reported that several non-endocrine cell linesexpress posttranslational processing enzymes including

peptidylglycine a-amidation activities. Maintenance of ex-pression of peptidylglycine a-amidation enzymes in non-endocrine tumor cells suggests thatthese cell types may havea requirement for previously undetected a-amidated peptide

hormones. Although this finding suggests that inhibition ofpeptidyl amidation and autocnine stimulation may be ap-plicable in tumor types not usually considered to be endo-cnine, it also raises the possibility that the side effects of sys-temic inhibition of peptide amidation may be wider thanwould otherwise be anticipated.

We have focused on peptidylglycine amidation enzymesin these studies as a-amidation is a common posttransla-tional modification of peptide hormones including auto-crine growth factors. The biochemical characteristics ofPAM enzymes described here have suggested several agentswhich we are testing for growth-inhibitory effects in lungcancer cell lines. Biochemical analyses of other enzymescommonly involved in peptide prohormone processing,such as senine endoproteases and basic amino acid carboxy-peptidases, may lead to further classes of agents which maybe useful as cancer therapeutic and/or intervention tools. Itis critical that the biochemistry of human tumor cell post-translational processing enzymes be well understood, to en-able design of inhibitors which will show maximum selec-tivity for the forms of the enzymes found in tumor cells andspare inhibition of processing in nonneoplastic endocrineorgans.

Materials and Methods

Cell Culture

Tumor cell lines were maintained in RPMI i 640 with addedL-glutamine and 5-1 0% fetal bovine serum, or in phenolred-free, hormone-free RPM1 1 640 with added L-glutamineand selenium (3 X 1 0_8 vs NaSeO4). Cells were washed withDulbecco’s phosphate-buffered saline prior to extraction.

Cell ExtractsWhole cell extracts were prepared from approximately 5 X106 cells homogenized in 1-2 ml ice-cold deionized waterwith a protease inhibitor cocktail of phenylmethylsulfonylfluoride, bacitracin, aprotinin, and leupeptin. The homog-enate was then sonicated at high power for 1 0 s with a Bran-son ultrasonic probe, allowed to sit on ice for 30 mm, resoni-cated, buffered with 100 mt�i sodium TES, pH 7.4, andultracentrifuged at 1 00,000 X g for 30 mm.

Subcellular fractionation was carried out by gentle ho-mogenization ofthe cells in 20 m�i sodium TES, pH 7.4, with0.25 M sucrose. Nuclei and unbroken cells were pelleted bycentrifugation at 3,000 X g for 1 0 mm. The supernatant wasthen ultracentrifuged at 100,000 x g for 30 mm. The pelletcontaining microsomes and secretory granules was ex-tracted with water and protease inhibitor mix as above. Ex-traction of whole cells or granules with 20 msa sodium TES,pH 7.4, containing 1 0 ms�i mannitol and 0.i % Triton X-1 00,yielded slightly more enzyme activity with a lower specificactivity.

Cell Growth & Differentiation 919

Peptidylglycine a-Amidation Enzyme Assays

Assay methods are variations of the procedures of Mizunoet a!. (23) and Perkins et a!. (44). Reactions were shown tobe linear with time (1-2 h) and amount of protein (up to5 pg/assay) over the ranges used here. For all three assaysdetailed below, the a-amidated product was extracted byshaking the reaction mix with 250 jil 1 .0 M sodium 4-(2-hydroxyethyl)-i -piperazineethanesulfonic acid buffer, pH7.5, saturated with ethyl acetate, and 650 .il ethyl acetatesaturated with the same 4-(2-hydroxyethyl)-i -piperazine-ethanesulfonic acid buffer. The extraction procedure wasvalidated with iodinated authentic substrate and amidatedproduct: approximately 1�/� of the radiolabeled substratecompared to 90% ofthe radiolabeled amidated product wasextracted into the organic phase. Enzymatic conversion wascorrected for nonenzymatic baseline controls. Results re-ported for PHM and PAM activities were determined at theconcentration of Cu2� which gave maximal activity. Spe-cific activities were calculated from enzyme activities andprotein concentrations determined using the Pierce bicin-choninic acid reagent in the microtiter plate format.

PAM Assay. AcYFG is converted to the COOH-terminala-amidated analogue, acYFamide, by sequential PHM andPAL activity. The assay was carried out in microcentnifugetubes with 35 p1 ofassay mix and 5 jil ofenzyme preparation.The pH maximum of PAM activity in crude cell extracts wasdetermined to be 6.0-6.5, similar to that reported by Mizunoet a!. (23) for frog peptidylglycine a-amidating enzyme usingthis substrate. The standard 3S-pI assay mix contained ap-proximately i 50 mM sodium 2-(N-morpholino)ethanesul-fonic acid, pH 6.25, with 20,000 cpm [125l-TyrIacYFG, 0.2�M acYFG, 0.5 jig/mI bovine catalase, i jiM Cu2� (as CuSO4)for each 0.5 pg of analyte protein, and 0.5 mvi ascorbate.

PHM Assay. In a parallel assay, any a-hydroxyglycine in-termediate [acYF(aHO)G] remaining in the pH 6.25 PAMreaction mix was converted to acYFamide by addition of 50p1 0.2 M NaOH for 5 mm at 37#{176}C.This base-catalyzed con-version step ensures that the assay is independent of PAL,and so is a measure of PHM activity only. Both acYFG andacYF(aHO)G are acidic and do not extract from the aqueousphase at pH 7.5 into the organic solvent.

PAL Assay. A modification ofthe method of Perkins et a!.(44) was used. An aliquot of purified bovine or human PHMwith no PAL activity was used with a PHM reaction mix atreduced pH [1 50 m� sodium 2-(N-morpholino)ethane-sulfonic acid, pH 5.51 to produce acYF(aHO)G. After 5 h at37#{176}C,the PHM reaction was terminated by reaction for 30mm with a 100 jiM concentration of the covalent PHM in-hibitor 4-phenyl-3-butenoate (1 2). Generally, about 2-3%of total radioactivity was extracted in the absence of addedPAL, whereas addition of NaOH before extraction resultedin extraction ofabout 85% ofthe radioactivity. This amountrepresents almost complete conversion of acYFG to the PALsubstrate acYF(aHO)G by purified PHM. The PAL reactionwas carried outwith 35 jil ofthis mix added to 5 uI of enzymepreparation for 1-2 h at 37#{176}C.

Northern Blot Analysis of Human Tumor PAM mRNATotal RNA was prepared from frozen cell pellets of indicatedcell lines using the guanidine isothiocyanate-cesium chlo-ride method of Glisin et a!. (45). Total RNA (1 0 jig) waselectrophoresed on a denaturing i .2% agarose gel and trans-fenred to a nitrocellulose membrane. For fragment size de-termination, a RNA ladder was fractionated in an adjacentlane. Integrity of RNA and equivalence of loading were

checked with ethidium bromide under UV light before trans-fer of the RNA. The cDNA probe used was a 0.8-kilobaseEcoRl fragment of human APAM-1 (1 7), which was labeledby random priming to a specific activity of i-2 X 1 06 cpm/j.il. Hybridization was carried out overnight at 42#{176}Cin 10%dextran sulfate-40% formamide. The blot was washed twicewith 2X standard saline citrate at room temperature for 30mm, and twice with 0.1 x standard saline citrate at 60#{176}Cfor30 mm. The blot was exposed to Kodak XAR film for 24 hwith an intensifying screen. Faint bands in the lanes ofMCF-7 and MoIt-4 are clearly visible on longer exposures.

Phenyl-Sepharose Hydrophobic InteractionChromatography

Approximately 5 X i07 cells were extracted by homogeni-zation and sonication in 6 ml of 0#{176}Cdeionized water con-taming 80 pg/mI Ieupeptin, 500 pg/mI bacitnacin, i 70 pg/mIaprotinin, and 0.5 mM phenylmethylsulfonyl fluoride. Theextract was then slowly made 0.5 M in ammonium sulfateand buffered to pH 7.4 in a final concentration of 20 msasodium TES. Cell debris and precipitated proteins were pel-leted by ultracentrifugation at i 00,000 x gfor 30 mm at 4#{176}C,and the supernatant was pumped onto a 10-mI phenyl-Sepharose (Pharmacia-LKB) column equilibrated in 0.5 M

ammonium sulfate in 20 m�a sodium TES, pH 7.4, at 0.5mI/mm. The column effluent was recirculated for approxi-mately 3 column volumes. For larger volumes, such as withcell-conditioned culture medium, the sample was made 0.5M in ammonium sulfate, centrifuged at 3,000 X gfor 30 mm,

and equilibrated with the phenyl-Sepharose resin by shakingin a flask. The resin was then washed in TES buffer con-taming 0.5 M ammonium sulfate and packed into an emptyglass column. After both loading methods, bound proteinswere eluted from the resin with a complex gradient of de-creasing concentration of ammonium sulfate, low ionicstrength TES buffer, and increasing concentrations of ethyl-ene glycol as indicated in legend to Fig. 6. The slope ofdifferent sections of the gradient was adjusted to increaseresolution in regions of interest.

Acknowledgments

Statistical analysis comparing PAM enzyme levels and neuroendocrine mark-ers was carried out by Drs. Barnett Kramer and Sylvan Green, Division of

Cancer Prevention and Control, National Cancer-Institute, Rockville, MD.

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