[CANCER RESEARCH 53, 5214-5218, November 1, 1993]

Thymosin al Down-regulates the Growth of Human Non-Small Cell Lung Cancer

Cells in Vitro and in VivoTerry W. Moody,1 MírelaFagarasan, Farah Zia, Mirjana Cesnjaj, and Allan L. Goldstein

Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington, DC 20037 ¡T.W. M., M. F., E Z.,A. L. G.I, and Laboratory of Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892[M. C.I

ABSTRACT

The elicci of thymosin al (THNal) and its NH2-terminal fragment(THN1-14) and COOH-terminal fragment (THIS15-2") on non-small cell

lung cancer (NSCLC) growth was evaluated. Using an anti-THNal anti

body, receptors were identified on NSCLC cells that were pretreated withIO-6 M THNal. | 'll| \niihidoMic acid was readily taken up by NSCLC

cells and THNal significantly increased the rate of arachidonic acid release. THN1-15 slightly stimulated but THN1"" and THNß4did not alter

arachidonic acid release troni NCT-H1299 cells. In clonogenic growthassays in vitro, THNal (10~* M) significantly decreased NSCLC colonynumber whereas THN'-'4, THN1"8, and THNß4were less potent. Usinggrowth assays in vivo, THNal (10 /ig s.c./day) but not THN1-14, THN15-28,

or IIIN/ÃŽ-Iinhibited significantly NSCLC xenograft formation in nude

mice. These data suggest that biologically active THNal receptors arepresent on NSCLC cells and that native THNal inhibits the growth ofhuman NSCLC.

INTRODUCTION

THNal,2 a 28-amino acid peptide which functions as a biological

response modifier by virtue of its immunomodulatory effects onT-cells, was initially isolated from a crude bovine thymus extract,THN fraction 5 (1). THNal is derived from a 113-amino acid precursor protein, prothymosin a; the NH2-terminal 28-amino acid se

quence of prothymosin a is identical to that of THNal (2, 3). Byradioimmunoassay THN-like peptides have been detected in numer

ous tissues including the rat thymus, pituitary, and brain (4). In addition to THNal, THNß4,a 43-amino acid peptide with pleotropic

effects as a biological response modifier, was isolated from THNfraction 5 (5). THNß4, which has little sequence homology toTHNal, is an actin-sequestering peptide, stimulates terminal deoxy-nucleotidyltransferase, and stimulates luteinizing hormone-releasing

factor secretion from the rat hypothalamus resulting in increasedluteinizing hormone secretion from the pituitary (5-7).

THNal has been localized to the surface of mouse thymic epithelialcells using anti-THN antibodies (8). While the second messenger for

THNal is not known, THN fraction 5 stimulated arachidonic acidrelease from anterior pituitary cells (9). Also, THNal has sequencehomology to VIP (10). THNal is reported to inhibit 125I-VIP binding

sites in rat blood mononuclear cells and liver plasma membranes withlow affinity (50% inhibitory concentration, 1 JAM).These studies suggest that THNal can interact with VIP receptors. Previously we found125I-VIP binding sites on human lung cancer cells (11, 12).

THN fraction 5 prolongs survival of some lung cancer patients (13).In postradiotherapy patients with NSCLC, synthetic THNal accelerated reconstitution of thymic-dependent immunity (14). Also, THNal

Received 5/24/93; accepted 8/27/93.The costs of publication of this article were defrayed in part by the payment of page

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

' To whom requests for reprints should be addressed, at Biomarkers and Prevention

Res. Br.. NCI, 9610 Medical Center Dr., Bldg. C, Rm. 3(K). Rockville, MD 20850.2 The abbreviations used are: THNal, thymosin al; VIP, vasoactive intestinal peptide;

PBS. phosphate-buffered saline; NSCLC, non-small cell lung cancer; BSA. bovine serumalbumin; EOF, epidermal growth factor; cAMP. cyclic AMP; TGFa, transforming growthfactor a.

increased survival especially in patients with nonbulky tumors (15):THNal in combination with interferon after cyclophosphamide treatment increased survival of mice with Lewis lung carcinomas (16).These data suggest that THNal inhibits lung cancer proliferation.Here the effects of THN-like peptides on NSCLC cells were investi

gated.

MATERIALS AND METHODS

Cell Culture. NSCLC cells were cultured in RPMI 1640 containing 10%heat-inactivated fetal bovine serum. When a monolayer formed, the adherent

cells were washed with PBS and treated with trypsin/EDTA. The cells werepelleted and resuspended in serum supplemented medium and incubated at37°Cin 5% COz/95% air (17). Routinely the cells were passed 1/1 weekly and

experiments were conducted when the cells were in exponential growth phase.The cells utilized included NCI-H157 (squamous cell carcinoma) and NCI-H1299 (large cell carcinoma), NC1-H727 (lung carcinoid), and NCI-H838 andNCI-HI264 (adenocarcinoma).

Immunocytochemistry. Antiserum (PR-1) to synthetic THNal-carbo-diimide-hemocyanin conjugates were elicited in rabbits. The antibodies wereimmunoaffinity purified (NSP) and PR-1 or NSP were used at 1:1000 dilution.NSCLC cells (IX IO4) were plated in Nunc chambers coated with fibronectin.

After 4 days the media were changed. After 16 h the cells were treated withTHN-like peptides for 30 min. The cells were washed twice with PBS con

taining 1% BSA, fixed with PBS containg 2% glutaraldehyde, and rinsed withPBS containing 5% BSA and 4% normal goat serum. Subsequently the cellswere treated with NSP or PR-1 at 25°C.After 16 h, the cells were washed twice

in PBS containing 5% BSA. The cells were treated with goat anti-rabbit serum

(1:60 dilution) for 60 min followed by incubation with PBS containing 5%BSA for 10 min. The washing with PBS/BSA was repeated three times. Thenthe samples were treated with water for 3 min and this process was repeated 3times. The samples were treated with Auroprobe LM and intense M (Amer-

sham) for light microscopic immunogold silver staining. The samples werethen treated with water for 5 min twice and the cells were visualized forimmunostaining.

Arachidonic Acid Release. NSCLC cells (5 x 10") were placed in 24-well

plates coated with human fibronectin (20 fig) as described previously (11).After a monolayer of cells formed (5 days) [5,6,8,9,11,12,14,15-3H]arachi-donic acid (2.5 x IO5 cpm) was added. After 16 h the cells were washed twicein 1 ml of SIT medium (RPMI 1640 containing 3 X IO'8 MNa2SeO3, 5 /J.g/ml

insulin and 10 fig/ml transferrin) containing 0.2% fatty acid-free bovine serumalbumin (S1T/BSA). New medium were added containing thymosin-like pep

tides. After 40 min. 100 fil of media were removed from each well and placedin a scintillation vial, scintillation fluid added, and the samples were countedin a beta counter.

Clonogenic Assay. NSCLC cell lines were harvested and tested in theagarose cloning system described previously (18). The base layer consisted of2 ml of 0.5% agarose in SIT medium containing 5% fetal bovine serum in6-well plates (Falcon, Oxnard, CA). The top layer consisted of 2 ml of SITmedium in 0.3% agarose, the THN-like peptides, and 1 X IO4 single viable

cells/ml. For each cell line and peptide concentration, triplicate wells wereplated. After 2 weeks 1 ml of 0.1% p-iodonitrotetrazolium violet was addedand after 16 h at 37°Cthe plates were screened for colony formation; the

number of colonies larger than 120 f¿min diameter were counted.Nude Mouse Assay. The ability of THNal to inhibit xenograft formation

in nude mice was investigated (19). Female athymic BALB/c nude mice, 4-5weeks old, were housed in a pathogen-free temperature-controlled isolation

room and the diet consisted of autoclaved rodent chow and autoclaved watergiven ad libitum. NCI-H157 or H727 cells (1 X IO7) were injected into the

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THYMOSIN ai DOWN-REGULATES GROWTH OF HUMAN NSCLC

right flank of each mouse by s.c. injection. Palpable tumors were observed inapproximately 90% of the mice after 2 weeks. PBS (100 fil) or THN-like

peptides (10 fig/day) were injected s.c. adjacent to the tumor. The tumorvolume (height x width x depth) was determined weekly by calipers andrecorded. When the tumor became necrotic, the growth studies were termi

nated.

RESULTS

Immunocytochemistry. Previously, we found that THNal antise-

rum recognized the COOH terminus of THNal whereas the NH2terminus was essential for biological activity (4). Here NSCLC cellswere fixed with glutaraldehyde and treated with a primary antibody(NSP or PR-1), followed by a secondary antibody (goat anti-rabbitserum) and silver enhancer. Using large cell carcinoma cells (NCI-

H1299) optimal staining was observed using a 1:1000 dilution of NSPor PR-1. Cells did not stain in the absence of THNal antiserum (Fig.L4) or THNal treatment (Fig. Iß).NCI-H1299 cells immunostained

if they were treated with l /ÃœMTHNal, followed by extensive washing, fixation, and treatment with primary and secondary antibody (Fig.1C). These data suggest that THNal binds to NSCLC cell surfacecomponent. Similar data were obtained using adenocarcinoma (NCI-H838), squamous cell carcinoma (NCI-H157), and lung carcinoidcells (NCI-H727).

Arachidonic Acid. Previously it was found that THN fraction 5stimulated arachidonic acid release from pituitary cells (9). Here theeffects of THN-like peptides on arachidonic acid release were inves

tigated using NSCLC cells. THNal had little effect on arachidonicacid release at a 10~9 M concentration using NCI-HI299 cells butsignificantly stimulated arachidonic acid release at IO"6 Mconcentration (Fig. 2). THNal (10~5 M) increased the rate of arachidonic acid

release approximately 3-fold and the half-maximal effective concentration was 0.7 X 10~7 M. Table 1 shows that TON1'14 (IQ-6 M)

slightly increased arachidonic acid release, respectively, whereasTHN'5-2* and THNß4had no effect. Similar results were obtained

using NCI-H727, 838, and 1264 cells.

NSCLC Proliferation. The effect of THNal on NSCLC growthwas investigated in vitro. Fig. 3 (top) shows that 150 NC1-H1299

colonies formed in the control culture. When 100 nMVIP was addedthe number of colonies formed increased significantly to 322 (data notshown). THNal inhibited NCI-HI299 colony formation in a dose-dependent manner with IO"7, IO"6, or W~5 but not IO"8 M THNal

causing significant inhibition. Fig. 3 (middle) shows that numerouslarge viable colonies formed. In the presence of l JU.MTHNal [Fig. 3(bottom)] the number of colonies was reduced as was the size. Similardata were obtained using NSCLC cell lines NCI-H157, H727, H838,

and H1264.Table 2 shows that other THN-like peptides inhibited NCI-H838

colony formation. THN1"14 and THNI5~28 inhibited colony formation

by approximately 25% whereas THNß4was inactive. These datasuggest that NH2- and COOH-terminal fragments of THNal have

some biological activity but neither fragment is as potent as is nativeTHNal.

Also, the effects of THNal on NSCLC growth was investigated invivo. Fig. 4 shows that tumors formed 1 week after s.c. injection ofNCI-H157 cells into nude mice. Fig. 4 (top) shows that the NSCLC

xenografts grew exponentially and after 5 weeks the xenograft volumewas 2306 mm1. When THN1-'4 or TON15-28 was administered s.c.

(10 ju,g/day) xenograft volume was reduced by approximately 20%. Incontrast, THNal (10 fig) significantly reduced tumor volume byapproximately 75%. A dose-response curve for THNal was deter

mined. Fig. 4 (bottom) shows that 10 or 1 /xg of THNal significantlydecreased NCI-H727 tumor volume whereas 0.1 jug of THNal or 10

p.g of THNß4were ineffective. The effects of THNal were reversible

<••«•

'

B

•¿�>•*

Fig. l. Immunocytochemistry of NSCLC cell lines. NCI-H1299 cells were treated with(B, C) or without (A) anti-THNal antisera after treatment with (C) or without (A, B) l^LMTHNal. The cells were then silver stained as described in "Materials and Methods."

This experiment is representative of 2 others.

in that if treatment was discontinued, the growth rate of the tumorreturned to normal values.

DISCUSSION

Previously, THNal was postulated to prolong survival of NSCLCpatients via effects of the immune system (15). Here THNal inhibitedthe growth of NSCLC cells in vitro and in vivo suggesting that THNa 1directly interacts with NSCLC cells.

(125I-Tyr")THNal binds with high affinity to THNal antiserum but

not NSCLC cells. Additional studies indicated that THNal andTHN1A~2I<but not THN1"'4 strongly cross-reacted with the antiserum.

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THYMOSIN al DOWN-REGULATES GROWTH OF HUMAN NSCLC

I-8 -7 -6

(THN alpha 1) , Log M

-5

Fig. 2. Effect of THNa-1 on arachidonic acid release. NCI-H1299 cells were loadedwith pHJarachidonic acid and the amount of radiolaheled arachidonic acid released wasdetermined with increasing concentrations of THNal {•).This experiment is representative of 2 others. Bars, SD.

Table 1 Effect of THN-like peptides on arachidonic acid release

Agent added 3H]Arachidonic acid released (cpm)

NoneTHNal, 10-6MTHN'-'4. 1(T6 MTHNi5-2« 10-6MTHN/34, IO"6 M

688 ± 90"1734 ± 72fc

855 ± 61602 ± 51608 ±168

" Mean ±SE of 4 determinations using NCI-HI299 cells.

bP< 0.01.

Table 2 Effect of THN-like peptides on NSCLC growth in vitro

AgentaddedNoneTHN

15-2« (1^MjTHN'-'4(1

/IM)

THN04 (1 (¿M)THNal (1 (¿M)Colony

no.54±4°

44 ±440±3fc

52 ±517±4C

" The mean ±SE of NCI-H838 colonies (n = 3) was determined.kP< 0.05.CP < 0.01.

4-

cvio

ooo

-8 -7 -6

(Peptide), Log M

-5

.v.

Fig. 3. NSCLC clonal growth assay. (A) The effect of THNal on NC1-H1299 colonyformation was determined in the absence (•)or presence (O) of increasing concentrationsof THNal. The mean value ±SE (bars) of 3 determinations is indicated: P < 0.05. *.

Large viable NCI-H838 colonies formed (B) whereas the colony size and number wasreduced in the presence of 1 ^tMTHNal (C).

Therefore a unique immunocytochemical approach was used to demonstrate interaction of THNal with NSCLC cells. Using this assay,THNal was bound to NSCLC cells and fixed with glutaraldehyde (theNH2 terminus of THNal may bind to a receptor protein whereas theCOOH terminus was in solution and consequently interacted withTHNal antibodies). Then THNal antiserum was added, followed bygoat anti-rabbit sera, and the bound antibodies were visualized by

silver staining. These data suggest that THNal may interact with aNSCLC cell surface component, possibly a THNal receptor. Also,THNal can bind to the VIP receptor inasmuch as THNal inhibits125I-VIP binding to NSCLC cells with low affinity (50% inhibitoryconcentration, 10 /iM3). Previously we found that almost all NSCLC

cell lines examined have high affinity binding sites for EOF (19) and

1T. Moody, unpublished observations.

VIP (20); the VIP and EOF receptor proteins are composed of 459-and 1186-amino acid residues, respectively (21, 22).

The ability of THNal to alter second messenger production wasdetermined. THNal (10 fj..w)did not alter intracellular cAMP, whereas10 nMVIP strongly increased cAMP levels (20). THNal did not altertyrosine phosphorylation whereas EOF (100 ng/ml) activated tyrosinekinase activity causing phosphorylation of the A/r 170,000 EOF receptor (23). Also, THNal had no effect on phosphatidylinositol turnover or cytosolic calcium but did significantly stimulate arachidonicacid release. The effect was dose dependent and THNal significantlystimulated [3H]arachidonic acid release. THN1"14 slightly stimulatedarachidonic acid release whereas THNI5~2K and THNß4did not alter

arachidonic acid release. These data suggest that THNal may stimulate phospholipase A2 but not adenylate cyclase activity or tyrosinekinase activity. Previously, it was found that NSCLC cells have cy-

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THYMOSIN al DOWN-REGULATES GROWTH OF HUMAN NSCLC

PBS

ÌTHNJ-1«THN15-28

THNalpha 1

3 4Time, Weeks

nio

B

PBS10 ug THN beta40.1ug THN alphal

and was less effective if injected i.p. or i.v. It is possible that THNalis degraded by serum proteases and we are currently investigating thehalf-life of THN-like peptides in the blood. Recently, we found that

THNamide, which has an amidated COOH terminus and is resistant todegradation by carboxypeptidases, strongly inhibits NSCLC growth.VIP, which has an amidated COOH terminus (28), is also resistant todegradation by carboxypeptidases.

It remains to be determined if THN-like peptides function as autocrine growth factors in NSCLC. By radioimmunoassay, THN-likepeptides are present in NSCLC extracts and conditioned medium.3

Previously, TGFa mRNA was found in NSCLC cells and TGFaimmunoreactivity was found in conditioned medium (29). TGFabinds with high affinity to EOF receptors, causes tyrosine kinaseactivity, and stimulates growth (23). Because EOF receptor monoclonal antibodies inhibit the growth of NSCLC, TGFa may be apostitive autocrine growth factor (19). Currently, we are investigatingif THNal is a negative autocrine growth factor for NSCLC.

THNal may also be effective at slowing the growth of othercancers. In small cell lung cancer, VIP elevates cAMP levels, stimulates early oncogene expression of c-fos, and increases the secretionrate of bombesin-like peptides resulting in increased small cell lung

cancer growth (30). THNal (10 JAM)inhibited the clonal growth ofseveral small cell lung cancer cell lines (20).

In summary, THNal stimulates arachidonic acid release and inhibits NSCLC growth in vitro and in vivo. Therefore THNal may function as a regulatory peptide in NSCLC.

*1 Ug THN alpha 1 REFERENCES

*lOug THN alpha 1

2345

Time, weeksFig. 4. NSCLC xenograft formation. (Top) NCI-H157 tumors formed after 2 weeks and

the mice were given daily injections of PBS (O), or 10 ug (s.c.) THN'-'4 (A), THN15-28

(A), and THNal (•).(Bottom) Nude mice bearing NCI-H727 tumors were given dailys.c. injections of PBS (•),10 ngTHNß4 (O), 0.1 /¿gTHNal (A), l /ig THNal (A), and10 (ig THNal (•).The mean tumor volume ±SD of 3 determinations is indicated; P <0.05, •¿�.

clooxygenase activity resulting in prostaglandin E2 production (24). Itremains to be determined if THNal alters prostaglandin levels inNSCLC cells.

THN-like peptides inhibited the growth of NSCLC cells in vitro.

The effect was dose dependent and THNal (l JU.M)significantlyinhibited colony formation. THN1"14 (1 pt,M)significantly inhibitedNSCLC growth but was less potent than was THNal. THN15~28

caused a slight inhibition of growth whereas THNß4was inactive.Previously it was found that THN1^14 was more potent at stimulatingT-cell function in aging mice than was THN15-28 (25). VIP (100 nM)

significantly increased NSCLC colony formation whereas if VIP plusTHNal was added there were few colonies.1 These data suggest that

the stimulatory effects of VIP on colony formation are overwhelmedby THNal. It remains to be determined if THNal alters NSCLConcogene expression. NSCLC cells have amplification and increasedexpression of the c-myc and k-ras oncogenes (26, 27).

THN-like peptides inhibited the growth of NSCLC in vivo. THNalinhibited NSCLC growth in a dose-dependent manner and the half-maximal concentration was 1 fig (0.04 mg/kg). In contrast, THN1"14,THN15-28, and THNß4had little effect. Preliminary data3 indicate

that THNal was most effective if injected s.c. adjacent to the tumor

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2. Grangou-Laxaridis. M., Clinton. M.. Goodall, G. J., and Horecker, B. L. Prolhymosina and parathymosin: amino acid sequences deduced from the cloned rat spleencDNAs. Arch. Biochem. Biophys.. 26.?: 305-310, 1988.

3. Eschenfeldt, W. H., and Berger. S. L. The human prothymosin a gene is polymorphicand induced upon growth stimulation: evidence using a cloned cDNA. Proc. Nati.Acad. Sci. USA, 83: 9403-9407. 1986.

4. Palaszynski, E. W., Moody, T. W., O'Donohue, T. L., and Goldstein, A. L. Thymosin

al-like peptides: localization and biochemical characterization in the rat brain andpituitary gland. Peptides, 4: 463-469, 1983.

5. Low. T. L. K., and Goldstein, A. L. Chemical characterization of thymosin (34. J. Biol.Chem., 257: 1000-1008, 1982.

6. Rebar, R. W.. Miyake, A., Low, T. K. L.. and Goldstein, A. L. Thymosin stimulatessecretion of luteinizing hormone releasing factor. Science (Washington DC), 214:669-671, 1981.

7. Sanders, M. S.. Goldstein. A. L., and Wang, Y-L. Thymosin ß4(Fx peptide) is a potent

regulator of actin polymerization in living cells. Proc. Nati. Acad. Sci. USA, 89:4678-4682, 1992.

8. Rabien. N., Auger. C. and Monier. J. C. Immunolocalization of thymosin ori, thy-

mopoietin and thymulin in mouse thymic epithelial cells at different stages of culture:A light and electron microscopic study. Immunology. 63: 721-727, 1988.

9. Spangelo, B. L.. Judd. A. M., Ross, P. C.. Login, I. S., Jarvis, D., Badamchian, M.,Goldstein, A. L., and MacLeod, R. M. Thymosin fraction 5 stimulates prolactin andgrowth hormone release from anterior pituitary cells in vitro. Endocrinology. 727:2035-2043, 1987.

10. Calvo, J. R., Guerrero, J. M.. and Goberna. R. Interactions of thymic peptide thymosinal with VIP receptors in rat intestinal epithelial cells: Comparison with PHI andsecretin. Gen. Pharmacol.. 20: 503-505. 1989.

11. Lee, M., Jensen, R. T.. Huang. S. C.. Bepler, G., Korman, L., and Moody. T. W.Vasoactive intestinal polypeptide binds with high affinity to non-small cell lungcancer cells and elevates cyclic AMP levels. Peptides, 77: 1205-1209, 1990.

12. Shaffer. M. M., Carney. D. N., Korman, L. Y, Lebovic. G. S., and Moody, T. W.High affinity binding of VIP to human lung cancer cell lines. Peptides, fi:1101-1106, 1987.

13. Cohen. M. H., Chretien, P. B., Inde, D. C., Fossieck, B. E., Makuch. R., Bunn, P. A.,Johnston. A. V, Shackney, S. E., Matthews, M. J., Lipson, S. D., Kenady. D. E., andMinna. J. D. Thymosin fraction 5 and intensive combination chemotherapy prolonging the survival of patients with small-cell lung cancer. JAMA. 2-*7: 1813-1821,

1979.14. Schulof, R. S.. Lloyd, J. J., Cox, J., Palaszynski, S. R., McClure, J. E., Incefy, G., and

Goldstein. A. L. An evaluation of two different schedules of synthtic thymosin aladministration in patients with lung cancer—preliminary results. In: M. Chirogos andA. L. Goldstein (eds.), Thymic Hormones and Lymphokines, pp. 601-613. New York:

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15. Schulof. R. S.. Lloyd. M. J., Cleary, P. A., Palaszynski. S. R.. Mai, D. A., Cox, !. W.,Alabaster. O.. and Goldstein. A. L. A randomized trial to evaluate the immunorestor-alive properties of synthetic thymosin al in patients with lung cancer. J. Biol.Response Modif.. 4: 147-158. 1985.

16. Garaci. E.. Mastino, A.. Pica. F.. and Favilli. C. Combination treatment using thymosin a 1 and Interferon after cyclophosphamide is able to cure Lewis lung carcinomain mice. Cancer Immunol. Immunother.. 32: 154-161), 1990.

17. Carney, D. N., Gazdar, A. F., Bepler, G., Guccion. J. G., Marangos, P. J., Moody. T.W., Zweig. M. H., and Minna, J. D. Establishment and identification of small cell lungcancer cell lines having classic and variant features. Cancer Res.. 45: 2913—2923.

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19. Lee. M., Draoui, M., Zia, F., Gazdar, A. F., Oie. H.. Tarr. C.. Bello!, F., Kris, R.. andMoody, T. W. Epidermal growth factor receptor monoclonal antibodies inhibit thegrowth of lung cancer cell lines. J. Nati. Cancer Inst. Monogr., 13: 117-123, 1992.

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23. Moody, T. W., Lee, M., Kris, R. M.. Belli«.F.. Bepler, G., Oie, H., and Gazdar, A. F.Lung carcinoid cell lines have bombesin-like peptides and EOF receptors. J. Cell.Biochem.. 43: 139-147. 1990.

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1993;53:5214-5218. Cancer Res   Terry W. Moody, Mirela Fagarasan, Farah Zia, et al.  

in Vivo and in VitroCell Lung Cancer Cells 1 Down-regulates the Growth of Human Non-SmallαThymosin

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