Vasopressin and Gastrin-Releasing Peptide Genes Pathobiology 1992;60:136-142

Rudolf Magnus Institute. Utrecht; Department of Clinical Immunology, University Hospital. Groningen: Department o f Medical Genetics. Stale University of Groningen. The Netherlands; Department of Medical Oncology. Mater Miscricordia Hospital. Dublin. Ireland: Department of Medicine. Duke University Medical Center. Durham. N.C.. USA: Department o f Biochemistry. University of Nijmegen. The Netherlands

Key Words

Vasopressin mRNA Gastrin-releasing peptide Small cell lung carcinoma

Expression of the Vasopressin and Gastrin-Releasing Peptide Genes in Small Cell Lung Carcinoma Cell Lines

Abstract

Various polypeptide hormones including vasopressin (VP) and gastrin-releas­ ing peptide (GRP) are produced by small cell lung carcinomas (SCLC). VP as well as GRP have mitogenic effects on several cell types and are proposed to be autocrine growth factors. In this study the presence of VP mRNA. oxytocin (OT) mRNA and GRP mRNA was investigated in cell lines derived from SCLCs. Out of 26 cell lines 3 contained low amounts of VP mRNA (GLC-8, SCLC-2IH and NCI-H345) and 7 contained abundant GRP mRNA (GLC16. GLC-1-M13. SCLC-22H. NC1-H249, NC1-H345, NCI-H449 and NCIH450). The GRP mRNA-containingcell lines belong to the classic SCLC type, whereas VP mRNA was found in two classic and one variant cell line. None of the SCLC cell lines contained detectable levels of OT mRNA. Of the three VP-expressing SCLC cell lines. GLC-8 had the highest level of VP mRNA. Both the length of the transcript and the hybridization with different probes containing exons A and C of the VP gene suggest that the detected transcript is a normal VP messenger. SCLC GLC-8 contained low levels of VP immunoreactivity and VP receptors. In GLC-8 an autocrine role of VP may be sus­ pected.

Introduction

20-30% of all newly diagnosed lung tumors are classi­ fied as small cell lung carcinomas (SCLC) [1], These tumors show neuroendocrine features typical for amine precursor uptake and decarboxylation (APUD) cells [2], like the presence of dense-core granules and the produc­ tion of peptide hormones, e.g. adrenocorticotropin (ACTH) [3-5] gastrin-releasing peptide (GRP) [6], calci­ tonin (CT) [7] and vasopressin (VP) [8. 9], Ectopic hor­

Received: January 14. 1991 Accepted: April 18.1991

mone synthesis leads to a paraneoplastic syndrome in some cases [ 10, 11 ]. The syndrome of inappropriate secre­ tion of antidiuretic hormone (S1ADH) occurs in 25% of patients with SCLC and is associated with VP production by the tumor, giving rise to elevated plasma VP levels and impaired diuresis [10-12], Although the incidence of VP synthesis in SCLC has been reported to be as high as 60% [8]. immunoreactive VP has been infrequently detected in cell lines derived from those tumors [3, 13]. Sausville et al. [14] described one SCLC cell line (NCI-H378) which

Dr. J.P.H. Burbach Rudolf Magnus Institute Vondcllaan 6 N I.-2135 GD Utrecht (The Netherlands)

© 1992 S. Karger AG. Basel 1015-2008/92/060.V-0I36 $2.75/0

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Marken A.E. Verbeecka Jack P.M. Elandsa Lou F.M.H. de Leijb Charles H.C.M. Buysc Desmond N. Carneyd Gerold BepleC Anton J.M. Roebroeck( Wim J.M. Van de Venf J. Peter H. Burbacha

Materials and Methods Cell Cultures Growth conditions and media for SCLC cell lines have been described by De Lcij ct al. [26] and Posimus et al. [27] for GLC-1. GLC-2, OLC-3, GLC-4 and GLC-8. and were also used for GLC-16. GLC-28. GLC-35. GLC-36. C.LC-42. GLC1-M13 and GLC-CI: by Gazdar et al. [ 15] and Carney ct al. [ 16] for NCI-H155. NCI-H125. NCI-HI46. NCI-H249. NCI-H345. NCI-N4I7. NCI-H429. NCiH446. NC1-H449. NCI-H450, NC1-H524 and NC1-H526. and by Bcplcr et al. [28. 29] for SCLC-I6HC. SCLC-16HV, SCLC-21H and SCLC-22H. Isolation o f Total Cellular RNA Cells (5 X 107) were washed in saline-buffered phosphate, pelletted and homogenized in 0.7 ml of 4 M guanidinium isothiocyanate, 25 mM sodium citrate. 0 .1 M [3-mercaptoethanol. pH 7.0. The DNA was sheared by passing through a 19-gauge needle. The homogenate was extracted twice with an equal volume of phenofchlorofomrisoamyl-alcohol (25:24:1). RNA was precipitated overnight at -2 0 °C after supplementing 0.6 vol of 45 mM acetic acid in 100% ethanol. The mixture was centrifuged at 4°C for 20 min. the pellet was washed with 70% ethanol, resuspended in 10 m l/ Tris-HCI, I mM EDTA, pH 7.4 and reprecipitated by adding 0.1 vol of 3 Msodium acetate and 2.5 vol 100% ethanol. After precipitation the pellets were kept in 75% ethanol at - 80 °C. Before use the pellets were dissolved in 20 mA/ Tris-HCI. pH 7.4, I mM EDTA and the amount of RNA was determined spectrophotometrically. Northern Blot Analysis Fifty micrograms of total RNA was denaturated with glyoxal and dimethylsulfoxide in 10 mM phosphate buffer, pH 6.5. at 55 °C for 60 min, cooled on ice, loaded on a 1.4% agarose -10 mM phosphate gel and run at 10 V/cm for 3 h. Transfer of RNA from the gel to a nylon membrane (Hybond-N, Amersham, UK) was carried out over­ night in 25 mM phosphate buffer pH 6.5. The blot was briefly washed in 2 X SSC (1 X SSC = 0.15 M NaCl, 0.015 M sodium citrate. pH 7.0) and baked at 80 °C for 2 h. Filters were examined for

RNA loading and integrity by UV shadowing after this step and later hybridized to a [3-actin probe (see below). Filters were prehybridized in 50% (v/v) formamide. 6 X SSC. 8% dextrane sulfate. 0.5 mg/ml denaturated herring sperm DNA and 0.5% (w/v) defatted milk powder (Refit. Campina. Eindhoven. The Netherlands) at 50 °C for at least 6 h. Denaturated ,2P-labeled DNA probes were added to the prehybridization mix to a final concentration of at least 2 X I06 dpm/ml and incubated overnight. The filters were washed twice in 2 X SSC. 0.5% Refit, 0 .1% SDS at room temperature for 5 min. fol­ lowed by one wash at 50 °C for 30 min. one wash at 60 °C for 20 min and a final wash at 65 °C in 0.1 X SSC. 0.1 % SDS for 20 min. The filters were then exposed to X-ray film (Kodak XAR-5 of Fuji-film) with an intensifying screen at -8 0 °C for various lengths of time. Based on Northern blot-containing standards, the procedure can detect approximately 5 pg VP mRNA per sample. Probes Human VP- and OT-specific sequences were subcloned from genomic clones of the VP and OT genes (gift from J. Battey. NCI. Bethesda, Md., USA) [14], The VP-specific probes were the Hgi AIBamHI fragment of 523 bp containing 255 bp of the third exon (probe VP-C: fig. 1). and the 344-bp BamHI-Rsal fragment contain­ ing 169 bp of the first exon (probe VP-A: fig. 1). The OT-specific probe was the 700-bp Pstl-EcoRI fragment with 252 bp from the third exon (probe OT-C: fig. I). The human GRP probe w'as the EcoRl-insen of clone pBI2 [25] (gift from E. Sausvillc, NCI. Bethesda). The (i-actin probe was a rat cDNA of 1.5 kb (gift from J. Drouin, CRIM. Montreal, Canada) [30].The DNA fragments were labeled by random priming, resulting in probes with a specific activity of 0.5I X 101Jdpm/pg DNA. Amplification ofcDNA by Polymerase Chain Reaction (PC'R) In a final volume of 20 pi I X PCR buffer (10 mM Tris-HCI pH 8.3. 50 mM KCI. 1.5 mM MgCk 0.001 % (w/v) gelatin) including I mM of each dNTP. 20 U RNAsin. 40 pmol VP-specific primer. 5 pg of denaturated GLC-8 total RNA and 200 U reverse transcriptase were incubated at 42 °C for 120 min. The mixture was heated for 5 min at 100 °C, quickly chilled on ice and new components were added to make a total volume of 50 pi containing 10% DMSO. 40 pmol of the 5 'VP-specific primer and I unit Taq-polymerase [31]. After 25 cycles of 95 °C denaturation for I min. 57 °C annealing for 2 min and 72 °C primer extension for 3 min. the PCR products were analyzed on an agarose gel. The 5' VP - specific primer was the 30-mer oligonu­ cleotide 5'-CACGTCGACCTGCTACTTCCAGAACTGCCC-3' lo­ cated in the first exon, and the 3'VP-specific primer was a 25-mer oligonucleotide 5'-GCCCGTCCAGCTGCGTGGCGTTGCT-3' lo­ cated in the third exon. Radioimmunoassay Washed cells (3.5-10 X 107) were extracted in boiling acetic acid and the VP content in the extract was determined by radioimmu­ noassay using the W 1E antibody, which recognizes the amidated Cterminus of VP. [I25I]-VP was used as tracer. Details have been described by Liu and Burbach [32], Receptor-Binding Assay The presence of VP receptor was assessed as described by Elands et al. [33]. Briefly, freshly harvested cells (106 cells/ml) were incu­ bated in RPM1. containing 0.4 mM MgCL, I mg/ml BSA and [l25I]i/(CH2)5[Tyr(Me)2, Thr4, Tyr-NH29]OVT (0.01-0.28 n.V) at 21 °C

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expressed abundant amounts of VP mRNA. This cell line was derived from an SCLC patient with SI ADH, and was characterized as a classic-type cell line [ 15, 16]. It has been reported that VP has trophic and mitogenic effects on a number of cells [17-21], However, its growth-promoting role in SCLC cells is not clear. It has been proposed that VP, like GRP, the mammalian counterpart of bombesin, functions as an autocrine factor promoting tumor growth [22], GRP has mitogenic effects and its production by SCLC and other tumors has been demonstrated [6, 23, 24]. The presence of GRP transcripts in SCLC cell lines has also been found [5, 25]. Therefore, we investigated the incidence of expression of the VP gene and the closely related oxytocin (OT) gene in a number of SCLC cell lines of both the classic and the variant types, in comparison to expression of the GRP gene.

S VP VP precursor peptide

I

I

probe VP-A

probe VP-C S OT

OT precursor peptide

Ô5 w

NP-I

1

m



cc

y**' / ' c o : — co : ; E ; E : co;

cl

(O :

;

: < ; —i

01 Gene exon A

exon B

exon C

100 bp

lL»____________ probe OT-C

for 60 min. The nonspecific binding was determined by adding 10-6 M nonlabeled VP. The reaction was stopped by adding 9 ml ice-cold isotonic phosphate-buffered saline, after which the cells were washed three times in the same buffer. The radioactivity in the resulting cells was counted in a gamma counter. A small fraction of cells was used to control for nonspecific uptake.

Results

Total RNA of different lung cancer cell lines was screened for the presence of transcripts of the VP, OT and GRP genes on Northern blots. The results are listed in table 1. Two non-SCLC cell lines GLC-C1 and NC1H125, 14 classic and 12 variant SCLC cell lines were included. Hybridizations with the VP-specific probe (VPC) derived from the third exon showed VP transcripts in three SCLC cell lines: GLC-8, SCLC-21H and NCI-H345 (see table 1. fig. 2). GLC-8 and NC1-H345 are classic type SCLC cell lines [16. 27], SCLC-21 H has been classified originally as a variant type [28]. Hybridizations to the GRP-specific probe revealed 7 cell lines of the classic type with GRP transcripts: GLC-16. GLC1-M13, SCLC-22H. NCI-H345. NCI-H249, NCI-H449 and NCI-H450 (see table 1, fig. 3). The average length of the GRP transcripts was approximately 800 nucleotides (fig. 2). Hybridization of total RNA of the same cell lines with the OT-specific

138

III,

probe did not provide evidence for the presence of OT transcripts. The GRP mRNA content in GRP-positive cell lines was always severalfold higher than the VP mRNA signal in the three VP-positive cell lines, as esti­ mated from the ratio of peptide mRNA signal to (3-actin mRNA signal. The VP mRNA content in SCLC GLC-8 is estimated to be around 10 pg VP mRNA per 5 X 107 cells. With a similar RNA load on Northern blots, the sig­ nals of VP mRNA in NCI-H345 and SCLC-21 H. but not in GLC-8 (fig. 2), were always too weak for proper photo­ graphic reproduction, indicating that the level of VP transcripts in NC1-H345 and SCLC-21 H was lower than in GLC-8. The VP transcripts in GLC-8 and SCLC-21 H were also detected by hybridization to probe VP-A con­ taining the first exon. NCI-H345 was not investigated with the VP-A probe. The length of the VP transcript in GLC-8 was about 750-850 nucleotides, which is the same as mature mRNAs in the human brain [34] and similar to rat VP mRNA from the hypothalamus (fig. 2). The pres­ ence of authentic human VP mRNA in the GLC-8 cell line was also confirmed by PCR amplification of cDNA with specific VP primers (fig. 4). Restriction enzyme (Sma I) digestion of the isolated PCR product produced two fragments of the expected length (fig. 1) confirming its identity as the human VP cDNA [results not shown]. Since GLC-8 was most abundant in VP transcripts, this cell line was selected to investigate other aspects

Vcrbeeck/Elands/de Leij/Buys/Carney/ Bepler/Roebroeck/Van de Ven/Burbach

VP Gene Expression in Human SCLC Cell Lines

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Fig. 1. Structure of the human VP and OT gene and the organization of the VP- and OT-precursor peptide. Both genes comprise three exons (A, B. C). Arrowed lines repre­ sent the genomic fragments used as probes. Probe VP-A: VP *BamHI-Rsal fragment containing exon A (*BamHl site originates from the vector). Probe VP-C: VP HgiAIBamH! fragment containing exon C. Probe OT-C: OT Pstl-EcoRI fragment containing exon C. S = Signal peptides; NP I = neurophysin I; NP II = neurophysin II: CPP = Cterminus of prepropressophvsin.

NP-II C C P

Table 1. Neuropcplidc expression in lung cancer cell lines

Type

GLC-8 GLC-I6 GLC-28 GLC-35 GLC-36 GLC-42 GLC-I-M13 GLC-16HC SCLC-22H NCI-H146 NCI-H249 NCI-H345 NCI-H449 NCI-H450

C C C C C C C C C C C C C C

GLC-I GLC-2 GLC-3 GLC-4 SCLC-2IH SCLC-I6HV NCI-H 155 NCI-N417 NC1-H429 NCI-H446 NCI-H524 NCI-H526

V V V V V V V V V V V V

NCI-H 125 GLC-CI

N N

Rat

Human

Expression VP

OT

GRP

+ (+) -

_

_

-

+++ ++ +-H(+) +++ ++ +



-

(+) -

-

-

-

-

-

C = Classic SCLC type; V = variant SCLC type; N = non-SCLC type; - = not detected; (+) = very low; + = low; ++ = high; +++ = very high.

required for autocrine growth control, i.e. the presence of VP peptide and VP receptors. As determined by radioim­ munoassay using a C-terminal-spccific antibpdy [32] the VP content was 52.5 ± 20.6 pg VP equivalent per 108 cells. VP immunoreactivity was undetectable ( < l pg/ml) in the culture medium. Low amounts of VP receptors were detected in GLC-8 cells by receptor assays using [l25I]-i/(CH2)s[Tyr(Me)2.Thr4.Tyr-NH29]OVT as a ligand [33]. A scatchard analysis on 9 determinations with ligand concentrations between 0.01 and 0.28 nM (rcorr = 0.795) showed low but significant specific binding with a maxi­ mal binding capacity of 0.08 fmo!/106 cells and a dissocia­ tion constant of 0.12 nM.

GLC 2

GLC 3

GLC 4

GLC 8

GLC 16

GLC Hypo28 thalamus 18S rRNA

— VP mRNA





A | -

j

|

— GRPmRNA

• • B



Fig. 2. Transcripts of the VP and GRP genes in SCLC cell lines. Fifty micrograms total RNA of SCLC lines GLC-2. GLC-3. GLC-4. GLC-8. GLC-16. GLC-28 and 5 pg of rat SON (supraoptic nucleus) RNA. was electrophoresed. transferred to a nylon membrane and hybridized at 50 °C. The washing conditions were such that the rat VP mRNA could be visualized. A Hybridization to probe VP-C. B Hybridization to probe GRP. For details see 'Materials and Meth­ ods'.

Discussion

SCLC is a lung tumor associated with the production of a variety of hormones, amongst which the antidiuretic hormone VP. North et al. [8] showed elevated levels of VP in plasma and tumors in 60% of the investigated patients with SCLC. The percentage of patients diagnosed for the syndrome of inappropriate secretion of antidiuretic hor­ mone (S1ADH), which has been reported to be 20% of the cases [8]. is presumably due to the tumor-associated over­ production of VP [ 11, 12], Our results show that the inci­ dence of VP gene expression in cultured cells derived from SCLC tumors is low, i.e. approximately 10% of all

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Cell line

Fig. 3. Transcripts of the human GRP gene in SCLC cell lines. Fifty micrograms total RNA of human NC1-H526. NCI-H524. NCI-H450, NCI-H449, NCI-H446. NCIN417. NCI-H345. NCI-H429. NCI-HI55 and NCI-H125 was electrophoresed, trans­ ferred to a nylon membrane and hybridized to the GRP probe at 50°C. For details see ‘Materials and Methods’.

140

1

2

3

Fig. 4. PCR amplification of GLC-8 VP mRN'A. Five micrograms total RNA of human GLC-8 was transcribed into cDNA by reverse transcriptase and subsequently submitted to PCR with two specific VP primers, located in the first and in the last exon of VP. After 25 cycles, 20% of the PCR sample was analyzed on a 2.5% agarose gel. Lane 1 contains a length marker (pGem4 cut by Hinf-I. small fragments are invisible on the gel): lane 2 shows the PCR prod­ uct of GLC-8 cDNA; lane 3 shows the PCR product of rat hypothala­ mus cDNA. Samples containing FLO or cDNA from the SK-N-MCIXC cell line were negative [not shown]. For details see ‘Materials and Methods-.

Verbceck/Elands/dc Leij/Buys/Carney/ Beplcr/Roebroeck/Van de Ven/Burbach

VP Gene Expression in Human SCLC Cell Lines

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tested cell lines. Hybridization with human VP-specific probes on Northern blots revealed 3 cell lines with VP transcripts out of 26 different SCLC cell lines. GRP is more frequently expressed in this collection of SCLC cells (7 out of the 26 tested SCLC cell lines) and has been shown to be a mitogenic factor for tumor [22, 23] as well as normal cells [35]. VP has also been proposed as a growth factor for SCLC. based on the trophic and mito­ genic effects on a number of cells [ 17-21 ]. However, from our data we conclude that VP expression is not a general feature of all SCLC cell lines, but is rather restricted to a minor population. It suggests that VP does not function as a general autocrine growth factor in all SCLC cell lines. The lack of a stimulating effect of exogenous VP on the growth of SCLC cell lines has been reported [13], On the other hand, it has recently been reported that VP mobil­ izes calcium in 5 SCLC cell lines [36], We have not sys­ tematically investigated whether VP has growth-promot­ ing activity per se in one particular VP-expressing SCLC cell line, e.g. GLC-8. The results that the level of VP immunoreactivity is low in GLC-8 extracts and a low number of VP receptors is present in GLC-8, shows that prerequisites for autocrine effects, i.e. the presence of pep­ tide and receptor, exist in this particular cell line. Allough the capacity of VP-binding sites was low, the value is com­ parable to binding capacities for neurohypophyseal pep­ tides on human blood peripheral mononuclear cells, in which VP reportedly has a biological effect, i.e. stimula­ tion of ACTH production [37],

The expression of the GRP gene was only detected in SCLC cell lines of the classic type. Classic cells are APUD cells, displaying L-dopa carboxylase and neuron-specific enolase activity. Generally, they have a long doubling time and no c-myc proto-oncogene amplification. This type of cells is characterized by the presence of elcctronmicroscopically detectable cytoplasmatic dense-core granules, resembling neurosecretory vesicles in which peptide hormones are processed and stored. The VP mRNA-positive cell lines GLC-8 and NCI-H345 have been classified as classic types. The GLC-8 cell line has no c-myc amplification but does show N-myc proto-onco­ gene amplification [26, 38], A low level of VP mRNA was also found in SCLC-21H. SCLC-21H has been originally typed variant [28] because of the amplificated c-myc pro­ to-oncogene, undetectable L-dopa decarboxylase activity and low neuron-specific enolase levels. However, this cell line contains dense-core granules and expresses the 7B2 polypeptide which is associated with neurons and endo­ crine cells [39, 40], Our results show that it also expresses a peptide hormone gene. It is evident from our results that not all of the classic SCLC cell lines produce substantial amounts of GRP, as has been suggested before [25]. Only 7 out of the 14 classic SCLC cell lines were positive for the presence of GRP mRNA. Various other peptides may be produced by the GRP- and VP-deficient cells, such as calcitonin/CGRP-related peptides [41]. A search for the expression of other peptide genes is in progress. Our results show a low to very low level of VP trans­ cripts in the VP-expressing cell lines. Several reasons may account for this finding. Firstly, the VP mRNA content per cell may be very low. Secondly. VP transcripts mayhave a high turnover rate. Thirdly, the VP gene may be expressed by only a subset of cells. The latter possibility seems likely since a rough estimation of the levels of VP

mRNA (about 10 pg per 5 X 107 cells) results in a copy number per cell which is lower than I. The expression of the GRP gene on the other hand was much more abun­ dant, which is consistent with the high content of GRP immunoreactivity in these cell lines, as previously de­ scribed [6. 13, 24]. The length of the VP mRNA in cell line GLC-8 was about 750-850 nucleotides, similar to the VP mRNA in the human hypothalamus [34]. Since abnormal VP trans­ cripts lacking exon A have been observed [42], the nature of the VP transcript in the cell line GLC-8 was investi­ gated in more detail. Hybridizations to probes derived from the first and the last exon of the human VP gene, as well as the amplification of a correct VP cDNA by PCR indicated that this VP gene transcript is the normal VP mRNA and may thus be functional for VP precursor syn­ thesis. However, the data indicate that the concentration of immunoreactive VP in GLC-8 is low. Since our antise­ rum is directed against the amidated C-terminus of VP [32,43], the VP precursor will not be detected. Recently, a SCLC cell line has been described which also expresses the VP gene but does not process the VP precursor [44], Another study also indicated the absence of immunoreac­ tive VP in a number of SCLC cell lines [13]. On the other hand, one SCLC cell line (NCI-H378) has been described having an abundance of both VP mRNA and immunore­ active VP [14], Yamaji et al. [12] showed that the VP pre­ cursor circulates in blood of patients with SIADH. There­ fore, it may be that the VP precursor is not or incom­ pletely processed in SCLC and derived cell lines. The nature of the VP gene products needs to be investigated. The VP-expressing SCLC cell line GLC-8 has provided a valuable model for studies on VP gene regulation in neo­ plastic tissue [45, 46].

1 Carney DN. De Lcij L: Lung cancer biology. Semin Oncol 1988:15:199-214. 2 Gazdar AF. Carney DN. Russell EK. et al: Establishment of continuous, clonable cultures of small cell carcinoma of the lung which have amine precursor uptake and decarboxylation cell properties. Cancer Res 1980:40:35023507. 3 Sorenson GD. Pettcngill OS. Brinck-Johnscn T. et al: Hormone production by cultures of small cell carcinoma o f the lung. Cancer 1981 : 47:1289-1296.

4 Rees LH: The biosynthesis of hormones by non-endocrinc tumors - A review. J Endocri­ nol 1975;675:143-175. 5 White A. Stewart MF. Farrel WE. et al: Pro­ opiomelanocortin gene expression and peptide secretion in human small cell lung cancer lines. J Mol Endocrinol 1989;3:65-70. 6 Moody TW, Pert CB. Gazdar AF. cl al: High levels of intracellular bombesin characterize human small cell lung carcinoma. Science 1981;214:1246-1248. 7 Silva OL. Becker K.L. Primack A. el al: Ectopic secretion of calcitonin in oat cell carcinoma. N Engl J Med 1974:290:1122-1124.

8 North WG. Maurer LH. O’Donnell JF: The neurophysins and small cell lung cancer; in Greco FA (ed): Biology and Management of Lung Cancer. Den Haag. NijhofT, 1983. pp 143-169. 9 Yamaji T, lshibashi M. Kalayama K: Nature of the immunoreactive neurophysins in ectopic vasopressin producing oat cell carcinomas of the lung. J Clin Invest 19 8 1;68:338-389. 10 Hamilton BP. Upton GV. Amatruda TT J r Evidence for the presence of ncurophvsin in tumors producing the sandrome of inappro­ priate antidiurcsis. J Clin Endocrinol Metab 1972;35:764-767.

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Expression of the vasopressin and gastrin-releasing peptide genes in small cell lung carcinoma cell lines.

Various polypeptide hormones including vasopressin (VP) and gastrin-releasing peptide (GRP) are produced by small cell lung carcinomas (SCLC). VP as w...
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