Vasopressin and vasopressin-receptor immunoreactivity in small-cell lung carcinoma (SCCL) cell lines: disruption in the activation cascade of V1a-receptors in variant SCCL

CANCER LETTERS Cancer Letters 82 (1994) 167-174

ELSEVIER

Vasopressin and vasopressin-receptor immunoreactivity in small-cell lung carcinoma (SCCL) cell lines: disruption in the activation cascade of V,,-receptors in variant SCCL Michael

J. Fay *, Andrew

S. Friedmann,

Xiao-ming

Yu, William G. North

Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA

Received 17 November 1993; revision received 9 May 1994; accepted I I May 1994

Abstract Four classical and three variant small-cell carcinoma of the lung (SCCL) cell lines were examined for vasopressin and vasopressin V,,-receptor immunoreactivity. One of these classical cell lines, NCIH345, and one variant cell line, NCI-H82, were further investigated for binding of Vi and V, vasopressin-receptor antagonists, vasopressin-induced calcium mobilization, and vasopressin-induced thymidine uptake. All classical and variant SCCL cell lines examined contained vasopressin and vasopressin-receptors as determined by immunocytochemistry. Both NCI-H82 and NCIH345 demonstrated similar binding patterns with the V, and V, vasopressin-receptor antagonists, indicating the presence of both receptor subtypes. For the classical cell line (NCI-H345), vasopressin (1 PM) induced an increase in cytosolic free calcium, while the peptide was ineffective at increasing cytosolic calcium in the variant cell line (NCIH82). However, vasopressin (0.1 or 1 PM) was unable to stimulate thymidine uptake in the classical (NCI-H345) or variant (NCI-H82) cell lines for the conditions used. These results indicate that both classical and variant SCCL produce vasopressin, and vasopressin V,, and V, receptors. In the variant cell line, there appears to be a disruption in the activation cascade for V,, receptors as indicated by the lack of vasopressin-induced calcium mobilization. Keywords:

Vasopressin;

Vasopressin-receptors;

Small cell lung carcinoma;

1. Introduction Small-cell carcinoma of the lung is believed to undergo a progression from a classical to a variant form. This transformation is associated with a loss of neuroendocrine features, increased cell proliferation, amplification of the c-myc proto-oncogene, * Corresponding author.

Calcium

mobilization;

Thymidine

uptake

and resistance to therapeutic intervention

[4,5,7,15]. One of the neuropeptides produced by SCCL in vivo and in vitro is vasopressin, and the vasopressin generelated product neurophysin [6,10,18,20,26,30]. In addition to well-established antidiuretic and vasoconstrictive effects, vasopressin is also known to act as a growth factor [27,31]. The growth promoting effects of vasopressin are believed to occur through activation

0304-3835/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3835(94)03398-3

of vasopressin

Vt,-receptors

with subse-

168

M.J. Fav et al. /Cancer

quent generation of inositol triphosphate, calcium mobilization, activation of protein kinase C, and the transcriptional activation of early genes such as cfos [22]. A number of studies have shown that vasopressin induces calcium mobilization and increased clonal growth of SCCL cell lines [2,3,13,22,28]. The goal of the present study was to investigate the production of vasopressin and vasopressin receptors by classical and variant SCCL cell lines, and to examine vasopressin-induced calcium mobilization and thymidine uptake in one classical and one variant cell line.

2. Materials and methods 2.1. Cell lines Classical SCCL cell lines NCI-H69 and NCIH345, and variant lines, NCI-H82 and NCI-H446, were obtained from the American Type Culture Collection (Rockville, MD). Classical cell lines, UMC-5, UMC-31, and the variant line, UMC-19, were generously supplied by Dr. Stephen L. Graziano (SUNY-Health Science Center, Syracuse, NY). These SCCL cell lines have been characterized in detail elsewhere [5,7,9]. All the cultures were maintained in RPMI-1640 growth medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FBS (Hyclone Labs., Logan, UT), and 50 pg/ml gentamicin (United States Biochemical Corp., Cleveland, OH). Every 2-3 days, the growth medium was changed or the cells were subcultured. For the non-adherent cell lines (NCI-H69, NCI-H82, NCI-H345, UMC-5, and UMC-31) cells were pelleted (500 x g, 5 min) and resuspended at the appropriate density in fresh RPM1 1640 medium. For the adherent cultures (NCI-H446 and UMC19), cells were removed from the flask by brief exposure to 0.25% trypsin (Hazleton Biologics, Inc., Lenexa, KS), pelleted (500 x g, 5 min) and resuspended at the appropriate density in fresh growth medium. For the immunocytochemical detection of vasopressin and vasopressin receptors, cells were seeded into g-well tissue culture chamber slides (Nunc, Inc., Naperville, IL) at a density of 104-lo5 cells per well. Slides were previously coated with poly-D-lysine (mol. wt. 70 000-150 000, Sigma) to facilitate cell adherence.

Lett. 82 (1994)

167-174

2.2. Antibodies The polyclonal antibody preparation directed against vasopressin (Gonzo-3) was generated here by injecting a rabbit with purified vasopressin coupled to thyroglobulin. This antibody preparation, which appears to be chiefly directed against the ring moiety of vasopressin, was shown to have low crossreactivity with oxytocin in RIA (< 0.01%). The polyclonal antibody preparation directed against the vasopressin Vi, receptor (SDSAVP) was generously provided by Dr. Carlos B. Gonzalez (Universidad Ausrtal de Chile, Valdivia, Chile). The generation and characterization of this antibody preparation has been described elsewhere in detail [8]. Antibodies were diluted (1: 1OO- 1:800) in PBS with 10% normal goat serum (Gibco BRL, Grand Island, NY) for use in immunocytochemistry. 2.3. Immunocytochemistry Cells were washed with phosphate-buffered saline (Sigma Chemical Co., St. Louis, MO) and fixed for 2 min in acetone. The fixative was removed with PBS (2 x 3 min) and the cells then stained at room temperature for vasopressin or vasopressin Vi, receptors, and reactivity visualized using the avidinbiotin complex (ABC) procedure [ 121. Non-specific interactions were blocked with 1.5% normal goatserum (20 min). After blocking, cells were washed (2 x 3 min) with PBS, and incubated for 1 h with either Gonzo-3 or SDSAVP. All preparations were washed with PBS (2 x 3 min), and incubated for 30 min with biotinylated goat anti-rabbit IgG (20 &ml, Vector Labs, Burlingame, CA). Unbound secondary antibody was removed by washing with PBS (2 x 3 min). Preparations were then incubated with avidin-peroxidase complex (25 &ml, Vector Labs) for 30 min, followed by 2 x 3 min washings with PBS. Pretreatment of the cells with peroxide was found to be unnecessary because these SCCL cells were found to have insignificant levels of endogenous peroxidase activity. Visualization of the antigen-antibody complex was performed by the peroxidase oxidation of 3,3’-diaminobenzidine (DAB) to an insoluble brown precipitate (DAB substrate kit, Vector Labs). Antibody specificity was determined by the identification of magnocellular neurons in acetone-fixed human hypothalamus using Gonzo-3, and hepatocytes in acetone-fixed

M.J. Fay et al. /Cancer Leti. 82 (1994) 167-174

human liver with SDSAVP. Negative controls incubated with 1.5% goat-serum instead of primary antibody confirmed that staining was not due to non-specific reactivity of the secondary antibody. 2.4. Radioligand binding assay The binding of [3H]V, and [3H]V, vasopressinreceptor antagonists was performed using a modification of a previously described method [29]. At 48 h prior to experimentation, NCI-H345 cells were subcultured 1:2, and NCI-H82 cells were subcultured 1:5, as previously described. At 24 h prior to experimentation, the cells were resuspended in serum-free RPMI- 1640 medium containing insulin (5 &ml), transferrin (5 &ml), and sodium selenite (5 ng/ml) (ITS, Sigma). Cells were washed with PBS, pelleted, and lo6 cells were placed in serum-free RPM1 1640 medium (0.5 ml) containing 0.1 mg/ml BSA (fraction V, Sigma), and 0.1 PM of the [3H]vasopressin antagonist (Vi antagonist, ( d(CHz)sTyrMe-[3H]AVP), 45.1 Ci/mmol; V2 antagonist, ( d(CH2)5D-Ile2,11e4, des-Gly--NH2 [3H]AVP)), 65.5 Ci/mmol; Dupont/NEN, Wilmington, DE). Specific details about these antagonists are described elsewhere [ 14,161. After a 30-min incubation at room temperature, the cells were pelleted and washed (3 x) with ice-cold PBS. The cell pellets were resuspended in 0.5 ml of PBS and added to 10 ml of HydrofluorrM scintillation cocktail (National Diagnostics, Manville, NJ). Samples were counted 3 x for 1 min using a Packard model 3255 liquid scintillation spectrometer. 2.5. Calcium assay Changes in cytosolic calcium were monitored using the cell permeable acetoxymethyl ester form of the dye Indo-l [l 11.Cells were subcultured as described for the radioligand binding assay. Cells (2 x lo6 cells/ml) were loaded with 5 PM Indo1AM (Molecular Probes, Inc., Eugene, OR) for 45 min at 37°C in serum free RPMI-1640. They were then pelleted (500 x g, 5 min), washed (2 x ) with PBS, and resuspended at lo5 cells/ml in PBS containing 1 mg/ml dextrose and 1 mg/ml bovine serum albumin (fraction V, Sigma). Calcium mobilization was monitored using a Facstar Plus flow cytometer (Becton Dickinson, San Jose, CA). Cells were excited at 356 nm, and emission monitored at 405 nm

169

(calcium bound Indo), and at 485 nm (unbound Indo). Vasopressin (Calbiochem, La Jolla, CA) was administered through the injection port of the cytometer after approximately 20 s of baseline had been established to give a final peptide concentration of 1 PM. Previous studies examining vasopressin-induced calcium mobilization in SCCL have shown a saturating effect at this concentration [ 131. In a follow-up study, vasopressin was administered to give a final concentration of 0.001 PM to 10 PM. As a positive control, the calcium ionophore, 4bromo A-23187 (Molecular Probes, Inc.) was administered in the same manner as peptide to give a final concentration of 1 PM. As a negative control, the same volume of PBS was administered through the injection port. The ratio of bound to unbound (405 nm485 nm) Indo and the percentage of cells responding was determined over time using Lysis II software (Becton Dickinson). 2.6. Thymidine uptake Cells were subcultured at lo5 cells/ml in RMPI1640 medium, supplemented with 0.1% BSA (Sigma) and ITS (Sigma). After 3 days, 5 x lo3 cells/well were seeded into 96-well plates. Cells were allowed to attach for 4 h, and vasopressin was added to give a final concentration of 0.1 PM or 1 PM. Cells were incubated with vasopressin for 48 h, and with 1 &i/well [3H]thymidine (82.4 Ci/mmol; Dupont/NEN) for the final 18 h. Cells were harvested using a Wallac cell harvester (Wallac Inc., Gaithersburg, MD) and counted for [3H]thymidine. 3. Results All four classical cell lines, NCI-H69, NCI-H345, UMC-5, and UMC-31 as well as the three variant cell lines, NCI-H82, NCI-H446, and UMC-19, demonstrated a qualitatively similar pattern of staining for vasopressin and vasopressin receptors. Fig. 1 demonstrates positive staining for vasopressin and vasopressin-receptors in the classical cell line NCIH345, and the variant cell line NCI-H82. Both NCI-H345 and NCI-H82 demonstrated similar binding patterns with the Vi and V2 vasopressin-receptor antagonists (see Fig. 2). Using Student’s t-test, there was no significant difference between the classical and the variant cell lines with

170

hf. J. Fay et al. /Cancer

Letr. 82 (1994)

167-174

Fig. 1. Representative staining for vasopressin and vasopressin Vt, receptors in a classical (NCI-H345) and a variant (NCI-H82) cell line using immunocytochemistry. The dilution of each primary antibody used was 1:200. These results are representative of duplicate or triplicate independent experiments. (A) Positive staining of NCI-H345 cells with anti-vasopressin polyclonal antisera; (B) positive staining of NCI-H345 cells with anti-vasopressin V,, receptor polyclonal antisera; (C) positive staining of NC1 H82 cells with antivasopressin polyclonal antisera; (D) positive staining of NCI-H82 cells with anti-vasopressin VI,-receptor polyclonal antisera (magnification x 1462).

M.J.

62-Vl

345.Vl

62-V2

Fay et al. I Cancer Leli. RZ (199441 167-174

345.V2

Fig. 2. Binding of 0.1 PM [3H]Vt and [3H]V, vasopressin antagonists to classical (NCI-H345) and variant (NCI-H82) SCCL cell lines. The data are presented as the mean counts/min per IOr’ cells f the standard error of the mean, N = 5 per group. These results are representative of duplicate independent experiments. Using Student’s r-test there was no significant difference between the classical and variant cell lines with the binding of V, antagonist. 400 -

regard to the binding of the Vi and V2 vasopressin receptor antagonists. As shown in Fig. 3A, vasopressin (1 PM) caused a transient rise in cytosolic calcium in the classical cell line, NCI-H345, with a 19% maximal increase in cytosolic calcium above baseline occurring 25 s following injection of the peptide. The variant cell line, NCI-H82, exhibited no such increase in cytosolic calcium when exposed to this concentration of vasopressin (Fig. 3B). In addition, 0.001, 0.01, 0.1, and 10 PM vasopressin were ineffective at causing calcium mobilization in the variant cell line, however, calcium mobilization was observed at all these concentrations of vasopressin with the classical cell line (data not shown). Also, in the classical line, a 1 PM concentration of the calcium ionophore, 4bromo A-23 187, rapidly increased cytosolic calcium - im

-I 100

A

350 - +

171

Ratio (405nm/465nm)

-

% Responding

-60

90

- 70

- 30

a

-20

ae

-60

F

-50

- 30

z :: a m

- 20

ae

- 40

300

- 10

.

150

0

, 50

.

, 100

.

, 150

.

1 200

.

/

- 10

0

0

250

50

Time (set)

600 r

100

150

200

250

Time (set)

im

650

C

90

I

70 60

550

600

50

450 400

4o 30

350

20

300

60

100

150

Time (set)

200

250

g c a

$

IO 0

250

0

4 z

0

50

100

150

200

250

Time (set)

Fig. 3. Effect of 1 PM vasopressin or I pM 4-bromo A-23187 on cytosolic free calcium in a classical (NCI-H345) and variant (NCIH82) SCCL cell line. Vasopressin or 4-bromo A-23 187 were injected after approximately 20 s of baseline, as indicated by the arrow. The data are expressed as the ratio of calcium bound indo-l AM (405 nm) to unbound indo-1AM (485 nm), and as the percentage of cells responding. (A) Response of the classical cell line (NCI-H345) to I PM vasopressin; (B) response of the variant SCCL cell line (NCI-H82) to I pM vasopressin; (C) response of the classical cell line (NCI-H345) to I gM 4-bromo A-23187; (D) response of the variant cell line (NCI-H82) to 1PM cl-bromo A-23 187. These values are representative of duplicate or triplicate independent experiments.

M.J. Fay et al. /Cancer

172

control

0.1

pM

AVP

1

pM

AVP

AVP

1

uM

AVP

B

control

Fig. 4. Effect on the uptake iant (B) SCCL as the mean

0.1

pM

of vasopressin (0. I and I PM) incubation (48 h) of [3H]thymidine (18 h) by classical (A) and var(NCI-H345 and NCI-H82). The data are presented countsimin (x IO’) f the standard error of the

mean, N = 12 per group. These results duplicate independent experiments.

are representative

of

with levels remaining elevated throughout the measurement period (Fig. 3C). In the variant cell line, 1 PM of the calcium ionophore, 4-bromo A-23 187, elicited a rapid and transient elevation in cytosolic calcium (Fig. 3D). The injection of PBS had no effect on calcium mobilization with either the classical or variant cell line (data not shown). Fig. 4 demonstrates that vasopressin had no effect on the uptake of [3H]thymidine by either the classical (Fig. 4A) or the variant (Fig. 4B) cell line. 4. Discussion Vasopressin and other neuropeptides are common products of small-cell carcinoma of the lung [6,10,17,18,20,23,26,30]. In addition to established physiological roles, many of these neuropeptides are

Lett. 82 (1994)

167-174

now known to act as growth modulating agents and growth factors [27]. The mitogenic action of vasopressin and many other neuropeptides is believed to be exercised through activation of the inositol phosphate pathway and an increase in intracellularfree calcium. The immunocytochemical studies here provide convincing evidence that classical and variant forms of SCCL produce vasopressin, or a vasopressin-like peptide. Bepler et al. [ 11found non-detectable levels of arginine vasopressin in SCCL cell lines. Differences between these findings and those of Bepler et al. could relate to differences in antibody specificity and/or the sensitivity of the immunological methods employed. Additionally, both variant and classical cell lines appear to have a similar capacity to generate immunoreactive forms of vasopressin receptors. Although the receptor antibody preparation used in the present studies was generated against the Vi, receptor, cross-reactivity of this antibody with other vasopressin receptor subtypes (Vlb, V,) on these cells cannot be ruled out since both the classical and variant cell lines exhibited binding with both the Vi and V2 vasopressinreceptor antagonists. Vasopressin induced a rise in cytosolic calcium in the classical cell line, NC1 345. This finding has been demonstrated in NCI-H345 and other classical SCCL cell lines by other investigators [2,3,13,21, 22,281. In contrast, our failure to demonstrate a change in cytosolic free calcium in response to vasopressin with the variant cell line, NCI-H82, is in agreement with published results of Bunn et al. [2] with other variant SCCL cell lines. In subsequent experiments, concentrations of vasopressin as high as 10 PM were unable to elicit calcium mobilization, while in the classical cell line, calcium mobilization was observed at concentrations as low as 0.001 PM (data not shown). However, both the classical and variant cell lines demonstrated a similar binding pattern with the [ 3H]V, vasopressin-receptor antagonist. Therefore, these studies suggest that the refractiveness of NCI-H82 does not seem to be due to a reduced number of vasopressin receptors, nor to small changes in receptor affinity for the ligand. Refractiveness of NCI-H82 and other variant SCCL cell lines to the actions of vasopressin could therefore be due to the expression of null receptors

M.J. Fay et ~1. /Cancer

by this form of the tumor. In this respect, it is of interest that a rat mammary tumor cell line has been reported to contain high affinity and low affinity vasopressin binding sites, and that the low affinity binding site is uncoupled to the inositol phosphate transduction cascade [29]. The preferential expression of low affinity vasopressin ‘binding sites’ by variant SCCL is readily testable through binding studies using radiolabelled ligand. An interesting observation from the present studies is that the variant cell line, NCI-H82, had higher basal cytosolic calcium levels than those found in the classical cell line, NCI-H345. This finding is in accordance with an earlier finding that SCCL cell lines selected for multidrug resistance have elevated cytosolic calcium [ 191. Also when compared to the classical cell line, the variant cell line appeared to have a greater capacity to sequester intracellular calcium as indicated by the rapid decrease in fluorescence over time when the ionophore was administered. The present results demonstrate that variant and classical SCCL cell lines produce immunoreactive vasopressin and vasopressin receptors. However, on the basis of the one variant line tested in this study and the study of Bunn et al. with other variant cell lines, refractiveness of variant SCCL to vasopressininduced calcium mobilization appears to be a common occurrence. Bunn’s results taken together with the results of the present study indicate that a refractiveness to peptide-induced calcium mobilization may be of importance in the progression of SCCL from the classical to the variant form. It is of interest to note that neoplastic progression of rat tracheal epithelial cells is associated with a decreased dependency on growth factors for clonal growth [25]. In the present studies, 0.1 and 1 PM vasopressin did not stimulate thymidine uptake in either the classical or the variant cell line. This apparent lack of a mitogenic effect of vasopressin, as measured by thymidine uptake, indicates that calcium mobilization alone is not necessarily an indication of a mitogenic effect. Similar results have been published by Takuwa et al. [24], who found that the peptides bombesin and tachykinin stimulated calcium mobilization but not thymidine uptake in SCCL cell lines. Possibly, other factors are required along with peptides like vasopressin for such a mitogenic effect to occur. Nevertheless, since SCCL

Lett. 82 (1994)

1’67-174

173

cell lines produce not only vasopressin, but also both V,and V2 vasopressin receptors, it is likely that vasopressin is performing an important autocrine function in these cells. Therefore, the data obtained in this study lead to the proposition that refractiveness to vasopressininduced calcium mobilization is due to the expression of non-functional or null, vasopressin Vi, receptors on variant SCCL. This could involve alterations in receptor structure, or a dislocation of the receptor from the transduction cascade in some manner. Acknowledgement This work was supported in part by PHS awards (CA 19613 and CA 46551). Michael J. Pay was supported as a postdoctoral fellow on NIH training grant T32 DK 07508. The Fannie E. Ripple flow cytometry facility at Dartmouth medical school is supported by the core grant of the Norris Cotton Cancer Center (CA 23108). We thank Dr. Alice Given and Gary Ward for their advice and technical assistance with the flow cytometry analysis, and Dr. Ann Griffin and Dr. Keith Wegmann for their assistance with the thymidine uptake studies. The technical assistance of Susan Gagnon and Sara Pai is gratefully appreciated. References 111 Bepler,

G., Rotsch, M., Jaques. G.. Haeder, M., Heymanns, J., Hartogh, G.. Kiefer, P. and Havemann, K. (1988) Peptides and growth factors in small cell lung cancer: production, binding sites, and growth effects. J. Clin. Res. Clin. Oncol., 114, 235-244. 121 Bunn, P.A., Dienhart, D.G., Chan, D. Puck, T.T., Tagawa. M., Jewett, P.B. and Braunschweiger, E. (1990) Neuropeptide stimulation of calcium flux in human lung cancer cells: delineation of alternative pathways. Proc. Natl. Acad. Sci. USA, 87, 2162-2166. [31 Bunn, P.A., Dienhart, D.G., Chan, D., Tagawa, M. and Jewett, P. (1992) Effects of neuropeptides on human lung and breast cancer cells. Monogr. Natl. Cancer Inst., 13, 145-151. 141 Carney. D.N., Mitchell, J.B. and Kinsella, T.J. (1983) In vitro radiation and chemotherapy sensitivity of established cell lines of human small cell lung cancer and its large cell morphological variants. Cancer Res., 43, 2806-28 I 1. 151 Carney, D.N., Gazdar, A.F., Bepler, G., Guccion, J.G., Marangos, P.J., Moody. T.W. Zweig. M.H. and Minna.

174

M.J. Fay et al. /Cancer

J.D. (1985) Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res., 45, 2913-2923. I61 Friedmann, AS., Malott, K.A., Memoli. V.A., Pai, S.I., Yu, X. and North, W.C. (1993) Products of vasopressin gene expression in small-cell carcinoma of the lung. Br. J. Cancer, 69, 260-263, 1994. 171 Gazdar, A.F., Carney, D.N., Nau, M.M. and Minna, J.D. (1985) Characterization of distinctive biochemical, morphological, and growth properties. Cancer Res., 45, 2924-2930. PI Gonzalez, C.B., Caorsi, C.E., Reyes, C.E., Troncoso, S. and Barra, V. (1993)Identification and purification of the vasopressin receptor from rat liver membranes. Ann. N.Y. Acad. Sci., 689. 526-529. [91 Graziano, S.L., Pfeifer, A.M., Testa, J.R., Mark, G.E., Johnson, B.E., Hallinan, E.J., Pettengill, O.S., Sorenson, G.D., Tatum, A.H., Brauch, H., Zbar, B. and Flejter. W.L. (1991) Band 3~25, in the 3p deletion of small-cell lung cancer. Genes Chromosomes Cancer, 3, 283-293. 1101 Gross, A.J., Steinberg, S.M., Reilly, J.G., Bliss, D.P., Brennan, J., Tramle, P. Simmons, A., Phelps, R., Mulshine. J.L., Ihde, D.C. and Johnson, B.E. (1993) Atrial natriuretic factor and arginine vasopressin production in tumor cell lines from patients with lung cancer and their relationship to serum sodium. Cancer Res., 53, 67-74. (111 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem., 260, 3440-3450. [I21 Guesdon, J.L., Ternynck, T. and Avrameas, S. (1979) The use of avidin-biotin interaction in immunoenzymatic techniques. J. Histochem. Cytochem., 27, 1131-I 139. u31 Hong, M. and Moody, T.W. (1991) Vasopressin elevates cytosolic calcium in small cell lung cancer cells. Peptides, 12, 1315-1319. 1141 Kruszynski, M., Lammek, B. and Manning, M. (1980) (I(P-Mercapto-P,P-cyclopentamethylenepropionicacid), 2(O-methyl)tyrosine]arginine-vasopressin and [ l-(pmercapto-fi,&cyclopentamethylenepropionic acid)] arginine-vasopressin, two highly potent antagonists of the vasopressor response to arginine-vasopressin. J. Med. Chem., 23, 364-368. [I51 Little, C.D., Nau. M.N., Carney, D.N., Gazdar, A.F. and Minna, J.D. (1983) Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature, 306, 194-196. M., Misicka, A., Olma. A., Klis, W.A., 1161 Manning, Bankowski, K., Nawrocka, E., Kruszynski, M., Kruszynski, M., Kolodziejczyk, A., Cheng, L., Seto, J., Wo, N.C. and Sawyer, W.H. (1987) C-Terminal deletions in agonistic and antagonistic analogues of vasopressin that improve their specificities for antidiuretic (V,) and vasopressor (V,) receptors. J. Med. Chem., 30, 2245-2252. 1171 Maurer, L.H. (1985) Ectopic hormone syndrome in small cell carcinoma of the lung. Clin. Oncol., 4, 1289-1296. 1181 North, W.C. (1991) Neuropeptide production by small cell carcinoma: vasopressin and oxytocin as plasma markers of disease. J. Clin. Endocrinol. Metab., 73, 1316-1320.

Len. 82 (1994)

167-I 74

(191 Nygren, P., Larsson, R., Gruber, A., Peterson, C. and Bergh, J. (1991) Doxorubicin selected multi-drug-resistant small cell lung cancer cell lines characterized-by elevated cytoplasmic Ca2+ and resistance modulation by verapamil in absence ofp-glycoprotein overexpression. Br. J. Cancer, 64, 1011-1018. m

Sausville, E., Camey, D. and Battey, J. (1985) The human vasopressin gene is linked to oxytocin gene and is selectively expressed in a cultured lung cancer cell-line. J. Biol.

Chem., 260, 10236- 10241. 1211 Sethi, T. and Rozengurt, E. (1991) Multiple neuropeptides stimulate clonal growth of small cell lung cancer: effects of bradykinin, vasopressin, cholecystokinin, galanin, and neurotensin. Cancer Res., 51, 3621-3623. WI Seth, T., Langdon, S., Smyth, J. and Rozengurt, E. (1990) Growth of small cell lung cancer cells: stimulation by multiple neuropeptides and inhibition by broad spectrum anv31

1241

[251

WI

1271 1281

1291

1301

1311

tagonists Cancer Res., 52, 2737s-2742s. Sorenson, D.D., Pettengill, OS., Brinck-Johnsen, T., Cate, C.C. and Maurer, L.H. (1981) Hormone production by cultures of small-cell carcinoma of the lung. Cancer, 47, 1289- 1296. Takuwa, N., Takuwa Y., Ohue, Y., Mukai, H., Endoh, K., Yamashita, K., Kumada, M. and Munekata, E. (1990) Stimulation of calcium mobilization but not proliferation by bombesin and tachykinin neuropeptides in human small cell lung cancer cells. Cancer Res., 50, 240-244. Thomassen, D.G. (1993) Neoplastic progression of rat tracheal epithelial cells is associated with a reduction in the number of growth factors required for clonal proliferation in culture. In Vitro Cell. Dev. Biol., 29A, 498-504. Verbeeck, M.A.E., Elands, J.P.M., de Leij, L.F.M.H., Buys, C.H.C.M., Carney, D.N., Bepler, G., Roebroeck, A.J.M., Van de Ven, W.J.M. and Burbach, J.P.H. (1992) Expression of the vasopressin and gastrin-releasing peptide genes in small cell lung carcinoma cell lines. Pathobiology, 60, 136-142. Woll, P.J. and Rozengurt, E. (1989) Neuropeptides as growth regulators. Br. Med. Bull., 45, 492-505. Wall, P.J. and Rozengurt, E. (1989) Multiple neuropeptides mobilise calcium in small cell lung cancer: effects of vasopressin, bradykinin, cholecystokinin, galanin and neurotensin. Biochem. Biophys. Res. Commun., 164, 66-73. Woods, D.J. and Monaco, M.E. (1988) Characterization of the vasopressin receptor on WRK-1 cells. Mol. Endocrinol., 2, 350-354, 1988. Yamaji, T., Ishibashi, M., Yamada, N. and Kondo, K. (1983) Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production. Endocrinol. Jpn., 30, 45 I-461. Zachary, I., Sinnett-Smith, J. and Rozengurt, E. (1993) Vasopressin regulation of cell growth in Swiss 3T3 cells. Regulatory Peptides, 45, 23 l-236.