4th LTBW Abstracts/Lung Cancer IO (1994) 347-373

secreted from NCI-H345 or NCI-H209 cells by agents which elevate intracellular CAMP levels such as VIP. High aff’nity binding sites for ( ‘%GRP) and ( i251-Tyro) NMB have been identified on NCI-H345 cells (B,, = 1500 and &IO/cell, respectively [I]). Specific ‘251-GRP but not (‘*‘I-Tyr”)NMB binding is inhibited with high affinity by (Psi’3*‘4, Leu14)BN (Psi-13,14) or (D-Phe6)BN6-‘3methylester (P)BN6-13ME (ICsO = 30 and 5 nM, respectively; [2,3]). Also, IO nM BN or NMB elevate the intracellular Ca2+ levels. Psi-13,14 and (P)BN6-13ME inhibit the increase in cytosolic Ca2+ caused by BN but not NMB. BN (IO nM) causes translocation of protein kinase C from the cytosol to the membrane and PKC activation is blocked by (P)BN6-13ME. Also, IO nM BN increases c-fos mRNA and the increased c-fos expression is blocked by (P)BN6-13ME. BN or NMB (IO nM) increased the growth of SCLC using a clonogenic assay and Psi-13,14 or (P)BN6-13ME inhibit the increase in clonal growth caused by IO nM BN. Also, Psi-l3,14 (0.4 mg/kg injected daily s.c.) inhibits SCLC xenograft formation in nude mice. Psi-13,14 and (P)BN6-13ME are slowly metabolized by SCLC endopep- tidases [4]. Therefore Psi-13,14 and (P)BN6-13ME function as SCLC GRP receptor antagonists.

High affinity ‘251-vasoactive intestinal peptide (VIP) binding sites were identified on almost all SCLC and NSCLC cell lines examined [5]. Low levels of VIP immunoreactivity were detected in lung cancer cells but NCI-H838 had moderate levels of VIP mRNA. Specific ‘251-VIP binding was inhibited with high affinity by VIP and VIPhybrid (VIPhyb; ICso = IO and 500 nM, respectively [6]). VIP elevated the CAMP levels IO-fold using NCI-H838. In contrast, VIPhyb had little effect on the basal CAMP levels but inhibited the increase in CAMP caused by IO nM VIP. VIP (0.1 PM) increased NCI-H838 clonal growth whereas VIPhyb (I PM) inhibited colony formation. Also, VIPhyb (0.04 mg/kg injected daily s.c.) inhibited NSCLC xenograft formation in nude mice. The data suggest that VIPhyb functions as a NSCLC VIP receptor antagonist. (Sup- ported by NC1 grants CA-48071 and 53477).

Moody TW,Staley J, Zia F, Coy DH, Jensen RT. J Phar- macol Exp Therapeutics 1992; 263: 31 I-317. Staley J, Coy DH, Taylor JE, Kim S, Moody TW. Peptides 1991; 12: 145-149. Mahmoud S, Staley J. Taylor J, Bogden A, Moreau JP, Coy DH, Avis I, Cuttitta F, Mulshine J, Moody TW. Cancer Res 1991; 51: 1798-1802. Davis TP, Crowell S, Taylor J, Coy D, Staley J, Moody TW. Peptides, 1992; 13: 401-407. Lee M, Jensen RT, Bepler G, Korman LY, Moody TW. Peptides 1990; II: 1205-1210. Moody TW, Zia F, Draoui M, Brenneman DE, Fridkin M, Davidson A, Gozes I. Proc Natl Acad Sci USA 1993. In press.

The influence of epidermal growth factor and transforming growth factor+ on the growth properties of human small cell lung caacer cell liaes. Correlation to invasivion and retiooblastoma gene expression Skovgaard Paulsen H, Damstrup L, Nggaard P, Rygaard K,

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Spang-Thornsen M, Hansen HH. Department of Oncology. Rigshospitalet and Department of Pathological Anatomy, Uni- versity qf Copenhagen, Denmark.

Introduction: The polypeptide growth factors epidermal growth factor (EGF), and transforming growth factor fl (TGFB) are known to modulate growth, DNA synthesis and gene expression of a wide range of human cells. Both EGF and TGF/3 exert their main actions after binding to the correspon- ding high aff’nity cell surface receptors. We have examined a panel of 21 human small cell lung cancer (SCLC) cell lines for the expression of EGF- and TGFj3 receptors, and the effects of exogenously added EGF and TGF& Furthermore we have examined for the in vitro invasion of the cells. To examine the possible involvement of retinoblastoma (Rb) protein in the TGFB mediated growth inhibition, we examined the cells for expression of functional Rb protein. TGFP receptor expression and growth inhibition was correlated to phosporylation and lo- calization of Rb protein.

Receptor expression: The EGF- and TGFB receptors were detected by radioreceptor assay, chemical crosslinking and Northern blotting. TGFj3 receptors were expressed by seven cell lines, in different combinations of the three known human receptor subtypes (I, II and III). EGF receptors were detected in I1 cell lines.

Growth modulation: For growth assays the cells were cultured in medium containing TGF& or EGF in the concentrations; 0, 20, 100, and 250 CM (TGF&) or 0, 50, 500, and 2.000 pM (EGF). Growth curves were constructed on the basis of regular samples assayed for protein or DNA content. Upon treatment with EGF in the few cell lines examined so far, a growth stimu- lation was observed, were as TGFB exerted a growth sup- pressive effect in the cell lines expressing TGFP receptors I and/or II.

In vitro invasion: The in vitro invasive potential was tested us- ing the Boyden invasion assay. A correlation between in vitro invasion and the expression of EGF receptors was detected, as all the EGF receptor positive cell lines were invasive. No corre- lation was observed between TGFS receptor expression and Boyden chamber invasion.

Retinoblastoma gene expression: The expression and degree of phosphorylation of Rb protein was examined by Western blotting, and the subcellular localization of Rb protein was evaluated by immunocytochemistry. Functional Rb protein, defined as localized in the nucleus and expressed with varying degree of phosphorylation, was found only in 4 cell lines. Two of these (CPH 54A 8r CPH 54B) also expressed all three sub- types of TGF@ receptors and were growth inhibited by TGFj3,, whereas TGFB had no influence on the growth properties of the other two cell lines (DMS 53 & DMS 114).

Conclusion: The results indicate that EGF and TGFj3 exert a growth regulatory effect on receptor positive SCLC cell lines and that the inhibition by TGFfi was mediated by the type II receptor. Rb protein was not an obligatory component of this pathway in SCLC, as proposed by others in different cell types. The invasion data suggest that EGF might be involved in mediating the invasive phenotype of small cell lung cancer.