011

EXPRESSION OF C-JUN AND CDC2 IN HUMAN PRIMARY LUNG CANCERS

S.S. Park, IS. Park, H.J. Yoon, D.H. Shin, D.H. Lee, J.D. Lee, J.H. Lee, Hanyang University Hospital, Seoul, Korea

C-jun has stn~chral and fwctional properties of transcriptional factor,activator

protein l(Science 238, 1386, 1987, Nature 332, 166, 1988) .C-jun has been found

to be expressed at high levels in small-cell, and non-small-cell lung cancer (Proc

Am Assoc Cancer Res 29, 455, 1988). The CDCZ kiiase is known to be a key

regulator of eukaryotic cell cycle and is believed to act both Gl and G2

phase(Nahue 298,391,1982, Proc Nat1 Acad Sci 87,5697, 1990). To evaluate the

role of c-j”” and cdc2 in human primary lung cancers, expression of c-j”” and

cdc2 was investigated in tissue sections oftwenty three cases with lung cancers and

seven cases with ““n-cancerous lung tissues by imnunohistochemical methods.

Recombinant clonal antibodies of c-jun/AP-1 and cdc2 were employed as primary

antibodies after dewaxing the formal&lixed, paraffiized tissue sections. C-j””

was expressed in the nuclei pf tlllllor cells of lung cancers in twenty cases of

twenty three primary lung cancers(87.0 %). The expression of c-jun was disclosed

in six cases of seven small cell lung carcinomas and fourteen cases of sixteen “on

small cell lung carcinomas. Also, expression of cdc2 was noted in the nuclei of

h”““I cells of lung cancers i” ninteen cases of twenty three prinxuy lung

cancers(82.6 %). The expression of cdc2 was disclosed in six cases of seven small

cell lung carcinomas and thirteen cases of sixteen n”” small cell lung carcinomas.

Expression of c-jun and cdc2 was not discernible in the fourteen with non-

cancerous lung tissues. These findings suggest that upregulation of c-j”” and cdc2

is involved during development and growth of primary lung cancers.

013

BRADYKININ ANALOG.9 STRUCTURAL AND FUNCTIONAL STUDIES IN LUNG CANCER CELL LINES. D. Ghan, J. Stewart, L. Gcra, B. Helfrich, K. Helm, M. Tagawa, P, Jew&t and P. Bun”. Jr. Univer.of Colorado Cancer Center, Denvcr.CO. We pteviously reported bmdykini” was the moat potent agonist and iu receptor most commonly expressed in lung cancer cell lines. In order to improve the therapeutic potentials of bradykinii antagonists for the treaoncnt of lung cancer, we have evaluated “hvo lwneratio”s” of bradvki”ii a”ahxs consiatina of 130 Deutides with various

amino acid cyclimtio” and pcptide dimuiaatio”. Peptide pot&& specificity and plasma stability was studied by intraceUular calcium flux in small cell lung cancer cell lines assessed with flow cytometry. Effects of peptide on cell proliferation in lung cancer cells were assess& by (3H)thymidi”c inuxporado”. Stmctares and results of a few representative bradykiain agonists and antagonists are shown BS follows: BradyLinin Arg-Pro-b Gly-Phe-Ser - Pm - Phc-Arg-CODH B4801 (Antagonist) DArg-Arg-Pm-HypGly-Phe-Ser-DPhe- Phe-Arg-COOH B6014(Agonist) Arg-Pro-HypGly-Thi-Ser*Pm- Thi-Arg-CODH B5872(Antago”iat) DArB-Ar8-~Hypcly-Phe-Ser-DPhelPhe-ArgH HOE14O(Antagonist) DArB-Arg-Pro-HypGly-Thi-Set-DTic- Oic-Arg-COOH CPOl27(Antagonist) DArg-Arp-pm-HwGly-Wscys-Dphe- L-z”-Arg-COOH

Peptides Activity ICSO Spcciticity Plasma Stability Bmdykinin Agonist 0.3nM blocks BK wly 2Smin B4801 Antagonist O&M blocksBKonly “d B6014 Agonist 0.5nM blocks BK only 22 min. B5872 Antagonist 1 “M blocks BKonly “d HOE140 Antagonist 0.1 urn blocks BKonly 24 hr 80127 Antagonist 0.5 aM blocks BK only 15 min. Even with the most potent, specific and long-acting bradykinii antagonists up to 40 ti we failed to detect any inhibitory effects in the (3H)thynddine incorporation assay. sqporting OUT prwious observation that interference with a single peptidcxcceptor interaction would have little ultility in the treatment of lung czawr. We suggest the altexnative of conjugating toxins or radio-isotopes with the potent, specifc and long- acting hadyki”in alalogs for tie trcatnlent of lung cancer.

012

ANTITUMOR ACTIVITY OF AN ANTI-GASTRIN RELEASING PEPTIDE (GRP) MURINE MONOCLONAL ANTIBODY (2All). PHASE II TRIAL AND IiV VITRO CORRELATION WITH PEPTIDE AND RECEPTOR EXPRESSION. H.-K. Yang, R.I. Linnoila, G.E. Richardson, F. Scott, I. Avis, C. Boland, J. Phares, F. Cutitta, J. Mulshine, B.E. Johnson, M.J. Kelley. National Cancer Institute and National Naval Medical Center; Bethesda, MD, USA

The murine monoclonal antibody, ZAll, has been shown to have anti- tumor activity in oifro and in athymic nude mice. We examined the activity of 2All (250 rng/m2, 3 doses/wk for 4 wks) in 12 patients (pts) with small cell lung cancer (SCLC) in a phase II trial. Prior treatment (tx) included at least one cisplatin-containing regimen for all pts. Median age was 57 y’s (range 44-69); male:female ratio was 93; and performance status was 0 (n=l), 1 (n=lO), or 2 (n=l). One pt (8%) had a compiete response documented by cl&t k-ray, chest CT scan and direct bronchoxooic visualization; 4 had stable disease. 6 had ore- gressive disease, and 1 was nbt evaluable. There were no side eifects. ihe completely responding pt was a 65 year old female with lung recurrence after a 3.5 vear disease-free interval followine initial tx with combined modalitv thekpy for limited stage SCLC. She wagtreated with a second course of 2Ali and maintained a CR for 5 months at which time she had local recurrence documented by fiberoptic bronchoscopy. The pt remained free of immunologi- cal evidence of human anti-mouse antibody (HAMA) but her tumor pro- gressed at the site of the initial recurrence despite a third course of 2All.. To determine if tumor exoression of bombesin-like ueotides (BLP) lGRP and neuromedii B (NMB)] &d their receptors (R) predi& ior respbnse i;ZAll, we analyzed 7 SCLC cell lines (H69, H82, H187, N417, H510, N592, H&O) for mRNA expression by RNase protection assay and RT-PCR and for peptide expression by RIA using 2All. mRNA expression of GRP was detected in 3 liis (H69, H345, H510), NMB in all liies, GPR-R in 2 (H69, H345) and NMB-R in aU but N417. RIA detected BLP in all cell lines but was higher in those with GRP mRNA (n=3; 3.96-59.9 pmol/mg) than in those without (n=4; 0.084.84). Clonogenic assay showed inhibition of cell growth @Xl%) after exposure to 5 pg/ml of 2All (a readily sustainable plasma level) for 2 weeks for all cell lines except N417. We conclude that 1) 2All has clinical antitumor activity; 2) 2All administration is non-toxic and sustained administration is possible; 3) honor cell expression of GRP-R or NMB-R is necessary, but not necessarily sufficient, to predict in vitro antitumor activity of 2All; and 4) further studies exploring the mec.hanism(s) of growth inhibition of 2All are needed to allow prospective identification of patients with SCLC who are likely to benefit from this therapy.

014

PROTEIN KINASE C ALPHA ABNORMALITIES IN HUMAN SMALL CELL LUNG CARCINOMA C.L.A. Jones, E.C. Dempsey, & M.A. Kane, Denver VAMC & Univ. Cola. HSC, Denver, CO 80220

Protein kinase C (PKC) is an important signal transduction protein in the bombesin induced growth response found in small cell lung cancer cells. Because PKC alpha has previously been implicated in both growth and transformation, we have screened SCLC cell lines for the presence of PKC alpha; a ubiquitous, Ca++ and lipid dependent isoenzyme. By Western blot analysis, we have identified the expected 82 kD PKC alpha protein in four out of six cell lines tested: NCI-H930, SHP77, UMCI 9 and NCI- H774. Neither NCI-N417 or NCI-H345 cells have detectable levels of the 82 kD PKC alpha. NCI-H345 cells do express a 57 kD PKC alpha protein, but PKC alpha protein appears to be absent in NCI-N417 cells. Since a low molecular weight PKC alpha may arise either from mutation or abnormal posttranslational processing, we have analyzed RNA from NCI-H345 cells by RT/PCR. Using PCR primers pairs for various regions of PKC alpha, we have obtained PCR products of predicted size for the first approximately 1200 bases of the sequence. These products span the proposed Ca++ ATP binding domains. Abnormal PKC’s may be involved in the abnormal growth of at least some SCLC cells.