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INTRODUCTION OF A MUTATED p53 GENE ALTERS NEUROENDOCRINE DIFFERENTIATION IN HUMAN SMALL CELL LUNG CANCER. Mabrv. M.. Baker, S., Nelkin, B.D.. Jasti, R., Barges, M., Percengill, O.', Sorenson, G.', Vogelstein, B. and Baylin, S.B. The Johns Hopkins Oncology Center, Baltimore, MD 21231, and 'Dartmouth Medical School, Hanover, NH 03755.

Work from our laboratory showed that v-r&' complementation with c-myc or N-myc but not L-myc models SCLC tumor progression (PNAS 85:6X23-6527. JCI 85:1740- 1745), as well as acquisition of differentiation- associated features (JCI &:194-199). Hence, these systems provide s context for assaying functional consequences of genetic abnormalities in SCLC. Since SCLC has s high frequency of p53 gene mutations (Science &:491-494, Nature j&:705-708), we postulated introduction of s mutated ~53 gene might result in loss of characteristics associated with neuroendocrine differentiation and acquisition of features consistent with tumor progression. To test this hypothesis, we inserted a mutant ~53 gene into DMS53 SCLC. This cell line has one normal allele and s mutated allele and has bee" shown to differentiate following v-r&' insertion (JCI &:194-199). Following insertion of a ??utsnt p53 gene, DMS53 SCLC developed altered morphology, decreased cloning efficiency, decreased calcitonin hormone secretion and decreased calcitonin mRNA production. Since these phenotypic alterations were reciprocal to those following v-rass induced differentiation of these CSllS. we asked whether v-r-as" induced differentiation was affected by mutant p53 insertion. The acquisition of differentiation associated ~ronerties followins . . insertion and expression of the v-r&' gene in DMS53 SCLC cells was blocked in cells containing the inserted mutated ~53 gene. These data show that introduction of a mutated ~53 results in phenotypic changes which suggest a role for p53 in SCLC tumor progression and suggest, in neuroendacrine cells, that interference with "euroendocrine differentiation ??sy mediate this effect.

IWDUCTIOH OT NOR-BMALL CELL LUNQ CANCBR (USCLC) PHmOTYPIC BEAWJRE8 BY ONCOGBNIC TRRHSPORMATION OF EHRLL CBLL LUNQ CWCGR (SCLC) CELLS. u Dovle, M. Mabry, F. cuttitta, and J. Fontana, University of Maryland Cancer center, Baltimore, MD 21201; the Johns Hopkins Oncology Center, Baltimore, MU, 21231; and the NCI-Navy Medical Oncology Branch, Bethesda, MB 20914.

rt has been discovered that variant SCLC cells infactod with a v-Ha-rag oncogene acquire manv features of NSCLC. We have used classic HZ09 SCLC Cells, transfected with c-my0 (H209- rnVCl. or co-transformed with c-m% and v-Ha-ras @i&tayo~ras), to examine the expression of SCLC surface antigens and secreted proteins. The H209- mvc-ram subline has lower expression of the r&ently defined SCLC surface antigens SC-I, SC- 2. and SC-S. The SC-1 antiaen has been reuorted to be identical with the &sure1 cell adhesion molecule (NCAM) , which is intereeting because the HZOP-myc-ras subline forms a surface adherent monolayer like NSCLC cells. Unusual variant SCLC cells, which grow as a monolayer, also have low expression of NCAM immunoreactivity.

SCLC and NSCLC cell lines cnn be adapted to growth in unsupplemented serum-free (RO) medium. We have adapted H209, H209-myC, and H203-myo-ras to RO medium, along with the NSCLC line H157. The conditioned medium (CM) of the H209-myc-ras sublinr demonstrated a Pattern of IGF-1 binding protein6 more similar to CM from the NSCLC line than to that of the H203 cells. Fractionated protein6 in the CM of each line were also identified with silver-stained gels. The N209- myc-ran subline expresses 2 proteins (50 and 55 kd) found in the CM of the NSCLC line Hl57, but not in CM of the HZ09 or H209-myc sublines. Both

proteins have been purified to homogeneity. We believe that the changes in surface and CM proteins by the H209-myc-ras subline supporte the concept of a phenotypic switch in lung cancer subtype by oncogene activation.

GKOWTH FACTUKS IN NON-SMALL CELL LUNG CANCER Jill M. Sieefried, Department of Pharmacology, Universrty of Pittsburgh School of Medicine, Pittsburgh, PA. USA 15261 Frank Cuttitta. Medical Oncology Branch, National Cancer Institute-Navy and Uniformed Services University of the Health Sciences. Bethesda. MD. USA 20814

Cell lines established from non-small cell lung carcinomas (NSCLC) were assessed for secretion of growth factors in vitro. The cell line A549 was found to secrete the highest level of growth-promoting peptides with the properties of Transforming Growth Factor a and Insulin-like Growth Factor 1. Radioimmunoassay and receptor binding studies were used to characterize peptides secreted by A549 cells Northern blot analysis demonstrated presence of messenger RNA with homology to IGF-I and TGFa cDNAs. Medium conditioned by A549 cells was found to be a potent stimulant of the growth of primary human malignant and "on- malignant epithelial cells derived from the lung. Between 2. and lo-fold stlmulatio" of anchorage-dependent and anchorage-independent growth was observed using A549 conditioned medium. Use af conditioned medrum has allowed the successful primary culture of cells from 67 of 76 tissue donors. Twelve cell lines have bee" established using this method. I" order to produce conditioned medium with the highest possible content of secreted growth factors, we have selected for A549 cells which can grow in serum-free medium supplemented only with selenium (% medium). A549 cells growing in R, medium must be synthesizing all the necessary factors to maintain cell growth, since no peptides are added to the medium. The conditioned medium produced by these cells is a" extremely potent stimulator of cell growth and allows early passage cells derived from lung tumors to grow in serum-free conditions with no other additives. HPLC separation of concentrated conditioned medium produced 12 peaks with growth-promoting activity. Efforts are underway to identify the peptides responsible for this growth stimulation. These results suggest that NSCLCs both produce and respond to autocrine growth factors.

This work was supported by grants from the American Cancer Society (IN-5628 and JFR&262), the Pharmaceutzical Manufacturers Ass". Foundation. and the NIH (CA-45145).

TREATMEMTOFSMALLCELLCARCINOMAOFTHE LUNG WITH MONOCLONAL ANTIBODIES. Edward D. Ball. Departments of Medicine and Microbiology, Dartmouth-Hitchcock Medical Center, Hanover, New Hampshite 03756

We have prepared or obtained several monoclonaI antibodies (tnAb) to antigens associated with small cell carcinoma of the lung (SCCL) for use in diagnosis and therapy including SCCL-1, SCCL 124, SCCL-175, TPS-2, TPS-4, HNK-1. PM-81, and AML-1-99. SCCL-175, an IgM mAb is of interest because this mAb macts with >90% of SCCL tumor specimens from patients and may identify a subset of patients with better prognosis. SCCL-1. an IgG2a mAb. reacts with the wnsferrin receptor. I-INK-l. an I&, reacts with a glycolipid antigen. TPS-4, an IgGl described by Okabe et al (Csncer Res 44: 5273, 1984) reacts with N-CAM. AU of these mAb react frequently with tumor cells from atients with SCCL. We are exploring therapeutic applications o P these SCCL-teactive mAb. In one set of studies immunomagnttic bead-mediated purging of SCCL tumor cells from bone marrow has been accomplished. In another study m4b-targeted liposomes have been used in a nude mouse SCCL xenopaft model to show spacifte in viva uugeting. We have developed an effective purging method for removing SCCL tumor cells from bone marrow for use in autologous bone marrow transplantation (ABMT) using immunomagnetic beads. Using SCCL-175, HNK-I. and TPS-4 and magnetic microsphehs @ynal) coated with sheep anti-mouse antibody we can remove 4 to 5 logs of clonogenic SCCL tumor cells from fwo SCCL cell lines (DMS 279