SIO

transferrin synthesis by a small cell lung cancer line (NCI-HSIO) that survives in serum-free media without added transferrin. Immunoassays for human transferrin demonstrated that these cells contained im- munoreactive human transferrin. Immunofluorescence studies showed that the protein is expressed on the surface of cells, presumably bound to transfctin receptor. Media conditioned by NCI-H-510 cells support

proliferation of human leukemic cells that would not survive in media lacking transferrin. [“S]Methionine incorporation documented tram-

ferrin synthesis by NCI-HSlOcellsas wellasthreeothersmallce111ines. Transferrin synthesis by NCI-HSlO cells increased more than IO-fold when cells entered active phases of the cell cycle, and this increase was seen before large increases in transfetin-receptor expression. Further experiments examining the effects of agents that affect iron metabolism show that the addition of agents that affect iron metabolism show that the ad&ion of wansfcrrin-iron or hemin to the media is associated with a more rapid initial rate of proliferation and lower rates of transferrin synthesis than control cells. Gallium salts, which inhibit iron uptake, inhibited proliferation of these cells. If the cells recovered from this effect, aansfcrrin synthesis remained greatly increased compared to control. We conclude that transferrin synthesis by these malignantce11s is ultimately related to an iron requirement for cellular proliferation. 11

appears that this synthcsizcd transferrin acts as part of an important autocrinc mechanism permitting proliferation of these cells, and per- haps permitting tumor cell growth in vivo in areas not well vascularized.

Human lung cancer nodules in organotypic culture: No evidence of correlation between the antiproliferative effects of interferoas and the induction of 2’,_5’-oligoadenylate synthetase. Martyrc M-C, Beaupain R, Falcoff E. C/ 196 INSERM, Instilut Curie, F- 75231 Paris Cedex 05. Tumor Biol 1988;9:263-9.

Alveolar II pulmonary tumor cells (A.549), maintained in continuous tridimcnsional organotypic culture, were used in an attempt to investi- gate whether there could be a relationship of the 2’S’-oligioadenylate (2,SA)synthetasepathway totheantiproliferativeactivity ofinterferons (IFNs) in this particular tumor ccl1 model. IF?+_,, -8 and -gamma were used separately and in combinations. IFN-_, and -gamma demonstrated an inhibitory effect on the nodule growth, whereas IFN-I3 did not. Moreover, combinations of IFN-_, and -gamma resulted in a significant synergisticanriproliferativeactivity;lFN-OonlypotentiatedslighUythe effect of IFN-gamma. All three IFNs induced an increase in the 2,ZA synthetase activity, indicating a discrepancy with the pattern of anticel- lular activity. Furthermore, whereas the combination of IFN-, and - gamma resulted in a synergistic antiproliferative effect, no synergism was observed in the induction of the enzyme. These results show that there is a lack of correlation between the sensitivity or the resistance to IFNs of AS49 tumor nodules and the induction of the 2,5A synthetase activity.

Methylation status of epidermal growth factor receptor gene in lung carcinoma cells. Gamou S, Shimosato Y, Merlin0 GT, Shimizu N. Deparlment of Molecular Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160. Jpn J Cancer Res, Gann 1988;79:989-95.

The methylation status of the epidermal growth factor receptor (EGFR) gene was compared in cell lines from four major types of lung carcinoma, small cell lung carcinoma (SCLC), large cell lung carci- noma, squamous cell carcinoma and adenocarcinoma, in order to examine whether DNA meth ylation is responsible for the suppression of EGFR gene in SCLC cells. Southern blot analysis revealed that the structural region of the EGFR gene is methylated in various degrees regardless of the expression of EGF receptor on the surface. An 8- kilobase EcoRl fragment which contains the EGFR gene promoter region is readily digested with various methylation-sensitive restriction enzymes in all types of cells, indicating that the EGFR gene 5’ region is

barely methylated. Thus, a mechanism other than DNA methylation appears to control EGFR gene expression and the lack of EGFR gene expression in SCLC cells may be caused by a paucity of some transcrip- tion regulatory factor(s).

Castrin-releasingpeptidegene-associatedpeptidesareexpressed in normal human fetal lung and small cell lung cancer: A novel peptide family found in man. CuttittaF,FedorkoJ,GuJ,Lebacq-VerheydenA-M,LinnoilaRl,Battey JF. Uniformed Services Universiry of the Health Sciences, Department of Defense, Division of Cancer Treatment. National Cancer Institute and National Naval Medical Center, Bethesda, MD 20814. J Clin Endocrinol Metab 1988;67:576-83.

Mammalian gastrin-releasing peptide (GRP) is found in cells of neuroendocrine and neural origin, and GRP mediates a variety of physiological and trophic responses when it binds to high affinity cell surface receptors on effector cells. Analysis of cDNA clones derived from prepro-GRP mRNAs predict the concurrent expression ofa unique series of peptide hormones, the GRP gene-associated peptides (GGAPs). AltemativeRNA splicing of the primary GRPgene transcript results in mRNAs that could encode 3 distinct forms of GGAPs. Using specific antisera directed against synthetic peptides representing par- tions of the predicted GGAPs, we found multiple GGAP forms in the endocrine cells of human fetal lung and in human small cell lung cancer cells (SCLC). Within a single pulmonary endocrine cell, at least 2 of these 3 predicted forms could be demonstrated using immunohisto- chemical techniques. In addition, concordant expression of GRP and GGAPs was found in 10 SCLC cell lines and 3 human SCLC tumors. These findings establish GGAPs as a novel peptide family in man and warrant further investigation into their potential role in normal and malignant growth.

Response of primary human lung carcinomas to autocrine growth factors produced by a lung carcinoma cell line. Siegfried JM, Owens SE. Carcinogenesis and Melabolism Section, Environmerual Health Research and Teaing. Inc., Research Triangle Park, NC 27709. Cancer Res 1988;48:4976-81.

Medium conditioned for 48 to 72 h by A549-1 lung carcinoma cells was used to culture primary solid lung tumors on feeder layers of inactivated Swiss 3T3 cells. Of 22 cases placed into culture, primary cultures of carcinoma cells were obtained in 20. Subcultures were obtained in 18 cases, and cell lines were established in nine cases. The neoplastic origin of the cultured cells was demonstrated by several criteria: tumorigenicity in athymic mice; anchorage-independent growth; expression of altered lactate dehydrogenase isoenzyme pro- tiles; and expression of the lung tumor marker pregnancy-specific glycoprotein 1. The epithelial nature of cultured carcinoma cells was demonstrated by expression of keratin. These characteristics were compared to normal epithclial cells established in culture from bron- chialexplantsfromthesamedonorsas tumortissue,orotherdonors.The growth-stimulating effect of conditioned medium toward primary or newly cultured tumor cells was quantitated by clonal assays in soft agar and in monolayer culture. Growth response in clonal assays of newly cultured carcinoma cells to the purified growth factors transforming growth factor-and insulin-likegrowthfactor 1, two knowncomponents of medium conditioned by A549-1 cells, was also demonstrated.

Effects of bombesin antagonists on the growth of small cell lung cancer cells in vitro. Layton JE, Scanlon DB, Soveny C, Morstyn G. Ludwig Insiitufe for CancerResearch,Melbourne TumourBiologyBranch,Melbourne.Vic. 3050. Cancer Res 1988;48:4783-9.

Small cell lung cancer (SCLC) produces several neuroendocrine peptides, including gasuin-releasing peptide (GRP), the mammalian

equivalent of bombesin. There is some evidence to support the sugges- tion that GRP is an autocrine regulator of SCLC growth. Therefore, we have tested the effect of hombesin and two antagonists of bombeain on SCLC cell growth in a serum-free liquid tissue culture system. The antagonists used were analogues of substance P: spantide and (D-Arg’, D-Pro*, D-T~TI”,~, Leu”) substance P. The cell lines used in this study all produced GRP-related peptides and one line had demonstrable GRP receptors. Exogenous bombesin did not cause any stimulation of growth in the liquid culture assay. The bombesin antagonists inhibited SCLC cellgrowth, butapparentlynotviathebombesinreceptor.Thebombesin used was biologically active because it stimulated the proliferation of Swiss 3T3 fibroblasts. The antagonists caused inhibition of this bomb- esin-induced proliferation, which was reversed by addition of excess bombesin. In addition, the antagonists and substance P alone stimulated proliferationof3T3 cells, indicating that they may intcractwithanother growth factor receptor on 3T3 cells. We conclude that growth of SCLC cells is not dependent on bombesin under all in vitro culture conditions because bombesin failed to stimulate growth in liquid cultures and the growth inhibition caused by bombesin antagonists was probably not mediated by the bombesin receptor.

myc family DNA amplification in small cell lung cancer patients’ tumors and corresponding cell lines. Johnson BE, Makuch RW, Simmons AD, Gazdar AF, Burch D, Cashell AW. NCI-Navy Medical Oncology Branch, National Cancer Inslilule, Naval Hospital, Berhesda, MD 20814. Cancer Res 1988;48:5163-6.

Tumor specimens procured from 38 different small cell lung cancer patients were studied for DNA amplification of the myc family of protooncogenes (c-myc, N-myc, and L-myc). Six of the 38 specimens (16%) had 4.fold or greater myc family DNA amplification (N-myc in 4 and L-myc in 2). All 6 tumors with amplification came from patients who had received combination chemotherapy. The myc family gene copy number of the DNA prepared from 9 tumor cell lines established from these 38 patients was similar to the myc family gene copy number in the DNA prepared from fresh tumor specimens from these same patients. myc family DNA amplification is present in 16% of small cell lung cancer patients’ tumors and the amplification pattern in the tumor cell lines is representative of the fresh tumors obtained from the same patients.

Restriction fragment length polymorphism studies show consistent loss of chromosome 3p alleles in small cell lung cancer patients’ tumors. Johnson BE, Sakaguchi AY,GazdarAFetal.Na~ionalCancerlns?itute- Navy Medical Oncology Branch, Narianal Cancer Instilute and Naval Hospilal. Bethesda, MD 20814. J Clin Invest 1988;82:502-7.

Previous karyotypic analysis of human small cell lung cancer cell lines hasdemonstratedaconsistentdeletionofaportionoftheshortarm of chromosome 3@14-23). DNA prepared from tumors and normal tissues obtained from 24 small cell lung cancer and two extrapulmonary small cell cancer patients was hybridized to four probes that detect restriction fragment length polymorphisms within chromosome region 3~14-21. Of the 25 patients who were heterozygous for at least one marker in this region in the DNA from normal tissue, 23 (92%) showed an unequivocal loss of heterozygosity in the DNA from their tumor tissue. From these studies we conclude that loss of alleles from the short arm ofchromosome 3 isaconsistent finding in unselected small cell lung cancer patients’ tumor DNA.

A glycopeptide from adenocarcinoma tissue-of human lung. Masuda H, Ozeki T. ThirdDepartment ofInternal Medicine, University of Occupational and Environmental Health, School of Medicine. Yahalanishi-ku, Kitakyushu. Fukuoka 807. Biochem Med Metab Biol 1988;40: l-7.

A glycopeptide from adenocarcinoma tissue of human lung was extracted by protein digestion with papain and trypsin. This component was isolated by Pevikon block electrophoresis. It possessed hexose, glucosamine, fucose, galactosamine, and sialic acid. In its carbohydrate composition and also the abundance of threonin in the peptide moiety it was quite similar to the glycoprotein from gastrointestinaI tract. The physical characteristic of the two, such as their optical rotation and viscositv. were also very similar to each other.

Growth factor effects on small cell lung cancer cells using a colori- metric assay: Can a transferrin-like factor mediate autocrine growth? Nakanishi Y, Cut&a F, Kasprzyk PG et al. NCI-Navy Medical Oncol- ogy Branch, Naval Hospital, Bethesda. MD 20814. Exp Cell Biol 1988;56:74-85.

A semiautomated calorimetric assay @ITT assay), based on the ability of live cells to reduce a tetrazolium-based compound, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MT-I’), to a purplish colored formazan product that can be measured spectro- photometrically, has recently been adapted for use in drug sensitivity analysis of cultured human tumor cell lines. We report the application of this assay for the evaluation of the growth factor requirements of humansmallcelllungcancer(SCLC)celllines.Specifically,thegrowth stimulation of each constituent of a previously reported serum-free defined medium system for SCLC including various concentrations of hydrocortisone, insulin, transfer&, 17B-estradiol, and selenium (HITES) was evaluated. The optimal concentrations for insulin, trans- ferrin, and selenium derived in the previously reportedexperiments with direct counting of viable cells were similar to optimal concentrations determined for the growth of three SCLC cell lines (NCI-H82. NCI- N417, NCI-H526) using the MTI assay. In contrast to the previous report, the growth-stimulating effects of hydrocortisone and 176estra- diol were negligible. Using the MTT we have been shown that a SCLC cell line, NCIH345 (which has been previously reported to produce a transferrin-like molecule), was growth-inhibited by an antitransferrin receptor antibody, when grown in transferrin-free media. The condi- tioned media from this cell line is stimulatory to other transferrin- sensitivecelllines,suggestingIhepossibilityofanau~rineroleforchis transferrin-likemoleculeat least in thatcellline. Withcarefullydefined conditionsforagivencelllinein whichcelldensityandotherparameters are within a range of constant MTI metabolism, the assay is well suited for precise analysis of growth factor effects.

Radiolaheled monoclonal antibody 15 and its fragment for locahzp- tion and imaging of xenografts of human lung cancer. Endo K, Kamma H, Ogata T. Departmenr of Surgery, InsrirrUe of Clinical Medicine, University of Tsukuba, Ibaraki 305. J Natl Cancer Inst 1988;80:835-42.

Monoclonal antibody (MAb) 15 and its F(ab’), and Fab fragments were radioiodinated, and their biodistribution and imaging were com- pared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for r”I-labeled MAb I5 IgG, F(ab’),, and Fab were 1.9 x 109, 1.8 x 109, and 3.7 x lb M-l, respectively.