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Expression of myc family onto-proteins in small cell lung cancer cell lines and xenografts

ICAre Rygaard & Mogens Spang-Thomsen University Institute of Pathological Anatomy, University of Copenhagen, Frederik V’s Vej 11, DK-2100 Copenhagen, Denmark.

Expression of myc family (c-myc, L-myc and N-myc) mRNA occurs frequently in small cell lung cancer (SCLC), but little is known about expression of the corresponding myc onco- proteins. We studied myc gene activity in a panel of 24 cell lines and xenografts established from patients with SCLC. Gene amplification was studied by Southern blotting, mRNA expression by Northern blotting and and protein expression by Western blotting with several commercially available mono- clonal and polyclonal antibodies. Amplification was found in 11 tumours, all of which expressed the corresponding myc mRNA. There was excellent correlation between the level of myc mRNA expression and onto-protein expression for c-myc and N-myc. c-myc once-protein was expressed as two bands with M,‘s of approx. 62.000 and 65.000 while N-myc onco- protein was expressed as three bands in the 60-65.000 range. Three tumours had Lmyc amplification and 7 expressed high levels of Lmyc mRNA, but as yet we have not been able to demonstrate the Lmyc protein, probably because the avail- able antibodies were not optimally suited for use in Western blotting. The majority of tumours were studied both as cell lines and as xenografts and revealed no difference in myc family activity between the two model systems.

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085

REFRACTORY SMALL CELL LUNG CANCER (SCLC) AND rnjc FAMILY ONCOGENES. m’, 1. Osaki’. S. Saito’, M.

Watanabe’. I. Tachibanaf, M. Matsunashil, T. Sakumal, S. Hayashil, S. ~osoel, 1. Kawasel, T. Masunol, N. Kodama*, K. Furuse* and T.Kishimotol. Department of Medicine III, Osaka University Medical Schooll and Deparfment of Medicine of National Kinki-Chuo Hospital*.

Most SCLC are sensitive to initial chemo-radiotherapy, but soon relapse and become refractory to any therapy. However, some SCLC are already resistant to initial therapy

To study the resistance mechanisms of SCLC we have established SCLC cell lines derived from untreated primary tumors biipsied by bronchofiirscopy. We analyzed 4 cell lines derived from 3 patients for their chemo-radiisensitii by cbnogenic assay. OS1 showed primary resislance to both anticancer drugs and radiation. OS8 was also resistant to drugs but partially sensitive to radiation. 0S2, which was established from untreated primary tumor, was quite sensitive to both drugs and radiation, but O!S2-R, which was derived from the same patient as OS2 at relapse, acquired resistance to both drugs and radiation. Autocrine growth factor, GRP, was elevated in both OS2 and OS*-R, and dense core granules were also detected in these 2 cell lines. In vitro doubling time was 86h for OS1,121 h for OS2,77h for OS2-R. and 49h for OS3.

Southern bbt analysis of mycfamily oncogenes (c-, N-, L-myc) revealed ; i) the N-mycwas amplified in OSl, if) the amplification of the Lmycwas shown in OS&R buf not in OS2, iii) OS3 had no myc ampfiiitbn. Dot blot analysii of mRNA also showed overexpression of the N-myc and the L-myc in OS1 and OS*-R respectively. However, the overexpressbn of the mdri or the c-ra~oncogene was not detected. These results suggested ; i) the amplification of the N-myc may be

associated wlh variant type of SCLC and its primary resistance. ii) the ampliiitiin of the L-myc may be related with classic type of SCLC and its acquired resistance. iii) resistance mechanisms of OS8 were not exdained bv the mvc abnormaliiv.

Receptors for Transforming Growth Factor @ and Epidermal Growth Factor in Small Cell Lung Cancer cell lines.

Lars Damstrup, are Rygaard, Mogens Spang-Thomsen and Hans Skovgaard Paulsen. Institute of Pathological Anatomy, University of Copenhagen and Department of Oncology, Finse.n/Rigshospitalet, 2103 Copenhagen, Denmark.

The aim of the present study was to investigate Small Cell Lung Cancer (SCLC)-cell lines for Transforming Growth Factor p (TGFb)- and Epidermal Growth Factor (EGF)- receptors. The panel of 26 SCLC-cell lines established from 22 patients in 5 different laboratories, is probably representative of SCLC-cell lines. The receptors were detected by the radioreceptor assay and further visualizing the ligand binding 1o the receptor by affinity labelling. For the EGF receptor we also screened for expression of the EGF receptor mRNA by Northern blotting. High- affinity, specific TGFB receptors was found in 11 of the 22 examined cell lines. Binding capacity ranged from 3 to 60 femtomol/mg protein. In all positive cell lines a specific band with M, of approximately 280.000 was found by affinity labelling and SDS polyacrylamide gel electrophoresis. High affinity, specific EGF receptors was found in 14R6 SCLC-cell lines. Binding capacity ranged from 3 10 52 femtomoUmg protein. In all positive cell lines specific receptors were also visualized after affinity labelling of the EGF-receptor complex. The resulting complex had a M, = 17s ~SWCJO. Northern blotting with the pE7 EGF-receptor mRNA probe demonstrated that 15/25 expressed mRNA. All cell lines found positive in the radioreceptor assay also displayed the EGF-receptor mRNk These. results maps the presence of TGFB and EGF receptors in SCLC-cell lines. This “mapping” can be used in future biological studies, in which the role of these receptors will be. investigated in cells carrying only one, as well as both receptors.

086

SUSCEPTlBlLllY OF A HUMAN SMALL CELL LUNG CANCER (SCLC) CELL LINE TO AUTOLOGOUS

PERIPHERAL LYMPHOCYTES AND INDUCTION OF TUMOR- SPECIFIC KILLER CELLS. LQsaki, Y.Tanio, M.Watanabe, LTachibana, SSaito, M.Matsunashi, T.Sakuma, S.Hosoe, S.Hayashi, I.Kawase. T.Masuno and TKiihimoto. Department of Medicine Ill, Osaka University Medical School.

Effective immunotherapy for SCLC has not been established. SCLC is usually resistant to chemo-radiotherapy on recurrence. In order to overcome its resistance we have established SCLC cell lines and devised to induce specific killer cells against refractory SCLC.

A ceil line (OS7) was established from the untreated primary tumor of a patient with extensive disease of SCLC. After 4 courses of chemotherapy followed by radiotherapy, partial remission was acheiied. But 8 months later the primary lesion regrew, then pleural exudative lynphocyles (PLEL) were obtained from the malignant pleural effusion.

While upregulation of weakly expressed HlA-class I antigen of OS7 was shown after treatment with IFN-y(lOOU/ml), neither HLA-class II antigen nor SCLC specific antigen expression changed. Spedfii killer cells were induced after 3 cycles of the 7day MLTC when PLEL were stimufatd with IFN-7 treated OS7 in the presence of IL2 (0.05 U/ml) and IL8 (lOU/mf). Tfw killer cells specifiilfy lysed both IFN- treated OS7 and untreated OS7, although the former was more suscef.%iie than the latter. Moreover the fylii adivii of the killer cells was enhanced when cuflured with IL-6 and IL2 conpared to IL-2 only.

The HLA-class I antigen expression of SCLC is mostly weak, which may be a cause of rapid recurrence or escape from host immunofogiil defense mechanisms. We showed the possibility of adoptive immunotherapy against recurrent SCLC with specifii killer cells, which could be induced by combfnatiinal use of cytokines, IL- 2 and IL-6.