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SCREENING OF POINT MUTATIONS ON p53 Secretary type and intracellular type of thiol pro- GENE IN TISSUES FROM PRIMARY LUNG tease inhibitors purified from human lung cancer

CANCER USING SINGLE-STRAND CONFORMATION POLYMORPHISMS ANALYSIS

cell line.

Kunihiro Matsuda’, Hideki Takahashi’. Kuniaki Seyama’, Akihiko Ohwada’. Toshihii Nukiwal, and Shiro Kim*. ‘Department of Respiratory Medicine, Juntendo University, School of Medicine,2-l-l Hongo, Bunkyo-ku. Tokyo, 113 Japan.

S.Yoshioka, P.Kuranishi, M.Nishiki, K.Dohi The 2nd Department of Surgery, Hiroshima University School of Hedicine, Hiroshima, Japan.

Wild-type p53 is considered to play an important role as a tumor suppressor gene in various organs, and mutations of the p53 gene occur commonly in lung, colon and breast cancers as well as less common malignancies. This study reports rapid and sensitive method to detect point mutations in p53 gene using single-strand conformation polymorphisms(SSCP) analysis (Orita et al. Proc Natl Acad Sci USA vol 86, 2766-70. 1989) with several modifications. cDNA was synthesized from total RNA of primary or metastatic tissues of lung cancer and non-cancerous tissues, using oligo(dT) primer and reverse uanscriptase. 35S-labelled partial cDNA of p53 gene(Alal%erml) was amplified by polymerase chain reaction using p53 specific primers and [35S]d-CTP. After denaturing with formamide, the amplified products were elecuophoresed on nondenaturing polyacrylamide gel and electrophoretic mobilities of the bands were compared. Among 15 samples from patients with primary lung cancer (adeno 11, small cell 1, epidermoid 2, adenosquamous l), three cancer samples showed different electrophoretic mobilities compared with the non-cancerous tissues. To confm the mutation, we determined the nucleotide squence of a sample with abnormal mobility and found a point mutation at residue 234(TyraAC)-HiscAC)) of the mature protein. These results suggest the SSCP method is useful for screening of a large number of clinical specimens to detect point mutations of p53 gene.

He have suggested that thiol protease inhibitors (TPIs) have SOW roles in growth, invasion and metasta- sis of lung cancer and breast cancer.

In this study we purified TPIs from the cells and medium of human lung cancer cell line ( LK-2 : squamous cell carcinoma ) we have established. The forner is named as intra-cellular type and the later is secretary type. Both of these TPIs were purified respectively by almost the saae methods that are amaoniuo salphate fractionation, Sephadex G-50 gel filtration, DEAE- Sephacel chronatography. No differences were found in molecular weights and spectruas of inhibitory activity between two types of TPIs. The molecular weight of them is about 10,000. And they inhibit thiol proteases esp- ecially and have no activity on serine proteases.

Secretary type of TPIs inhibits the cell growth of human lung cancer cell line. And some of well-known TPIs also inhibit cell growth. On the other hand, some of thiol proteases accelarate cell growth.

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BFFECTOFJMMUNOMODULATORONNEUIROPHIL.S MIGRAnONTOPULMONARYALVEOLIANDONALVEOLAR MACROPHAGEFUN(JI1ONSlNLUNGMJ?l'ASTASISMODEL

YokoIshiharaaadJunKagawapeptofHygieneBpUblichealth,To~o Wo~'sMcdicalcollege,To~o,Japan

Previously we reported that immunomodulator induced neutrophils (PMNs) migration to pulmonary alveoli in lung metastasis model of BDF 1 mice (Proceeding Japan Cancer Associ. 1990, p.409). The interaction of PMNs and alveolar macrophages @MS) may be of crucial importance in inflammatory process of lung metastasis. Nevertheless, little is knowa about fimction and its regulation of AMs and PMNs in hmg metastasis.

Lung metastasis was induced by intravenous injection of LlO cells (a diethyhdtrosamine-induced tmnsplantable guinea pig hepatoma) into SPF strain 2 guinea pigs. The hnmunomodulator treated guinea pigs (PSK group) were administrated PSK ( a protein-bound polysaccharide,500mg/l@ay, o.s.) everyday after the instillation of LlO cells. Bronchoalveolar lavage fluid (BALF) was collected and analyzed at 2 or 3 weeks after L 10 cells administration. 1) In LlO cells treated guinea pigs (control group) and PSK group,

total ceil counts in BALF showed a significant increase compared with those in normal guinea pigs. 2) Control group revealed a normal cellular profiles of greater than 90% AMs , but the cellular composition consisted of 63.1% AMs, 32.0% PMNs, 2.5% lymphocytes in PSK group. 3) Giant cells were observed in BALF of PSK group. 4) The functions of AMs which were phagocytosis (2um latex beads) vd su?rerde anion generation were not atTectedbyPSK_

These results suggeatid that PSK might induce PMNs migration to hmg metastasis and accelerated inflammatory reaction in the process of cell killing. In addition to that PSK could affect monocyte differmtiation or cell fusion of AMs which resulted in the appcruancc of giant cells.

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PRODUCTION OF TDMOR NECROSIS FACTOR ALPHA (TNF-c*) IN LUNG CANCEB PATIENTS. Giorgio V. SCAGLIOTTI*,

Emiliano GATTI. Giovanni FElUURI, Prauco MIcBELoTTO. Ernest0 POZZI. University of Turin, Department of Cl? nical and Biological Sciences. Orbassauo, ITALY.

TNF-tilevel was determined: a)iu the serum of 31 heal- thy controls (mear+.E.=7.8+0.6 pg/ml) aud 68 Lung Can car (LC) patients (16.2+1.1 pg/~)(t=4.93;p<0.001); bj in the Pleural Fluid (PP) of 31 Neoplastic Effusions (NE)(115+87 pg/ml) and 12 exudative Benign Effusions (BE)(246+198 pg/ml)(p=u.s.). Spontaneous release of _ TNF-1in short time cultures of blood mouocytes and pleural macrophages was investigated in 5 cases of LC with NE (release in macrophages > monocytes)(t=3.105; p<O.O3). In PF additional evaluation included IL-Z.IL- 2 receptor aud prostaglaudin E2 levels. No correlation between TNF-*level and age, sex. and histology was seen. In LC, raised levels of serum TNF-(were usually associated with far advanced disea- se stage, lower perfo-ce status and inversely rela- ted to serum triglycerides level. In PF there was uo relationship between TN@-qproduction and the level of other cytokines investigated. Independently by the ty- pe of the underlying disease TNF-&level was greatly raised in PP and its production seems to be relatively selective at the site of disease.