S 49 VBR 22

VBR 24 COLONY INHIBITION OF HUMAN TUMOR XENOGRAFTS IN VITRO BY FACTOR AF2 G.H. Leder, H.H. F i e b i g , K.H. Widmer, H. Arnold

CALCITONIN BINDING SITES AND CALCITONIN-RESPONSIVE ADENYLATE CYCLASE ACTIVITY IN SMALL CELL LUNG CARCINOMA CELL LINES M. Rotsch, G. Bepler, M. H~der and G. Jaques The incidence of paraneoplastic syndromes in lung cancer, however, is only 3% although elevated peptide hormone levels in sera are found in up to 80% of all patients (Oropp et al., Verh. Dtsch. Krebsges. 4, 778, 1983). Small cell lung'cancer shares a variety of neuroendocrine properties with these described normal cells (Carney et al., Cancer Res. 45, 2913, 1985). This endocrine differentiation can also be demonstrated by the production of various peptides, neuron-specific enolase, L-dopa decarboxylase, and the BB-isoenzyme of creatine kinase. The role of these peptides as tumor growth factor is currently under intensive investigation. First results have been obtained for bombesin and physalamin (Cuttitta et al., Nature 316, 823, 1985; Beplar et al., J. Cancer Res. Clin. Oncol. 109, A 21, 1985). The hypothesis proposes that carcinoma cells produce and secrete their own proliferation factors which interact with specific membrane receptors on their surface. We and others have established a large number of continuously growing cell lines derived from small cell lung carcinoma. These cell lines were cultivated from biopsy material of patients with histologically proven small cell lung carcinoma. The study we present here is restricted to calcitonin, a peptide hormone that is frequently elevated (60%) in the sera of small cell lung carcinoma patients. Several of our cell lines produce calcitonin. We have examined whether they have specific calcitonin binding sites and have tried to obtain data concerning the second messenger. Our results indicate that small cell lung carcinoma cell lines which produce calcitonin have a high affinity binding site for calcitonin but a low number of binding sites. We also found an increase of cyclic AMPconcentrations in cell preparations following incubation with increasing amounts of calcitonin. These results are strong evidence for the presence of a receptor which uses the adenylate cyclase system as second messenger. Preliminary results indicate that calcitonin does not effect the cell growth directly. Whether this hormone effects the proliferation indirectly, i.e. through interaction with other growth factors, remains to be determined. Zentrum f6r Inhere Medizin der Philipps-Universit~t, Abt. H~matologie/ Onkologie, Baldingerstrasse, D-3550 Marburg

Effects

of Factor AF2 in the c o l o n y assay

Tumor 30

A F 2 300

(#g/m!) 1000

3000

Lt210

1/1

I/1

1/1

Lung small c e l l non small c e l l Colon Melanoma Mesothelioma

1/1 4/4 0/1 2/2 0/I

1/1 1/1

0/I

0/1 2/7 0/2 0/2 0/I

0/I

0/I

I/I

Stomach

0/5 0/2

1/1

Mammary 0/I 0/I Renal 0/I 0/I 0/I Testicle 0/I 0/I 0/I Sarcoma 0/I 0/3 I/3 2/3 Human tumors 0/12 2/20 9/15 5/7 Total 0% 10% 60% 71% E f f i c a c y : Colony No. in t e s t < 30% of c o n t r o l group H.H. F i e b i g , M e d i z i n i s c h e U n i v e r s i t ~ t s k l i n i k , H u g s t e t t e r S t r . 55, D-7800 F r e i b u r g i . B r .

VBR 25

VBR 23 HOMOGENEOUS AGAR CONTAINING

L i v e r and spleen e x t r a c t from lamb m a i n l y cons i s t i n g of p o l y p e p t i d e s ( F a c t o r AF2, Fa. b i o s y n , Ludwigsburg) showed tumor response in some p a t i ents w i t h m a l i g n a n t d i s e a s e as s i n g l e agent or t o g e t h e r w i t h chemotherapy ( R b h r e r , p e r s o n a l c o m m u n i c a t i o n ) . At p r e s e n t the mechanism of the p o s s i b l e e f f i c a c y of AF2 is q u i t e o b s c u r e . Besides a s t i m u l a t i o n of the immune system a d i r e c t c y t o t o x i c e f f e c t can be presumed. As demons t r a t e d in the t a b l e a m o d i f i e d Hamburger & S a l mon assay r e v e a l e d some dose r e s p o n d i n g e f f e c t s of AF2. For t h e d e t e r m i n a t i o n of t h e r e l e v a n t dose i n - v i t r o the most s e n s i t i v e tumors are being s t u d i e d i n - v i v o in nude mice.

GROWTH GLAS

OF TUMOR CAPILLARIES

CELL

COLONIES

IN

B . L a t h a n . K. K e r k h o f f . W. S c h e i t h a u e r . V. D i e h l The human tumor c l o n i n g s y s t e m by Hamburger and Salmon has shown i t s p o t e n t i a l as a m e t h o d f o r p r e d i c t i o n of r e s p o n s e of an i n d i v i d u a l p a t i e n t s tumor to an a n t i t u m o r a g e n t . Maurer ( N a t u r w i s s . 67:381-382, 1981) a n d Von H o f f ( P r o c . A A C r 2 4 : 3 1 0 , 1983) h a v e p r o p o s e d t h e u t i l i s a t i o n of agar containing glas capillaries i n s t e a d of p e t r i d i s h e s to u t i l i z e l e s s tumor c e l l s and to i n c r e a s e the c l o n i n g e f f i c i e n c y (CE, no.of c o l o n i e s f o r m e d / n o . o f c e l l s p l a t e d ) . Since the o p t i mal c o n d i t i o n s f o r colony g r o w t h in c a p i l l a r i e s have not been e s t a b l i s h e d y e t , the s t u d y p r e s e n t e d h e r e was i n i t i a t e d . The s t u d y f o c u s e d on 5 human tumor c e l l l i n e s to p r o v i d e r e p r o d u c a b l e c o n d i t i o n s . The volume of the a g a r / c e l l s u s p e n s i o n f i l l e d i n t o each c a p i l l a r y tube had a m a j o r i m p a c t on t h e CE. S u f f i c i e n t c o l o n y g r o w t h was a c h i e v e d a t b o t h ends of t h e c a p i l l a r i e s , but due to p o o r g r o w t h in t h e m i d d l e . o f t h e t u b e s t h e CE was low when t h e t u b e s w e r e c o m p l e t l y filled. In c o n t r a s t , h o m o g e n e o u s g r o w t h was achieved utilizing low f i l l i n g volumes ( i . e . 30%) w i t h s u b s e q u e n t i n c r e a s e in t h e CE. The s i z e of tumor c o l o n i e s was s i m i l a r e l y i n f l u e n c e d . The CE was h i g h e r in s m a l l t u b e s ( i . e . 0.9 and 1.2 mm i . d . ) c o m p a r e d t o l a r g e t u b e s ( i . e . 1.4 to 1.9 mm i . d . ) . H i g h e s t CE w i t h homogeneous g r o w t h was a c h i e v e d when c a p i l l a r y tubes of 1.2 mm i.d. f i l l e d w i t h 30 pl were u t i l i z e d . S e a l i n g both ends of the c a p i l l a r y tubes d i d not i n f l u e n c e t h e CE, p r o v i d e d t h a t 10 mM HEPES b u f f e r was u s e d . T h e s e f i n d i n g s a r e of i n t e r e s t f o r f u t u r e work w i t h the c a p i l l a r y c l o n i n g s y s t e m , s i n c e homogeneous g r o w t h is e s s e n t i a l f o r in v i t r o drug s e n s i t i v i t y t e s t i n g . I. M e d i z i n i s c h e K l i n i k der U n i v e r s i t ~ t Kbln, J o s e p h - S t e l z m a n n - S t r . 9, D-5000 Kbln 41

M E C H A N I S M O F T P A - R E L E A S E FROM T U M O R C E L L S A N D T H E S I G N I F I C A N C E FOR M E A S U R E M E N T OF T P A - P L A S M A V A L U E S P. Oehr, M. Kr~mer, B. Schult, G. Kamp and H.-J. Biersack In order to u n d e r s t a n d the release of T P A from tissue w e studied tissue cultures, solid t u m o r s in animals a n d h u m a n tumors and p e r f o r m e d TPA-determinations in e a c h group. W e observed a sequential r e l e a s e of TPA from sync h r o n i z e d HeLa cells immediately after cell d i v i s i o n and e l e v a t e d TPA-levels in supernatants of n o n - s y n c h r o n i z e d HeLa cells after cell d e a t h caused b y irradiation. Similar results w e r e found in animal experiments a n d studies w i t h patients. Nude rats w i t h r a p i d l y g r o w i n g tumors w i t h a size , 0 , 4 g showed a n increase of TPA-levels in plasma. Irradiation of these tumors r e s u l t e d in a short increase of TPA-values w i t h i n the first two days. Later, the TPAlevels d e c r e a s e d or returned to normal. Similar results w e r e found in studies of patients. In c o n t r a s t to tissue cultures w h e r e small numbers of cells can b e a s s o c i a t e d w i t h h i g h p l a s m a T P A - c o n c e n trations w i t h i n a short time, a g r e a t e r a m o u n t of solid tumor t i s s u e is n e c e s s a r y to induce e l e v a t e d T P A in plasm a of t u m o r - b e a r i n g a n i m a l s a n d patients. In c o n t r a r y t o T P A f r o m the c l o s e d tissue c u l t u r e system in w h i c h t h e half life of T P A m i g h t be m a i n l y influenced b y proteolytic enzymes, p l a s m a T P A in the animal s y s t e m is cleared from peripheral b l o o d b y liver metabolism. Elevated levels of T P A will only b e p r e s e n t in plasma w h e n the r e l e a s e rate of T P A from the tumor tissue to the blood is higher than the clearance rate from the blood. Our results include the fact that e l e v a t e d TPA-values c a n appear i n a n i m a l s w i t h tumors 9:0,4 g after irradiation. A n increase in TPA-levels in this case is a result of cell d e a t h and n o t of tumor proliferation. In patients this m i g h t indicate that the effectiveness of treatment should also b e m o n i t o r e d in those individuals w h i c h do n o t e x h i b i t elevated plasma values b u t have T P A - p o s i t i v e tumors. Institut fUr klin. u n d exp. N u k l e a r m e d i z i n d e r Universit~t Bonn, S i g m u n d - F r e u d - S t r a S e 25, D-5300 B o n n 1