Apoptosis in the APO-1 System

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Apoptosis in the APO-1 System

P.H. Krommert I. Behrmann,^] V. Bier,² P. Don/e/,¹ J. Dhein,^] M.H. FcrWc,³ G . Gcrrc/n,¹ C . K/as,¹ E. Knipping,^]

K.-M. Lücking-Famira,^] S. Matzku* A. Oehm,^] S. Richards,^] B.C. Trauth,^] G.W. Bornkamm,³ W. Falk,^] P. Möller*

and K.-M. Debatin²

institute for Immunology and Genetics, German Cancer Research Center, Heidelberg, Germany

2Children's Hospital, University of Heidelberg, Germany

^Institute for Clinical Molecular Biology/GSF, Munich, Germany

^Institute for Radiobiology and Pathophysiology, German Cancer Research Center, Heidelberg, Germany

^Institute for Pathology, University of Heidelberg, Germany

Cell-surface molecules are c r u c i a l i n lymphocyte growth c o n - trol. S u c h molecules m a y f u n c t i o n as receptors for growth - s t i m u l a t i n g cytokines or m a y be associated w i t h receptors a n d t r a n s m i t signals essential for growth regulation. Receptor blockade or removal of the s t i m u l a t i n g cytokines m a y lead to decreased lymphocyte growth (Duke a n d C o h e n 1986). W i t h - d r a w a l of i n t e r l e u k i n s slows h u m a n lymphocyte growth a n d Anally leads to the characteristic form of " p r o g r a m m e d cell death" or apoptosis. A p o p t o s i s is the m o s t c o m m o n form of eukaryotic cell death and o c c u r s i n embryogenesis, metamor- p h o s i s , tissue atrophy, a n d t u m o r regression. It is also i n - d u c e d b y cytotoxic T lymphocytes a n d n a t u r a l killer cells, b y cytokines like t u m o r necrosis factor (TNF) a n d l y m p h o t o x i n (LT), a n d b y glucocorticoids. T h e m o s t characteristic signs of apoptosis are segmentation of the n u c l e u s , c o n d e n s a t i o n of the c y t o p l a s m , m e m b r a n e blebbing, a n d D N A fragmentation into m u l t i m e r s of a b o u t 180 b p (called a D N A ladder) (see K e r r a n d H a r m o n , t h i s volume). To analyze m e c h a n i s m s of lymphocyte growth c o n t r o l a n d to interfere w i t h the replication of l y m p h o i d t u m o r cells, we r a i s e d m o n o c l o n a l antibodies against cell-surface molecules involved i n these processes.

M o n o c l o n a l antibodies were u s u a l l y tested a n d selected b y

Apoptosis: The Molecular Basts of Cell Death

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virtue of their b i n d i n g to cell-surface antigens of test cells. O u r a i m w a s to define reactive m o n o c l o n a l antibodies b y f u n c t i o n a l assays, n a m e l y b y abrogation of growth of m a l i g n a n t test cells i n vitro. M o n o c l o n a l antibodies were r a i s e d against the h u m a n B - l y m p h o b l a s t cell line S K W 6 . 4 . O n e m o n o c l o n a l antibody, a n t i - A P O - 1 , s h o w e d the strongest f u n c t i o n a l activity a n d reacted w i t h a n antigen (APO-1) of - 5 0 k D o n a set of activated h u m a n lymphocytes, o n m a l i g n a n t h u m a n lymphocyte lines, a n d o n some patient-derived l e u k e m i c cells. A n t i - A P O - 1 w a s of the I g G 3/K isotype a n d h a d a h i g h affinity of K^D = 1.9 x 1 0 "^{1 0}. Despite m a n y cell fusions u n d e r t a k e n i n o u r laboratory, the h y b r i d o m a w i t h a n t i - A P O - 1 activity h a s r e m a i n e d the only one i n a b o u t 2 5 , 0 0 0 tested. N a n o g r a m quantities of a n t i - A P O - 1 completely b l o c k e d proliferation of cells b e a r i n g A P O - 1 i n vitro i n a m a n n e r characteristic of apoptosis {Fig. 1). C e l l death w a s preceded b y changes i n cell morphology a n d fragmentation of D N A . T h i s process w a s d i s t i n c t from antibody- a n d complement-dependent cell lysis a n d w a s mediated b y the a n t i b o d y alone (Trauth et a l . 1 9 8 9 ; K r a m m e r 1989; K r a m m e r et a l .

1989; Köhler et a l . 1990).

Purification of the APO-1 Antigen

It w a s i m p o r t a n t to further characterize the A P O - 1 molecule w i t h the a i m of l e a r n i n g more a b o u t its f u n c t i o n . Therefore, we purified the A P O - 1 antigen from m e m b r a n e s of S K W 6 . 4 cells.

The purified A P O - 1 antigen w a s f o u n d to be a glycoprotein w i t h apparent M^r of a p p r o x i m a t e l y 5 0 , 0 0 0 , w i t h a b o u t 8 0 0 0 of the M^r a c c o u n t e d for b y sugars. Purified A P O - 1 b l o c k e d a n t i - A P O - 1 - i n d u c e d apoptosis of S K W 6 . 4 cells i n vitro, p r o v i n g i t s

serological identity w i t h t h e A P O - 1 - m e m b r a n e antigen. Large quantities of the A P O - 1 antigen enabled u s to o b t a i n a se- quence of the A P O - 1 p r o t e i n . A c o m p u t e r s e a r c h revealed that A P O - 1 w a s a n e w cell-surface antigen. Motifs i n the A P O - 1 se- quence m a y provide u s w i t h a clue to the as yet elusive physiological f u n c t i o n of the antigen.

The APO-1-mediated Signal

I n d u c t i o n of apoptosis w a s mediated b y a n t i - A P O - 1 alone a n d w a s complement-independent. Nevertheless, the F(ab ' )² frag-

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a n t i » A P O - l 2 4 0 m i n a n t i - A P O - 1 24 h

FIGURE 1 I n d u c t i o n of apoptosis of S K W 6 . 4 cells b y a n t i - A P O - 1 . The time of i n vitro i n d u c t i o n w i t h c o n t r o l m o n o c l o n a l a n t i b o d y or a n t i - A P O - 1 (1 ng/ml) is i n d i c a t e d .

m e n t of the IgG3 a n t i - A P O - 1 d i d not i n d u c e apoptosis. W h e n c r o s s - l i n k e d , however, b y F ( a b ' )² sheep a n t i - m o u s e Ig a n t i - bodies, apoptosis w a s observed. To further s t u d y the role of the F c region of a n t i - A P O - 1 , we isolated a n t i b o d y class s w i t c h v a r i a n t s from the IgG3 a n t i - A P O - 1 - s e c r e t i n g h y b r i d o m a cell line. We obtained a n t i - A P O - 1 antibodies of the IgG3, I g G l , IgG2b, IgG2a, a n d IgA isotypes. These antibodies showed the following effects: (1) a different degree of i n d u c t i o n of apoptosis of S K W 6 . 4 cells o c c u r r e d i n the following order: IgG3, I g G l , IgG2a, IgA, IgG2b. (2) C r o s s - l i n k i n g of the less effective class s w i t c h v a r i a n t IgG2b a n t i - A P O - 1 b y P r o t e i n A showed the same degree of growth i n h i b i t i o n as IgG3 a n t i - A P O - 1 . These r e s u l t s suggested that i n d u c t i o n of apoptosis w a s dependent

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o n c r o s s - l i n k i n g of the A P O - 1 cell-surface antigen. IgG3 a n t i - A P O - 1 b o u n d to the cell surface might have self-aggregating capacity v i a F c - F c interactions or b i n d to F c receptors a n d therefore efficiently c r o s s - l i n k the A P O - 1 antigen. IgG2b a n t i - A P O - 1 might show fewer F c - F c interactions, be a less efficient cross-linker, a n d therefore be less effective i n i n d u c t i o n of apoptosis. C r o s s - l i n k i n g of A P O - 1 o n the cell m e m b r a n e m a y be essential for A P O - 1 - m e d i a t e d signal t r a n s d u c t i o n across the m e m b r a n e .

We also a s k e d whether i n t e r n a l i z a t i o n of A P O - 1 a n d / o r a n t i - A P O - 1 m i g h t be a prerequisite for apoptosis i n o u r system. T h e following experiments suggested that this is n o t the case. We c h e m i c a l l y c o u p l e d a n t i - A P O - 1 to s i l i c a beads several times larger t h a n cells a n d i n c u b a t e d S K W 6 . 4 cells w i t h these beads. W e f o u n d that bead-coupled a n t i - A P O - 1 w a s a n efficient i n d u c e r of apoptosis i n S K W 6 . 4 cells. These results r e i n - force o u r a s s u m p t i o n that the A P O - 1 antigen m a y produce a genuine t r a n s m e m b r a n e signal, the n a t u r e of w h i c h r e m a i n s to be investigated. These results also p r o m p t e d u s to develop systems that might allow u s to s t u d y the a n t i - A P O - 1 apoptosis process i n m o l e c u l a r terms. T h u s , we looked for cellular systems that m i g h t be informative i n this respect.

Selection of Cell Variants That Express the APO-1 Antigen but Are Resistant to Anti-APO-1 -induced Apoptosis

After screening a large p a n e l of h u m a n B - a n d T-cell lines, we f o u n d that expression of the A P O - 1 antigen is a prerequisite, a l t h o u g h n o t sufficient b y itself for a n t i - A P O - 1 - i n d u c e d cell death. T h u s , we identified several strongly A P O - 1⁺ cell lines resistant to a n t i - A P O - 1 - i n d u c e d apoptosis. To s t u d y this phe- n o m e n o n further, we selected several cell v a r i a n t s that differed i n the sensitivity to a n t i - A P O - 1 . T h e B - c e l l line S K W 6 . 4 ( s I g M⁺, A P O - l⁺, sensitive to 2 n g / m l anti-APO-1) w a s c u l t u r e d w i t h i n c r e a s i n g a m o u n t s of a n t i - A P O - 1 for about 1 year. We obtained a stable v a r i a n t that expressed the A P O - 1 antigen b u t was resistant to at least 5 0 ng/ml a n t i - A P O - 1 . In a d d i t i o n , the T-cell line C C R F was cloned u n d e r l i m i t i n g d i l u t i o n conditions.

R e p l i c a c u l t u r e s of s u b c l o n e s were screened for susceptibility to a n t i - A P O - 1 . Two s u b c l o n e s were selected that b o t h expressed the A P O - 1 antigen b u t differed i n sensitivity to a n t i -

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A P O - 1 at least b y a factor of 1000. It is conceivable that the m e c h a n i s m of resistance to apoptosis i n S K W 6 . 4 a n d C C R F variant cells i s different. In a n y case, however, this pair of cell lines shows very clearly that two requirements for a n t i - A P O - 1 - i n d u c e d apoptosis are important: the cell-surface expression of the A P O - 1 antigen a n d a n intact apoptosis s i g n a l pathway. W e p r e s u m e that these findings m a y be of great future relevance to the putative u s e of the apoptosis concept i n t u m o r therapy.

Apoptosis in Human T Lymphocytes

Another informative set of cells w i t h respect to the A P O - 1 - m e - diated s i g n a l of apoptosis are n o r m a l h u m a n T lymphocytes.

A l t h o u g h we have d a t a suggesting that, i n contrast to resting B cells, activated B cells also undergo a n t i - A P O - 1 - m e d i a t e d apoptosis, i n t h i s paper, we focus p r i m a r i l y o n T cells. T h e m a - jority of n o r m a l h u m a n resting T lymphocytes do n o t express the A P O - 1 antigen. After activation, however, b o t h the C D 4⁺ a n d C D 8⁺ s u b p o p u l a t i o n s of T cells become positive for the A P O - 1 antigen. A l t h o u g h n o significant difference i n the a m o u n t of A P O - 1⁺ T cells a n d i n the epitope density of A P O - 1 antigens between T cells early (e.g., 1 day) or late (e.g., 6 days) after activation w a s observed, apoptosis w a s o n l y i n d u c e d b y a n t i - A P O - 1 i n the latter cell p o p u l a t i o n . Hence, the s u s c e p - tibility for i n d u c t i o n of apoptosis i n activated T lymphocytes i s dependent o n the stage of differentiation of these cells. A c o m - p a r i s o n of the set of A P O - 1⁺ T cells early or late after activation might help to elucidate the e n i g m a of "death genes" involved i n a n t i - A P O - 1 - m e d i a t e d apoptosis. In a d d i t i o n , t h i s p h e n o m e n o n might help to u n d e r s t a n d i n m o l e c u l a r terms the e l i m i n a t i o n of peripheral T cells at the cessation of a n i m m u n e response.

Anti-APO-1 -mediated Tumor Regression

A s d i s c u s s e d above, a n t i - A P O - 1 i n d u c e d apoptosis i n v a r i o u s T- a n d B - c e l l lines i n vitro. T h i s result led u s to test the a n t i - A P O - 1 efficiency i n a n experimental t u m o r s y s t e m i n vivo (Fig.

2). The h u m a n B - l y m p h o m a line B J A B w a s c h o s e n for these i n vivo experiments. Xenografts of this line i n n u / n u mice were

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DAYO mab anti-APO-1 DAY 7

control m a b DAY 14 m a b anti-APO-1 DAY 14 FIGURE 2 A n t i - A P O - 1 - m e d i a t e d t u m o r regression of B J A B l y m p h o - b l a s t o i d t u m o r x e n o t r a n s p l a n t s i n n u / n u mice. The p i c t u r e s s h o w prototype m i c e from e a c h group, n u / n u m i c e w i t h h u m a n B J A B l y m - p h o b l a s t o i d t u m o r s - 1 . 5 - 2 . 5 c m i n diameter (day 0) were i.v. injected w i t h 5 0 0 isotype m a t c h e d c o n t r o l m o n o c l o n a l a n t i b o d y or a n t i - A P O - 1 (IgG3/ K ) o n day 0. M i c e w i t h t u m o r s were p h o t o g r a p h e d 7 a n d

14 days after m o n o c l o n a l a n t i b o d y injection.

previously s h o w n to a c c u m u l a t e radiolabeled m o n o c l o n a l a n t i - bodies only i n the outer layer of the t u m o r , whereas c e n t r a l areas of n o d u l e s were v i r t u a l l y inaccessible. U s i n g a n t i - A P O - 1 i n B J A B - b e a r i n g n u / n u mice, we a s k e d three questions: (1) Is a n t i - A P O - 1 as effective i n vivo as i n vitro? (2) Does a n t i - A P O - 1 affect the whole t u m o r despite preferential a c c u m u l a t i o n i n

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the periphery? (3) Does a n t i - A P O - 1 -mediated t u m o r cell death i n vivo alter the accessibility b a r r i e r s of the B J A B t u m o r ? The results were clear-cut. A n t i - A P O - 1 antibodies, like a l l other antibodies tested, a c c u m u l a t e d exclusively i n the periphery of n o d u l e s even i f u p to 5 0 0 \ig of a n t i b o d y w a s injected per m o u s e . Nevertheless, established t u m o r s - 1 . 5 - 2 . 5 c m i n diameter regressed i n 10/11 n u d e mice w i t h i n a few days.

Histological t h i n sections performed before complete t u m o r regression showed that as i n vitro, a n t i - A P O - 1 also i n d u c e d apoptosis i n vivo. T h e action of the antibody, however, d i d n o t result i n a d i s t u r b a n c e of the accessibility barrier. We c o n - c l u d e d from these experiments that t u m o r s m a y be efficiently t a c k l e d b y m o n o c l o n a l antibodies, p a r t i c u l a r l y a n t i - A P O - 1 , despite restriction of accessibility, provided the cytolytic a c - tivity of the antibody is h i g h a n d the residence time of the antibody i n the t u m o r is long e n o u g h to "melt d o w n " the t u m o r nodules from the outside (Trauth et a l . 1989). In a d d i t i o n , the outcome of these experiments suggested that a n t i - A P O - 1 - i n - d u c e d apoptosis is a v a l i d concept w o r t h testing for t u m o r treatment i n a c l i n i c a l s i t u a t i o n , provided putative systemic toxicity of the a n t i b o d y c a n be controlled.

One i m p o r t a n t result s h o u l d be m e n t i o n e d at this point. In p r e l i m i n a r y experiments, we tested the i n vivo therapeutic efficiency of a n t i - A P O - 1 o n large S K W 6 . 4 t u m o r s . In vitro a n t i - APO-1-sensitive (S) a n d -resistant (R) S K W 6 . 4 cells b o t h ex- p r e s s i n g A P O - 1 o n the cell surface were grown to t u m o r s of a b o u t 2 c m i n diameter i n S C I D mice. A n t i - A P O - 1 treatment of these a n i m a l s resulted i n complete t u m o r regression of the S K W 6 . 4^S t u m o r s only. A n i m a l s w i t h S K W 6 . 4^R t u m o r s were k i l l e d b y the t u m o r . These results suggested that two requirements for a n t i - A P O - 1 - m e d i a t e d t u m o r regression b y i n d u c t i o n of apoptosis also exist i n vivo: (1) expression of the A P O - 1 antigen and (2) a n intact apoptosis signal pathway. A s already stated, these results m a y have far-reaching i m p l i c a t i o n s for therapy u s i n g r a t i o n a l intervention strategies i n the clinic.

Preclinical Applications of Apoptosis in the APO-1 System

The above i n vivo experiments p r o m p t e d u s to test A P O - 1 ex- p r e s s i o n i n v a r i o u s t u m o r systems a n d to test i n vitro i n d u e -

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tion of apoptosis i n m a l i g n a n c i e s that m a y be candidates for future a n t i - A P O - 1 treatment i n the clinic.

Expression of the APO-1 antigen on acute lymphoblastic leuke- mia cells. In T-acute l y m p h o b l a s t i c l e u k e m i a (ALL), A P O - 1 is expressed constitutively, especially i n cases c o r r e s p o n d i n g to stages of very early T-cell differentiation. Cells of the c o m m o n A L L phenotype representing the m a l i g n a n t p r e c u r s o r s of B cells w e a k l y express A P O - 1 i n a m i n o r i t y of cases. However, i n these cells, A P O - 1 expression is i n d u c e d i n vitro b y p h o r b o l myristate acetate (PMA) a n d cytokines s u c h as IL-4. In a d d i - tion, the constitutive expression of A P O - 1 o n p r e - T - A L L cells is m o d u l a t e d b y mitogens a n d cytokines. The A P O - 1 antigen m a y therefore be of i m p o r t a n c e for growth regulation i n m a l i g n a n t lymphocytes a n d m a y also serve a f u n c t i o n i n the development of n o r m a l p r e c u r s o r cells. In a d d i t i o n , APO-1-positive m a l i g - n a n t cells m a y be a n e w s u b g r o u p of A L L a n d m a y be a target for A P O - 1 - d i r e c t e d therapeutic approaches i n vitro a n d i n vivo u s i n g the a n t i - A P O - 1 antibody.

Anti-APO-1 antibody-mediated apoptosis in adult T-cell leuke- mia. We have described that the A P O - 1 antigen is expressed o n activated T cells a n d that sensitivity to i n d u c t i o n of apoptosis b y a n t i - A P O - 1 is a c q u i r e d d u r i n g long-term c u l t u r e of activated T cells i n the presence of IL-2. Since a d u l t T-cell leuke- m i a (ATL) cells are the transformed c o u n t e r p a r t of m a t u r e T lymphocytes, we were interested to see whether these cells express the A P O - 1 antigen a n d whether they are sensitive to growth i n h i b i t i o n a n d i n d u c t i o n of apoptosis b y a n t i - A P O - 1 . E x p r e s s i o n of the antigen a n d sensitivity to the i n d u c t i o n of cell death b y a n t i - A P O - 1 were s t u d i e d i n h u m a n T-cell lines transformed b y h u m a n l e u k e m i a v i r u s type 1 (HTLV-1 ) a n d i n c u l t u r e d cells from patients w i t h A T L . A P O - 1 w a s strongly expressed o n b o t h types of cells, a n d i n c u b a t i o n of the cells w i t h a n t i - A P O - 1 resulted i n i n h i b i t i o n of proliferation a n d apoptosis. I n d u c t i o n of apoptosis m a y therefore be a therapeutic tool i n H T L V - 1 - a s s o c i a t e d m a l i g n a n t disorders (Debatin et a l .

1990).

Expression of the APO-1 phenotype in BurkitVs lymphoma cell lines correlates with a phenotype shift to a lymphoblastoid phe-

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notype. We h a d previously f o u n d that A P O - 1 was also ex- pressed o n n o r m a l activated B cells (Trauth et a l . 1989). F u r - thermore, a s m a l l subset of follicle center B cells r e s i d i n g at a location i n w h i c h m a t u r a t i o n , proliferation, a n d e l i m i n a t i o n b y apoptosis of B cells takes place h a d been s h o w n b y i m m u n o - h i s t o c h e m i s t r y to be A P O - l⁺. Therefore, we tested whether m a l i g n a n t counterparts of s u c h g e r m i n a l center B cells, B u r k i t t ' s l y m p h o m a (BL) cells, expressed A P O - 1 a n d were sensitive to a n t i - A P O - 1 - i n d u c e d apoptosis. T a k i n g together the evaluation of a large n u m b e r of tests of B L cells a n d B L lines phenotypically r e s e m b l i n g i n vivo B L a n d cell lines s h o w i n g a phenotype of E p s t e i n - B a r r virus-positive l y m p h o b l a s t o i d cells (LCL), the following results were obtained. B L cells directly isolated from t u m o r biopsies were A P O - 1 " . B L type cell lines were A P O - 1 " , a n d L C L type cell lines were A P O - 1⁺. Cells of the B L / L C L phenotype showed a heterogeneous A P O - 1⁺ pattern.

Some b u t not a l l cells of the A P O - 1⁺ phenotype were sensitive to a n t i - A P O - 1 - i n d u c e d apoptosis. The phenotypic shift of B L cell lines m a y correlate w i t h the one i n B - c e l l activation.

Therefore, these cell lines m a y represent a u s e f u l s y s t e m to s t u d y A P O - 1 expression a n d f u n c t i o n i n B cells.

Expression of the APO-1 antigen on glioblastoma cell lines and their susceptibility to apoptosis. To assess the potential u s e f u l - ness of a n t i - A P O - 1 for therapy i n other t u m o r systems, we also tested h u m a n glioblastoma cell lines for expression of the A P O - 1 antigen a n d susceptibility to a n t i - A P O - 1 - i n d u c e d apoptosis. M o s t cell lines expressed A P O - 1 at least at a low level.

Some cell lines showed growth i n h i b i t i o n a n d apoptosis if i n - c u b a t e d w i t h a n t i - A P O - 1 . T h u s , a l t h o u g h A P O - 1 w a s expressed o n most cell lines tested, only a few responded to a n t i - A P O - 1 . S u b c l o n i n g a partially responsive cell line yielded A P O - 1⁺, anti-APO-1-sensitive a n d A P O - 1 \ a n t i - A P O - 1 - r e s i s - tant subclones. The d a t a i n t h i s cellular system, therefore, stress again that expression of the A P O - 1 antigen a n d a n i n - tact apoptosis s i g n a l p a t h w a y are necessary for s u c c e s s f u l a n t i - A P O - 1 - m e d i a t e d apoptosis. Presently, we are investigating w h i c h parameters determine the s u s c e p t i b i l i t y of s u c h clones to i n d u c t i o n of apoptosis, a n d whether l o c a l a n t i - A P O - 1 therapy might be considered i n s u c h a disease where s u r v i v a l

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after relapse is short a n d no therapeutic possibilities exist.

APO-1 expression in colorectal carcinomas correlates with poor prognosis. A l l above d a t a o n v a r i o u s m a l i g n a n t cells s h o w a c o m m o n trait. A P O - 1 expression o n the same type of t u m o r varies. In a d d i t i o n , s i m i l a r v a r i a b i l i t y is observed as to s u s c e p - tibility to a n t i - A P O - 1 - i n d u c e d apoptosis o n A P O - 1⁺ m a l i g n a n t cells. T u m o r s are either sensitive, resistant, or composed of sensitive a n d resistant cells. T h i s observation also extends to s a r c o m a s a n d m a m m a r y c a r c i n o m a s not extensively d i s c u s s e d here. A l t h o u g h the physiological f u n c t i o n of A P O - 1 is still u n - clear, one m a y speculate that the observed heterogeneity is m e a n i n g f u l for the biology of the t u m o r a n d t h u s also for the c l i n i c a l course of the m a l i g n a n t disease. These considerations led u s to investigate A P O - 1 expression o n colorectal car- c i n o m a s a n d to correlate o u r findings w i t h the c l i n i c a l p a r a m e - ters of this m a l i g n a n t disease.

B y m e a n s of i m m u n o h i s t o c h e m i s t r y , we f o u n d that A P O - 1 is expressed i n n o r m a l colon e p i t h e l i u m . In a m i n o r fraction of colon a d e n o m a s a n d i n 3 9 . 6 % of colorectal c a r c i n o m a s , however, A P O - 1 expression w a s d i m i n i s h e d . In 4 8 . 3 % of car- c i n o m a s , p r e d o m i n a n t l y of the n o n m u c i n o u s type, A P O - 1 w a s completely abrogated. The n o r m a l level of A P O - 1 expression i n c a r c i n o m a w a s correlated w i t h the m u c i n o u s type (p<0.0001).

R e d u c e d or lost antigen e x p r e s s i o n w a s more frequent i n c a r c i - n o m a s localized i n the r e c t u m (p<0.0001). In a g r o u p of 149 patients w h o h a d undergone potentially curative surgery for colorectal c a r c i n o m a , the physiological level of A P O - 1 expres- s i o n w a s correlated w i t h a shorter s u r v i v a l after relapse (p = 0.031) a n d w i t h a n increased r i s k of tumor-related death (p = 0.051) (P. Möller et a l . , i n prep.). T h i s suggested that the A P O -

1 antigen is i m p o r t a n t for signals i n growth c o n t r o l of n o r m a l a n d m a l i g n a n t cells. T h u s , A P O - 1 m a y confer growth a d v a n - tage to m a l i g n a n t cells a n d determine the grade of m a l i g n a n c y . F u r t h e r m o r e , this first set of c l i n i c a l d a t a u n d e r s c o r e s the i m - portance of A P O - 1 testing a n d correlation w i t h patient h i s t o - ries i n other m a l i g n a n c i e s . T h i s applies p a r t i c u l a r l y to those i n w h i c h heterogeneous A P O - 1 expression is already observed. It w o u l d not be s u r p r i s i n g if the A P O - 1 antigen also c o n s t i t u t e d a valuable prognostic parameter i n s u c h diseases.

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DISCUSSION AND OUTLOOK

We showed that a n t i - A P O - 1 specifically b l o c k e d growth a n d triggered p r o g r a m m e d cell death, apoptosis, of a set of activated n o r m a l lymphocytes a n d cells from m a l i g n a n t l y m p h o i d a n d n o n l y m p h o i d lines after b i n d i n g to the cell-surface protein antigen A P O - 1 . The A P O - 1 antigen does not seem to be part of the T N F receptor complex, since its representation o n the s u r - face of v a r i o u s cells does not c o r r e s p o n d to the d i s t r i b u t i o n of T N F receptors; i.e., macrophage cell lines tested so far are A P O - 1 " . Nevertheless, it w i l l be i m p o r t a n t to test whether v a r i - o u s apoptosis p a t h w a y s s u c h as the one triggered b y T N F a n d a n t i - A P O - 1 have c o m m o n features.

A p o p t o s i s is f o u n d i n a l l t i s s u e s a n d also i n cells from lower o r g a n i s m s . It is conceivable, therefore, that several d i s t i n c t cell-surface antigens w i t h a different tissue d i s t r i b u t i o n are i n - volved i n the i n d u c t i o n of apoptosis. E l u c i d a t i o n of the s t r u c - ture of A P O - 1 , its possible c o n n e c t i o n to the cytoskeleton, a n d the m o l e c u l a r events following a n t i - A P O - 1 b i n d i n g m a y resolve some of these i s s u e s .

Since A P O - 1 is expressed o n m a t u r e activated lymphocytes, a d d i t i o n a l experiments w i l l be needed to determine whether the antigen might p l a y a role i n the down-regulation of the i m - m u n e response a n d be involved i n selection a n d e l i m i n a t i o n of lymphocytes. It h a s previously been s h o w n that LT, T N F , a n d killer cells w i t h their effector molecules i n d u c e apoptotic cell death. B e c a u s e a n t i - A P O - 1 also i n d u c e s apoptosis, a n u m b e r of possibilities m i g h t be considered for the physiological role of the A P O - 1 antigen. A P O - 1 m i g h t be a receptor for cytotoxic molecules or for a u t o c r i n e growth factors. Alternatively, it c o u l d be a molecule essential for vertical or lateral growth sig- n a l t r a n s d u c t i o n . T h u s , a n t i - A P O - 1 m i g h t trigger receptors for lytic molecules or b l o c k receptors for growth signals. Putative signals given b y A P O - 1 m a y r e m a i n a n e n i g m a u n t i l the s t r u c - ture of the antigen reveals its secrets. In a n y case, the e l u c i d a - tion of the A P O - 1 - m e d i a t e d apoptosis p a t h w a y w i l l constitute a challenge for o u r r e s e a r c h a n d w i l l provide a b a s i s for the development of a r a t i o n a l intervention strategy i n v a r i o u s diseases, p a r t i c u l a r l y cancer.

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O u r d a t a also have c l i n i c a l relevance. A n t i - A P O - 1 m a y be useful as a diagnostic tool to define subsets of n o r m a l a n d m a l i g n a n t lymphocytes a n d other t u m o r types. In a d d i t i o n , i n - d u c t i o n of apoptosis m a y have i m p l i c a t i o n s for a n t i t u m o r therapy. A n t i b o d i e s have frequently been u s e d as heteroconjugates w i t h toxins or d r u g s to destroy t u m o r cells. O u r data, however, s h o w that m o n o c l o n a l antibodies alone c a n be lethal to target cells, provided these cells express A P O - 1 a n d have a n intact apoptosis pathway. A n t i - A P O - 1 might, therefore, be considered for ex vivo or i n vivo therapy, u n d e r conditions where reactivity w i t h vital n o r m a l cells c a n be excluded or tolerated. T h u s , i n the immediate future, careful toxicity studies i n S C I D mice reconstituted w i t h a h u m a n i m m u n e system, i n primates, a n d i n patients w i l l be necessary.

It is easily i m a g i n e d that a s u c c e s s f u l putative a n t i - A P O - 1 therapy m i g h t go b e y o n d a therapy of cancer a n d m i g h t i n - volve e l i m i n a t i o n b y apoptosis, e.g., of activated lymphocytes i n a u t o i m m u n e diseases. It s h o u l d also be considered that apoptosis m a y be involved i n the p a t h o m e c h a n i s m of the e l i m - i n a t i o n of T-helper lymphocytes i n A I D S , a process that is still largely not u n d e r s t o o d . In t h i s context, we tested the presence of A P O - l⁺ lymphocytes a n d of a n t i - A P O - 1 autoantibodies i n A I D S . We f o u n d the n u m b e r of A P O - l⁺ cells increased i n H I V⁺ donors. In a d d i t i o n , i n the s e r u m of H I V⁺ donors, a n t i - A P O - 1 autoantibodies were detected. These findings m a y suggest a role for apoptosis i n the depletion of T cells i n A I D S a n d clearly w a r r a n t further studies.

Finally, the m o l e c u l a r investigation of cell death i n d u c e d b y a n t i - A P O - 1 m i g h t lead to a general u n d e r s t a n d i n g of apoptosis. In this case, the use of modified or normad physiological ligands to the cell-surface antigen i n i t i a t i n g apoptosis or of c h e m i c a l s interfering w i t h the apoptotic s i g n a l might be en- visaged.

T a k e n together, the A P O - 1 apoptosis system might help to find "death genes" a n d clarify whether death o c c u r s i n steps, is a single-hit event, or c a n be reversed once its i n i t i a l signals are triggered. T h u s , the investigation of apoptosis shows that essential questions of death are l i n k e d a n d c a n be as exciting as the essential questions of life.

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ACKNOWLEDGMENTS

We t h a n k K. Hexel, G . Hölzl, M . Kaiser, J . Köllner, R. Kühnl, C . M a n d l , S. Menges, J . Moyers, a n d W . Müller for t e c h n i c a l assistance; H . Sauter for expert secretarial assistance; D . H a l l for organization of the patient follow-up; T. Gernet for help w i t h the biostatistics; a n d U . A b e l , R. B a m f o r d , R. B r a u n , H.W. Dörr, H . F i s c h e r , C . K G o l d m a n n , E . B . H e l m , M . K i e s s l - ing, K. Koretz, M . M e r c e p , H . Näher, A . Peters, D . Petzold, H . Rübsamen-Waigmann, P. S c h l a g , a n d T.A. W a l d m a n n for v a r i - o u s s u p p o r t a n d c r i t i c i s m s t h r o u g h o u t this s t u d y . T h i s s t u d y w a s s u p p o r t e d b y grants from the t u m o r center Heidelberg/

M a n n h e i m , the D e u t s c h e Krebshilfe (989-91), the B u n d e s - regierung ( P l . l - A i d s - 1 0 7 5 . 0 1 , A I 0 2 11-044-88), a n d the A i d s P r o g r a m m Baden-Württemberg (11-740.l-Aids/41).

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