- Email: [email protected]
Inhibition of the mixed lymphocyte proliferative response by phorbol esters
246
Biochimica et Biophysica Acta, 517 ( 1 9 7 8 ) 2 4 6 - - 2 5 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical P r e s s
BBA 9 9 0 8 6
INHIBITION OF THE MIXED LYMPHOCYTE PROLIFERATIVE RESPONSE BY PHORBOL ESTERS
A N D R E A M. M A S T R O * a n d G E R A L D C. M U E L L E R **
McArdle Laboratory for Cancer Research, The University of Wisconsin, Madison, Wisc. 53706 (U.S.A.) ( R e c e i v e d April 2 6 t h , 1977) (Revised m a n u s c r i p t received S e p t e m b e r 2 0 t h , 1977)
Summary The phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, a p o t e n t cocarcinogen in mice, blocks the induction of DNA synthesis in lymphocytes undergoing the mixed l y m p h o c y t e response . At 10 -7 M diester, the induced DNA synthesis is inhibited almost completely (99%). This action of the diester affects some early step in the response which is necessary for the triggering of cell replication; on-going DNA replication is not significantly affected. Phorbol 12,13-diacetate, a less p o t e n t analogue in t u m o r p r o m o t i o n in vivo, is also a less p o t e n t inhibitor of the mixed l y m p h o c y t e response (75% inhibition at 10 .6 M). Phorbol, the parent alcohol, is n o t effective in either system. The use of phorbol diesters in the molecular dissection of mixed l y m p h o c y t e responses is discussed.
Introduction When genetically dissimilar l y m p h o c y t e s are cultured together for several days there is a striking stimulation of DNA synthesis and cell replication [ 1 ]. This proliferative reaction, the mixed l y m p h o c y t e response, is thought to result from interactions of surface antigens from the donor cell with receptors on the surface of the responding cell. The degree of the response has been shown to correlate with the degree of histoincompatibility at certain loci of the major histocompatibility complex. In fact, in humans, the reaction provides a means for identifying the alleles responsible for the rejection of transplanted tissues [2]. Accordingly, the response is useful as an in vitro model of in vivo allograft * Present address: The Pennsylvania State University, Department of Biochemistry and Biophysics, 6 1 8 Life Sciences Building, University Park, Pa. 16802, U.S.A. ** T o w h o m reprint requests should be addressed.
247 rejection [3] and as a system for dissection of immune cell-cell interactions [41. In the present study we report that 12-O-tetradecanoyl-phorbol-13-acetate, is a highly effective inhibitor of the proliferative response in mixed l y m p h o c y t e cultures. This c o m p o u n d is the most active member of a series of phorbol diesters derived from croton oil which are promoters of chemical carcinogenesis in vivo [5--8]. In addition to being co-carcinogens, these c o m p o u n d s function acutely as activators of cell membrane metabolism. In vitro, 12-O-tetradecanoylphorbol-13-acetate at low doses (10-s--10 -6 M) produces changes in cell morphology, stimulates lipid synthesis in cell membranes, and promotes cell division [9--12]. These actions at the cell membrane (see also refs. 13, 14) appear to lead to the induced synthesis of a spectrum of enzymes among which are ornithine decarboxylase [15], 5-adenosyl methyltransferase [15], and plasminogen activator protease [16]. The observation that the phorbol diester blocks the mitogenic response in mixed l y m p h o c y t e cultures, yet is comitogenic in cultures of human peripheral lymphocytes [17], rat thymic lymphoblast [18] and bovine lymph node lymphocytes stimulated with lectins [19], raises the possibility that an enzyme induced by this c o m p o u n d may selectively disrupt the mitogenic sequence where histocompatibility antigens are involved. In this case phorbol esters might be extremely useful tools in dissecting this response and providing basic insights into the practical control of such cellular reactions. Materials and Methods
Isolation and culture of lymphocytes Procedure A. L y m p h o c y t e s were isolated from bovine retropharyngeal lymph nodes as previously described [19]. Freshly isolated cells were suspended at 4 • 106 -- 5 • 106 ml in modified Eagle's medium containing 10% bovine serum [20]. Either 5 or 15 ml were planted in 12 or 40 ml conical centrifuge tubes, respectively, and kept on a slant at 37°C in a 5% CO2 atmosphere. The cultures contained either cells from one animal or a mixture of l y m p h o c y t e s from t w o or more animals. A two-way or multiple response was allowed to occur; no sets of cells were prevented from dividing. 12-O-Tetradecanoyl-phorbol-13-acetate at a final concentration of 10-TM and 0.5% dimethyl sulfoxide, was added to the medium of cultures at the start of the incubation and fresh Eagle's medium plus 10% bovine serum was added back at each medium change (every second day) unless otherwise specified. In some experiments the addition of the diester was delayed, or it was present only part of the time. Phorbol 12,13
248 1640 medium containing 10% calf serum. The compounds to be tested (12-Otetradecanoyl-phorbol-13-acetate, phorbol 12,13-diacetate or phorbol) were added to the cells so that the final dimethyl sulfoxide concentration was 0.1%. The suspension was pipetted into round b o t t o m microtiter plates (linbro), 0.2 ml (1 • 106 cells) per well. Cultures were pipetted in triplicate. The plates were kept at 37°C in a 5% CO2 atmosphere without a medium change. [3H]Thymidine (0.2 gCi in 10 pl, 3.4 Ci/mM) was added to each culture well 2 h before harvesting with a Skatron multiple cell harvester [ 21].
Chemicals 12-O-Tetradecanoyl-phorbol-13-acetate and phorbol 12,13-diacetate were synthesized by Dr. Tucker Helmes, who generously provided us with samples. Phorbol was purchased from Consolidated Midlands Corporation, Brewster, N.Y. Results
The mixed lymphocyte response with lymphocytes from bovine retropharyngeal lymph nodes To test if the lymphocytes isolated from bovine lymph nodes gave a characteristic response, cells from the lymph nodes of two animals or from several animals were cultured together in equal numbers and tested for [3H]thymidine incorporation every 24 h for 8 d a y s . In mixed cultures, the l y m p h o c y t e s differentiated into blast cells and began to synthesize DNA after 6--8 days in culture (Fig. 1, dashed lines). Isologous cells, isolated from a single node, did not respond. The highest rate of [3H]thymidine incorporation occurred on day 6, 7 or 8 depending on the experiment. Fig. 1, a composite of the results of two experiments, shows data typical of at least 10 such trials. Similar kinetics are seen with cells cultured in microwells. Within the limits of the cell numbers used, the kinetics and character of the mixed l y m p h o c y t e responses were similar despite minor variations on the cell densities in i~.4ividual experiments. The effect of 12-O-tetradecanoyl-phorbol-13-acetate on the mixed lymphocyte response A mixture of lymphocytes from several animals was cultured with 10 - ~ 10-9M 12-O-tetradecanoyl-phorbol-13-acetate present throughout the entire incubation period. Under these conditions the increased DNA synthesis characteristic of the mixed l y m p h o c y t e response was prevented by 10-SM diester (Fig. 2). This result is not due simply to cell death because the decline in the number of viable cells (trypan blue exclusion test) in cultures with the diester remained approximately the same as in non-treated controls. Furthermore, a direct cell count on day 6 of a typical experiment revealed that 64 and and 50% of the starting control and diester-treated cell populations remained, respectively; on day 2 this cell survival was 66 and 67%, respectively. Since we have shown previously that this concentration of diester also enhances blast division in cultures stimulated with phytohemagglutinin or with concanavalin A [19], these numbers can only be viewed as summing up the population
249
tM
15
'_o
~ B+C
•
ii I \
I%j = IQ U ~.
/ //
~'5 b.I
,
II
x ] pA+B+C ~ I/ \>,,, J/ /z
/
/
///" //.11/i/I ~1 / --/ I X
/" //" / /i
_z
~:S >" -i-
//0 ....
I / 0//
/
~A+B
/ ////
•
/
^^
I-
0"
2
3
4
5
6
7
8
DAYS Fig. 1. K i n e t i c s o f m i x e d l y m p h o c y t e r e s p o n s e w i t h l y m p h o e y t e s f r o m b o v i n e r e t r o p h a r y n g e a l l y m p h n o d e s . L y m p h o c y t e s f r o m o n e l y m p h n o d e o f single a n i m a l s ( A , B, or C) or e q u a l m i x t u r e s o f l y m p h o c y t e s f r o m t h e n o d e s o f t w o o r t h r e e a n i m a l s as i n d i c a t e d w e r e c u l t u r e d at 4 • 105 c e l l s / m l in 15 m l E a g l e ' s m e d i u m w i t h 10% b o v i n e s e r u m . A t 24-h i n t e r v a l s , [ 3 H ] t h y m i d i n e w a s a d d e d t o a set o f c u l t u r e t u b e s w h i c h w e r e i n c u b a t e d f o r 2 h m o r e a n d p r o c e s s e d as d e s c r i b e d in P r o c e d u r e A o f M a t e r i a l s a n d M e t h o d s . [ 3 H ] T h y m i d i n e i n c o r p o r a t i o n is s h o w n f o r c u l t u r e s o f l y m p h o c y t e s f r o m single a n i m a l s (solid l i n e s ) , a n d f r o m m i x t u r e s o f t w o or t h r e e a n i m a l s ( d a s h e d lines). S i m i l a r r e s u l t s w e r e o b t a i n e d f r o m cells c u l t u r e d on m i c r o t i t e r p l a t e s ,
dynamics. Thus, while a small subpopulation of cells may have been dest royed selectively by p h o rbol diester, an interesting possibility in its own right, these results do rule o u t a massive killing of the cells. We f u r th er tested the phorbol diester over the range of concentrations 10 -9 , 10 .8 and 10 -7 M (Fig. 2}. At 10 -7 or 10 -8 M the inhibition of [3H]thymidine incorporation was greater than 97% on the day of the peak DNA synthesis in u n t reated cultures. However, 10 -9 M diester did n o t inhibit incorporation, and in fact stimulated it somewhat. In three out of four experiments it was observed th at 10 -9 M phorbol diester shortened the lag period of the response by a b o u t one day.
The effect of the time of addition of 12-O-tetradecanoyl-phorbol-13-acetate on the mixed lymphocyte response In an a t t e m p t to define the period during which the l y m p h o c y t e s are sensitive to the phorbol diester, the c o m p o u n d was added 2, 4 or 6 days after establishing the mixed cultures and in each case was kept in the medium until the end of the experiment. One set of cultures had the diester present from day 0. Control cells were never exposed to the c o m p o u n d . Cultures to which the diester was added on the second day after planting failed to show an
250
q.
9 -
b K
UJ
6 ft. W Z u
"1I--
10-8
0
I
2
;5
4 DAYS
~
~
r
Fig. 2. L y m p h o c y t e s f r o m t w o a n i m a l s w e r e c u l t u r e d t o g e t h e r in m u l t i w e l l dishes as d e s c r i b e d in P r o c e d u r e B, Materials a n d M e t h o d s . 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e ( T P A ) was p r e s e n t f r o m the b e g i n n i n g o f c u l t u r e at 10 -9 o r 10 -8 M w i t h 0.1% d i m e t h y l s u l f o x i d e . A c o n t r o l set o f m i x e d cells received only d i m e t b y l sulfoxide (DMSO). [ 3 H ] T h y m i d i n e was added for 2 h each day to triplicate c u l t u r e s f r o m e a c h s e t o f cells. R e s u l t s are e x p r e s s e d as d p m -+ S.E. This e x p e r i m e n t w a s r e p e a t e d f o u r t i m e s in t r i p l i c a t e , e a c h w i t h cells f r o m d i f f e r e n t a n i m a l s . T h e results are essentially t h e s a m e in all e x p e r i m e n t s e x c e p t t h a t in t h r e e o f t h e f o u r e x p e r i m e n t s , 10 -9 M p h o r b o l d i e s t e r s h o r t e n e d the t i m e o f t h e m i x e d l y m p h o c y t e r e s p o n s e b y a b o u t 1 d a y . P b o r b o l d i e s t e r at 0 c o n c e n t r a t i o n (o); 10 -9 M (o); 10 -8 M (m). D a t a f o r 10 -7 M p h o r b o l d i e s t e r ( n o t s h o w n ) w e r e similar to 10 -8 M.
increased [3H]thymidine incorporation (Fig. 3); however, if the diester was added from day 4 or 6, the cells still synthesized DNA on days 6 or 7. In a similar experiment, 10 -~ M phorbol diester was added to cells in microculture plates from days 0, 2, 3 and 4. Compared to untreated cells, addition of the diester on day 2 caused a 75% inhibition of DNA synthesis; however, diester added on day 3 or day 4 no longer blocked the response. In a separate study to further examine the time of action of phorbol diester, cultures of mixed cells were incubated in the presence of 10 -v M diester for the first 2 days of culture. The medium was then changed and the diester was n o t added back. A n o t h e r set of cultures had phorbol diester present throughout the 9 days of incubation while the control cells were incubated w i t h o u t it. The cells t h a t were exposed to the c o m p o u n d for the first 2 days only, failed to synthesize DNA at the characteristic time as did the cells exposed continuously (Fig. 4). In some cultures, cells incubated with the phorbol diester showed a burst of DNA synthesis at 48 h (Figs. 3 and 4). This response could be eliminated by preincubating the cells for 18 h prior to adding the diester. While the explana-
251 NO TPA
=' 4
\
J
~
, / II
I
,.,-=
'~,
'~\
;
//,"
"'TPA,DAY 6
",,.
i/,,
',.
#
I'-
, I
~
~TPA , DAY 4 .
&----&--.
TPA, DAY 0
,'S
0
" ....
"ATPA, DAY 2
I
I
I
l
l
I
I
2
3
4
5
6
7
8
DAYS Fig. 3. T h e e f f e c t of t h e d e l a y e d a d d i t i o n of 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a e e t a t e ( T P A ) o n t h e m i x e d l y m p h o c y t e r e s p o n s e . R e p l i c a t e sets o f c u l t u r e s o f m i x t u r e s o f l y m p h o c y t e s w e r e p l a n t e d a t 5 • 10 6 cells/ m l in 5 m l E a g l e ' s m e d i u m w i t h 10% b o v i n e s e r u m . T h e p h o r b o l d i e s t e r at 10 -7 M w a s a d d e d at t h e indicated times. • e c o n t r o l c u l t u r e s n o p h o r b o l dicster; o o, p h o r b o l d i e s t e r f r o m d a y 0; • "-, p h o r b o l d i e s t e r f r o m d a y 2; ~ ~, p h o r b o l d i e s t e r f r o m d a y 4; ~ D, p h o r b o l d i e s t e r f r o m d a y 6. Similar r e s u l t s w e r e seen w h e n t h e e x p e r i m e n t w a s p e r f o r m e d as in P r o c e d u r e B.
TPA I
o_
o - - o O - 2 DAYS = = 0 - 9 DAYS
x
"'4
~
0
-.3 =E (L C~ v
T~)
"'2
z
,,.,..
2E
>" "11" I--
I
.m. 0
H
I
I
I
i
I
t
~
~,TPA
2
3
4
5
6
7
8
9
DAYS Fig, 4. T h e e f f e c t s o f a n initial t w o - d a y e x p o s u r e o f l y m p h o c y t e s t o 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 a c e t a t e ( T P A ) o n t h e m i x e d l y m p h o c y t e r e s p o n s e . L y m p h o c y t e s w e r e c u l t u r e d as d e s e r l b e d in t h e l e g e n d t o Fig. 1. T h e p h o r b o l d i e s t e r a t 1 0 - ? M w a s a d d e d t o c u l t u r e s o n d a y 0. F r o m o n e set it w a s r e m o v e d b y a m e d i u m c h a n g e o n d a y 2. C o n t r o l c u l t u r e s h a d n o p h o r b o l d i e s t e r a d d e d , o . . . . . . o, p h o r b o l d l e s t e r d a y s 0 - - 2 ; -" e, phorbol diester days 0--9; • • , control, no p h o r b o l diester.
252
~ O -7 M
°-9,,
/ ~ o -au
o= b
i?;i ?o / II
ILl
I.~ !
I
a.--.-~OMSO
'
z_
0
I
2
3
4 DAYS
5
6
7
8
Fig. 5. T h e e f f e c t s of p h o r b o l 1 2 , 1 3 - d i a c e t a t e o n t h e m i x e d l y m p h o c y t e r e s p o n s e . L y m p h o c y t e s f r o m t w o a n i m a l s w e r e c u l t u r e d in m u l t i w e n dishes as d e s c r i b e d in Materials a n d M e t h o d s . R e s u l t s are e x p r e s s e d as d p m -+ S.E. f o r m i x e d cells c u l t u r e d w i t h n o a d d i t i o n s (ee ) , or with phorbol diacetate at 10 -8 M (© o), 10 -7 M ( i I ) a n d 10 -6 M (o ~) a n d 0.1% d i m e t h y l s u l f o x i d e . Cells w i t h 0.1% d i m e t h y l s u l f o x i d e ( D M S O ) a l o n e are s h o w n ( a a ) as are cells f r o m o n e a n i m a l only w i t h no a d d i t i o n s (X X). This e x p e r i m e n t , w h e n r e p e a t e d w i t h cells f r o m t w o d i f f e r e n t a n i m a l s , gave essentially t h e s a m e results. P h o r b o l d i a c e t a t e at 10 -6 M i n h i b i t e d t h e m i x e d l y m p h o c y t e r e s p o n s e b y 76 a n d 72% in t h e t w o e x p e r i m e n t s , r e s p e c t i v e l y .
tion of this response is not completely established, it appears likely that lymphocytes from lymph nodes of slaughter house animals may have received a significant, y e t limiting, level of endogenous mitogenic stimulation, and that the diester may act co-mitogenically on such lymphocytes as reported earlier for its action on lectin-treated cells [19]. In any event the response appears n o t to be directly related to the mixed l y m p h o c y t e response and can be eliminated by delaying the mitogenic interaction of the mixed lymphocytes or the addition of the phorbol diester.
The effects of 12-O-tetradecanoyl-phorbol-13-acetate analogues on the mixed lymphocyte response While 12-O-tetradecanoyl-phorbol-13-acetate is the most p o t e n t t u m o r promoter of the phorbol diester series, certain analogues of this compound, such as phorbol 12,13-diacetate are weaker promoters [5]. To test if the activity of this ester, which is about one-tenth as effective as a t u m o r promoter, is reflected in its effects on lymphocytes, we tested it in mixed cell cultures (Fig. 5). Phorbol 12,13-diacetate (10 -8, 10 -7 and 10 -6 M) or dimethyl sulfoxide
253 {0.1%) was added to mixed cultures at d a y 0 . Unlike 12-O-tetradecanoylphorbol-13-acetate, 10-SM and 10-TM phorbol 12,13-diacetate and 0.1% dimethyl sulfoxide had no inhibitory effect on the uptake of [3H]thymidine, However, 10 -6 M phorbol 12,13-diacetate blocked the incorporation by about 75%. Thus, ten times the concentration of phorbol 12,13-diacetate was required to inhibit to nearly the same level as 10 -7 M 12-O-tetradecanoylphorbol-13-acetate. Similarly, ten times as much phorbol 12,13-diacetate is required to give the same level of co-mitogen action as 12-O-tetradecanoylphorbol-13-acetate [ 17 ]. Phorbol, the parent c o m p o u n d of the series, was also tested at 10 -6, 10 -v and 10 -8 M in the mixed l y m p h o c y t e culture. At these concentrations, it did not inhibit the response {data n o t shown}. Discussion
The experiments in this paper show that 12-O-tetradecanoyl-phorbol-13acetate (10 -7, 10 -8 M} is a highly effective agent for blocking the induction of DNA synthesis of the mixed l y m p h o c y t e response in bovine l y m p h o c y t e cultures. This phorbol diester was not generally toxic since cells which were triggered for or already in the process of DNA replication were n o t affected significantly. The selective killing of a small group of lymphocytes which are essential for the mixed lymphocyte response cannot be ruled o u t in these experiments; however, this result in itself would point to an interesting new characteristic of this population of cells. The observation that the same levels of the diester are co,mitogenic when added to lectin-treated cultures [19] further supports this view and suggests that the diester may induce changes in the target cells which selectively interrupts the chain of mitogenic events where histocompatibility antigens are involved. The finding that 12-O-tetradecanoylphorbol-13-acetate was effective when added as late as day 2 after the start of the mixed l y m p h o c y t e cultures implies that an early, but y e t n o t the initial step in the response, was affected. While one-way mixed l y m p h o c y t e responses were not carried o u t in these studies, it seems most likely, in the view of the delayed addition studies, that the responding cell is the target for phorbol diester action. This question will be explored directly in future studies. Since the phorbol diester has been already shown to be a highly effective inducer of certain enzymes, it also appears quite probable that its effect on the mixed l y m p h o c y t e response is mediated as well through its inductive effects in gene expression. If this turns o u t to be the case the product of the induction will likely be an enzyme or a metabolic state which is uniquely opposed to the progression of mitogenic events set in motion by antigens involved in histocompatability. In this case 12-O-tetradecanoyl-phorbol-13-acetate and related phorhol esters should be useful tools in dissecting the molecular processes which control this type of lymphocyte response and possibly open the way to new approaches to the practical control of such cellular reactions in transplantation biology. Acknowledgements This study was supported by U.S. Public Health Service Grants T01-CA5002 and CA-07175 to G.C.M., and in part by a grant from NIH (GM 22953}
254
to A.M.M.G.C.M. is the recipient of a Research Career Development Award, U.S.A. Public Health Service. A.M.M. was a Damon R u n y o n Postdoctoral Fellow. The authors thank Dr. Tucker Helmes for his gift of the phorbol esters and Ms. Mary LeMahieu for her help with preparation of this manuscript. References 1 Bach, F., Bock, H., Graupner, K., Day, E. and Klostermann, H. (1969) Proc. Natl. Acad. Sci. U.S. 62, 377 --384 2 Bach, F.H., Bach, M.L. and Sondel, P.M. (1976) Nature 259, 273--281 3 H~'yry, P. and Defendi, V. (1970) Science 168, 133--135 4 Cohen, L. and Howe, M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2 7 0 7 - - 2 7 1 0 5 Baird, W.M., Sedgwiek, J.A. and Boutwell, R.K. (1971) Cancer Res. 31, 1434--1439 6 Boutwell, R.K. (1964) in Progress in E x p e r i m e n t a l Tumor Research (Homberger, F., ed.), Vol. 4, pp. 207--250, Karger, New York 7 Hecker, E. (1971) in Methods in Cancer Research (Busch, H., ed.), Vol. 6, pp. 439--484, Academic Press, New Yo rk 8 Van Duuren, B.L. (1964) in Progress in E x p e r i m e n t a l Tumor Research (Homberger, F., ed.), Vol. 11, pp. 31--68, Karger, New York 9 Sivak, A., Ray, F. and Van Duuren, B.L. (1969) Cancer Res. 29, 624--630 10 Sivak, A., Mossman, B.T. and Van Duuren, B.L. (1972) Biochem. Biophys. Res. C ommun. 46, 605-609 11 Sfiss, R., Kreibich, G. andKinzel, V. (1972) Eur. J. Cancer 8 , 2 9 9 - - 3 0 4 12 Estensen, R.D., Hadden, J.W., Hadden, E.M., Touraine, F., Touraine, J., Haddox, M.K. and Goldberg, N.D. (1974) in Control of Proliferation in Animal Cells, CSH Conference on Cell Proliferation (Clarkson, B. and Baserga, R., eds.), Vol. 1, pp. 627--634, Cold Spring Harbor, New Y ork 13 Wenner, C.E., Hackney, J., Kimelberg, H.K. and Mayhew, E. (1974) Cancer Res. 34, 1731--1737 14 Rohrschneider, L.R. and Boutweil, R.K. (1973) Nature New Biol. 243, 212--213 15 O'Brien, T.G., Simsiman, R.C. and Boutwell, R.K. (1975) Cancer Res. 35, 1 6 6 7 - - 1 6 7 0 16 Wigler0 M, and Weinstein, I.B. (1976) Nature 259, 232--233 17 MueBer, G.C. and Kajiwara, K. (1965) in Developmental and Metabolic Control Mechanisms and Neoplasia, pp. 452--474, Wflkens and Wilkens, Baltimore 18 Whitfield, J.F,, MacManus, J.P. and Gillan, D.J. (1973) J. Cell. Physiol. 82, 151--156 19 Mastro, A.M. and Mueller, G.C. (1974) Exp. Cell Res. 88, 40--46 20 Mueller, G.C., Kajiwara, K., Stubblefield, E. and Rueckert, R. (1962) Cancer Res. 22, 1084--1090 21 Hirschberg, H. and Thorsby, E. (1973) J. I m m u n o l . Methods 3, 2 5 1 = 2 6 4
Biochimica et Biophysica Acta, 517 ( 1 9 7 8 ) 2 4 6 - - 2 5 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical P r e s s
BBA 9 9 0 8 6
INHIBITION OF THE MIXED LYMPHOCYTE PROLIFERATIVE RESPONSE BY PHORBOL ESTERS
A N D R E A M. M A S T R O * a n d G E R A L D C. M U E L L E R **
McArdle Laboratory for Cancer Research, The University of Wisconsin, Madison, Wisc. 53706 (U.S.A.) ( R e c e i v e d April 2 6 t h , 1977) (Revised m a n u s c r i p t received S e p t e m b e r 2 0 t h , 1977)
Summary The phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, a p o t e n t cocarcinogen in mice, blocks the induction of DNA synthesis in lymphocytes undergoing the mixed l y m p h o c y t e response . At 10 -7 M diester, the induced DNA synthesis is inhibited almost completely (99%). This action of the diester affects some early step in the response which is necessary for the triggering of cell replication; on-going DNA replication is not significantly affected. Phorbol 12,13-diacetate, a less p o t e n t analogue in t u m o r p r o m o t i o n in vivo, is also a less p o t e n t inhibitor of the mixed l y m p h o c y t e response (75% inhibition at 10 .6 M). Phorbol, the parent alcohol, is n o t effective in either system. The use of phorbol diesters in the molecular dissection of mixed l y m p h o c y t e responses is discussed.
Introduction When genetically dissimilar l y m p h o c y t e s are cultured together for several days there is a striking stimulation of DNA synthesis and cell replication [ 1 ]. This proliferative reaction, the mixed l y m p h o c y t e response, is thought to result from interactions of surface antigens from the donor cell with receptors on the surface of the responding cell. The degree of the response has been shown to correlate with the degree of histoincompatibility at certain loci of the major histocompatibility complex. In fact, in humans, the reaction provides a means for identifying the alleles responsible for the rejection of transplanted tissues [2]. Accordingly, the response is useful as an in vitro model of in vivo allograft * Present address: The Pennsylvania State University, Department of Biochemistry and Biophysics, 6 1 8 Life Sciences Building, University Park, Pa. 16802, U.S.A. ** T o w h o m reprint requests should be addressed.
247 rejection [3] and as a system for dissection of immune cell-cell interactions [41. In the present study we report that 12-O-tetradecanoyl-phorbol-13-acetate, is a highly effective inhibitor of the proliferative response in mixed l y m p h o c y t e cultures. This c o m p o u n d is the most active member of a series of phorbol diesters derived from croton oil which are promoters of chemical carcinogenesis in vivo [5--8]. In addition to being co-carcinogens, these c o m p o u n d s function acutely as activators of cell membrane metabolism. In vitro, 12-O-tetradecanoylphorbol-13-acetate at low doses (10-s--10 -6 M) produces changes in cell morphology, stimulates lipid synthesis in cell membranes, and promotes cell division [9--12]. These actions at the cell membrane (see also refs. 13, 14) appear to lead to the induced synthesis of a spectrum of enzymes among which are ornithine decarboxylase [15], 5-adenosyl methyltransferase [15], and plasminogen activator protease [16]. The observation that the phorbol diester blocks the mitogenic response in mixed l y m p h o c y t e cultures, yet is comitogenic in cultures of human peripheral lymphocytes [17], rat thymic lymphoblast [18] and bovine lymph node lymphocytes stimulated with lectins [19], raises the possibility that an enzyme induced by this c o m p o u n d may selectively disrupt the mitogenic sequence where histocompatibility antigens are involved. In this case phorbol esters might be extremely useful tools in dissecting this response and providing basic insights into the practical control of such cellular reactions. Materials and Methods
Isolation and culture of lymphocytes Procedure A. L y m p h o c y t e s were isolated from bovine retropharyngeal lymph nodes as previously described [19]. Freshly isolated cells were suspended at 4 • 106 -- 5 • 106 ml in modified Eagle's medium containing 10% bovine serum [20]. Either 5 or 15 ml were planted in 12 or 40 ml conical centrifuge tubes, respectively, and kept on a slant at 37°C in a 5% CO2 atmosphere. The cultures contained either cells from one animal or a mixture of l y m p h o c y t e s from t w o or more animals. A two-way or multiple response was allowed to occur; no sets of cells were prevented from dividing. 12-O-Tetradecanoyl-phorbol-13-acetate at a final concentration of 10-TM and 0.5% dimethyl sulfoxide, was added to the medium of cultures at the start of the incubation and fresh Eagle's medium plus 10% bovine serum was added back at each medium change (every second day) unless otherwise specified. In some experiments the addition of the diester was delayed, or it was present only part of the time. Phorbol 12,13
248 1640 medium containing 10% calf serum. The compounds to be tested (12-Otetradecanoyl-phorbol-13-acetate, phorbol 12,13-diacetate or phorbol) were added to the cells so that the final dimethyl sulfoxide concentration was 0.1%. The suspension was pipetted into round b o t t o m microtiter plates (linbro), 0.2 ml (1 • 106 cells) per well. Cultures were pipetted in triplicate. The plates were kept at 37°C in a 5% CO2 atmosphere without a medium change. [3H]Thymidine (0.2 gCi in 10 pl, 3.4 Ci/mM) was added to each culture well 2 h before harvesting with a Skatron multiple cell harvester [ 21].
Chemicals 12-O-Tetradecanoyl-phorbol-13-acetate and phorbol 12,13-diacetate were synthesized by Dr. Tucker Helmes, who generously provided us with samples. Phorbol was purchased from Consolidated Midlands Corporation, Brewster, N.Y. Results
The mixed lymphocyte response with lymphocytes from bovine retropharyngeal lymph nodes To test if the lymphocytes isolated from bovine lymph nodes gave a characteristic response, cells from the lymph nodes of two animals or from several animals were cultured together in equal numbers and tested for [3H]thymidine incorporation every 24 h for 8 d a y s . In mixed cultures, the l y m p h o c y t e s differentiated into blast cells and began to synthesize DNA after 6--8 days in culture (Fig. 1, dashed lines). Isologous cells, isolated from a single node, did not respond. The highest rate of [3H]thymidine incorporation occurred on day 6, 7 or 8 depending on the experiment. Fig. 1, a composite of the results of two experiments, shows data typical of at least 10 such trials. Similar kinetics are seen with cells cultured in microwells. Within the limits of the cell numbers used, the kinetics and character of the mixed l y m p h o c y t e responses were similar despite minor variations on the cell densities in i~.4ividual experiments. The effect of 12-O-tetradecanoyl-phorbol-13-acetate on the mixed lymphocyte response A mixture of lymphocytes from several animals was cultured with 10 - ~ 10-9M 12-O-tetradecanoyl-phorbol-13-acetate present throughout the entire incubation period. Under these conditions the increased DNA synthesis characteristic of the mixed l y m p h o c y t e response was prevented by 10-SM diester (Fig. 2). This result is not due simply to cell death because the decline in the number of viable cells (trypan blue exclusion test) in cultures with the diester remained approximately the same as in non-treated controls. Furthermore, a direct cell count on day 6 of a typical experiment revealed that 64 and and 50% of the starting control and diester-treated cell populations remained, respectively; on day 2 this cell survival was 66 and 67%, respectively. Since we have shown previously that this concentration of diester also enhances blast division in cultures stimulated with phytohemagglutinin or with concanavalin A [19], these numbers can only be viewed as summing up the population
249
tM
15
'_o
~ B+C
•
ii I \
I%j = IQ U ~.
/ //
~'5 b.I
,
II
x ] pA+B+C ~ I/ \>,,, J/ /z
/
/
///" //.11/i/I ~1 / --/ I X
/" //" / /i
_z
~:S >" -i-
//0 ....
I / 0//
/
~A+B
/ ////
•
/
^^
I-
0"
2
3
4
5
6
7
8
DAYS Fig. 1. K i n e t i c s o f m i x e d l y m p h o c y t e r e s p o n s e w i t h l y m p h o e y t e s f r o m b o v i n e r e t r o p h a r y n g e a l l y m p h n o d e s . L y m p h o c y t e s f r o m o n e l y m p h n o d e o f single a n i m a l s ( A , B, or C) or e q u a l m i x t u r e s o f l y m p h o c y t e s f r o m t h e n o d e s o f t w o o r t h r e e a n i m a l s as i n d i c a t e d w e r e c u l t u r e d at 4 • 105 c e l l s / m l in 15 m l E a g l e ' s m e d i u m w i t h 10% b o v i n e s e r u m . A t 24-h i n t e r v a l s , [ 3 H ] t h y m i d i n e w a s a d d e d t o a set o f c u l t u r e t u b e s w h i c h w e r e i n c u b a t e d f o r 2 h m o r e a n d p r o c e s s e d as d e s c r i b e d in P r o c e d u r e A o f M a t e r i a l s a n d M e t h o d s . [ 3 H ] T h y m i d i n e i n c o r p o r a t i o n is s h o w n f o r c u l t u r e s o f l y m p h o c y t e s f r o m single a n i m a l s (solid l i n e s ) , a n d f r o m m i x t u r e s o f t w o or t h r e e a n i m a l s ( d a s h e d lines). S i m i l a r r e s u l t s w e r e o b t a i n e d f r o m cells c u l t u r e d on m i c r o t i t e r p l a t e s ,
dynamics. Thus, while a small subpopulation of cells may have been dest royed selectively by p h o rbol diester, an interesting possibility in its own right, these results do rule o u t a massive killing of the cells. We f u r th er tested the phorbol diester over the range of concentrations 10 -9 , 10 .8 and 10 -7 M (Fig. 2}. At 10 -7 or 10 -8 M the inhibition of [3H]thymidine incorporation was greater than 97% on the day of the peak DNA synthesis in u n t reated cultures. However, 10 -9 M diester did n o t inhibit incorporation, and in fact stimulated it somewhat. In three out of four experiments it was observed th at 10 -9 M phorbol diester shortened the lag period of the response by a b o u t one day.
The effect of the time of addition of 12-O-tetradecanoyl-phorbol-13-acetate on the mixed lymphocyte response In an a t t e m p t to define the period during which the l y m p h o c y t e s are sensitive to the phorbol diester, the c o m p o u n d was added 2, 4 or 6 days after establishing the mixed cultures and in each case was kept in the medium until the end of the experiment. One set of cultures had the diester present from day 0. Control cells were never exposed to the c o m p o u n d . Cultures to which the diester was added on the second day after planting failed to show an
250
q.
9 -
b K
UJ
6 ft. W Z u
"1I--
10-8
0
I
2
;5
4 DAYS
~
~
r
Fig. 2. L y m p h o c y t e s f r o m t w o a n i m a l s w e r e c u l t u r e d t o g e t h e r in m u l t i w e l l dishes as d e s c r i b e d in P r o c e d u r e B, Materials a n d M e t h o d s . 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e ( T P A ) was p r e s e n t f r o m the b e g i n n i n g o f c u l t u r e at 10 -9 o r 10 -8 M w i t h 0.1% d i m e t h y l s u l f o x i d e . A c o n t r o l set o f m i x e d cells received only d i m e t b y l sulfoxide (DMSO). [ 3 H ] T h y m i d i n e was added for 2 h each day to triplicate c u l t u r e s f r o m e a c h s e t o f cells. R e s u l t s are e x p r e s s e d as d p m -+ S.E. This e x p e r i m e n t w a s r e p e a t e d f o u r t i m e s in t r i p l i c a t e , e a c h w i t h cells f r o m d i f f e r e n t a n i m a l s . T h e results are essentially t h e s a m e in all e x p e r i m e n t s e x c e p t t h a t in t h r e e o f t h e f o u r e x p e r i m e n t s , 10 -9 M p h o r b o l d i e s t e r s h o r t e n e d the t i m e o f t h e m i x e d l y m p h o c y t e r e s p o n s e b y a b o u t 1 d a y . P b o r b o l d i e s t e r at 0 c o n c e n t r a t i o n (o); 10 -9 M (o); 10 -8 M (m). D a t a f o r 10 -7 M p h o r b o l d i e s t e r ( n o t s h o w n ) w e r e similar to 10 -8 M.
increased [3H]thymidine incorporation (Fig. 3); however, if the diester was added from day 4 or 6, the cells still synthesized DNA on days 6 or 7. In a similar experiment, 10 -~ M phorbol diester was added to cells in microculture plates from days 0, 2, 3 and 4. Compared to untreated cells, addition of the diester on day 2 caused a 75% inhibition of DNA synthesis; however, diester added on day 3 or day 4 no longer blocked the response. In a separate study to further examine the time of action of phorbol diester, cultures of mixed cells were incubated in the presence of 10 -v M diester for the first 2 days of culture. The medium was then changed and the diester was n o t added back. A n o t h e r set of cultures had phorbol diester present throughout the 9 days of incubation while the control cells were incubated w i t h o u t it. The cells t h a t were exposed to the c o m p o u n d for the first 2 days only, failed to synthesize DNA at the characteristic time as did the cells exposed continuously (Fig. 4). In some cultures, cells incubated with the phorbol diester showed a burst of DNA synthesis at 48 h (Figs. 3 and 4). This response could be eliminated by preincubating the cells for 18 h prior to adding the diester. While the explana-
251 NO TPA
=' 4
\
J
~
, / II
I
,.,-=
'~,
'~\
;
//,"
"'TPA,DAY 6
",,.
i/,,
',.
#
I'-
, I
~
~TPA , DAY 4 .
&----&--.
TPA, DAY 0
,'S
0
" ....
"ATPA, DAY 2
I
I
I
l
l
I
I
2
3
4
5
6
7
8
DAYS Fig. 3. T h e e f f e c t of t h e d e l a y e d a d d i t i o n of 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a e e t a t e ( T P A ) o n t h e m i x e d l y m p h o c y t e r e s p o n s e . R e p l i c a t e sets o f c u l t u r e s o f m i x t u r e s o f l y m p h o c y t e s w e r e p l a n t e d a t 5 • 10 6 cells/ m l in 5 m l E a g l e ' s m e d i u m w i t h 10% b o v i n e s e r u m . T h e p h o r b o l d i e s t e r at 10 -7 M w a s a d d e d at t h e indicated times. • e c o n t r o l c u l t u r e s n o p h o r b o l dicster; o o, p h o r b o l d i e s t e r f r o m d a y 0; • "-, p h o r b o l d i e s t e r f r o m d a y 2; ~ ~, p h o r b o l d i e s t e r f r o m d a y 4; ~ D, p h o r b o l d i e s t e r f r o m d a y 6. Similar r e s u l t s w e r e seen w h e n t h e e x p e r i m e n t w a s p e r f o r m e d as in P r o c e d u r e B.
TPA I
o_
o - - o O - 2 DAYS = = 0 - 9 DAYS
x
"'4
~
0
-.3 =E (L C~ v
T~)
"'2
z
,,.,..
2E
>" "11" I--
I
.m. 0
H
I
I
I
i
I
t
~
~,TPA
2
3
4
5
6
7
8
9
DAYS Fig, 4. T h e e f f e c t s o f a n initial t w o - d a y e x p o s u r e o f l y m p h o c y t e s t o 1 2 - O - t e t r a d e c a n o y l - p h o r b o l - 1 3 a c e t a t e ( T P A ) o n t h e m i x e d l y m p h o c y t e r e s p o n s e . L y m p h o c y t e s w e r e c u l t u r e d as d e s e r l b e d in t h e l e g e n d t o Fig. 1. T h e p h o r b o l d i e s t e r a t 1 0 - ? M w a s a d d e d t o c u l t u r e s o n d a y 0. F r o m o n e set it w a s r e m o v e d b y a m e d i u m c h a n g e o n d a y 2. C o n t r o l c u l t u r e s h a d n o p h o r b o l d i e s t e r a d d e d , o . . . . . . o, p h o r b o l d l e s t e r d a y s 0 - - 2 ; -" e, phorbol diester days 0--9; • • , control, no p h o r b o l diester.
252
~ O -7 M
°-9,,
/ ~ o -au
o= b
i?;i ?o / II
ILl
I.~ !
I
a.--.-~OMSO
'
z_
0
I
2
3
4 DAYS
5
6
7
8
Fig. 5. T h e e f f e c t s of p h o r b o l 1 2 , 1 3 - d i a c e t a t e o n t h e m i x e d l y m p h o c y t e r e s p o n s e . L y m p h o c y t e s f r o m t w o a n i m a l s w e r e c u l t u r e d in m u l t i w e n dishes as d e s c r i b e d in Materials a n d M e t h o d s . R e s u l t s are e x p r e s s e d as d p m -+ S.E. f o r m i x e d cells c u l t u r e d w i t h n o a d d i t i o n s (ee ) , or with phorbol diacetate at 10 -8 M (© o), 10 -7 M ( i I ) a n d 10 -6 M (o ~) a n d 0.1% d i m e t h y l s u l f o x i d e . Cells w i t h 0.1% d i m e t h y l s u l f o x i d e ( D M S O ) a l o n e are s h o w n ( a a ) as are cells f r o m o n e a n i m a l only w i t h no a d d i t i o n s (X X). This e x p e r i m e n t , w h e n r e p e a t e d w i t h cells f r o m t w o d i f f e r e n t a n i m a l s , gave essentially t h e s a m e results. P h o r b o l d i a c e t a t e at 10 -6 M i n h i b i t e d t h e m i x e d l y m p h o c y t e r e s p o n s e b y 76 a n d 72% in t h e t w o e x p e r i m e n t s , r e s p e c t i v e l y .
tion of this response is not completely established, it appears likely that lymphocytes from lymph nodes of slaughter house animals may have received a significant, y e t limiting, level of endogenous mitogenic stimulation, and that the diester may act co-mitogenically on such lymphocytes as reported earlier for its action on lectin-treated cells [19]. In any event the response appears n o t to be directly related to the mixed l y m p h o c y t e response and can be eliminated by delaying the mitogenic interaction of the mixed lymphocytes or the addition of the phorbol diester.
The effects of 12-O-tetradecanoyl-phorbol-13-acetate analogues on the mixed lymphocyte response While 12-O-tetradecanoyl-phorbol-13-acetate is the most p o t e n t t u m o r promoter of the phorbol diester series, certain analogues of this compound, such as phorbol 12,13-diacetate are weaker promoters [5]. To test if the activity of this ester, which is about one-tenth as effective as a t u m o r promoter, is reflected in its effects on lymphocytes, we tested it in mixed cell cultures (Fig. 5). Phorbol 12,13-diacetate (10 -8, 10 -7 and 10 -6 M) or dimethyl sulfoxide
253 {0.1%) was added to mixed cultures at d a y 0 . Unlike 12-O-tetradecanoylphorbol-13-acetate, 10-SM and 10-TM phorbol 12,13-diacetate and 0.1% dimethyl sulfoxide had no inhibitory effect on the uptake of [3H]thymidine, However, 10 -6 M phorbol 12,13-diacetate blocked the incorporation by about 75%. Thus, ten times the concentration of phorbol 12,13-diacetate was required to inhibit to nearly the same level as 10 -7 M 12-O-tetradecanoylphorbol-13-acetate. Similarly, ten times as much phorbol 12,13-diacetate is required to give the same level of co-mitogen action as 12-O-tetradecanoylphorbol-13-acetate [ 17 ]. Phorbol, the parent c o m p o u n d of the series, was also tested at 10 -6, 10 -v and 10 -8 M in the mixed l y m p h o c y t e culture. At these concentrations, it did not inhibit the response {data n o t shown}. Discussion
The experiments in this paper show that 12-O-tetradecanoyl-phorbol-13acetate (10 -7, 10 -8 M} is a highly effective agent for blocking the induction of DNA synthesis of the mixed l y m p h o c y t e response in bovine l y m p h o c y t e cultures. This phorbol diester was not generally toxic since cells which were triggered for or already in the process of DNA replication were n o t affected significantly. The selective killing of a small group of lymphocytes which are essential for the mixed lymphocyte response cannot be ruled o u t in these experiments; however, this result in itself would point to an interesting new characteristic of this population of cells. The observation that the same levels of the diester are co,mitogenic when added to lectin-treated cultures [19] further supports this view and suggests that the diester may induce changes in the target cells which selectively interrupts the chain of mitogenic events where histocompatibility antigens are involved. The finding that 12-O-tetradecanoylphorbol-13-acetate was effective when added as late as day 2 after the start of the mixed l y m p h o c y t e cultures implies that an early, but y e t n o t the initial step in the response, was affected. While one-way mixed l y m p h o c y t e responses were not carried o u t in these studies, it seems most likely, in the view of the delayed addition studies, that the responding cell is the target for phorbol diester action. This question will be explored directly in future studies. Since the phorbol diester has been already shown to be a highly effective inducer of certain enzymes, it also appears quite probable that its effect on the mixed l y m p h o c y t e response is mediated as well through its inductive effects in gene expression. If this turns o u t to be the case the product of the induction will likely be an enzyme or a metabolic state which is uniquely opposed to the progression of mitogenic events set in motion by antigens involved in histocompatability. In this case 12-O-tetradecanoyl-phorbol-13-acetate and related phorhol esters should be useful tools in dissecting the molecular processes which control this type of lymphocyte response and possibly open the way to new approaches to the practical control of such cellular reactions in transplantation biology. Acknowledgements This study was supported by U.S. Public Health Service Grants T01-CA5002 and CA-07175 to G.C.M., and in part by a grant from NIH (GM 22953}
254
to A.M.M.G.C.M. is the recipient of a Research Career Development Award, U.S.A. Public Health Service. A.M.M. was a Damon R u n y o n Postdoctoral Fellow. The authors thank Dr. Tucker Helmes for his gift of the phorbol esters and Ms. Mary LeMahieu for her help with preparation of this manuscript. References 1 Bach, F., Bock, H., Graupner, K., Day, E. and Klostermann, H. (1969) Proc. Natl. Acad. Sci. U.S. 62, 377 --384 2 Bach, F.H., Bach, M.L. and Sondel, P.M. (1976) Nature 259, 273--281 3 H~'yry, P. and Defendi, V. (1970) Science 168, 133--135 4 Cohen, L. and Howe, M. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2 7 0 7 - - 2 7 1 0 5 Baird, W.M., Sedgwiek, J.A. and Boutwell, R.K. (1971) Cancer Res. 31, 1434--1439 6 Boutwell, R.K. (1964) in Progress in E x p e r i m e n t a l Tumor Research (Homberger, F., ed.), Vol. 4, pp. 207--250, Karger, New York 7 Hecker, E. (1971) in Methods in Cancer Research (Busch, H., ed.), Vol. 6, pp. 439--484, Academic Press, New Yo rk 8 Van Duuren, B.L. (1964) in Progress in E x p e r i m e n t a l Tumor Research (Homberger, F., ed.), Vol. 11, pp. 31--68, Karger, New York 9 Sivak, A., Ray, F. and Van Duuren, B.L. (1969) Cancer Res. 29, 624--630 10 Sivak, A., Mossman, B.T. and Van Duuren, B.L. (1972) Biochem. Biophys. Res. C ommun. 46, 605-609 11 Sfiss, R., Kreibich, G. andKinzel, V. (1972) Eur. J. Cancer 8 , 2 9 9 - - 3 0 4 12 Estensen, R.D., Hadden, J.W., Hadden, E.M., Touraine, F., Touraine, J., Haddox, M.K. and Goldberg, N.D. (1974) in Control of Proliferation in Animal Cells, CSH Conference on Cell Proliferation (Clarkson, B. and Baserga, R., eds.), Vol. 1, pp. 627--634, Cold Spring Harbor, New Y ork 13 Wenner, C.E., Hackney, J., Kimelberg, H.K. and Mayhew, E. (1974) Cancer Res. 34, 1731--1737 14 Rohrschneider, L.R. and Boutweil, R.K. (1973) Nature New Biol. 243, 212--213 15 O'Brien, T.G., Simsiman, R.C. and Boutwell, R.K. (1975) Cancer Res. 35, 1 6 6 7 - - 1 6 7 0 16 Wigler0 M, and Weinstein, I.B. (1976) Nature 259, 232--233 17 MueBer, G.C. and Kajiwara, K. (1965) in Developmental and Metabolic Control Mechanisms and Neoplasia, pp. 452--474, Wflkens and Wilkens, Baltimore 18 Whitfield, J.F,, MacManus, J.P. and Gillan, D.J. (1973) J. Cell. Physiol. 82, 151--156 19 Mastro, A.M. and Mueller, G.C. (1974) Exp. Cell Res. 88, 40--46 20 Mueller, G.C., Kajiwara, K., Stubblefield, E. and Rueckert, R. (1962) Cancer Res. 22, 1084--1090 21 Hirschberg, H. and Thorsby, E. (1973) J. I m m u n o l . Methods 3, 2 5 1 = 2 6 4