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Chemical xenogenization of tumor cells
485
TIPS - December 1985
Chemical xenogenization of tumor cells Paolo Puccetti, Luigina Romani and Maria C. Fioretti Chemical xenogenization occurs when tumor cells cultured in the presence of selected drugs or chemicals become immunogenic, i.e., acquire antigenic foreignness to the host of origin. Unlike modifications induced by haptens, changes in cell immunogenicity associated with chemical xenogenization are heritable as a result of drug interference with the genetic code. Xenogenized tumor cells elicit in-vivo strong immunological antitumor responses which Maria C. Fioretti and colleagues describe as having been successfully exploited in experimental models of immunotherapy of tumors. It is w i d e l y believed that experimental tumors i n d u c e d b y oncogenic viruses or polycyclic h y d r o carbons carry t u m o r specific transplantation antigens w h i c h m a y function as a target for host a n t i t u m o r i m m u n e responses. Yet, these reactions - which have occasionally also been detected against h u m a n cancer - fail for the major part part to control t u m o r growth. They are p r e s u m a b l y too w e a k to b r i n g a b o u t an efficient i m m u n o l o g i c a l rejection. In recent years, the c o n d i t i o n of low imm u n o g e n i c i t y of malignant cells has b e e n the subject of extensive studies in experimental models of t u m o r i m m u n o l o g y and i m m u n o therapy. Strategies to induce i m p r o v e d i m m u n i t y to neoplastic cells have p r i m a r i l y focused on nonspecific stimulation of the host i m m u n e system or m a n i p u l a t i o n of the t u m o r in order to increase its i m m u n o g e n i c potential. In the latter case, evidence suggests that neoplastic cells m o d i f i e d such that they acquire n e w surface antigens often induce stronger i m m u n e reactions than the original tumor. A m o n g the m a n y examples of this type, those involving viral infection of t u m o r cells, somatic h y b r i d ization w i t h antigenically distinct cells, treatment w i t h m e m b r a n e m o d i f y i n g e n z y m e s or exposure to h a p t e n i z i n g chemicals a p p e a r particularly encouraging. Changes in the antigenic makeup of a cell, and thus its i m m u n o genicity, m i g h t also be expected to be part of the p h e n o t y p i c variaMaria Cristina Fioretti is Head of the Institute of Pharmacology of the University of Perugia, Via del Giochetto, 06100 Perugia, Italy. Paolo Puccetti and Luigina Romani are senior research workers at the same Institute.
tions i n d u c e d b y mutagens. In a few well d o c u m e n t e d examples, this a s s u m p t i o n has p r o v e d to be true and thus suggested that mutagenic c o m p o u n d s could be u s e d to increase the i m m u n o g e n i c potential of t u m o r cells. In particular, the extent of i m m u n o g e n i c changes i n d u c e d b y triazene and n i t r o s o g u a n i d i n e derivatives is such that the cells are regarded as 'chemically x e n o g e n i z e d ' i.e. they d i s p l a y a high degree of antigenic foreignness to the host of origin. The triazene derivatives
Many cytoreductive drugs, mostly triazene derivatives, have n o w b e e n s h o w n to possess xenogenizin~ properties. The triazenylimidazole agent dacarbazine, a drug c o m m o n l y used in the treatm e n t of malignant melanoma, is p e r h a p s the m u t a g e n whose xenogenizing properties in vivo have been most extensively s t u d i e d 1. Repeated exposure of tumor cells to dacarbazine in vivo or in vitro leads to the appearance of nontumorigenic variants which will only grow in i m m u n o d e f i c i e n t hosts, and w i l l ' b e rejected b y normal recipients after an apparent initial take 2'3. The i m m u n o genic character of the dacarbazine t u m o r variants has b e e n p r o v e n b y a n u m b e r of observations, including protection experiments in w h i c h resistance to tumor challenge is conferred u p o n i m m u n o deficient hosts b y transfer of i m m u n e lymphocytes 4. One crucial and still much d e b a t e d issue in this regard is the nature of the antigens a p p e a r i n g on the imm u n o g e n i c tumor variants. It n o w seems that the dacarbazine tumor variants acquire n e w transplantation antigens that are, as a rule, u n i q u e to each monoclonal vari-
ant. Thus, a dacarbazine line w h i c h is m a d e up of a highly polyclonal cell p o p u l a t i o n generally consists of a large n u m b e r of variants with different antigens. All the variants, however, retain and share tumor-associated transplantation antigens which might pre-exist on the original t u m o r (see Fig. 1). Interestingly, animals that have rejected a decarbazine-xenogenized line are in some instances found to resist s u b s e q u e n t challenge with the original noni m m u n o g e n i c t u m o r 3"s. It appears, therefore, that even such parental tumors carry antigenic structures that can trigger efficient antitumor responses w h e n they are generated in the context of the strong i m m u n i t y i n d u c e d b y a xenogenized variant. Since most h u m a n tumors show little, if any, immunogenicity, these findings might open up n e w perspectives in the i m m u n o t h e r a p y of h u m a n neoplasia. The n i t r o s o g u a n i d i n e derivatives
A high frequency of clones with reduced tumorigenicity is also observed w h e n t u m o r cell lines are treated in vitro with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) or other mutagens 6. As in the case of the dacarbazine-xenogenized lines, all tumors seem to acquire n e w antigens that account for the decreased transplantability of these so-called ' t u m - ' variants (as o p p o s e d to the 'tum ÷' initial tumorigenic line7). In line with dacarbazine-treated tumors, a tumor cell population m u t a g e n i z e d by M N N G is a spectrum of antigenic variants, containing a range of clones extending continuously from those that are only slightly less tumorigenic than the original tumor to those that grow in only a very small minority of recipient hosts. Like dacarbazine tumor variants, all tum clones show an oncogenic potential similar to that of the original tumor w h e n grafted into i m m u n o c o m p r o m i z e d hosts. Although each turn clone carries a u n i q u e set of antigenic determinants, all turn- variants of a tumor share a common antigen with tum+ cells. Thus, the only cross-reactivity detectable between any pair of tum- variants must be due to the turn + antigen 8. In agreement with results o b t a i n e d with dacarbazinexenogenized tumors is the finding
~) 1985, Elsevier Science Publishers B.V., Amsterdam
0165
6147/85/$02.00
486
TIPS - December 1985
that tum- cells have been found to successfully vaccinate against the original nonimmunogenic tumor 9. In this regard, it has been postulated that tum- determinants may serve as helper antigens for the response against turn ÷ antigen in a sort of nonspecific immunopotentiation in situations of tum--induced immunity 1°. More specific cooperative effects in the immunogenicity of tum- and tum+ determinants have also been hypothesized. In view of the analogy between dacarbazine- and MNNG-treated tumors, it is very likely that the two drugs effect chemical xenogenization of tumor cells through an essentially unitary mechanism.
Changes occurring in chemical xenogenization There is little doubt that the immunogenic changes associated with chemical xenogenization are the consequence of truly heritable modifications resulting from direct action of dacarbazine or MNNG on tumor cells. A series of alternative explanations, including selection of pre-existing immunogenic clones, has been ruled out on the basis of compelling evidence 11. On the other hand, the nature of the genetic interference of the drugs with the susceptible tumor target cell is still very
Number of treatments
Tumor ceil
controversial. In principle, such interference could be mutational (involving heritable changes in nucleotide sequence) or epigenetic (involving any other mechanism of hereditary variation) 6. In theory, the high frequency of variants seems to be more compatible with an epigenetic event. Indeed, the number of histocompatibility genes theoretically required in the mouse to account for the observed frequency of mutational events leading to immunogenic variants is considerably higher than is generally believed. Nonetheless, all of the epigenetic models that have been proposed in the past often involve highly elaborate mechanisms and are thus, not completely satisfactory. Recently, a rather simple epigenetic model was proposed in which the major target for the xenogenizing mutagens was represented by the enzyme that methylates the base cytosine in DNA 12. It is well established that levels of cytosine methylation regulate the expression of several gene functions and thus possibly affect the degree of cell immunogenicity. In this regard, it has been postulated that chemically xenogenized tumor variants might have decreased levels of methylcytosine which would increase the activity of genes involved in the
Tumor antigens Tumor specific
Degree of immunogenicity +
transplantation antigens (TSTA) ~ T A A single treatment results in increased density of TSTA (?) and the ++ appearance of drug mediated transplantation antigens (DMTA) Multiple treatments result in increased density of TSTA and DMTA
3--6 TSTA
+++~
DMTA
Fig. 1. Scheme illustrating the events occurring in chemical xenogenization of tumor cells by triazene derivatives. Strong immunogenicity is acquired following 3 - 6 in-vivo or in-vitro exposures to the drug. After a single treatment with a triazene derivative, the newly acquired antigens are not, as a rule, very strong (dotted line). It cannot be that, at this very early stage of xenogenization, a role is also played in the increased immunogeneity by an augmented expression of pre-existing TSTA. On subsequent exposures to the drug, the increased density of TSTA remains uncertain, while DMTA become stronger and more • numerous.
expression of immunogenicity. Although this hypothesis is appealing and fits well with observations on frequency and stability of immunogenic tumor variants, preliminary studies conducted in our laboratory failed to reveal decreased cytosine methylation in tumor cell lines treated with triazene derivatives 13. Therefore, a mutational origin still remains the most plausible explanation for the induction of new antigenicity following treatment of tumor cells with mutagens. Interestingly, the antimutagenic compound quinacrine can inhibit the xenogenizing properties of dacarbazine in vivo when administered concurrently, without affecting its cytoreductive potential 14. The mutational hypothesis could perhaps be further elaborated so as to better reconcile data on the frequency of mutation with the number of histocompatibility genes. Along this line, it has been recently hypothesized that mutagens may cause DNA rearrangements in genes, forming hypermutable regions which would produce a wide variety of new antigens at very high frequencies 6. Despite the lack of any direct evidence, the above mechanism is at present the only one that justifies most of the data on genetic interaction between xenogenizing chemicals and tumor cells.
Response against xenogenized tumors The nature of the immune response triggered by immunogenic tumor variants has been investigated in terms of both cellular and humoral reactions. Xenogenized variants promote antitumor responses mediated by cytotoxic T lymphocytes in vitro 8As as well as in vivo 7"16. Lymphocytes recovered from animals that have rejected an immunogenic tumor variant will specifically lyse the relevant variant in vitro 17. Similarly, primary cytotoxic T lymphocytes raised in vitro against a xenogenized tumor show strong cytolytic activity against the same variant TM and, to a lesser extent, against the original nonimmunogenic tumor if particular assay conditions are employed. It is interesting to note that both the invivo- and in-vitro-generated cytotoxic T lymphocytes can be used to adoptively confer protection against a xenogenized tumor vari-
487
T I P S - D e c e m b e r 1985
~
N
CONH2
genic t u m o r variants and possibly the location of the mutations responsible for xenogenization.
N=N-N(CHs)2
Exploitation of chemical xenogenization for immunotherapy
dacarbazine / CH~
R
~
N=N-N
aryl-triazenederivatives
HsC ~ _ ~ _
O=N /
N,.-/H
~
NOa
N-methyl-N'-nitrosoguanidine Fig. 2. Chemical structure of some xenogenizing compounds.
ant u p o n i m m u n o d e f i c i e n t mice challenged with the same t u m o r 4"18. Varied results, on the other hand, are o b t a i n e d w h e n i n - v i t r o - g e n e r ated cytotoxic T lymphocytes specific for a tumor variant are tested in v i v o for protection against the original n o n - i m m u n o g e n i c tumor. O n testing long-term cytotoxic T lymphocyte clones raised against a turn- variant for cytotoxic activity in v i t r o against the corresp o n d i n g turn + cells, it was found that some clones showed reactivity whereas others did not 19. It is possible that i n - v i v o testing of the polyclonal population of i n - v i t r o primed cytotoxic T lymphocytes for protection against the original tumor, is influenced by the crucial factor of the a m o u n t of t u n + reactive lymphocytes present in the transferred p o p u l a t i o n with respect to the conditions of challenge with the original tumor. This would account for the varied results o b t a i n e d by our group in protection experiments based on different experimental conditions. Humoral a n t i b o d y reactions can a l s o be found to occur against chemically-xenogenized tumors. Recently, antisera that specifically recognize antigenic d e t e r m i n a n t s which are not shared b y the original l y m p h o m a have been raised 2°. Should these determin a n t s represent transplantation antigens, these antisera could provide valuable m e a n s to further explore the nature of i m m u n o -
It is obvious that the ultimate goal of experimental studies on chemical xenogenization is its application to h u m a n cancer. In this regard, a n u m b e r of different approaches might reasonably be envisioned. (1) T u m o r - b e a r i n g hosts could be treated with xenog e n i z i n g drugs i n order to increase the i m m u n o g e n i c potential of the malignancy. (2) The host could be treated with i m m u n o g e n i c tumor variants obtained in vitro through exposure of the original tumor to m u t a g e n i z i n g chemicals. This might be particularly appropriate w h e n a p r i m a r y tumor has been surgically removed and tumor cells are available to be adapted to culture. These cells, once xenogenized and reinfused into their original host, could elicit a rejection response directed against residual tumor cells which possibly escape classical treatment. (3) The adoptive transfer of cytotoxic lymphocytes raised in vitro against i m m u n o g e n i c variants of the original t u m o r could be exploited. Although all of these approaches have been successfully attempted in m u r i n e l y m p h o m a models 4,21,22, m a n y obstacles still prevent their application to h u m a n neoplasia. O n e basic problem is the lack of definitive evidence that h u m a n tumors carry antigenic determin a n t s and that these cells can u n d e r g o i m m u n o g e n i c changes. A major technical problem would be the necessity of detecting i n - v i t r o i m m u n o g e n i c changes of h u m a n cancer cells as well as their loss of tumorigenicity. The approach of using repeated mutagenesis in vivo, as in the case of the dacarbazine lines, would dispense with the need to recognize i m m u n o g e n i c tumor variants in vitro. However, even i n such conditions, the dramatic suppressive effects dacarbazine exerts on the host imm u n e system would create a considerable obstacle. For this reason, a major goal of research in this area should be to find molecules with equivalent, or even enhanced, ability to induce chemical xenogenization b u t w i t h o u t any severe immunotoxicity. A series of aryl-triazene derivatives
presently u n d e r investigation in our laboratory seems to provide encouraging results in this direction 23. These n e w e r c o m p o u n d s m i g h t prove to be particularly useful towards the clinical application of chemical xenogenization,
References 1 Fioretti, M. C., Romani, L. and Bonmassar, E. (1983) in Biology of Cancer (Mirand, E. A,, Hutchinson, W, B. and Mihich, E., eds), Vol. 1, pp. 435-445, Alan R. Liss, Inc., New York
2 Bonmassar, E., Bonmassar, A., Vadlamudi,S. and Goldin,A. (1970)Proc. Natl Acad. Sci. USA 66, 1089--1095
3 Contessa, A. R., Bonmassar, A.,
Giampietri, A., Circolo, A., Goldin, A. and Fioretti,M. C. (1981)Cancer Res. 41, 2476-2482 4 Romani,L., Bianchi,R., Puccetti, P. and Fioretti,M. C. (1983)Int.]. Cancer31,477482 5 Riccardi,C., Giampietri,A., Puccetti,P., Biancifiori,F. and Fioretti, M. C. (1977) Pharmacol. Res, Commun. 9, 349-358 6 Boon, T. (1983) Adv. Cancer Res. 39, 121-151 7 Boon,T. and Kellerman,O. (1977)Proc. Natl Acad. Sci. USA 74, 272-275 8 Boon, T., Van Snick, l., Van Pel, A., Uyttenhove,C. and Marchand,M. (1980) J. Exp. Med. 152, 1184-1193 9 Boon,T. and Van Pe[, A. (1978)Proc. Natl Acad. Sci. USA 75, 1519-1523 10 Mitchison,N. A. (1970)Transplant. Proc. 2, 92-96 11 Fioretti, M. C., Bianchi, R., Romani, L. and Bonmassar,E. (1983)J. Natl Cancer Inst. 71, 1247-1251 12 Frost,P., Liteplo,R. G., Donaghue,T. P. and Kerbel,R. S. (1984)]. Exp. Med. 159, 1491-1501 13 Puccetti, P., Romani, L., Dominici, P., Tancini, P. and Fiore~ti, M. C. (1985) Cancer Detect. Prev. 8, 547 14 Giampietri, A., Fioretti, M. C., Goldin, A. and Bonmassar,E. (1980)J. Natl Cancer Inst. 64, 297-301 15 Romani, L., Fioretti, M.C. and Bonmassar, E. (1979)Transplantation 28, 218--222
16 Campanile, F., Houchens, D., Gaston, M.,Goldin,A. and Bonmassar,E. (1975)]. Natl Cancer Inst. 55, 207-209 17 Nicolin, A., Bini, A., Coronetti, E. and Goldin, A. (1974) Nature (London) 251, 654--655 18 Romani, L., Nardelli, B., Bianchi, R., Puccetti,P., Mage,M. and Fioretti,M. C. (1985) Int. J. Cancer 35, 659-665 19 Maryanski, J., Van Snick, J., Cerottini, J, C. and Boon,T. (1982) Eur. ]. Immunol. 12, 401406 20 Romani, L., Puccetti, P., Fioretti, M. C. and Mage, M, G. (1985)Int. }. Cancer 36, 225-231 21 Romani, L., Fioretti, M. C., Bianchi,R., Nardelli, B. and Bonmassar,E. (1982)J. Natl Cancer Inst. 68, 817-822 22 Giampietri,A., Bonmassar,A., Puccetti, P., Circolo, A., Goldin, A. and Bonmassar,E. (1981)Cancer Res. 41,681687 23 Nardelli, B., Puccetti, P,, Romani, L., Sava, G., Bonmassar, E. and Fioretti, M.C. (1984) Cancer Immunol. lmmunother. 17, 213-217
TIPS - December 1985
Chemical xenogenization of tumor cells Paolo Puccetti, Luigina Romani and Maria C. Fioretti Chemical xenogenization occurs when tumor cells cultured in the presence of selected drugs or chemicals become immunogenic, i.e., acquire antigenic foreignness to the host of origin. Unlike modifications induced by haptens, changes in cell immunogenicity associated with chemical xenogenization are heritable as a result of drug interference with the genetic code. Xenogenized tumor cells elicit in-vivo strong immunological antitumor responses which Maria C. Fioretti and colleagues describe as having been successfully exploited in experimental models of immunotherapy of tumors. It is w i d e l y believed that experimental tumors i n d u c e d b y oncogenic viruses or polycyclic h y d r o carbons carry t u m o r specific transplantation antigens w h i c h m a y function as a target for host a n t i t u m o r i m m u n e responses. Yet, these reactions - which have occasionally also been detected against h u m a n cancer - fail for the major part part to control t u m o r growth. They are p r e s u m a b l y too w e a k to b r i n g a b o u t an efficient i m m u n o l o g i c a l rejection. In recent years, the c o n d i t i o n of low imm u n o g e n i c i t y of malignant cells has b e e n the subject of extensive studies in experimental models of t u m o r i m m u n o l o g y and i m m u n o therapy. Strategies to induce i m p r o v e d i m m u n i t y to neoplastic cells have p r i m a r i l y focused on nonspecific stimulation of the host i m m u n e system or m a n i p u l a t i o n of the t u m o r in order to increase its i m m u n o g e n i c potential. In the latter case, evidence suggests that neoplastic cells m o d i f i e d such that they acquire n e w surface antigens often induce stronger i m m u n e reactions than the original tumor. A m o n g the m a n y examples of this type, those involving viral infection of t u m o r cells, somatic h y b r i d ization w i t h antigenically distinct cells, treatment w i t h m e m b r a n e m o d i f y i n g e n z y m e s or exposure to h a p t e n i z i n g chemicals a p p e a r particularly encouraging. Changes in the antigenic makeup of a cell, and thus its i m m u n o genicity, m i g h t also be expected to be part of the p h e n o t y p i c variaMaria Cristina Fioretti is Head of the Institute of Pharmacology of the University of Perugia, Via del Giochetto, 06100 Perugia, Italy. Paolo Puccetti and Luigina Romani are senior research workers at the same Institute.
tions i n d u c e d b y mutagens. In a few well d o c u m e n t e d examples, this a s s u m p t i o n has p r o v e d to be true and thus suggested that mutagenic c o m p o u n d s could be u s e d to increase the i m m u n o g e n i c potential of t u m o r cells. In particular, the extent of i m m u n o g e n i c changes i n d u c e d b y triazene and n i t r o s o g u a n i d i n e derivatives is such that the cells are regarded as 'chemically x e n o g e n i z e d ' i.e. they d i s p l a y a high degree of antigenic foreignness to the host of origin. The triazene derivatives
Many cytoreductive drugs, mostly triazene derivatives, have n o w b e e n s h o w n to possess xenogenizin~ properties. The triazenylimidazole agent dacarbazine, a drug c o m m o n l y used in the treatm e n t of malignant melanoma, is p e r h a p s the m u t a g e n whose xenogenizing properties in vivo have been most extensively s t u d i e d 1. Repeated exposure of tumor cells to dacarbazine in vivo or in vitro leads to the appearance of nontumorigenic variants which will only grow in i m m u n o d e f i c i e n t hosts, and w i l l ' b e rejected b y normal recipients after an apparent initial take 2'3. The i m m u n o genic character of the dacarbazine t u m o r variants has b e e n p r o v e n b y a n u m b e r of observations, including protection experiments in w h i c h resistance to tumor challenge is conferred u p o n i m m u n o deficient hosts b y transfer of i m m u n e lymphocytes 4. One crucial and still much d e b a t e d issue in this regard is the nature of the antigens a p p e a r i n g on the imm u n o g e n i c tumor variants. It n o w seems that the dacarbazine tumor variants acquire n e w transplantation antigens that are, as a rule, u n i q u e to each monoclonal vari-
ant. Thus, a dacarbazine line w h i c h is m a d e up of a highly polyclonal cell p o p u l a t i o n generally consists of a large n u m b e r of variants with different antigens. All the variants, however, retain and share tumor-associated transplantation antigens which might pre-exist on the original t u m o r (see Fig. 1). Interestingly, animals that have rejected a decarbazine-xenogenized line are in some instances found to resist s u b s e q u e n t challenge with the original noni m m u n o g e n i c t u m o r 3"s. It appears, therefore, that even such parental tumors carry antigenic structures that can trigger efficient antitumor responses w h e n they are generated in the context of the strong i m m u n i t y i n d u c e d b y a xenogenized variant. Since most h u m a n tumors show little, if any, immunogenicity, these findings might open up n e w perspectives in the i m m u n o t h e r a p y of h u m a n neoplasia. The n i t r o s o g u a n i d i n e derivatives
A high frequency of clones with reduced tumorigenicity is also observed w h e n t u m o r cell lines are treated in vitro with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) or other mutagens 6. As in the case of the dacarbazine-xenogenized lines, all tumors seem to acquire n e w antigens that account for the decreased transplantability of these so-called ' t u m - ' variants (as o p p o s e d to the 'tum ÷' initial tumorigenic line7). In line with dacarbazine-treated tumors, a tumor cell population m u t a g e n i z e d by M N N G is a spectrum of antigenic variants, containing a range of clones extending continuously from those that are only slightly less tumorigenic than the original tumor to those that grow in only a very small minority of recipient hosts. Like dacarbazine tumor variants, all tum clones show an oncogenic potential similar to that of the original tumor w h e n grafted into i m m u n o c o m p r o m i z e d hosts. Although each turn clone carries a u n i q u e set of antigenic determinants, all turn- variants of a tumor share a common antigen with tum+ cells. Thus, the only cross-reactivity detectable between any pair of tum- variants must be due to the turn + antigen 8. In agreement with results o b t a i n e d with dacarbazinexenogenized tumors is the finding
~) 1985, Elsevier Science Publishers B.V., Amsterdam
0165
6147/85/$02.00
486
TIPS - December 1985
that tum- cells have been found to successfully vaccinate against the original nonimmunogenic tumor 9. In this regard, it has been postulated that tum- determinants may serve as helper antigens for the response against turn ÷ antigen in a sort of nonspecific immunopotentiation in situations of tum--induced immunity 1°. More specific cooperative effects in the immunogenicity of tum- and tum+ determinants have also been hypothesized. In view of the analogy between dacarbazine- and MNNG-treated tumors, it is very likely that the two drugs effect chemical xenogenization of tumor cells through an essentially unitary mechanism.
Changes occurring in chemical xenogenization There is little doubt that the immunogenic changes associated with chemical xenogenization are the consequence of truly heritable modifications resulting from direct action of dacarbazine or MNNG on tumor cells. A series of alternative explanations, including selection of pre-existing immunogenic clones, has been ruled out on the basis of compelling evidence 11. On the other hand, the nature of the genetic interference of the drugs with the susceptible tumor target cell is still very
Number of treatments
Tumor ceil
controversial. In principle, such interference could be mutational (involving heritable changes in nucleotide sequence) or epigenetic (involving any other mechanism of hereditary variation) 6. In theory, the high frequency of variants seems to be more compatible with an epigenetic event. Indeed, the number of histocompatibility genes theoretically required in the mouse to account for the observed frequency of mutational events leading to immunogenic variants is considerably higher than is generally believed. Nonetheless, all of the epigenetic models that have been proposed in the past often involve highly elaborate mechanisms and are thus, not completely satisfactory. Recently, a rather simple epigenetic model was proposed in which the major target for the xenogenizing mutagens was represented by the enzyme that methylates the base cytosine in DNA 12. It is well established that levels of cytosine methylation regulate the expression of several gene functions and thus possibly affect the degree of cell immunogenicity. In this regard, it has been postulated that chemically xenogenized tumor variants might have decreased levels of methylcytosine which would increase the activity of genes involved in the
Tumor antigens Tumor specific
Degree of immunogenicity +
transplantation antigens (TSTA) ~ T A A single treatment results in increased density of TSTA (?) and the ++ appearance of drug mediated transplantation antigens (DMTA) Multiple treatments result in increased density of TSTA and DMTA
3--6 TSTA
+++~
DMTA
Fig. 1. Scheme illustrating the events occurring in chemical xenogenization of tumor cells by triazene derivatives. Strong immunogenicity is acquired following 3 - 6 in-vivo or in-vitro exposures to the drug. After a single treatment with a triazene derivative, the newly acquired antigens are not, as a rule, very strong (dotted line). It cannot be that, at this very early stage of xenogenization, a role is also played in the increased immunogeneity by an augmented expression of pre-existing TSTA. On subsequent exposures to the drug, the increased density of TSTA remains uncertain, while DMTA become stronger and more • numerous.
expression of immunogenicity. Although this hypothesis is appealing and fits well with observations on frequency and stability of immunogenic tumor variants, preliminary studies conducted in our laboratory failed to reveal decreased cytosine methylation in tumor cell lines treated with triazene derivatives 13. Therefore, a mutational origin still remains the most plausible explanation for the induction of new antigenicity following treatment of tumor cells with mutagens. Interestingly, the antimutagenic compound quinacrine can inhibit the xenogenizing properties of dacarbazine in vivo when administered concurrently, without affecting its cytoreductive potential 14. The mutational hypothesis could perhaps be further elaborated so as to better reconcile data on the frequency of mutation with the number of histocompatibility genes. Along this line, it has been recently hypothesized that mutagens may cause DNA rearrangements in genes, forming hypermutable regions which would produce a wide variety of new antigens at very high frequencies 6. Despite the lack of any direct evidence, the above mechanism is at present the only one that justifies most of the data on genetic interaction between xenogenizing chemicals and tumor cells.
Response against xenogenized tumors The nature of the immune response triggered by immunogenic tumor variants has been investigated in terms of both cellular and humoral reactions. Xenogenized variants promote antitumor responses mediated by cytotoxic T lymphocytes in vitro 8As as well as in vivo 7"16. Lymphocytes recovered from animals that have rejected an immunogenic tumor variant will specifically lyse the relevant variant in vitro 17. Similarly, primary cytotoxic T lymphocytes raised in vitro against a xenogenized tumor show strong cytolytic activity against the same variant TM and, to a lesser extent, against the original nonimmunogenic tumor if particular assay conditions are employed. It is interesting to note that both the invivo- and in-vitro-generated cytotoxic T lymphocytes can be used to adoptively confer protection against a xenogenized tumor vari-
487
T I P S - D e c e m b e r 1985
~
N
CONH2
genic t u m o r variants and possibly the location of the mutations responsible for xenogenization.
N=N-N(CHs)2
Exploitation of chemical xenogenization for immunotherapy
dacarbazine / CH~
R
~
N=N-N
aryl-triazenederivatives
HsC ~ _ ~ _
O=N /
N,.-/H
~
NOa
N-methyl-N'-nitrosoguanidine Fig. 2. Chemical structure of some xenogenizing compounds.
ant u p o n i m m u n o d e f i c i e n t mice challenged with the same t u m o r 4"18. Varied results, on the other hand, are o b t a i n e d w h e n i n - v i t r o - g e n e r ated cytotoxic T lymphocytes specific for a tumor variant are tested in v i v o for protection against the original n o n - i m m u n o g e n i c tumor. O n testing long-term cytotoxic T lymphocyte clones raised against a turn- variant for cytotoxic activity in v i t r o against the corresp o n d i n g turn + cells, it was found that some clones showed reactivity whereas others did not 19. It is possible that i n - v i v o testing of the polyclonal population of i n - v i t r o primed cytotoxic T lymphocytes for protection against the original tumor, is influenced by the crucial factor of the a m o u n t of t u n + reactive lymphocytes present in the transferred p o p u l a t i o n with respect to the conditions of challenge with the original tumor. This would account for the varied results o b t a i n e d by our group in protection experiments based on different experimental conditions. Humoral a n t i b o d y reactions can a l s o be found to occur against chemically-xenogenized tumors. Recently, antisera that specifically recognize antigenic d e t e r m i n a n t s which are not shared b y the original l y m p h o m a have been raised 2°. Should these determin a n t s represent transplantation antigens, these antisera could provide valuable m e a n s to further explore the nature of i m m u n o -
It is obvious that the ultimate goal of experimental studies on chemical xenogenization is its application to h u m a n cancer. In this regard, a n u m b e r of different approaches might reasonably be envisioned. (1) T u m o r - b e a r i n g hosts could be treated with xenog e n i z i n g drugs i n order to increase the i m m u n o g e n i c potential of the malignancy. (2) The host could be treated with i m m u n o g e n i c tumor variants obtained in vitro through exposure of the original tumor to m u t a g e n i z i n g chemicals. This might be particularly appropriate w h e n a p r i m a r y tumor has been surgically removed and tumor cells are available to be adapted to culture. These cells, once xenogenized and reinfused into their original host, could elicit a rejection response directed against residual tumor cells which possibly escape classical treatment. (3) The adoptive transfer of cytotoxic lymphocytes raised in vitro against i m m u n o g e n i c variants of the original t u m o r could be exploited. Although all of these approaches have been successfully attempted in m u r i n e l y m p h o m a models 4,21,22, m a n y obstacles still prevent their application to h u m a n neoplasia. O n e basic problem is the lack of definitive evidence that h u m a n tumors carry antigenic determin a n t s and that these cells can u n d e r g o i m m u n o g e n i c changes. A major technical problem would be the necessity of detecting i n - v i t r o i m m u n o g e n i c changes of h u m a n cancer cells as well as their loss of tumorigenicity. The approach of using repeated mutagenesis in vivo, as in the case of the dacarbazine lines, would dispense with the need to recognize i m m u n o g e n i c tumor variants in vitro. However, even i n such conditions, the dramatic suppressive effects dacarbazine exerts on the host imm u n e system would create a considerable obstacle. For this reason, a major goal of research in this area should be to find molecules with equivalent, or even enhanced, ability to induce chemical xenogenization b u t w i t h o u t any severe immunotoxicity. A series of aryl-triazene derivatives
presently u n d e r investigation in our laboratory seems to provide encouraging results in this direction 23. These n e w e r c o m p o u n d s m i g h t prove to be particularly useful towards the clinical application of chemical xenogenization,
References 1 Fioretti, M. C., Romani, L. and Bonmassar, E. (1983) in Biology of Cancer (Mirand, E. A,, Hutchinson, W, B. and Mihich, E., eds), Vol. 1, pp. 435-445, Alan R. Liss, Inc., New York
2 Bonmassar, E., Bonmassar, A., Vadlamudi,S. and Goldin,A. (1970)Proc. Natl Acad. Sci. USA 66, 1089--1095
3 Contessa, A. R., Bonmassar, A.,
Giampietri, A., Circolo, A., Goldin, A. and Fioretti,M. C. (1981)Cancer Res. 41, 2476-2482 4 Romani,L., Bianchi,R., Puccetti, P. and Fioretti,M. C. (1983)Int.]. Cancer31,477482 5 Riccardi,C., Giampietri,A., Puccetti,P., Biancifiori,F. and Fioretti, M. C. (1977) Pharmacol. Res, Commun. 9, 349-358 6 Boon, T. (1983) Adv. Cancer Res. 39, 121-151 7 Boon,T. and Kellerman,O. (1977)Proc. Natl Acad. Sci. USA 74, 272-275 8 Boon, T., Van Snick, l., Van Pel, A., Uyttenhove,C. and Marchand,M. (1980) J. Exp. Med. 152, 1184-1193 9 Boon,T. and Van Pe[, A. (1978)Proc. Natl Acad. Sci. USA 75, 1519-1523 10 Mitchison,N. A. (1970)Transplant. Proc. 2, 92-96 11 Fioretti, M. C., Bianchi, R., Romani, L. and Bonmassar,E. (1983)J. Natl Cancer Inst. 71, 1247-1251 12 Frost,P., Liteplo,R. G., Donaghue,T. P. and Kerbel,R. S. (1984)]. Exp. Med. 159, 1491-1501 13 Puccetti, P., Romani, L., Dominici, P., Tancini, P. and Fiore~ti, M. C. (1985) Cancer Detect. Prev. 8, 547 14 Giampietri, A., Fioretti, M. C., Goldin, A. and Bonmassar,E. (1980)J. Natl Cancer Inst. 64, 297-301 15 Romani, L., Fioretti, M.C. and Bonmassar, E. (1979)Transplantation 28, 218--222
16 Campanile, F., Houchens, D., Gaston, M.,Goldin,A. and Bonmassar,E. (1975)]. Natl Cancer Inst. 55, 207-209 17 Nicolin, A., Bini, A., Coronetti, E. and Goldin, A. (1974) Nature (London) 251, 654--655 18 Romani, L., Nardelli, B., Bianchi, R., Puccetti,P., Mage,M. and Fioretti,M. C. (1985) Int. J. Cancer 35, 659-665 19 Maryanski, J., Van Snick, J., Cerottini, J, C. and Boon,T. (1982) Eur. ]. Immunol. 12, 401406 20 Romani, L., Puccetti, P., Fioretti, M. C. and Mage, M, G. (1985)Int. }. Cancer 36, 225-231 21 Romani, L., Fioretti, M. C., Bianchi,R., Nardelli, B. and Bonmassar,E. (1982)J. Natl Cancer Inst. 68, 817-822 22 Giampietri,A., Bonmassar,A., Puccetti, P., Circolo, A., Goldin, A. and Bonmassar,E. (1981)Cancer Res. 41,681687 23 Nardelli, B., Puccetti, P,, Romani, L., Sava, G., Bonmassar, E. and Fioretti, M.C. (1984) Cancer Immunol. lmmunother. 17, 213-217