Cancer, Differentiation and Embryonic Antigens: Some Central Problems

CANCER. DIFFERENTIATION AND EMBRYONIC ANTIGENS: SOME CENTRAL PROBLEMS J . H. Coggin. Jr . and N. G . Anderson Department of Microbiology.

' University

.

of Tennessee. Knoxville. Tennessee. and

The Molecular Anatomy ( M A N ) Progrom. O a k Ridge National Laboratory: O a k Ridge. Tennessee

I. Introduction . . . . . . . . I1. Transformation-Associated Cellular Alterations . A . Tumor- Associated Autoanti gem . . .

I11.

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V. VI . VII .

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B. Tumor-Associated Embryonic Antigens or Factors . . C . Isozynies in Tumors . . . . . . . . . 1). Hormones in Tumors . . . . . . . . E . Surface Changes in Tumor Cells . . . . . . F. Miscellaneous Changes . . . . . . . . G . Conclusions . . . . . . . . . . The Organization of Differentiation . . . . . . A . General Rules of Differentiation . . . . . . B. Inducers and Capacitation . . . . . . . C . Complexity of the Program . . . . . . . D . Reversibility of Differentiation and Metaplasia . . . E . Molecular Evolution and Embryogenesis . . . . F. Phase-Specific Substances . . . . . . . . G . Surveillance . . . . . . . . . . H . Maternal-Embryo Separation . . . . . . . Differentiation and Cancer . . . . . . . . A . Embryomas and Teratomas . . . . . . . B. Retrogenesis and Residues of the Differentiative Programs C . New Approaches to Tamor Classification . . . . D . Mutagenesis, Teratogenesis, and Oncogenesis . . . Biology of Maternal-Fetal Differences . . . . . . A . Evidence for Obligatory Embryonic Autoantigens . . B . Tumor and Fetal Escape Mechanisms . . . . . Molecular Basis of Differentiation and Cancer . . . . Conclusions . . . . . . . . . . . References . . . . . . . . . . .

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106 107 108 130 133 133 134 135 135 136 137 139 139 140 140 141 142 142 143 144 146 147 148 149 150 153 155 157 159

Supported by the United States Atomic Energy Commission AT( 40.1)3645 . 'The Molecular Anatomy Program at Oak Ridge and at the University of Tennessee is supported, in part. by the Virus Cancer Program. National Cancer Institute. Contract CP 73.210 . Operation for the United States Atomic Energy Commission by Union Carbide Corporation .

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J. H. COGGIN, JR. AND N. G. ANDERSON

I. Introduction

We examine here in full detail the theory that cancer is a disease of the mechanism( s ) of differentiation and that the compositional and behavioral changes observed in the majority of human tumors are due to changes in the programming of normal genes. For this to be true, it must be shown that all observed gene products and most behavioral traits of neoplastic cells result from the renewed expression of genes normal to some stage of development, that mutant proteins are not essential to the maintenance of neoplastic transformation, and that viral infection or viral gene function is not necessarily an obligatory requirement for continued malignancy. Lest we seem too heretical, we stress that many biological, chemical, and physical carcinogens serve to induce or promote these changes in genetic expression. However, if alteration of the products of structural genes is not required in neoplasia, then it must be postulated that the control of structural gene expression in cancer cells occurs via altered regulating substances which are coded for by a class of DNA solely concerned with regulation (Britten and Davidson, 1969). Control of differentiation must also reside in this class of DNA. In this view the basic problems therefore lie in factors regulating gene expression. The idea that cancers result from diseases of the cellular differentiation process is not new (see Conheim, 1889; Boyse et al., 1968; Markert, 1968; Potter, 1969) . The basic problem is approached in four ways. First, by inquiring into the compositional changes which accompany malignant transformation and asking whether the changes observed involve normal gene products (i.e., proteins that occur normally at some stage of development). Should nonnormal proteins be found, one may then ask whether they are the products of mutations or viral genes. We leave for later discussion the question of whether genes that are universal i a a species should (or can) be considered exclusively viral. Second, we may examine available information on the differentiation program itself and ask whether changes seen in cancer cells are those that might be expected from a master schedule gone partially awry but with some evidence of the original program still present. Third, if only normal substances (embryo or phase specific) and functions appear in most human malignancy, then we must inquire how these are dealt with when they appear normally. For example, if cancer-associated autoantigens appear normally during fetal development, is an immune response mounted, and how does the fetal cell exhibiting such antigens escape immune destruction? This raises the question of whether host responses to cancer are extensions or caricatures of normal immunologic responses.

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

107

Fourth and last, we ask whether present knowledge of the complexity of the mammalian genome and of mechanisms for the control of gene expression are compatible with the basic thesis. For example, is there evidence that carcinogens selectively effect the transcription of nonmessenger RNA which may be involved in the control of differentiation. This review examines a large body of information from one viewpoint. It is evident that quite different interpretations may be placed on some of the data by other investigators. The importance of divergent views is that they lead to different, and often new, experiments. For this reason alone, it is important to follow each concept or theory of cancer to the end of its productivity. We have obviously not attempted an in depth review of all published results in all areas discussed but have deliberately selected results that focus attention on the proposal at hand. II. Transformation-Associated Cellular Alterations

The search for characteristic molecular alterations associated with malignant transformation has been diligently pursued for decades and has been, until recently, relatively unrewarding. Changes in the pattern of enzyme activity were reported, but for each generalization some exception was usually found. However, with the discovery that tumors possessed cellular isozymes (see Weinhouse, 1972), multiple forms of certain RNA species (see Yang, 1971), many embryonic and fetal analogs of adult proteins (see Abelev, 1971) and tumor-associated autoantigens (see Anderson and Coggin, 1971) different from true transplantation antigens, the picture changed rapidly. Special interest has surrounded tumor antigens, especially those occurring in autochthonous tumors, because of the immunotherapeutic and detection possibilities which they appear to raise. Tumors are generally antigenic in their hosts (Old and Boyse, 1964; Klein, 1966). High interest has developed over the past decade in the source of genetic information for these antigens. Both intrinsic and extrinsic sources have been proposed. The past 8 years have witnessed an incredible search for viral gene contributions to malignancy in hopes of identifying extrinsic genes associated with malignancy. Few data have been available for meaningful exploitation of the intrinsic sources of human tumor antigen expression. In considering the genetic origin of tumor antigens in 1968, Boyse and his associates stated, “If suppression of the embryonic antigen were complete in adult life, then its reappearance (in tumors) might be expected to provoke an immune response. This criterion, which has not been met in any experimental system, would provide the best evidence for qualitative acquisition of antigen rather than simply increased syn-

108

J. H.

COGGIN,

JR. AND N. G . ANDERSON

thesis accompanying a change in the profile of tissue antigen: consequent on malignancy.” Shortly after this statement was published these “missing” data began to appear. A. TUMOR-ASSOCIATED AUTOANTIGENS Model systems in rodents established a pattern of thought about the character, specificity, and potential usefulness of the “neoantigens” appearing on tumors which has prevailed since the early 1960’s. Briefly stated, tumors of rodents induced by a given virus, whether in the same individual, in different individuals of a species or in different species, carry the same tumor-specific transplantation antigen (TSTA ) (Table I). Tumors induced by different viruses, even within the same strain of inbred rodent, carry distinct TSTA’s and the antigens did not crossprotect (see Haughton and Nash, 1969). Fink et al. (1966) reported a notable early exception describing a cross-reaction betwee:i leukemias induced by the closely related Friend, Moloney, Rauscher viruses, but the generalization otherwise seemed valid. As we shall discuss later, many of these tumors are now recognized to possess an additional embryonic or fetal antigen (Table I ) . TABLE I A LISTOF ONCORNAVIRUSES .4ND ONCODNhVIRUSES THATCARRY VIILUS-SPECIFIC TUMOR TRANSPLANTATION ANTIOEN(S) (TSTA’S) AND FETALANTIOEN(S) Antigen identified0 Viruses Oncornviruses Sarcomas Rous (Schmidt-Ruppin) Moloney Leukemias Gross GrafTi Friend, Moloney, Rauscher Mammary tumor virus Onwdnaviruses Polyoma SV40 Adenoviruses

7, 12, 18, 31

Shope papilloma

TSTA

+ + + + +

Fjtal

Nl’

+

+ (?)

+ + + +

+

NT

.NT, Not known to have been t,ested to date; ?, interpretattionof result mag be questionable.

CANCER, DIFFERENTIATION, AND EMBRYONIC ANTIGENS

109

By definition TSTA’s are demonstrated by the capacity to induce a state of heightened immune reactivity of the transplantation rejection type ( delayed-type hypersensitivity) against subsequent grafts of syngeneic tumor or autochthonous virus tumor. Radiation-inactivated, syngeneic or allogeneic tumor cells (Sjogren, 1964) transformed by the homologous virus or living virus (Sjogren et al., 1961) have traditionally been used to stimulate transplantation immunity. Animals cured of their tumors by surgery or remaining tumor free after challenge with subthreshold tumors also demonstrate the tumor resistance phenomenon and confirm the presence of TSTA on the corresponding vaccine preparation or tumor. In contrast, tumors induced by a variety of chemicals or by physical agents (plastic films, ultraviolet light, or radiation) possess individually specific TSTA’s that do not seem to cross-protect against similar tumors induced in other syngeneic animals with the identical carcinogens. Each new tumor would appear to possess a new antigenic individuality. The molecular basis for the diversity of the specific transplantation antigens of chemically and physically induced tumors is still a matter of considerable speculation. As study progressed, an increasing number of reports suggested that independently arising tumors ( spontaneous and chemically induced) showed some small degree of cross-reactivity (Prehn and Main, 1957; Stern, 1960; Pasternak et al., 1962; Koldovsky and Svoboda, 19f33; Globerson and Feldman, 1964; Takeda et al., 1966; Reiner and Southam, 1969). Hanging over the entire field of tumor immunology only 5 years ago was the concept of the so-called “tumor-specific” antigens. For more than a decade TSTA’s have received major attention in cancer immunology. Some thought them to be of potential usefulness for cancer therapy, although their use in cancer detection, if they were individually specific, would be nil. What type of antigens were human cancers observed to carry? Cross-reactive autoantigens and tumor-associated antigens in human tumors (Morton et al., 1970; Hellstrom et al., 1971; Abelev, 1971; Gold and Freedman, 1965), such as melanomas, hepatomas, and adenocarcinomas of the colon and other tumors received little attention initially. The immunologic implications associated with the application of cross-reactive antigens to human cancer control were somewhat confusing, and the results seemed at odds with the accumulated data regarding the specificity and non-cross reactivity of TSTA’s derived from many animal model studies. There were, in fact, no animal models with the exception of the a-fetoprotein ( AFP) -excreting rat hepatomas or occasional cross-reacting chemically induced sarcomas to predict or investigate such results. However, with the development of

TABLE I1 S n a a r a ~ ~OFr EFFORTSTO DETECTEMBRYONIC OR FETAL ANTIGENS IN MODELTUMOR SYSTEMS PRIORTO 1970 Date

Model

1906

Mice-spontaneous sarcoma (?)

1962

Rat-human sarcoma H51

1964

Mice-chemically induced sarcomas

1967

Mice-3-MCA sarcomas

1968

Mice-polyoma tumor

1968

1970 1970 1970

i970

0

Mice-polyoma tumor

Mice-72 mouse tumors Pregnant mice-3-MCA sarcomas Mice-MTV SVG-uuiertiiized mouse ova

Fetal antigen demonstrated

+ + + + + -

+

+ - (?I

+

Comment Fetal immunization prevented tumor transplantation Cortisone treated rats Only detected with nonsyngeneic fetus Moderate to weak protection Alloantisera used No protection observed against polyoma tumor challenge Many tumors crossreact with antifetal sera Pregnant effector cells destroy tumor cells Attempted to prevent MTV tumon Noantisera reacted against SV40 tumor cells

Reference

Assay

Allogeneic

Schone (1906)

TR

+

Buttle et al. (1962)

TRa

Buttle et al. (1964); Buttle and Frayn

TR

(1967) Prehn (1967)

TR

Pearson and Freeman

As*

(1968) Ting (1968)

TR

Stonehill and Bendich (1970)

As

Brawn (1970)

MC.

Blair (1970)

TR

Bttrusktt el ill. (1970)

As

Tumor rejection assay. AS, Allogenic serum prepared against mouse fetus, absorbed and tested. MC, Microcytotoxicity test.

Syngeneic or inbred

+ +

+

n

+ +

"Q z cl

+ + + t-

8

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

111

more sensitive tumor antigen and autoantigen assays and a greater variety of animal model systems, it has become possible to examine the question of cross-reactive antigens in different tumors and to determine their relatedness to embryonic and fetal tissue autoantigens in greater detail. Again, the concept of the tumor-specific antigen generally implies that the antigenic determinant is found only on the tumor and absolutely nowhere else in the entire natural life history of the host. The test battery of control antigens to establish specificity is difficult to imagine if the mammalian genome contains several million structural genes, many of which are only transiently active during development. The demonstration of such specificity is and will continue to be enormously difEcult, as we shall discuss. Many workers began to search for embryonic antigens on tumor cells to fill the data void cited earlier in the quotation from Boyse and his colleagues. Experimentally, the problem of searching for tumor-associated antigens in normal tissues may be approached by attempts to immunize adult syngeneic or inbred animals against tumor challenge with fetal tissues employing conventional immunologic methods. Some early efforts, outlined chronologically in Table I,I,were controversial and, in the view of many, not particularly encouraging. Nevertheless, several promising leads were evident from a close inspection of the data and suggested more direct and probing experiments. Brawn’s discovery that normal pregnant mice (inbred) possessed lymph node cells ( LNC‘s ) cytotoxic for several “non-cross-reactive” 3-methylcholanthrene (3-MCA) sarcomas of the same strain of mouse was, in our view, most significant. Why should tumors carrying only TSTA’s be antigenically reactive with effector cells which were apparently sensitized to normal embryonic autoantigens? Effector cells from these same pregnant donors did not destroy normal fibroblasts, only the several “non-cross-reacting 3-MCA cancers.” Clearly Brawn’s data suggested that mouse tumors possessed antigens cross-reactive with antigens on fetal cells which could elicit a potent cell-mediated response in histocompatible mothers. Prehn ( 1967) had made a related observation earlier, noting that adult mice challenged with syngeneic embryo implants were weakly resistant to challenge with mouse sarcomas induced by 3-MCA. Negative attempts to perform similar immunization were soon reported by R. C. Ting (1968) using polyoma tumors and vaccination with unirradiated fetal cells. Pearson and Freeman (1968) reported results contradictory to those of Ting, however, in the same year having observed an apparent relationship between polyoma TSTA and a normal embryonic antigen albeit in an xenogenic test model. Using the mouse mammary tumor model, Blair (1970) attempted to decrease

112

J. H. COGGIN, JR. AND N. G. ANDERSON

the occurrence of autochthonous mammary tumors in mice by immunization with either fetal tissue extracts or fetal fragment implants. Although Blair concluded that “pretreatment with embryonic tissue extracts or implants had little if any effect on the development of tumors in the test females,” it is most interesting to reexamine her data. In one experiment she reported 60-70% occurrence of tumors in female mice infected as neonates with mammary tumor virus and immunized with syngeneic control tissue material from normal lactating mammary gland, but the data show that only 2622% of tumors were observed in a large number of mice primed with early embryo or late embryo fragments. This protection level ( approximately 68%) held in embryo-primed animals for over 14 months. Although Blair feels she failed, we suggest that in fact she may have succeeded. These and similar findings, together with the overriding observation that many human tumors cross-reacted immunologically within histologic types ( Hellstrom et al., 1971; Gold and Freedman, 1965), prompted us to initiate a search for similar embryonic or fetal antigens on tumors induced by the oncodnaviruses.

1. Zmmunization against Tumor with Fetal Cells Prehn (1967) had observed, at best, weak and sometimes unreliable protection against chemically induced tumors using fetus as immunogen. Other workers (Ting, 1968; Blair, 1970) had failed to report significant transplantation immunity in syngeneic animals following immunization with fetus, in agreement with the earlier report of Buttle et al. (1964). Anticipating serious difficulties in achieving strong immune reactions with autoantigens, we were convinced that oncodnavirus tumors possessed fetal antigens that might induce cell-mediated as well as humoral immunity. Several considerations supported this belief, The reported coding capacity of the small oncodnaviruses was an obvious theoretical lead, and the SV40 hamster model was particularly attractive in this regard. At best this virus possessed 5-8 genes, most of them devoted to “virogene” activity ( genes needed for virus replication in permissive cells). Only one or two genes, at most, seemed to be available for transforming potential of the virus. Hence it seemed implausible that this small virus could code directly for elaborate, virus-specified changes in the surface membrane of transformed cells. In particular, it is difficult to conceive of how the agent could code directly for what appeared to be a unique antigenic determinant in the plasma membrane which could be identified as a TSTA (see Coggin et al., 1970, 1971). It seemed more plausible that the production of the unique TSTA might result from

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

113

specific regulatory changes in normal host biosynthesis under virus control. Against this interpretation it must be pointed out, however, there was the clear situation wherein SV40 produces the same TSTA in several species of cells transformed by the virus, suggesting that, indeed, SV40 does code for TSTA directly. Further supportive evidence for our view however came from studies in the course of which we observed that a specific immunoglobulin ( cytostatic antibody), believed to react only with tumor-specific neoantigens in the plasma membrane (Coggin and Ambrose, 1969), occurred transiently in the serum of normal pregnant hamsters (Coggin et al., 1970). Similar reports given by Duff and Rapp ( 1970) for suiface or S-antibody simultaneously appeared. An alternative explanation for these findings could be that the extrinsic, regulatory genes of the infecting virus served to activate intrinsic, cellular DNA expression common to many mammalian species, possibly important to an early phase of embryogenesis and resulted in “neoantigen” appearance. Such a consideration raises difficult questions about the specificity exhibited by TSTA’s which do not cross-protect against other tumors. Individually specific viral regulation of tumor clones is possible and such tumors might carry similar fetal antigens. Virus-induced derepression of host-specified protein synthesis is a common phenomenon in molecular virology and requires no precedent. The first probe in our studies was to test SV40 tumors for fetal antigens by a direct test. Could one prevent SV40 tumors in adult hamsters by sensitizing the rodents to syngeneic fetal tissues and subsequently challenge the animals with living syngeneic tumor cells? In several attempts we were able to obtain, reproducibly, reasonable levels of tumor resistance following immunization of inbred LSH hamsters with mid-gestation fetal homogenates containing living cells (Table 111; also see Coggin et al., 1970). Similar results were obtained in the unusual syngeneic but random-bred LVG hamster strain, which demonstrates no detectable histoincompatibility or sex-linked antigens by many parameters of test ( microcytotoxicity test, primary and second-set graft tolerance, organ transplantation, lymphocyte stimulation, radioimmuno antibody test, cytostatic antibody test). The LVG strain was derived from sibling matings many years ago. This strain may exhibit a graft vs. host reaction ( P. Koldovsky, personal communication). Most significantly, we were able in subsequent work to demonstrate the interruption of autochthonous SV40 or adenovirus tumor formation in neonatally infected male hamsters primed with syngeneic, midgestation fetal cells at 3 5 weeks of life, before the first virus-induced tumors appeared in control animals ( Coggin et al., 1971). Again, pro-

114

J . H. COGGIN, JR.

AND N. G. ANDERSON

TABLE I11 TYPICAL RESULTSDEMONSTRATINQ THE INDUCTION OF TRANSPLANTATION IMMUNITY TO SV40 HAMSTER TUMORCELLCHALLENQE EMPLOYINQ FETAL TISSUES AS IMMUNOQEN No. tumors detected"

Antigen Unvaccinated controls SV40 tumor cells-irradiated (5000 R) Adult hamster tissue homogenate (5000 R) Syngeneic hamster fetal cells0 9-Day gestation (5000 R) 10-Day gestation (5000 R) 14-Day gestation (5000 R) 9-Day gestation (0 R) 14-Day gestation (0 R) Mouse fetal cells RALB/c 12-Day gestation (5000 R) Term (5000 R) Human embryonic kidney cells (5000 R) Adult kidney homogenate (5000 R)

No. animals challenged (% tumors)

Percent protectionb against SV40 tumor challenge

14/15 (93) 0/15 (0) 15/15 (100)

Control 100 0

6/15 (40) 5/15 (33) 13/15 (86) 13/14 (92) 9/10 (90)

53 67 7

4/10 5/5 7/10 9/9

(40)

(100)

(70)

(100)

-

53

-

23 -

0 Animals challenged with 5 X lo4 SV40 tumor cells (syngeneic) (50% tumors obtained in controls at day 68). * Test result compared to unvaccinated control in each case to obtain percentage of protection. c Viability of cells >40% as determined by the dye-exclusion test using trypan blue.

tection in these early studies was not absolute, but was statistically significant and reproducible. Term-fetus and adult tissues of many types were without protective effect. In these early studies many intriguing and sometimes baffling results were obtained. The following summary outlines some of the more challenging observations which relate to the expression of fetal antigens on tumor cells with particular reference to the SV40 fibrosarcoma of hamsters. 1. Normal, virgin female hamsters did not show transplantation resistance or cell-mediated immunity against SV40 target cells in oitro when primed intrapmitoneally with syngeneic fetal cell homogenates, whereas male vaccinees did have immunity when so treated (Coggin et al., 1971; Dierlam et al., 1971). 2. Female recipients did have a demonstrable humoral response after fetal tissue sensitization following parenteral immunization ( Coggin et al., 1971). Both fetal cells and tumor cells react with the immunoglobulin elicited in females after immunization with fetal cells.

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

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3. Irradiation of the fetal cell homogenates was essential to antigen retention among the living (but nonreplicating) fetal cells comprising the homogenate ( Coggin et al., 1970, 1971). 4. Living fetal cells were essential for proper vaccine efficacy, and immunogenicity of the fetal vaccines diminished with increasing loss of fetal cell viability ( Coggin and Anderson, 1972). 5. Fetal tissues were not immunogenic unless obtained from primiparous donors. Multiparous donors have proven to be unsuitable sources of fetal tissues (Girardi et al., 1973; Coggin and Anderson, 1972). 6. Mid-gestation fetus was immunogenic. Late or term fetus was nonprotective ( Coggin et al., 1970). Phasing of the antigen ( s ) involved was indicated ( Coggin and Anderson, 1972). 7. Mouse (fetal homogenates) and human fetal cells (kidney) also gave cross-protection against SV40 tumor induction in hamsters ( Coggin et al., 1970, 1971; Ambrose et al., 1971a). Adult tissues from the same species were nonprotective. 8. Pregnant hamsters yielded cytotoxic effector cells reactive or protective against several oncodnavirus tumors in oiuo ( Coggin and Anderson, 1972; Girardi et al., 1973) and in uitro (Dierlam et al., 1971; Coggin and Anderson, 1972). 9. Pregnant hamsters show transient humoral immune responses to SV40 tumors during pregnancy which were not detectable postpartum ( Coggin et al., 1971). These early observations, many of which have since been confirmed and/or extended in other laboratories, show quite clearly that SV40 and adenovirus tumor cells possess embryonic or fetal antigens. These autoantigens, present transiently on fetal cells during development, were capable of eliciting both antibody and cell-mediated responses against tumor cells bearing the antigen( s ) . In reviewing work completed in our own laboratories and reports from other laboratories since 1970, we have collected information showing that some 40 distinct tumors of rats, mice, guinea pigs, and hamsters have fetal antigens by one or several test parameters (Table IV and Table VI). It is important to note that immunization with one of these tumors will generally not protect against challenge with any other in a given animal system with the exception of tumors induced by the same virus. Hence, each tumor would appear to have fetal antigen( s ) present as well as TSTA(s). Humoral studies, conducted in mice, further support this contention (Ting et al., 1972). We will return to this point later, as it is a most important one. Many investigators are justifiably concerned with one apparent dissimilarity between TSTA and fetal antigens reexpressed on cancer cells.

116

J. H. COGGIN, JR. AND N. G . ANDERSON

TABLE IV TUMORS IN SYNGENEIC SYSTEMS RECOGNIZISD TO POSSESS EMBRYONIC OR FETAL ANTIGENS’ Antigen detected Animal model and tumor type Hamster Sarcoma

Carcinoma

Lymphoma Rat Sarcoma Carcinoma Lymphoma Mouse Sarcoma Leukemia Lymphoma Carcinoma Plasma cell tumor Guinea pig Carcinoma Sarcomas

Inducer or tumor origin (No. different tumors tested) Adenovirus 7-Td Adenovirus 31 DMBA-T Moloney sarcoma virus-T 3-Methylcholanthrene (MCA)-T (2) Polyoma SV40-T (3) Spontaneous sarcoma-T Gross virus 3-MCA-T (13) DMBA (4) Gross SV40 MKS-A-T FMR-Induced-T ?-Spontaneous 3-MCA T Spontaneous-T 3-MCA (4)-T Liposarcoma-3-MC A-T 0 steogenic-sarcoma-?

In vivd

+ + + + + f + + NT + (?) ? + + + NT

NT

+ + NT NT

In vitro“

+ + + NT + + +

NT

+ + + +

t

+ + + + +

NT

+ +

a References: List was compiled from findings in our own laboratory and from the following sources: Brawn (1970); Bendich et al. (1973); Baldwin et al. (1971); Hanna et a!. (1972); Herberman et al. (1971); Grant et al. (1973); Borsos and Leonard (1972); Girardi et al. (1973). * Immunity was induced by fetal immunization, detected by transplantation resistance test or interruption of oncogenesis. Immunity detected in vitro using antibody or cell mediated tests. T, Tumor transplant.

Tumor cells will generally confer strong protection against homologous tumor challenge, yet the reported efficacy of fetal vaccines against similar challenge doses is often modest. Many conclude that TSTA must be “more” immunogenic ( antigenic) than fetal antigen because of this disparity in the strength of the cell-mediated immune reaction evoked. This may or may not be the case. If we conclude that tumor cells possess at least two antigens at the cell surface which elicit cellular immune reactions in the host (TSTA and fetal antigen), both may be equally

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

117

antigenic but may present differently at the target cell surface as binding determinants for lymphocyte and/ or macrophage recognition. Fetal antigens seem more autosoluble than TSTA in studies in our laboratories and thus may provide poorer target antigens for the cell-cell interactions leading to tumor cell destruction. Carcinoembryonic antigen and afetoprotein are autoreleased from human tumors. It is very important that we remember that neither TSTA nor fetal antigen serves the host as an effective rejection antigen in animals dying from malignant tumors. In another view, fetal antigens may be more potent inducers of humoral immune factors than TSTA’s, and although they may induce comparable cellular immune sensitization, fetal constituents may simultaneously elicit potent blocking reactions produced to a comparable or lesser degree, by TSTA’s. Theories such as these are in keeping with the observed induction of antifetal cellular reactivity observed in pregnancy. Pregnant hamsters and mice have potent cellular immunity against tumor cells measured in vivo in passive transfer experiments or in uitro using the microcytotoxicity assay; immunity quantitatively equivalent to that observed in control females immunized directly against homologous tumor cells (Dierlam et al., 1971; Anderson and Coggin, 197213; Girardi et al., 1973). Yet, pregnant animals have blocking factors which prevent immune destruction of SV40 target cells in the microcytotoxicity test by effector cells sensitized to SV40 tumor ( Coggin et al., 1973). In another view, modulation of fetal antigen expression in vivo might occur. Similar modulation might also exist in vivo for TSTA’s. Two mechanisms could be involved. In one situation, fetal antigens cross-reactive between tumors might be expressed in uitro in microcytotoxicity tests, for example, but not be expressed qualitatively or quantitatively to the same degree in the autochthonous host bearing the tumor cells. Another mechanism suggested by George Klein is that the immune response against the antigens, particularly the cross-reactive fetal antigens on tumor cells, could potentiate a change ( modulation) in the expression (repression?) of fetal antigens by interaction with antibody or even cellular effector cells in a fashion similar to that described for the TL-system. In brief then, the apparent disparity between the antigenicity of TSTA and fetal antigen may be due to biological factors (solubility of antigen in plasma, blocking factor, location of antigen in the membrane) other than relative antigenicity. 2. Preparation of Fetal Cell Vaccines It is important to review briefly some general “rules” for making fetal vaccines which are effective inducers of tumor immunity against the SV40 or adenovirus tumors using hamsters. Table V summarizes our preparative techniques to date.

118

J. H. COGGIN, JR. AND N. G . ANDERSON

TABLE V ON PREPARATION AND USE OF “FETAL” VACCINEFOR GENERAL GUIDELINES ACHIEVINO PROTECTION AOAINST ONCODNAVIRUS TUMORS IN HAMSTERS 1. Employ time-mated females where fetal age is accurately known ( kO.tj day). 2. We have not used trypsin t,o dispense fetal tissues, but tissues have been disag-

gregated by passing through small-orifice needles. 3. Use disaggregated cell preparat,ions of fetus with high viability to achieve maximal immunogenicity (40-60%). 4. Use fetus from primiparous females only (fetus from multiparous females seems to be coated with IgG which “masks” immunogenicity). 5. Give multiple intraperitoneal injections of fetus. 6. X-Irradiate the fetus to prevent differentiation of the tissue in uiuo and to prevent embryoma induction in vaccinees. 7. Keep the time from fetal harvest to injection less than 1 hour to avoid plasma membrane antigen degradation by hydrolytic enzymes released from cells destroyed during fetal disaggregation. 8. Avoid the use of passaged tissue-cultured fetal cells, which undergo apparent in nitro differentiation. 9. Select the correct age fetus for maximal immunogenicity by actually testing each day of fetal age possible (do not make an empirical selection of a given fetal age for convenience’ sake); for example, in the SV40 model system, fetal antigen expression is “silenced” between 10.5 and 11.0 days. 10. Use the most sensitive challenge assay possible (e.g., TPD)u until all factors above are determined.

Are results obtained with the hamster true for the mouse or guinea pig under different conditions with different tumors? Table VI summarizes some of the recent observations which clearly indicate that direct immunization against many types of tumors can be obtained employing fetus as the antigen source. Several researchers have reported that direct immunization of mice (Ting et al., 1971) or rats (Baldwin et al., 1971) with syngeneic fetus (irradiated) did not elicit cellular resistance to tumors. Since other workers (Table VI) have recently reported contradictory results showing protection against similar tumors in mice and rats, we must conclude that technique or methodology are the variables and not the immunogenic capacity or lack of antigenicity in the fetal vaccine. Bendich et al. (1973) recently reported that 18-day mouse embryo cells (near term in gestation) were protective (approximately 80% reduction in tumor takes) when used to immunize syngeneic female C57B1 mice against 3-MCA induced tumors. In this study the embryo tissue was dispersed by exposure to trypsin and hyaluronidase and irradiated; the embryos were derived from mothers of undetermined parity. Enzymatic treatment may render term hamster fetus immunogenic (personal communication from A. Girardi), The observation that female mice were protected against tumor challenge after immunization with

TABLE V I REPORTED OBSERVATIONS OF INDUCTION OF TUMOR IMMUNITY FOLLOWING DIRECTIMMUNIZATION WITH FETAL TISSUE. Tumor immunity induced against

Males

Mouse Mouse

QUA MCA-10

N.D. N.D.

Mouse Mouse

MCA-10

System

Rat Guinea pig

PCT MCA-R MCA-A, MCA-25

s

Fetal vaccine effective in

+ + ++

r

Route of vaccination

Females

++

+

N.D.

++

S.C. ?

I.P. I.P. ? I.D. (+CFA) I.P.

1

h a y

Reference

E

Direct challenge Direct challenge, serum cytotoxicity with iododeoxyuridme-1451 Cell challenge Spleen colony assay (FCFU) Direct challenge DTHR’s Direct challenge

Bendich et al. (1973) LeMevel and Wells (1973)

=1 0

2 ~

;P

Castro et al. (1974) F. A. Salinas et al. (1972) Grant et al. (1973) Grant et al. (1973)

Abbreviations. N.D. = Not done. S.C. = Subcutaneous injection. I.P. = Intraperitoneal injection. I.D. (CFA) = Intradermal injection (complete Freund’s adjuvant). FCFU = Fetal colony forming unit. DTHR’s = Delayed type hypersensitivity reactions. a

@m

3 M

z

2

8 3 ;P

8

m

120

J. H. COGGIN, JR. AND N. G. ANDERSON

irradiated fetal cells is a most interesting finding in view of our observations reported for in hamsters. Grant et al. (1973), using inbred guinea pigs, observed highly significant protection against a 3-MCA sarcoma in female guinea pigs and male guinea pigs primed with Xirradiated guinea pig fetus. These workers used sul?cutaneowi and intraperitoneal routes for multiple immunization of the females, Delayed type hypersensitive reactions were detected in fetal primed animals and in female exbreeders using fetal extracts as the challenge antigen. Similar protection against 3-MCA tumor transplants in mice has been reported (Wells et al., 1973; LeMevel and Wells, 1973) further demonstrating that many tumors carry fetal antigens and that these antigens can serve as target antigens to render tumor cells susceptible to cell-mediated destruction under the proper conditions of test. As we shall discuss in Section II,A,4, all these results obtained in females immunized with fetus raise basic questions regarding the immunology of reproduction. The techniques for achieving protection against tumor with fetal tissues are obviously in a primitive, if not embryonic, state. Much is to be learned. Which fetal cells in the homogenates harbor the autoantigen? Where are the fetal antigens located within the cell? Can we establish that TSTA's and fetal antigens are distinct using pure antigen preparations in transplantation tests? Are the antigenic detenninants for antibody production the same as those for the activation of cellular immune responses? Many similar questions remain to be answered. What is clear from the data which are available is that fetal antigens can elicit significant, reproducible cell-mediated immune responses under specified conditions. What is unclear is why fetal antigens, like the so-called TSTA's, fail to elicit immune responses in a proper toay in individuals with progessive cancer and whether fetal antigens contribute to the survival of the tumor. In searching for the role that fetal antigens play in tumor progression or rejection we may reveal the role of these autoantigens in ontogeny, fetal survival, and developmental biology. We leave open the question of whether true tumor-specific antigens not coded for by normal genes exist in man and whether any such antigens are of viral origin. Since the genes of viruses probably have their origin from cells like those in which they reproduce, this argument may be circuitous at best. A recent report indicates that the cross-reactive S2 antigen which occurs in many types of human sarcomas is a fetal antigen (Mukherji and Hirshaut, 1973).

3. Embryomas Nonirradiated fetal cells have proved to be ineffective as inducers of cell-mediated tumor immunity in our hands and elsewhere (e.g., Herber-

121

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

man et al., 1971; Castro and Medawar, 1974) (see Table 111). We proposed ( Coggin et al., 1970) that ,radiation-impaired fetal cells expressing the fetal autoantigens might be retarded in their ability to undergo maturational changes when injected into adult tissues. If this reasoning were correct, the autoantigens would continue to be displayed at the plasma membrane surface when in the adult recipient. Using precisely timed fetus, we were able to determine that fetal antigen phasing occurs between day 10 and day 11 of gestation in the hamster (Table VII). Similar studies have not been completed in other species and should be done. No doubt different phasing “schedules” will exist in each species for each fetal autoantigen. A number of immunologic tests were employed to detect the disappearance or silencing of the antigen(s) cross-reactive with SV40 sarcomas and excellent correlation among all assays was obtained. What seems clear from these data is that fetal tissues undergo maturational changes over brief time periods in gestation which result in antigen phasing. If the injected fetus (10-day gestation) is within 8-24 hours in its genetic “program” from undergoing natural phasing of the fetal antigen( s ) , it is reasonably obvious why X-irradiation is essential to fetal antigen expression in fetal vaccines. No published data are available to prove that the “silenced fetal antigens reside cryptically TABLE VII THE PHASING OF FETAL ANTIGENS .4s DEMONSTRATED RY VARIOUS IMMUNOLOGIC TESTSA N D AGAINST SV40 TUMORCEI~LSO Fetal age (days post coitus) Immune reaction Induction of cytostatic antibodyb Induction of cell-mediated immunity In vivoc In vitrod Induction of tumor resistance4 Interruption of oncogenesisf 4

b-j.

9

10

11

+ + -

+ ++ +

+ + + + -

12

13

14

15

Birth

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

The techniques3 employed to obtain these data are described in detail in references

By diffusion chambers (Ambrose d al., 1969). By adoptive transfer test (Dierlam et al., 1971). By microcytotoxicity test-colony inhibition (Dierlam el a!., 1971). By transplantation immmity (Coggin el a!., 1970). f By prevention of 8V40 tumor induction in neonatally infected animals (Coggin el al., 1971).

122

J. H. COGGIN, JR. AND N. G . ANDERSON

in the older or mature fetal membranes where antigen expression is not detected; these studies too must be done. Biochemical evidence raising this possibility is available (Rogan et al., 1973; Hannon et al., 1974). Additional data on the phasing of fetal antigen were obtained several years ago when adult male and female hamsters which had received nonirradiated, whole cell embryo homogenates from syngeneic donors were autopsied several months later (Ambrose et al., 1971). Small embryomas containing well defined, highly differentiated conglomerations of teeth, cartilage, bone, muscle, gonad, neural tissues, hair, etc., were found at the site of fetal cell deposition intraperitoneally. When challenged with fairly large challenge doses of tumor cells ( 0 11 group in Table 111) we had detected little resistance to SV40 tumor appearance in these embryoma-bearing test animals. The well differentiated embryomas were rarely observed to be invasive, although several hamsters with rather large embryomas in the abdomen (site of original injection) did develop physical obstruction from the “benign” tumor load and die. Similar results were obtained in inbred mice injected with syngeneic mouse embryo. We attempted to titrate the minimum number of syngeneic hamster fetal cells required to produce an “inoculation” embryoma and to test these hamsters with very low challenge doses of SV40 tumor cells to determine whether embryoma-bearing animals had some degree of tumor resistance. We observed that 5 X lo5 live unirradiated fetal cells could produce 100%embryomas when injected subcutaneously into adult hamsters of either sex (Ambrose et al., 1971b). Injection of lo5 live fetal cells yielded only 20%embryomas indicating a sharp dependency on fetal cell dose for embryoma induction, as would be expected if embryonic cells had to surmount a weak immunological surveillance barrier to survive. Hamsters bearing subcutaneous embryomas were tested for resistance to SV40 tumor challenge at another subcutaneous site, and it was found that 50%of the male recipients and 24%of the female recipients with embryomas resisted tumor challenge, whereas all the age-matched normal control hamsters developed tumors (Ambrose et al., 1971). These data were reproducible with another SV40 hamster tumor cell lint: and show that animals bearing subcutaneous ( sc) embryomas (not intraperitoneal embryomas ) had concomitant immunity to SV40 tumors. Several reasons may account for the failure of nonirradiated fetal cells to produce tumor immunity within 10 to 14 days post-immunization (Table 111). It is apparent that the fetal cells differentiati: since the embryomas they produce contain only “mature” cells; however, the fetal cells producing the sc embryomas may retain antigenicity sufficiently

CANCER, DIFFERENTIATION AND Eh4BRYONIC ANTIGENS

123

long to activate cellular immunity, whereas fetal cells placed intraperitoneally ( ip ) do not do so. Alternatively, subcutaneous sensitization may be more effective than the ip route, as is suggested by the observation by Grant et al. (1973) that female guinea pigs respond to immunization with fetal cells when given subcutaneous injections. Other equally plausible explanations are clearly possible. Can one affect fetal cell growth into embryomas by immunization with tumor? If both tumor and fetal cells share common fetal autoantigens and if fetal cells are immunosensitive to cell-mediated immune destruction, then such an experimental result might be possible. It is apparent that this question must be answered to establish the true, crossreactive nature of fetal antigen, provided some technical difficulty or biological barrier does not exist (i.e., inability to get the fetal cell to retain its antigenicity and not undergo antigen phasing). In immunofluorescence studies we observed that intact 10-day (antigen positive) but not 14-day ( antigen negative ) hamster fetal cells reacted with antitumor ( SV40), antifetal, and multiparous hamster serum, suggesting such crossreactivity. The real test, of course, is to prove that cellular immunity, induced by tumor surface antigens, will function against fetal target cells. In some initial experiments we could not demonstrate that hamsters immunized against SV40 tumor could reject embryomas induced by challenge with 5 X loGlive syngeneic fetal cells from whole-embryo homogenates (Coggin and Anderson, 1972; Ambrose et al., 1971b). Perhaps the hamster fetal cell differentiated too quickly in vivo to present an antigenic target to sensitized effector cells in the tumor-immune host. Again, other explanations are possible. Clearly a new approach was required. Salinas et al. (1972) reported that mice primed to a high state of tumor resistance with irradiated plasma cell tumor, and subsequently irradiated and challenged intravenously with living, mouse fetal liver cells, formed fewer embryomas than did control mice. Such an observation, which has now been extended to several other tumors ( Hanna, personal communication) including xenogeneic tumors (i.e., SV40 hamster tumor), affords the needed proof of true cross-reactivity between tumor-associated fetal antigens and fetal autoantigens in situ. Even human tumors might be examined for fetal antigens in this way ( Coggin and Anderson, 1972). Recently Granatek (1974) and her associates indeed established such a relationship. 4. Pregnancy and Tumm Immunity in Animals If direct immunization with fetal antigens elicits cell-mediated immune reactions to tumor cells as previously described, can one detect sensitized effector cells cytotoxic for tumor cells in pregnant females?

124

J. H. COGGIN, JR.

AND N. G . ANDERSON

Brawn (1970) had observed activated lymph node cells (LNC‘s) in pregnant mice which inhibited the growth of non-cross-reactive 3-MCAinduced sarcomas in uitro. In similar studies we observed LNC‘s in pregnant hamsters which were cytotoxic for SV40, polyoma, and adenovirus target tumor cells in uitro ( Coggin et al., 1973; Girardi et al., 1973). The activity of these immune effector cells could be detected in viuo also. As early as 10 days into the first pregnancy, pregnant female donors yielded LNC‘s capable of protecting normal recipient hamsters against SV40 tumor cell challenge (Table VIII ) . Primipurous LNC’s ( washed 3 times before testing following harvest) collected during day 10 of gestation were two times more cytotoxic than LNC‘s from pregnant multiparous donors in the same test. In other studies employing the microcytotoxicity test we were able to establish that serum from pregnant hamsters would abrogate the cytotoxic interaction between either multiparous LNC‘s or LNC‘s from hamsters rendered hyperimmune to SV40 tumor using SV40 target cells (Anderson and Coggin, 1972; Coggin et al., 1973). Brawn (1970) observed that extracts from fetal cells of the mouse would “desensitize” cytotoxic effector cells preventing their destructive action on 3-MCA tumors in uitro. In the rat system Baldwin et al. (1972) did not detect cross-blocking between several rat sarcomas and pregnant serum in vitro (Baldwin et al., 1974). Whitley (1972) in our laboratory has recently observed that multiparous hamster serum would enhance SV40 tumor cell growth in vitro and survival by 2- to 3-fold over primiparous serum. TABLE VIII TRANSFER OF RESISTANCE AGAINST SV40 TUMORS EMPLOYING PRIMIPAROUS AND MULTIPAROUS LYMPHNODECELLS”EMPLOYING THE h O P T I V E TR.4NSFER METHOD Percent hamsters with SV40 tumors Days post-challenge with lo4 SV40 tumor cellsb Effector cell source

10

50

100

Final 150

protection

90 55 30 0 0

39 67 77 100

~~

Virgins 10-Day pregnant multiparous Not pregnant multiparous 10-Day pregnant primiparous SV40 immune

0 0

0 0 0

50 20 10 0

80 55 30 0

0

0

One hundred effector cells per target cell. Cells (effector and target) incubated in vilro for 30 minutes at 37°C prior to injection into normal recipients, sc.

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

125

Primiparous serum, collected at term, was found to produce 302 “enhancement” (abrogation?) of SV40 tumor cell survival in the presence of cytotoxic, multiparous LNCs. Although considerable technical difficulty has been encountered in our laboratory in reliably measuring “blocking or abrogating” factors in pregnant hamster serum, sufficient data are available, similar to those just presented, to establish that increased impairment (abrogation) (see Coggin and Anderson, 1972; Anderson and Coggin, 1972b ) of sensitized LNC cytotoxicity against tumor cells carrying fetal antigens correlated with increased parity. In other quantitative studies using the microcytotoxicity test procedure to compare the relative cytotoxicity of LNCs from donors of increasing parity, we have observed that LNC cytotoxicity diminishes with increased numbers of pregnancies. Extensive washing of LNC‘s was necessary to obtain in vitro cytotoxicity of effector cells from multiparous compared to primiparous donors using SV40 tumor target cells. Challenge of female hamsters (sc) with SV40 tumor cells following zero through four pregnancies usually results in a graded enhancement of tumor appearance coordinate with increased pregnancy experience ( Winslow, 1972). Parmiani and Della Porta (1973) recently reported that immunization of female rodents with tumor causes significant alterations in neonate size and litter number and conception frequencies. These data promote some interesting speculation regarding immune reactions against fetus in pregnancy and against fetal antigens on tumor cells in cancer-bearing hosts. If it can be firmly established that serum from tumor-bearing animals and likewise serum from pregnant donors can abrogate the cytotoxic action of “specifically” sensitized effector cells from tumor immune or tumor-bearing donors against tumor or fetal target cells, several points become clear. The natural immunologic interactions associated with fetal “tolerance” where the fetus carries histologically dissimilar transplantation-type antigens and fetal or embryonic autoantigens would certainly be involved in tumor cell survival. Abrogation of tumor cell destruction by sensitized effector cells by factors (antibody, antigen, complexes of antigen and antibody) in the plasma of tumor-bearing animals and humans seems to be a general reality (see K. E. Hellstrijm et al., 1969; Sjogren et al., 1961). Ignoring for the moment that animal tumor cells may contain both TSTA‘s and fetal antigens and concentrating on the human “model,” where only cross-reacting antigens borne on histologically similar tumors have been reported, let us consider the immunologic implications of the maternofetal analogy to the host-cancer situation. Suppose, for the sake of discussion, that most cancers arise from carcinogen-activated ( chem-

126

J. H. COGGIN, JR. AND N. G . ANDERSON

ical, biological, or physical), retrogenic processes resulting from altered intrinsic gene programming. Wallach (1968) and others have proposed that specific membrane alterations within a cell could account for most, if not all, of the physiological and social cellular characteristics of neoplasia. Suppose further that these induced programming changes lead phenotypically to the aberrant synthesis of plasma membrnne in the transformed cell. Many rational models are conceivable for these molecular changes (discussed in a later section). We favor a simple model which suggests that the transforming adult cell experiences biosynthetic changes which result in “immature” membrane synthesis yielding tumor cell membranes with many features of membranes found in an earlier stage of normal fetal development. The invasive, migrating traits associated with fetal cells bearing such membranes (see Anderson and Coggin, 1971, 1972a) are conferred to the transformed adult cell, and malignant growth characteristics are established. Autoantigens are present at the tumor membrane as they were in the precursor fetal or embryonic cells which normally displayed these antigens. In normal fetal development expression of these antigens on immature membranes is highly regulated, and complete phasing is possible for at least some of these antigens within a few hours of gestation (Table VII). In tumor cells the regulatory control is defective (nonnormal) because of carcinogen-induced damage to the regulator genes, or structural genes, or heritable changes in translating mechanisms within the cell or resulting from the persistence of virus-coded (probably derived originally from intrinsic cellular DNA from the cells in which the virus genome originated in evolution ) regulators which repress ( or derepress ) normal, adult cellular DNA expression. The net result, if reprogramming is successful and leads to viable, functional tumor cells, is the production of autoantigenic, malignant cells. The essential role that fetal autoantigens once played in fetal survival as a “homograft” now serves a new master, the neoplastic cell. Immunologic impairment which occurs in pregnancy, especially after the second pregnancy, may play a parallel role in cancer cell survival. Multiple mechanisms of blocking seem imperative ( Coggin et al., 1974). Antibody appears to coat fetal cells, masking their imtigenicity (Girardi et al., 1973). Soluble fetal autoantigens from fetal tissues or tumor cells may desensitize the cytotoxic effector cells of the host (Brawn, 1970; Coggin and Anderson, 1972; Baldwin et al., 1972; Currie and Basham, 1972). Complexes of soluble antigen and antibody may further impair the immunity of the host to the developing tumor. These possibilities exist, and the data are accumulating which strongly support them. If true, the survival of a tumor cell may be a simplified caricature

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

127

of the immunology of reproduction (omitting here consideration of the trophoblast ) . We have reviewed rather extensively elsewhere the similarities between many properties of malignant cells and those of embryonic fetal cells (Anderson and Coggin, 1971, 19724. Data supporting this view for fetal survival were reviewed by Billington (1969).

5. Organizational Aspects

of Immunity to SV40 Tumors and Fetal Antigens

Hamster cells transformed by SV40 or other oncodnaviruses possess several antigenic sites (determinants) at the tumor cell membrane (see Butel et al., 1972). Each is identified by a distinct immunologic test, hence each has the possibility of being distinct from the others. The TSTA is identified by its ability to induce tumor immunity either to tumor transplant or to virus tumor formation. Surface or S antigen on the SV40 tumor cell is measured by indirect immunofluorescence and may or may not be distinct from TSTA; certainly this point is moot since neither test procedure is quantitative (Collins and Black, 1973). Animals rendered hyperimmune to SV40 tumor were observed to possess a specific immunoglobulin which selectively inhibited the growth of tumor target cells in diffusion chambers in viuo, and the term “cytostatic” or C antibody was coined to denote the functional nature of the antibody (Coggin and Ambrose, 1969; Ambrose et al., 1969). The antibody could be detected transiently in the circulation of hamsters developing autochthonous SV40 tumors until such time as the tumor mass became palpable (Ambrose et al., 1971a). At that time no C antibody could be detected. Surgical removal of the tumor resulting in tumor cure permitted a recrudescence of functional C antibody. Normal hamsters receiving a single, protective dose of X-irradiated SV40 tumor cells developed C antibody. It is important to remember that the C antibody is measured by a functional test, not by a binding antibody assay. The presence of this immunoglobulin (IgG) is an index of free, unbound circulating antibody. In summary, this antibody reflected or served as an index of the presence of tumor-specific immunity in immunized animals or surgically cured animals, and loss of the antibody preceded tumor appearance in SV40-infected animals under the conditions of test employed. In 1969, we observed the antibody to occur transiently during normal pregnancy (Table IX) and in male and female hamsters injected with X-irradiated syngeneic or xenogeneic fetus, which induced protection against SV40 tumors. Since C antibody seemed to be elicited to fetal antigens present on SV40 tumor cell surfaces, we followed the time course of development of C antibody and several other similar antibodies [ S

128

J. H. COGGIN, JR. AND N. G. ANDERSON

TABLE IX DETECTIONOF C ANTIBODY IN MULTIPAROUS FEMALE HAMSTERS FOLL~WINQ CONCEPTION Days post conceptionn

Cytostasisb

Virginse 9 14

-

18

19 20

+ + +

Chambers containing 15,000 viable SV40 tumor cells were placed into the peritoneal cavity 5 days before the indicated days below. "ercentage of cytostasis was determined by removing the chamber at 5 days implantation a t the indicated dates from 10 hamsters and determining the average number of viable cells in the pregnant hamsters or mothers. This figure WW compared with cell counts from chambers implanted simultaneously into virgin hitmsters and counted on the same sampling date to determine the percentage of cytosta'k. c Virgin hamsters were the same age as the multiparous animals, and cell counts over 5 days in these animals are identical to those obtained in male hosts (-550,000 viable cells).

antibody and antibody (IAT) detected by the radioimmuno assay (Ting et al., 1971)l reactive with antigen( s ) on the SV40 tumor cell surface during the time course of SV40 induced tumor formation in hamsters. Simultaneously, we tested the serum donors for the advent of cellmediated immune responses by three assay procedures. Typical results from the SV40-neonate hamster model are shown in Table X. As previously reported, C antibody could be detected prior to tumor appearance in virus-infected animals as long as they remained free of tumor ( Ambrose et al., 1971a). Tumor bearers do not have detectable C antibody. S or IAT antibody are not present in these animstls. S antibody is difficult to produce in the hamster, requiring multiple injections of inactivated and, later, living tumor cells. Similar methods are required to induce IAT antibody (6-10 injections with 5 million tumor cells). Hamsters rendered immune to a single injection of X-irradiated SV40 tumor develop C antibody by 10 days and coordinate transplantation resistance but fail to show IAT or S antibody using the procedure described by Ting et al. ( 1971) or Tevethia et al. ( 1968). Significantly, cytotoxic LNC's were detected in the pre-tumor-bearing and small tumor-bearing animals by 5 weeks using the inicrocytotoxicity procedure of Takasugi and Klein (1970). More difficulty was encountered using this in vitro test in detecting CMI as the tumors became larger, but washed effector cells were generally cytotoxic. Con-

129

CANCER, DIFFERENTIATION AM) EMBRYONIC ANTIGENS

TABLE X

COMP.4RATIVE

STUDY O F IMMUNOLOGIC RESPONSES AGAINST CELLS I N THE SV40-NEONATE MODEL

SV40 TARQET

Weeks post infection with SV40 Response”

0

5

10

15

20

25

Percent hamsters with SV40-induced sarcomas C ant,ibody S antibody IAT antibody Cytotoxic LNC’s Protective LNC’s Challenge immunity Blocking serum

0

0

5

30

65

100

f

f

+

a C antibody, cytostatic antibody; S antibody, surface antibody; IAT antibody, antibody detected by radioimmune test; cytdoxic LNC, in microcytotoxicity test using SV40 target cells; protective LNC, in adoptive transfer test; challenge immunity, concomitant immunity.

comitant resistance can be detected to graded tumor challenge doses. We are generally unable to obtain LNC‘s or exudate cells from animals with tumors which are able to confer long-term protection to adult normal recipients challenged simultaneously with SV40 tumor cells, although transient protection ( 2040%) was noted. Two points derive from these data. First, the decline of C antibody with the increase in tumor load suggests that a period of antigen excess occurs when tumor mass exceeds a certain critical size, Since the tumor is extremely localized initially, metastasizing much later, we can assume that the early diminution in antibody is the result of systemic antigen excess suggesting that fetal antigens and perhaps TSTA’s are shed from the tumor cells. Blocking activity (measured in the microcytotoxicity test; see Coggin and Anderson, 1972; Anderson et al., 1973) in the hamster serum was present coordinate with the decline in C antibody level. The decline in demonstrable cell mediated immunity in those tests where reactivity was initially present further establishes the growing ineffectiveness of the cellular response against the tumor coordinate with palpable tumor detection at the site of SV40 infection. Recognizing the possibility that serum factors from multiparous animals can abrogate SV40 tumor target cell destruction by tumor immune as well as multiparous effector cells, the analogies between fetal tolerance in utero and tumor cell survival “beg” for recognition. Several interesting differences should be noted, however. For example, in the

130

J. H.

COGGIN,

JR. AND N. G. ANDERSON

course of pregnancy we have not observed periods when C antibody declined to zero values suggesting that fetal antigen excess in the mother is never achieved in pregnancy. In opposition to the natural situation in pregnancy, antigen excess seems to be the status quo in tumor-bearing animals if C antibody disappearance reflects this condition as we suspect. In addition, we were always able to detect cytotoxic effector cells in the adoptive transfer test and in the microcytotoxicity test in pregnant animals regardless of the parity state albeit to a diminishing degree whereas neither test parameter yields highly cytotoxic or protective LNC or PEC (peritoneal exudate cells) in animals with growing tumors. What we point to here is degree of reactivity; in pregnancy and in the pretumorous or hyperimmune state, a condition of antibody excess seems to prevail whereas in tumor-bearing hamsters a condition of antigen excess and impaired effector cells is detected, befitting the biological tragedy that is occurring. If similar results can be obtained in other autochthonous animal models and in humans, a major advance in understanding cancer immunology will have been made. If fetal autoantigens are cross-reacting determinants on human cancer cells and can participate in cancer cell survival as described above, they are most important antigens. This seems to be the situation for human sarcomas (Mukherji and Hirshaut, 1973) .

B. TUMOR-ASSOCIATED EMBRYONIC ANTIGENSOR FACTORS The rapidly increasing number of different embryonic and fetal antigens found to be associated with tumors precludes an exhaustive review here. Emphasis is placed on those described in man. A few of the antigens are reported to be autoantigens; however, none described in this section have been demonstrated to be important in tumor rejection. 1. Fetal Serum Proteins The first reexpressed fetal antigen characteristically associated with cancer which was discovered was a-fetoprotein (Abelev et al., 1963) originally found associated with a mouse hepatoma. Since Tatarinov’s discovery of an analogous protein associated with human liapatomas (Tatarinov, 1964), an extensive search has been made to determine whether other tumors also exhibit the antigen, and whether more sensitive tests might show a higher number of positive instances (Abelev, 1971). The antigen has been found associated with human testicular and ovarian teratocarcinomas and with gastric cancer metastatic to the liver (Alpert et al., 1971). The percentage of human hepatoma cases found to be a-fetoprotein positive has varied with ethnic background ( Abelev,

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

131

1971) and with the sensitivity of the test used. When the serum a-fetoprotein was too low to be detected by immunodiffusion, the antigen could be detected in tumor cells using the indirect immunofluorescent technique (Nishioka et al., 1972). When a very sensitive radioimmunoassay was employed (Sells, 1973; Thomson et al., 1969), the percentage of human hepatoma patients found to be positive rose sharply, and low levels of antigen were found associated with other types of tumors. An azH-globulin, a ferroprotein, normally found in fetal organs and in fetal serum, has been found in 81%of children with tumors, using a radioimmunodiffusion method. Out of 122 sera obtained from children with benign diseases, only 8%were positive (Buffe et al., 1970). Edynak et al. (1970) reported an additional fetal serum protein which was present in a high percentage of cancer patients. In a very few instances, patients showed circulating antibodies against this antigen. In rats, an a,-glycoprotein ( Darcy’s antigen), present in embryonic sera, is found in the sera of all rats given sufficiently high doses of chemical carcinogens irrespective of the tissue alterations seen ( Stanislawski-Birenswajz et al., 1967). 2. Hemoglobin Many different fetal hemoglobins have been described in animals. In man the sequence of appearance of the predominant forms is from embryonic Gower 1 ( c 4 ) and Gower ( a z - c 2 ) through fetal ( a 2 - y 2 ) (through fetal cuz-yz) to the adult a2--p2 (Huehns d al., 1964). In leukemia and a few other hematological diseases, fetal hemoglobin may recur (Miller, 1969; Singer et al., 1951a,b). 3. Placental Antigens Seven major antigens have been identified in normal human placenta which do not occur in serum from normal nonpregnant adults (Hofmann et al., 1969). Five of these have been examined in greater detail (Hofmann et al., 1969), and three have been shown to occur in the serum of pregnant women. One of these is probably identical with the placental antigen reported by Tal to be found in pregnancy serum and in the serum of a wide variety of cancer patients (Tal et al., 1964; Tal, 1965, 1972; Tal and Halperin, 1970; Tal et al., 1971) . As purified monospecific antibodies against a spectrum of placental antigens become available and are employed to radioimmunoassays, it is not unlikely that additional instances of reexpression of placental antigens will be found. The antigens mentioned in this section do not include known placental hormones, but may include the placental alkaline phosphate described in a subsequent section.

132

J. H. COGGIN, JR. AND N. G. ANDERSON

4. Antigens of the Gut The carcinoembryonic antigen( s ) (CEA) found in human fetal gut (Gold and Freedman, 1965) and in a high percentage of human colon cancers (Gold, 1967; Moore et al., 1971; Zamcheck et al., 1972) has now been found to recur in a wide variety of tumors of both endodermal and nonendodermal origin ( LeGerfo et al., 1971). Very low levels of this antigen are found in normal human serum (Chu et al., 1972). At least six different molecular species are found in CEA as currently used ( Eveleigh, 1972). Although such findings diminish the usefulness of the test as presently employed, it does raise the possibility of greater specificity when more highly purified reagents become available. Two additional antigens of the gastrointestinal tract have been described; one of these, called the “gastric antigen,” persists throughout adult life (de Boer et al., 1969). The second, or “intestinal antigen,” while present in both stomach and intestine in the fetus disappears from the stomach soon after birth. In gastric neoplasia, metaplasia, and senescence, it recurs in gastric mucosal cells while the level of the “gastric” antigen decreases ( Nairn et al., 1962). A fetal sulfoglycoprotein apparently distinct from the “intestinal” antigen has been described by Hakkinen et al. (1968a,b) and Hakkinen and Vikari ( 1969). It occurs in the fetal alimentary tract, recurs in gastric juice and tissues in gastric cancer and in some nonmalignant diseases of the stomach.

5 . Leukemia and Hodgkins Disease Antigens associated with human leukemia cells which cross-react with antigens found in the fetus have been described by Viza (1971), and an antigen found associated with Hodgkin’s tissue is also found in fetal cells (Katz et al., 1973). 6. Other Antigens Some of the antibodies present in Burkitt tumor convalescent sera and in convalescent infectious mononucleosis sera are absorbed by fetal cells (Harris et al., 1971; Harris and Harrell, 1972). Fetal antigen( s ) from mouse tissues were reported by Stonehill et al. (Stonehill and Bendich, 1970) to cross react with a variety of mouse tumors and with adult skin. Comparable results with human fetus have been recently reported (Klavins et al., 1971; Mesa-Tejada et al., 1971). A variety of other tumor antigens have been described which cross react with antigens present on fetal cells and are either absent from adult tissues or present at trace levels; they will not be reviewed in detail here.

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

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C. ISOZYMES IN TUMORS The tendency for tumors to express the isozyme patterns and the total enzyme patterns of fetal tissues has been discovered and rediscovered in many different laboratories (see review by Criss, 1971, listing 425 references; Weinhouse, 1972; Potter, 1969; Knox, 1972), and the number of enzymes observed to be similarly involved is increasing rapidly. Of greatest interest here are isozymes that are characteristic of embryonic or extraembryonic tissues and are not normally found in the adult except in pregnancy. A heat-stable alkaline phosphatase (the socalled Regan isozyme) noimally found in the placenta recurs in a small percentage of cancer patient sera or tumor tissues (Fishman et al., 1968a,b). Diamine oxidase levels rise rapidly during pregnancy increasing by a factor of approximately 100 during the first 8-9 weeks of pregnancy (Weingold and Southern, 1968). Since the enzyme is not present in patients with trophoblastic tumors, these authors propose that high concentrations of histamine in the fetus induce the enzyme in the mother. It will be of interest to determine whether this enzyme is ever associated with cancer. IN TUMORS D. HORMONES

1. Ectopic Synthesis of Hormones Normally Present in the Adult Many tumors of endocrine glands continue to produce hormones or to respond to hormone stimulation, demonstrating that these functions are compatible with malignancy. Many instances are also known in which tumors produce hormones not characteristic of the cell or tissue of origin but which are indistinguishable from the normal adult hormone. These include ACTH, PTH, arginine vasopressin, erythropoietin, gastrin, gonadotropins, thyrotropin ( Omenn, 1970). 2. Placental Hormones The growth hormone of the placenta, human placental lactogen or chorionic somatomammotropin, was found in 9%of 128 cancer patients with nontrophoblastic cancers ( Weintraub and Rosen, 1971). Chorionic gonadotropin production also occurs in a variety of tumors including bronchiogenic carcinoma ( Rosen et al., 1968), esophageal cancer ( McKechnie and Fechner, 1971) , tumors of the kidney ( Castleman et al., 1972), primary bladder cancer in a male (Ainsworth and Gresham, 1960), a chorionepithelioma of the stomach ( Regan and Cremin, 1960), in tumors of the pineal and pituitary (Edmonds and Cerrera, 1%),

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mediastinum (Bennington et al., 1964), thymus (Jernstrom and McLaughlin, 1962), ovaries and testis (Rubin, 1970), and breast and in melanomas ( McArthur, 1963). Tumors have long been known to produce a factor which stimulates the host to provide a blood supply (Greenblatt and Shubik, 1968). This angiogenesis factor (TAF) appears to be crucial to the growth of many tumors and is also found in the placenta ( Folkman, 1972). As other characteristic placental hormones are described, it is important that these be sought in a wide variety of tumors. 3. Evidence for Additional Factors

A variety of effects of tumors on the host remain to be explained, including ill-defined changes in host metabolism, growth of hair, pigmentation, taste, and affect. These have been described and are included with the effects of ectopic hormone production under the general heading of paraneoplastic syndromes (see symposium edited by Hall, 1974). Tumor-bearing animals exhibit a higher rate of turnover of DNA in some nontumor tissue than do control age- and sex-matched animals (Griffin, 1957; Kelly et al., 1951; Stewart and Begg, 1953), while other tissues show a decrease in proliferation and DNA turnover (Morgan and Cameron, 1973). These results suggest that a number of as yet undiscovered factors may be produced by tumor cells which affect host tissues. E. SURFACECHANGES IN TUMOR CELLS While many intracellular changes have been described in tumor cells, the concept persists that the most important alterations accompanying transformation occur at the cell surface and are concerned with cell-cell interactions and with invasiveness. Since, during embryogenesis, many cells disassociate themselves from surrounding cells and migrate through tissues to find a new position the question arise$ whether tumor cell surfaces resemble those of fetal cells in this important respect.

1. Lectin Binding Plant lectins resemble antibodies to blood group substances in possessing specific sites binding to carbohydrates. A variety of neoplastic cells have been shown to bind lectins such as concanavalin A or wheat germ agglutinin to a much greater extent than do normal adult cells and to then exhibit a more normal growth pattern in culture ( Burger and Noonan, 1970). Concanavalin A will agglutinate transformed and embryonic cells, but not untransformed adult cells. Wheat germ agglutinin behaves similarly with respect to normal and trans-

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formed cells, but proteolytic treatment of embryonic cells is required to uncover the binding sites on embryonic cells ( Moscona, 1971).

2. Electrophoretic Mobility Rabbit antiserum against rat fetal liver, after absorption with adult rat liver cells, reacted with fetal liver cells to decrease their electrophoretic mobility by 51%and rat hepatoma cells by 45%,indicating that a cross-reactive antigen was present (Woo and Cater, 1972). These authors also showed that €eta1 liver and hepatoma cells had higher electrophoretic mobilities before treatment than normal adult cells.

F. MISCELLANEOUS CHANGES 1. Radiogal lium Uptake Radiogallium localizes in some human neoplasms to an extent which is useful for tumor localization (Edwards and Hayes, 1972). The mechanisms of this localization are unknown. However, the observation that this element also localizes in embryonic tissues (Otten et al., 1973) suggests that the mechanisms involved may be common to embryonic and tumor cells. 2. tRNA Embryos exhibit tRNA’s which differ from those found in the adult. Some of these have been demonstrated to occur in tumors (Yang, 1971) and may be the source of methylated bases observed in the urines of many cancer patients ( Waalkes et al., 1973). 3. Putrescine, Spermine, and Spermidine Polyamines generally occur in higher concentration in rapidly dividing cells, in embryonic tissues (Heby and Lewan, 1971; Raina et al., 1970; Russell et al., 1970), and in association with cancer cells (Russell and Snyder, 1968; Williams-Ashman et al., 1972). The function of these substances in such tissues may be to condense chromatin during mitosis ( Davidson and Anderson, 1960).

G. CONCLUSIONS The evidence presented thus far suggests that a variety of alterations are associated with neoplastic transformation and raises the question of whether these are totally random changes, are selected from a wide selection of possible random changes, or represent the end result of a large variety of different accidents to a complicated program or schedule of differentiation containing many homeostatic mechanisms. Should the

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latter be true, then there is hope that the program can be deciphered to an extent that will allow the causal lesions to be localized. It is important therefore to examine present concepts of differentiation and to ask whether any evidence of the program persists in the neoplastic cell. As noted certain specific genes appear to be activated with high frequency in human embryonic or fetal cells-including the genes concerned with either the production of CEA or the enzymes synthesizing it, with a-fetoprotein, with placental alkaline phosphatase, with one or more pregnancy associated antigens, and with a variety of other enzymes, antigens, and tRNA's. In contrast, there appears to be a rather large library of embryonic or fetal antigens which may be reexpressed in tumors, as demonstrated in a previous section. The probability that a given gene will be reexpressed in a tumor by a purely random process depends on the total number of genes present and on the frequency of the random or triggering event. The fact that some genes are reexpressed with a high frequency and others almost never in tumor cells argues strongly for the view that cancer is a disease of differentiation itself as stressed by Markert (1968) and that large segments of the differentiation program are still functional in tumor cells, and that some portions of the program are characteristically activated in cancer. Ill. The Organization of Differentiation

In this section we are concerned with the organization of differentiation from a descriptive viewpoint and with the question of whether remnants of differentiation so described persist in tumor cells. This is a part of a larger question of whether gene expression in cancer is a completely random, or whether the complex, differing and changing patterns seen during tumor progression in man are understandable as specific pathologies superimposed on a highly ordered system. It is in theory possible to inventory the cellular proteins of each of the approximately 100 different cell types found in the human adult and to do similar inventories (though at a much greater expenditure of effort) of embryonic and extraembryonic tissues even back to eggs and sperm. With such information, the order of appearance and disappearance of gene products, and hence a description of the differentiation program itself, could be written in detail. There is little likelihood that it will be attempted in a systematic manner in the near future. Yet many basic questions concerning human cancer cannot be answered in the absence of much more detailed information concerning gene programming. If a simple analogy may be employed, the launch program of a space vehicle represents an elaborate schedule involving many inputs accepted at specific times, many orders to execute tasks, :md built-in

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decision points where the entire operation may be stopped and the program reset to an earlier time and, if necessary, held at that point to await some new signal. It is conceivable that the program may be desynchronized with part of the program reset to one time (or condition), while another section may proceed to or be held at another time, thus disjoining the entire operation. A very detailed examination of the program may be required to show where the errors were, and it may be extremely difficult to deduce the nature of errors from the havoc which may ensue because the possibilities for such errors, accidents, malfunctions, or failures are legion. We stress the importance of the simple question: Does all evidence of the program vanish when malfunctions occur? With space vehicles the answer is almost always no. Rather the great variety of malfunctions possible is a direct reflection on the complexity of the program and of the total system. With respect to cancer and differentiation, we ask whether the same conclusion holds. Will cancer cells ultimately be understandable in terms of one or more definable errors or perturbations whose results could be predicted if the program and the rules governing it were known in detail? The experimental data and theoretical concepts available in this area are meager. Nevertheless they are briefly reviewed here because they bring into focus the types of information that may be required to understand cancer. A. GENERAL RULESOF DIFFERENTIATION In early embryogenesis, structure and order at the tissue and organ levels appear as a gradually unfolding process proceeding from simple structures to complex ones. At the molecular level many gene products appear which are unique to, and characteristic of, differentiated cells. It might be thought that differentiation therefore involves the gradual activation of an increasing number of genes until all those characteristic of adult cells are active. These “differentiation” activities or properties might be superimposed on a “minimal cell” exhibiting only those enzymes and functions required for bare survival, namely a stripped down undifferentiated cell. It has been suggested that cancer cells may approach such a condition. The view of differentiation as a gradual restriction in competence associated with the appearance of differentiationassociated molecules results directly from the fact that the majority of biochemical studies have looked at the appearance of end products and have not sought substances that may be present transiently (Moog, 1965; Rutter et al., 1964; Gross, 1968). Experimentally, a surprising number of adult proteins have quite different fetal counterparts, which form part of an array of “phase-

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specific” substances that appear transiently in different tissues, organ anlage, and in plasma at different stages during development (see review by Holleman and Palmer, 1972). It appears, therefore, that development involves selective expression of genes in a carefully controlled sequence. (Discussion of the number of genes which may be involved and concepts of their control at the molecular level are reserved for Section VI.) The more general principles are presented here as follows. 1. Genes Are Turned On and Ofl in Sets This conclusion is based on both theory and observation. From the theoretical viewpoint, if each of a very large number of genes is controlled individually ( i.e., there are no signals controlling, activating, or repressing more than one gene), then an integrated differentiation program is difficult if not impossible to model in terms of control theory, because no internal systems convergence exists. Experimentally many genes are known which appear to be always coexpressed. These include the subunits of many proteins (Klotz and Darnall, 1969) and the crystallins of adult lens. While gene sets usually include two or more genes, the sets may contain only one gene. Inclusion rules define set members, and may have a physical basis in either polycistronic mRNA’s or multiple gene activation by a single derepressor or activator.

2. Gene Sets Are Nonexclzcsive A given gene may appear in more than one gene set. 3. Exclusion Rules Exclusion rules exist forbidding the cotemporal expression of certain gene sets. Exclusion rules are suggested (but not proved) by the many instances where two proteins or cell products characteristic of fully different cells have not been found together such as hemoglobin and melanin, myosin and a-crystallin. Greater suppoit for the concept is provided by the instances of preprogrammed cell death occurring during embryogenesis which may be due to the deleterious effects of coexpression of certain gene sets. In the simplest case of exclusion, gene Set A cannot be active in the presence of an active gene Set C producing a repressor for Set A.

4. Sequencing Rules A hypothetical gene set A may yield a set of gene products including an inducer (derepressor) for gene Set B. Gene Set B, when active, may result in the expression of gene Set C, one of whose prodiicts is a re-

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pressor for Set A. If Set C also contains a gene for self inducer to hold Set C on, then the observed sequence of events will be as follows. The products of gene Set A will appear, followed by those of B, then of C. This in turn will be followed by a decline in Set A products, a subsequent decline in Set B products, but continued expression of Set C. Such interlinked gene sets are termed chains.

5. “And Gates” Link Chains of Gene Sets This concept merely states that some events require that more than one prior event occur. For example Wolffian regeneration of lens from iris tissue in tissue culture requires both the presence of lung and a retinal tissue in addition to iris tissue, hence at least two different signals appear to be involved. Another example is the requirement for both a hormone receptor and a hormone for a hormone-specific reaction to occur.

6 . Unindirectionality Gene sets may be organized for sequential activation in only one direction, and orderly dedifferentiation in which each step in the differentiation process is reversed may not be possible. For purposes of regeneration and repair, however, the capacity for limited dedifferentiation may be preprogrammed into some cells.

B. INDUCERS AND CAPACITATION During embryogenesis a tissue may respond to a nonspecific stimulus with a specific and complex response at one time and be refractory to the same stimulus at another. This effect is best described as being a property of an “and gate,” i.e., of a decision making system requiring more than one input to give a response. If three chemical signals are required, for example, no effect may be observed from the first two until addition of a third produces a response. The third, in the absence of the first two, produces no effect. The differential effect of simple evocators on different times is thus best described in terms of “and gates.”

C. COMPLEXITY OF THE PROGRAM Without a detailed molecular mapping of human development, it is difficult to obtain a notion of the program complexity or of the redundancy which may have been built into it. However, the fact of the production of a large number of intergrades serially during development with few if any sudden compositional changes argues for both a very complex program and for the possibility of a very large number of different patterns of gene expression compatible with life. The reason for

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interest in program complexity here is this: many additional patterns of gene expression are possible in addition to those observed. But many can be postulated which are in fact not observed. Is the program of development sufficiently complex to contain internal barriers to different patterns of gene expression, that is, to the formation of new phenotypes from existing genomes?

D. REVERSIBILITY OF DIFFERENTIATION AND METAPLASIA One way to minimize the possibility of forming neophenotypes would be to make differentiation totally unindirectional. However, in response to injury, many tissues and cell types will resume activities that appear to involve dedifferentiation; for example, cells may round up, divide, move, and finally redifferentiate back to the original state, Could this in fact be due to a transdifferentiation instead, to progression to a new state followed by return to the differentiated one? The fact of metaplasia argues for true dedifferentiation and redifferentiation along a new line. The point here is that retrograde movement along differentiation pathways, albeit to a limited extent, is possible, and even if transdifferentiation is invoked, return to the original differentiated state would then involve a retrograde movement. It appears that a large number of “stops” exist in the program of development which limit the extent to which a cell can retrace its steps to an earlier state. This in turn suggests that damage to the differentiation control mechanisms could produce a very large number of different phenotypes depending on where in the program the injury occurred. We have reviewed evidence for the reactivation of embryonic or fetal genes in cancer cells, and have presented evidence for these genes as a source of tumor-associated antigens, hormones, isozymes, tRNA’s, and other substances, and have noted that normal genes could be programmed in suicidal or malignant arrays. We have thus far avoided the central question of the ultimate origin of the large number of differences observed between fetal and adult cells at the molecular level and for much of molecular phasing. One may invoke special fetal requirements for these differences, for example fetal hemoglobin is better suited to supplying oxygen to the fetus from the placenta than is adult hemoglobin. Similar reasoning is difficult to apply, to all the other embryounique proteins, especially autoantigens. AND EMBRYOGENESIS E. MOLECULAR EVOLUTION Evolution selects advantageous phenotypes. Adult cells of different species would, therefore, be expected to exhibit compositional differences, the proteins to have sequence differences, and orgims to have

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different morphologies. In contrast, as has long been stressed by embryologists, the events of very early embryogenesis have common features that persist throughout the entire animal kingdom, and the embryos of different species in the same class or order are almost indistinguishable until relatively late in development. Since, as has been discussed, many of the proteins involved are transient and are characteristic of the embryo or fetus, the question arises, what factors exist to alter these transient proteins when their function has not changed; and when few selective pressures are brought to bear on them? If ontogeny tends to repeat phylogeny at the molecular level, then the embryo may resemble in sequence many different ancestral species. We have stressed that malignant cells reexpress embryonic and fetal genes, and that the embryo appears to possess all the constituents and characteristics necessary for malignancy. The possibility of accidental reexpression of these genes would appear to be a universal threat extending over the entire animal kingdom.

F. PHASE-SPECIFIC SUBSTANCES The available information on phase-specific substances during development has been recently reviewed, as previously noted, by Holleman and Palmer (1972). Many examples of these substances exist in addition to those cited as recurring in cancer in a previous section. We return repeatedly to the question of the existence, number, and autoantigenicity of transient gene products of early development. This is in part because most biochemical studies have been concerned with the time of appearance and level of known substances almost always characteristic of one or more differentiated cells. Technically there has been little choice, since few substances were known which were characteristic of only one stage in development, largely because only very small amounts of organ anlagen could be obtained, and because assays for these materials were not widely available. Hence, the type of data that could be obtained unavoidably led to the impression that differentiation consisted almost entirely in a scheduled turning on of genes characteristic of fully differentiated cells. In many cases the substances examined have been enzymes which are part of the base set of substances present in all nucleated cells of a species. Approximately 1000 enzymes are known, and the number of genes in the mammalian genome is thought to be approximately 3 orders of magnitude larger. It would appear therefore, that the sampling of molecular species during development has been vanishingly small relative to what may be there. In addition the terms “differentiated” and “undifferentiated” as used in pathology refer to visible structures that may not correctly reflect

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the compositional complexity of cells. Morphogenetic move ments associated with early development may require very complex changes in membrane composition and structure which are not apparent in the light or electron microscope. One method of approaching the problem of phase-specific substances is to prepare antisera against extracts from early embryos or from various fetal organs, and then absorb these with extracts of adult tissues, serum, or with extracts from embryos of stages other than those used to prepare the original antiserum. When this is done, phase- or stage-specific antigens may be demonstrated during embryonic development of Ram temporaria ( Romanovsky, 1964a,b), R a m pipiens (Spar, 1953), and Triturus pyrrhogaster (Inoue, 1961). If differentiation is controlled by a large spectrum of phase specific substances, thcn the likelihood of isolating any given one from embryonic materials may be inversely proportional to the number present. For this reason the recurrence of these substances in tumors which may be grown in mass in tissue culture offers the best hope for their ultimate isolation. While embryology has much to contribute to oncology, fractionation of tumors may yield key factors of central importance in understanding development. Not only enzymes and antigens vary during development. An extract from early frog embryo strongly inhibits amino acid incorporation by a cell-free extract of adult frog liver ( Strittmatter, 1968) demonstrating that enzyme inhibitors may be present as well.

G. SURVEILLANCE Cancer has been reported for nearly all animal phyla ( Huxley, 1958). Evidence for a cell-mediated immune response has been presented for many invertebrates and is thought to represent the most ancient form of immunity. Transplantation of many embryonic cells into adult tissue has been shown to give rise to embryomas that may exhibit invasiveness and be fatal. It has been long postulated that cell-mediatcd immunity originated to recognize and destroy malignant cells as rapidly as they appeared. We have carried this concept one step further and proposed that surveillance may be directed at cells reexpressing embryonic genes associated with malignancy. We term the mechanisms whereby only adult characters are allowed as enforcement mechanisms and the possibility that such mechanisms exist prompts a reexamination of maternalembryo relations over the whole animal kingdom.

H. MATERNALEMBRYO SEPARATION The yolked egg and barriers to maternal-embryo interactions in the form of shells, jelly coats, or physical externalization of eggs, appear to

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have been accepted without question by generations of biologists as the inevitable consequence of sexuality. However, in some of the first bisexual organisms, such as hydra, the embryo develops embedded and protected in maternal tissues, a mechanism any efficient designer would, in our view, have adopted more widely. However, this is not the case, and a recent careful review of maternal-embryonic interactions in all animal phyla suggests that such interactions are rare indeed and either transient or subject to special barriers (Anderson and Coggin, 1972a; Lynn and Anderson, unpublished studies ) , This original generation gap may have persisted for more than one reason. Not only transient autoantigens, but also diffusible hormones are different in fetal and adult tissues, and the interchange of these might be deleterious. This simple concept, that embryogenesis involves the transient expression of archaic or antique genes, that the modification of these to be compatible with the rapidly evolving adult phenotype is infeasible, that the solution to the problem has been physical separation, and that the immune system developed for surveillance and enforcement, brings two problems into sharp focus. The first is whether cancer be more closely related to the process of differentiation itself and to specific damage to specific portions of the differentiation program. The second problem, which cuts to the heart of tumor immunology, relates to how placental animals cope with both embryonic and fetal development on one hand, and retrogenesis in potentially malignant cells, on the other. One must be allowed, the other recognized and reacted to. In cancer does one in fact fight his own genes? IV. Differentiation and Cancer

A unitary theory of cancer is one in which activation of one or a few genes (oncogenic virus genes or oncogenes) might be expected to result in the formation of a demonstrable gene product found in many different tumors. Should the gene product produce its effect by genomic interaction, then some specificity in the resultant pattern of gene expression could be observed. This is the case with many tumors of inbred animals produced by oncogenic viruses. The observed pattern may be dependent in whole or in part on the pattern existing before infection, resulting in different tumors produced in different organs or tissues by one type of virus. It would be predicted that receptivity to oncogenic virus transformation would be highly susceptible to small genetic differences, and that experimental animals could be selectively bred for high sensitivity to oncogenic virus infection, as appears to be the case. Recent results, showing that tumor sensitivity to oncogenic virus tumor formation may be bred out of high incidence strains, are therefore hardly unexpected.

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In an outbred diploid animal two slightly different copies of the differentiation program exist ( two complete copies in females, two copies differing by the differences in x and y chromosomes in males). This nonidentical redundancy may serve as a natural barrier to viral oncogenesis, while identical redundancy, as occurs in inbred animals, may serve to promote it. If such observations are confirmed, this may explain why virally produced tumors are seen in highly inbred laboratory animals and in domesticated animals, but are more rarely seen in outbred strains. In marked contrast to tumors produced by unitary transitional events, a large fraction of human solid tumors involved progression through a series of premalignant stages, often including quite large tissue areas. In addition, a great number of variations in growth rate, morphology, and malignancy are observed, giving rise to great individuality. Thus, if the number of initial or triggering changes were restricted to only a few genes, the end result would involve changes in a very large number of them, with such variable results that cancer has been considered to involve uncontrolled random gene expression. The opposite view is presented here, namely, that the program of differentiation is so complex that hundreds if not thousands of programming errors causing massive changes in the pattern could occur. Should this be true, then a large number of so-called genetic cancers might be possible, but may be difficult to observe because the mutations involved may be lethal to the cell in which they occur.

A. EMEG~YOMAS AND TERATOMAS Tumors have been described in tissues at all stages of development, most of which retain some residual characteristic of the stage of development of the cell origin, and none of which appear (to the knowledge of these reviewers) to contain gene products from a later stage of development. The phrase “oncogeny is blocked ontogeny” ( Potter, 1969) would appear therefore to apply best to embryonic tumors. Experimentally it is difficult to demonstrate that an embryonic tum0.r is blocked at a specific point in the program since we do not have detailed information on the total profile of gene products appearing in development. Teratomas may provide the requisite model for stage 0:‘ pathwayspecific blockage. Transplantable teratomas continue to prodiice a number of differentiated cell types and also tumor stem cells which continue to give rise to these differentiated cells as well as more stem cdls (Kleinsmith and Pierce, 1964). During differentiation, division points must exist where one cell gives rise to two daughter cells which ultimately differentiate along different pathways, i.e., express different parts of the

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differentiation program. If the possibility of differentiation along one pathway is blocked while development along another remains open, then a single division could give rise to one blocked and one unblocked cell as is illustrated in Fig. 1 (Anderson and Coggin, 1971). This appears to be the case with teratomas, which cannot be caused to breed true and to give rise to only one differentiated cell type, but rather continue to generate stem cells and many differentiated tissues. Embryomas produced by transplanting embryonic tissues into adults have long been studied by embryologists, and many of them have been reported to be malignant. From the viewpoint of this review embryomas are important because ( a ) as shown in a previous section, they may produce concomitant and sinecomitant immunity to tumor challenge, ( b ) they, like most tumors, require that a large number of cells be implanted to produce growth, ( c ) inoculation of a subcritical number of embryonic cells immunizes against embryoma formation by cell challenge doses which normally give rise to embryomas, and ( d ) the presence of a growing embryoma may also enhance growth of transplantable tumors (Castro et al., 1973). If, as postulated, early embryonic cells are “foreign” to themselves at a later stage in development and possess special mechanisms to escape immune destruction, then it might be

FIG. 1. Schematic diagram of blockage of one developmental pathway while leaving an alternate one open. Division of the teratoma stem cell (TSC), which would normally give rise to cell types A and B, yields instead B and another TSC. Cell B continues to develop along normal pathways and gives rise to cell types C and D, which in turn differentiate into cell types E and F and G and H. Thus a continuing supply of both differentiated cells (E-H) and teratoma stem cells is provided.

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thought that early embryonic tissue could be transplanted across species barriers more easily than could tissues from later stages. As noted by Green (1955), the interval of transplantability extends through the first third to half of pregnancy. The mechanism by which transplantation of embryonic tissues into an adult environment produces embryomas that may become malignant may be by disordering the program of development without damage to the genome itself (Braun, 1969). The disordering may be due to failure to receive proper signals at times when the cells were competent to receive them. It is of special interest therefore, that when tissues from embryos produced from aged eggs are transplanted into adult frogs, malignant growths are more often observed than when tissues from normal embryos are used (Allison, 1955).

B. RETAOGENESIS AND RESIDUESOF

THE

DIFFERENTIATIVE PRC~GRAMS

The expression of embryonic genes in cancer may be due to ( a ) blocking of development at an early stage to freeze a cell :it a certain stage of development or ( b ) reactivation of genes of early development resulting in collapse of part of the program. These ideas are not in conflict and apply to tumors occurring at different stages of development. However, the end results may be quite similar. If, as proposed, development involves interconnected arrays of events which proceed for the most part in one direction, then the uncontrolled reactivation of a gene concerned with the control of early stages of development may result in a wide variety of changes which could fall into three major classes which are: First, repression of gene:; characteristic of the cell of origin resulting in the loss of an enzyme or antigen. This would occur when the activity of these genes depends in some direct or indirect manner on the absence of the products of the embryonic gene. Second, the reexpressed embryonic gene may activate genes characteristic of precursors of the cell of origin. Thus cells differentiating from the endoderm may give rise to tumors which exhibit embryonic germ layer-characteristic antigens which have disappeared from adult tissues. Third, the reexpressed embryonic gene may be related to development along pathways quite different from that traversed by the precursors of the cell of origin. The result, therefore, may be the activation of genes characteristic of extraembryonic tissues such as the placenta, or of antigens or hormones characteristic of tissues derived from the same or even a different germ layer. The fact that a very large number of different types of tumors occur

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in man, with an enormous number of variations of many of them, and with gradual changes in the appearance, composition, and behavior with time in many instances argues strongly for the conclusion that the human genome can be activated in many different ways to produce new viable phenotypes not found normally during development. While there may be many suicidal combinations of genes, and very complex and effective means for preventing the appearance of nonprogrammed phenotypes, we must conclude that a large number of otherwise viable phenotypes are excluded normally, not by deleterious interactions among structural gene products, but by the operation of the intact program. C. NEW APPROACHESTO TUMOR CLASSIFICATION These considerations raise the possibility of a new approach to human tumor classification based on specific molecular deletions, on molecules associated with linear retrogenesis, and with molecular metaplasia. Sherbet ( 1974) has recently attempted such a classification and divides neoplasms associated with ectopic endocrinopathies into three major groups as follows: Group I. Subgroup A: Neoplasms which have acquired a preceding competence as evidenced by syndromes of gynecomastia, hyponatremia, hypercalcemia, and adrenocorticism associated with thymic and bronchial carcinoma. All the endocrine organs that normally produce the hormones involved originate from the bronchial clefts; these in turn arise from the bronchial endoderm. Subgroup B: Neoplasms which have acquired what Sherbet terms a precessive competence; i.e., they have undergone more than one stage in de-determination. These may include hepatomas producing hypoglycemia, pancreatic carcinomas associated with hyperadrenocorticism, and hypercalcemia or gynecomastia produced by primary hepatomas. Group ZZ. These tumors have nothing in common except that dedifferentiation may be more extensive than in Group I. Examples include the formation of erythropoietin by cerebellar haemangioblastomas, uterine fibroids, benign renal lesions, and renal carcinomas. Group ZZZ. This group includes instances in which tumors derived from one germ layer produce a hormone characteristic of an endocrine organ delivered from another germ layer. Examples are adrenocortical tumors causing hypoglycemia or gynecomastia, or breast carcinomas producing parathyroid hormone. In an exhaustive review of ectopic hormone syndromes in cancer, Metz (1972) has also presented a tumor classification based on embryological relationships. These efforts open up new approaches to tumor classification; however, they are based on observation of a limited class

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of substances, namely, hormones. If, as now appears likely, one or more placental genes are reactivated in most tumors, then classification based on gene derepression will turn out to be much more complex and will have to be written in terms of the total program of differentiation. The point stressed here is that retrogenesis is already of practical clinical importance in understanding some aspects of host reactioc to cancer as evidenced by paraneoplastic syndromes.

D. MUTAGENESIS,TERATOGENESIS, AND ONCOGENESIS The view that cancer is a disease of the mechanisms of difl'erentiation is strongly supported by the close relationships between teratogenesis and oncogenesis. That the increased incidence of cancer accompanies congenital malformations is well known, and the increased incidence of leukemia in patients with Down's syndrome for example, is 20 times higher than in the general population (Gunz and Fitzgerald, 1964). Chemical carcinogens are nearly always teratogenic, and teratogens are nearly always carcinogenic. In the wide range of compounds listed by DiPaolo and Kotin (1966), only ethionine was listed as being oncogenic, but not teratogenic. This result is quite surprising in view of the fact that many carcinogens appear to act locally at the site of implantation and may be metabolized, or not transported, so that appreciable concentrations are not seen elsewhere. Teratogenicity is usually assayed as a systemic effect, and the drug in question must therefore pass into the maternal circulation, traverse the placenta, enter the fetal circulation, and finally reach the site of action. Mutagens appear to invariably cause cancer and developmental defects; for example, ionizing radiation uniformly produces mdformation and cancer (Upton, 1968); however, a large fraction of all known teratogens and oncogens have not been uniformly shown to produce mutations. Mutagenicity is tested in a variety of ways, but the end result is usually a structural alteration in a protein. The possibility therefore, exists that apparently nonmutagenic teratogens or carcinogens act either by producing mutations in genes concerned with the production of nontranslated RNA (rRNA, tRNA, etc.) or by interfering with gene regulation or control without affecting base sequence in DNA. Evidence for the former possibility is provided by experiments in Dnxophila in which mutations of loci involved in the transcription of ribosomal RNA were seen in the form of bobbed mutations as measured by the effect on the heterochromatic X-nuclear organizer region (Fahmy and Fahmy, 1970). Four benzanthracene derivatives (two carcinogenic and two noncarcinogenic) were used; all proved virtually inactive with respect to the induction of point mutations, visible sex-linked and autosomal recessives and lethals. Chromosome breaks leading to major rearrange-

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ments including X-fragments that were viable were not seen, and specific mutability tests on loci within X-chromosome euchromatin showed all compounds to be inactive. Only the carcinogenic derivatives produced the mRNA-associated mutations. The authors concluded that the results provide strong evidence in favor of a selective somatic mutation theory of cancer, with different compounds tending to act selectively on specific loci, and that the loci affected by some carcinogens involve the transcription of specific categories of RNA. If a special class of genes exists to receive either intra- or extranuclear signals, such as the receptor genes proposed by Britten and Davidson (1969), and if the number of such genes were very large, and if many of the signals are in the form of unique chemical substances, and if the receptor genes are key controlling elements in differentiation, then it would not be unexpected to find a large number of compounds that interfered with such genes. It would also be likely that few common denominator properties would be apparent in lists of such compounds since the substances reacted with might include the postulated intranuclear activator RNA, allosteric proteins binding hormones, or a wide range of effector substances, or small signal molecules themselves. That an enormous range of substances and physical agents could influence the very complex mechanisms involved in differentiation is not difficult to accept. That a similarly large range of substances should cause cancer strongly argues that cancer can be produced by functional errors occurring at very many different points in the differentiation program, and that these compounds are not all managing to activate one or a few specific oncogenes whose function is necessary for cancer to occur. That virus infection produces marked effects on the pattern of gene expression is well documented; infection of human embryos with rubella, cytomegalovirus, or herpes hominis produces a variety of malformations (Sever, 1971). It would be of interest to examine the effects of infection of early embryonic cells with both oncogenic and a variety of nononcogenic viruses in a systematic manner to determine whether oncogenicity is here also linked with teratogenicity. V. Biology of Maternal-Fetal Differences

The evidence thus far reviewed suggests that a large library of genes exist concerned solely with early embryogenesis, that some of these genes may be reexpressed in cancer (concept of retrogenesis) and that the mechanisms which allow the embryo to escape immune destruction are also employed by tumors to the same end. If the tumor-embryo parallels which have been proposed are correct, then there should be evidence for a spectrum of autoantigens in the human embryo, and there should exist experiments of nature ( diseases ) which provide such evidence.

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We contrast two views of the nature of tumor-associated antigens that occur in cancer. The first is that transiently active or phase specific genes of early development are associated with ancient functions common to early embryogenesis in many different species, that until the advent of the placenta the products of these genes were never seen by the adult immune system except in potential tumor cells, and that many were, therefore, autoimmunogenic. (Note that tolerance here is not an issue since it appears to be produced not by antigens that have appeared at any time during development, but rather by antigens present during the transition to immunocompetence from the fetal condtion. ) The contrasting view is that antigens or autoantigens found in tumors and in embryos are the products of viral genes, and that the embryo may have found these genes to be useful during early development and hence has retained them so that they have become universal ( H ~ e b n e rand Todaro, 1969). The concept that appearance of these antigens in the embryo renders the adults tolerant has not been borne out by experimental studies (Hanna et al., 1972). Some crucial issues emerge. The first is: Can a librarj- of phasespecific antigens and autoantigens be demonstrated in the embryo? The second concerns the question of the mechanisms of escape from immune destruction by tumors and embryos: Are they truly parallel in detail and different by degree? In one important historical respect the tumor-embryo parallelism may be exact. The existence of autoantigens in tumors was obscured for a long period by transplantation antigens that produced tumor rejection in nonisogenic transplants. Similarly, the presence of true transplantation antigens in the products of conception in man and in all nonisogenic animals may have similarly obscured the presence of obligatory and phase specific autoantigens in the embryo. We stress here some of the technical problems associated with work in this area. Differentiation begins with the fertilized egg, and evidence for selective gene activation shortly after this time is accumulating. It is therefore, not unlikely that genes exist that are active in only a few cells for a short period of time. In many instances, therefore, minuscule amounts of a gene product may be formed and the product may be unstable and not be present in sufficient amounts or for a long enough time to reach the maternal immune system.

A. EVIDENCE FOR OBLIGATORY EMBRYONIC AUTOANTIGENS 1. Sperm and E g g At the very outset, sperm, including human sperm, ccntain autoantigens. These have been exhaustively reviewed by Shulmart ( 1971). In

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the guinea pig four aspermatogenic factors termed S, P, Z, and T have been demonstrated, and except for Z, have been purified and shown to be biochemically distinct (Voisin and Toullet, 1968). Four autoantigens have been demonstrated on human sperm ( Hjort and Hansen, 1971). One or more sperm autoantigens persists through the early stages of development since the treatment of rat embryos with an isoantiserum against rabbit sperm causes a highly significant decrease in embryo implantation rate as compared with rabbit embryos treated with normal rabbit serum ( Menge, 1968). These results could not be due to sperm coating antigens which originate in the seminal plasma since isoantiserum against seminal plasma was without effect. The persistance of sperm autoantigens in the early embryo is confirmed by the work of Sokolovskaya and Reshetnikova (1968), who found no offspring in female rabbits which had been immunized with washed rabbit sperm. Although some eggs were fertilized, embryonic development was abnormal. Much less work has been done on the autoantigens of mammalian eggs. Some evidence of these in man is provided by the observation that in Addison’s or Cushing’s disease autoantibodies may be made against ova, lutein tissue, membrana granulosa, and theca interna ( Irvine et al., 1968). Primordial follicles are apparently not affected during the early stages of the disease; however they are later destroyed. It was proposed that the autoantibodies were against enzymes common to the ovaries and adrenals which might be concerned with steroid synthesis. Fluorescent antibody staining patterns and immunoabsorption studies indicate that a multiplicity of antigens are involved (Irvine et al., 1969). In both sperm and eggs the autoantigens involved appear to have nothing in common with classical transplantation antigens. 2. Euidence from Gynecology Reproductive physiologists have previously noted that a variety of autoantigens are present during development and that the pregnant female responds to them. As noted by Volkova and Maysky (1968), “The existence of a phasic immune interaction between mother and fetus is confirmed by the appearance in the blood of pregnant women of antibodies which enter into a specific reaction with antigens of the tissues of the fetuses at particular stages of development.” However, as noted by Bratanov (1968), “In the normal course of the various periods of the reproductive process, the antibodies induced by these alien antigens do not disturb the natural tolerance between mother and fetus.” The early view that the embryo and fetus are not rejected immunologically because the placenta constituted an impermeable physical barrier is no longer accepted. While a free exchange of protein does not occur, there appears to be sufficient leakage in both directions to

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allow immunological responses to take place. Circulating antibodies against paternal transplantation antigens are often present ( Billingham and Silvers, 1971). The suggestion has been repeatedly made that the gestoses of late pregnancy may have an autoimmune basis (Levanon et al., 1968; Montemagno et al., 1968; Noschel and Kuhnert, 1970; Klink et al., 1970; Muhe, 1970; Krieg, 1970). The antibodies do not appear to be against paternally derived transplantation antigens since the autoantibodies cross-react with an antigen in the blood vessels of pregnant women producing hypertension, edema, glomerulonephritis, and other symptoms. Hulka and Mohr (1969) reported that postpartum antibodies, conjugated with fluorescein, stained all the cellular elements in the trophoblastic villus. Gusdon et al. (1970) reported that circulating antibodies appear postpartum against placental lactogen; since more highly purified placental lactogen did not show this effect, however, the antibodies may be against a different placental antigen. The problem of the role of constitutive autoantigens in human pregnancy deserves much more careful study, especially in view of negative findings in attempts to demonstrate antiplacental antibodies ( Sinha et al., 1968). It has also been suggested that an immune response to the placenta may be necessary for pregnancy (Patillo, 1974). It is therefore, interesting that antilymphocyte serum has no effect on pregnancy, while antithymocyte serum almost completely ablates it ( Gusdon, 1971). Kurloff cells are produced in part of the thymus, increase in numbers during pregnancy, and contain mucopolysaccharide inclusion bodies which appear to be delivered to the placental trophoblasts (Marshall et al., 1970, 1971). The mucopolysaccharide is toxic to macrophages. The authors suggest that this system may be concerned with a defense mechanism against immunological rejection of the placenta. Possibly the antithymocyte serum of Gusdon interferes with it. When homogenized human placenta is extracted with acid, IgG is recovered which reacts with an unidentified material present on the thickened trophoblast basement membrane of some villi and in fibrinoid ( McCormick et al., 1971). No complement is bound in the reaction.

3. Evidence for Embryonic Autoantigens from Studies of Pregnancy in Animal Models The evidence for autoimmunity to placental or embryonic antigens in animal models is, in many cases, indirect and most of the work requires repetition in isogenic systems. It has been repeatedly suggested that immune reactions are important in reproduction, regulating placental size, enhancing or stimulating

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fetal growth, or providing a stimulus to birth (see Billington, 1969; Patillo, 1974). The fact of reproduction in isogenic strains means that if functional postulates are correct, then the immune reactions must involve autoantigens. Group-specific antigens of C-type viruses are expressed in the embryo, and it has been thought that the presence of such antigens rendered the bearer tolerant (Huebner et al., 1970). However, recent studies demonstrate autogenous immunity to endogenous RNA tumor virus antigens in mice with a low natural incidence of leukemia (Hanna et al., 1972) suggesting that such antigens may be considered as fetal autoantigens. Bratanov et al. (1965) showed that during pregnancy cows were allergic to extracts of chorion membranes, placental cotyledons, and fetus, but not to adult control tissue extracts. The reaction reached its peak during the last month of pregnancy. In pregnant ewes, in contrast, an anaphylactic reaction was observed (Bratanov et al., 1967), but no reaction was seen in pregnant sows. Recently Castro et al. (1973) have provided definitive evidence for an autoimmunological or transplantation reactions to syngeneic fetal antigens. Tissue from 12-day-old fetuses were implanted under the kidney capsule of adult mice and grew poorly with lymphocytic infiltration in mice, but grew in great profusion with no lymphatic infiltration in immunologically deprived mice. When normal mice were implanted with syngeneic fetal tissue, a second fetal implant attained greater size and showed more histological diversity, suggesting an enhancing effect.

B. TUMORAND FETAL ESCAPEMECHANISMS The concept of placental impermeability to proteins and cells as the mechanism by which immune rejection of the fetus is avoided is no longer tenable. Kaliss (1968) showed that pregnant females produced antibodies against fetal H-2 antigens of paternal origin, demonstrating that these antigens reached the maternal immune system. In a subsequent pregnancy the antibodies were shown to traverse the placenta and appear in the fetus, thus demonstrating movement of the antigen in one direction and antibody in the other. Maternal cells have been found in the mouse embryo up to an age of 8.5 weeks (Tuffrey et al., 1969). The uterus is not an immunologically privileged site ( Schlesinger, 1962), and immunological escape is not due to the effect of known placental hormones ( Hulka and Mohr, 1969). The ease of transplantability of trophoblasts suggests that they are immunologically inert or are shielded by a sialomucin coat, or that a cell-mediated immune response may be blocked. Normal trophoblast cells undergo cytolysis in culture in the presence of allogeneic or ma-

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ternal lymphocytes, and choriocarcinoma cells are likewise lysed in uitro (Currie and Bagshawe, 1967). Using a migration inhibition test a specific plasma factor has been demonstrated which blocks human cellmediated immune reactions to placental antigens (Youtanar ukorn and Matangkasombut, 1973), in confirmation of earlier reports ( I . Hellstrom et aZ., 1969). Thus rejection, blocking, and enhancement may all be parallel phenomena in pregnancy, cancer, and in the transplantation of normal tissues. That the escape mechanism is quite effective is illustrated by the development, nearly to term, of rat fetuses in m ce (Kirby, 1968). The close relation between tumor immunology and the evolution of the placenta was first explicitly pointed out by Sinkovics, DiSaia, and Rutledge in 1970. Humoral immunity presumably developed during evolution to block cell-mediated destruction of the fetal allograft. Placentation and circulating immunity appeared at approximately the same time during evolution (Anderson and Coggin, 1972a), thus opening, as proposed by Sinkovics et al. (1970), “a loophole in the highly integrated structure of mammalian adaptive immunity.” Some tumors, imitating embryonic growth morphologically and biologically by dere pression of fetal genes and by resynthesis of fetal substances and antigens may continue to take advantage of immunological enhancement. As reviewed in previous sections the tumor-embryo parallelism appears to be complete, and tumor bearer and pregnancy lymphocytes have both been shown to be cytotoxic for tumor and for fetal cells, and serum from both tumor bearers and pregnant animals have been shown to block. Tumors, however, may reach one stage not seen during pregnancy for many fetal antigens, namely, a stage of circulating antigen excess. From these considerations has evolved the threshold concept ( Anderson and Coggin, 1971), which proposes that the immune system of placental animals has been so poised that small numbers cf cells expressing fetal autoantigens are destroyed while larger mas: es escape. Since autochthonous tumors appear to rise from single cells, this mechanism ordinarily would suffice; however, once a critical mass is exceeded for reasons of low antigenicity, impaired response, or anatomical shielding, the tumor can survive. It is notable that most transplantable tumors, especially those not highly selected for nialignancy, require thousands of cells for transplantation, and that the same is true of embryonic cells used to produce embryomas. As noted previously, the chief, common characteristics of iumor cells to be explained are: (1) appearance of surface antigens different from classical transplantation antigens; ( 2 ) ability to escape destruction by

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the host immune response; (3) invasiveness and the ability to, metastasize; (4) alteration in the pattern of expression of genes for isozymes, tRNA's, and hormones; ( 5 ) stimulation of an adequate blood supply and supporting tissue; ( 6 ) an increased rate of growth and cell division (in most instances). In each case we have noted that the portions of the gene library devoted to early development may contain the requisite genes. Invasiveness we have noted to be a property of many embryonic cells, but is a prime characteristic of the trophoblast. Since invasiveness is the major distinguishing feature of many forms of cancer it is important to inquire into the mechanisms controlling trophoblastic invasiveness during pregnancy. In both the mouse and man, highly invasive trophoblastic tissue erodes and invades the decidua until maternal blood vessels are reached and entered (Kirby and Cowell, 1968). Invasiveness then ceases abruptly at about day 9 in the mouse. In the uterus, implantation stimulates the formation of a mass of decidua, which in turn limits trophoblast invasiveness. Since melanoma cells in a pseudopregnant mouse invaded nondecidualized portions of the uterus, but were not able to penetrate decidual tissue (Wilson, 1963), the possibility exists that the decidua contains or produces substances which may limit invasiveness of both trophoblast and tumor cells. The relationship between cancer and pregnancy deserves additional comment. Pregnancy might be thought to affect cancer incidence, as indeed it does in a number of cases (reviewed in Anderson and Coggin, 1971), and immunization with tumor cells to affect pregnancy, as previously described. However, it is necessary to place these matters in perspective. The findings of many different tumor-associated substances suggests a large library of genes for potential autoantigens. If this library is very large, then maternal exposure to one or two autoantigens in pregnancy may be minimal, and the effects of pregnancy on cancer may be predictably small and transient. Many data are badly needed in this area before meaningful conclusions can be drawn. VI. Molecular Basis of Differentiation and Cancer

The sequence of events by which information coded for by DNA is transcribed into RNA and translated into protein is understood in considerable detail. In contrast, little is known of the mechanisms whereby structural genes are orchestrated during mammalian development. From the viewpoint of this review two questions predominate: (1) Does the genome contain enough genes of sufficient diversity to account for all the antigens and properties exhibited by tumor cells? ( 2 ) Is gene

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phasing a rare event, or is a large fraction of the genome transiently active during development, leaving all differentiated cells with a library of previously active but now silent genes? The average mammalian cell contains approximately 6 <: gni of DNA, or 4.5-5.0 X lo9 nucleotide pairs ( McCarthy, 1967). The DNA is present both as unique or single-copy DNA, thought to represent structural genes (i.e., genes for individual proteins), and repeating DNA (i.e., DNA sequences present in hundreds or thousands of copies). The latter is thought to be important in genome regulation (Ilritten and Davidson, 1969). All mammalian DNA's which have been examined thus far contain 10430%repetitious DNA, the result depending, in part, on the techniques employed. The first question, concerning the number of genes producing mRNA actually transcribed into protein, cannot be answered definitely since it is not known whether all nonrepeating DNA serves this funciion. However, in the mouse approximately 60% of the DNA is nonrepeating; this suggests that the mouse has the equivalent of about two million structurally unique genes (defining a gene quite arbitrarily as 1000 riucleotides of DNA). In the rabbit blastocyst 1.8%of the unique DNA is transcribed, which is equivalent to 60,000 genes (Schultz et al., 1973). Similar values are reported for the preimplantation mouse embryo (Church and Brown, 1972). If the theory of Davidson and Britten (1971) is correct, then large changes in the pattern of expression of repeating DNA measured using RNA-DNA hybridization techniques should occur during development, as is indeed the case (Church and McCarthy, 1967a,b; Denis, 1966; Hahn and Church, 1970). This may represent the actual playing out of the program of development. Coordinate with this, and presumably as a direct result, different families of unique DNA should be transcribed. The key question from our viewpoint is: Are more and more unique DNA's transcribed during development until the adult number is reached, or does phasing occur at this level? Experimentally, phasing and transcriptional overlap occur ( Schultz and Church, 1973),with some genes common to several stages, others unique to one. In newborn rat liver, 2.5%of unique DNA is transcribed; in the adult, 2.0%is transcribed. However, in competition experiments it is found that these two stages account for 3.5%of the unique DNA. This means that 1.5%is turned off between birth and adulthood and this would be equivalent to as many as 40,000 genes. Much work remains to be done in the study of gene phasing during development; however, all results based on enzymological, immunological, and hybridization studies agree that phasing is an important aspect of development, and that a sufficient number of genes are present to account for the effects observed. In fact, the num-

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ber of genes appears to be so large that one wonders how immunization with fetal autoantigens described in an earlier section could have been detected in the first place. If repeating DNA is concerned with the orchestration of development, then an important question is, how many difierent repeating DNA's are there? Each different repeating DNA may be the receptor gene which in turn may be linked to a producer gene as suggested by Britten and Davidson ( 1969). Davidson ( 1973) has found that in Xenopus, which has a genome of approximately 3 X lo9 genes 700 nucleotides long, that the repeating DNA averages 1600 copies of approximately 2000 different repeating sequences, 300 nucleotides long. This suggests the organizational dimensions of the control system which may be involved in differentiation. We conclude that present knowledge of the composition and organization of the genome is compatible with the concepts of retrogenesis reviewed here. VII. Conclusions

Approximately 1000 different mammalian enzymes, serum proteins and structural proteins have been described. If all unique DNA sequences are ultimately translated into protein, then for each known protein there remains roughly 1000 to be discovered. It therefore cannot be concluded that new or mutant genes are necessary constituents of cancer cells merely because the observed changes and antigenic properties cannot be explained in terms of the minor fraction of cellular molecules with which we are presently familiar. A review of the major characteristics of cancer cells suggests that all essential characteristics for neoplasia are found in embryonic cells and that selective reexpression of some of the genes involved in early development occurs in cancer. The major difficulty with this concept has been that of accounting for the appearance of, the diversity of, and the host response to, tumor antigens. The finding that adult animals can be immunized against challenge with a wide variety of tumors using irradiated fetal cells as a vaccine has opened a new era of exploration into the origin, number, and mode of maternal response to embryonic autoantigens. Early morphogenesis is thought to be mediated in large measure by molecular changes on cell surfaces. A sizable fraction of the genome appears to be concerned with these events, and, therefore, with coding for different cell surface molecules. Because these early events produced no or few changes in cell morphology observable with the light or electron microscope, they have not been widely appreciated. Further-

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more, views of cellular proteins have been largely conditioned by the study of enzymes common to many different cell types, and of proteins characteristic of adult functions which are present during all of adult life and which are present in excess. The existence of a truly enormous gene library, a large fraction of which is concerned with embryogenesis, has only recently been considered. Associated with this gene library is a control program whose complexity is only dimly perceived, with many indigenous homeostatic mechanisms and much redundancy. :Embryonic cells of very similar appearance may thus have very many molecular differences. We propose, as have many previous investigators, ihat cancer is one end result of any one of very many errors that may occur in the differentiation program. Since the differentiation program iippears to have been largely built by the addition of new genes actiire in later development and in adult life, many of the early scaffolding and sculpturing genes have been retained through many families, classes, and orders. Since, through most of evolution, these early genes were transiently expressed during early development in eggs completely sep irate from contact with maternal tissue, the opportunity existed to develop a mechanism-cell mediated immunity-to delete cells that night have reexpressed fetal antigens leading to behavior that was unacceptable in an adult environment. These early transient antigens are therefore, in many cases, autoantigens. With the advent of placentation, the protective mechanisms required modification in the form of humoral immunity to block cell-mediated destruction of embryonic cells or of cells reexpressing embryonic antigens. Thus a threshhold appears to have been set allowing destruction of small numbers of either embryonic: or tumor cells, but allowing larger masses of cells to survive. As noted, all the predictions of cross-reactivity and cross-blocking between turnor bearer and pregnancy lymphocytes, tumor and embryonic cells, and tumor bearer and pregnancy serum have thus far been met, suggesting that the escape mechanisms of the embryo have also been used by tumors. The central problem in cancer research now, in our view, is the development of separations and identification systems capable cd handling the very complex mixtures of molecules found in normal emb:ryonic and transformed cells (see Anderson et al., 1973). Coordinate with the need to develop separations systems to obtain pure fetal or tumor materials or autoantigens is the requirement for detection systems wii h suitable sensitivity to measure the unique constituents which tumor and fetal cells have in common. These considerations suggest that few truly tumorspecific antigens will be found in human tumors, that most will be found at some stage in development, occurring in all probability, in normal, cancer-free subjects at low levels and in some diseases other than cancer,

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or with advancing age. When sufficient numbers of human tumor-associated embryonic autoantigens have been isolated and assays for them developed, early detection, tumor typing, immunotherapy, and the estimation of residual tumor mass may become possible.

REFERENCES Abelev, G. I. (1971). Aduan. Cancer Res. 14, 295. Abelev, G. I., Perova, S. D., Khramkova, N. I., Postnikova, Z. A., and Irlin, I. S. ( 1963). Transplantation 1, 174. Ainsworth, R. W., and Gresham, G. A. (1960). J. Pathol. Bacteriol. 79, 185. Allison, J. E. (1955). Anat. Rec. 122, 561. Alpert, E., Pinn, V. W., and Isselbacher, K. J. (1971). New Engl. J. Med. 285, 1058. Ambrose, K. R., Candler, E. L., and Coggin, J. H. (1969). PTOC.SOC. Erp. Biol. Med. 132, 1013. Ambrose, K. R., Anderson, N. G., and Coggin, J. H. (1971a). Nature (London) 233, 321. Ambrose, K. R., Anderson, N. G., and Coggin, J. H. (1971b). In “Embryonic and Fetal Antigens in Cancer” ( N . C. Anderson and J. H. Coggin, Jr., eds.), Vol. 1, p. 281. U. S. Dep. Commerce, Springfield, Virginia. Anderson, N. G., and Coggin, J. H. (1971). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 1, p. 7. U. S. Dep. Commerce, Springfield, Virginia. Anderson, N. G., and Coggin, J. H. (1972a). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.),Vol. 2. U. S. Dep. Commerce, Springfield, Virginia. Anderson, N. G., and Coggin, J. H. (1972b). In “Membranes and Viruses in Immunopathology” (S. B. Day and R. A. Good, eds.), p. 217. Academic Press, New York. Anderson, N. G., Holladay, D. W., Caton, J. E., and Peterson, L. B. (1973). Fed. Proc., Fed. Amer. SOC. E x p . Biol. 32,961. ( Abstr.) Baldwin, R. W., Glaves, D., and Pimm, M. V. (1971). In “Progress in Immunology” ( B. Amos, ed.), p. 907. Academic Press, New York. Baldwin, R. W., Glaves, D., and Vose, B. M. (1972). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 1, p. 193. U. S. Dep. Commerce, Springfield, Virginia. Baldwin, R. W., Graves, D., and Vose, B. M. (1974). Brit. J. Cancer 29 (in press). Baranska, W. P., Kolodovsky, P., and Koprowski, H. (1970). Proc. Nut. Acad. Sci. U.S. 67, 193. Bendich, A., Borenfreund, E., and Stonehill, E. (1973). J. lmmunol. 111, 285. Bennington, J. L., Haber, S. L., and Schweid, A. (1964). Dis. Chest 46, 623. Billingham, R., and Silvers, W. ( 1971 ). “The Immunobiology of Transplantation.” Prentice-Hall, Englewood Cliffs, New Jersey. Billington, W. D. (1969). Clin. Deuelop. Med. 3 4 89. Blair, P. B. ( 1970). Cancer Res. 30, 1199. Borsos, T., and Leonard, E. J. (1972). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 2, p. 206. U. S. Dep. Commerce, Springfield, Virginia. Boyse, E. A., Old, L. J., Stocked, E., and Shigeno, N. (1968). Cancer Res. 28, 1280.

160

J. H. COGGIN, JR. AND N. C. A N D W O N

Bratanov, K., Batchev, M., and Danov, D. (1965). Vet. Med. Nauki 2, 1133. Bratanov, K., Batchev, M., Danov, D., and Pavlov, A. (1967). Vet. M c d . Nauki 4, 17. Bratanov, K. (1968). “The Cancer Problems.” Columbia Univ. Press, New York. Braun, A. C. (1969). In “Immunology and Reproduction” (R. G. Edwards, ed.), pp. 175-189. Collings Int. Publ., New York. Brawn, R. J. (1970). lnt. J . Cancer 6, 245. Britten, R. J., and Davidson, E. H. (1969). Science 165, 349. Buffe, D., Rimbaut, C., Lemerle, J., Schweisguth, O., and Burtin, P. (1970). Int. J. Cancer 5, 85. Burger, M. M., and Noonan, K. D. (1970). Nature (London) 228, 512. Butel, J. S., Tevethia, S. S., and Melnick, J. L. (1972). Aduan. Cancer Res. 15, 1. Buttle, G. A. H., and Frayn, A. (1967). Nature (London) 215, 1495. Buttle, G. A, H., Eperon, J. L., and Kovacs, E. ( 1962). Nature ( L o n d m ) 194, 730. Buttle, G. A. H., Eperon, J., and Menzies, D. N. (1964). Lancet ii, 14. Castleman, B., Sculley, R. E., and McNeely, B. W. (1972). New EngZ. .1. Med. 286, 713. Castro, J. E., Lance, E. M., Medawar, P. B., Zanelli, J., and Hunt, R. (1973). Nature (London) 243,225. Castro, J. E., Hunt, R., Lance, E. M., Medawar, P. B., and Zanelli, J. (1974). Cancer Res. (in press). Chu, T. M., Reynoso, G., and Hansen, H. J. (1972). Nature (Lmdori) 238, 152. Church, R. B., and Brown, I. R. (1972). In “Results and Problems in Cell Differentiation” (H. Ursprung, ed.), Vol. 3, p. 11. Springer-Verlag, Berlin and New York. Church, R. B., and McCarthy, B. J. (1967a). J. MoZ. BioZ. 23, 459. Church, R. B., and McCarthy, B. J. (1967b). J. MoZ. BioZ. 23, 476. Coggin, J. G., and Ambrose, K. R. (1969). PTOC.SOC. E x p . Biol. Med. 1132, 246. Coggin, J. H., and Anderson, N. G., eds. (1972). In “Embryonic and Fetal Antigens in Cancer,” Vol. 2, p. 72. U. S. Dep. Commerce, Springfield, Virginia. Coggin, J. H., Ambrose, K. R., and Anderson, N. G. (1970). J . lmmund. 105, 524. Coggin, J. H., Ambrose, K. R., Bellamy, B. B., and Anderson, N. G. ( 1971). J. Immunol. 107, 528. Coggin, J. H., Ambrose, K. R., and Anderson, N. G. (1973). Aduan. Exp. Med. Btol. 29, 483. Coggin, J. H., Ambrose, K. R., Dierlam, P. J., Whitley, C. Y., and Anderson, N. G. ( 1974). Cancer Res. (in press). Collins, J. J., and Black, P. H. (1973). J . Nut. Cancer Inst. 51, 95. Conheim, J. F. (1889). Lect. Gen. Puthol. 2, 1. New Sydenham SOC.,I’ondon. Criss, W. E. (1971). Cancer Res. 31, 1523. Currie, G. A., and Bagshawe, K. D. (1967). Lancet i, 708. Currie, G. A., and Basham, C. (1972). Brit. J . Cancer 26, 427. Davidson, D., and Anderson, N. G. (1960). Exp. Cell Res. 20, 610. Davidson, E. H. (1973). Personal communication. Davidson, E. H., and Britten, R. J. (1971). J . Theor. BioZ. 32, 123. de Boer, W. G. R. M., Forsyth, A,, and Nairn, R. C. (1969). Brit. J. Med. 3, 93. Denis, H. (1966). J. MoZ. BioZ. 22, 285. Dierlam, P., Anderson, N. G., and Coggin, J. H. (1971). In “Embryonic and Fetal Antigens in Cancer” ( N. G. Anderson and J. H. Coggin, Jr., eds. ), Vo1. 1, p. 203. U. S. Dep. Commerce, Springfield, Virginia.

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

161

DiPaolo, J. A., and Kotin, P. (1966). Arch. Pathol. 81, 3. Duff, R. J., and Rapp, F. (1970). J. Immunol. 105, 522. Edmonds, L. C., and Cerrera, G. M. (1965). J. Pediat. 67, 94. Edwards, C. L., and Hayes, R. L. (1972). “Clinical Uses of Radionuclides: Critical Comparison With Other Techniques,” Conf. 711101, p. 618. Edynak, E. M., Old, L. J., Vrana, M., and Lardis, M. (1970). Proc. Amer. Ass. Cancer Res. 11, 22. Edynak, E. M., Old, L. J., Vrana, M., and Lardis, M. (1972). New Engl. J . Med. 286, 1178. Eveleigh, J. W. ( 1972). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 2, p. 133. U. S. Dep. Commerce, Springfield, Virginia. Fahniy, 0. G., and Fahmy, M. J. (1970). Int. J . Cancer 6, 250. Fink, M. A,, Rauscher, F. J., and Chirigos, M. (1966). In “Viruses Inducing Cancer” (W. J. Burdette, ed.), Vol. 25, p. 25. Univ. of Utah Press, Salt Lake City. Fishman, W. H., Inglis, N. I., Stolbacii, L. L., and Krant, M. (1968a). Cancer Res. 28, 150. Fishman, W. H., Inglis, N. R., Green, S., Anstiss, C. L., Gosh, N. K., Reif, A. E., Rustigian, R., Krant, M. J., and Stolbach, L. L. (196813). Nature (London) 219, 697. Folkman, J. (1972). Ann. Surg. 175, 409. Girardi, A. J., Repucci, P., Dierlam, P., Rutala, W., and Coggin, J. H. (1973). Proc. Nat. Acad. Sci. U.S . 70, 183. Globerson, A., and Feldman, M. (1964). J. Nut. Cancer Inst. 32, 1229. Gold, P. (1967). Cancer (Philadelphia) 20, 1163. Gold, P.,and Freedman, S. 0. (1965). J . E x p . Med. 121, 439. Granatek, C. H., Hanna, M. G., Hersh, E. M., Gutterman, J. U., and Mavligit, G. M. ( 1974) . J . Immunol. ( in press ) . Grant, J., and Wells, S. A. (1973). J. Surg. Res. (in press). Grant, J., and Wells, S. A. ( 1974 ). Surgery (in press). Grant, J., Ladisch, S., and Wells, S. A. (1973). Cancer (Philadelphia) (in press). Green, H. S. N. (1955). Ann. N . Y. Acad. Sci. 59, 311. Greenblatt, M.,and Shubik, P. (1968). J. Nut. Cancer Inst. 41, 111. Griffin, A. C. (1957). Tex. Rep. Biol. Med. 15, 161. Gross, P. R. (1968). Annu. Reo. Biochem. 37, 631. Gunz, F. W., and Fitzgerald, P. H. (1964). Blood 23, 394. Gusdon, J. R., Jr. ( 1971). Obstet. Gynecol. 37, 192. Gusdon, J. R., Jr., Leake, N. H., and Burt, R. L. (1970). J . Obstet. Gynecol. 108, 1056. Hahn, W. E., and Church, R. B. (1970). In “Cell Differentiation” (0. A. Schjeide and J. devellis, eds. ), pp. 119-140. Van Nostrand-Reinhold, Princeton, New Jersey. Hakkinen, I., and Vikari, S. (1969). Ann. Surg. 169, 277. Hakkinen, I., Jarvi, O., and Gronroos, J. ( 1968a). Int. J . Cancer 3, 572. Hakkinen, I., Korhonen, L. K., and Saxen, L. (1968b). Int. J . Cancer 3, 582. Hall, T. C. (1974). Ann. N . Y. Acad. Sci. (in press). Hanna, M. G., Tennant, R. W., Yuhas, J. M., Clapp, N. K., Batzing, B. L., and Snodgrass, M. J. (1972). Cancer Res. 39, 2226. Hannon, W. H., Anderson, N. G., and Coggin, J. H. (1974). In “World Symposium

162

J. H. COGGIN, JR. AND N. G . ANDERSON

on Chemical Carcinogenesis” (J. DiPaolo, ed.). Academic Press New York. ( in press ) . Harris, W. W., and Harrell, B. W. (1972). In “Embryonic and Fetal Antigens in Cancer” (N. G. Anderson and J. H. Coggin, Jr., eds.), Vol. 2, p. 127. U. S. Dep. Commerce, Springfield, Virginia. Harris, W. W., Harrell, B. W., and Anderson, N. G. (1971). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., i:ds.), Vol. 1, p. 129. U.S. Dep. Commerce, Springfield, Virginia. Haughton, G., and Nash, D. R. (1969). Progr. Med. Virol. 11, 248. Heby, O., and Lewan, L. (1971). Virchow’s Arch. B 8, 58. Hellstrom, I., Hellstrom, K. E., Evans, C. A., Heppner, G. H., Pierce, G. E., and Yang, J. P. S. (1969). Proc. Nut. Acad. Sci. U. S. 62, 362. Hellstrom, I., Hellstrijm, K. E., Sjogren, H. O., and Warner, G. A. (1971). Int. J. Cancer 7 , 1. Hellstrom, K. E., Hellstrom, I., and Brawn, J. (1969). Ndure (London) 224, 914. Herberman, R. B., Ting, C.-C., and Lavrin, D. H. (1971). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 1, p. 259. U. S . Dep. Commerce, Springfield, Virginia. Hjort, T., and Hansen, K. (1971). Clin. Em. Immunol. 8, 9. Hofmann, R., Friemel, H., and Brock, J. (1969). Arch. Gynuekol. 208, 187. Holleman, J. W., and Palmer, W. G. (1972). In “Embryonic and Fetal Antigens in Cancer” ( N. G. Anderson and J. H. Coggin, Jr., eds. ), Vol. 2, p. 11;’.U. S. Dep. Commerce, Springfield, Virginia. 1087. Huebner, R. J., and Todaro, G. J. (1969). Proc. Nut. Acad. Sci. U. S . 1-4 Huebner, R. J., Kelloff, G. J., Sarma, P. S., Lane, W. T., and Turner, H C. (1970). Proc. Nut. Acad. Sci. U. S. 67, 366. Huehns, E. R., Dance, N., Beaven, G. H., Keil, J. V., Hecht, F., and ldotulsky, G. (1964). Nuture (London) 201, 1095. Hulka, J. F., and Mohr, K. (1969). Amer. J. Obstet. Gynecol. 104, 889. Hulka, J. F., Hsu, K. C., and Beiser, S. M. ( 1961). Nature (London) 191, 510. Huxley, J. S. ( 1958). “Biological Aspects of Cancer.” Harcourt, New I.ork. Inoue, K. (1961). Deoelop. BioZ. 3, 657. Irvine, W. J., Chan, M., Searth, L., Kolb, P. O., Hartog, M., Bayliss, It. I. S., and Diury, M. I. (1968). Lancet ii, 883. Irvine, W. J., Moira, M., Chan, W., and Searth, L. (1969). Clin. Exp. Immunol. 4, 489. Jernstrom, P., and McLaughlin, N. (1962). J. Amer. Med. Ass. 182, 147. Kaliss, N. (1968). Proc. SOC. E x p . Biol. Med. 129, 83. Katz, D. H., Order, S. E., Graves, M., and Benacerraf, B. (1973). Proc. Nut. Acad. Sci. U.S. 70, 396. Kelly, L. S., Payne, A. H., White, M. R., and Jones, H. B. (1951). Cuficer Res. 11, 694. Kirby, D. R. S. ( 1968). In “Immunology and Reproduction” (R. G. Edwards, ed.), p. 43. Int. Planned Parenthood Found., London,and New York. Kirby, D. R. S., and Cowell, T. P. (1968). In “Epithelial-Mesenc1,ymal Interactions” (R. Fleishmajer and R. E. Billingham, eds.), pp. 64-77. Williams & Wilkins, Baltimore, Maryland. Klavins, J. V., Mesa-Tejada, R., and Weiss, M. (1971). Nature ( L o i d o n ) , New Biol. 234, 153. Klein, G. (1966). Annu. Reu. Microbiol. 20, 223. Kleinsmith, L. J., and Pierce, G. B. (1964). Cancer Res. 24, 1544.

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

163

Klink, R., Kessler, M., and Schneider, J. (1970). Arch. G y w k o l . 211, 300. Klotz, I. M., and Darnall, D. W. (1969). Science 166, 126. Knox, W. E. (1972). “Enzyme Patterns in Fetal, Adult, and Neoplastic Rat Tissues.” Karger, Basel. Koldovsky, P., and Svoboda, J. ( 1963). Folia Biol. (Prague) 9, 233. Krieg, H. (1970). Arch. Gynaekol. 211, 298. LeGerfo, P., Krupey, J., and Hansen, H. J. (1971). New Engl. J. Med. 285, 138. LeMevel, B. P., and Wells, S. A. (1973). Nature (London),New Biol. 244, 183. Levanon, Y., Stela, M., and Rossetini, 0. (1968). 2. Immunitaetsforsch., Allerg. Klin. Immunol. 136, 178. McArthur, J. W. (1963). Progr. Gynecol. 4, 146. McCarthy, B. J. (1967). Bacteriol. Reu. 31, 215. McCormick, J. N., Faulk, W. P., Fox, H., and Fudenberg, H. H. (1971). J. Ezp. Med. 133, 1. McKechnie, C., Jr., and Fechner, R. ( 1971). Cancer (Philadelphia) 27, 694. Markert, C. L. (1968). Cancer Res. 28, 1908. Marshall, A. H. E., Swettenham, K. V., and Vernon-Roberts, B. (1970). J. A m t . 106, 414. Marshall, A. H. E., Swettenham, K. V., Vernon-Roberts, B., and Revell, P. A. (1971). Int. Arch. Allergy Appl. Immunol. 40, 137. Menge, A. C. (1968). Proc. SOC. Erp. Biol. Med. 127, 1271. Mesa-Tejada, R., Fierer, J. A., Klavins, J. V., Weiss, M., and Berkman, J. L. (1971). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 2, p. 177. U. S. Dep. Commerce, Springfield, Virginia. Metz, S. A. (1972). Ph.D. Thesis, Yale Univ. Press, New Haven, Connecticut. Miller, D. R. ( 1969). Int. J. Haemutol. 17, 103. Montemagno, N., De Stefano, M., and Cardone, A. (1968). Minema Ginecol. 20, 1737. Moog, F. (1965). In “Biochemistry of Animal Development” ( R . Weber, ed.), Vol. 1, p. 307. Academic Press, New York. Moore, T. L., Kupchik, H. Z., Marcon, N., and Zamcheck, N. (1971). Amer. 1. Dig. Dis. 16, 1. Morgan, W. W., and Cameron, I. L. (1973). Cancer Res. 33, 441. Morton, D. L., Eilber, F. R., Joseph, S . L., Wood, W. C., Trahan, E., and Ketcham, A. S. (1970). Ann. Surg. 172, 740. Moscona, A. A. ( 1971). Science 171, 905. Muhe, E. (1970). 2. Geburtsh. Gynaekol. 172, 80. Mukherji, B., and Hirshaut, Y. (1973). Science 181, 441. Nairn, R. C., Fothergill, J. E., McEntegart, M. G., and Richmond, H. G. (1962). Bn’t. Med. J. 1, 1791. Nishioka, M., Ibata, T., Okota, K., Harada, T., and Fujita, T. (1972). Cancer Res. 32, 162. Noschel, H., and Kuhnert, A. (1970). Zentralbl. Gynuekol. 92, 1384. Old, L. J., and Boyse, E. A. (1964). Annu. Reu. Med. 15, 167. Omenn, G. S. (1970). Ann. Intern. Med. 72, 136. Otten, J. A., Tyndall, R. L., Estes, P. C., Gude, W. D., and Swartzendruber, D. C. (1973). Proc. SOC. Exp. Biol. Med. 142, 92. Parmiani, G., and Della Porta, G. (1973). Nature (London),New Biol. 241, 26. Pasternak, G., Horn, K. H., and Graffi, A. (1962). Acta Biol. Med. Ger. 9, 306. Pattillo, R. A. ( 1974). In “Endocrine and Immunological Factors in Trophoblastic Cancer Models.” Academic Press, New York. In press.

164

J. H. COGGIN, JR. AND N. G. ANDERSON

Pearson, G., and Freeman, G. (1968).Cancer Res. 28, 1665. Potter, V. R. (1969).Proc. Can. Cancer Res. Conf. 8, 9. Prehn, R. T. (1967).In “Cross-Reacting Antigens and Neoantigens” (1. J. Trentin, ed. ), p. 105. Williams & Wilkins, Baltimore, Maryland. Prehn, R. T., and Main, J. M. (1957).J. Nut. Cancer Inst. 18, 768. Raina, A., Janne, J., Hannonen, P., and Holtta, E. (1970).Ann. N . 1 . Acad. Sci. 171,697. Regan, J. F., and Cremin, J. H. (1960).Amer. J. Surg. 100, 224. Reiner, J., and Southam, C. M. (1969).Cancer Res. 29, 1814. Rogan, E.,Schafer, M. P., Anderson, N. G., and Coggin, J. H. (1973).Differentiation 1,199-204. Romanovsky, A. (1964a).Folia B i d . (Prague) 10, 1. Romanovsky, A. (1964b).Folia Biol. (Prague) 10, 12. Rosen, S . W., Becker, C. E., Schlaff, S., Easton, J., and Gluck, M. C. (1968).New Engl. J . Med. 279, 640. Rubin, P. (1970).1. Amer. Med. Ass. 213, 89. Russell, D. H.,and Snyder, S . H. (1968).Proc. Nut. Acad. Sci. U. S . Cu), 1420. Russell, D.H., Medina, V. J., and Snyder, S . H. (1970).1. Bid. Chem. 245, 6732. Rutter, W. J., Wessells, N. K., and Grobstein, C. (1964).Nut. Cancer Iwt. Monogr. 13,51. Salinas, F. A., Smith, J. A., and Hanna, M. G. (1972).In “Embryonic and Fetal Antigens in Cancer” ( N. G. Anderson and J. H. Coggin, Jr., eds.), Vol. 2,p. 187. U. S. Dep. Commerce, Springfield, Virginia. Salinas, F. A., Smith, J. A., and Hanna, M. G. (1973).Nature (Londtm) 240, 41. Schlesinger, M. (1962).J . Nut. Cancer Inst. 28, 927. Schone, G. (1906).Muenchen. Med. Wochemchr. 51, 1. Schultz, G. A,, and Church, R. B. (1973).In “Biochemistry of Animal Development” (R. Weber, ed.), Vol. 3. Academic Press, New York. Schdk, G. A., Manes, C., and Hahn, W. E. (1973).Biochm. Genet. ‘7, 67. Sells, S . (1973).Cancer Res. 33, 1010. Sever, J. L. (1971).Fed. Proc., Fed. Amer. Soc. Exp. Biol. 30, 114. Sherbet, G. V. (1974).Ann. N. Y. Acad. Sci. (in press). Shulman, S. ( 1971). Crit. Reo. Clin. Lab. Sci. 2, 393. Singer, K., Chernott, A. I., and Singer, L. (1951a).Blood 6,413. Singer, K., Chernott, A. I., and Singer, L. (1951b).Blood 6, 429. Sinha, H. B., Mukherji, A. K., Mallick, H., and Achari, A. G. (1968).Ind. Med. Ass. J . 50, 456. Sinkovics, J. G., DiSaia, P. J., and Rutledge, F. N. (1970).Lancet ii, 1190. SjLigren, H. 0.(1964).J. Nut. Cancer Inst. 32, 645. Sjagren, H. O.,Hellstrom, I., and Klein, G. (1961).Ezp. Cell Res. 32, 618. Sjogren, H. O.,Hellstrom, I., Bansal, S. C., and Hellstrom, K. E. ( 1971). Proc. Nut. Acad. Sci. U.S . 68, 1372. Sokolovskaya, I. I., and Reshetnikova, N. M. (1968).In “Immunology and Reproduction” (R.G. Edwards, ed.), p. 192. Collings Int. Publ., New ’J’ork. Spar, I. L. (1953).J. Ezp. Zool. 123, 467. Stanislawski-Birencwajz,M.,Uriel, J., and Grabar, P. (1967).Cancer Res. 27, 1990. Stem, K. 0.( 1960). Nature (London) 185, 787. Stewart, A. G.,and Begg, R. W. (1953).Cancer Res. 13, 556. Stonehill, E. H.,and Bendich, A. (1970).Nature (London)228, 370. Strittmatter, C. F. (1968).Proc. Soc. Erp. B i d . Med. 128, 366.

CANCER, DIFFERENTIATION AND EMBRYONIC ANTIGENS

165

Takasugi, M., and Klein, E. (1970). Transplantation 9, 219. Takeda, K., Aizawa, M., Kikuctti, Y., Yamawaki, S., and Nakamura, K. (1966). Gann 57,221. Tal, C. (1965). Proc. Nut. Acad. Sci. U.S. 54, 1318. Tal, C. ( 1972). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 2, p. 53. U. S. Dep. Commerce, Springfield, Virginia. Tal, C., and Halperin, M. (1970). Isr. J. Med. Sci. 6, 708. Tal, C., Dishon, T., and Gross, J. (1964). Brit. J. Cancer 18, 111. Tatarinov, J. S. (1964). Vop. Med. Khim. 10, 90. Tevethia, S. S., Convillion, L. A., and Rapp, F. (1968). J. Immunol. 100, 358. Thonison, D. M. P. J., Krupey, J., Freedman, S. O., and Gold, P. (1969). Proc. Nut. Acad. Sci. U . S . 64, 161. Ting, C. C., Herberman, R. B., Lavrin, D. H., and Shiu, G. (1971). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 1, p. 223. U. S. Dep. Commerce, Springfield, Virginia. Ting, C. C., Lavrin, D. H., Shiu, G., and Herberman, R. B. (1972). Proc. Nut. Acad. Sci. U . S. 69, 1664. Ting, R. C. (1968). Nature (London) 217, 858. Tuffrey, M., Bishun, N. P., and Barnes, R. D. (1969). Nature (London) 221, 1029. Upton, A. C. (1968). Methods Cancer Res. 4, 54. Viza, D. ( 1971). In “Embryonic and Fetal Antigens in Cancer” ( N . G. Anderson and J. H. Coggin, Jr., eds.), Vol. 1, p. 369. U. S. Dep. Commerce, Springfield, Virginia. Voisin, G. A., and Toullet, F. (1968). Ann. Inst. Pusteur, Paris 114, 727. Volkova, L., and Maysky, I., (1938). In “Immunology and Reproduction” (R. G. Edwards, ed.), pp. 211-230. Collings Int. Publ., New York. Waalkes, T. P., Dinsmore, S. R., and Mrochek, J. E. (1973). J. Nut. Cancer Inst. In press. Wallach, D. F. H. (1968). Proc. Nut. Acad. Sci. U . S . 61, 868. Weingold, A. B., and Southern, A. L. (1968). Obstet. Gynecol. 32, 593. Weinhouse, S. ( 1972). Cancer Res. 32,2007. Weintraub, B. D., and Rosen, S. W. (1971). J. Clin. Endocrinol. 32, 94. Wells, S., Grant, J., and LeMevel, B. (1973). PTOC.Amer. Ass. Cancer Res. 14, 83. Whitley, C. (1972). Master’s Thesis, p. 1. Univ. of Tennessee, Univ. of Tennessee Press, Knoxville. Williams-Ashman, H. G., Coppoc, G. L., and Weber, G. (1972). Cancer Res. 32, 1924. Wilson, I. B. (1963). Proc. Zool. SOC. London 141, 139. Winslow, S. (1972). Master’s Thesis, p. 1. Univ. of Tennessee, Univ. of Tennessee Press, Knoxville. Woo, J., and Cater, D. B. (1972). Biochem. J. 128, 1273. Yang, W. K. ( 1971). Cancer Res. 31, 639. Youtananukorn, V., and Matangkasombut, P. ( 1973). Nature (London), New Biol. 242,110. Zamcheck, N., Moore, T. L., Dhar, P., and Kupchik, H. (1972). New Eng. J . Med. 286, 83.