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Biochimica et Biophysiea Acta, 5 6 0 ( 1 9 7 9 ) 2 4 3 - 2 8 0 © E l s e v i c r / N o r t h - t t o l l a n d B i o m e d i c a l Press

BBA 87060

DEVELOPMENTAL

GENE

EXPRESSION

IN CANCER

K E N N E T H H. I B S E N a a n d W I L L I A M H. F I S H M A N b

a Department o f Biological Chemistry, California College o f Medicine, UnNersity o f California, Irvine, CA 9271 7, and b La Jolla Cancer Research Foundation, 2945 Science Park Road, Post Office Box 1376, La Jolla, CA 92038 (U.S.A.J ( R e c e i v e d A u g u s t 1st, 1 9 7 8 )

Contents

I°

Introduction

I1.

Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. O n c o d e v e l o p m e n t a l P r o t e i n s . . . . . . . . . . . . . . . . ". . . . . . . . . . . . . . . . . . 1. c ~ - F e t o p r o t e i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. A l d o l a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. P y r u v a t e k i n a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. G l y c o g e n p h o s p h o r y l a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. H e x o k i n a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. L - G l y c e r o l - 3 - p h o s p h a t e d e h y d r o g e n a s e s . . . . . . . . . . . . . . . . . . . . . . . . . 7. A l c o h o l d e h y d r o g e n a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. B r a n c h e d - c h a i n a m i n o a c i d t r a n s f e r a s e s . . . . . . . . . . . . . . . . . . . . . . . . . 9. G l u t a m i n a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. H i s t a m i n a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. C a r b a m y l - p h o s p h a t e s y n t h e t a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. O r n i t h i n e d e c a r b o x y l a s e ( s ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. t R N A m e t h y l a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14. R i b o n u c l e o t i d e r e d u c t a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15. T h y m i d i n e k i n a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16. U r i d i n e k i n a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17. D N A p o l y m e r a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18. d C M P d e a m i n a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19. G l u c o s a m i n e - 6 - p h o s p h a t e s y n t h e t a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . 20. H e x o s a m i n i d a s e s ( N - a c e t y l - j 3 - g l u c o s a m i n i d a s e s ) . . . . . . . . . . . . . . . . . . . . 21. E s t e r a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. A l k a l i n e p h o s p h a t a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. T h e t e r m p l a c e n t a l ( R e g a n ) i s o z y m e . . . . . . . . . . . . . . . . . . . . . . . . . b. T h e e a r l y p l a c e n t a l ( n o n - R e g a n ) i s o z y m e . . . . . . . . . . . . . . . . . . . . . . c. H e p a t o m a i s o z y m e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. ~,-Glutamyl transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24. I s o f e r r i t i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25. A r y l a m i d a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

: . . .

244 245 245 246 246 247 248 249 250 250 251 251 251 251 252 252 252 252 253 253 254 254 254 254 255 255 255 255 255 256 256

244 26. Antigens of unknown function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. The carcinoembryonic antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. CS-antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Embryonic hamster antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. Fetal antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Gastrointestinal antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f. Acute lymphocytic leukemic antigen . . . . . . . . . . . . . . . . . . . . . . . . g. Leukemic antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . h. Embryonic pancreatic antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . i. Transformed mouse L-cell antigen . . . . . . . . . . . . . . . . . . . . . . . . . . j. Oncofetal antigcq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Ectopic production o f peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Peptide hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. lntestinalization o f gastric mucosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Phosphofructokinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Creatine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Glycogen synthetascs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Fructose 1,6-bisphosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Glucose-6-phosphatc dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . d. Enolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Aldehyde dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Modulation versus primary gene activation (lactate dehydrogenases) . . . . . . . . . . . D. Loss o f adult forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

256 256 256 256 256 256 256 257 257 257 257 257 257 257 258 258 258 259 259 259 259 260 260 260 262

III. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Gene activation involving retrodifferentiation . . . . . . . . . . . . . . . . . . . . . . . . 1. Reversible differentiation as a normal response . . . . . . . . . . . . . . . . . . . . . . 2. Post-transcriptional regulation of the rate of protein synthesis . . . . . . . . . . . . . 3. Secondary modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B, Gene activation not involving retrodifferentiation . . . . . . . . . . . . . . . . . . . . . . 1. Oncoembryonic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Carcinoplacental proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. An oncoamnionic protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Ectopically expressed adult proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Relationship to neoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

262 262 262 266 269 270 270 271 271 27l 272

Acknowledgements

272

References

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272

I. Introduction T h e t e n d e n c y f o r m a l i g n a n c i e s t o lose t h e final d i f f e r e n t i a t e d m o r p h o l o g y o f t h e i r tissue o f origin (i.e. t o d e d i f f e r e n t i a t e ) w a s r e c o g n i z e d s o m e 150 y e a r s ago (cf. R e f . 1); m o r e t h a n 3 0 y e a r s ago, G r e e n s t e i n first p o i n t e d o u t t h a t t h e b i o c h e m i c a l p h e n o t y p e o f c a n c e r also d e v i a t e s t o w a r d a c o m m o n p a t t e r n h a v i n g t r a i t s s h a r e d w i t h f e t a l issue (cf. R e f s . 2, 3); a n d , f o r at least t h e p a s t d e c a d e , it h a s b e e n w i d e l y c o n c e d e d t h a t a b n o r m a l g e n e expression, rather than mutation, was the basic mechanism responsible for these observed p h e n o t y p i c s h i f t s (cf. R e f s . 4 - 8 a ) . Y e t , d e s p i t e e x t e n s i v e r e s e a r c h e f f o r t s a n d t h e p u b l i c a t i o n o f m a n y t h o u g h t p r o v o k i n g articles, t h e r e is n o well d e f i n e d c o n c e p t o f w h a t t h e s e s h i f t s in g e n e e x p r e s s i o n m a y m e a n , h o w t h e y c o m e a b o u t , o r w h a t s h o u l d b e t h e direct i o n o f f u t u r e r e s e a r c h in t h i s area.

245 Some confusion arises from inexact definitions. For example: whereas in fact, development covers all the events from the pairing of the gametes to at least the attainment of sexual maturity, the implication in the title is that only prenatal development is relevant. The stage of development need be further characterized as to its site and time, e.g. yolk sac, trophoblast, blastocyst, placenta, embryo or fetus. In recognition of this need the term oncodevelopmental gene product is used for the general case while ternts such as oncofetal or carcinoplacental are used for the more specific case. Moreover, since the sequence of development in one cell line may differ from that of another, the cell type must also be considered; a fetal gene product in one cell line may not be expressed in another. For example, ~-fetoprotein (yolk sac) is not expressed in the trophoblast. Unfortunately, too little is known about the specific developmental patterns of almost all proteins. Similar ambiguity arises from the term gene expression. Many 'fetal' gene products are found in low levels in adult tissues. Nonetheless, to be meaningful oncodevelopmental forms these genes need be essentially shut-off in the adult. That is: it is important to recognize the distinction between primary gene activation and further amplification of expression by previously activated genes. While conceding our present state of ignorance makes it impossible to fully avoid these ambiguities, an attempt will be made to remain cognizant of them while briefly reviewing observed changes in developmental-gene expression reported for cancers. The relevant literature is huge and diffuse. In order to reduce the number of references to manageable limits, previous reviews and monographs have been extensively used in place of primary literature sources. The remainder of the review will be used to develop hypotheses considered to be compatible with the observed results and which, right or wrong, may provide directions for further research. The hypotheses promulgated are: (1) most, but not all, of the shifts in gene expression are shifts between post-natal ones and late fetal genes; (2) these shifts represent a normally reversible, proliferation induced, retrodifferentiation associated with recovery from stress and, it is only in cancer that the shift is irreversible; (3) once the genes involved in such reversible shifts in differentiation are first activated during normal development, their expression is subsequently primarily regulated at a post-transcriptional level; and (4) some additional factor may cause a secondary amplification which is responsible for the high levels of expression typically associated with undifferentiated tumors. These same mechanisms may be used to explain the examples of the re-expression of still earlier developmental forms, provided a common ancestry is involved. However, other mechanisms need to be invoked to explain those relatively few examples of the oncological induction of expression of gene products that seem to be outside the normal developmental history of the particular cell. It seems, however, that even such expression is not a random event.

lI. Scope 11.4. Oncodevelopmental proteins Table 1 lists oncodevelopmental forms found in the literature. In each case there was some reason to believe the peptide was a distinct gene product (i.e.: one not produced from an adult gene product by post-translational effects) and that it was not expressed, or only expressed at a very low level, in the adult cells (i.e.: that its increased level of expres-

246 sion was less a matter of amplification but rather a circumstance of changed gene expression). These oncodevelopmental gene products are discussed more fully in the following paragraphs. 1. e~-Fetoprotein: As befitting its early recognition (cf. Ref. 9) and widespread clinical use, a-fetoprotein is a relatively well characterized oncodevelopmental protein. In nomlal development, the yolk sac is derived from the endodermal germ layer of the blastocyst and, in the mouse it produces a-fetoprotein within 3 days of conception [ 10]. From the earliest morphologically recognizable stages of development, the liver also produces a-fetoprotein and depending upon the degree to which a particular species retains the yolk sac, the liver becomes a, or the, major producer ofa-fetoprotein until birth. At about this time, albumin synthesis increases and a-fetoprotein production declines to the extremely low [11-13], but still detectable, adult levels [14]. The fetal gastrointestinal tract also produces low levels of a-fetoprotein [15]. Other fetal organs may produce traces [15]. In neoplasia, elevated a-fetoprotein levels are found to be associated with: germ cell tumors of yolk sac origin [16 21]; hepatomas [9,13,21-23]; and, some gastrointestinal malignancies [22]. a-Fetoprotein is rarely found associated with cancers arising from other sources. Clearly, deregulation of expression of the a-fetoprotein gene in cancer is more readily accomplished in the homologous tissue than in non-homologous tissues. a-Fetoprotein production by adult liver cells is not limited to neoplasms. The level of expression is also increased in: regenerating liver [13,24 26], toxin stimulated liver [13, 26 28]; hyperplastic preneoplastic liver [13,29 -32];in proliferating, but not in quiescent, hepatocytes in culture [33]; and in mature differentiated cells bordering necrotic liver cells [25]. Thus cellular growth and/or DNA synthesis may promote a-fetoprotein expression. Of possible relevance to this conclusion is the observation that a-fetoprotein is only synthesized in the G1 through the S phase and the synthesis is very susceptible to Actinomycin-D. The latter observation was interpreted as evidence for a short lived mRNA [34,35]. There is also a loose correlation between tumor growth rates and levels of a-fetoprotein [ 13]. However, a-fetoprotein production can be stimulated prior to or in the absence of DNA syntheses [25,26,36]. The increased expression of a-fetoprotein observed in liver diseases, such as: hepatitis, cirrhosis, and tyrosinosis (of. Ref. 37) is, no doubt analogous to the effect caused by experimentally induced injury. All non-neoplastic stimuli, except perhaps the CCI4poisoned liver, raise a-fetoprotein levels less than cancer can. 2. Aldolases: The aldolase isozyme system is also relatively well characterized. It consists of three distinct gene products, tire A, B and C subunits. These are most stable as tetramers and readily form hybrids [6 -8,38,39]. In the chick [38,40,42] and amphibia [41], the C subunit is very prominent in the early embryo and it is likely to be the first fonn expressed in development. In mammals the pattern is not as clear. Earlier work suggested the pattern in man [42] and rat [43] is from A and B or from A to C. Subsequent studies clearly show the C gene always to be expressed during development [39,44 47] and it is tile most prominent form in the early embryo of guinea pigs and rabbits [44,45]. On the other band, the C subunit may only be expressed fleetingly during the development of the human kidney, where tile basic trend is to replace the A form by the B type [48]. However, it still seems that expression of the C subunit is important during early emblyogenesis and that it may be the primordial form. Except in ruminants [49], the C subunit is strongly repressed in most mammalian adult tissues. It remains, however, as the major isozyme of brain, and as a minor form in

247 the lens, gonads, spleen and erythrocytes. The A subunit is the adult muscle isozyme, and is found in most other adult organs [6--8,38,39,42]. However, relatively late in developmen{ the A subunit (which presumably earlier completely replaced the more primitive C form) is itself largely replaced by the B subunit in liver and intestine and to some degree in kidney [6 -8,38,39,42,48,50]. While studies on homogenates of liver can give the impression of possible complete repression of A subunit expression in most of the liver cells [6-8,42,50,51], histological studies, using the very sensitive immunoperoxidase method, clearly show repression is not complete, i.e.: hepatocytes predominantly producing B-subunits also produce small amounts of the A-subunit [52]. Studies conducted on kidney and intestinal homogenates also indicate the simultaneous synthesis of B and A subunits in the same cells [6-8,42,48] ; however, it has been alleged that in both organs some cells only express the B subunit ]43]. Spleen undergoes a late developmental reversal in the normal pattern. That is: after B subunits are expressed there is a subsequent increase in A subunit activity which ends up the dominant adult form [53,54]. There is an increase in the absolute amount of aldolase A in slow and fast growing hepatomas and some fast growing hepatomas also express the C subunit [6 -8,55]. Histochemical studies show no morphological difference between cells producing A or B subunits [55] and the presence of AB hybrids indicate that the same cells are producing both subunits. Furthermore, immunoperoxidase studies show all three subunits can be present in a single hepatoma cell [52]. These observations suggest that the changed pattern of expression in hepatomas is due to retrodifferentiation rather than to a change in cell population due to stem cell activation, etc. A relatively large [51], small [55-58], and no [42] increase in the ratio of A to B subunits has been reported in regenerating liver. Aldolase A undergoes a resurgence in the preneoplastic stage of carcinogenesis [59-63], and in livers of organisms bearing nonhepatic tumors ]64,65]. Whereas the C subunit is expressed in fast growing dedifferentiated hepatomas it seems not usually expressed in the slower growing ones (cf. Ref. 55), suggesting the gene is less readily activated. The occasional report of C subunit expression in regenerating liver seems to be due to reticuloendothelial cell expression, not hepatocytes [66]. The retrodifferentiation-like changes in the aldolase pattern is not limited to liver. The report of AC hybrids in human rhabdomyosarcomas [67], involves activation of the mechanism for muscle C gene expression and the appearance of A and C subunits in duodenal adenocarcinomas [68] indicates the mechanisms responsible for the expression of both these subunits have been stimulated. Even the increase of non-fetal B subunit in a mouse spleen reticulosarcoma seems to represent a type of reversal of normal development, i.e. a reversal of the late modulation of A-subunit activity ]53,54]. 3. ~,ruvate kinases: The pyruvate kinase isozyme system also consists of three distinct subunits which may well be products of distinct genes. These have been called the K-, L-, and M-forms. They too favor tetrameric forms and will form hybrids ]4-7,39,69]. The K-isozyme is prominent in the mammalian fetus [69-77]. It is also the first isozyme expressed in the chick ]78,79] and at least the major early form observed in developing Xenopus eggs (unpublished observations). Thus it is likely to be the primordial form. The L-isozyme, however, is expressed early in the developing mammalian liver, and it appears to completely replace the K-isozyme in the normal adult hepatocyte [80-84]. Its level increases markedly after weaning [84a]. The L-isozyme is also expressed in adult kidney, intestine and red cells (cf. Refs. 6, 7, 69). In rat kidney it first becomes expressed near term [71,74] and the presence of KL hybrids in several species [85,86], suggests that unlike tile hepatocyte the K-isozyme is never fully repressed in any kidney cell. The L-sub-

248 units appear in the same kidney cells as aldolase B [85]. The human red cell enzyme appears to be a heterotetramer consisting of 2 L-subunits and 2 pro-subunits, suggesting the L-isozyme is synthesized as an inactive precursor [87]. The red cell enzyme may be expressed in the hemopoietic tissue of fetal liver [63] and in rodents, unlike in the human, it may have electrophoretic properties similar to the M-type [63,88]. The M-isozyme is first expressed before term and it becomes predominant in adult muscle, brain and mammalian heart; however, the K-isozyme continues to be expressed at low levels in these tissues [72,84]. The K-isozyme also continues to be expressed as the isozyme of most other adult cells. Interestingly, apparent KM hybrids are observed not only in adult tissues expressing the M-isozyme but also in those expressing the K-isozyme as the major form [88]. This would suggest the M-gene is at least minimally activated in all these tissues. Evidence for transient M-subunit expression was also obtained in regenerating hepatocytes [89]. This again suggests M-gene activation in association with K-isozyme synthesis. Alternatively, it is conceivable that the M-subunit is derived posttranscriptionally from the K-type [73]. In contrast to normal hepatocytes, rapidly growing, dedifferentiated hepatomas fail to express the L-isozyme or, at best, express it at low levels, while they express the K-isozyme at a very high specific activity [4,5,70,76,77,90]. Well-differentiated hepatomas generally express both isozymes at low levels and show a lower combined specific activity than normal liver [4,5,76,90]. Some minimal deviation tumors show little change from the normal liver pattern but always do express some K-isozyme [4,5,76,90]. Since hepatocytes express no measurable K-isozyme, this is a real increase, provided the tumors consist only of transformed hepatocytes. The rat hepatoma 9618A [76] and a human minireal deviation-type of hepatoma [91] have higher L-isozyme activity than normal liver, but they still express some K-isozyme. The K-isozyme is also expressed in regenerating liver [47,63,92,93] toxin stimulated liver [94,95], preneoplastic carcinogen treated liver [95-97], and in proliferating but not quiescent hepatocytes in culture [33]. Except in the case of CC14-poisoned liver [94,95] the level of expression of the K-isozyme is more than an order of magnitude lower than that obtained in dedifferentiated hepatomas. The level of K-isozyme expression in the liver of the intact organism can also be increased by tumor growth at non-hepatic sites or by injection of cell free extracts [69,98 103]. A switch from predominant M-to predominant K-isozyme expression was observed in a ;ariety of human brain tumors [104] and cultured rat brain tumors [105]. In some cultured tumors a partial reversal could be observed upon the addition of low levels of bromodeoxyuridine or by lowering the serum levels [105]. Histologically normal brain adjacent to removed gliomas also showed increased K-isozyme levels [104]. Adult rat skeletal muscle expresses the M-subunit at a high specific activity, but a rhabdomyosarcoma was found to express mainly K-isozyme at about I/8 the normal specific activity. Thus the tumor lost its capacity to express the M-isozyme and activated mechanisms responsible for K-isozyme expression [51 ]. 4. Glycogen phosphorylases: There appear to be three forms of dephosphorylated glycogen phosphorylase having different isoelectric points, electrophoretic mobilities, kinetic properties, immunological properties and tissue distribution patterns [4,5,7]. These distinguishing features plus the fact that hereditory deficiencies of the liver or muscle enzyme are independently inherited suggest these forms are distinct gene products. The lowest pl form, which also has the highest electrophoretic mobility, is the only type found in the 10 or 14 day fetus and in placenta. This form is completely replaced by

249 the highest pI, slowest migrating form in muscle and by a form having intermediate focusing and electrophoretic properties in liver. The fetal form persists in most other tissues, often to be expressed with'one or the other isozymes. Evidence for fetal-muscle type hybrids has been adduced [ 106,107]. The low pl form reappears in hepatomas and is sometimes the only form present, particularly in highly undifferentiated tumors. Some such tumors, as the Yoshida ascites hepatoma also express the adult liver enzyme at high activity [ 106,107]. Regenerating liver expresses the fetal enzyme at low levels [ 106]. 5. Hexokinases: Although the hexokinases have been relatively intensively studied, the relationships among them is not clear. It is generally held that there are four forms called I to IV or sometimes A to D. In addition, intermediate or 'split' forms are often observed. Type IV differs from the others by being a glucose-specific, high Km enzyme and it is also known as glucokinase [6-8,108,109]. Although glucokinase has been observed to migrate as immunologically distinct slow and fast forms [110], it may in fact have four distinct forms called a through d [111 ]. These have different tissue distribution and developmental patterns. The three low Km hexokinases have molecular weights of about 100 000 and hexokinase I and II, at least, are not broken into subunits by SDS; glucokinase has a molecular weight of about 50 000 and seems not to be formed from the others by proteolysis [6-8,108,109,112]. Because the four primary hexokinases have distinct immunological and kinetic properties, are separable by several techniques and seem generally to be noninterconvertible, they are tentatively assumed to be products of distinct genes [6--8,108,109,114-115]. This view is supported by the observation that individuals having allelic variants of the type III enzyme show no changes in type I or II enzymes [115] and that while isozymes I, II and IV have related amino acid compositions the compositions are different [ 112]. Studies conducted on organisms during different stages of fetal development suggest the type I isozyme is the primordial form [109,116,117]. It continues, however, to be expressed in adult tissues. A band between isozyme I and II is often found in early fetal liver, but it gradually decreases with development [116,118,119]. Hexokinase II and/or II1 have been reported to first be expressed relatively late in the developing rat liver [109, 118] but hexokinase llI is expressed early in the guinea pig liver [! 17]. Quantitative data obtained in the rat show these two isozymes undergo a transient period of high specific activity near the time of gestation [95,109]. This may account for contradicting reports of isozyme II as being the most prominent fetal form [95,121,122], or that isozyme I! is weak or not present in fetal liver [116,120]. Expression of isozymes II and IV is also dependent upon diet and insulin in the normal animal and therefore may not be expressed in all species [117]. Hexokinase IV has been reported in fetal tissues at low levels [121,123] and it has been alleged that the fetal form is IVa [111]. All four isozymes have been reported to be present in placenta throughout a large period of development [118,124], but isozyme lI [120] or isozyme I [119] have also been reported to be the primary placental forms. The adult liver is characterized as having a low specific activity for all three low K m forms, with the type II enzyme being present at very low levels; hexokinase IV predominates in the normal well fed animal [6-8,108,109,121 ]. Dedifferentiated hepatomas have just the reverse pattern [4,5] with most of the increase in lowKm hexokinase usually being found in type II enzyme activity [7,116,120,123-125]. Type II enzyme has a high affinity for mitochondria and it has been postulated that this reversible association is metabolically significant with the bound form being responsible for the Crabtree effect character-

250 istic of tumors [ 1 2 6 - 1 2 9 ] . Although the similarity of expression in minimal deviation tumors and normal liver has been emphasized [4,5], examination of DEAE-chromatographic patterns show that even in these tumors the type II enzyme undergoes a slight increase in activity relative to normal liver [123]. This increase would even be more marked if the type II activity of normal liver does not reside within the hepatocyte. Type II activity is also slightly increased in the regenerating liver [96,121,122]. Toxins, CC14 in particular, can cause large increases in type lI and Ill hexokinase [94,95,122]. This pattern of high isozyme I11 activity is said to distinguish the toxin damaged liver from the neoplastic one [ 122]. Expression is also altered in non-hepatic neoplasms and in some physiological circumstances. Thus, in the rat mammary gland, type 11 enzyme is increased relative to the virgin gland during pregnancy and lactation [ 116]. In rat breast cancers, the type I1 enzyme seems sometimes to be filrther elevated, but the type Ill enzyme is also elevated [116,123]. In kidney tumors either type 11 and/or type III enzyme activity is increased [116,123]. Type 111 enzyme activity has also been reported to increase in a rat rhabdonlyosarcoma, although this isozyme was not found in the 17 day fetal muscle [116]. The type II enzyme also was reported to predominate in a variety of other human cancers [120]. In these latter studies no type 11 enzyme was found in the 2 month fetus but it was found to predominate in placenta. Thus, it was suggested the enzyme belonged to the carcinoplacental group [ 120]. tfowcver, as discussed the type II enzyme dues appear in fetal liver. 6. L-Glycerol-3-phosphate dehydrogenases: A developmental foml of L-glycerol-3phosphate dehydrogenase, distinguishable from the adult form by its lower Km value, greater lability, immunological properties and electrophoretic character, has been observed [130 132]. These fomls have been suggested to be products of distinct genes [130,1311. Whereas the developmental form has been reported to persist in low activity in adult brain, kidney and heart [132], it has also been observed to persist only to the 9th day in brain, to the 13th day in liver, and to the 16th day in kidney [130]. Thus it may be more of an embryonic than a fetal gene product. It is not found in all tumors. For example: it is only present in the embryoid body of a teratoma [130] and it is not re-expressed in hepatomas. Since the activity of tile adult form is lost in hepatomas, glycerol-3-phosphate dehydrogenase is generally classified as an adult type of enzyme, i.e.: one which is not present in the fetus and is lost in hepatomas [133]. The isozymic forms can switch depending on the growth status of a tumor, for instance the embryonic isozyme was expressed when a neuroblastoma was grown in vitro, while the adult form was expressed in such cells in vivo [131]. In general, solid tumors express the adult from regardless of growth rate, while ascites cells derived from these same tumors express the developmental form [131]. Z Alc()hol deh3,drogenases: There may be three distinct genes coding for human alcoh~,l dehydrogenases. As the enzymes hybridize and as allelic variants are common, additional forms may be found [134]. During normal development the early fetal liver form becomes less dominant, while the adult gene product is activated [135,136]. This form was also expressed by early fetal hmg where it remains active throughout life [135]. A third gene is activated in liver around puberty, although the degree to which this product is t\)rmed seems variable [ 136]. Although the rat enzymes have not been well studied, late fetal liver and well differentiated hepatomas share a common electrophoretic form, rarely found (and then as a trace) in adult liver [137]. Hybrids between hepatoma cells and cells which do not

251 express the adult form repress expression of alcohol dehydrogenase until a large number of genes have been expelled, suggesting a "repressor' gene was lost [ 137]. 8. Branched-chain amino acid transferases: The branched amino acid transferases catalyze tile transfer of the ee-amino group of leucine, valine or isoleucine to ~-ketoglutarate. Three forms (I, II, III) have been isolated from cellular supernatants, others may exist in the mitochondria. The three forms have different kinetic properties; the type II enzyme is a high Km leucine specific enzyme: the other two are non-specific low K m types. They also have distinctive molecular weights, tissue distribution patterns and immunological properties. Thus they are likely to be coded for by different genes [138-139]. Fetal liver, from as early as the 16th day after conception to birth, expresses only type I enzyme. However, the specific activity decreases with development. The liver specific, type I1 enzyme appears after birth, although it can be induced in the late fetal liver by glucagon or glucocorticosteroids. The adult rat liver phenotype is about 75% type II and 25% type [ enzyme. The type III enzyme is the major adult brain enzyme. Placenta and ovary express roughly equal proportions of the type I and III forms [138-140]. Slow growing Morris hepatomas retain isozyme I and lI, some also express isozyme Ill. In those that do not, the proportion of isozyme I relative to II increases and the Morris hepatoma 7777 expresses only tire type I enzyme. This is thus a reversion to the fetal pattern involving loss of the adult type. Moreover, the specific activity of the type I enzyme increases, which is also a reversal of the developmental pattern. However, since the type 1 enzyme is undoubtably also expressed in the hepatocytes, this reversal does not involve gene activation. The faster growing more undifferentiated tumors all fail to express type II enzyme and express type I and III enzymes at high activity levels. Treatment of a cultured liver cell line, which only expresses type I enzyme, with carcinogens causes the cells to express the type III enzyme as well. Benign adenomas induced by carcinogens retain the normal adult pattern of expression of type I and II enzyme, while most carcinogen induced hepatomas lose enzyme II and gain enzyme III. Regenerating liver also retains isozyme I and [l and does not express type III enzyme. It was suggested that tumors expressing isozyme Ill arise from primative stem cells [ 138--140]. 9. (;lutaminases: There are two forms of glutaminase. They have distinctive kinetic, structural and inmmnological properties [ 141,142]. The adult kidney enzyme, K-type, is also the fetal liver enzyme. Its expression is completely [133] or largely [143] repressed in adult liver, but it reappears in hepatomas. There appears to be fair correlation between rate of growth and level of expression [ 133]. Low levels of expression of the K isozyme also occur in regenerating liver, and this appears to be a marginal increase over the normal liver level [ 133,143 ]. Regenerating liver does repress L-isozyme expression to a large degree [ 133]. K-isozyme activity is increased about 10 fold in kidney tumors, relative to normal kidney indicating further amplification occurs [143]. Mammary tumors also express K-glutaminase while the normal adult gland does not. The specific activity also increases with the growth rate [133]. 10. Histaminase: Several human cancers from various organs, including colon, thyroid and ovary produce high levels of histaminase as does the placenta. The placental and neoplastic histaminases have properties in common [144]. Thus, the ectopic production of histaminase may be another example of a carcinoplacental type response. 11. CarbarnyI-phosphate synthetases: Two types of carbamyl-phosphate synthetase are known. The one (type I), located in liver mitochondria, is involved in urea synthesis, the other (type II), located in the cytosol of rapidly dividing cells, is involved in pyrimidine

252 biosynthesis [145]. While type II enzyme is found in fetal liver, hepatomas and mammary tumors, normal adult mammary gland expresses neither isozyme. There is general correlation between tumor growth rate and the level of expression of type II enzyme [146,147]. 12. Ornithine decarboxylase(s): Nonproliferating cells, in general have low ornithine decarboxylase activity. Stimulation of division by hormones or mitogens causes a remarkable but brief enhancement of activity, apparently due to new synthesis. Fetal and neoplastic tissues also have high activities, although minimal deviation hepatomas show but a small increase relative to normal liver. Thus the enzyme belongs to the onco-fetal group. Regenerating liver also shows a marked increase in activity [ 148-152]. Isozymes of this enzyme may exist, since decarboxylase activity in different tissues were noted to respond differently to cyclic AMP [153]. More recently, two fractions were separated on thiol-activated Sepharose 4B columns [154]. Both forms are dimeric with subunit molecular weights of about 50 000; they have similar antigenic properties but different affinities for ornithine. Both have short but different half-lives [155]. Also, two forms having different affinities for pyridoxin phosphate were observed in 3T3 cells and only the low Km form increased with proliferation [156]. Therefore, the low level of activity associated with quiescent cells may be caused by a different gene product than the one responsible for the proliferation associated activity. This would enhance the possibility that gene activation is responsible for the increased activity observed with proliferation. The presence of two isozyme forms may also help explain the absence of a direct relationship between ornithine decarboxylase levels and mitotic index [148-153]. In both liver and intestine the ornithine decarboxylase activity increase caused by carcinogens occurred long before tumors could be formed [153]. 13. tRNA methylases: The tRNA methylases are a family of some 50 enzymes which modify preformed tRNA. The level of these methylating enzymes is higher in fetal and neoplastic cells than in adult liver. This is due in part to competing enzyme systems present in normal adult tissue but not in fetal or tumor cells. Tumor methylases also have kinetic properties which distinguish them from the liver types. Moreover, the tRNA species isolated from tumor or fetal tissue are altered to a greater extent and in different ways than their counterparts in normal liver. Hence, it seems probable that fetal and tumor cells share isozymic forms not found in normal adult liver [157-160]. 14. Ribonucleotide reductases: Although evidence has been adduced to suggest CDP and ADP are reduced to the deoxy forms by different enzymes [161], most work has been done on the CDP enzyme. This enzyme is expressed at very high levels in the early fetal liver and decreases in amount with development until levels become unmeasurably low. However, the enzyme reappears in hepatomas in a manner which correlates with growth: in activities increasing up to 140 000 fold. Plots of level of expression versus growth rate of hepatomas are sigmoidal. Activity is also increased in regenerating liver but maximal activity is low in comparison to tumors having similar growth rates [162,163]. Unlike developing liver, maximal activity in developing spleen occurs after birth, at a period corresponding to intensive cellular proliferation [ 162]. 15. Thymidine kinases: Adult liver expresses low levels of thymidine kinase activity which is associated with mitochondria. In contrast fetal liver expresses high levels of activity in the cytoplasm. The two forms have distinct properties. The fetal form is also expressed in hepatomas and in regenerating liver [ 133,164-166]. Total activity may increase several hundred fold in undifferentiated hepatomas [133] and plots of growth rate of hepatomas versus level of expression are complex, with an order greater than one [ 166]. Activity levels also increase in regenerating and fetal liver,

253 but these are low relative to that found in neoplasms with similar rates of proliferation [133]. Presumably all the increases are due only to the oncofetal form of the enzyme. Similar phenomena occur in nonhepatic cancers including: HeLa cells, KB cells, SV40 transformed fibroblasts, rhabdomyosarcoma, Wilm's tumor and bladder adenocarcinoma. Fetal gut, gastrointestinal tumors and placenta also express a different type of thymidine kinase than does adult gut. Moreover the activity in fetal gut and gastrointestinal cancers is much higher. The flat mucosal cells which have a high thymidine uptake in the adult also express the fetal enzyme, suggesting expression is growth related. The activity was also elevated in the premalignant phase when treated With carcinogens [153,167]. Two forms of the enzyme can also be obtained from lymphocytes. One is found in both quiescent cells and phytohemagglutin stimulated cells. The other is found only in the stimulated cells at a very high activity level [168]. 16. Uridine kinases: Krystal and Webb [169] separated two forms of uridine kinase on Sepharose-6B. A 30 000 dalton enzyme (form II) is the major form present in the 18 day fetal liver, but by the 7th post-natal day it is virtually absent, as it is from adult liver. It is, however, prominent in the dedifferentiated Novikoff hepatoma, but not in a minimal deviation tumor or in regenerating liver. A 120 000 dalton form (form I) is also present in fetal liver, but it is retained in adult liver at a reduced activity level. The activity of this form appears to increase in regenerating liver and in both types ofhepatoma [169]. That 5-azocytidine treatment increases type I activity in normal liver, but increases type I I activity in hepatomas (even if little type II enzyme was expressed initially) suggests the two forms are not in simple equilibrium [170]; thus the forms were tentatively considered to be different isozymes. Moreover it has been alleged that type II enzyme from some, but not all sources has kinetic properties which could distinguish it from the type I enzyme [171]. Both forms are also found in several transplantable leukemia lines, but only type I enzyme was found in primary tumors of lymphoid origin as well as in a variety of normal leucocytes. Species I was increased by phytohemagglutinin in normal lymphocytes [ 172]. If two gene products are involved even the gene coding for the adult type I enzyme responds to growth needs; however, the mechanism would involve modulation rather than activation. On the other hand, the type II enzyme increase in certain tumors could involve gene activation. It is also possible that the two forms are products of a single gene and that they share a common subunit. Their similar location, the ratio of their molecular weights (1 to 4) and their usually similar kinetic properties are observations compatible with the two forms being different quaternary variants. During kinetic assay they may usually, but not necessarily always, aggregate or dissociate to form the same species. However, different cellular environments might induce different degrees of association and thus explain their distributions and variable properties. 17. DNA polymerases: There are three non-mitochondrial eukaryotic DNA polymerases, called the a, ~ and ~ isozymes. Immunological, kinetic and structural studies suggest they are products of distinct genes. All three are probably nuclear, even though the a-form is isolated in the cytoplasm when cells are fractionated in aqueous media [173]. Fetal liver expresses high levels of the 7-isozyme. A 30 fold or so drop in activity occurs rapidly after gestation and levels remain low. Slow growing hepatomas show a significant increase ( 3 - 6 fold) and the more rapidly growing ones a still larger increase in activity. An activity increase also occurs in the regenerating liver [173,174] and in the carcinogen treated liver, prior to irreversible transformation [174]. The c~-form is expressed in fetal, but not adult cerebellum [175]. Its activity increases

254 in regenerating liver [175] and in the activated lymphocyte [176]. It has also been found in various tumors and in fact seems to be ubiquitous in growing cells [174]. Therefore, although seemingly not investigated from this point of view, it is likely to have oncofetal character. The/3-form seems not to increase ill activity in growth stimulated cells [174,175]. 18. dCMP dearninase: Potter found dCMP deaminase in fetal liver and hepatomas but not in adult liver (cited in Ref. 6). Plots of activity versus growth rate are sigmoidal [ 166] and its expression is enhanced in regenerating liver [ 163,1661. 19. Glucosamine-6-1)hosphate synthetase: Tile reaction catalyzed by this enzyme (which is also known as glucosaminephosphate isomerase and L-glutamine: D-fructose-6phosphate aminotransferase) is an early committed step in membrane synthesis. Five forms were obtained in rat tissues, with pI values of 4. l, 4.3, 4.5, 4.8 and 5.0. This general behavior (see below) and the symmetry of the pI values suggest there may be two basic forms. That is, the pH 4.1 and 5.0 forms may be distinct gene products and the others' hybrids, ttowever, speaking against this idea is the observation that the plt 4.5 and 5.0 forms show little imnmnological cross-reactivity. This suggests they may not share subunits. However, this lack of immunological cross-reactivity could also be a functi,)n of conformation. The 3 forms partially characterized (pH 4.1, 5.5 and 5.0)have distinctive physical and kinetic properties. Thus it seems probable that at least two, but perhaps more distinct gene products may be involved. The behavior of the various forms during development, in neoplasia and in regenerating liver is as follows. The 12 day fetus has almost only pH 4.1 enzyme, but by the 14th day the whole fetus expresses pH 4.1 and 4.5 forms with some pH 4.3 enzyme. The 19 day fetal liver expresses mainly pH 4.8 and 5.0 enzymes and adult liver extracts contain mainly ptt 5.0 enzyme. The adult brain primarily expresses the pH 4.1 variant, ttepatomas re-express low pI forms to a degree and in a step wise sequence which co~relates with the degree of dedifferentiation, and even minbnal deviation tumors show some change. Regenerating liver also shows a rather large but transient shift toward lower pI fl)rms. The switch toward lower pl forms in regenerating liver and hepatoma is accompanied by an increase in total activity. In regenerating liver this increase occurs after the peak of thymidine uptake. However DNA synthesis can be stinmlated without causing enzyme activity to increase. Thus the two events may be separable [177,178]. 20. Hexosarninidases (N-acetyl-~-glucosaminidases): Three electrophoretic forms of hexosaminidase have been observed in human tissues. These are known as A, B and C. Forms A and C are both deficient in Tay Sachs disease. Form A is also more thermolabile than B [179]. It seems likely that forms A and C are products of a single gene having a locus distinct from the gene coding for the B form. Two electrophoretic forms of hexosaminidase were observed in adult rat liver extracts. The slower band (B) predominates. It is more thermostable and is found in the lysosome. The faster band (A) is more thennolabile and more readily extracted. These properties suggest these forms are analogous to the human A and B forms. Although a trace form in liver, band A predominates in adult brain, in fetal liver and in fast growing hepatomas. In the latter, the slower band B diminishes or disappears. The CC]4 treated and regenerating liver are almost normal but show minor changes tending toward the fetal pattern [55,180,181]. 21. L~sterases: The esterases represent an assortment of groups of isozymes [39]. Various reports of oncodevelopmental forms have been made (cf. Ref. 6). That the rapidly

255 migrating isozyme-V of 5'-nucleotide phosphodiesterase can be found in sera of patients with hepatomas and in cord blood suggests it too is an oncofetal protein [182]. 22. Alkaline phosphatases: There are at least three probably different genotypes, plus scores of phenotypes of the alkaline phosphatases. The interrelationships among these forms is still not fully understood and several reviews have been published recently (cf. Refs. 5, 183-192). Therefore, only the more salient aspects will be reviewed here. The three seemingly distinct genotypes are: (a) The term placental (Regan) isozvme: This isozyme is normally expressed in full tern1 placenta and testes. A great number of phenotypical variants are known, the Regan isozyme being tile most common form. The Regan isozyme is most frequently expressed in cancers of ovarian, testicular and pancreatic origin, although it is also found ill cancers at other sites. The Nagao variant frequently expressed in ovarian cancer appears to be a different phenotype identical to the relatively rare placental D-form. It has not been observed in embryoblastic development. (b) The early placental (non-Regan) isozyme: This isozyme shares antigenic determinants with the adult liver and testes forms. During normal development it is expressed during the early chorionic phase of trophoblastic development, and as a generalized fetal form in embryonic development. It commonly reappears in a wide variety of cancers. (c) Hepatoma isozyme: This form, commonly found in hepatomas, appears to be identical to the normal adult intestinal isozyme. During normal development it is found in fetal intestine, but it is not a general fetal form, and as an isozyme expressed in the FL Amnionic cell line, but not in normal term amnion. Common variants of the hepatoma form include Kasahara isozyme and Warnock's variant. The ability to discriminate anlong the various alkaline phosphatase isozymes led to the discovery of interesting modulation phenomena. For example: Singer and Fishman demonstrated that corticosteroid induces expression of the placental-type (Regan) isozyme in HeLa cells [ 186], and that the intestinal (hepatoma) isozyme diminishes, while the placental one increases, in HeP-2 and FL-amnion cells treated with prednisolone, n-butyrate or hyperosomolar medium [185]. Perhaps related to these examples of experimentally induced changes in expression are the observations that BeWo choriocarcinoma cells produce both early and term placental forms in addition to high levels of human chorionic gonadotropin [193] and that several human breast cancer cell lines also express the two chorionic alkaline phosphatase bands of early placenta [194]. The use of such model systems enables one to study modulation of expression of developmental phase specific gene products. The placental alkaline phosphatases comprise an allelic population of over 40 phenotypes, as seen on starch gel electrophoresis. In human tumors, the most frequently seen phenotypes are also the most common F, FS and S phenotypes of normal placenta. However, the rare L-leucine-sensitive slow moving o-phenotype (Nagao variant) is often observed in patients with ovarian cancer. The reason for this is unknown. If, as suspected, the large number of allelic isoproteins is due to point mutations, it will become necessary to explain why this gene is especially susceptible in placenta and in neoplasia and whether such susceptibility occurs in other genes [194a]. 23. 7-Glutamyl transferase: This membrane enzyme whose physiological role is related to amino acid transport has recently been recognized as a sensitive marker of early hepatocarcinogenesis. The 14 day fetal liver expresses high levels of this enzyme, but the specific activity continues to decrease with development; the activity of the adult liver is about 2% of the 14 day fetal liver. Up to several hundred fold increases in activity can occur in both slow and rapidly growing hepatomas. Increases are also observed in preneo-

256 plastic liver and in regenerating liver. However, the change observed in the latter is small, less than 2.5 fold [195-197]. Mitogens were observed to produce increased expression in human and rat but not in mouse or guinea pig lymphoidal cells. This increase occurred prior to DNA synthesis and appeared to be due to de novo synthesis [198]. 24. Isoferritins: The ferritins are a complex family of iron containing proteins, with a molecular weight of some 450 000 in the native state. The native proteins are composed from two different subunits with molecular weights of 21 000 and 19 000. The different distribution patterns and diverse structural and immunological properties of these subunits indicate they are likely to be products of different genes. The subunits hybridize and form various tissue specific ferritins. Since tire native form contains 2 2 - 2 4 subunits, many different combinations may occur. The subunit with the lower pl value prevails in placenta, fetal liver, hepatoma, HeLa cells, and adult heart, but represents only a very small fraction of the subunits found in normal adult liver [199-201 ]. 25. Arylamidase: Hepatoma membrane bound arylamidase is not formed in normal liver but has the same electrophoretic mobility as placental membrane bound arylamidase. Renal cancer also expresses the placental form. Despite their different electrophoretic properties the molecular weights and kinetic properties are indistinguishable 12021. 26. Antigens of unknown function: Several onco-developmental antigens which have no known biochemical functions have "also been described, briefly these include: (a) The carcinoembryonic antigen: The carcinoembryonic antigen (CEA) was originally reported by Gold and Freedman [203] as a glycoprotein specific for neoplastic and embryonic tissue of the digestive tract. Although of particular value because of the clinical interest stimulated by these subsequent observations, it has proven not to he as specific as hoped. Whether the absence of specificity is a biological or methodological problem is not clear (cf. Refs. 204, 205). Nonetheless the antigen is more strongly expressed in fetal gut and gastrointestinal malignancies than in adult gastrointestinal tissue and therefore has oncofetal character. (b) CS-antigen: These antigens are found in fetal tissues and in C-type virus induced tumors [206]. These observations have been used to support the concept of vertical transmission of viral RNA genetic information. Since the genetic information is presumed to be incorporated within the host cells genome, expression of the antigens fits tire definition of an oncodevelopmental gene product. This also would be true if the virus induced the tumor cell to express a fetal fonn. (c) Embryonic hamster antigens: These are antigens found in embryonic hamsters and in the SV-40 virus transformed hamster cells [207]. (d) Fetal antigens: A mixture of antibodies prepared against fetal antigens at various stages of development show developmental phase specificity, and in general crossreact with antigens on cancer cells, long term tissue culture cells and rapidly proliferating adult cells [208]. In a similar fashion serum prepared against fetal antigens specifically react with leukemic cells and proliferating bone marrow cells [209]. (e) Gastrointestinal antigens: A wide variety of antigens (in addition to CEA) prepared from normal and cancerous gut tissues have been studied (cf. Ref. 210). Although the literature is confusing to the nonspecialist some of these appear to have oncodevelopmental characteristics. This seems certainly to be true of an antigen reported by Goldenberg et al. [211]. (f) Acute lymphocytic leukemic antigen: White cells from acute lymphatic leukemic

257 patients have an antigen in common with early fetal thymus cells, which is not found in white cells in remission, in spleen, or in bone marrow cells [212]. (g) Leukemic antigens: Antibody prepared against leukemic cells, reacts with fetal hematopoietic stem cell antigen, but not with adult cell preparations [213]. (h) Embryonic pancreatic antigen: Antibody prepared against embryonic pancreatic antigen also reacts with pancreatic tumor antigens, but fetal colon liver, gut cancers, hepatomas or pancreatitis tissue do not express this antigen [214]. Thus specificity between fetal source and cancer type was noted with this oncodevelopmental antigen. (i) Transformed mouse L-cell antigen: Rabbit autisera to mouse teratoma cells reacts with three heteroantigens not found on any normal adult tissue except ovary. Of these, antigen I is detected on several transplantable tumors and on the ova, morulae, and inner cell mass through implantation. Antigen II is secreted by hepatomas and teratomas and is found on the trophectoderm. Antigen Ill appears to be teratoma-specific [215,216]. (/) Oncofetal antigen: Membrane antigen prepared from a cultured human melanoma line has immunological determinants in common with melanomas, other types of skin cancer, some no-skin cancers and fetal brain, skin, muscle, lung and intestinal tissue. It does not react with adult tissues including skin when obtained from biopsy but would react when grown in culture [217].

liB. Ectopic production of peptides 1. General: It was established in the early 1960s that tumors arising from non-~endocrine sources sometimes synthesize hormones. This phenomenon was called ectopic hormone production and the term has been applied to the appearance of any gene product inappropriate for that tissue. 2. Peptide hormones: The ectopic production of hormones attracted attention first because of their dramatic clinical effects and later for their theoretical implications. These phenomena were first cited as evidence for the random nature of gene activation in cancer. [t was then established that whatever their significance, expression was not random. Certain peptides are almost invariably produced by a specific, limited set of cancer cells, e.g.: the calcitonin producing C cells of the thyroid. Thus the genes involved must be more readily available in these particular cell types and the concept of statistical variance loses validity. It has also become evident that the peptides produced by these tumors tend not to be the normal circulating forms of the hormones. The available evidence suggests the tumors accumulate pro-hormones and/or degradation products and it is possible that certain normal cell lines that do not manufacture active hormone still synthesize inactive peptides. The neoplastic transformation may somehow amplify this synthesis causing the excretion of active hormone at physiologically significant levels. Indeed, in at least the relatively well studied ACTH system the relative concentration of nonactive ACTH derivatives in tumors parallels the patterns obtained in fetal pituitary cells. Therefore ectopic hormone production may be another example of oncofetal gene expression and the expression of some of these hormones in placenta has led to their classification as oncoplacental forms [218-221 ]. Indeed, human chorionic gonadotropin is one of the earlier oncodevelopmental proteins to be recognized. That it was first classified as an oncodevelopmental hormone rather than as an ectopic product relates to the fact it was known to be a gene product of the syncitiotrophoblast and because its expression was thought to be limited to trophoblastic tumors. It (or its a- or/3-subunit or both) is now felt to be distributed in a wider variety of cancer, such as cancer of the stomach,

258 lung, ovary, pancreas, etc. In these sites chorionic gonadotropin expression is ectopic [222,223]. Nonetheless, it must be classified as all oncodevelopmental form. From the preceding the general question arises whether ectopic gene products may be oncodevelopmental in nature. 3. Intestinalization o f gastric rnucosa: The area surrounding gastric cancer is usually hyperplastic and it expresses one or more enzymes normally found limited to intestinal nmcosa. These enzymes include sucrase, maltase, trehalase and alkaline phosphatase. However, except for alkaline phosphatase, the cancer itself does not express these enzymes. Nonetheless, the phenomena is an example of abnormal gene expression associated with cancer and it is not known whether intestinalization precedes neoplasia or if the two events occur in parallel [224]. In neither case would this be an example of a developmental form being expressed. However, tile gastric cells and the intestinal cells involved largely share a common developmental pattern. They only diverge at last steps in differentiation. 4. Phosphofructokinases: Four forms of phosphofructokinase have been separated by chromatographic and electrophoretic techniques. The lack of interconvertibility and different kinetic and immunological properties suggest that at least three are products of distinct genes. Adult hepatocytes contain a form (isozyme IV) not found in any other normal adult tissue but erythrocytes [225-228]. This form tends to decrease in hepatoma, and a kidney type, or type lI and I11, isozyme(s) appear(s) [225-227]. Newborn liver [225], 'embryonic' liver [227], and regenerating liver [225] expressed the adult liver form but not the hepatoma enzyme(s). An unusual feature is that the specific norreal adult liver form is 'ectopically' expressed in non-hepatic tumors, including: metastic lymph node, gastric cancer, and Ehrlich ascites tumor cells [225]. lsozyme shifts are not limited to the liver type enzyme since leukemic cells express a form similar to muscle phosphofructokinase but different from the normal leukocyte enzyme [229]. The expression of liver enzyme by neoplasms is a reversal of the usual pattern since this would be an example of an adult tissue specific enzyme being expressed in cancer. It also seems that cancer forms are not expressed in developmental tissues. It is possible that further investigation would uncover suitable developmental isozymes or it may be that epigenetic effects are involved. The observation that the addition of mitogenic agents to cultured liver cells caused a protein synthesis independent increase in total activity [230], could be considered as evidence for potential post-translational regulation. Binding with an insulin induced, antidegradative, peptide [231], may also affect electrophoretic mobility. At this time however, phosphofructokinase would appear to be a marked exception to the general rule and must be classified as a nondevelopmental gene product aberrantly expressed in cancer. 5. Creatine kinase: It seems to be generally accepted that the dimeric creatine kinase can have either or both of two distinct subunits, i.e.: the B and the M subunits. Mthough amino acid composition studies, fingerprint analysis and immunochemical reactions indicate a high degree of homology between the subunits, they appear to be distinct forms coded by different genes [232]. However, evidence has also been adduced for two distinct forms of the M-subunit and for the possible existence of a mitochondrial form [2331. The young fetus expresses only BB forms, but in the adult the M form predominates in muscle and heart and is also found in other tissues. Despite the fall in activity of the BB

259 enzyme in muscle the rate of synthesis seems not to decline during development and ezymatically inactive BB protein has been reported to exist in the striated muscle of rabbit and man [232]. M forms appear to be replaced by the B subunit in several carcinomas [234], and in two rhabdomyosarcomas [67] and it has been suggested the enzyme is oncofetal in nature [5,234]. More recently, Weinhouse and his coworkers [235] have performed an in-depth study of the creatine kinase isozyme patterns in normal rats and mice. Except for muscle (MM), heart (MM and MB), mammary gland (MM and BB), lung (MM and BB) and aorta (MM and BB) all other rat tissues including the whole 13-15 day embryo express only the BB form. Although adult liver had only BB enzyme, late !brenatal, neonatal and early regenerating liver express both MM and BB forms. Adult mouse kidney and liver expressed significant amounts of the M-subunit. The specific activity of creatine kinase did not increase with tumor growth rate nor was an ordered pattern of isozyme expression observed. Among 4 rat hepatomas the proportion of MM isozyme ranges from 0 to 65% of the total. Among kidney tumors 2 had barely detectable levels of MM isozyme and 1 2 0 55%. Two rat hepatomas and the mouse sarcoma 37 ascites and Ehrlich ascites tumors expressed only BB enzyme. In contrast, a mouse sarcoma, a neuroblastoma and rhabdomyosarcoma all had a predominance of MM activity. No tumors expressed MB forms even though both BB and MB enzyme was present [235]. In some cases the trend was for the isozyme alteration to deviate toward the developmental form. Thus adult muscle expresses only MM but the mouse BW 10139 rhabdomyosarcoma does express some BB enzyme, and all the tumors of connective tissue origin expressed some BB enzyme. Indeed the highly undifferentiated ascites sarcoma 37, had only the BB form. However, when the BB form was the sole isozyme of the tissue of origin as in rat liver, kidney, and rat and mouse brain, the corresponding neoplasm expressed MM as well as BB activity. Thus, as with phosphofructokinase a shift toward a tissue specific adult from the prhnordial fetal form occurs in neoplasia. Hence creatine kinase may be classified as a gene product which is ectopically expressed by neoplasms [235]. It is significant to note, however, that the M subunit is transiently expressed during normal liver development as a very late fetal gene. It is also expressed by the regenerating liver [235]. Thus among the hepatomas, at least, expression of the M subunit may be considered a recapitulation of a late and transient stage in development. As is so often the case this stage was also reflected by the regenerating liver. As noted above, and as speculated might be the case for phosphofructokinase, posttranslational regulation may also play a role in determining patterns of expression. 6. Miscellaneous: Under this category are listed proteins which appear in cancers, which have not been found in development, but about which too little information was uncovered to permit even tentative classification. This group of enzymes includes the following: (a) Glycogen synthetases: Muscle and liver glycogen synthetases are known to be different and are probably different gene products. Fast growing hepatomas express muscle type. Apparently fetal forms have not been studied (cf. Refs. 7,236). (b) Fructose 1,6-bisphosphatases: Hepatomas express the muscle type rather than normal liver enzyme. Again no developmental form seems to have been looked for (cf. Refs. 6, 7,236). (¢)Glucose-6-phosphate dehydrogenases: Total glucose-6-phosphate dehydrogenase

260 activity increases markedly in rat hepatoma [237] and in rat [238] or human [239] breast carcinoma. In the rat breast the increase results in a new (a third) electrophoretic band. In the human some activity can be observed in this 'cancer' band in the absence of neoplasia. In hepatoma the increased activity is associated with the appearance of four glucose 6-phosphate specific bands of activity not found in normal liver [237]. An extra band was also found in renal tumors. Although the cancer associated bands may represent epigenetic variants, they may also be different genotypes since three different genotypes may exist in mammals. They are: the commonly recognized sex linked, cytoplasmic, glucose 6-phosphate specific type, a somatic, cytoplasmic type with broad substrate specificity [39] and mitochondrial form [240]. Evidently no relevant developmental studies have been done. The presence of a peptide which binds to and alters the electrophoretic behavior of glucose-6-phosphate dehydrogenase suggests observed changes in electrophoretic mobility of the dehydrogenases could in fact be a reflection of changes in the peptide [241]. (d) Enolases: The enolases are a family of dimeric proteins composed from three distinct subunits probably coded at different genetic loci. The fetus expresses the a-subunits, which is replaced in adult tissues by either the/~- or ")'-subunits [242-244]. Kamel and Schwarzfisher [245] also describe 3 isozymic variants and claim that while tumor tissues differ from their adult counterparts fetal tissues do not. The data seem contradictory, but this latter observation, if true, would indicate that fetal and tumor forms diverge. (e) Aldehyde dehydrogenases: Carcinogen induced hepatomas undergo a five fold increase in activity of aldehyde dehydrogenase. This induced enzyme differs from the normal liver enzyme in that it is more stable, a greater proportion is formed in the cytoplasm as opposed to the microsomes, and it can use NADP as well as NAD as a co-substrate. This form was not found in well or poorly differentiated Morris hepatomas, tumors induced in other organs, spontaneous hepatomas, 1 6 - 2 0 day fetal liver, or regenerating liver. It was suggested that the isozyme modification was due to the activation of an 'archeogene', i.e.: a gene repressed for ages [246]. However, it seems possible it would be expressed in early embryogenesis or trophoblastic development.

IIC. Modulation versus primary gene activation (lactate dehydrogenases) Modulation as defined herein is a change in levels of the gene product once a gene is expressed and it is distinguished from primary gene activation. Since gene products are commonly observed to be present at low levels under conditions in which they are norreally not expressed (e.g. a-fetoprotein in adult liver) the difference can be ambiguous and it is not impossible that some of the phenomena previously discussed do not involve changed gene expression but some other modulating effect, for example a decreased rate of degradation. The following paragraphs argue the point that this is the case for the oncological and developmental changes observed in the lactate dehydrogenase isozymes. This isozyme set consists of three distinct gene products: the A (M), B (H) and C subunits. The C subunit is expressed only in testes, but the A and B subunits and their hybrids are expressed in the other adult tissues [6,7,247]. The tendency for both the A and B subunits to be simultaneously expressed carries back into early development. This is perhaps most clearly observed in fish, where not only are both subunits expressed early, but the relative quantity of each varies from species to species [248]. Although thoroughly studied in only a few developing mammalian species the same generalization seems to hold. Thus, whereas the mouse oocyte expresses the B subunits, the activity drops to near zero by the time of implantation [249,250]. At this time a surge of new activity

261 appears which is mainly A type, but is said to include some increase in B subunits [ 2 5 0 255]. Thus both forms may be produced from the beginning; however, the A subunit continues to predominate through development and little, if any, B subunit was observed prior to the 16th day of development [256,257]. Other rodents seem to show a similar developmental pattern [258-260]; tile early fetal rat, in particular, has been shown to express a large excess of A-subunits [261-266]. The trends toward the adult patterns start between the last half to third of fetal development; the B subunit tends to become more prevalent, until just before birth when the A subunit of liver and skeletal muscle undergoes a rapid increase in specific activity [51,256,261-265]. Thus in adult rodent these two tissues characteristically express mainly A4 isozyme, but they do not necessarily repress B subunit synthesis from the fetal level. Other mammalian species show different developmental patterns. The rabbit, while still expressing predominantly A subunits in the early embryo [258,266,267] seems always to h~ve more B subunit than the mouse or rat. The late surge in A subunit activity in the liver does not occur and the adult liver expresses a high proportion of B subunits [262]. The pig seems to follow the rabbit pattern but expresses still higher levels of the B subunit [261]. Human fetal tissues express the B subunit as the major form [260,261, 262,268]. That a hereditary deficiency of B subunits has been observed and seems to be symptomless [269], suggests the enzyme is normally present in excess and it matters little which isozyme is present through the course of development and life. As in the rodent, the human liver undergoes a late surge in A subunit expression [262]. All species show the high A-type activity in skeletal muscle due to the late developmental increase. The general trend in rodent and human neoplasms is to increase the proportion of A subunits, usually due to a net increase in activity (cf. Refs. 6, 7,239, 270). However, this pattern is not universal [271]. Thus in four different lung carcinomas an increase in the fraction of A subunit was observed, but: in two cases the specific activity of both subunits decrease; in one tumor the activities of both subunits is increased; while, in the fourth carcinoma the activity of the B subunit decreased [272]. That non-specific modulation was involved seems evident. Similarly a relative increase in B subunits was observed in a human leiomyoma [273] and rhabdomyosarcoma [67]. Since rhabdomyosarcomas show a decrease in total lactate dehydrogenase activity [274], the changed pattern likely represents a disproportionate loss of the A subunit. This would seem to be a reversal of the late modulation occuring during fetal development. Rat hepatomas sometimes show a small but significant increase in the proportion of the B subunit and the change has been called fetal-like [275-278]. That is, it seems to reverse the late fetal surge of A subunit activity. Indeed, both fetal liver [51] and hepatoma [51,279] have lower specific activity levels than does adult rat liver. Moreover, the slower growing tumors tend to have the lowest specific activities among the hepatomas; while the faster growing hepatomas tend to show an increase due to a greater proportion of B subunits [279]. Again the changes appear to represent modulation. Thus the lactate dehydrogenase system would seem to be one in which the genes coding for the A and B subunits are always turned-on and in which therefore changes in levels are due to modulation. This conclusion has received experimental support by the recent work of Nadal-Ginard [280] who measured lactate dehydrogenase subunit turnover rates in various adult tissues and found the levels expressed correlated inversely to rates of degradation rather than directly to rates of synthesis. It is important to note that the usually stringently repressed C gene has not been reported to be activated in cancer. Since so many different neoplasms have been investi-

262 gated by so many workers, this is good evidence against the idea that gene activation is randonl. liD. Loss oJ'adult forms The re-expression of developmental gene products usually correlates with the loss of those products normally expressed by the tissue of origin. Presumably these two phenomena share common mechanisms. Knox [133] lists 67 adult liver enzyme forms in his Table XVlII. Of these 23 have their total activity reduced more than 95% in fast growing hepatomas. That is, expression was stringently repressed. All 23 of these enzymes were also not expressed in fetal liver. This list of 23 proteins is by no means complete since it excludes several isoforms as well as other proteins. Thus it may be concluded that this phenomena of loss of normal adult gene products by hepatomas is widespread. As a rule slow growing hepatomas repress expression of the same proteins, but to a lesser degree. Values for regenerating liver were reported R~r 19 of the 23 cases [133]. Thirteen of these 19 proteins were also significantly reduced in activity in regenerating liver, indicating that the phenomena is not limited to neoplasia. This is also shown by the facts that the levels of glucokinase and pyruvate kinase-L decline in the preneoplastic carcinogen treated liver [97] ; the levels of several adult forms are reduced in the toxin poisoned liver [94,95] ; and the levels of glucokinase, pyruvate kinase-L and ligandin diminish in proliferating hepatocytes in culture [33]. That the activities and/or levels of these latter two enzymes drop to near zero, indicates that even stringent repression is not limited to neoplasms. Their re-expression when the cultured cells pass back into the quiescent phase indicates that the mechanism responsible for the loss of these enzymes is reversible and therefore probably does not involve a changed cell population, mutation, chromosomal loss, etc. Thus the loss of adult gene products is a phenomenon commonly observed in hepatoma but it is a trait shared by non-neoplastic cells stimulated to divide. Of the remaining 44 adult enzymes listed by Knox [133] which are not completely repressed in hepatomas, 5 (malate dehydrogenase decarboxylation, xanthine oxidase, ornithine transaminase, pyrophosphatase and arginosuccinate synthetase) were reported as being completely repressed in fetal tissue but not in fast growing hepatomas. That 'all of the adult fornls listed in Knox's table are not fully repressed in the fetal liver is not surprising since most of the data is derived at one point late in development. However, it would be expected that the dedifferentiated tumors have reached the limit of their capacity to repress tire activity. In some cases such residual activity may reflect the presence of unknown isoforms. This is the case for adenylate kinase where the adult form is repressed in hepatoma but the residual activity is due to a distinct isozyme which is also the fetal form [281 ]. 111. Conclusions IliA. Gene activation &volving retrodifferent&tion 1. Reversible differentiation as a normal response: At least 29 of the 39 oncodevelopmental proteins listed in Table 1 are expressed late in fetal development in tissues homologous to tile neoplastic ones. That tumors gain these proteins in preference to the large number of other repressed gene products available in the mammalian genome and that a large number of late gene products are lost under the same conditions, suggest that in effect the tumor has retrodifferentiated one step. That is, it has reverted back to a late

263 TABLE 1 ONCO-DEVELOPMENTAL PROTEINS Protein

Stage of development expressed

Neoplasms expressed in

a-t:etoprotein

Yolk Sac Fetal G.I. tract Fetal liver

Germ-cell Upper Gastrol intestinal tract Hepatomas

Fetal liver

Hepatoma

Fetal gut

Rat duodenal adenocarcinoma

Aldolase C

Embryonic liver Fetal muscle

Dedifferentiated hepatoma Rhabdomyosarcoma

Pyruvate kinase K

Fetal liver

Hepatoma

Fetal muscle Fetal brain

Rhabdomyosarcoma Various brain tumors

Brain adjacent to gliomas

Glycogenphosphorylase (low pI form)

Fetal liver Placenta

Hepatoma

Regenerating liver

Hexokinase-II

Fetal liver Placenta

ttepatoma

Regenerating liver Preneoplastic liver Toxin damaged liver

Aldolase A

Non-neoplastic conditions in which normally repressed protein is re-expressed

Regenerating liver Preneoplastic liver Toxin stimulated liver Proliferating hepatocytes in culture Liver cells bordering necrosis Regenerating liver Preneoplastic liver laver of organisms bearing tumors

Regenerative liver Preneoplastic liver Toxin stimulated liver Proliferating hepatocytes in culture Tumor extract injection

Kidney tumors Mammary tumors tlexokinase-III

Neo-natal liver Placenta

CC14-damaged liver Kidney tumors Mammary tumors Rhabdomyosarcoma

k-Glycerol-3phosphate dehydrogenase-anodal form

Embryo, early fetus

Rapidly growing (ascites) forms of tumors

Alcohol dehydrogenase (anodal form)

Fetal liver

Hepatoma

Branched-chain amino acid transferase-IlI

Placental

Hepatomas (mainly rapidly growing)

(not in regenerating liver)

(continued on next page)

264 TABLE I (continued) Protein

Stage of development expressed

Neoplasms expressed in

Non-neoplastic conditions in which normally repressed protein is re-expressed

Glutaminase-K

Fetal liver

ttepatoma Mammary tumors

Regenerating liver

Histaminase

Placental

Various cancers

Carbamylphosphate synthetase I1

General fetal

lfepatoma Mammary tumors

Ornithine Decarb ox ylase (low K m type?)

General fetal

Various

tRNA methylases

Fetal liver

Hepatomas Other tumors

CDPribonucleotide reductase

Fetal liver

flepatoma

Regenerating liver

Thymidine kinase cytoplasmic

Fetal liver Fetal gut

ltepatoma Gastrointestinal tumors

Placenta

Various

Regenerating liver Preneoplastic Phytohaemagglutinin Stimulated lymphocytes

Uridine kinase 11

Fetal liver

Hepatoma Leukemia

(not regenerating liver)

DNA polymerase 7

Fetal liver

Hepatoma

Regenerating liver Preneoplastic liver

dCMP deaminase

Fetal liver

tlepatoma

Regenerating liver

Glucoseamine 6-phosphate synthetase (low pl form)

Fetal liver

Hepatoma

Regenerating liver

ltexoseaminidase (-fast)

Fetal liver

Hepatoma

Regenerating liver CC14 treated liver

5'-Nucleotide phosphodiestcrase-(V)

Cord blood

Sera of hepatoma patients

Alkaline phosphatase placental (Regan) isozyme

Placenta

Many

Alkaline phosphatase hepatoma isozyme

FL Amnionic cells Fetal intestine

Itepatoma

Alkaline phosphatase non-Regan isozyme

Chorion General fetal

Many

Regenerating liver Preneoplastic Growth stimulation in general

Not observed in regenerating liver

265 TABLE I (continued) Protein

Stage of development expressed

Neoplasms expressed in

Non-neoplastic conditions in which normally repressed protein is re-expressed

3,-Glutamyl transferase (fetal form)

Fetal liver

Hepatoma

Regenerating liver Preneoplastic liver

lsoferritins low pI subunits

Placenta Fetal liver

Hepatoma Other tumors

Arylamidase

Placenta

Hepatoma

Carcinoembryonic antigen

Fetal gut

Gastrointestinal cancer others?

CS antigens

Fetal

C-type virus Transformed cells

Embryonic hamster antigen

Embryo

Virus transformed cells

'Antifetal' antigens

Fetal

Various leukemic cells

A 'new' gastroin testinal oncofetal antigen

Fetal colon

Gastric and colonic tumors

Acute lymphocytic leukemic antigen

Fetal thymus

Leukemic cells

Leukemic antigens

Fetal hematopoietic cells

Leukemic cells

Embryonic pancreatic antigen

Embryonic (fetal?) Pancreatic antigen

Pancreatic tumor

Transformed Mouse L-cell antigen I

Early embryogenesis

Several

Antigen II

Trophectoderm

Hepatoma and teratoma

Oncofetal antigen

Fetal brain and other fetal tissue

Skin cancers Some others

Human chorionic gonadotropin

Trophoblastic

Various

Cultured cells, other proliferating adult cells Proliferating bone marrow cells

Ova

Cultured skin

fetal stage. A similar retrogression occurs in non-neoplastic proliferative states, including: regenerating liver, toxin poisoned liver, carcinogen induced hyperplastic but preneoplastic liver, and in liver cells in culture. Under these non-neoplastic conditions, the adult gene products reappear, and the fetal ones disappear when proliferation ceases. The cancer cell does not redifferentiate, presumably because enhanced proliferation never stops. Although the majority of observations involve hepatomas, evidence for similar regressive changes has been observed in muscle, brain, gastrointestinal, lymphoid, skin and pan-

266 creatic tissues. In at least lymphoidal, gastrointestinal and skin tissues some fetal genes have been observed to be re-expressed when adult cells were stimulated to proliferate. Thus it would seem that proliferation associated retrodifferentiation is a generalized phenomenon. Indeed some oncofetal forms, such as ornithine decarboxylase and thymidine kinase are more widely recognized as growth stimulated proteins. The idea of retrodifferentiation in cancer is not new: it goes back over 100 years and seemingly can be attributed to Durant-e [ 1,97,282-284]. That reversible differentiation is a normal response of cells has been clearly expressed less often. Yet, it is logical to suppose that such a phenomenon is required to permit the cell to carry out its specialized functions without losing the capacity to regenerate when damaged. Uriel [285] had previously arrived at a similar conclusion using different types of evidence while Leffert and his coworkers have called attention to the close correlation between c~-fetoprotein synthesis, and proliferation in vitro and in vivo [ 2 8 6 - 2 8 8 ] . Since the growth related and adult functions of the cell require different sets of proteins, the alternatives to retrodifferentiation-redifferentiation are: to simultaneously maintain both sets of gene products with their functional intermediates, which would be very expensive in terms of the energy and space needed; to give up the capacity to heal after injury; or, to maintain a population of primative cells having the potential of becoming activated and thereby filling the breech. Only the stem cell concept can be considered a logical alternative to retrodifferentiation and even it would be less efficient since a population of non-functional cells needs to be maintained. Also, the reversible changes between adult and fetal gene products observed in a cultured hepatocyte system [33] is particularly difficult to explain in terms of stem cell proliferation since it needs to be argued that an unobservably small number of stem cells completely dilutes out the hepatocyte population and then differentiates. To explain the loss of L-isozyme activity on the basis of dilution when expressed on a per plate basis [33], it is required that within four days the whole cell population switched from at least 95% mature hepatocytes to at least 95% stem cells. Assuming a stable population of hepatocytes, this would require a 380 fold increase in the stem cell population (i.e., 5 stem cells in an original population of 100 would need to increase to 1900 cells). This would be equivalent to a generation time of about 11 h which, while conceivable, is unusually rapid for a mammalian cell. It is also hard to use the stein cell concept to explain the appearance of fetal gene products in stimulated lymphocytes. Moreover, tile simultaneous presence of the three different subunits of aldolase in a single hepatoma cell as well as the presence of AB hybrids are observations which suggest the isozyme pattern arises via retrodifferentiation rather than by abnormal development of a stem cell [52]. Thus it seems fair to conclude that proliferative control of gene expression is the most viable mechanism available, best able to explain the observations under all conditions observed. This does not necessarily preclude activation of stem cells under some conditions. 2. Post-transcriptional regulation of the rate of protein synthesis: If expression of some late fetal and adult genes is in somc way coupled to the proliferative status of a cell, it may follow that expression of the genes involved in the switch-over is regulated by mechanisms relatively unique to them. It is reasonable to speculate that these genes are primarily controlled at the translational level, provided there is also a secondary feedback loop to the genetic level which would function to avoid accumulation of unused mRNAs. Such a mechanism may help explain the complexity of eucaryotic initiation and o f mRNA processing, the relatively free reversibility involved in the expression of these

267 genes, and the tendency the genes involved to be 'leaky'. More specific evidence for this concept is presented in the following paragraphs. The most widely accepted specific example of translational control is the heroin regulated control of protein synthesis in reticulocytes. Heroin supports synthesis by inhibiting the activation of a protein kinase, which acts to inactivate an initiation factor (elF2) by catalyzing its phosphorylation. This sytem has widespread occurrence [289-293]. A more relevant system involves catalase synthesis in liver. Catalase is an adult enzyme often lost in hepatomas and hepatoma is alleged to be a rich source of a cytoplasmic factor which inhibits the translation of catalase mRNA by binding with polyribosomes synthesizing catalase. Normal liver, in contrast, has an excess of a factor antagonistic to the hepatoma factor [294-295]. Another relevant specific example is ornithine decarboxylase, the expression of which can be induced in enucleated cells [296-298]. Ferritin which undergoes oncodevelopment isoprotein shifts seems also to be regulated at the translational level; iron causes the aggregation of free ferritin subunits, which otherwise complex with message and inhibits its translation [299]. Relevant but more generalized phenomena explained by this hypothesis include the following. Some cells transformed in vitro undergo large shifts in protein products [300]. That a similar large shift in protein products was not noted when embryonic Syrian hamster cells were transformed [301] could be explained by the proposed hypothesis, since the embryonic cells would presumably be in the proliferative state prior to transformation. Moreover, the observation that when such shifts occur they are accompanied by the synthesis of relatively few new mRNA species even though the total mRNA content is increased [302-304], would also be predicted by this hypothesis. The very relevant molecular hybridization studies of Fausto et al. [305], led to the conclusion that widespread activation of repressed genes in the repetitive portion of the genome is unlikely to occur in regenerating as opposed to quiescent liver, but rather that the transport of RNA molecules from the nucleus to the cytoplasm is altered. That is: these workers concluded, as predicted by the concepts promulgated in this review, that most of the changes in repetitive RNA transcripts occurring in response to hepatectomy reflect changes in RNA concentration rather than qualitative alterations in gene expression. In addition to these more mechanistically revealing studies there are examples of proliferation-quiescent regulated proteins being more influenced by inhibitors of translation than by inhibitors of transcription. Indeed, on the basis of such data Pitot [306] promulgated the concept that changed messenger stability is of prime importance in explaining altered regulation in cancer, while Roth [307] pointed out that growth stimulated enzymes seem refractory to transcriptional inhibitors. Examples of proliferative dependent hepatoma enzymes, whose expression was found to be relatively insensitive to transcriptional blockage, include: deoxycytidylate deaminase [307], thymidine kinase [306, 307], thymidylate kinase [307] and ribonucleotide reductase [162]. The expression of ornithine decarboxylase in non-neoplastic growth stimulated cells was also found to be insensitive to actinomycin [308-310]. The observation that cyclic AMP induced ornithine decarboxylase is less susceptible to actinomycin D inhibition, than is the dexamethasone induced enzyme [309] may be particularly significant in view of the previously mentioned possible role of isozymes. Similarly, while spleen ribonucleotide reductase was susceptible to inhibitors of transcription and translation, the tumor enzyme was only sensitive to the latter [162]. As would also be expected from the hypothesis, the expression of adult proteins in well-differentiated hepatomas becomes more sensitive relative to normal liver to translational inhibitors, examples include: tryptophan pyrrolase

268 [306], threonine dehydrase [306], tyrosine transaminase [306] and serine dehydratase in the Reubner H-35 but not in the Morris 5123 hepatoma [311]. These observations could suggest that as a consequence of transformation the mRNAs progressively become more labile, or that they become progressively less efficiently translated. In either case when the effect reaches a critical level, measurable expression ceases and the tumor is said to have dedifferentiated with respect to the missing function. Accumulation of untranslated mRNA would trigger a feedback mechanism inhibiting further transcription of that message. If the critical point is not reached and the effect is brief, as may be the case in regenerating liver, the net change will be minimal. The CC14 induced increase of glucose-6-phosphate dehydrogenase in liver was shown by immunological techniques to be due to de novo protein accumulation and the accumulation was prevented by cycl0heximide but not by actinomycin [95]. Thus at least this non-neoplastic proliferative type response may be regulated at the translational level.. Lodish [312] developed a mathematical description of the effects of various conditions on the rates of translation of mRNA and predicted that a change which decreases the probability of initiation would have a more pronounced effect on the poorer initiation species of mRNA. This concept has received some experimental verification. An example of particular relevance was the use of Lodish's concept to explain the rapid cessation of production of several proteins without a concommitant loss in their translatable messengers, when Dictyosteliurn discoideurn is induced to differentiate [313]. This idea suggests that a group of proteins could be simultaneously affected if their affinity for the initiation complex was altered. Moreover, it could explain the type of progressive phenomena often observed. One signal could either cause reciprocal effects (e.g.: increasing the affinity of fetal forms and decreasing the affinity of adult forms) or two or more separate signals could be involved. Moreover a limited number of signals could shift the expression of many products. Even if such a relatively simple kinetic mechanism did account for the observations, it would still be necessary to identify: the signal(s), the molecular alteration caused by a signal and the mechanism by which the effect was fed back to the gene. The feedback loop could involve inactive messenger ribonucleoprotein (mRNP) particles. Such particles have been observed in several cell types and they have been called informosomes [314]. It seems likely that RNA-protein complexes have more than one function. For example: some may be required for translation, some may inhibit transloation and some may serve to hold the mRNA in 'protective storage' and/or promote transport of the mRNA, In some eggs such particles do act as storage forms and active mRNA becomes released after fertilization (cf. Refs. 314, 315). Similar mRNP complexes have been reported for the e~-globin [316]. Proteins that may be part of this complex are released from reticulocyte polysomes by EDTA [317,318] or puromycin [319]. These proteins appear to be bound to the poly(A) sequence and the complex is relatively refractory to nuclease digestion (cf. Ref. 320). Thus it seems possible that in the present context these particles could serve as a storage form of transcribed but untranslatable mRNAs. The accumulated cytoplasmic mRNP could, then, in some way inhibit transport of nuclear messenger or pre-messenger and then transcription itself. These concepts are supported by the following observations: An RNA-protein complex able to switch the expression of the K- and L-pyruvate kinase isozymes, has been isolated from tumor cells [ 103]; the stimulation of growth by serum in 3T3 cells is accompanied by the association of pre-existing cytoplasmic poly(A) rich mRNA with ribosomes [321]; changes in ratios of poly(A) to non-adenylated message are found in the growing liver for c~-fetoprotein and albumin

269 [322]; and total ~-fetoprotein mRNA declines during development but some mRNA is present at all stages, even in the adult [322]. 3. Secondary modulation: In every documented case but one (uridine kinase II)some degree of expression of oncofetal proteins was observed in regenerating liver (Table l). However, the change is always much smaller than for hepatomas having similar growth rates. The effect has therefore sometimes been dismissed as being insignificant. In a similar fashion, the decrease in adult forms is less than for fast growing hepatomas. The statistical result of the sum of these two quantitative differences led Knox [133] to group regenerating liver with normal liver rather than with hepatoma. In contrast, the qualitative relationships point to the similarities between regenerating liver, other proliferative states and hepatoma. Nonetheless, there must be an explanation for the quantitative differences; obviously it is not the rate of proliferation since regenerating liver grows more rapidly than most tumors [ 1 3 3 ] . Slow growing tumors also express oncofetal proteins at a low level. In many cases there is a rather abrupt increase to the levels expressed in the more rapidly growing dedifferentiated tumors. Log-log plots of the activity of several enzymes against growth rates of hepatomas are sigmoidal or almost sigmoidal. These enzymes include: ribonucleotide reductase, aspartate transcarbamylase, total DNA polymerase, thymidylate kinase, thymidylate synthetase, dCMP deaminase, aspartate transcarbamylase and dehydroorotase [162,163]. Sigmoidal log-log plots would remain sigmoidal if plotted on a linear scale. Thus it seems that some type of cooperative effect may be in operation. That is, the much higher levels expressed in the more rapidly growing tumors is due to the activation of some type of amplification phenomenon. Presumably this amplification does not occur in regenerating liver. It is possible that gene duplication is the method of amplification used by the dedifferentiated tumors [323,324], but such amplification is not necessarily a neoplastic dependent phenomenon. Fetal tissue can have activity levels as high or higher than the most rapidly growing dedifferentiated tumors. A case in point is CDPribonucleotide reductase [162]. Moreover, the CC14 damaged liver also expresses fetal proteins at relatively high levels in a manner perhaps even divorced from proliferative activity [95]. Thus an amplification mechanism may be activated by CC14. Although the literature reviewed offers no obvious clue concerning possible mechanisms responsible for the suggested, separate amplification effect, the highly modulated, well studied lactate dehydrogenase-A subunit may provide some examples. As discussed, expression of this protein is amplified in muscle during late development and this late modulation is reversed in rhabdomyosarcomas. The expression of this subunit is also modulated by oxygen. High oxygen tension levels inhibit a shift toward increased A-subunit expression while low tension promotes A-subunit expression. These effects are not mediated by pH or lactate and the increase is prevented by inhibitors of transcription or translation [325-329]. That the oxygen effect is negated by chelators has led to the suggestion that a metal ion acts as a mediator [329]. Perhaps similar mechanisms exist for other proteins. It has also been noted that the high levels of activity sustained in adult muscle appears to be a function of its long half-life [280]. Conceivably control of levels by modulation generally lies in changed rates of degradation rather than synthesis. The hypothesized amplification effect could also occur at the same site as the activation effect. If so modulation and primary activation could be a continuum. For example: fetal enzymes, such as hexokinase I, branched chain amino acid transferase I, etc.. which continue to be expressed at a low but significant level in adult liver and then become

270 re-expressed at high levels in hepatomas may be responding to the same mechanisms as those proteins more fully repressed in the adult. In terms of the kinetic mechanisms for regulating translation discussed above~ it need only be postulated that such partially repressed proteins retain higher affinity for the initiation complex, than do their more completely excluded brethren. It is also possible that all three mechanisms for amplifying levels of expression, i.e.: gene duplication, decreased rates of degradation and increased affinity ofmRNA, coexist to different degrees in different cells.

IIIB. Gene activation not involving retrodifferentiation Eight of the oncodevelopmental proteins listed in Table I were not found to be late fetal proteins of the homologous tissue. These could be subdivided into four classes, early embryonic, amnionic and placental gene products and products seemingly not expressed during development. Since these latter forms appear outside the usual definition of development, they are called ectopically expressed forms. Although some of these products may yet be found in the homologous fetal tissue, it seems probable that they represent examples of genes not being expressed directly before the adult genes become expressed. Neither do these gene products seem to be expressed in proliferation stimulated nonneoplastic cells. Thus it seems likely that the mechanism involved in their expression differs from that of the retrodifferentiation type. These non-fetal oncodevelopmental enzymes will be briefly discussed below. 1. Oncoembryonic proteins: This represents a usually unrecognized type of gene product that can only be distinguished from onco-fetal forms with some ambiguity. The primary criteria for their classification is that the gene involved be expressed during embryogenesis but be essentially silent for a period before birth. Three gene products are tentatively classified as oncoembryonic forms, liver aldolase C, the labile form of L-glycerol-3phosphate dehydrogenase and transformed mouse L-cell antigen. The adult liver aldolase-B isozyme replaces the late fetal A form. However, the C isozyme was present earlier in fetal liver. Although gaps in the known development sequence exist, it is postulated that the A form replaced the more primordial C type. This conclusion is consistant with the apparent lack of expression of the C isozyme in non-neoplastic proliferating liver and in slower growing hepatomas. That mainly A and C isozyme is expressed in highly dedifferentiated tumors suggests that in these tumors another level of control is removed. However, the C isozyme is the homologous fetal form in muscle and therefore, is an oncofetal form in this tissue. It may be that expression of the B subunit in liver somehow decreases the affinity of C subunit messenger for the initiation complex in liver and re-expression of the C subunit requires inactivation of B-subunit message. Thus, in fact, the basic mechanism involved could remain a matter of the relative affinity for the initiation complex. In this way the same mechanism could explain pseudo-sequential effects in neoplasia as well as the sequential order of expression which occurs during normal development. In somewhat analogous fashion it has been reported that the labile form of L-glycerol3-phosphate dehydrogenase ceases to be expressed 13 days after conception in mouse liver [130] and does not appear in hepatomas and other slow growing tumors, but it can be expressed in the same tumors when they are growing in vitro or in the ascites form. That is, it seems to be on the verge of being expressed in tumors, requiring only an added stimulus to be expressed. This stimulus may be an increased proliferative rate. This could also suggest that the type of kinetic regulation postulated by Lodish is functioning as a

271 continuum and these carcinoembryonic forms have a relatively low efficiency with respect to initiation in the late fetus and adult. 2. Carcinoplacental proteins: Five peptides were expressed in the developing or mature placenta and were not found during embryogenesis. These are: branched chain amino acid transferase-III, histaminase, the Regan-isozyme of alkaline phosphatase and its alleleotypes, arylamidase and the/3-subunit of human chorionic gonadotropin (HCG). It is not at all clear why these gene products should be expressed in cancer. These peptides seem to be expressed most often in endocrine and colon cancers. Whatever the case, they seem not to be expressed in non-neoplastic conditions, but they can be induced in cell lines in which they are not normally expressed, e.g.: the treatment of dedifferentiated culture liver cells with carcinogens causes expression of branched-chain amino acid transferase-IlI, and the Regan isozyme can be induced in several cell lines by butyrate, steroids or hyperosmolarity. Thus, there may be some characteristic shared by these gene products, which permits their genes to be more readily activated. 3. An oncoarnnionic protein: One gene product was recognized as a possible oncoamnionic form. This is the hepatoma isozyme of alkaline phosphatase. There is some ambiguity, since this isozyme is not produced in normal term amnion, but only in cultured FL-amnionic cells. Moreover it is expressed in fetal intestine. However, it seems not to be expressed in fetal liver, which of course is usually the homologous developmental form. 4. Ectopically expressed adult proteins: Recognizing the artificiality of presuming that development stops at birth, it becomes necessary to term oncological gene products not yet observed in developmental tissues as being ectopically expressed adult ones. The word adult is used in an operational sense to define products which are not early developmental or late fetal ones. That is: aside from recognizing that oncofetal gene expression represents a unique response, there is no a priori reason why the mechanisms of activation of abberant adult products need differ from those operating for oncoplacental or any other non-fetal developmental form. Thus despite the fact, four possible examples of ectopic protein production by cancer cells were listed (namely: peptide hormones, intestinalization of gastric tissue, phosphofructokinases and creatine kinases), it may be possible to identify common themes. In at least the cases of the peptide hormones and the intestinalized gastric cells, it would appear that the ectopic response is limited to cancers arising from a limited number of cell types. Moreover, the relationship between gastric and intestinal cells is particularly close. Both have essentially the same developmental ancestry until the last step in differentiation. Thus it would seem probable that either would retrodifferentiate to essentially the same form. If the cells then redifferentiate under somewhat abnormal conditions they might take the wrong route. Thus, since normal cells commonly retrodifferentiate under the influence of neoplasms, or prior to the neoplastic transformation, it seems reasonable to suppose that non-neoplastic gastric cells in the area of overt carcinoma cells would undergo a reversible retrodifferentiation and may in redifferentiating take a wrong turn, and thereby express a package of gene products normally expressed only by their first cousins in the mucosa. Whereas this explanation requires the reversible redifferentiation concept, the other cases do not since the ectopic gene product is expressed by the neoplastic cells themselves. If a common ancestry is the explanation in these cases, it would seem that the mechanism would involve either the pealing back off successive layers of 'repression', perhaps as postulated for the embryonic gene products, or a concept of stem-cell activation,

272 such as Potter's [63,97] idea of oncogony being blocked ontogeny. It is also possible that the examples do not involve gene activation per se. That is: the activated genes were never truly repressed and the events involve posttranslational phenomena. Examples of observations consistant with this possibility were presented in the discussion of the specific gene product. Also as previously considered, it is possible that developmental phenomena are involved. This may particularly be true in the cases of phosphofructokinase, where a true developmental study has not yet been conducted. With respect to creatine kinase, the developmental nature of the transient expression of the M subunit by liver was de-emphasized because the B-subunit is so clearly predominant in slightly earlier phases of development. However, the transient nature of the expression of the M-subunit, may be similar to the behavior of isozyme II and III of hexokinase or the C subunit of aldolase, i.e.: creatine kinase could perhaps also be classified as an oncodevelopmental form.

IIIC. Relationship to neoplasia How does oncodevelopmental gene expression relate to neoplasia? It is not possible to provide a direct answer to this question. However, it would seem reasonable to refer the retrodifferentiation-redifferentiation expression to the 'promotor' phase of carcinogenesis (also see Ref. 306). The latter requires that the target cells be maintained in constant cycling and that up to a certain point can revert to normal. It would also be reasonable to suggest that cells which are constantly cycling have a statistically better chance to become neoplastic. On the other hand, the ectopic synthesis, for example, oftrophoblast protein is a phenomenon which may contribute to the malignant phenotype and thus may have a somewhat different significance. Genes which may participate in retro-redifferentiation may be expressed in neoplasia or organs other than the ones which are usually studied. For example, 3,-glutamyltransferase ordinarily associated with preneoplasia and neoplasia of liver is expressed in bronchogenic cancer and testicular teratocarcinoma, a-Fetoprotein is associated with germ cell tumors as well as hepatoma. Clearly this chapter points to the fact that in order to interpret the significance of oncodevelopmental gene expression, it will be necessary to understand the controls of gene expression during normal development and differentiation. The advantages of investigating enzymes as the gene products rather than undefined antigens are illustrated in this chapter. It is also our view that it is important to identify developmental gene products in terms of their developmental phase and specific tissue of origin rather than to homogenize their significance into the term fetal antigens.

Acknowledgements Work cited from the authors' laboratories was supported by funds from The American Diabetes Association, NIH Grant PHS 5-R01-CA 07883-12 (KHI) and from NIH Grant PHS 2-R01-CA 21967-03 (WHF). The authors would also like to thank Sherry McGee and Mary Richey for checking the bibliography and Elfriede Whiteside and Rhonda Gasper for typing the many drafts.

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