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Oncofetal proteins in marine animals
Comp. Biochem. Physiol., Vol. 61B, pp. 499 to 500 © Per~tamon Press Lid 1978. Printed in Great Britain
0305-0491/78/1115-0499502.00/0
ONCOFETAL PROTEINS IN MARINE ANIMALS* ALBERT C. SMITH Oceanic Institute, Makapuu Point, Waimanalo, HI 96795, U.S.A. (Received 10 April 1977) Abstract--1. Oncofetal proteins are produced by developing mammalian and avian fetuses. They are
also produced in adults with certain types of disease, particularly malignancy. 2. In this study, three common marine animals were tested for the presence of two oncofetal proteins, alpha fetal protein (AFP) and carcinoembryonic antigen (CEA). 3. The animals were two fish species, 771apia mossambica and Chanos chanos, and one sea cucumber species, Holothuria cinerascens. The latter, as an echinoderm, is widely considered to be close to the vertebrate evolutionary line. 4. Only CEA (or CEA-like substance) could be quantitatively identified. It was found in one of the fish species and the sea cucumber, in which it was in highest concentration. The presence of CEA or CEA-like substance in these animals indicates it is evolutionarily old. 5. The finding of CEA or CEA-like substance in the sea cucumber suggests: (a) In some malignant as well as nonmalignant disorders, there is not only developmental but also phylogenetic regression. In short, pathology (like ontogeny) may recapitulate phylogeny. (b) The sea cucumber may provide a readily available source of CEA or CEA-like substance for production of test antisera and cancer research.
INTROD UCTION Oncofetal proteins have in recent years been the subject of a great deal of basic and clinical medical research, articles, and symposia (Anderson & Coggin, 1974; Hirai & Alpert, 1975; Fishman & Sell, 1976; Gold et al., 1977). The great interest has been due principally to a high association of these proteins with certain types of malignancy and, therefore, their use (1) as clinical indicators of malignancy; and (2) for identifying any recurrence of malignancy in the patient under treatment for the disease. Oncofetal proteins were also found as normal components of developing mammalian and avian fetuses (Sanders & Kline, 1975, 1977). It, therefore, appears that in certain malignancies and some nonmalignant disorders there is an embryological regression resulting in the production of oncofetal proteins, i.e. a recapitulation of ontogeny in pathology. Since these proteins are normally produced by the fetus, they might also be phylogenetically old in accordance with Haeckel's biogenetic law which states, in brief, that ontogeny recapitulates phylogeny. This law was mainly derived from studies of anatomical structures with little attention given to chemical substances. A full discussion of this law can be found in Gould (1977). The present research was conducted to test the possibility that two of the most clinically useful oncofetal proteins, alpha fetal protein (AFP) and carcinoembryonic antigen (CEA), or substances related to these, might be phylogenetically old. The tests were carried out on the coelomic fluid of an invertebrate, a sea cucumber species, and on the serum or plasma of two lower vertebrates, both fishes. As a member of the class Holothuroidea (Borradaile et al., 1961) and
phylum Echinodermata (Kukalowi-Peck, 1973; Barrington & Jefferies, 1975), the sea cucumber is widely considered to be close to the evolutionary line leading to man and the other vertebrates.
* Oceanic Institute Contribution No. 78-144. 499
MATERIALS AND METHODS
The fish species studied were the tilapia, Tilapia mossambica, and the milkfish, Chanos chanos. The sea cucumber species was a common Hawaiian variety, Holothuria cinerascens. All animals, which had been feeding, growing, and behaving normally in circulating seawater aquaria, appeared healthy. Fish For the AFP tests, blood was aspirated from the haemal arch of six tilapia and the heart of three milkfish. It was collected as pooled samples from each species. Serum was obtained by allowing the blood to clot for 1 hr at ambient temperature (25°C), "rimming" the clot with an applicator and lightly centrifuging. For the CEA tests, blood was obtained by the same methods from six tilapia and five milkfish. The blood from each species was collected as pooled samples in EDTA Vacutainers (Becton-Dickinson, Rutherford, N J, Catalog No. 3206Q). Plasma was obtained by centrifugation. Sea cucumbers For the AFP tests, coelomic fluid from five individuals was aspirated and pooled in test tubes. For the CEA tests, coelomic fluid from these same individuals was pooled in EDTA Vacutainers. All samples were centrifuged to remove coelomocytes and then, to decrease salt concentration, subjected to overnight dialysis in the refrigerator (5°C) against a solution of 0.9 g ~ NaCI. Testing of samples All samples were tested for AFP and CEA at a commercial medical laboratory. A counterelectrophoretic procedure was used for the AFP test; and the CEA-Roche assay, a radioimmunoassay method was used for the CEA test (Bio-Science Laboratories 1976).
ALBERT C. SMITH
500 RESULTS
AFP test results for all species were reported to be under 25 ng/ml. For CEA, the test results were as follows: tilapia, under 1 ng/ml; milkfish, 1.5 ng/ml; sea cucumber, 13.2 ng/ml. DISCUSSION AND CONCLUSIONS
The normal level for AFP in human males and nonpregnant females occurs below 25ng/ml (BioScience Laboratories, 1977). The values reported in the fishes and sea cucumber are comparable to this level. An absolute value for AFP (or AFP-like substance) in these animals may exist at levels below the sensitivity of current methodology. Studies on the CEA levels in humans (HoffmannLa Roche, 1973) have indicated that values between 0-2.5 ng/ml are very common in healthy people. However, levels above 10ng/ml are almost never found except in association with certain types of malignant disease, particularly colorectal cancer, pancreatic cancer and pulmonary cancer. Thus, the CEA value of 13.2 ng/ml in the sea cucumber is high by human standards. Although the test results for CEA did not give an absolute reading in tilapia, definite values were obtained for the milkfish and sea cucumber. The identification of CEA or CEA-like substance in the lower marine forms indicates that it is evolutionarily old. The concentrations of CEA or CEA-like substance in the three animals studied suggest a possible evolutionary pattern. Tilapia, with the lowest value, is considered higher on the evolutionary tree (Lagler et al., 1977) than the milkfish, a primitive teleost, that had a higher value. Sea cucumber, with the highest CEA value is, of course, more primitive than either fish. From the three species studied, it is possible that concentrations of CEA are higher in more primitive animals. Although the proportion of humans showing a CEA level as high as the sea cucumber is greatest in association with certain malignancies, elevated levels may also occur in a number of nonmalignant disorders (Hoffmann-La Roche, 1973). Thus, in a variety of pathological conditions, there seems to be a tendency for reappearance of proteins seen not only in normal fetal development but also in distant ancestral relations such as the sea cucumber. Stated as a modification of Haeckel's biogenetic law: pathology, like ontogeny, may recapitulate phylogeny. Another example of this concept is myeiofibrosis, a condition in which the mammalian kidney takes up the primitive function of hematopoiesis (Smith, 1978). That proteins of early development can reappear in the adult in association with disease has become familiar in medicine through the many research studies and increasingly widespread use of oncofetal protein tests as aids to diagnosis and management. However, the concept of these fetal and pathologyassociated proteins having a phylogenetic component does not appear to be well known. From a practical point of view, it might also be noted that the sea cucumber provides a readily available source of CEA or a similar substance. This source of the antigen might facilitate production of CEA test antisera and open up new opportunities for cancer research utilizing marine invertebrates. For
example, a basic question that awaits this research is what is the function, if any, of CEA or CEA-like substance in the sea cucumber?
Acknowledgements--1 would like to thank Ms Herlinda M. Broadgate, Pathology, Research Assistant, and Mr David E. Coleman, Jessie Smith Noyes Foundation Fellow, Oceanic Institute, Waimanalo, Hawaii, for excellent technical services: and Dr Colin E. Nash, Director, Oceanic Institute and Dr Jonathan Morse, Assistant Professor of English, University of Hawaii, Honolulu, for valuable criticism of the manuscript. This research was supported under a grant to the Oceanic Institute (No. AID/ TA-C-1189, on "Research in artificial propagation of milkfish") from the United States Agency for International Development, as part of their program in marine pathology.
REFERENCES ANDERSON N. G. & COGGIN J. H. JR (Co-chairmen) (1974) Third Conference on Embryonic and Fetal Antigens in Cancer, Hyatt Regency Hotel, Knoxville. Tennessee, November 4-7, 1973. Cancer Res. 34, 1767-2142. BARRINGTON E. J. W. & JEFFERIES R. B, S. (Eds) (1975) Protochordates. Syrup. zool. Soc. Lond., No. 36, l-16, 105 127, 253-318. Academic Press, New York. q-SCIENCE LABORATORIES (1976) The BiD-Science Handbook. Specialized Diagnostic Laboratory Tests, l l t h edn. BiD-Science Laboratories, Van Nuys, CA. BIO-SCIENCE LABORATORIES(1977) BiD-Science Laboratories Research Notebook, No. 1308. BiD-Science Laboratories, Van Nuys, CA. BORRADAILE L. A., Porrs F. A., EASTHAM L E. S. & SAUNDERS J. T. (1961) The Invertebrata, 4th edn. (revised by KERKUT G. A.). Cambridge University Press, London. FISHMAN W. H. & SELL S. (Eds) (1976) Onco-Developmental Gene Expression. Papers presented at a conference sponsored by the International Research Group for Carcinoembryonic Proteins, San Diego, CA, May 29-June 1, 1976. Academic Press, New York. GOLD P., GOLDENBERG D, M., HANSEN H. J., HERBERMAN R. B., NEVILLE A. M., SNYDER J. & ZAMCHECK N. (Programming Planning Committee) (1977) International Conference on the Clinical Uses of Carcinoembryonic Antigen, College of Medicine, University of Kentucky, Lexington, June 1-3, 1977. GOULD S. J. (1977) Ontogeny and Phylogeny, pp. 1-501. The Belknap Press of Harvard University, Cambridge, MA. HIRA; H. & ALPERT E. (Eds) (1975) Carcinofetal proteins: biology and chemistry. Ann. N.Y. Acad. Sci. 259, 1-452. HOFFMANN-LA ROCHE (1973) Third Conference, Carcinoembryonic Antigen CEA Test Collaborative Study. Hoffman-La Roche Inc., Nutley, NJ, April 21, 1973. KUKALOV/~-PECK J. (1973) A Phyloyenetic Tree of the Animal Kingdom (1ncludin9 Orders and Higher Categories ). Publications in Zoology, No. 8. National Museum of Natural Sciences, National Museums of Canada, Ottawa. LAGLER K. F., BARDACH J. E., MILLER R. R. & PASSINO D. R. M. (1977) Ichthyology. 2nd edn. John Wiley, New York. SANDERS B. G. & KLINE K. (1975) Fetal-tumor membrane associated antigens:genetic and immunological implications. Trans. Am. Microsc. Soc. 94, 470-479. SANDERS B. G.. & KLIr,~ K. (1977) Expression of serum proteins in the developing chick embryo. Comp. Biochem. Physiol. 58, 97-101. SMITH A. C. [1978) A proposed phylogenetic relationship between sea cucumber Polian vesicles and the vertebrate lymphoreticular system. J. Invert. Pathol. 31, 353-357.
0305-0491/78/1115-0499502.00/0
ONCOFETAL PROTEINS IN MARINE ANIMALS* ALBERT C. SMITH Oceanic Institute, Makapuu Point, Waimanalo, HI 96795, U.S.A. (Received 10 April 1977) Abstract--1. Oncofetal proteins are produced by developing mammalian and avian fetuses. They are
also produced in adults with certain types of disease, particularly malignancy. 2. In this study, three common marine animals were tested for the presence of two oncofetal proteins, alpha fetal protein (AFP) and carcinoembryonic antigen (CEA). 3. The animals were two fish species, 771apia mossambica and Chanos chanos, and one sea cucumber species, Holothuria cinerascens. The latter, as an echinoderm, is widely considered to be close to the vertebrate evolutionary line. 4. Only CEA (or CEA-like substance) could be quantitatively identified. It was found in one of the fish species and the sea cucumber, in which it was in highest concentration. The presence of CEA or CEA-like substance in these animals indicates it is evolutionarily old. 5. The finding of CEA or CEA-like substance in the sea cucumber suggests: (a) In some malignant as well as nonmalignant disorders, there is not only developmental but also phylogenetic regression. In short, pathology (like ontogeny) may recapitulate phylogeny. (b) The sea cucumber may provide a readily available source of CEA or CEA-like substance for production of test antisera and cancer research.
INTROD UCTION Oncofetal proteins have in recent years been the subject of a great deal of basic and clinical medical research, articles, and symposia (Anderson & Coggin, 1974; Hirai & Alpert, 1975; Fishman & Sell, 1976; Gold et al., 1977). The great interest has been due principally to a high association of these proteins with certain types of malignancy and, therefore, their use (1) as clinical indicators of malignancy; and (2) for identifying any recurrence of malignancy in the patient under treatment for the disease. Oncofetal proteins were also found as normal components of developing mammalian and avian fetuses (Sanders & Kline, 1975, 1977). It, therefore, appears that in certain malignancies and some nonmalignant disorders there is an embryological regression resulting in the production of oncofetal proteins, i.e. a recapitulation of ontogeny in pathology. Since these proteins are normally produced by the fetus, they might also be phylogenetically old in accordance with Haeckel's biogenetic law which states, in brief, that ontogeny recapitulates phylogeny. This law was mainly derived from studies of anatomical structures with little attention given to chemical substances. A full discussion of this law can be found in Gould (1977). The present research was conducted to test the possibility that two of the most clinically useful oncofetal proteins, alpha fetal protein (AFP) and carcinoembryonic antigen (CEA), or substances related to these, might be phylogenetically old. The tests were carried out on the coelomic fluid of an invertebrate, a sea cucumber species, and on the serum or plasma of two lower vertebrates, both fishes. As a member of the class Holothuroidea (Borradaile et al., 1961) and
phylum Echinodermata (Kukalowi-Peck, 1973; Barrington & Jefferies, 1975), the sea cucumber is widely considered to be close to the evolutionary line leading to man and the other vertebrates.
* Oceanic Institute Contribution No. 78-144. 499
MATERIALS AND METHODS
The fish species studied were the tilapia, Tilapia mossambica, and the milkfish, Chanos chanos. The sea cucumber species was a common Hawaiian variety, Holothuria cinerascens. All animals, which had been feeding, growing, and behaving normally in circulating seawater aquaria, appeared healthy. Fish For the AFP tests, blood was aspirated from the haemal arch of six tilapia and the heart of three milkfish. It was collected as pooled samples from each species. Serum was obtained by allowing the blood to clot for 1 hr at ambient temperature (25°C), "rimming" the clot with an applicator and lightly centrifuging. For the CEA tests, blood was obtained by the same methods from six tilapia and five milkfish. The blood from each species was collected as pooled samples in EDTA Vacutainers (Becton-Dickinson, Rutherford, N J, Catalog No. 3206Q). Plasma was obtained by centrifugation. Sea cucumbers For the AFP tests, coelomic fluid from five individuals was aspirated and pooled in test tubes. For the CEA tests, coelomic fluid from these same individuals was pooled in EDTA Vacutainers. All samples were centrifuged to remove coelomocytes and then, to decrease salt concentration, subjected to overnight dialysis in the refrigerator (5°C) against a solution of 0.9 g ~ NaCI. Testing of samples All samples were tested for AFP and CEA at a commercial medical laboratory. A counterelectrophoretic procedure was used for the AFP test; and the CEA-Roche assay, a radioimmunoassay method was used for the CEA test (Bio-Science Laboratories 1976).
ALBERT C. SMITH
500 RESULTS
AFP test results for all species were reported to be under 25 ng/ml. For CEA, the test results were as follows: tilapia, under 1 ng/ml; milkfish, 1.5 ng/ml; sea cucumber, 13.2 ng/ml. DISCUSSION AND CONCLUSIONS
The normal level for AFP in human males and nonpregnant females occurs below 25ng/ml (BioScience Laboratories, 1977). The values reported in the fishes and sea cucumber are comparable to this level. An absolute value for AFP (or AFP-like substance) in these animals may exist at levels below the sensitivity of current methodology. Studies on the CEA levels in humans (HoffmannLa Roche, 1973) have indicated that values between 0-2.5 ng/ml are very common in healthy people. However, levels above 10ng/ml are almost never found except in association with certain types of malignant disease, particularly colorectal cancer, pancreatic cancer and pulmonary cancer. Thus, the CEA value of 13.2 ng/ml in the sea cucumber is high by human standards. Although the test results for CEA did not give an absolute reading in tilapia, definite values were obtained for the milkfish and sea cucumber. The identification of CEA or CEA-like substance in the lower marine forms indicates that it is evolutionarily old. The concentrations of CEA or CEA-like substance in the three animals studied suggest a possible evolutionary pattern. Tilapia, with the lowest value, is considered higher on the evolutionary tree (Lagler et al., 1977) than the milkfish, a primitive teleost, that had a higher value. Sea cucumber, with the highest CEA value is, of course, more primitive than either fish. From the three species studied, it is possible that concentrations of CEA are higher in more primitive animals. Although the proportion of humans showing a CEA level as high as the sea cucumber is greatest in association with certain malignancies, elevated levels may also occur in a number of nonmalignant disorders (Hoffmann-La Roche, 1973). Thus, in a variety of pathological conditions, there seems to be a tendency for reappearance of proteins seen not only in normal fetal development but also in distant ancestral relations such as the sea cucumber. Stated as a modification of Haeckel's biogenetic law: pathology, like ontogeny, may recapitulate phylogeny. Another example of this concept is myeiofibrosis, a condition in which the mammalian kidney takes up the primitive function of hematopoiesis (Smith, 1978). That proteins of early development can reappear in the adult in association with disease has become familiar in medicine through the many research studies and increasingly widespread use of oncofetal protein tests as aids to diagnosis and management. However, the concept of these fetal and pathologyassociated proteins having a phylogenetic component does not appear to be well known. From a practical point of view, it might also be noted that the sea cucumber provides a readily available source of CEA or a similar substance. This source of the antigen might facilitate production of CEA test antisera and open up new opportunities for cancer research utilizing marine invertebrates. For
example, a basic question that awaits this research is what is the function, if any, of CEA or CEA-like substance in the sea cucumber?
Acknowledgements--1 would like to thank Ms Herlinda M. Broadgate, Pathology, Research Assistant, and Mr David E. Coleman, Jessie Smith Noyes Foundation Fellow, Oceanic Institute, Waimanalo, Hawaii, for excellent technical services: and Dr Colin E. Nash, Director, Oceanic Institute and Dr Jonathan Morse, Assistant Professor of English, University of Hawaii, Honolulu, for valuable criticism of the manuscript. This research was supported under a grant to the Oceanic Institute (No. AID/ TA-C-1189, on "Research in artificial propagation of milkfish") from the United States Agency for International Development, as part of their program in marine pathology.
REFERENCES ANDERSON N. G. & COGGIN J. H. JR (Co-chairmen) (1974) Third Conference on Embryonic and Fetal Antigens in Cancer, Hyatt Regency Hotel, Knoxville. Tennessee, November 4-7, 1973. Cancer Res. 34, 1767-2142. BARRINGTON E. J. W. & JEFFERIES R. B, S. (Eds) (1975) Protochordates. Syrup. zool. Soc. Lond., No. 36, l-16, 105 127, 253-318. Academic Press, New York. q-SCIENCE LABORATORIES (1976) The BiD-Science Handbook. Specialized Diagnostic Laboratory Tests, l l t h edn. BiD-Science Laboratories, Van Nuys, CA. BIO-SCIENCE LABORATORIES(1977) BiD-Science Laboratories Research Notebook, No. 1308. BiD-Science Laboratories, Van Nuys, CA. BORRADAILE L. A., Porrs F. A., EASTHAM L E. S. & SAUNDERS J. T. (1961) The Invertebrata, 4th edn. (revised by KERKUT G. A.). Cambridge University Press, London. FISHMAN W. H. & SELL S. (Eds) (1976) Onco-Developmental Gene Expression. Papers presented at a conference sponsored by the International Research Group for Carcinoembryonic Proteins, San Diego, CA, May 29-June 1, 1976. Academic Press, New York. GOLD P., GOLDENBERG D, M., HANSEN H. J., HERBERMAN R. B., NEVILLE A. M., SNYDER J. & ZAMCHECK N. (Programming Planning Committee) (1977) International Conference on the Clinical Uses of Carcinoembryonic Antigen, College of Medicine, University of Kentucky, Lexington, June 1-3, 1977. GOULD S. J. (1977) Ontogeny and Phylogeny, pp. 1-501. The Belknap Press of Harvard University, Cambridge, MA. HIRA; H. & ALPERT E. (Eds) (1975) Carcinofetal proteins: biology and chemistry. Ann. N.Y. Acad. Sci. 259, 1-452. HOFFMANN-LA ROCHE (1973) Third Conference, Carcinoembryonic Antigen CEA Test Collaborative Study. Hoffman-La Roche Inc., Nutley, NJ, April 21, 1973. KUKALOV/~-PECK J. (1973) A Phyloyenetic Tree of the Animal Kingdom (1ncludin9 Orders and Higher Categories ). Publications in Zoology, No. 8. National Museum of Natural Sciences, National Museums of Canada, Ottawa. LAGLER K. F., BARDACH J. E., MILLER R. R. & PASSINO D. R. M. (1977) Ichthyology. 2nd edn. John Wiley, New York. SANDERS B. G. & KLINE K. (1975) Fetal-tumor membrane associated antigens:genetic and immunological implications. Trans. Am. Microsc. Soc. 94, 470-479. SANDERS B. G.. & KLIr,~ K. (1977) Expression of serum proteins in the developing chick embryo. Comp. Biochem. Physiol. 58, 97-101. SMITH A. C. [1978) A proposed phylogenetic relationship between sea cucumber Polian vesicles and the vertebrate lymphoreticular system. J. Invert. Pathol. 31, 353-357.