Patterns of Cellular Proliferation in Normal and Tumor Cell Populations Stanley M. Gartler, PhD
Three types of cell mosaics have been used in mammalian studies: hemopoietic chimeras, mosaics formed by aggregation of preimplantation embryos, and mosaics resulting from X-chromosome inactivation. The problems investigated with these cell mosaics have included normal tissue organization, cell selection, primordial cell pool sizes, and tumor cell kinetics. The emphasis in this review is on the application of Xchromosome inactivation mosaics to the analysis of tumor cell proliferation. The first application of mosaicism to tumor ontogeny involved leiomyomas and demonstrated single cell and independent origin of the tumors. Other tumor studies are reviewed including those of presumed multiple cell origin, especially those of hereditary origin and viral etiology. The concept of target size is invoked to explain these multiple cell origin tumors. The recent reports on the clonal nature of atherosclerotic plaques is also discussed. Emphasis is placed on resolving the relationship between the multiclonal underlying fatty streak and the clonal plaque in order to understand the implications of the clonal plaques. (Am J Pathol 86:685-692. 1977)
IN THIS PRESENTATION I will describe some of the techniques available for analyzing cell proliferation and discuss some of the results obtained using X-chromosome inactivation mosaics. In order to study the cellular origins of a structure, the investigator must have some way of marking or distinguishing cells. The earlv embryologist used externally applied markers, such as particles of India ink, for this purpose. Because of dilution by cell division, this approach is restricted in its applications. Built in markers-those that replicate with cell division-are not limited bv cell multiplication, and cell mosaics are examples of such systems. A mosaic individual is one who is composed of two or more cell types that differ in genetic expression. The cell types may be derived from one or more zygotes; in the latter case, the term chimera is preferred bv some workers. To be useful, the mosaicism should occur early in development, and the different cell tvpes making up the mosaic should be distinguished by a simple and sensitive assay. Such a mosaic cell population can provide a built-in set of cell markers that may be used to investigate a variety of developmental phenomena, both normal and abnormal. The earliest use of cell mosaics for developmental studies was carried From the Departments of Medicine and Genetics. University of Washington. Seattle. W\ashington Presented at the Sixtieth Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, Calif., April 14. 1976. Address reprint requests to Dr. Stanley M. Gartler. Department of Medicine. Univ ersity of Washington. Seattle, WA 98195 685
686
GARTLER
American Journal of Pathology
out by Sturtevant 1 in 1929 to analyze cell lineage relationships between different morphologic structures in Drosophila. He used nondisjunctional chromosomal segregation occurring early in development to produce an XO/XX mosaic. The cell types were distinguished by sexual phenotypes, and marked segregation of the two cell types during growth and development was observed. Three differTgnt mosaic systems have been used in mammalian studies. Hemopoietic chimeras are produced by injecting bone marrow into lethally irradiated recipients, and such mosaics have been used to answer questions regarding development of the hemopoietic system and the recurrence of leukemia in humans.2'3 A second system involves the experimental production of generalized mosaicism in laboratory mice. Preimplantation embryos of two or more different genotypes are surgically manipulated to form a single embryo of mixed origin. The embryo is then injected into the uterus of a pseudopregnant female where it can develop into a normal animal containing cells of all the parental types. These tetraparental or allophenic mice have been utilized to study a variety of problems.4'5 It is interesting that when the first allophenic mice were constructed in the early 1960s, a human allophenic individual, consisting of half XX and half XY cells,6 was discovered. The third source of mosaicism is that due to X-chromosome inactivation. In the mammalian female, cytodifferentiation of the Xchromosomes occurs early in development so that one X in each cell is inactivated and one X chromosome remains active. The event is random from cell to cell, so that in some cells the paternal X -is active and the maternal X inactive, while in other cells the reverse is true. Once X chromosome differentiation takes place, it becomes a fixed part of the cell's somatic heredity.7 Thus in a person heterozygous at an X-linked locus, a natural mosaic is made available for developmental investigations. The X-linked glucose-6-phosphate dehydrogenase (G6PD) electrophoretic variants are excellent markers for this purpose and have been used in both normal and pathologic applications.8 What kinds of questions can one ask with such mosaics? Our initial interest was the nature of intratissue growth; that is, do the cells of a tissue grow in a highly organized pattern (coherent clonal growth) or do the cells of a tissue populate that tissue in a more or less random manner, migrational forces predominating over any specific pattern. Insect development is an example of extremely organized growth; it is as though every cell originated with specific three-dimensional coordinates. The organization results in clearly discernable patterns when visible mosaic markers are present.
Vol. 86, No. 3 March 1977
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687
Normal tissue growth in humans and mammals appears in general to be much less ordered. In mosaic tissues the two cell types are well mixed, that is, there is little tendency for cells of like type to be contiguous (to form patches or be variegated). This lack of pattern was observed in fetal and adult specimens of dermis, liver, kidney, and a number of other soft tissues and organs. Pure patches in these tissues were never observed even though some specimens were of the order of a 1 cu mm. Even in the case of tissue substructures such as hair follicles, variegation seems minimal. Hair follicles can be seen to originate from a small number of adjacent cells early in fetal life. Our marker studies indicate the starting numbers to be three or four cells;"0 even with this small number most hair follicles tumed out to be of mixed phenotype, indicating that the patch size in the parental tissue is very small. The same is true for the follicular cells surrounding individual oocytes: these also start from a small sample of two or three cells, and again we find that they are often of mixed origin."' Marked variegation may be seen in phenotypes determined by migrating cells such as pigment precursors. A small number of such cells populate relatively large areas of skin, producing a major clonal effect.7 Cell selection occurring in a mosaic can lead to marked variegation. The development of a tumor is an example of cell selection with one or a few abnormal cells overgrowing a mass of nonmalignant cells. However, cell selection can also occur in normal mosaic systems without pathologic effect. This is best illustrated by the heterozygote for the Lesch-Nyhan disease [deficiency for the X-linked controlled hypoxanthine guanine phosphoribosyl transferase (HPRT)], where the hemopoietic cells expressing the normal allele overgrow the cells expressing the mutant gene.12 The phenomenon of cell selection in normal mosaics appears to take place primarily in blood,13 but it must be kept in mind as a mechanism for normal tissue variegation. Since, in the great majority of cases, normal tissue development in mosaics is characterized by minimal varigation, abnormal growth patterns characterized by significant coherent clonal growth are clearly distinguishable. The first abnormal growth studied using mosaics was the leiomyoma of the uterus.14 Leiomyomas are relatively frequent, benign neoplasms that are homogeneous in structure and easily separable from adjoining normal tissues. Over 200 nonnecrotic tumors from 25 C6PD heterozygotes have been examined. All the leiomyomas were singlephenotype tumors (either all electrophoretic band A or B); furthermore, both A and B tumors were found in individual patients. Normal myometrial specimens as small as 1 cu mm and immediately adjacent to the leiomyomas were found to contain both A and B bands, whereas tumors
688
GARTLER
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of Pathology
measuring up to several centimeters in diameter were consistently of a single phenotype throughout.'5 These observations are consistent with the hypothesis of single cell origin of leiomyomas. It is possible that a single-phenotype tumor could start from multiple cells simply by chance sampling of all cells of the same G6PD type. A statistical analysis of the leiomyoma data excludes the possibility that the more than 200 tumors could all be single phenotype tumors simply due to chance sampling of two or more adjacent cells of like type. It is also possible that the single G6PD phenotype in these tumors results from selective overgrowth of one cell type in an originally mixed population. This possibility seems very unlikely in view of the fact that both A and B tumors are found in individual patients and that microdissection of both small and large tumors gives no evidence of more than one G6PD type in the tumor. Thus, these tumors would appear to be truly of single cell origin, suggesting a mutational type induction and an independence of cell-cell interaction in initiation and development. The results of most other tumor studies with mosaic systems are also compatible with a single-cell origin of the tumor. However, in none of these cases has there been sufficient data to exclude other kinetic explanations. It is particularly difficult to resolve the situation with malignant cells where clonal evolution is known to occur repeatedly (e.g., chromosomal changes in CML progression). However, single-phenotype tissues or structures are not found normally and their detection is a strong signal of an abnormal cellular proliferation pattern.'5 The hypothesis that sequential mutational events are required for cancer predicts that tumors should always be of single-cell origin.'6 However, we now know of several examples of tumors of multicellular origin, indicating that mutations are not the only route of tumor origin. One of the most interesting examples of multicellular tumor origin are the hereditary tumors. Both trichoepithelioma and neurofibromas have been studied in mosaics and in both cases the data indicate that the tumors originated from a large number of cells. In fact, the mosaic composition of the tumors is the same as that of the normal surrounding tissue. In hereditary tumors an initial susceptibility exists in every cell of the subject, and therefore, the initiating tumor event would have a large target. Presumably the initiating event is not mutational.'15"17"8 Several other neoplasms have been reported to have double phenotypes in mosaic studies and therefore a multicellular origin. The virus-induced or virus-associated tumors may be informative. The clearest case is that of herpesvirus induction of malignant lymphoma in marmosets.'9 Most marmosets are natural hemopoietic chimeras as a result of bone marrow
Vol. 86, No. 3 March 1977
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^ no%
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exchange between litter mates. A lymphoma cell line was established by herpesvirus infection of XX/XY mosaic cells from a mesenteric lymph node. The transformed cell line had both XX and XY cells in a 1: 1 ratio and appeared to be a stable mosaic, demonstrating multicellular origin."9 Friedman and Fialkow 2 have recently reported on a study of condylomata acuminata in 2 patients heterozygous for the G6PD electrophoretic variant. This neoplasm results from infection of epidermal tissue with a papova virus. Most of the individual verrucous subunits were pure A or B, with both kinds found in each growth. About 5% of the subunits were mixed A and B, indicating multiple cell origin of these units. The mixed verrucous units were found in a region of pure A and B verrucae. The tumor appears to be a blow-up of a small region of epidermis from which the tumor originated, with the mixed subunits indicating the border between adjoining patches. From what is known of normal patch size and the proportion of mixed verrucae, it was estimated that each verruca starts from about three cells. The interesting question that arises from this study is whether the multicellular origin of each unit is a requirement for successful transformation or does it simply reflect the expected outcome with any infectious agent? A previous study on several isolated skin warts in G6PD heterozygotes showed only single phenotypes.21 In view of the small number of warts studied, the finding of single phenotypes is not at variance with a three-cell origin. The only other virus-associated tumor studied in mosaics is the Burkitt lymphoma.' These Epstein-Barr virus-associated malignant tumors are all single phenotypes even in metastases. These results have been interpreted as indicating single-cell origin of these tumors; however, these cases are only studied late in their development, which means there has been considerable opportunity for clonal evolution from a possible original multicellular origin. Radiation is another known carcinogenic factor. Assuming that radiation acted via a mutagenic mechanism, we would expect only neoplasms of single-cell origin. Unfortunately no irradiation experiments in mosaic animals have been carried out or reported. However, a recent statistical analysis of irradiation-induced mammary tumors in mice suggests a multicellular origin. The essence of this work is that, at low doses, a much higher incidence of tumors was observed than expected. This result suggests that irradiation does not necessarily act in a mutagenic fashion in the case of tumor induction." A recent and most stimulating study utilizing G6PD mosaicism is that of Benditt and Benditt 24 on atherosclerotic plaques from C6PD heterozygotes. The normal arterial wall exhibited a fine mixture of A and B cells,
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while the plaques were distinctly different, with some apparently pure A and others pure B. A small porportion of plaques exhibited double phenotypes. These findings on plaques and normal arterial wall have been confirmed by Pearson et al.25 and extended to include a study of the mosaic composition of fatty streaks. The fatty streaks, which some workers consider to be the forerunners of plaques, exhibited a mosaic composition intermediate to plaques and normal arterial wall samples (that is, mainly mixed samples but more single phenotype specimens than found in normal tissue). These observations demonstrate an unusual, if not abnormal, proliferative response as the basis of the fibrous plaque. Is this finding at variance with current ideas regarding the origin of the atherosclerotic lesion? One notion of plaque origin involves wound repair following mechanical or chemical injury.26 It seems impossible that macroscopic wound repair could be initiated from a few cells, let alone a single cell. However, repeated microinjuries or a cyclic injury-repair pattern could result in single phenotype lesions. Another pertinent factor in wound repair is cell senescence.27 If due to senescence there are few cells capable of regeneration in a wound area, then a single-phenotype plaque could result from a wound repair process. In this respect it would be interesting to look at plaques in young subjects with hyperchlolesterolemia where cell senescence would not be a factor. Since abnormal proliferative responses are characteristic of neoplasms, Benditt and Benditt 24 have suggested that consideration should be given to tumorigenic factors in the development of atherosclerotic plaques. More specifically Benditt28 has recently suggested that derivatives of cholesterol could be involved in a mutagenic or cell stimulatory role. A mutational origin for atheromas predicts only single-phenotype plaques in the mosaic. Both groups of workers on this problem have reported some double-phenotype plaques and it remains to be seen whether these are truly of multicell origin or represent overlapping single-phenotype growths. Also pertinent to a mutational interpretation is the report of Pearson et al.26 that the fatty streaks are primarily double phenotypes. If the fatty streak is the percursor of the plaque, then a mutation type origin is excluded. Rather, a tumor progression type of development might be indicated as has been suggested for cervical cancer. This question might be resolved by a sectional analysis of individual plaques beginning from the underlying fatty streak. If the single phenotype fibrous plaque derives by progression from an original multicellular origin, then a gradual transition from a double phenotype to a single phenotype should be found in such an analysis. An abrupt transition, on the other hand, would indicate contamination with underlying normal tissue in plaque dissection.
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The utilization of mosaic systems in cellular proliferation studies has demonstrated that clonal growth is indicative of an abnormal proliferative response. Most tumors studied with mosaic markers exhibit single phenotvpes. The single phenotvpe may indicate true single-cell origin or tumor progression from a multicell origin. Some tumors show a mixed phenotype, demonstrating a multicellular origin. Of special interest in this respect are the hereditary tumors. True single-cell origin of a tumor would suggest a mutational origin. Other carcinogenic agents, depending on titer, can produce neoplasms of multicellular origin. The mosaic studies of atherosclerotic plaques discussed in the symposium clearly show that the plaques represent an abnormal cellular proliferative response. Whether they are of true clonal origin or represent a multicell to singlecell tvpe progression is an important point that remains to be clarified bv further studies. References 1. Sturtevant AH: The claret mutant type of Drosophila simulans, a study of chromosome elimination and cell lineage. Z Wiss Zool 135:323, 1929 2. Wu AM, Till JE, Siminovitch L, McCulloch EA: Cytological evidence for a relationship between normal hematopoietic colony-forming cells and cells of the lvmphoid system. J Exp Med 127:455464, 1968 3. Fialkow PJ, Thomas ED, Bryant JI, Neiman PE: Leukaemic transformation of engrafted human marrow cells in vivo. Lancet 1:251-255, 1971 4. Tarkowski AK: Mouse chimeras developed from fused eggs. Nature 190:857-860, 1961 5. Mintz B, Custer RP, Connellv AJ: Genetic diseases and development defects analyzed in allophenic mice. Int Rev Exp Pathol 10: 143-179, 1971 6. Gartler SM, Waxman SH, Giblett E: An XX,'XY human hermaphrodite resulting from double fertilization. Proc Natl Acad Sci USA 48:332-335. 1962 7. Lyon MF: Mechanism and evolutionary origins of variable X-chromosome activity in mammals. Proc R Soc Lond [Biol] 187:243-268, 1974 8. Gartler SM. Linder D: Selection in mammalian mosaic cell populations. Cold Spring Harbor Svmp Quant Biol 29:253-260, 1964 9. Linder D, Gartler SM: Distribution of glucose-6-phosphate dehydrogenase electrophoretic variants in different tissues of heterozygotes. Am J Human Genet 17:212-220, 1965 10. Gartler SM, Gandini E, Hutchison HT, Campbell B, Zechhi G: Utilization of glucose-6-phosphate dehydrogenase mosaicism in the study of hair follicle variegation and development in man. Am Hum Genet 35:1-7, 1971 11. Gartler SM, Andina R: Mammalian X-chromosome inactivation: A review. Adv Hum Genet (In press) 12. Nvhan WL, Bakay B, Connor JD, Marks JF, Keele DK: Hemizvgous expression of glucose-6-phosphate dehvdrogenase in erythrocytes for the Lesch-Nyhan syndrome. Proc Natl Acad Sci USA 65:214-218, 1970 13. Gandini E, Gartler SM, Angioni G, Argiolas N, Dell'Acqua G: Developmental implications of multiple tissue studies in glucose-6-phosphate dehydrogenase deficient heterozygotes. Proc Natl Acad Sci USA 61:94-948, 1968 14. Linder D, Gartler SM: Glucose-6phosphate dehvdrogenases mosaicism: Utilization as a cell marker in the studv of leiomvomas. Science 150:67-69, 1965
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15. Gartler SM: Utilization of mosaic systems in the study of the origin and progression of tumors. Chromosomes and Cancer. Edited by J German. New York, John Wiley & Sons, 1974, pp 314-334 16. Knudson AG: Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 68L820-823, 1971 17. Gartler SM, Ziprkowsky L, Krakowski A, Ezra R, Szeinberg A, Adam A: Glucose-6phosphate dehydrogenase mosaicism as a tracer in the study of hereditary multiple trichoepithelioma. Am J Human Genet 18:282-287, 1967 18. Fialkow PJ, Sagebiel RW, Gartler SM, Rimoin NL: Multiple cell origin of hereditary neurofibromas. N Engl J Med 284:298-300, 1971 19. Chu EW, Rabson AS: Chimerism in lymphoid cell culture line derived from lymph node of marmoset infected with Herpesvirus saimiri. J Natl Cancer Inst 48:771-773, 1973 20. Friedman JM, Fialkow PJ: Viral "tumorigenesis" in man: Cell markers in condylomata acuminata. Int J Cancer 17:57-61, 1976 21. Murray RF, Hobbs J, Payne B: Possible clonal origin of common warts (Verruca vulgaris). Nature 232:51-52, 1971 22. Fialkow PJ, Klein E, Klein G, Clifford P, Singh S: Immunoglobulin and glucose-6phosphate dehydrogenase as markers of cellular origin in Burkitt's lymphoma. J Exp Med 138:89-102, 1973 23. Rossi HH, Kellerer AM: Radiation carcinogenesis at low doses. Science 175:200-202, 1972 24. Benditt EP, Benditt JM: Evidence for a monoclonal origin of human atherosclerotic plaques. Proc Natl Acad Sci USA 70:1753-1756, 1973 25. Pearson TA, Wand A, Solez K, Heptinstall RH: Clonal characteristics of fibrous plaques and fatty streaks from human aortas. Am J Pathol 81:379-387, 1975 26. Ross R, Glomset JA: Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science 180:1332-1339, 1973 27. Martin GM, Sprague CA: Symposium on in vitro studies related to atherogenesis: Life histories of hyperplastoid cell lines from aorta and skin. Exp Mol Pathol 18:125-141, 1973 28. Benditt EP: Evidence for a monoclonal origin of human atherosclerotic plaques and some implications. Circulation 50:650-652, 1974
Three types of cell mosaics have been used in mammalian studies: hemopoietic chimeras, mosaics formed by aggregation of preimplantation embryos, and mosaics resulting from X-chromosome inactivation. The problems investigated with these cell mosaics have included normal tissue organization, cell selection, primordial cell pool sizes, and tumor cell kinetics. The emphasis in this review is on the application of Xchromosome inactivation mosaics to the analysis of tumor cell proliferation. The first application of mosaicism to tumor ontogeny involved leiomyomas and demonstrated single cell and independent origin of the tumors. Other tumor studies are reviewed including those of presumed multiple cell origin, especially those of hereditary origin and viral etiology. The concept of target size is invoked to explain these multiple cell origin tumors. The recent reports on the clonal nature of atherosclerotic plaques is also discussed. Emphasis is placed on resolving the relationship between the multiclonal underlying fatty streak and the clonal plaque in order to understand the implications of the clonal plaques. (Am J Pathol 86:685-692. 1977)
IN THIS PRESENTATION I will describe some of the techniques available for analyzing cell proliferation and discuss some of the results obtained using X-chromosome inactivation mosaics. In order to study the cellular origins of a structure, the investigator must have some way of marking or distinguishing cells. The earlv embryologist used externally applied markers, such as particles of India ink, for this purpose. Because of dilution by cell division, this approach is restricted in its applications. Built in markers-those that replicate with cell division-are not limited bv cell multiplication, and cell mosaics are examples of such systems. A mosaic individual is one who is composed of two or more cell types that differ in genetic expression. The cell types may be derived from one or more zygotes; in the latter case, the term chimera is preferred bv some workers. To be useful, the mosaicism should occur early in development, and the different cell tvpes making up the mosaic should be distinguished by a simple and sensitive assay. Such a mosaic cell population can provide a built-in set of cell markers that may be used to investigate a variety of developmental phenomena, both normal and abnormal. The earliest use of cell mosaics for developmental studies was carried From the Departments of Medicine and Genetics. University of Washington. Seattle. W\ashington Presented at the Sixtieth Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, Calif., April 14. 1976. Address reprint requests to Dr. Stanley M. Gartler. Department of Medicine. Univ ersity of Washington. Seattle, WA 98195 685
686
GARTLER
American Journal of Pathology
out by Sturtevant 1 in 1929 to analyze cell lineage relationships between different morphologic structures in Drosophila. He used nondisjunctional chromosomal segregation occurring early in development to produce an XO/XX mosaic. The cell types were distinguished by sexual phenotypes, and marked segregation of the two cell types during growth and development was observed. Three differTgnt mosaic systems have been used in mammalian studies. Hemopoietic chimeras are produced by injecting bone marrow into lethally irradiated recipients, and such mosaics have been used to answer questions regarding development of the hemopoietic system and the recurrence of leukemia in humans.2'3 A second system involves the experimental production of generalized mosaicism in laboratory mice. Preimplantation embryos of two or more different genotypes are surgically manipulated to form a single embryo of mixed origin. The embryo is then injected into the uterus of a pseudopregnant female where it can develop into a normal animal containing cells of all the parental types. These tetraparental or allophenic mice have been utilized to study a variety of problems.4'5 It is interesting that when the first allophenic mice were constructed in the early 1960s, a human allophenic individual, consisting of half XX and half XY cells,6 was discovered. The third source of mosaicism is that due to X-chromosome inactivation. In the mammalian female, cytodifferentiation of the Xchromosomes occurs early in development so that one X in each cell is inactivated and one X chromosome remains active. The event is random from cell to cell, so that in some cells the paternal X -is active and the maternal X inactive, while in other cells the reverse is true. Once X chromosome differentiation takes place, it becomes a fixed part of the cell's somatic heredity.7 Thus in a person heterozygous at an X-linked locus, a natural mosaic is made available for developmental investigations. The X-linked glucose-6-phosphate dehydrogenase (G6PD) electrophoretic variants are excellent markers for this purpose and have been used in both normal and pathologic applications.8 What kinds of questions can one ask with such mosaics? Our initial interest was the nature of intratissue growth; that is, do the cells of a tissue grow in a highly organized pattern (coherent clonal growth) or do the cells of a tissue populate that tissue in a more or less random manner, migrational forces predominating over any specific pattern. Insect development is an example of extremely organized growth; it is as though every cell originated with specific three-dimensional coordinates. The organization results in clearly discernable patterns when visible mosaic markers are present.
Vol. 86, No. 3 March 1977
CELLULAR PROLIFERATION PATTERNS
687
Normal tissue growth in humans and mammals appears in general to be much less ordered. In mosaic tissues the two cell types are well mixed, that is, there is little tendency for cells of like type to be contiguous (to form patches or be variegated). This lack of pattern was observed in fetal and adult specimens of dermis, liver, kidney, and a number of other soft tissues and organs. Pure patches in these tissues were never observed even though some specimens were of the order of a 1 cu mm. Even in the case of tissue substructures such as hair follicles, variegation seems minimal. Hair follicles can be seen to originate from a small number of adjacent cells early in fetal life. Our marker studies indicate the starting numbers to be three or four cells;"0 even with this small number most hair follicles tumed out to be of mixed phenotype, indicating that the patch size in the parental tissue is very small. The same is true for the follicular cells surrounding individual oocytes: these also start from a small sample of two or three cells, and again we find that they are often of mixed origin."' Marked variegation may be seen in phenotypes determined by migrating cells such as pigment precursors. A small number of such cells populate relatively large areas of skin, producing a major clonal effect.7 Cell selection occurring in a mosaic can lead to marked variegation. The development of a tumor is an example of cell selection with one or a few abnormal cells overgrowing a mass of nonmalignant cells. However, cell selection can also occur in normal mosaic systems without pathologic effect. This is best illustrated by the heterozygote for the Lesch-Nyhan disease [deficiency for the X-linked controlled hypoxanthine guanine phosphoribosyl transferase (HPRT)], where the hemopoietic cells expressing the normal allele overgrow the cells expressing the mutant gene.12 The phenomenon of cell selection in normal mosaics appears to take place primarily in blood,13 but it must be kept in mind as a mechanism for normal tissue variegation. Since, in the great majority of cases, normal tissue development in mosaics is characterized by minimal varigation, abnormal growth patterns characterized by significant coherent clonal growth are clearly distinguishable. The first abnormal growth studied using mosaics was the leiomyoma of the uterus.14 Leiomyomas are relatively frequent, benign neoplasms that are homogeneous in structure and easily separable from adjoining normal tissues. Over 200 nonnecrotic tumors from 25 C6PD heterozygotes have been examined. All the leiomyomas were singlephenotype tumors (either all electrophoretic band A or B); furthermore, both A and B tumors were found in individual patients. Normal myometrial specimens as small as 1 cu mm and immediately adjacent to the leiomyomas were found to contain both A and B bands, whereas tumors
688
GARTLER
American Journal
of Pathology
measuring up to several centimeters in diameter were consistently of a single phenotype throughout.'5 These observations are consistent with the hypothesis of single cell origin of leiomyomas. It is possible that a single-phenotype tumor could start from multiple cells simply by chance sampling of all cells of the same G6PD type. A statistical analysis of the leiomyoma data excludes the possibility that the more than 200 tumors could all be single phenotype tumors simply due to chance sampling of two or more adjacent cells of like type. It is also possible that the single G6PD phenotype in these tumors results from selective overgrowth of one cell type in an originally mixed population. This possibility seems very unlikely in view of the fact that both A and B tumors are found in individual patients and that microdissection of both small and large tumors gives no evidence of more than one G6PD type in the tumor. Thus, these tumors would appear to be truly of single cell origin, suggesting a mutational type induction and an independence of cell-cell interaction in initiation and development. The results of most other tumor studies with mosaic systems are also compatible with a single-cell origin of the tumor. However, in none of these cases has there been sufficient data to exclude other kinetic explanations. It is particularly difficult to resolve the situation with malignant cells where clonal evolution is known to occur repeatedly (e.g., chromosomal changes in CML progression). However, single-phenotype tissues or structures are not found normally and their detection is a strong signal of an abnormal cellular proliferation pattern.'5 The hypothesis that sequential mutational events are required for cancer predicts that tumors should always be of single-cell origin.'6 However, we now know of several examples of tumors of multicellular origin, indicating that mutations are not the only route of tumor origin. One of the most interesting examples of multicellular tumor origin are the hereditary tumors. Both trichoepithelioma and neurofibromas have been studied in mosaics and in both cases the data indicate that the tumors originated from a large number of cells. In fact, the mosaic composition of the tumors is the same as that of the normal surrounding tissue. In hereditary tumors an initial susceptibility exists in every cell of the subject, and therefore, the initiating tumor event would have a large target. Presumably the initiating event is not mutational.'15"17"8 Several other neoplasms have been reported to have double phenotypes in mosaic studies and therefore a multicellular origin. The virus-induced or virus-associated tumors may be informative. The clearest case is that of herpesvirus induction of malignant lymphoma in marmosets.'9 Most marmosets are natural hemopoietic chimeras as a result of bone marrow
Vol. 86, No. 3 March 1977
CELLULAR PROLIFERATION PATTERNS
^ no%
b68
exchange between litter mates. A lymphoma cell line was established by herpesvirus infection of XX/XY mosaic cells from a mesenteric lymph node. The transformed cell line had both XX and XY cells in a 1: 1 ratio and appeared to be a stable mosaic, demonstrating multicellular origin."9 Friedman and Fialkow 2 have recently reported on a study of condylomata acuminata in 2 patients heterozygous for the G6PD electrophoretic variant. This neoplasm results from infection of epidermal tissue with a papova virus. Most of the individual verrucous subunits were pure A or B, with both kinds found in each growth. About 5% of the subunits were mixed A and B, indicating multiple cell origin of these units. The mixed verrucous units were found in a region of pure A and B verrucae. The tumor appears to be a blow-up of a small region of epidermis from which the tumor originated, with the mixed subunits indicating the border between adjoining patches. From what is known of normal patch size and the proportion of mixed verrucae, it was estimated that each verruca starts from about three cells. The interesting question that arises from this study is whether the multicellular origin of each unit is a requirement for successful transformation or does it simply reflect the expected outcome with any infectious agent? A previous study on several isolated skin warts in G6PD heterozygotes showed only single phenotypes.21 In view of the small number of warts studied, the finding of single phenotypes is not at variance with a three-cell origin. The only other virus-associated tumor studied in mosaics is the Burkitt lymphoma.' These Epstein-Barr virus-associated malignant tumors are all single phenotypes even in metastases. These results have been interpreted as indicating single-cell origin of these tumors; however, these cases are only studied late in their development, which means there has been considerable opportunity for clonal evolution from a possible original multicellular origin. Radiation is another known carcinogenic factor. Assuming that radiation acted via a mutagenic mechanism, we would expect only neoplasms of single-cell origin. Unfortunately no irradiation experiments in mosaic animals have been carried out or reported. However, a recent statistical analysis of irradiation-induced mammary tumors in mice suggests a multicellular origin. The essence of this work is that, at low doses, a much higher incidence of tumors was observed than expected. This result suggests that irradiation does not necessarily act in a mutagenic fashion in the case of tumor induction." A recent and most stimulating study utilizing G6PD mosaicism is that of Benditt and Benditt 24 on atherosclerotic plaques from C6PD heterozygotes. The normal arterial wall exhibited a fine mixture of A and B cells,
690
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American Journal of Pathology
while the plaques were distinctly different, with some apparently pure A and others pure B. A small porportion of plaques exhibited double phenotypes. These findings on plaques and normal arterial wall have been confirmed by Pearson et al.25 and extended to include a study of the mosaic composition of fatty streaks. The fatty streaks, which some workers consider to be the forerunners of plaques, exhibited a mosaic composition intermediate to plaques and normal arterial wall samples (that is, mainly mixed samples but more single phenotype specimens than found in normal tissue). These observations demonstrate an unusual, if not abnormal, proliferative response as the basis of the fibrous plaque. Is this finding at variance with current ideas regarding the origin of the atherosclerotic lesion? One notion of plaque origin involves wound repair following mechanical or chemical injury.26 It seems impossible that macroscopic wound repair could be initiated from a few cells, let alone a single cell. However, repeated microinjuries or a cyclic injury-repair pattern could result in single phenotype lesions. Another pertinent factor in wound repair is cell senescence.27 If due to senescence there are few cells capable of regeneration in a wound area, then a single-phenotype plaque could result from a wound repair process. In this respect it would be interesting to look at plaques in young subjects with hyperchlolesterolemia where cell senescence would not be a factor. Since abnormal proliferative responses are characteristic of neoplasms, Benditt and Benditt 24 have suggested that consideration should be given to tumorigenic factors in the development of atherosclerotic plaques. More specifically Benditt28 has recently suggested that derivatives of cholesterol could be involved in a mutagenic or cell stimulatory role. A mutational origin for atheromas predicts only single-phenotype plaques in the mosaic. Both groups of workers on this problem have reported some double-phenotype plaques and it remains to be seen whether these are truly of multicell origin or represent overlapping single-phenotype growths. Also pertinent to a mutational interpretation is the report of Pearson et al.26 that the fatty streaks are primarily double phenotypes. If the fatty streak is the percursor of the plaque, then a mutation type origin is excluded. Rather, a tumor progression type of development might be indicated as has been suggested for cervical cancer. This question might be resolved by a sectional analysis of individual plaques beginning from the underlying fatty streak. If the single phenotype fibrous plaque derives by progression from an original multicellular origin, then a gradual transition from a double phenotype to a single phenotype should be found in such an analysis. An abrupt transition, on the other hand, would indicate contamination with underlying normal tissue in plaque dissection.
Vol. 86, No. 3 March 1977
CELLULAR PROLIFERATION PATTERNS
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The utilization of mosaic systems in cellular proliferation studies has demonstrated that clonal growth is indicative of an abnormal proliferative response. Most tumors studied with mosaic markers exhibit single phenotvpes. The single phenotvpe may indicate true single-cell origin or tumor progression from a multicell origin. Some tumors show a mixed phenotype, demonstrating a multicellular origin. Of special interest in this respect are the hereditary tumors. True single-cell origin of a tumor would suggest a mutational origin. Other carcinogenic agents, depending on titer, can produce neoplasms of multicellular origin. The mosaic studies of atherosclerotic plaques discussed in the symposium clearly show that the plaques represent an abnormal cellular proliferative response. Whether they are of true clonal origin or represent a multicell to singlecell tvpe progression is an important point that remains to be clarified bv further studies. References 1. Sturtevant AH: The claret mutant type of Drosophila simulans, a study of chromosome elimination and cell lineage. Z Wiss Zool 135:323, 1929 2. Wu AM, Till JE, Siminovitch L, McCulloch EA: Cytological evidence for a relationship between normal hematopoietic colony-forming cells and cells of the lvmphoid system. J Exp Med 127:455464, 1968 3. Fialkow PJ, Thomas ED, Bryant JI, Neiman PE: Leukaemic transformation of engrafted human marrow cells in vivo. Lancet 1:251-255, 1971 4. Tarkowski AK: Mouse chimeras developed from fused eggs. Nature 190:857-860, 1961 5. Mintz B, Custer RP, Connellv AJ: Genetic diseases and development defects analyzed in allophenic mice. Int Rev Exp Pathol 10: 143-179, 1971 6. Gartler SM, Waxman SH, Giblett E: An XX,'XY human hermaphrodite resulting from double fertilization. Proc Natl Acad Sci USA 48:332-335. 1962 7. Lyon MF: Mechanism and evolutionary origins of variable X-chromosome activity in mammals. Proc R Soc Lond [Biol] 187:243-268, 1974 8. Gartler SM. Linder D: Selection in mammalian mosaic cell populations. Cold Spring Harbor Svmp Quant Biol 29:253-260, 1964 9. Linder D, Gartler SM: Distribution of glucose-6-phosphate dehydrogenase electrophoretic variants in different tissues of heterozygotes. Am J Human Genet 17:212-220, 1965 10. Gartler SM, Gandini E, Hutchison HT, Campbell B, Zechhi G: Utilization of glucose-6-phosphate dehydrogenase mosaicism in the study of hair follicle variegation and development in man. Am Hum Genet 35:1-7, 1971 11. Gartler SM, Andina R: Mammalian X-chromosome inactivation: A review. Adv Hum Genet (In press) 12. Nvhan WL, Bakay B, Connor JD, Marks JF, Keele DK: Hemizvgous expression of glucose-6-phosphate dehvdrogenase in erythrocytes for the Lesch-Nyhan syndrome. Proc Natl Acad Sci USA 65:214-218, 1970 13. Gandini E, Gartler SM, Angioni G, Argiolas N, Dell'Acqua G: Developmental implications of multiple tissue studies in glucose-6-phosphate dehydrogenase deficient heterozygotes. Proc Natl Acad Sci USA 61:94-948, 1968 14. Linder D, Gartler SM: Glucose-6phosphate dehvdrogenases mosaicism: Utilization as a cell marker in the studv of leiomvomas. Science 150:67-69, 1965
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15. Gartler SM: Utilization of mosaic systems in the study of the origin and progression of tumors. Chromosomes and Cancer. Edited by J German. New York, John Wiley & Sons, 1974, pp 314-334 16. Knudson AG: Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 68L820-823, 1971 17. Gartler SM, Ziprkowsky L, Krakowski A, Ezra R, Szeinberg A, Adam A: Glucose-6phosphate dehydrogenase mosaicism as a tracer in the study of hereditary multiple trichoepithelioma. Am J Human Genet 18:282-287, 1967 18. Fialkow PJ, Sagebiel RW, Gartler SM, Rimoin NL: Multiple cell origin of hereditary neurofibromas. N Engl J Med 284:298-300, 1971 19. Chu EW, Rabson AS: Chimerism in lymphoid cell culture line derived from lymph node of marmoset infected with Herpesvirus saimiri. J Natl Cancer Inst 48:771-773, 1973 20. Friedman JM, Fialkow PJ: Viral "tumorigenesis" in man: Cell markers in condylomata acuminata. Int J Cancer 17:57-61, 1976 21. Murray RF, Hobbs J, Payne B: Possible clonal origin of common warts (Verruca vulgaris). Nature 232:51-52, 1971 22. Fialkow PJ, Klein E, Klein G, Clifford P, Singh S: Immunoglobulin and glucose-6phosphate dehydrogenase as markers of cellular origin in Burkitt's lymphoma. J Exp Med 138:89-102, 1973 23. Rossi HH, Kellerer AM: Radiation carcinogenesis at low doses. Science 175:200-202, 1972 24. Benditt EP, Benditt JM: Evidence for a monoclonal origin of human atherosclerotic plaques. Proc Natl Acad Sci USA 70:1753-1756, 1973 25. Pearson TA, Wand A, Solez K, Heptinstall RH: Clonal characteristics of fibrous plaques and fatty streaks from human aortas. Am J Pathol 81:379-387, 1975 26. Ross R, Glomset JA: Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science 180:1332-1339, 1973 27. Martin GM, Sprague CA: Symposium on in vitro studies related to atherogenesis: Life histories of hyperplastoid cell lines from aorta and skin. Exp Mol Pathol 18:125-141, 1973 28. Benditt EP: Evidence for a monoclonal origin of human atherosclerotic plaques and some implications. Circulation 50:650-652, 1974
