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Somatic cell hybrids: Applications relevant to genetic disease
MEDICAL
PROGRESS
Somatic cell hybrids: Applications relemnt genetic disease
to
Techniques introduced for the genetic analysis o[ microorganisms are being adapted to the analysis o[ mammalian cells. Methods [or the production of both somatic cell heterokaryons and mononucIear hybrid cells have already been sufficiently developed so that one can appreciate their potential as a probe for the study of the genetic basis o[ a number o[ important biological phenomena. The ability to hybridize somatic cells from individuals o[ diverse genetic qualities not only gets around the limitation imposed by man's long generation time, breeding habits, and small family size but also permits the analysis of genetic differences between species so distantly related that breeding is not possible. Therefore, studies of cell hybrids will contribute significantly to knowledge of gene action in our species, thus providing information essential to the institution of effective treatment for genetic disease.
Barbara Ruben Migeon, M.D. BALTIMORE~ MD.
O N E A P P R O A C H to the treatment of genetic disease has been to modify the phenotype of the affected individual. Treatments have been designed to substitute for, to minimize the need for, or to enhance the activity of the defective gene product. Examples of this kind of environmental manipulation are numerous and include the administration of cortisone to patients with congenital adrenal hyperplasia, the instituFrom the Department o/Pediatrics, Johns Hopkins University School o/Medicine, and the Harriet Lane Service of the Johns Hopkins Children's Medical Center. Supported in part by United States Public Health Service Grant No. HD 05465. Abbreviations: HAT = h~poxanthlne amlno~terln, and thymidlne. TK -~ thymldlne klnase, HGPRT = hypoxa~thine-guanlne phosphoribosyt transferase, A = amlnoOterln, H = hypoxanthlne, T = thymidlne, BUDR ~ 5-bromodeoxyuridlne, PGK = r kinase, G-6-PD = glueose-6-phosphate dehydrogenase.
tion of a low-phenylalanine diet for infants with phenylketonuria, and the use of pharmacologic doses of vitamin BI~ in some patients with methylmalonic aeidemia. 1 The success of this kind of genetic intervention increases with the specificity of the treatment and therefore ideally requires some knowledge of the genetic basis for the disease. Another therapeutic approach which has only recently left the realm of science fiction is the possibility of replacing "defective" genes with "normal" ones. Although the task is formidable, it may eventually be feasible to introduce into appropriate cells an exogenously supplied gene, either as naked DNA or as part of a nonlethal virus. In this case, it is obviously necessary that the gene which will replace the mutant one have a Vol. 79, No. 6, pp. 887-897
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function similar to the normal or "wildtype" gene. Therefore, whether the attempt is to change environment or genes, the prerequisite is knowledge of what the "defective" gone is doing and the ability to detect its presence in the fetus or newborn infant. The possibility of gone transfer implies an even more rigorous understanding of the mechanisms of gene action and the need for a degree of sophistication in human genetic methodology not yet achieved. We need to know in detail the nature of human genes, which of these are concerned with the specification of protein structure, which are concerned instead with control of gene expression, and how this control is accomplished. Since loci adjacent to "replacement" genes may be administered along with the pertinent genetic material, we must also know their functions. Therefore, we should be able to assign genes to chromosome pairs and identify those in close proximity (linkage groups). Two features are critical to the analysis of the human genetic constitution. The first is the existence of variation among individuals, for it is the analysis of these phenotypic differences which provides insight into their genotypic basis. The second is the ability to observe the transmission of the variant phenotype from parents to progeny. The preferred phenotype for study is the primary product of the pertinent gone, i.e., messenger RNA. Since this is not generally feasible, the best alternative phenotype is the protein specified by the RNA. The approach is to determine whether a variant protein is specified at the same locus as the wildtype protein, in which case the genes are alleles. The test of allelism is accomplished through the analysis of the distribution of the two proteins (segregation) among progeny of parents differing in these proteins. The pattern of segregation indicates whether these proteins are specified by genes occupying the same or different chromosomal locus or, as in the case of nonallelic genes, the same or different chromosome (linkage group).
The Journal o[ Pediatrics December 1971
Since microorganisms have short generation times and an unlimited number of offspring, formal genetic analysis has been carried out most successfully in these organisms. Moreover, in contrast to the diploid nature of mammalian organisms, microorganisms are haploid, having a single copy of each gene. Consequently each gone is dominant and its mode of action easier to analyze. The classic approach to genetic analysis in diploid organisms had been to follow the segregation of hereditary traits in families in which two forms of a specific trait happen to occur. The method is sensitive enough to distinguish human X chromosomal variants from autosomal ones, but there has been only minimal success in defining linkage groups of autosomal genes and less success in assigning individual genes or linkage groups to a specific chromosome. The problem is that man's long generation time, small family size, and ethics preclude the kind of genetic analysis based on controlled matings and observations of large numbers of progeny. Recent efforts have been directed toward bypassing sexual reproduction and have involved the propagation of somatic cells in vitro. Human cells in culture have a relatively brief generation time, reproducing themselves every 24 hours, thereby transmitting their unique genetic constitution to literally millions of progeny. Enzyme deficiencies or protein variants present in skin cells or lymphocytes of individuals persist in vitro and provide a source of biochemical markers useful for genetic analysis. Cultivated somatic cells have already been applied successfully to the study of hereditary disease, 2, ~ detection of heterozygotes,4 and prenatal diagnosis of genetically abnormal fetuses. 5 Studies of the progeny of single skin fibroblasts have provided the best evidence supporting the hypothesis of X chromosomal inactivation. 6, ~ Furthermore, somatic cells in culture can be manipulated so that some of the elegant experimental designs developed for the genetic analysis of microorganisms can now be ap-
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Fig. 1. Photomicrographs of human skin fibroblasts following exposure to inactivated Sendal virus. A, Multinueleated cells resulting from cell fusion 14 hours previously. (Original magnification x200.) B, Fibroblasts each with two nuclei, 24 hours following fusion. (Original magnification • plied to mammalian cells. Analogous to their bacterial prototypes, the growth of somatic cells can be selectively disfavored or favored by changes in their in vitro environment, and one of the most promising developments is the ability of cells with different genetic constitutions to hybridize with one another. Prospects for the genetic analysis of mammalian somatic cells have been advanced considerably by the demonstration that two unrelated cells in culture can fuse into a single one (Fig. 1). In its simplest form, the product of such fusion is a heterokaryon consisting of the nuclei of both parental cells encased in a new common cytoplasm. The facts are that heterokaryons, even when derived from cells of widely divergent spe-
cies,* can be induced with regularity, s, 9 These multinucleated cells carry out specific functions including synthesis of DNA, RNA, and proteins, for about one week before degenerating. The heterokaryon, therefore, can be useful for the study of cytoplasmic-nuclear relationships. Should the nuclei fuse, the heterokaryon may then become a mononuclear hybrid cell containing the genome of both parental cells and frequently capable of prolonged cultivation. Barski and colleagues 1~ were the first to describe the appearance of mononu*Chick erythrocyte nuclei have been introduced into the cytoplasm of human HeLa ceils and the resultant heterokaryon, therefore, is derived from cells of different classes of vertebrates) Interphylum heterokaryons (mosquitohuman [HeLa]) have also been induced. 52
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clear cell .hybrids. The parents were two clones of mouse somatic cells with a common origin, but each had evolved in culture so that they could be distinguished from each other by morphologic, karyotypic, and neoplastic properties. It was expected that something analogous to the mitotic segregation in cultures of filamentous fungi a~ might occur, so that the hybrid cell would subsequently give rise to daughter nuclei, each with a reassortment of the original parent genes. On the contrary, these interspecific hybrids maintained a relatively stable karyotype with a total number of chromosomes equal to the sum of the parental complements. On continued propagation, the chromosome number decreased slightly, but there was no evidence of any significant production of daughter cell segregants. Ephrussi and Weiss ~2 showed that the ability of cells to fuse was not dependent upon similarities in their genotype, by isolating the first interspecific hybrids (rat X mouse). Hybrids derived from cells of unrelated species carry out all essential cell functions and represent the result of a remarkable degree of integration of information contained in the parental genomes. There seem to be neither intracellular incompatibility nor species-specific signals controlling events in the cell cycle of these interspecific hybrids. Since the spontaneous occurrence of hybrid cells in mixed cultures of parent cells is infrequent, and since the hybrids rarely are empowered with sufficient "hybrid vigor" to overgrow parental ceils, it is necessary to either increase the frequency of fusion or provide the means to isolate the occasional spontaneous hybrid. Okada ~3 provided a method for the former by demonstrating the ability of Sendai viruses to promote wholesale cell fusion,* while the latter has usually been in the form of a nutrient medium selectively disfavoring the ~'The virus, which has been previously inactivated by ultraviolet light or beta propriolactone, adsorbs to the surface of the cell a n d promotes fusion, presumably by increasing cell-to-cell contact.
The ]ournal o[ Pediatrics Decembe~ 1971
growth of at least one variety of parental cells.,14, 1~ As one might expect, the phenotype of the hybrid cell is determined by both parents and for the most part can vary anywhere between the parental extremes. Hybrids can be propagated indefinitely as long as one of the parents is capable of infinite growth. At least one parent of most of the hybrids isolated to date has been an "immortal" heteropoloid line. The tendency to chromosome loss observed in the initial hybrids 1~ has also characterized the subsequent ones. The extent of this loss generally depends on whether the hybrids were derived from cells of the same or different species. The loss of chromosomes in intraspecific hybrids is usually not greater than 20 per cent of the sum of parental sets? 6 The disappearance of chromosomes in interspecific hybrids, however, tends to be more extensive and is attributable to preferential exclusion of those of one parental type, resulting in an unusual type of chromosomal segregation. Fortuitously, it is the human chromosomes which are shed from hybrids derived from mouse 1~ or hamster ~s cells, so that one can attempt to relate the loss of human chromosomes with the loss of human gene products as a means of assigning genes to chromosomes. The invariable association of any two gene products would suggest that these are closely linked one to another. LINKAGE
The application of hybridization which has been most exploited so far has to do *The H A T selective system 14 is the one most frequently used and is based on the inability of parental cells lacking T K or H G P R T to grow in the presence of A, even when provided with exogenous H and T . T h e hybrid cell, by complementation, has both enzymes, so it can divide in the H A T selective m e d i u m . Available p a r e n t lines include mouse and h u m a n cells ~ i t h H G P R T defieiencies, as well as mouse and hamster cells with T K deficiency. O n e or more of these drug-resistant mutants have been the parent cells for most of the hybrids isolated to date. Useful, in conjunction with H A T m e d i u m which disfavors cells with H G P R ' I or T K deficlency~ are nutrient media favoring these m u t a n t cells at the expense of the wild type. Media containing azaguanlne and B U D R promote tile growth of t t G P R T - ant T K ~ cells respectively. I t is possible, therefore, with the use of these two varieties of m e d i u m , to obtain clones of cell.' with or without the pertinent chromosomes.
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with the assignment of genes to specific linkage groups. One of the more immediate benefits to be derived from linkage studies is the ability to determine the genotype of an individual at a locus not amenable to direct study by its association with another which can be typed. For example, the linkage group which includes the gene causing myotonic dystrophy also includes the secretor (Se) and Lutheran (Lu) loci. 19 Although it is not yet possible to detect parenterally the fetus affected with myotonic dystrophy, it might be possible to do so on the basis of his secretor phenotype, which is detectable in amniotie fluid? ~ Alternative forms of a gene must be present to reveal the pattern of its distribution from parents to progeny. Although there is extensive variation between proteins within our own species, intraspecific differences are rare when compared to interspecific variation. Protein made by cells of nonrelated species are likely to differ from each other, so that interspecific hybrids are particularly useful for linkage analysis. The model for linkage studies, therefore, has usually been the mouse-human hybrid; it is dependent upon the extensive loss of human chromosomes from the hybrid gehome. Karyotypes of newly isolated mousehuman hybrids include almost the entire mouse chromosome complement but, at best, only 25 per cent of the human complement. 1~ The loss of human chromosomes from hamster-human hybrids seems to be even more extensive, is so that these hybrids will also be particularly useful for gene linkage assignments. Linkage relationships have been determined in two ways, selectively and nonselectively. In the selective method, the mouse parent lacks a product which is made essential for survival by selective medium, therefore favoring the hybrid cells which retain the human choromosome that provides the essential product. The human genome in the hybrid, under selection pressure, can be reduced to the single relevant chromosome and the coincidence of other human proteins in such hybrids indicates that these
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also must be located on the relevant chromosome. The selective method has permitted the assignment of the gene specifying the structural locus for T K to the h u m a n chromosome number 17, * as well as confirming the X linkage of the gene specifying PGK, 25 previously suggested on the basis of its segregation in the only two informative families available. The nonselective method is the attempt to correlate the presence or absence of human proteins with changes in the hybrid karyotype. If two human genes are linked, once the chromosome is lost both the gene products should disappear simultaneously. The invariable association of two human enzymes has revealed the linkage of the B subunit of lactate dehydrogenase to peptidase B,26, 27 as well as linkage of lactate dehydrogenase A with arylesterase? s However, the genes specifying the two lactate dehydrogenase subunits have been shown to be unlinked, since one is present without the other in various clones of mouse-human hybrids.26, 27 The likelihood that normal linkage groups may occasionally be disrupted must be considered in the interpretation of data derived from hybrids. Chromosomal rearrangements have been demonstrated cytologically and further evidence of fragmentation is the occasional lack of association between human ~The first suggestion of an association between this enzyme and chromosome was derived from observing that the chromosome was present in mouse-human hybrid cells having T K activity, but absent from hybrid cells lacking this enzyme. ~7 Since the mouse parent cell lacked the enzyme, the hybrid cell must have obtained its T K f r o m one of the h u m a n chromosomes. T h e isolation of T K + hybrid clones having only a single h u m a n chromosome was further evidence that this submetacentric human chromosome had contributed this function. ~1, 22 Electrophoretic demonstration that the T K in the hybrid cell is of the h u m a n variety indicates that the pertinent chromosome provides the structural locus for T K . 2s Recently, it has become possible to distinguish individual chromosomes in the human karyotype on the basis of their pattern of fluorescence with qulnacrlne, and as a consequence the solitary submetacentric group E chromosome in these clones has been identified as a n u m b e r 17. ~ Unfortunately the usefulness of the H A T - B U D R - 8 azaguanlne media is limited to those mutations involving the H G P R T a a d T K loci, since these media favor or disfavor only chromosomes specifying these enzymes (the h u m a n X and I7, respectively). A variety of selective media is required for the exploitation of this m e t h o d for linkage studies.
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G-6-PD and H G P R T phenotypes in some hybrid clones? 9 Although both enzymes are specified by the X chromosome, the loci are probably not closely linked? ~ Fragmentation, therefore, may actually be useful to identify close linkage groups, and to define the sequence of the genes on the chromosome as well. Evidence of this kind suggests that PGK is also not closely linked with either G-6-PD or H G P R T , and their positions relative to the centromere can be determined using cells with X-chromosome deletions, as Human cells with chromosome deletions and rearrangements will be invaluable for determining precisely the chromosomal location of linkage groups. GENE R E G U L A T I O N We have innumerable clues that some genes function differently at various times in development. One of the best examples of this is the synthesis of y chains of hemoglobin which combine with a chains to form the fetal variety of hemoglobin. Hemoglobin F, containing y chains, predominates during fetal life, while hemoglobin A, with B chains, comprises most of the hemoglobin after birth. However, small amounts of /3 chains are produced as early as 9 weeks' gestation a= and small amounts of fetal hemoglobin are present in the adult. If we knew what is involved in the turning down of the y-chain locus and how this control could be manipulated, this gene conceivably could be turned up again postnatally in individuals with sickle cell anemia or thalassemia who cannot make normal/3 chains. The conditions limiting our knowledge of gene-chromosome linkage relationships have also deterred the elucidation of control mechanisms in human cells. Although the study of haploid microorganisms has provided insight into possible regulatory mechanisms, the systems involved in the control of gene action in one-cell organisms may not be equally appropriate for an organism as complex as man. The results of recent linkage studies suggest that genetic regulation has some unique features in mammalian cells. Genes specifying unlike subunits of proteins, such as hemoglobin and lactate de-
The Journal o[ Pediatrics December 1971
hydrogenase are not linked to one another, so that control of their synthesis probably differs from the operon type of regulation mechanisms which serve to control a group of contiguous genes. Hybrid cells can be used to probe the means by which the phenotype of the cell is determined and how the expression of the parental genes is regulated. Are the genes of one parent dominant or recessive to those of the other? Many of the proteins in the hybrid cell are specified by the genes of both parent cells. For example, the mouse-hamster hybrid cell will produce the mouse form of malate dehydrogenase, as well as the hamster variety of the enzyme, distinguishable on the basis of differences in electrophoretic migration. In addition, there is a third form of malate dehydrogenase, the novel "hybrid" heteropolymer, produced as a result of the association of mouse and hamster enzymes, a3 There are instances, however, in which the genes of one parent have not been expressed in the hybrid and which cannot be attributed to loss of the pertinent locus. Hamster melanoma cells, when fused with nonpigmented mouse cells, result without exception in nonpigmented hybrids which have lost DOPA oxidase? ~' ~5 The precise mechanism is undefined, but there appears to be some block to pigment formation in these hybrids; study of this phenomenon is likely to reveal the nature of the influence of one genome on the pigment production by the other. COMPLEMENTATION
It has become apparent that the amount of genetic heterogeneity discovered at any locus is proportional to the amount of effort involved in the search, and certainly is sufficient to indicate that in regard to variants which occur rarely, each family may have its unique nmtation. The amount of genetic heterogeneity in terms of enzyme kinetics is considerable, even in diseases which show a relatively uniform phenotype, as for example the Lesch-Nyhan syndrome? 6 Our ability to detect differences in mutations, which may not lead to differences in enzyme phenotype demonstrable by the usual
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biochemical techniques, may be enhanced by complementation analysis of pairs of mutant cell strains. Enzyme-deficient cells from two unrelated, but clinically similar, individuals may be fused together and the phenotype of their hybrid determined. If the mutations in the cells of the probands are similar, the hybrid derived from these cells should also be enzyme deficient. Therefore, restoration of normal enzyme function in the hybrid is evidence that the two parental mutations are not the same. Analysis of hybrids derived from glycine-dependent Chinese hamster cells have indicated that there are at least four loci associated with this phenotype and that complementation analysis is feasible in mammalian somatic cells? r Recently heterogeneity within the group of patients with galactosemia has been reported on the basis of studies of cultures containing mixed populations of fibroblasts. Cells from one of seven patients tested were able to complement those of the other six and the hybrid enzyme differed from the wild type in specific activity and some kinetic properties, as This kind of complementation analysis can be carried out in short-term cultures of Sendai-induced heterokaryons, especially when the normal enzyme phenotype is demonstrable in the intact ceil. Heterokaryons, however, usually represent a minority of the cells present in the mixed culture. The proportion of heterokaryons can be enriched and hybrid ceils can be isolated with the use of culture medium which disfavors the growth of mutant parent cells. Theoretically it should be possible to select against any mutant cell and the ability to devise appropriate selective media will enhance our ability to carry out complementation analysis of all kinds of human mutations, provided these are demonstrable at the level of the cultivated cell. OTHER APPLICATIONS THE METHOD
OF
One of the most exciting potential applications of the method is the use of cell hybrids for immunization against malignant tumors. The highly malignant Ehrlich's as-
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cites tumor is lethal to a variety of mouse strains because it is only weakly antigenic. However, if the mouse ascites tumor is hybridized with a hamster cell, the resulting hybrid cell, when injected into mice, is able to protect the host animal during subsequent exposure to the tumor cells? 9 The mice, by rejecting the hybrid cells immunologically because of the hamster component, apparently acquire an immunity to the Ehrlich's tumor antigens. Therefore, it is conceivable that a hybrid formed between a tumor with weak transplantation antigens and cells with strong antigens on the basis of species differences could be used to immunize the host animal against his own tumor. Since the tumor cells can be fused directly, not requiring cultivation, the method is applicable to the problem of human metastatic tumors. Even if immunization with interspecific hybrid cells does not fulfill its promise, the hybrid cell certainly has a significant role in the study of the malignant process. Highly malignant mouse tumor cells, when fused with less virulent cells, give rise to hybrids which do not express the malignant phenotype of their tumor cell parent. 4~ These experiments suggest that tumogenicity in the hybrid is suppressed by the presence of the genome of the nontumor cell parent. The similarities between the characteristics of malignant cells and the changes in cells following viral transformation leave little doubt that virus-induced neoplastic transformation is an excellent model for the study of malignancy. Cell hybridization has already been applied to the study of some aspects of the virus-host relationship and has been particularly useful in demonstrating the continuous presence of the viral genome in the transformed cells, even when these cells do not produce virus particles. 41, 42 In some cases the cell which harbors the virus is not a good indicator of its presence, and fusion of the host cells with susceptible indicator cells is necessary to reveal the presence of infectious virus. .8 In one instance, fusion of two parental cells, neither of whieh produced infectious virus, resulted in hybrid cells capable of producing virus, possibly by
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means of complementation of defective particles juxtaposed in the same heterokaryon. ~4 This approach to the recovery of infectious viruses could be extremely fruitful for the isolation of elusive viruses in diseases suspected to have a viral etiology. THE
TASK
AHEAD
Somatic hybridization is an excellent means to genetic analysis in man. It seems that interspecific hybrids will have their greatest value for gene localization, whereas hybrids between cells of the same species, because of their relatively stable karyotype, may be preferable for complementation analysis and studies of control of cell phenotype. Ideally we would like to study hybrids derived from nonheteroploid human cells of a variety of tissues and differentiated states. A beginning has been made by Siniscalco and associates, 4s who have observed tetraploid cells in mixed cultures of human diploid cells with different X-linked mutations. Since no one has yet isolated a clone of hybrids from parents who are both euploid, we do not know what it woutd took like. Judging from the efforts to isolate such a hybrid, it seems that the fused human cell does not have an obvious selective advantage over its diploid parents. Hence the senescence in culture which characterizes diploid fibroblasts may unfortunately also characterize the hybrid. Lymphocytes maintained in long-term culture, however, seem to have gained immortality, so that these may be ideal parents for human hybrids. Somatic hybridization has resulted from the development of methodology in many areas of research. Techniques for the in vitro cultivation of discrete mammalian cells have contributed the milieu for cell mating. Developments in cytogenetics and enzymology have provided the initial supply of cells having biochemical and cytogenetie markers. Further developments are required for the exploitation of this means of genotypie anaIysis. These should include the discovery of the means to prolong the life of cultivated diploid cells, so that studies of progeny of
single cells necessary for genetic analysis can be facilitated. We must develop methods to propagate cells from normal differentiated tissues, so that their in vivo function is maintained; such cells will provide a source of genetic markers invaluable for the study of the control of differentiation. Although the number of available cell lines of this type is small, more will be developed. The ingenious methods of Sato and colleagues46 for the cultivation of cells from hormone-producing mouse tumors will contribute a host of mouse cell markers not available in cells of connective tissue origin. For every aspect of genetic analysis, there is the need to increase the supply of cells with stable genetic markers. Among the lines available for use are those whose karyotypes include stable deletions and rearrangements resulting in morphologically distinctive chromosomes. The recent development of techniques for the identification of individual chromosomes on the basis of their patterns of fluorescence with various quinacrine compounds 4~ will enhance the supply of these variants by revealing chromosomal rearrangements not visible on the basis of altered gross morphology. Furthermore, the precise identification of individual chromosomes will obviously increase the specificity of linkage assignments. The extent of protein variation between species is significant and the number of relatively common variants of human proteins already demonstrated suggests that there is an abundance of heterogeneity within our own species which can be utilized. *s In more than 20 of the inborn metabolic errors, the enzyme deficiency related to the disease has also been demonstrated in skin fibroblasts derived from patients; as more of these errors are defined, more biochemically marked human cells will become available. In addition, the naturally occurring mutations can be supplemented with those inducible by various mutagenie agents. ~.9, 50 ~Since mutation~ in diploid cells are usually recessive, the ideal candidates for exposure to mutagens are: (1) male cells (haploid for X-linked genes), and (2) cells with chromosomal deletions (haploid for genes on the deleted segment),
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Since recombination is the essence of genetic analysis, efforts should be directed tow a r d the d e v e l o p m e n t of methods to promote mitotic segregation of the classical type, or at least to induce chromosome elimination from intraspecific cell hybrids. Pontecorv@ 1 has suggested that sensitivity to certain treatments, effective in p r o m o t i n g chromosome loss, m a y vary for i n d i v i d u a l chromosomes according to their replication pattern. If true, then these sensitivity differences provide the means for eliminating specific h u m a n chromosomes.
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3. 4. 5. 6.
7.
CONCLUSION T o change the genetic constitution of an individual is at best a f o r m i d a b l e task. Even if it were technically possible to transfer gone or gene products to the a p p r o p r i a t e host cells, the correction of the deleterious m u t a t i o n m a y be less t h a n perfect. T h e r e fore, when possible, p r e n a t a l detection of an affected fetus a n d subsequent abortion m a y prove to be the most effective t r e a t m e n t for most genetic diseases. However, in some instances p r o v i d i n g essential gene products or even D N A m a y be fully efficacious a n d the preferable alternative. In either event we must significantly increase our knowledge of the details of gene action in o u r species, as well as elucidate the genetic basis for inherited diseases. Preliminary results indicate that somatic cell hybrids are an excellent means for obtaining information regarding the sequence of genes on h u m a n chromosomes and the control of protein synthesis. Disease-related potential applications of the m e t h o d s are virtually unlimited a n d include p r e n a t a l detection of genetic disease, the t r e a t m e n t of m a l i g n a n t disease, the u n m a s k i n g of the viral etiology of some infectious diseases, and ultimately the r e p l a c e m e n t of defective genes.
8. 9.
10.
11. 12. 13.
14.
15. 16.
17.
KEFEKENCES
I. Hsia, Y. E., Lilljeqvist, A.-Ch., and Rosenberg, L. E.: Vitamin BI~ dependent methylmalonic aciduria, Pediatrics 46: 497, 1970. 2. Fratantonl, J. C., Hall, C. W,, and Neufeld, E. F.: The defect in Hurler and Hunter
18. 19.
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syndromes. II. Deficiency of specific factors involved in mucopolysaccharide degradation, Proc. Natl. Acad. Sci. 64: 360, 1969. Danes, B. S., and Bearn, A. G.: Cystic fibrosis of the pancreas. A study in cell culture, J. Exp. Med. 129: 775, 1969. Migeon, B. R.: X-linked HGPRT deficiency: Detection of heterozygetes by selective medium, Biochem. Genet, 4: 387, 1970. Nadler, H. L.: Antenatal detection of hereditary disorders, Pediatrics 42" 912, 1968. Davidson, R. G., Nitowsky, H. M., and Childs, B.: Demonstration of two populations of cells in the human female heterozygous for glucose-6-phosphate dehydrogenase variants, Proc. Natl. Aead. Sci. 50: 481, 1963. DeMars, R., and Nanee, W. E.: Electrophoretie variants of glucose-6-phospbate dehydrogenase and the single active X in cultured human cells, in Defendi, V,, editor: Retention of function differentiation in cultured cells, The Wistar Institute Symposium Monograph No. t, Philadelphia, t964, Wistar Institute Press, p. 35. Harris, H.: Behavior of differentiated nuclei in heterokaryons of animal cells from different species~ Nature 206: 583, 1965. Harris, H., and Watkins, J. F.: Hybrid cells derived from mouse and man: Artificial heterokaryons of mammalian cells from different species, Nature 205: 640, 1965. Barski, G., Sorieul, S., and Cornefert, F.: "Hybrid" type cells in combined cultures of two different mammalian cell strains, J. Natl. Cancer Inst. 26: I269, 196t. Ponteeorvo, G.: Trends in genetic analysis, New York, 1958, Columbia University Press. Ephrussi, B., and Weiss, M. C.: Interspecific hybridization of somatic cells, Proc. Natl. Acad. Sei. 53: 1040, 1965. Okada, Y.: Analysis of giant polynuclear cell formation caLlsed by HVJ virus from Erllch's aseites tumor cells, Exp. Cell Res. 26: 98, 1962. Szybalski, W., Szybalska, E. H., and Ragni, G.: Genetic studies with human cell lines, Natl. Cancer Inst. Monograph No. 7, 1962, p. 75. Littlefield, J. W.: Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants, Science 145: 709, 1964. Ephrussi, B., Scaletta, L. G., Stenehever, M. A., and Yoshida, M. C.: Hybridization of somatic cells in vitro, in Harris, R. J. C., editor: Cytogenetlcs of cells in culture, London, 1964, Academic Press, Inc., p. 13. Weiss, M. C., and Green, H.: Human-mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes, Proc. Natl, Acad. Sei. 58: 1104, 1967. Kao, F. T., and Puck, T. T.: Linkage studies with human-Chinese hamster cell hybrids, Nature 228: 329, 1970. Mohr, J,: A study of linkage in man, Monograph, Munkogaard (Copenhagen), 1954.
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20. Harper, Peter, Bias, Wilton B., Hutchinson, Judith R., and McKusick, Victor A.: ABH secretor status of the foetus; a genetic marker identifiable by amniocentesis, J. Med. Genet. In press_ 21. Migeon, B. R., and Miller, C. S.: Humanmouse somatic cell hybrids with single human chromosome (Group E): Link with thymidine kinase activity, Science 162: 1005, 1968. 22, Matsuya, Y., Green, H., and Basilico, C.: Properties and uses of human-mouse hybrid cell lines, Nature 220: 1199, 1968. 23. Migeon, B. R., Smith, S., and Leddy, C.: The nature of thymidine kinase in mousehuman hybrid cells, Biochem. Genet. 3: 583, 1969. 24. Miller, O. J., Allderdice, P. W., Miller, D. A., Breg, W. R., and Migeon, B. R.: Assignment of human thymidine kinase gene locus to chromosome 17 by identification of its distinctive quinacrine-fluorescenee in man/ mouse somatic hybrid, Science 173: 244, 1971. 25. Meera Khan, P., Westerveld, A,, Grzeschik, K.-H., Deys, B. F., Garson, O. M., and Sinlscalco, M.: X-linkage of human phosphoglycerate kinase confirmed in man-mouse and man-Chinese hamster somatic, cell hybrids, Nature. In press. 26. Santaehiara, A. S., Nabholz, M., Miggiano, V., Darlington, A. J., and Bodmer, W.: Genetic analysis with man-mouse somatic ceil hybrids: Linkage between human lactate dehydrogenase B and peptidase B genes, Nature 227: 248, 1970. 27. Ruddle, F. H., Chapman, V. M., Chen, T. R., and Klebe, R. J.: Genetic analysis with man-mouse somatic cell hybrids: Linkage between human lactate dehydrogenase A and B and peptidase B, Nature 227: 251, 1970. 28. Shows, T. B.: Human gene expression, llnkage relationships and control properties in human/mouse somatic hybrids, Abstract Am. Soc. Human Genet., 1970. 29. Miller, O. J., Cook, P. R., Meera Kahn, P., Shin, S., and Siniscalco, M.: Mitotic separation of two human X-linked genes in man-mouse somatic cell hybrids, Proc. Natl. Acad. Sci. 68: 116, 1971. 30. Nyhan, W. L., Bakay, B., Connor, J. D., Marks, J~ F., and Keele, D. K.: Hemizygous expression of glucose-6-phosphate dehydrogenase in erythrocytes of heterozygotes for the Lesch-Nyhan syndrome, Proc. Natl. Acad. Sci. 65: 214, 1970. 31. Sinlscalco, M.: Personal communication. 32. Hollenberg, M. D., Kaback, M. M., and Kazazian, H. H., Jr.: Synthesis of adult hemoglobin by reticulocytes from the human fetus at midtrimester, Science. In press. 33. Migeon, B. R.: Hybridization of somatic cells derived from mouse and Syrian hamster: Evolution of karyotype and enzyme studies, Biochem. Genet. h 305, 1968. 34. Davidson, R. L., Ephrussi, B., and Yama-
The Journal o] Pediatrics December 1971
35. 36. 37.
38.
39.
40.
4I.
42.
43.
44.
45.
46.
47.
48. 49,
moto, K.: Regulation of pigment synthesis in mammalian cells, as studied by somatic hybridization, Proc. Natl. Acad. ScL 56: 1437, 1966. Silagi, S.: Hybridization of a malignant melanoma celt line with L cells in vitro, Cancer Res. 27: 1953, 1967. McDonald, J. A., and Kelley, W. N.: LeschNyhan syndrome: Altered kinetic properties of mutant enzyme, Science 171: 689, 1971. Kao, F. T,, Chasin, L., and Puck, T. T.: Genetics of somatic mammalian cells, X complementation analysis of glycine-requiring mutants, Proc. Natl. Acad. Sci, 64: 1284, 1969. Nadler, H. L., Chacko, C. M., and Rachreeler, M.: Interallelic complementation in hybrid cells derived from human diploid strains deficient in galactose-l-phosphate urldyl transferase activity, Proc. Natl. Aead. Sci. 67: 976, 1970, Watkins, J. F., and Chen, L.; Immunization of mice against Ehrlich ascites tumour using a hamster/Ehrlich ascltes tumour hybrid cell line, Nature 223: 1018, 1.969. Harris, H., Miller, O. J., Klein, G., Worst, P., and Tachibana, T.: Suppression of malignancy by cell fusion, Nature 223: 363, 1969. Weiss, M. C., Ephrussi, B., and Scaletta, L. J.: Loss of T-antigen from somatic hybrids between mouse cells and SV-40 transformed human cells, Proc. Natl. Acad. Sci. 59: 1132, 1968. Weiss, Mary C.: Further studies on Ioss of T-antigen from somatic hybrids between mouse cells and SV~0-transformed human cells, Proc. Natl. Aead. Sci. 66: 79, 1970. Koprowski, H., Jensen, F. C., and Steplewski, Z.: Activation of production of infectious tumor virus SV~, in heterokaryon cultures, Proc. Natl. Acad. Sci. 58: 127, 1967. Knowles, B. B., Jensen, F. C., Steplewskl, Z., and Koprowski, H.: Rescue of infectious SV~0 after fusion between different SV4otransformed cells, Proc. Natl. Acad. Sci. 61: 42, 1968. Sinisealco, M., Klinger, H. P., Eagle, H., Koprowski, H., Fujlmoto, W. Y., and Seegmiller, J. E.: Evidence for intergenic complementation in hybrid cells derived from two human diploid strains each carrying an X-llnked mutation, Proc. Natl. Acad. Sci. 62: 793, 1969. Sato, G., Augusti-Tocco, G., Posner, M., and Kelly, P.: Hormone-secreting and hormoneresponsive cell cultures, Recent Progr. Horm. Res. 26: 539, 1970. Caspersson, T., Zech, L., and Johansson, C.: Analysis of human metaphase chromosome set by aid of DNA-binding fluorescent agents, Exp. Cell Res. 62: 490, 1970. Childs, B., and Der Kaloustian, V. M.: Genetic heterogeneity, N. Engl. J. Med. 279: 1205, I267, 1968. Kao, F., and Puck, T. T.: Genetics of
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somatic mammalian cells. IX. Quantltation of mutagenesis by physical and chemical agents, .1- Cell Physiol. 74: 245, 1969. 50. Albertini, R. J., and DeMars, R.: Diploid azaguanine-resistant mutants of cultured human fibroblasts, Science 169: 482, 1970. 51. Pontecorvo, G.: Induction of directional
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chromosome elimination in somatic cell hybrids, Nature 230: 367, 1971. 52. Zepp, H. D,, Conover, J. H., Hischhorn, K., and Hodes, H. L.: Human-mosquito somatic cell hybrids induced by ultraviolet inactivated Sendal virus, Nature 229: 119, 1971.
PROGRESS
Somatic cell hybrids: Applications relemnt genetic disease
to
Techniques introduced for the genetic analysis o[ microorganisms are being adapted to the analysis o[ mammalian cells. Methods [or the production of both somatic cell heterokaryons and mononucIear hybrid cells have already been sufficiently developed so that one can appreciate their potential as a probe for the study of the genetic basis o[ a number o[ important biological phenomena. The ability to hybridize somatic cells from individuals o[ diverse genetic qualities not only gets around the limitation imposed by man's long generation time, breeding habits, and small family size but also permits the analysis of genetic differences between species so distantly related that breeding is not possible. Therefore, studies of cell hybrids will contribute significantly to knowledge of gene action in our species, thus providing information essential to the institution of effective treatment for genetic disease.
Barbara Ruben Migeon, M.D. BALTIMORE~ MD.
O N E A P P R O A C H to the treatment of genetic disease has been to modify the phenotype of the affected individual. Treatments have been designed to substitute for, to minimize the need for, or to enhance the activity of the defective gene product. Examples of this kind of environmental manipulation are numerous and include the administration of cortisone to patients with congenital adrenal hyperplasia, the instituFrom the Department o/Pediatrics, Johns Hopkins University School o/Medicine, and the Harriet Lane Service of the Johns Hopkins Children's Medical Center. Supported in part by United States Public Health Service Grant No. HD 05465. Abbreviations: HAT = h~poxanthlne amlno~terln, and thymidlne. TK -~ thymldlne klnase, HGPRT = hypoxa~thine-guanlne phosphoribosyt transferase, A = amlnoOterln, H = hypoxanthlne, T = thymidlne, BUDR ~ 5-bromodeoxyuridlne, PGK = r kinase, G-6-PD = glueose-6-phosphate dehydrogenase.
tion of a low-phenylalanine diet for infants with phenylketonuria, and the use of pharmacologic doses of vitamin BI~ in some patients with methylmalonic aeidemia. 1 The success of this kind of genetic intervention increases with the specificity of the treatment and therefore ideally requires some knowledge of the genetic basis for the disease. Another therapeutic approach which has only recently left the realm of science fiction is the possibility of replacing "defective" genes with "normal" ones. Although the task is formidable, it may eventually be feasible to introduce into appropriate cells an exogenously supplied gene, either as naked DNA or as part of a nonlethal virus. In this case, it is obviously necessary that the gene which will replace the mutant one have a Vol. 79, No. 6, pp. 887-897
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function similar to the normal or "wildtype" gene. Therefore, whether the attempt is to change environment or genes, the prerequisite is knowledge of what the "defective" gone is doing and the ability to detect its presence in the fetus or newborn infant. The possibility of gone transfer implies an even more rigorous understanding of the mechanisms of gene action and the need for a degree of sophistication in human genetic methodology not yet achieved. We need to know in detail the nature of human genes, which of these are concerned with the specification of protein structure, which are concerned instead with control of gene expression, and how this control is accomplished. Since loci adjacent to "replacement" genes may be administered along with the pertinent genetic material, we must also know their functions. Therefore, we should be able to assign genes to chromosome pairs and identify those in close proximity (linkage groups). Two features are critical to the analysis of the human genetic constitution. The first is the existence of variation among individuals, for it is the analysis of these phenotypic differences which provides insight into their genotypic basis. The second is the ability to observe the transmission of the variant phenotype from parents to progeny. The preferred phenotype for study is the primary product of the pertinent gone, i.e., messenger RNA. Since this is not generally feasible, the best alternative phenotype is the protein specified by the RNA. The approach is to determine whether a variant protein is specified at the same locus as the wildtype protein, in which case the genes are alleles. The test of allelism is accomplished through the analysis of the distribution of the two proteins (segregation) among progeny of parents differing in these proteins. The pattern of segregation indicates whether these proteins are specified by genes occupying the same or different chromosomal locus or, as in the case of nonallelic genes, the same or different chromosome (linkage group).
The Journal o[ Pediatrics December 1971
Since microorganisms have short generation times and an unlimited number of offspring, formal genetic analysis has been carried out most successfully in these organisms. Moreover, in contrast to the diploid nature of mammalian organisms, microorganisms are haploid, having a single copy of each gene. Consequently each gone is dominant and its mode of action easier to analyze. The classic approach to genetic analysis in diploid organisms had been to follow the segregation of hereditary traits in families in which two forms of a specific trait happen to occur. The method is sensitive enough to distinguish human X chromosomal variants from autosomal ones, but there has been only minimal success in defining linkage groups of autosomal genes and less success in assigning individual genes or linkage groups to a specific chromosome. The problem is that man's long generation time, small family size, and ethics preclude the kind of genetic analysis based on controlled matings and observations of large numbers of progeny. Recent efforts have been directed toward bypassing sexual reproduction and have involved the propagation of somatic cells in vitro. Human cells in culture have a relatively brief generation time, reproducing themselves every 24 hours, thereby transmitting their unique genetic constitution to literally millions of progeny. Enzyme deficiencies or protein variants present in skin cells or lymphocytes of individuals persist in vitro and provide a source of biochemical markers useful for genetic analysis. Cultivated somatic cells have already been applied successfully to the study of hereditary disease, 2, ~ detection of heterozygotes,4 and prenatal diagnosis of genetically abnormal fetuses. 5 Studies of the progeny of single skin fibroblasts have provided the best evidence supporting the hypothesis of X chromosomal inactivation. 6, ~ Furthermore, somatic cells in culture can be manipulated so that some of the elegant experimental designs developed for the genetic analysis of microorganisms can now be ap-
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Fig. 1. Photomicrographs of human skin fibroblasts following exposure to inactivated Sendal virus. A, Multinueleated cells resulting from cell fusion 14 hours previously. (Original magnification x200.) B, Fibroblasts each with two nuclei, 24 hours following fusion. (Original magnification • plied to mammalian cells. Analogous to their bacterial prototypes, the growth of somatic cells can be selectively disfavored or favored by changes in their in vitro environment, and one of the most promising developments is the ability of cells with different genetic constitutions to hybridize with one another. Prospects for the genetic analysis of mammalian somatic cells have been advanced considerably by the demonstration that two unrelated cells in culture can fuse into a single one (Fig. 1). In its simplest form, the product of such fusion is a heterokaryon consisting of the nuclei of both parental cells encased in a new common cytoplasm. The facts are that heterokaryons, even when derived from cells of widely divergent spe-
cies,* can be induced with regularity, s, 9 These multinucleated cells carry out specific functions including synthesis of DNA, RNA, and proteins, for about one week before degenerating. The heterokaryon, therefore, can be useful for the study of cytoplasmic-nuclear relationships. Should the nuclei fuse, the heterokaryon may then become a mononuclear hybrid cell containing the genome of both parental cells and frequently capable of prolonged cultivation. Barski and colleagues 1~ were the first to describe the appearance of mononu*Chick erythrocyte nuclei have been introduced into the cytoplasm of human HeLa ceils and the resultant heterokaryon, therefore, is derived from cells of different classes of vertebrates) Interphylum heterokaryons (mosquitohuman [HeLa]) have also been induced. 52
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clear cell .hybrids. The parents were two clones of mouse somatic cells with a common origin, but each had evolved in culture so that they could be distinguished from each other by morphologic, karyotypic, and neoplastic properties. It was expected that something analogous to the mitotic segregation in cultures of filamentous fungi a~ might occur, so that the hybrid cell would subsequently give rise to daughter nuclei, each with a reassortment of the original parent genes. On the contrary, these interspecific hybrids maintained a relatively stable karyotype with a total number of chromosomes equal to the sum of the parental complements. On continued propagation, the chromosome number decreased slightly, but there was no evidence of any significant production of daughter cell segregants. Ephrussi and Weiss ~2 showed that the ability of cells to fuse was not dependent upon similarities in their genotype, by isolating the first interspecific hybrids (rat X mouse). Hybrids derived from cells of unrelated species carry out all essential cell functions and represent the result of a remarkable degree of integration of information contained in the parental genomes. There seem to be neither intracellular incompatibility nor species-specific signals controlling events in the cell cycle of these interspecific hybrids. Since the spontaneous occurrence of hybrid cells in mixed cultures of parent cells is infrequent, and since the hybrids rarely are empowered with sufficient "hybrid vigor" to overgrow parental ceils, it is necessary to either increase the frequency of fusion or provide the means to isolate the occasional spontaneous hybrid. Okada ~3 provided a method for the former by demonstrating the ability of Sendai viruses to promote wholesale cell fusion,* while the latter has usually been in the form of a nutrient medium selectively disfavoring the ~'The virus, which has been previously inactivated by ultraviolet light or beta propriolactone, adsorbs to the surface of the cell a n d promotes fusion, presumably by increasing cell-to-cell contact.
The ]ournal o[ Pediatrics Decembe~ 1971
growth of at least one variety of parental cells.,14, 1~ As one might expect, the phenotype of the hybrid cell is determined by both parents and for the most part can vary anywhere between the parental extremes. Hybrids can be propagated indefinitely as long as one of the parents is capable of infinite growth. At least one parent of most of the hybrids isolated to date has been an "immortal" heteropoloid line. The tendency to chromosome loss observed in the initial hybrids 1~ has also characterized the subsequent ones. The extent of this loss generally depends on whether the hybrids were derived from cells of the same or different species. The loss of chromosomes in intraspecific hybrids is usually not greater than 20 per cent of the sum of parental sets? 6 The disappearance of chromosomes in interspecific hybrids, however, tends to be more extensive and is attributable to preferential exclusion of those of one parental type, resulting in an unusual type of chromosomal segregation. Fortuitously, it is the human chromosomes which are shed from hybrids derived from mouse 1~ or hamster ~s cells, so that one can attempt to relate the loss of human chromosomes with the loss of human gene products as a means of assigning genes to chromosomes. The invariable association of any two gene products would suggest that these are closely linked one to another. LINKAGE
The application of hybridization which has been most exploited so far has to do *The H A T selective system 14 is the one most frequently used and is based on the inability of parental cells lacking T K or H G P R T to grow in the presence of A, even when provided with exogenous H and T . T h e hybrid cell, by complementation, has both enzymes, so it can divide in the H A T selective m e d i u m . Available p a r e n t lines include mouse and h u m a n cells ~ i t h H G P R T defieiencies, as well as mouse and hamster cells with T K deficiency. O n e or more of these drug-resistant mutants have been the parent cells for most of the hybrids isolated to date. Useful, in conjunction with H A T m e d i u m which disfavors cells with H G P R ' I or T K deficlency~ are nutrient media favoring these m u t a n t cells at the expense of the wild type. Media containing azaguanlne and B U D R promote tile growth of t t G P R T - ant T K ~ cells respectively. I t is possible, therefore, with the use of these two varieties of m e d i u m , to obtain clones of cell.' with or without the pertinent chromosomes.
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with the assignment of genes to specific linkage groups. One of the more immediate benefits to be derived from linkage studies is the ability to determine the genotype of an individual at a locus not amenable to direct study by its association with another which can be typed. For example, the linkage group which includes the gene causing myotonic dystrophy also includes the secretor (Se) and Lutheran (Lu) loci. 19 Although it is not yet possible to detect parenterally the fetus affected with myotonic dystrophy, it might be possible to do so on the basis of his secretor phenotype, which is detectable in amniotie fluid? ~ Alternative forms of a gene must be present to reveal the pattern of its distribution from parents to progeny. Although there is extensive variation between proteins within our own species, intraspecific differences are rare when compared to interspecific variation. Protein made by cells of nonrelated species are likely to differ from each other, so that interspecific hybrids are particularly useful for linkage analysis. The model for linkage studies, therefore, has usually been the mouse-human hybrid; it is dependent upon the extensive loss of human chromosomes from the hybrid gehome. Karyotypes of newly isolated mousehuman hybrids include almost the entire mouse chromosome complement but, at best, only 25 per cent of the human complement. 1~ The loss of human chromosomes from hamster-human hybrids seems to be even more extensive, is so that these hybrids will also be particularly useful for gene linkage assignments. Linkage relationships have been determined in two ways, selectively and nonselectively. In the selective method, the mouse parent lacks a product which is made essential for survival by selective medium, therefore favoring the hybrid cells which retain the human choromosome that provides the essential product. The human genome in the hybrid, under selection pressure, can be reduced to the single relevant chromosome and the coincidence of other human proteins in such hybrids indicates that these
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89 1
also must be located on the relevant chromosome. The selective method has permitted the assignment of the gene specifying the structural locus for T K to the h u m a n chromosome number 17, * as well as confirming the X linkage of the gene specifying PGK, 25 previously suggested on the basis of its segregation in the only two informative families available. The nonselective method is the attempt to correlate the presence or absence of human proteins with changes in the hybrid karyotype. If two human genes are linked, once the chromosome is lost both the gene products should disappear simultaneously. The invariable association of two human enzymes has revealed the linkage of the B subunit of lactate dehydrogenase to peptidase B,26, 27 as well as linkage of lactate dehydrogenase A with arylesterase? s However, the genes specifying the two lactate dehydrogenase subunits have been shown to be unlinked, since one is present without the other in various clones of mouse-human hybrids.26, 27 The likelihood that normal linkage groups may occasionally be disrupted must be considered in the interpretation of data derived from hybrids. Chromosomal rearrangements have been demonstrated cytologically and further evidence of fragmentation is the occasional lack of association between human ~The first suggestion of an association between this enzyme and chromosome was derived from observing that the chromosome was present in mouse-human hybrid cells having T K activity, but absent from hybrid cells lacking this enzyme. ~7 Since the mouse parent cell lacked the enzyme, the hybrid cell must have obtained its T K f r o m one of the h u m a n chromosomes. T h e isolation of T K + hybrid clones having only a single h u m a n chromosome was further evidence that this submetacentric human chromosome had contributed this function. ~1, 22 Electrophoretic demonstration that the T K in the hybrid cell is of the h u m a n variety indicates that the pertinent chromosome provides the structural locus for T K . 2s Recently, it has become possible to distinguish individual chromosomes in the human karyotype on the basis of their pattern of fluorescence with qulnacrlne, and as a consequence the solitary submetacentric group E chromosome in these clones has been identified as a n u m b e r 17. ~ Unfortunately the usefulness of the H A T - B U D R - 8 azaguanlne media is limited to those mutations involving the H G P R T a a d T K loci, since these media favor or disfavor only chromosomes specifying these enzymes (the h u m a n X and I7, respectively). A variety of selective media is required for the exploitation of this m e t h o d for linkage studies.
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G-6-PD and H G P R T phenotypes in some hybrid clones? 9 Although both enzymes are specified by the X chromosome, the loci are probably not closely linked? ~ Fragmentation, therefore, may actually be useful to identify close linkage groups, and to define the sequence of the genes on the chromosome as well. Evidence of this kind suggests that PGK is also not closely linked with either G-6-PD or H G P R T , and their positions relative to the centromere can be determined using cells with X-chromosome deletions, as Human cells with chromosome deletions and rearrangements will be invaluable for determining precisely the chromosomal location of linkage groups. GENE R E G U L A T I O N We have innumerable clues that some genes function differently at various times in development. One of the best examples of this is the synthesis of y chains of hemoglobin which combine with a chains to form the fetal variety of hemoglobin. Hemoglobin F, containing y chains, predominates during fetal life, while hemoglobin A, with B chains, comprises most of the hemoglobin after birth. However, small amounts of /3 chains are produced as early as 9 weeks' gestation a= and small amounts of fetal hemoglobin are present in the adult. If we knew what is involved in the turning down of the y-chain locus and how this control could be manipulated, this gene conceivably could be turned up again postnatally in individuals with sickle cell anemia or thalassemia who cannot make normal/3 chains. The conditions limiting our knowledge of gene-chromosome linkage relationships have also deterred the elucidation of control mechanisms in human cells. Although the study of haploid microorganisms has provided insight into possible regulatory mechanisms, the systems involved in the control of gene action in one-cell organisms may not be equally appropriate for an organism as complex as man. The results of recent linkage studies suggest that genetic regulation has some unique features in mammalian cells. Genes specifying unlike subunits of proteins, such as hemoglobin and lactate de-
The Journal o[ Pediatrics December 1971
hydrogenase are not linked to one another, so that control of their synthesis probably differs from the operon type of regulation mechanisms which serve to control a group of contiguous genes. Hybrid cells can be used to probe the means by which the phenotype of the cell is determined and how the expression of the parental genes is regulated. Are the genes of one parent dominant or recessive to those of the other? Many of the proteins in the hybrid cell are specified by the genes of both parent cells. For example, the mouse-hamster hybrid cell will produce the mouse form of malate dehydrogenase, as well as the hamster variety of the enzyme, distinguishable on the basis of differences in electrophoretic migration. In addition, there is a third form of malate dehydrogenase, the novel "hybrid" heteropolymer, produced as a result of the association of mouse and hamster enzymes, a3 There are instances, however, in which the genes of one parent have not been expressed in the hybrid and which cannot be attributed to loss of the pertinent locus. Hamster melanoma cells, when fused with nonpigmented mouse cells, result without exception in nonpigmented hybrids which have lost DOPA oxidase? ~' ~5 The precise mechanism is undefined, but there appears to be some block to pigment formation in these hybrids; study of this phenomenon is likely to reveal the nature of the influence of one genome on the pigment production by the other. COMPLEMENTATION
It has become apparent that the amount of genetic heterogeneity discovered at any locus is proportional to the amount of effort involved in the search, and certainly is sufficient to indicate that in regard to variants which occur rarely, each family may have its unique nmtation. The amount of genetic heterogeneity in terms of enzyme kinetics is considerable, even in diseases which show a relatively uniform phenotype, as for example the Lesch-Nyhan syndrome? 6 Our ability to detect differences in mutations, which may not lead to differences in enzyme phenotype demonstrable by the usual
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biochemical techniques, may be enhanced by complementation analysis of pairs of mutant cell strains. Enzyme-deficient cells from two unrelated, but clinically similar, individuals may be fused together and the phenotype of their hybrid determined. If the mutations in the cells of the probands are similar, the hybrid derived from these cells should also be enzyme deficient. Therefore, restoration of normal enzyme function in the hybrid is evidence that the two parental mutations are not the same. Analysis of hybrids derived from glycine-dependent Chinese hamster cells have indicated that there are at least four loci associated with this phenotype and that complementation analysis is feasible in mammalian somatic cells? r Recently heterogeneity within the group of patients with galactosemia has been reported on the basis of studies of cultures containing mixed populations of fibroblasts. Cells from one of seven patients tested were able to complement those of the other six and the hybrid enzyme differed from the wild type in specific activity and some kinetic properties, as This kind of complementation analysis can be carried out in short-term cultures of Sendai-induced heterokaryons, especially when the normal enzyme phenotype is demonstrable in the intact ceil. Heterokaryons, however, usually represent a minority of the cells present in the mixed culture. The proportion of heterokaryons can be enriched and hybrid ceils can be isolated with the use of culture medium which disfavors the growth of mutant parent cells. Theoretically it should be possible to select against any mutant cell and the ability to devise appropriate selective media will enhance our ability to carry out complementation analysis of all kinds of human mutations, provided these are demonstrable at the level of the cultivated cell. OTHER APPLICATIONS THE METHOD
OF
One of the most exciting potential applications of the method is the use of cell hybrids for immunization against malignant tumors. The highly malignant Ehrlich's as-
893
cites tumor is lethal to a variety of mouse strains because it is only weakly antigenic. However, if the mouse ascites tumor is hybridized with a hamster cell, the resulting hybrid cell, when injected into mice, is able to protect the host animal during subsequent exposure to the tumor cells? 9 The mice, by rejecting the hybrid cells immunologically because of the hamster component, apparently acquire an immunity to the Ehrlich's tumor antigens. Therefore, it is conceivable that a hybrid formed between a tumor with weak transplantation antigens and cells with strong antigens on the basis of species differences could be used to immunize the host animal against his own tumor. Since the tumor cells can be fused directly, not requiring cultivation, the method is applicable to the problem of human metastatic tumors. Even if immunization with interspecific hybrid cells does not fulfill its promise, the hybrid cell certainly has a significant role in the study of the malignant process. Highly malignant mouse tumor cells, when fused with less virulent cells, give rise to hybrids which do not express the malignant phenotype of their tumor cell parent. 4~ These experiments suggest that tumogenicity in the hybrid is suppressed by the presence of the genome of the nontumor cell parent. The similarities between the characteristics of malignant cells and the changes in cells following viral transformation leave little doubt that virus-induced neoplastic transformation is an excellent model for the study of malignancy. Cell hybridization has already been applied to the study of some aspects of the virus-host relationship and has been particularly useful in demonstrating the continuous presence of the viral genome in the transformed cells, even when these cells do not produce virus particles. 41, 42 In some cases the cell which harbors the virus is not a good indicator of its presence, and fusion of the host cells with susceptible indicator cells is necessary to reveal the presence of infectious virus. .8 In one instance, fusion of two parental cells, neither of whieh produced infectious virus, resulted in hybrid cells capable of producing virus, possibly by
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The ]ournal o[ Pediatrics December 1971
means of complementation of defective particles juxtaposed in the same heterokaryon. ~4 This approach to the recovery of infectious viruses could be extremely fruitful for the isolation of elusive viruses in diseases suspected to have a viral etiology. THE
TASK
AHEAD
Somatic hybridization is an excellent means to genetic analysis in man. It seems that interspecific hybrids will have their greatest value for gene localization, whereas hybrids between cells of the same species, because of their relatively stable karyotype, may be preferable for complementation analysis and studies of control of cell phenotype. Ideally we would like to study hybrids derived from nonheteroploid human cells of a variety of tissues and differentiated states. A beginning has been made by Siniscalco and associates, 4s who have observed tetraploid cells in mixed cultures of human diploid cells with different X-linked mutations. Since no one has yet isolated a clone of hybrids from parents who are both euploid, we do not know what it woutd took like. Judging from the efforts to isolate such a hybrid, it seems that the fused human cell does not have an obvious selective advantage over its diploid parents. Hence the senescence in culture which characterizes diploid fibroblasts may unfortunately also characterize the hybrid. Lymphocytes maintained in long-term culture, however, seem to have gained immortality, so that these may be ideal parents for human hybrids. Somatic hybridization has resulted from the development of methodology in many areas of research. Techniques for the in vitro cultivation of discrete mammalian cells have contributed the milieu for cell mating. Developments in cytogenetics and enzymology have provided the initial supply of cells having biochemical and cytogenetie markers. Further developments are required for the exploitation of this means of genotypie anaIysis. These should include the discovery of the means to prolong the life of cultivated diploid cells, so that studies of progeny of
single cells necessary for genetic analysis can be facilitated. We must develop methods to propagate cells from normal differentiated tissues, so that their in vivo function is maintained; such cells will provide a source of genetic markers invaluable for the study of the control of differentiation. Although the number of available cell lines of this type is small, more will be developed. The ingenious methods of Sato and colleagues46 for the cultivation of cells from hormone-producing mouse tumors will contribute a host of mouse cell markers not available in cells of connective tissue origin. For every aspect of genetic analysis, there is the need to increase the supply of cells with stable genetic markers. Among the lines available for use are those whose karyotypes include stable deletions and rearrangements resulting in morphologically distinctive chromosomes. The recent development of techniques for the identification of individual chromosomes on the basis of their patterns of fluorescence with various quinacrine compounds 4~ will enhance the supply of these variants by revealing chromosomal rearrangements not visible on the basis of altered gross morphology. Furthermore, the precise identification of individual chromosomes will obviously increase the specificity of linkage assignments. The extent of protein variation between species is significant and the number of relatively common variants of human proteins already demonstrated suggests that there is an abundance of heterogeneity within our own species which can be utilized. *s In more than 20 of the inborn metabolic errors, the enzyme deficiency related to the disease has also been demonstrated in skin fibroblasts derived from patients; as more of these errors are defined, more biochemically marked human cells will become available. In addition, the naturally occurring mutations can be supplemented with those inducible by various mutagenie agents. ~.9, 50 ~Since mutation~ in diploid cells are usually recessive, the ideal candidates for exposure to mutagens are: (1) male cells (haploid for X-linked genes), and (2) cells with chromosomal deletions (haploid for genes on the deleted segment),
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Since recombination is the essence of genetic analysis, efforts should be directed tow a r d the d e v e l o p m e n t of methods to promote mitotic segregation of the classical type, or at least to induce chromosome elimination from intraspecific cell hybrids. Pontecorv@ 1 has suggested that sensitivity to certain treatments, effective in p r o m o t i n g chromosome loss, m a y vary for i n d i v i d u a l chromosomes according to their replication pattern. If true, then these sensitivity differences provide the means for eliminating specific h u m a n chromosomes.
Somatic cell hybrids
3. 4. 5. 6.
7.
CONCLUSION T o change the genetic constitution of an individual is at best a f o r m i d a b l e task. Even if it were technically possible to transfer gone or gene products to the a p p r o p r i a t e host cells, the correction of the deleterious m u t a t i o n m a y be less t h a n perfect. T h e r e fore, when possible, p r e n a t a l detection of an affected fetus a n d subsequent abortion m a y prove to be the most effective t r e a t m e n t for most genetic diseases. However, in some instances p r o v i d i n g essential gene products or even D N A m a y be fully efficacious a n d the preferable alternative. In either event we must significantly increase our knowledge of the details of gene action in o u r species, as well as elucidate the genetic basis for inherited diseases. Preliminary results indicate that somatic cell hybrids are an excellent means for obtaining information regarding the sequence of genes on h u m a n chromosomes and the control of protein synthesis. Disease-related potential applications of the m e t h o d s are virtually unlimited a n d include p r e n a t a l detection of genetic disease, the t r e a t m e n t of m a l i g n a n t disease, the u n m a s k i n g of the viral etiology of some infectious diseases, and ultimately the r e p l a c e m e n t of defective genes.
8. 9.
10.
11. 12. 13.
14.
15. 16.
17.
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