Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.     

Activation of production of mouse liver enzymes in rat hepatoma-mouse lymphoid cell hybrids

The production of four liver-specific enzymes (tyrosine aminotransferase or TAT, alanine aminotransferase, aldolase B, and alcohol dehydrogenase) has been analyzed in rat hepatoma-mouse lymphoid cell hybrids containing a 1s or 2s complement of rat chromosomes. The hybrid clones which retain a nearly 2s complement of rat chromosomes show high activity of all four enzymes; those which contain a 1s rat complement show partial or complete extinction of these enzymes. The tyrosine aminotransferase produced by most of the hybrid clones is identifiable as being of both rat and mouse origin, based upon criteria of temperature sensitivity and electrophoretic mobility; antiserum to the rat liver enzyme was used to distinguish the pseudo-TAT produced by the lymphoid parent from liver-TAT produced by hepatoma and hybrid cells. By the criteron of electrophoretic mobility, the liver form (B) of aldolase, produced by only some of the hybrid clones, appears to be composed of both rat and mouse subunits. We conclude that when extinction of tissue-specific proteins does not occur or is only partial in hybrid cells (due to gene dosage effects), the genomes of both parents may be active in directing synthesis of these proteins.     

A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells

Extinction is an operational term that refers to the lack of expression of tissue-specific traits that is generally observed in hybrid cells formed by fusing dissimilar cell types. To define the genetic basis of this phenomenon, a series of rat hepatoma x mouse fibroblast hybrids has been isolated and characterized. We report here that the extinction of hepatic marker traits in these clones was strictly correlated with the retention of five particular fibroblast chromosomes (autosomes 8, 9, 10, 11, and 13). In order to dissect this correlation into its component parts, hepatoma microcell hybrids containing single, specific fibroblast chromosomes were constructed. Hepatoma clones retaining only fibroblast chromosome 11 were specifically extinguished for liver-specific tyrosine aminotransferase (TAT) expression, while expression of four other hepatic traits and of numerous constitutive markers was unaffected. Furthermore, removal of fibroblast chromosome 11 from the populations by back-selection resulted in reexpression of TAT activity to full parental levels. These data define and localize a genetic locus, tissue-specific extinguisher-1 (Tse-1), which regulates hepatic TAT expression in trans. We also provide evidence that human Tse-1 resides on the homologous chromosome (human chromosome 17), and that hybrids retaining active Tse-1 loci lack TAT-specific mRNA.     

Hormonal regulation of TSE1-repressed genes: evidence for multiple genetic controls in extinction.

Somatic cell hybrids formed by fusing hepatoma cells with fibroblasts generally fail to express liver functions, a phenomenon termed extinction. Previous studies demonstrated that extinction of the genes encoding tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and argininosuccinate synthetase is mediated by a specific genetic locus (TSE1) that maps to mouse chromosome 11 and human chromosome 17. In this report, we show that full repression of these genes requires a genetic factor in addition to TSE1. This conclusion is based on the observation that residual gene activity was apparent in monochromosomal hybrids retaining human TSE1 but not in complex hybrids retaining many fibroblast chromosomes. Furthermore, TSE1-repressed genes were hormone inducible, whereas fully extinguished genes were not. Analysis of hybrid segregants indicated that genetic loci required for the complete repression phenotype were distinct from TSE1.     

Dedifferentiated variants of a rat hepatoma: analysis by cell hybridization

Two independent dedifferentiated variants, H5 and FaoflC2, derived from the Reuber H35 hepatoma, produce trans-acting diffusible substances(s) that extinguish the expression of liver-specific proteins when hybridized with a well-differentiated cell line of the same origin (Fao and Fu5-5, respectively). H5 x Fao hybrids show total and stable extinction of four liver functions and clonal variability in the expression of three others. FaoflC2 x Fu5-5 hybrids are initially flat (like FaoflC2 cells), and die in glucose-free medium where survival requires expression of hepatic gluconeogenic enzymes, but then evolve to hepatoma-like and finally round morphology; these latter cells express all liver functions analyzed including the gluconeogenic enzymes. Two exceptional clones that remained flat long enough for complete analysis showed extinction of all hepatic functions not expressed by FaoflC2 cells. We conclude that this transitory extinction reflects the action and then loss of extinguishing factor(s) contributed by FaoflC2. When crossed with BW1-J mouse hepatoma cells. FaoflC2 causes stable extinction of mouse aldolase B. We propose that production of extinguishing factor(s) is the rule for dedifferentiated variants.     

Somatic cell hybrids between Friend erythroleukemia cells and mouse hepatoma cells.

Somatic cell hybrids between hepatoma and Friend erythroleukemia parental cells were studied for the expression of liver-specific and erythroid properties. Several independent clones were isolated using HAT selection and were shown to be true hybrids by isozyme and chromosome analysis. All displayed a complete extinction of hemoglobin and globin mRNA production, but a retention of albumin and transferrin secretion. The data suggest that erythroid differntiation is being actively inhibited by the hepatoma genome. Possible mechanisms that might explain these results are discussed in the light of current hypotheses regarding the mechanism of cell differentiation.     

Expression of differentiated functions in dexamethasone-resistant hepatoma cells

Abstract. The expression of liver-specific functions of different dexamethasone-resistant variants derived from a well-differentiated dexamethasone-sensitive Reuber H35 rat hepatoma cell line (Faza 967) was examined during long-term cultivation. The dexamethasone-sensitive Faza 967 cells are characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenic enzymes, secretion of serum albumin, and the presence of liver isozymes of alcohol dehydrogenase (L-ADH), aldolase (aldolase-B), and five isoenzymes of lactate dehydrogenase (LDH). The hormone-resistant cells undergo a very dramatic change in expression of most liver-specific functions (dedifferentiation) during long-term culture, in contrast to the sensitive cells in which only certain functions (TAT activity, inducibility, and synthesis of serum albumin) exhibit considerable changes. The hormone-dependent growth sensitivity and the expression of other differentiated functions is not controlled in coordinated way in Faza 967 cells. The time course of the expression of liver-specific functions shows that the cells are resistant before they became 'dedifferentiated', i.e., loss of these liver-specific functions is not a prerequisite of the establishment of the hormone-resistant state.     

Expression of differentiated functions in dexamethasone-resistant hepatoma cells

The expression of liver-specific functions of different dexamethasone-resistant variants derived from a well-differentiated dexamethasone-sensitive Reuber H35 rat hepatoma cell line (Faza 967) was examined during long-term cultivation. The dexamethasone-sensitive Faza 967 cells are characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenic enzymes, secretion of serum albumin, and the presence of liver isozymes of alcohol dehydrogenase (L-ADH), aldolase (aldolase-B), and five isoenzymes of lactate dehydrogenase (LDH). The hormone-resistant cells undergo a very dramatic change in expression of most liver-specific functions (dedifferentiation) during long-term culture, in contrast to the sensitive cells in which only certain functions (TAT activity, inducibility, and synthesis of serum albumin) exhibit considerable changes. The hormone-dependent growth sensitivity and the expression of other differentiated functions is not controlled in coordinated way in Faza 967 cells. The time course of the expression of liver-specific functions shows that the cells are resistant before they became 'dedifferentiated', i.e., loss of these liver-specific functions is not a prerequisite of the establishment of the hormone-resistant state.     

Expression and extinction of glial properties in glioma X neuroblastoma cell hybrids

Rat glioma cells (clone C₆TK⁻) were hybridized with mouse neuroblastoma cells (clone NA), and 18 primary and secondary hybrid clones containing one chromosome set from each parent were isolated. The hybrids were assayed for the glial marker enzymes 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH). In many of the hybrid clones, the levels of CNP and GPDH were reduced to 5–20% of the activity of C₆TK⁻, as has been observed in other classes of glial X non-glial cell hybrids. In some hybrid clones, however, GPDH and CNP were expressed at high activity. Rat (glial) GPDH activity was not reduced in these clones, but mouse GPDH activity remained low, and was not “de-repressed” or “activated”. This suggests that the controls governing differentiation in neuroblastoma cells and extinction in hybrids may differ in some important details. There was a strong positive correlation between the specific activities of CNP and GPDH in the hybrid clones, suggesting that a mechanism regulates the activity of these two glial enzymes coordinately.     

Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.     

Microsomal epoxide hydrolase activity in human x mouse hybrid cells

Microsomal epoxide hydrolase (E.C.3.3.2.3) activity has been measured in human x mouse hybrid cells prepared from human cells expressing 6-7 x the activity of the mouse cells. Rabbit antihuman and antimouse antisera raised against purified enzymes were used to discriminate between human and mouse enzymes. All twenty five clones examined did not express human enzyme and this correlated with the loss of human chromosome 6 from each cell line. Four hybrids expressed 2-3 x the activity expressed by the mouse cell parent and these all retained more human chromosomes, specifically chromosome 19, than those with low activity. It is concluded that the human gene for epoxide hydrolase may be on chromosome 6 and that other gene products can affect the level of activity expressed by a cell.     

Re-expression of hepatic functions in mouse hepatoma X rat hepatoma hybrids

Abstract. Neonatal hepatic functions are selectively extinguished in hybrids between mouse hepatoma cells, that express only fetal hepatic functions, and rat hepatoma cells expressing neonatal as well as fetal functions. A search for hybrid cells reexpressing these neonatal functions was undertaken to determine; (1) whether the selective extinction of neonatal functions is reversible and at what frequency, and (2) whether the reexpression of neonatal functions would be accompanied by modifications in the expression of fetal functions. The criterion used to obtain hybrids showing reexpression was glucose-free medium (G-) where growth requires the presence of the extinguished gluconeogenic enzymes. Even though the parental cells are of the same histotype it proved difficult to obtain re-expression. Survivors in G- were obtained only from hybrids containing a greater than Is complement of rat chromosomes; they reexpress not only gluconeogenic enzymes but also basal tyrosine aminotransferase activity, and the fetal hepatic function a-fetoprotein continues to be expressed in most of the clones. All survivors in G- display a significant loss of chromosomes and this loss concerns essentially mouse chromosomes.     

Analysis of myogenesis by somatic cell hybridization : I. Myogenic competence of homotypic hybrids derived from rat L6 myoblasts

Several authors have described the extinction of myogenic competence in hybrids produced by fusion of myogenic and non-myogenic cells. Interpretations of such experiments rest upon the assumption that extinction does not occur with any appreciable frequency as a non-specific consequence of the cell hybridization process itself. In order to test this assumption we have analyzed the myogenic competence of over 140 independent homotypic hybrid clones produced by PEG-mediated fusion of rat L6 myoblasts. Based upon an evaluation of myotube formation in hybrid colonies, we demonstrate that 99% of primary hybrid clones are myogenic. The fact that 97% of secondary hybrid colonies also differentiate indicates that myogenic competence is a stable characteristic of the hybrids. Four hybrid clones were isolated and expanded for analyses of chromosome numbers, myotube formation, creatine kinase activities, and microfluorimetric DNA determinations of myotube nuclei. Our results demonstrate that polyploid homotypic hybrid cells produced by fusion of non-neoplastic, developmentally determined rat myoblasts retain and express their program of differentiation. This work provides a foundation for future studies which will investigate the expression of myogenic properties in hybrids between myogenic and non-myogenic cells.     

Re-expression of hepatic functions in mouse hepatoma X rat hepatoma hybrids

Neonatal hepatic functions are selectively extinguished in hybrids between mouse hepatoma cells, that express only fetal hepatic functions, and rat hepatoma cells expressing neonatal as well as fetal functions. A search for hybrid cells reexpressing these neonatal functions was undertaken to determine; (1) whether the selective extinction of neonatal functions is reversible and at what frequency, and (2) whether the re-expression of neonatal functions would be accompanied by modifications in the expression of fetal functions. The criterion used to obtain hybrids showing re-expression was glucose-free medium (G) where growth requires the presence of the extinguished gluconeogenic enzymes. Even though the parental cells are of the same histotype it proved difficult to obtain re-expression. Survivors in G- were obtained only from hybrids containing a greater than 1s complement of rat chromosomes; they reexpress not only gluconeogenic enzymes but also basal tyrosine aminotransferase activity, and the fetal hepatic function alpha-fetoprotein continues to be expressed in most of the clones. All survivors in G- display a significant loss of chromosomes and this loss concerns essentially mouse chromosomes.     

Suppression of Immunoglobulin Synthesis by Cellular Hybridization

WHEN two cells which show differentiated characteristics are fused, the hybrids generally do not show these characteristics. Ephrussi et al.¹ have interpreted such trans dominant loss to show that the cells contain a diffusible repressor and this idea is supported by the finding that some mouse/human hybrids can spontaneously redifferentiate when the C10 human chromosome is lost². We now report another example of redifferentiation in hybrid cells; when IT mouse fibroblasts are fused with MPC 11 mouse myeloma cells which produce immunoglobulin, the hybrids neither produce nor excrete detectable amounts of immunoglobulin.     

Analysis of myogenesis by somatic cell hybridization. II. Retention of myogenic competence and suppression of transformed properties in hybrids between differentiation competent and incompetent rat L6 myoblasts

This study describes the characteristics of hybrids between two closely related rat myoblast lines, which differ both in the ability to express their program of differentiation and in the expression of neoplastic properties. Myogenic, nonneoplastic L6J1-S cells were hybridized with nonmyogenic, neoplastic L6J1-N1 cells. Six hybrid clones were isolated and expanded for analysis of myogenic competence, and four of these clones were also evaluated for parameters of transformation, including tumorigenicity, ability to clone in agar, and surface fibronectin. In addition to our analysis of isolated clones, we also assessed myogenic differentiation in colonies representing 226 early hybrid clones. Results of all these analyses demonstrate that the myogenic phenotype is retained and that the tumorigenic/transformed phenotype is suppressed in the hybrids. Furthermore, our results indicate that when the programs for myogenesis and neoplastic transformation are confronted within a single cell, they are expressed as mutually exclusive alternatives. In contrast to these results on myogenic X nonmyogenic L6 hybrids, it has been reported that isolated clones of A9 X L6 exhibited extinction of myogenic competence and retention of transformed properties. We have evaluated myotube formation in over 300 early hybrid clones between A9 and either diploid or subtetraploid L8 rat myoblasts. Our results demonstrate that all of these hybrid clones exhibit extinction regardless of the ploidy of the myoblast parent, and they further indicate that extinction is not a consequence of chromosome loss. These results support the conclusion that in A9 X L6 hybrids, the nonmyogenic, transformed phenotype is dominant.     

Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells.

Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.     

Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid fibroblasts

Hybrids were generated between mouse hepatoma cells which exhibit a transformed phenotype, and rat normal diploid fibroblasts. Most isolated hybrid clones contain a single set of chromosomes from each parent. Such clones grow to low saturation densities and are unable to grow or to form colonies in soft agar. The transformed phenotype of the parental hepatoma cells is thus suppressed in these hybrids. Suppression is very stable; however, subclones which have regained a transformed phenotype could be selected; these subclones show a significant reduction of their chromosome number. Amongst the hybrid clones isolated after fusion, a few are characterized by an excess of mouse chromosomes and a reduced number of rat chromosomes. Such clones exhibit a transformed phenotype. Our results show that, provided the hybrids contain an almost complete single set of chromosomes of each parent, spontaneous transformation behaves as a recessive trait in hybrids formed with normal diploid cells.