Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a 'luxury function' of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.     

Extinction, Retention and Induction of Serum Protein Secretion in Hepatoma-fibroblast Hybrids

The production of four serum proteins has been analysed in several hepatoma-fibroblast hybrids. Extinction of albumin and alpha-foetoprotein production occurs systematically in intra and interspecific (rat X mouse) hybrids derived from mouse hepatoma cells (BW). Similar hybrids derived from two related clones of rat hepatoma cells either do not produce albumin (Fa32-derived hybrids), as the BW-derived hybrids, or retain the capacity to produce it, but at a reduced rate (Fu5-derived hybrids); some differences in the control of albumin production thus seem to exist between clonal hepatoma cell lines. The mouse hepatoma cell hybrids retain the capacity to secrete transferrin at a reduced rate, and C3 (the third component of complement) at a high rate. Further analysis of C3 production in interspecific hybrids showed that both parental genomes actively contribute to C3 production: induction of C3 secretion is thus observed in these hybrids.     

Expression of differentiated functions in hepatoma cell hybrids. II. Aldolase

A study of aldolases in rat hepatoma clones and subclones has revealed that they synthesize all three forms of aldolase monomers: A (the ubiquitous glycolytic isozyme), B (the form characteristic of the liver) and C, and that in vitro–in vivo passage results in a reversible modulation in aldolase A activity. Three kinds of somatic hybrids, between rat hepatoma cells and either mouse fibroblasts or rat epithelial cells, have been studied. In each case, aldolase B, found only in the hepatoma parent, was absent in the hybrid cells. The absence of aldolase B in the somatic hybrids seems not to be due to trivial factors (species differences, inactivation of all hepatoma aldolase genes, increase in ploidy or loss of chromosomes); it is concluded that extinction of this differentiated function of the hepatoma parent reflects a genetic regulatory phenomenon.     

The control of serum protein synthesis in hepatoma-fibroblast hybrids.

Hybrids between mouse hepatoma cells (which secrete several serum proteins) and mouse or rat fibroblasts (which do not secrete these proteins) produce transferrin and the third component of complement (C3) like the parental hepatoma cells, while they do not secrete either albumin or alpha-fetoprotein (AFP). This lack of albumin and AFP secretion is probably due to a lack of synthesis, rather than to a simple defect in secretion. The cessation of albumin and AFP production is not dependent upon the parental fibroblast nor upon the selection conditions; it is best explained by a shut-off synthesis and could thus reflect the existence of a regulatory mechanism. This would imply a difference between the control of albumin and AFP synthesis and that of transferrin and C3 synthesis. On the other hand, in agreement with Peterson and Weiss (1972), hybrids between rat hepatoma cells and mouse fibroblasts continue to product rat albumin. This suggests that the mouse hepatoma cells differ from the rat hepatoma cells in the way they control albumin production.     

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.     

Long-range chromatin reorganization of the human serpin gene cluster at 14q32.1 accompanies gene activation and extinction in microcell hybrids

The genes encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of six serine protease inhibitor (serpin) genes located on human chromosome 14q32.1. Both genes are actively transcribed in the liver and in human hepatoma cells, but they are not expressed in most other cell types. In this study we mapped DNase I-hypersensitive sites (DHSs) in an approximately 130-kb region of 14q32.1 that includes both genes. The distributions of DHSs in expressing (HepG2) vs nonexpressing (HeLa S3) cells were very different: HepG2 cells displayed 29 DHSs in this interval, but only 7 of those sites were present in HeLa cells. To determine the chromatin organization of activated or extinguished serpin alleles, we transferred human chromosome 14 into rat hepatoma cells or fibroblasts, respectively. Human alpha1AT and CBG gene expression was activated in rat hepatoma microcell hybrids containing human chromosome 14, but extinguished in rat fibroblast hybrids with the same genotype. DHS mapping in these microcell hybrids demonstrated that the chromatin structure of the entire 130-kb region was reorganized in microcell hybrids, and the distributions of DHSs in activated and extinguished alleles recapitulated those of expressing and nonexpressing cells, respectively. Thus, microcell hybrids provide a system in which reproducible changes in gene activity and long-range chromatin organization can be induced experimentally. This provides a basis for studying the effects of targeted modifications of the alpha1AT and CBG loci on the regulation of gene activity and chromatin structure.     

Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma- mouse fibroblast heterokaryons and hybrids

We have used a combination of a sensitive immunocytochemical stain for intracellular albumin, and Hoechst 33258 dye for identification of parental nuclei to investigate the time-course of extinction, reexpression, and activation of albumin production in fusion products of 1s (hyperdiploid) or 2s (hypertetradiploid) rat hepatoma cells with mouse fibroblasts (L cells or embryonic cells). In all combinations, the initial event is extinction of albumin production. Extinction occurs immediately after fusion when the mouse fibroblast is a normal embryonic (senescent?) cell. In the case of an L cell, rat albumin is synthesized and secreted during the first 12 h after fusion; no production of mouse albumin occurs. Thereafter, albumin production ceases. 8-12 d after fusion, young hybrid colonies are found to resume the synthesis of rat albumin (reexpression), and several days later the production of mouse albumin begins (activation). The patterns of reexpression and activation indicate (a) that chromosome loss is not necessary for either event to occur and (b) that the cells active in the synthesis of mouse albumin are a subpopulation of those cells already engaged in the production of rat albumin. We conclude that (a) extinction is mediated by diffusible factor(s) from the L-cell parent that act in the hepatoma nucleus to prevent the formation of new albumin messenger RNA; (b) reexpression and activation are gene dosage- dependent but extinction is not; and (c) previously active genes are more rapidly expressed than previously silent ones.     

Regulation of expression of tyrosine aminotransferase in somatic cell hybrids between rat hepatoma cells and mouse fibroblasts

Somatic cell hybrids between rat hepatoma cells and mouse 3T3 fibroblasts fail to produce the liver-specific enzyme tyrosine aminotransferase. A novel approach using gamma-irradiation to induce chromosome loss from the non-expressing parent cell was applied to dissect genetically the factors in 3T3 cells that interact with the regulation of expression of tyrosine aminotransferase in these hybrids. Suppression of basal and steroid-inducible tyrosine aminotransferase activities was progressively relieved with increasing dose of radiation. The wide range in degree of reexpression suggests a complex of regulatory mechanisms. Suppression of steroid-inducibility was not linked to the mouse X-chromosome. Nor did the mouse genome affect the modulation of enzyme activity induced by insulin and by serum.     

Cytoplasmic suppression of malignancy

Summary Using both normal and transformed rat liver epithelial cells to prepare cytoplasmic hybrids (cybrids) we have found evidence to support the theory that the cytoplasm from a normal cell can suppress tumorigenicity. A unique aspect of this study is that all of the cells utilized, both normal and malignantly transformed, were derived from an original cloned cell. We found that fusing cytoplasts from normal cells to malignantly transformed whole cells resulted in cybrid clones which, when injected into newborn rat pups, isogenic with those from which the cell culture was initiated, yielted tumors in 51% of the animals injected compared to 92% of the animals injected with the tumorigenic parent. Those animals that did develop tumors from the cybrid cells survived longer than those injected with cells from the tumorigenic parent. Thus, the cybrid, formed of cytoplasm from both parents, was less tumorigenic than the malignantly transformed parent cell. When reconstituted cells were prepared by fusing cytoplasts from normal cells with karyoplasts from malignantly transformed cells, a situation in which essentially all of the cytoplasm of the reconstituted cell is derived from normal cells, the tumorigenic phenotype was extinguished. This work was supported in part by United States Public Health Service grant CA12056, and grant CA09100 from the National Cancer Institute, Bethesda, MD. This work is partial fulfillment for the degree of Doctor of Philosophy for B.A.I.     

Immortalization of normal liver functions in cell culture: rat hepatocyte-hepatoma cell hybrids expressing ornithine carbamoyltransferase activity.

Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver-specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H-ornithine into protein arginine, and can be selectively grown in arginine-free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue-specific phenotypes.     

Phenotypic and molecular expression of albumin in rat hepatoma X L cell hybrids

A rat hepatoma cell line (H₄A_ZC₂) was characterized with respect to seven liver-specific phenotypes. Ten clones from the fusion of H₄A_ZC₂ and mouse L cell were analyzed for the expression of these phenotypes. The only hepatic function retained by the hybrid clones was rat albumin synthesis which continued at reduced levels relative to the hepatoma parent. Rat albumin cDNA analysis of RNA from parental and hybrid cells indicated that the reduction in albumin production observed in the hybrids was reflected in coordinate reduction of cytoplasmic rat albumin mRNA.     

Phenotype and surface antigens of mouse teratocarcinoma x fibroblast cell hybrids

Numerous colonies of hybrids between PCC4-aza 1 teratocarcinoma cells and fibroblasts of the heteroploid Cl.1D cell line were examined. All of the hybrids were fibroblasts showing extinction of the multiple developmental potentialities of the teratocarcinoma cell parent, irrespective of whether the teratocarcinoma parent was diploid or tetraploid. The hybrids did not show loss of any specific chromosome contributed by the PCC4-aza 1 cell parent. In contrast with the PCC4 parental cells which carry F9 antigens and do not express H-2b, the hybrids do not express F9 antigens and carry H-2 alloantigens of both parental specificities. These results suggest that in hybrids whose phenotype is that of the Cl.1D parent, a change may occur in the genetic program of the teratocarcinoma cells.     

Albumin extinction followed by de novo methylation of its gene in somatic hybrids of a rat hepatoma

We have previously identified an Msp I site at the 5′ end of the rat albumin gene whose undermethylation is necessary but not sufficient for stable albumin expression in rat hepatoma cells [1]. We have also shown that the extinction of albumin expression in somatic hybrids is not the result of methylation at this site, since for two different crosses, rapid extinction was found to occur in the absence of any de novo methylation of the previously active gene[2]. In the present study, we examine albumin expression and albumin gene methylation for independent hybrid clones isolated from crosses between albumin expressing rat hepatoma cells and cells of two different non-expressing lines. The cells from hybrid clones of both crosses are characterized by stable extinction of albumin expression. Moreover, we find that de novo methylation of the “extinguished” albumin gene can occur in somatic hybrids, but only some weeks after the gene has ceased to be expressed.     

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.     

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.     

Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells.

A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin . The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.     

Extinction of Albumin Gene Expression in a Panel of Human Chromosome 2 Microcell Hybrids

Expression of the serum albumin gene is extinguished in rat hepatoma microcell hybrids that retain mouse chromosome 1. These data define atrans-dominant extinguisher locus,Tse-2,on mouse chromosome 1. To localize the human TSE2 locus, we prepared and characterized rat/human microcell hybrids that contained either human chromosome 1 or chromosome 2, the genetic homologues of mouse chromosome 1. Rat hepatoma microcell hybrids retaining a derivative human chromosome 1 [der 1 t(1;17)(p34.3;q11.2)] expressed their serum albumin genes at levels similar to those of parental hepatoma cells. In contrast, microcell transfer of human chromosome 2 into rat hepatoma recipients produced karyotypically heterogeneous collections of hybrid clones, some of which displayed dramatic albumin extinction phenotypes. For example, albumin mRNA levels in several extinguished microcell hybrids were reduced at least 500-fold, similar to albumin mRNA levels in hepatoma × fibroblast whole-cell hybrids. Expression of several other liver genes, including α1-antitrypsin, aldolase B, alcohol dehydrogenase, and phosphoenolpyruvate carboxykinase, was also affected in some of the microcell hybrids, but expression of these genes was not concordant with expression of albumin. Hybrid segregants were prepared from the albumin-extinguished hybrids, and reexpression of albumin mRNA and protein was observed in sublines that had lost or fragmented human chromosome 2. Finally, expression of mRNAs encoding the liver-enrichedtransactivators HNF-1, HNF-4, HNF-3α, and HNF-3β was not affected in any of the chromosome 2-containing hybrids. These data define and map a genetic locus on human chromosome 2 that extinguishes albumin gene expression intrans,and they suggest that TSE2-mediated extinction is independent of HNF-1, -4, -3α, and -3β expression.     

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.     

Expression of the ASV src gene in hybrids between normal and virally transformed cells: Specific suppression occurs in some hybrids but not others

Somatic cell hybrids have been made between clones of rat cells transformed by avian sarcoma virus and rat or mouse cells that are untransformed. Intraspecies hybrids were either predominantly morphologically normal or predominantly transformed, some clones that formed transformed intraspecies hybrids yielding normal interspecies hybrids. Untransformed hybrids usually showed no detectable alteration in the structure or location of the integrated provirus, but viral RNA and pp60^src kinase activities were much reduced. No decrease in viral gene expression was seen in transformed hybrids. Thus hybrid suppression of viral transformation, mediated in trans by the untransformed parent, is a specific event that depends on both untransformed and transformed parental parameters.