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.     

Conditions required for activation of the mouse albumin or α-fetoprotein gene in hybrids between mouse lymphoblastoma and rat hepatoma cells

Activation of two previously silent mouse hepatic genes has been investigated in hybrid cells between pseudodiploid mouse lymphoblastoma cells and hyperdiploid or hypertetraploid rat hepatoma cells. In this material, activation of the mouse albumin gene is a frequent event, whereas activation of mouse alpha-fetoprotein (AFP) occurs only in those cells that produce large amounts of albumin. Quantitative tests of hybrid populations for the activated proteins and their mRNAs revealed the expected sizes and structures: moreover, as in hepatoma cells, the amount of both rat and mouse albumin produced was directly proportional to the intracellular concentration of the corresponding mRNA. The cellular environment required for activation of the liver-specific genes was investigated by cell-by-cell analysis of each hybrid clone. Immunostaining for the presence of rat and mouse albumin and mouse AFP revealed unexpected heterogeneity in the phenotypes of the hybrid populations, which were found to contain cells that: (a) failed to express either of the proteins; (b) produced all three; (c) produced both rat and mouse albumin; or (d) produced rat albumin only. Karyotypic analysis indicated that the hybrid-cell phenotype depended on parental chromosome ratios rather than absolute numbers of chromosomes. It was found for albumin and mouse AFP that the fraction of immunostained cells was equal to the fraction of metaphases that contained a minimal rat-to-mouse chromosome ratio of 2.5 and 9, respectively. It is concluded that in those hybrids, expression of liver-specific genes is regulated by extinguishers, but in a dose-dependent fashion, suggesting the intervention of antagonistic activators from the rat hepatoma chromosomes.     

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.     

Selective detection of rat and mouse specific albumin and alpha-fetoprotein mRNA molecules under highly stringent hybridization conditions

The molecular analysis of some important interactions observed between the parental genomes in interspecific cell hybrids requires the availability of highly specific hybridization assays to selectively quantitate mRNA sequences coding for the same protein but transcribed from the two different genomes. Specific hybridization techniques which should permit the selective detection of rat and mouse albumin and alpha-fetoprotein (AFP) mRNA molecules in a mixture of the two types of mRNAs are presented here. The high degree of homology existing between the AFP mRNA sequences coding for mouse and rat AFP, and, presumably, albumin, results in extensive cross-hybridization with the cDNA probes under standard hybridization conditions. No size differences could be detected between the two types of mRNA molecules from the two species. A Tm difference of 7 degrees C between the intra- and interspecific mRNA:rat cDNA hybrids allowed the establishment of highly stringent solution hybridization conditions necessary to measure separately the contents of rat albumin and AFP mRNAs. Mouse albumin and AFP cDNA clones were then isolated from mouse liver and yolk sac cDNA libraries, and used to show the usefulness of highly stringent washing conditions to discriminate between rat and mouse albumin and AFP mRNA molecules in conventional "Northern blotting" techniques. In combination with the solution hybridization assay, these filter hybridization techniques can be used to specifically quantitate the content of rat and mouse albumin and AFP mRNA molecules in interspecific cell hybrids.     

A mouse hepatoma cell line which secretes several serum proteins including albumin and alpha-foetoprotein.

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces alpha-foetoprotein (AFP). Three clones were isolated from the uncloned culture: BW1, BW2 and BWTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both BW1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least five serum proteins into the culture medium: albumin, AFP, and alpha 2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 mug/24 h/10(6) cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: pigment and albumin production are not reexpressed simultaneously.

Hybridization of cells of defined and different histotypes has been carried out to investigate whether the expression (or reexpression) of parental functions is mutually exclusive, as is expected if the generally assumed rule of discreteness of differentiation applies to hybrid cells. A cross of pigmented mouse melanoma cells and albumin-producing rat hepatoma cells gave rise to hybrids containing essentially one set of chromosomes from each parent and producing neither melanin nor albumin. Cells of one hybrid clone are shown to retain the potential to reexpress both parental differentiations. Successive subclonings of this hybrid have shown that cells which reexpress one function may retain the potential to reexpress the other, and that freshly isolated, morphologically homogeneous subclones may produce pigment or albumin, but not both; there successive and exclusive shifts of phenotype are documented, and in these cases, chromosome loss is very slight. The use of immunoadsorbed antisera has revealed that most (if not all) of the albumin produced by the hybrid cells is of the mouse type. We conclude that both parental determinations are retained by the hybrid cells, and that the parental differentiations are reexpressed only in a mutually exclusive fashion.     

Somatic cell hybrids of mouse myeloma cells and B lymphocytes from a patient with agammaglobulinemia: failure to secrete human immunoglobulin.

Somatic cell hybrid clones were isolated from the fusion of RPC5.4 mouse myeloma cells and B lymphocytes from a patient with common varied agammaglobuinemia. The patient has B lymphocytes that synthesize immunoglobulin but fail to secrete immunoglobulin. The hybrid character of the six clones was established by examination of metaphase chromosome spreads. Most of the hybrid clones expressed mouse and human surface immunoglobulin. All of the clones synthesized immunoglobulin of mouse and human parental origin. Mouse parental immunoglobulin was secreted, whereas the human parental immunoglobulin was not secreted. Human light chain molecules were secreted as part of hybrid H2L2 molecules formed with mouse heavy chains. Human heavy chains had a reduced m.w. in comparison to the mouse heavy chains. Kinetic experiments indicated that human Ig was synthesized in amounts that were comparable to the mouse Ig. Pulse-chase experiments showed that that the intracellular human Ig was removed from the cytoplasm, probably by degradation. These experiments demonstrate that the hybrid cells are an in vitro model of naturally occurring failure of immunoglobulin secretion from agammaglobulinemia. The failure of fusion with mouse myeloma cells to complement the secretion defect suggests that these B cells produce an altered immunoglobulin molecule that is not programmed for secretion.     

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.     

Expression of liver phenotypes in cultured mouse hepatoma cells: synthesis and secretion of serum albumin

A permanent cell line, designated Hepa, has been isolated from a mouse hepatoma, BW 7756. The cell line synthesizes and secretes albumin at rates appreciably higher than previously reported hepatomas adapted to in vitro conditions. Monospecific antimouse serum albumin was produced in rabbits, and mouse serum albumin secreted by the hepatoma cells was identified by double diffusion, immunoelectrophoresis, and radioimmunodiffusion. A quantitative immunoassay was used to measure albumin secretion and to study the effects of culture conditions on albumin secretion. A subclonal analysis was performed to study the homogeneity and stability of cloned hepatoma lines in respect to albumin secretion. Different secretion rates were observed during the culture cycle. Significant clonal variation in respect to albumin secretion was found among ten subclones.The significance of clonal variation is discussed in relation to the study of epigenetic control of albumin expression in somatic hybrid cells.     

Molecular mechanism of extinction of liver-specific functions in mouse hepatoma x rat fibroblast hybrids: extinction of the albumin gene

Hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse albumin and alpha-fetoprotein synthesis. Karyotype analyses suggest that all parental chromosomes are present in the hybrids. The extinction, therefore, of mouse hepatocyte genes is attributed to the inhibitory action of the rat genome. In these studies, we show that these hybrids possess and express the mouse beta-glucuronidase gene (which is encoded on the same chromosome as the mouse albumin and alpha-fetoprotein gene), and we present data of Southern blot analysis which demonstrate that such hybrids have indeed retained both mouse and rat albumin DNA sequences. In addition, using mouse albumin cDNA, we have shown by cDNA-RNA reassociation kinetics that albumin mRNA is virtually absent in these hybrids. We conclude from these studies that the extinction of albumin synthesis involves a mechanism which results in the loss of cytoplasmic albumin mRNA.     

Secretion of proteins by the fertilized mouse ovum

Fertilized one-cell mouse ova were injected with messenger RNA (mRNA) for chicken ovalbumin or rat albumin. The ova were labeled with [³H]amino acids, and endogenous proteins as well as proteins in the culture medium were separated by two-dimensional electrophoresis. Following injection of either message, ova were able to synthesize and secrete the appropriate protein. However, when the message for globin was injected, globin appeared only among the endogenous ovum proteins but was not secreted into the culture medium. The location of the secreted proteins, albumin and ovalbumin, on two-dimensional gels suggests that ova process the proteins before secretion by removing the amino-terminal signal sequence from rat albumin and glycosylating ovalbumin. Rat albumin may be exported into the culture medium more rapidly than ovalbumin. In addition, at least one endogenous ovum protein was secreted at the one-cell stage. The studies establish the ability of the newly fertilized mouse ovum to synthesize and secrete proteins from injected as well as endogenous messages.     

Coexistence of expressed and non-expressed alpha-fetoprotein genes in somatic cell hybrids

Hybrids have been generated between mouse hepatoma cells, which actively synthesize alpha-fetoprotein (AFP), and adult hepatocytes, where AFP production is shut off. These hybrids maintain an active synthesis of mouse AFP. Using a specific radioimmunoassay, we found that rat AFP production is not activated. Southern blot analysis showed that mouse and rat AFP DNA sequences can be distinguished and that hybrid clones possessing something close to the complete chromosome sets of both parents have retained both parental AFP DNA sequences. Thus expressed and non-expressed AFP genes coexist in these hybrid cells as if their expression were dependent on a cis-acting event.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.     

Isolation and characterization of rat-mouse somatic cell hybrids secreting growth hormone and prolactin

Interspecific somatic cell hybrid clones have been isolated and characterized in order to study growth hormone (GH) and prolactin (PRL) gene expression. Rat pituitary tumor cells (GH3, 69 chromosomes) secreting rat GH and PRL were grown for 48 h together with nonhormone secreting, aminopterin-sensitive murine fibroblast cells (LMTK-, 55 chromosomes) and fused using polyethylene glycol. Resultant heterokaryons were selected in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned. Five clones produced rat GH and PRL. Hormone-producing hybrids morphologically resembled the mouse parent fibroblast. Hybrids grew in monolayers and contained 80-142 chromosomes, and marker chromosomes for both rat (small submetacentric) and mouse (bi-armed and large true metacentric) were identified. The interspecific nature of the hybrids was further confirmed by the presence of both rat and mouse adenosine deaminase and superoxide dismutase isozymes. Using specific antisera and indirect immunoperoxidase staining, both hybrid clones and GH3 rat parental cells stained positively for rat GH and PRL, while the murine fibroblast parental cells were negative. Hormone production by the hybrids has been sustained for over twenty subcultures; secretion rates were initially 150 ng PRL and 321 ng GH/10(6) cells/24 h and are currently 100 ng PRL and 90 ng GH/10(6) cells/24 h. Parental GH3 rat cells secreted 720 ng PRL and 660 ng GH/10(6) cells/24 h. Exposure of hybrids to KCl (50 mM) resulted in acute stimulation of rat PRL, but not rat GH release, and long-term incubation with thyrotropin-releasing hormone (TRH, 80 nM) stimulated PRL secretion. Hormone-dependent modulation of PRL secretion was transferred to the hybrid cell thus enabling the model to be used in studying regulation of PRL gene expression.     

Gene mapping in the rat by mouse-rat somatic cell hybridization: synteny of the albumin and alpha-fetoprotein genes and assignment to chromosome 14

The methods of somatic cell genetics and molecular hybridization were applied to a panel of mouse X rat hepatocyte hybrids segregating rat chromosomes to assign the rat genes coding for two serum proteins, albumin and alpha-fetoprotein (Alb and Afp). The molecular hybridization of DNAs from different hybrids with cloned DNA probes showed that all the hybrid clones possessing the rat Alb gene and expressing it also retained the rat Afp locus, which is not expressed in these hybrids. So the Alb and Afp genes are syntenic in the rat, as in the mouse. Furthermore, the cytogenetic analysis allowed the assignment of these two loci to rat chromosome 14.     

Somatic cell hybrids between totipotent mouse teratocarcinoma and rat hepatoma cells.

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinomas or hepatomas when injected in experimental animals.     

Hormonal regulation during secretion of alpha-fetoprotein in hepatoma cells grown in synthetic medium

The variant cell line of H4-II-E-C3 cells derived from the Reuber H-35 hepatoma cells has been established using protein- and lipid-free synthetic medium. This H4-II-E-C3-V line can synthesize and secrete considerable amounts of alpha-fetoprotein (AFP) and albumin. The addition of 5 X 10(-7) M dexamethasone to the medium stimulated the excretion of AFP without increasing total AFP synthesis, whereas 8.7 X 10(-8) M insulin inhibited the excretion of AFP without a significant inhibition of intracellular AFP synthesis. However, neither dexamethasone nor insulin altered either the cellular or secreted levels of albumin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. AFP appeared in the medium after 10 min, and 50% of the protein was secreted after 110 min. The rate of secretion of AFP was much slower than that of albumin, 50% of which was secreted after 25 min. Dexamethasone, 5 X 10(-7) M, caused a marked enhancement in the rate of AFP secretion, with 50% released after 75 min. Insulin, 8.7 X 10(-8) M, by contrast, caused a marked delay in AFP secretion with only 20% released after 180 min and then a plateau was approached. Since the intracellular AFP was excreted 55% after 180 min the remaining 25% of newly made AFP was suggested to be degraded during secretion. The kinetics of movement of AFP during secretion and endoglycosidase H treatment of intracellular and secreted AFP suggested that insulin impeded the transport of AFP from the rough endoplasmic reticulum to the Golgi apparatus.