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.     

Evidence for a trans-dominant regulator of purine nucleoside phosphorylase expression in rat hepatoma cells

Purine nucleoside phosphorylase (PNP) levels are modulated during the growth cycle of rat hepatoma cells and increase two- to three-fold as cells go from early exponential growth phase to stationary growth phase. A mutant of these hepatoma cells has been isolated which is deficient in PNP activity. Quantitative immunoprecipitation tests indicate that the decrease in enzyme activity is due to a decrease in the number of PNP molecules. The low level of PNP enzyme produced by the mutant, however, is indistinguishable from the wild-type enzyme, suggesting that the mutant may be defective in the ability to modulate PNP levels. Fusion of the mutant cells to wild-type parental cells results in hybrids that express the mutant phenotype. Segregants that arise from the hybrids show chromosome loss and reexpression of the wild-type parental phenotype, the mutant parental phenotype, and a 2S wild-type phenotype. These indicate the following about the defect in modulation in the mutant PNP-100: (1) it is trans dominant to the wild-type; (2) its effect is negative; (3) some genomic element is required for its continued effect; and (4) it does not act by obliterating its functioning counterpart in hybrid cells.     

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.     

A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue-specific extinguisher locus Tse-1

Tyrosine aminotransferase (TAT) gene expression is liver specific and inducible by glucocorticoids and via the cAMP signaling pathway. In fibroblasts and other nonliver cells the gene is subject to negative control by the trans-dominant tissue-specific extinguisher locus Tse-1. We identified a hepatocyte-specific enhancer that is repressed by Tse-1. Two distinct sequence motifs are absolutely essential for function of this enhancer: a cAMP response element (CRE), which is the target for repression by Tse-1, and a hepatocyte-specific element. The specificity of the enhancer is generated by the combination of these two essential elements, which are fully interdependent. In vivo footprinting indicates that Tse-1 acts by affecting protein binding at the CRE. A direct antagonism between Tse-1 and the cAMP signaling pathway suggests that Tse-1 plays a role in control of developmental activation of the TAT gene.     

Extinction and expression of the ribose-positive phenotype in hybrid Novikoff hepatoma cells

Expression of the ribose-positive phenotype was examined in hybrids obtained from the fusion of parental pentose-negative Novikoff hepatoma cells and ribose-positive variants. The two ribose-positive variants used differed phenotypically in their ability to use pentoses other than ribose for growth. One variant used D-ribose, D-xylose, and L-arabinose for growth, while the other variant used only D-ribose. Each variant was fused to pentose-negative parental hepatoma cells, and resultant hybrids were tested for the ability to use ribose. In both instances extinction of ribose utilization was the primary event, suggesting the existence of a trans-acting negative control element in the parental cells. In addition, hybrids from both fusion experiments eventually reexpressed the ribose phenotype. The rate of reexpression, however, was different for the two fusion experiments. Reexpression of ribose utilization in hybrids derived from the nonspecific variant occurred at approximately 10(-3) segregants/cell/day. Reexpressing segregants arose from the specific-derived hybrids at a rate of 0.5 segregants/cell/day. Possible reasons for this difference include a differential rate in chromosomal segregation or a difference in the regulation of ribose metabolism.     

Effect of protease inhibitors on albumin secretion in hepatoma cells.

Albumin biosynthesis and secretion have been studied in hepatoma cell cultures. The “in vivo” addition of the protease inhibitors antipain or leupeptin caused a fifty per cent reduction in the secretion of albumin without any effect on the total protein synthesis. Pulse and chase experiments in the absence or presence of protease inhibitors have also shown that these substances decrease markedly the rate of albumin secretion.     

Growth and hepatospecific gene expression of human hepatoma cells in a defined medium

Summary The production of albumin, α-fetoprotein (AFP), and α-1 antitrypsin has been compared among human hepatoma cells cultured in medium containing serum, medium containing hormones and growth factors, and a basal medium containing selenium as the only supplement. Growth is sustained in all three media, and the expression of all three proteins was maintained for over 4 mo. in the various media. However, the quantitative production of albumin and AFP were dramatically different in the three media. Two hormones, insulin and triiodothyronine, influenced the level of secreted proteins. Triiodothyronine increases the amount of secreted albumin whereas insulin at 10 μg/ml reduced the level of total secreted protein.     

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.     

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.