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.     

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.     

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.     

Binding of AlF-C, an Orc1-binding transcriptional regulator, enhances replicator activity of the rat aldolase B origin

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.     

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.     

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.     

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.     

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.     

Expression of rat hepatocyte plasma membrane antigens in hybrid clones: assignment of genes coding for two antigens of the basolateral domain to chromosomes 11 and 13

Expression of three rat hepatocyte plasma membrane antigens defined by monoclonal antibodies (mAbs) was examined by immunofluorescence in mouse hepatoma x rat hepatocyte hybrid clones segregating rat chromosomes. The antigen defined by mAb B1, a marker of the lateral domain of the hepatocyte plasma membrane in vivo, was expressed in hybrids retaining the rat chromosome 11. The antigen defined by mAb A39, mainly located on the sinusoidal (basal) domain of the plasma membrane in vivo, was expressed when chromosome 13 was present. The genes coding for these two antigens were thus assigned to chromosomes 11 and 13, respectively. The antigen defined by mAb B10, exclusively located on the canalicular (apical) domain of the plasma membrane in vivo, was not expressed in most hybrid clones, and the chromosome location of the gene could not be determined.     

Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro.

1. The induction of metallothionein (MT) protein by TPA (O-tetradecanoyl phorbol acetate), a protein kinase C activator, was demonstrated in vivo in rat liver and in vitro in rat hepatocytes in primary culture. In vivo half maximal induction at 25 hr was seen at 26 nmol TPA/kg body wt. Five- to seven-fold inductions were seen in vivo. De novo protein synthesis was required for this induction as demonstrated by cycloheximide inhibition of [35S]cysteine incorporation into MT protein. 2. TPA induction of MT protein in primary cultures of rat hepatocytes reached levels of 2.6-4.1-fold, as assessed by [35S]cysteine incorporation, 1.34-2.20-fold, as assessed by 109Cd binding in a metal displacement/HPLC assay, and 2.5-5-fold, as assessed by 109Cd binding in a metal displacement/Sephadex G-75 Superfine assay. 3. The induction of MT mRNA by TPA was demonstrated in vivo in rat liver and in vitro in 2 rat hepatoma cell lines, EC3 and 2M. MT mRNA was quantitated using dot blot and Northern gel assays. In vivo TPA induced hepatic MT mRNA 2.36-5.88-fold (dot blot) and 7.4-22-fold (Northern gels). In vitro TPA induced MT mRNA 1.71-15.26-fold in EC3 cells and 2.23-8.43-fold in 2M cells. MT mRNA was 0.54 kb, and alpha-tubulin mRNA was 1.62 kb in size on Northern gels. 4. TPA induction of MT protein and mRNA in vivo and in vitro is rapid and persistent and occurs at low concentrations. The 2 rat hepatoma cell lines provide a useful system in which to study MT induction in vitro without confounding secondary effects which can occur in vivo.     

Modifications of the expression of liver-specific and non-specific messenger RNAs during azo-dye hepatocarcinogenesis

The expression of specific and non-specific rat liver messenger RNAs has been studied during 3'-methyl-4-(dimethylamino)azobenzene (3'-MeDAB) carcinogenesis, using cDNA probes complementary to mRNAs encoding aldolase A and B, L-type pyruvate kinase, albumin, alpha-fetoprotein, transferrin and an unidentified 2.7 X 10(3)-base mRNA. mRNAs specific for undifferentiated cells, such as those encoding aldolase A and the unidentified 2.7 X 10(3)-base species were re-expressed very early, being easily detectable at the 1st week of 3'-MeDAB treatment. They reached a maximum of expression at the 4th week. Simultaneously the levels of aldolase B and L-type pyruvate kinase mRNAs dramatically decreased as compared to controls, but remained responsive to induction by a high-carbohydrate diet. Albumin and transferrin mRNA levels were only slightly modified in the course of the carcinogenic diet. At the terminal stage of hepatocarcinogenesis, i.e. in malignant hepatoma cells, expression and inducibility of aldolase B and L-type pyruvate kinase mRNAs were similar to those in normal adult rats while mRNAs specific for undifferentiated or foetal stages were also synthesized. The very early changes in gene expression for aldolases A and B, L-type pyruvate kinase and the 2.7 X 10(3)-base mRNA species could indicate that carcinogenic diet modifies gene control mechanisms long before inducing hepatoma.     

Expression of monoamine oxidase A and B activities in mouse cybrids and hybrids

The inheritance of monoamine oxidase (MAO; EC1.4.3.4) was studied in cultured cells using techniques of somatic cell genetics. Cells of a mouse neuroblastoma variant line lacking MAO activity were fused to cytoplasts prepared from a mouse L cell line which expresses MAO activity and is resistant to chloramphenicol (a cytoplasmically inherited trait). The resulting cybrids were resistant to chloramphenicol, but did not recover MAO activity, indicating that the loss of activity in the neuroblastoma parent was not the result of an inherited lesion in a cytoplasmically transmitted gene. This cybrid cells were then fused to rat hepatoma cells expressing both A and B types of MAO activity. A resulting hybrid line, grown in medium containing hypoxanthine, aminopterin and thymidine (HAT) to select cells that had retained HPRT activity and hence the rat X chromosome, expressed both types of activity, but at a reduced level compared to the hepatoma parent. This finding indicates that the genetic lesion in the neuroblastoma cells resulting in loss of MAO activity is not phenotypically dominant, and that both A and B types of activity can be conferred together to neuroblastoma cells which normally express only the A type of MAO activity.     

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.     

Starvation-induced lysosomal degradation of aldolase B requires glutamine 111 in a signal sequence for chaperone-mediated transport

Aldolase B is an abundant cytosolic protein found in all eukaryotic cells. Like many glycolytic enzymes, this protein was sequestered into lysosomes for degradation during nutrient starvation. We report here that the degradation of recombinant aldolase B was enhanced two-fold when rat and human hepatoma cells were starved for amino acid and serum. In addition, starvation-induced degradation of aldolase B was inhibited by chloroquine, an inhibitor of lysosomal proteinases and by 3-methyladenine, an inhibitor of autophagy. Aldolase B has three lysosomal targeting motifs (Q(12)KKEL, Q(58)FREL, and IKLDQ(111)) that have been proposed to interact with hsc73 thereby initiating its transport into lysosomes. In this study, we have mutated the essential glutamine residues in each of these hsc73-binding motifs in order to evaluate their roles in the lysosomal degradation of aldolase B during starvation. We have found that when glutamines 12 or 58 are mutated to asparagines enhanced degradation of aldolase B proceeded normally. However, when glutamine 111 was mutated to an asparagine or a threonine, starvation-induced degradation was completely suppressed. These mutations did not appear to alter the tertiary structure of aldolase B since enzymatic activity was not affected. Our results suggest that starvation-induced lysosomal degradation of aldolase B requires both autophagy and glutamine 111. We discuss the possible roles for autophagy and hsc73-mediated transport in the lysosomal sequestration of aldolase B.     

Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli

Rat aldolase C cDNA was inserted in an Escherichia coli expression vector to construct the rat aldolase C expression plasmid, pRAC42. This plasmid produces active rat aldolase C in the transfected E. coli host cells. The characteristics of the purified enzyme, e.g. mol. wt, electrophoretic mobilities and kinetic parameters, are indistinguishable from those of authentic rat brain aldolase C. Three different tetrameric hybrid forms, C3A, C2A2 and CA3, in addition to C4 and A4, were found to be produced in the host cell when E.coli was co-transfected with expression plasmids for rat aldolase C and for human aldolase A. Similarly, the hybrid forms, C3B, C2B2 and CB3, in addition to C4 and B4, were also produced in the cells when co-transfected with the plasmids for rat aldolase C and for human aldolase B.     

Purification and characterization of hepatic glutathione S-transferases of rhesus monkeys. A family of enzymes similar to the human hepatic glutathione S-transferases.

The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system.     

High recurrence of rearrangements involving chromosome 14 in an ataxia telangiectasia lymphoblastoid cell line and in its mutagen-treated derivatives

Summary The cytogenetic characterization of CH cell line obtained by Epstein-Barr-virus transformation of the lymphocytes of a patient affected by ataxia telangiectasia is reported. Control CH cells and 2 subcultures treated with the mutagens R7000 or NQO were developed in parallel and studied. A common chromosome anomaly, a der(14) t(11;14) (q13.2;q32), was found in all the studied karyotypes, indicating that it occurred either in vivo or early in vitro. In non-treated cultures, additional anomalies were present in 6 derived subclones. All R-7000 treated cells had the same karyotype corresponding to one of the subclones observed without prior treatment. All NQO-treated cells acquired 2 common anomalies, and could be differentiated into 2 subclones because of the addition of a t(7;14) or a t(11;14). Chromosome 14 was involved in various rearrangements after breakage in band q11.2 or q12 in 6/8 subclones. This was not correlated with tumorigenicity, which was clearly increased in mutagen-treated cells as tested by in vitro growth in semi-solid medium and in vivo by grafts into nude mice or growth on the chorio-allantoic membrane of chick embryos. The CH cell line and its derivatives appear to be a promising in vitro system, showing various stages progressing towards malignancy, and reproducing a number of chromosome anomalies spontaneously occurring in AT patients.     

Expression of the hepatitis B virus surface antigen gene by rat hepatocyte X mouse hepatoma hybrid cells

The expression of the S gene of hepatitis B virus has been studied in the somatic hybrid cells resulting from the fusion between rat hepatocytes in primary culture and cells of the mouse hepatoma line BWTG3, and in the parental line BWTG3. The DNA of the S gene inserted into the plasmids pAC Tk+ and pNY4 has been co-transfected into these cells with a plasmid DNA bearing a resistance gene to aminoglycoside. The level of expression of the S gene among the co-transfected resistant clones was estimated by radioimmunoassay. The results show that a high number of the co-transfected cellular hybrid clones express the S gene, whereas it is found, by contrast, that the S gene is poorly expressed in the mouse hepatoma cells. The level of expression of the S gene (as the amount of HBs Ag synthesized) is high in the hybrid clones and the synthesis of the HBs antigen is stable in time. These observations suggest for the first time in cell cultures in vitro, the role which is probably played by the normal hepatocyte genome in the expression of the S gene of HBV.