Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. V. Occurrence in dialyzed fetal bovine serum of macromolecules having both positive and negative growth regulatory functions.

Dialyzed fetal bovine serum contains two distinct growth-controlling macromolecular fractions: one stimulates and the other inhibits proliferation of primary cultured differentiated fetal rat hepatocytes. Both fractions are precipitated by ammonium sulfate (50% saturation, pH 7.4, 4 degrees C). Serum fraction I (SFI, mol wt >/= 120,000 daltons estimated by gel filtration with Bio-gel P200) appears to contain at least two factors which function, respectively, to initiate DNA synthesis (activity pH 4-10 stable) and to increase the rate at which initiated cells traverse the cell cycle (activity pH 4 and pH 10 labile). Intraperitoneal injections of SFI into adult rats have produced detectable stimulation of hepatic but not renal DNA synthesis. Serum fraction II (SFII, mol wt 40,000-80,000 daltons) suppresses in vitro incorporation of CH(3)-[(3)H]thymidine into DNA under conditions which diminish neither cell viability nor cell attachment. Mixing experiments indicate that SFI and SFII mutually antagonize each other with respect to DNA synthesis and cell multiplication. Thus, both the relative and absolute serum levels of multiple factors control in vitro fetal hepatocyte proliferation.

Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. VII. Hormonal control of DNA synthesis and its possible significance to the problem of liver regeneration.

The initiation of DNA synthesis has been studied in quiescent primary cultures of fetal rat hepatocytes using defined hormones and chemically defined medium. Preparations of crystalline insulin (0.01-10 microg/ml) or 20,000-fold purified somatomedin (0.01-1 microg/ml) are stimulatory. Growth hormone (0.025 microg/ml) and hydroxycortisone (0.025 microg/ml), 3':5'-GMP! (10(-5) M) fail by themselves to initiate DNA synthesis but added together with insulin, enhance the stimulatory response by 50-100%. Thyroid hormones (L-T(3), L-T(4), 7.5-30 ng/ml) are by themselves without effect, but when they are added to thyroid hormone-depleted serum, the reconstituted mixtures show an enhanced capacity to initiate DNA synthesis. In contrast, glucagon (0.01 microg/ml) inhibits the insulin-stimulated response by about 50% without reducing basal DNA synthesis rates. The results described here and in the accompanying two reports indicate that environmental control over the initiation of DNA synthesis is complex, and can involve the participation of many factors. The in vitro findings are consistent with the concept that liver regeneration is hormonally controlled and raise the possibility that alterations of the intrahepatic ratio of insulin to glucagon are growth regulatory.

Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. VI. Studies with conditioned medium and its functional interactions with serum factors.

Serum-deficient

Studies on primary cultures of differentiated fetal liver cells.

A method for culturing non- or slowly growing, differentiated fetal rat liver cells is described. It involves the use of collagenase as a digesting agent and of a selective medium deficient in arginine which suppresses the growth of nonparenchymal liver cells. Evidence is presented that surviving cells (a) retain liver-specific urea cycle functions measured by their capacity to transform ornithine into arginine, (b) synthesize DNA in glucose-deficient medium, and (c) synthesize and secrete albumin. This primary cell culture responds to partially hepatectomized rat serum and may be an appropriate assay system for the study of mechanisms which regulate liver regeneration.

Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. IX. Specific inhibition of DNA synthesis initiation by very low density lipoprotein and possible significance to the problem of liver regeneration.

Rat serum very low density lipoprotein (VLDL) inhibits initiation of DNA synthesis in fetal rat hepatocyte cultures; cells engaged in synthesizing DNA resist inhibition. VLDL action is specific and apparently blocks prereplicative protein synthesis. These and other results, from studies of altered blood VLDL levels and [3H] thymidine incorporation into isolated liver nuclei in 70% hepatectomized normal and mutant hyperlipoproteinemic rats, as well as from infusion studies with a "mitogenic" hormone solution, suggest that hepatic VLDL metabolism is linked to the suppression of hepatocyte proliferation.

Activation of Jun kinase is an early event in hepatic regeneration.

Compensatory hepatic regeneration after partial hepatectomy (PH) is dependent upon the extent of resection. This study analyzes the regulation of the AP-1 transcription factor c-Jun during hepatic regeneration. There is a progressive increase in c-jun mRNA levels after sham operation, one-third PH, and two-thirds PH. A concomitant increase in AP-1 binding activity is also observed. The c-Jun protein is a major constituent of the AP-1 complex in quiescent and early regenerating liver. The activity of c-Jun nuclear kinase (JNK), which phosphorylates the activation domain of the c-Jun protein, is markedly stimulated after one-third PH. JNK1 or an immunologically related kinase is a constituent of this stimulated JNK activity after PH. When primary cultures of adult rat hepatocytes are incubated with epidermal growth factor or transforming growth factor-alpha, AP-1 transcriptional activity is increased and the activation domain of the c-Jun protein is further potentiated. Phosphopeptide mapping of the endogenous c-Jun protein in proliferating cultured hepatocytes demonstrates phosphorylation of the c-Jun activation domain. Combining the results of these in vivo and culture studies, we conclude that the minimal stimulation of one-third PH activates JNK, which phosphorylates the c-Jun activation domain in hepatocytes, resulting in enhanced transcription of AP-1-dependent genes.

DNA-mediated gene transfer into adult rat hepatocytes in primary culture.

Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity. Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C. Chen and H. Okayama (Mol. Cell. Biol. 7:2745-2752, 1987). Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells). The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration [optimal at 2.4 micrograms of DNA per ml] and duration of exposure [optimal between 5 and 10 h]), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha. Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation. These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.

Expression of an oncodevelopmental gene product (alpha-fetoprotein) during fetal development and adult oncogenesis.

The expression of an "oncodevelopmental" protein, alpha-fetoprotein (AFP), has been systematically studied in rats during normal development and during regeneration of the liver by fetal rat hepatocytes in vitro, in rats bearing transplantable hepatomas, in rats fed chemical carcinogens, and in mice that spontaneously develop hematomas. AFP is a serum protein made normally during fetal and neonatal stages by liver and yolk sac cells. In newborn rats at approximately 4 weeks of age, the production of AFP is abruptly terminated, a process which is closely associated with cessation of liver cell proliferation. In adult rats, AFP production recurs following the reinitiation of hepatic DNA synthesis induced by partial hepatectomy or by the administration of heaptotoxic chemicals. Detailed metabolic and direct labeling studies of fetal rat hepatocytes in vitro also demonstrate a kinetically similar pattern of hepatocyte DNA synthesis and AFP production. In vitro studies utilizing combined autoradiography for DNA-synthesizing cells and immunofluorescence for AFP-containing cells demonstrates that replicating hepatocytes produce AFP, however, available data do not yet permit a distinction between G1 (pre- or postmitotic) and/or G2 production. During growth of an AFP- producing tumor, the serum concentration of AFP may be used as a accurate index of tumor growth, and, if a transplanted tumor is removed, as a marker for metastatic growth of the tumor. Using this model, we have shown that radiation to the lung at the time of surgical removal of a growing tumor in the leg will prevent establishment and growth of pulmonary metastases and that anti-AFP serum treatment may inhibit growth of a transplantable hepatoma that produces AFP. The exposure of rats to chemical hepatocarcinogens results in the appearance of evaluated serum AFP concentration as early as within 1 week of feeding; noncarcinogenic chemical analogs do not cause an elevation. AFP elevation also occurs with low doses of the hepatocarcinogen in the absence of detectable cell injury (by morphological examination of serum enzyme levels) or any other known morphological or biochemical change. This may represent a highly selective derepression of protein synthesis that occurs following the formation of a complex between the metabolites of the carcinogen and specific chromatin loci. Although every rat so far treated with even subcarcinogenic doses of hepatocarcinogens has elevated serum AFP concentrations, many primary carcinogen-induced hepatomas do not produce detectable AFP. Either there is a subsequent change in the preneoplastic AFP-producing cell that occurs prior to irreversible neoplastic alteration, or the hepatocytes originally influenced by the carcinogens to produce AFP are not necessarily the same cells that are the progenitors of the hepatoma produced by more prolonged exposure...

Inactivation of plasmid reporter gene expression by one benzo(a)pyrene diol-epoxide DNA adduct in adult rat hepatocytes.

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene diol-epoxide (BPDE-I) cause hepatocellular carcinoma. To identify short-term carcinogen effects, we studied hepatocytes transfected with nonreplicating plasmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE inactivated gene expression as a first-order function of BPDE concentration in adduction reactions. No evidence of cytotoxicity, diminished coprecipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was observed. At low BPDE:plasmid ratios, inactivation occurred with 1 adduct/plasmid within a target 23-27% of plasmid bases. Using nuclear extracts and BPDE-adducted G-free cassette-encoding plasmids, the fraction of full-length RNA polymerase II-initiated transcripts also declined as a first-order function of BPDE concentration when approximately 3 adducts were distributed among 48% of plasmid bases. These observations suggest that carcinogens such as BPDE block mRNA transcription along DNA templates by forming limited numbers of persistent adducts at coding or noncoding sites.

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes.

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.

Attenuation of gene expression by a trinucleotide repeat-rich tract from the terminal exon of the rat hepatic polymeric immunoglobulin receptor gene.

A 359 bp terminal exon fragment of the rat polymeric immunoglobulin receptor gene has been tested for biological effects. The fragment contains an S1 nuclease-sensitive microsatellite with d(GGA) and d(GAA) trinucleotide repeats that are expressed discordantly in the 3'UTRs of liver mRNAs encoded by the single copy gene. When human A293 cells are transfected with expression plasmids carrying this fragment in forward orientations, flanking or replacing poly(A) cassettes in the 3' ends of the transcription units, luciferase reporter gene expression is attenuated 47 to 59% or 98.5%, respectively. In contrast, when the fragment is tested similarly in reverse orientation, there is significantly less or no attenuation of gene expression. These observations, and computer models of partial triplet repeat DNA tertiary and RNA secondary structures, suggest that this fragment might regulate gene expression by orientation and position-dependent mechanisms at transcriptional and post-transcriptional levels.

Functional pleiotropy of an intramolecular triplex-forming fragment from the 3'-UTR of the rat Pigr gene.

A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3'-untranslated region (3'-UTR) and inserted into 3'-UTR or 3' flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5' flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5' and 3' flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3'-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.

Selective effects of portal blood diversion and glucagon on rat hepatocyte rates of S-phase entry and deoxyribonucleic acid synthesis.

Hepatocyte growth transition constants were measured during liver regeneration induced by 70% hepatectomy in portacaval shunted rats and in pair-fed sham-operated controls. Portal blood diversion 48 h before 70% hepatectomy coordinately reduced DNA synthetic and mitotic responses 60-70%. Both responses reflected preferential changes in overall rates of S- and M-phase entry (S delta and M delta, respectively); marked differences were not seen between experimental or control DNA synthesis and mitotic onset times (St = approximately 12 h; and Mt = approximately 20 h, for each group). Proliferative defects were manifested progressively across the liver lobule, from portal (approximately 44% of control) to midzonal (approximately 20%) to central areas (approximately 5%). In addition, radioautography and studies of [3H]thymidine uptake into nuclear DNA showed that in shunted rats, average DNA synthesis rates per hepatocyte fell 44-86% between 16-30 h. RIAs of glucagon and insulin in portal and aortic plasma obtained from 0-72 h showed overall reductions of 37-41% in hepatic hormone gradients of shunted rats. The greatest decreases occurred 8-24 h after 70% hepatectomy. Early (0-2 h) or late prereplicative (8-10 h) administration of insulin (0.02-1 mg kg-1) failed to restore [3H]thymidine uptake rates at 22-24 h to normal levels. However, exogenous glucagon (20 micrograms kg-1) given at 8-10 h increased these rates to 50% of control values. Higher doses (0.2-1 mg glucagon kg-1) were inhibitory. By contrast, no effects on DNA synthesis were found when glucagon was injected between 0-2 h. These findings suggest that hepatocyte growth transition constants are modulated selectively by different mitogens. Glucagon may control S delta and average hepatocellular DNA synthesis rates apart from other endocrine factors that regulate St.

Thyroid hormone metabolism during liver regeneration in rats.

The metabolism of thyroid hormones was studied during the prereplicative period of liver regeneration. After partial hepatectomy, serum thyroxine (T4) and triiodothyronine (T3) levles progressively fell, and reached a nadir at 12 h proportional to the quantity of liver tissue exised. The diminution (60-80%) in serum iodothyronines was related specifically to partial hepatectomy because laparotomy, ether anesthesia, and other stressful surgical procedures did not induce similar changes. At least 3 phenomena appear to be involved: 1) increased utilization and turnover of thyroid hormone by the regenerating liver remmant. 2) diminished hormone secretion by the thyroid gland between 6-12 h after surgery, and 3) a slightly reduced concentration of serum iodothyronine carrier proteins. The results support the concept that the liver participates in the metabolic regulation of T2 and T4 which in turn, control hepatocellular growth. It is suggested, however, that additional unknown factors control increased hepatic thyroid hormone turnover after partial hepatectomy.

Site-specific integration of targeted DNA into animal cell genomes.

Novel genetically engineered retroviral vectors and targeting plasmids are described that enable the site-specific targeting of exogenous DNA into the genomes of cultured animal cells. The protocol involves the transduction of competent cells by a chimeric retroviral vector containing a transcription unit composed of two linked cassettes: an upstream marker gene under the control of the viral 5' LTR; and a downstream reporter trap containing a strong promoter 5' to a 48bp yeast FRT element. When cells containing such integrated units are co-transfected with a plasmid encoding yeast FLP recombinase and a promoterless targeting plasmid containing a reporter cDNA tract 3' to an homologous FRT element, the targeting plasmid recombines at the chromosomally preconfigured FRT site, and a new hemizygous function is introduced into the downstream cassette. These reagents provide a new portable system for site-specific targeting of chemically modified genes into uniform and unique sites in genomically integrated transcription units.

Construction of a fusion protein between protein A and green fluorescent protein and its application to western blotting.

Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE. However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA-GFP, was used in Western and dot blots. The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening.

Relationship of the biosynthesis of alpha-fetoprotein, albumin, hemopexin, and haptoglobin to the growth state of fetal rat hepatocyte cultures.

AFP and albumin are produced by arginine-synthesizing fetal rat hepatocytes in vitro. AFP and hemopexin production are coupled to hepatocellular proliferation, whereas albumin and haptoglobin production are not. During the cell cycle, AFP is synthesized prior to S and released prior to M. AFP may play a role in regulation of hepatocellular growth through estradiol binding and modulation of the intracellular concentration of lipoprotein (VLDL).