Regulation of mouse haptoglobin synthesis.

A cloned line of mouse hepatoma cells (Hepa-1) responded to treatment with dexamethasone by a 30-80-fold increase in synthesis and secretion of functional haptoglobin. Under the same conditions, the production of albumin was only slightly elevated whereas that of alpha 1-fetoprotein was reduced by 50%. The hormone concentration for half-maximal stimulation of haptoglobin synthesis was between 1 and 2 X 10(-8) M. The time course of induction is characteristic for a glucocorticoid-regulated protein. Cell-free translation of RNA indicated an increase in the amount of functional haptoglobin mRNA that can account for the change in the protein production. To correlate our findings on Hepa-1 cells with those on nontransformed liver cells, we tested the hormonal response of isolated hepatocytes in tissue culture. Haptoglobin was first synthesized and secreted by hepatocytes from 17-19-d-old fetuses. But neither prenatal nor adult hepatocytes showed a dexamethasone-dependent increase in haptoglobin synthesis. However, when several independent clones of hybrid cells formed from adult mouse hepatocytes and rat hepatoma cells were treated with dexamethasone, the synthesis of mouse haptoglobin was in all cases elevated. It appears that haptoglobin expression in mouse liver cells is potentially sensitive to glucocorticoids, but this modulation is manifested only in transformed cells and their derivatives.

Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes.

Adult mouse hepatocytes respond in vivo to experimentally induced acute inflammation by an increased synthesis and secretion of alpha 1-acid glycoprotein, haptoglobin, hemopexin, and serum amyloid A. Concurrently, the production of albumin and apolipoprotein A-1 is reduced. To define possible mediators of this response and to study their action in tissue culture, we established primary cultures of hepatocytes. Various hormones and factors that have been proposed to regulate the hepatic acute phase reaction were tested for their ability to modulate the expression of plasma proteins in these cells. Acute phase plasma and conditioned medium from activated monocytes influenced the production of most acute phase plasma proteins, and the regulation appears to occur at the level of functional mRNA. Purified hormones produced a significant anabolic response in only a few cases: dexamethasone was found to be effective in maintaining differentiated expression of the cells; and glucagon produced a specific inhibition of haptoglobin synthesis. When cells were treated with a combination of conditioned monocyte medium and dexamethasone, secretion of proteins was markedly reduced. The carbohydrate moieties of all plasma glycoproteins were incompletely modified, apparently as a result of decreased intracellular transport of newly synthesized plasma proteins. Although primary hepatocytes were not phenotypically stable in tissue culture, the cells nevertheless retained a broad response spectrum to exogenous signals. We propose this as a useful system to study the production of plasma proteins and thereby pinpoint the nature and activity of effectors mediating the hepatic acute phase reaction.

Preferential degradation of the terminal carbohydrate moiety of membrane glycoproteins in rat hepatoma cells and after transfer to the membranes of mouse fibroblasts.

Glycoproteins in the plasma membrane of rat hepatoma cells were labeled at their externally exposed tyrosine residues with 131I and at their galactose and sialic acid residues with 3H. The degradation of both isotopes in the total cell protein fraction, in glycoproteins purified by concanavalin A, and in glycoproteins separated on two-dimensional gels was determined. Similarly, the total cellular membrane glycoproteins were metabolically labeled with [35S]methionine and [3H]fucose. The fate of both incorporated labels was followed by lectin chromatography or by precipitation of the proteins with specific antibodies followed by electrophoretic gel separation. In both labeling experiments, the carbohydrate markers were lost from the ligand-recognized fraction with similar kinetics as from the total cell protein fraction. In some glycoprotein species which were separated by two-dimensional gel electrophoresis, the polypeptide portion exhibited up to a twofold slower rate of degradation relative to that of the carbohydrate moiety. This difference is most pronounced in carbohydrate-rich glycoproteins. To corroborate this finding, double-labeled membrane glycoproteins were incorporated into reconstituted phospholipid vesicles which were then transferred via fusion into the plasma membrane of mouse fibroblasts. Both the polypeptide and carbohydrate moieties of the transferred membrane glycoproteins were degraded with the same relative kinetics as in the original hepatoma cells. The rate of degradation is mostly a function of the structural properties of the membrane components as shown by the preservation of metabolically stable fucogangliosides of Reuber H-35 hepatoma cells transferred onto the fibroblasts. The technique of insertion of membrane components into the plasma membrane of another cell should assist in the elucidation of the exact route and mechanism of membrane protein destruction.

Regulation of major acute-phase plasma proteins by hepatocyte-stimulating factors of human squamous carcinoma cells.

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.

Distinct regulation of the interleukin-1 and interleukin-6 response elements of the rat haptoglobin gene in rat and human hepatoma cells.

The transcription rate of the haptoglobin (Hp) gene is stimulated by interleukin-1 (IL-1), IL-6, and dexamethasone in rat hepatoma (H-35) cells. To identify the cis-acting regulatory elements responsive to these hormones, various lengths of 5' Hp gene-flanking regions, including the promoter, were inserted into chloramphenicol acetyltransferase gene expression vectors and transiently introduced into H-35 cells. The first 4 kb of 5' region mediated a severalfold increase in expression after treatment with IL-6 and dexamethasone. No response to IL-1 was detectable. When, however, upstream sequences were deleted to position -165 relative to the transcription start site, a significant stimulation by IL-1 was gained without appreciably affecting the IL-6 response. With the apparent removal of an inhibitory sequence, the promoter-proximal 165-bp region also displayed a severalfold enhanced response to the combination of dexamethasone, IL-1, and IL-6. The sequence from -165 to -147, termed the A-element, was found to be crucial for all hormone regulatory functions. Two copies of the A-element linked to a heterologous promoter responded to the three hormones, but to a lesser degree than in the Hp gene promoter context. The regulatory elements of the rat Hp gene were similarly active in human hepatoma cells. Optimal regulation by IL-6 in HepG2 cells was, however, independent of the A-element. The A-element functioned in these cells exclusively as an IL-1 response sequence. The results suggest that genomic sequences upstream of the rat Hp gene suppress the regulation by specific cytokines more prominently in transient expression assays than in the normal chromosomal context. Moreover, the functional comparison indicated that specific regulatory regions of the rat Hp gene do not function identically in different hepatic cell types.

Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome-wide allelotyping and inter-(simple sequence repeat) PCR.

We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loci exist for intrachromosomal genomic instability in human colorectal cancer.

Absence of 60-Hz, 0.1-mT magnetic field-induced changes in oncogene transcription rates or levels in CEM-CM3 cells.

Our objective was to assess the reproducibility of the 60-Hz magnetic field-induced, time-dependent transcription changes of c-fos, c-jun and c-myc oncogenes in CEM-CM3 cells reported by Phillips et al. (Biochim. Biophys. Acta, 1132 (1992) 140-144). Cells were exposed to a 60-Hz magnetic field (MF) at 0.1 mT (rms), generated by a pair of Helmholtz coils energized in a reinforcing (MF) mode, or to a null magnetic field when the coils were energized in a bucking (sham) mode. After MF or sham exposure for 15, 30, 60 or 120 min, nuclei and cytoplasmic RNA were extracted. Transcription rates were measured by a nuclear run-on assay, and values were normalized against either their zero-time exposure values, or against those of the c-G3PDH (housekeeping) gene at the same time points. There was no significant difference, at P=0.05, detected between MF and either sham-exposed or control cells at any time point. Transcript levels of the oncogenes were measured by Northern analysis and normalized as above. No significant difference (P=0.05) in transcript levels between MF and either sham-exposed or control cells was detected.

Diltiazem and superoxide dismutase modulate hepatic acute phase response in gram-negative sepsis.

This study assessed the hepatic acute phase response and cellular Ca2+ regulation in septic animals and in hepatoma cell lines in vitro. Sepsis was induced in male Sprague-Dawley rats by implanting in their abdominal cavities fecal pellets impregnated with live Escherichia coli and Bacteroides fragilis. 8 h after implantations, rats were treated with diltiazem (1.2 mg/kg) or superoxide dismutase (SOD) (5 x 10(3) units/kg). After 24 h, plasma acute phase proteins (APP) were determined by immunoelectrophoresis, and hepatic APP-mRNAs by Northern blot hybridization. Effects of diltiazem, verapamil, or SOD on hepatic cells were determined in rat Reuber H-35 and human HepG2 hepatoma cells. Sepsis induced a significant increase in plasma APP and their hepatic mRNAs. Diltiazem and SOD reduced the sepsis-induced elevations in plasma lactate, the febrile response and mortality. APP expression in H-35 and HepG2 cells, stimulated by interleukin 1 (IL-1), IL-6, and dexamethasone, was inhibited by diltiazem or verapamil but not SOD. The results suggest that a heightened hepatic APP response in septic animals accompanies systemic/metabolic derangements and a significant animal mortality. Because diltiazem was previously shown to prevent sepsis-related disturbances in hepatic cellular Ca2+ regulation, its mediation of decrease in APP, systemic/metabolic response, and mortality may be effected through modifications in cellular Ca2+ regulation. The data from hepatoma cells show an attenuation of the AAP can result from direct effects of a calcium blocker. However, whether the blocker primarily modifies cellular Ca2+ regulation and secondarily effects APP gene expression, or directly effects gene expression remains unknown.

Increased DNA synthesis in INIT/10T1/2 cells after exposure to a 60 Hz magnetic field: a magnetic-field or a thermal effect?

This study was designed to test the hypothesis that a 0.1-0.8-mT 60 Hz magnetic field may act as a promoter of carcinogenesis. C3H 10T1/2 mouse fibroblasts initiated with the carcinogen methylcholanthrene (INIT/10T1/2 cells) were used; in these cells, expression of the carcinogenic phenotype is suppressed indefinitely by the presence of retinyl acetate in the culture medium. After withdrawal of retinyl acetate, expression of the carcinogenic phenotype may be observed as the loss of contact inhibition. Cells grown without retinyl acetate were exposed to 0.1-0.8-mT (rms) 60 Hz magnetic fields or to sham fields. Eight days after exposure, magnetic-field and sham-exposed cells showed the same levels of incorporation of [3H]thymidine, and both had counts significantly higher than those of unexposed cells. The rate of incorporation of [3H]thymidine was very sensitive to small (0.1-0.8 degrees C) and transient (60 min) increases in incubation temperature during the first few days of withdrawal of retinyl acetate. Exposure of Jurkat (human acute T-cell lymphoma) and GH3 (rat pituitary tumor) cells to magnetic fields and sham conditions yielded similar results. INIT/10T1/2 cells cultured in the presence of retinyl acetate showed no effect of exposure conditions. Both magnetic-field and sham exposures caused a slight increase in temperature within the exposure zone in the incubator. Thus the differences between rates of incorporation of [3H]thymidine in magnetic field-exposed, sham-exposed and unexposed cells seem to be attributable at least in part to a slight elevation in temperature during exposure. Since some cells appear to be extremely sensitive to small increases in temperature, measurements of magnetic-field effects must be made and interpreted with caution.

Regulation of acute phase protein genes by hepatocyte-stimulating factors, monokines and glucocorticoids.

The synthesis of all the major acute phase plasma proteins is stimulated in rat hepatoma and primary cultures of hepatocytes by three, structurally and functionally distinct groups of hormones: 1) hepatocyte-stimulating factors (HSF) and interleukin-6 (IL-6); 2) interleukin-1 (IL-1) and tumor necrosis factor (TNF); and 3) glucocorticoids. Each plasma protein gene requires a specific combination of these 3 hormone types for maximal expression. One set of acute phase proteins, including alpha 2-macroglobulin, alpha 1-antichymotrypsin ( = contrapsin), cysteine protease inhibitor ( = thiostatin), alpha 1-antitrypsin, ceruloplasmin and fibrinogens are predominantly regulated by the keratinocyte-derived HSF-III/-II or IL-6, while a second set of proteins, including alpha 1-acid glycoprotein (AGP), haptoglobin and complement C3 are predominantly regulated by keratinocyte-derived HSF-I, IL-1 or TNF. In conjunction with the above peptide hormones, glucocorticoids synergistically enhance the stimulated expression of most, but not all, acute phase proteins. An exceptionally strong synergy between HSF (or IL-6), IL-1 and glucocorticoids is noted for the activation of the AGP gene. To elucidate the molecular mechanisms of regulation, we have identified the cis-acting genetic elements through which all these hormones control the transcriptional activity of the AGP gene. It appears that acute phase activates a specific nuclear binding protein in the rat liver that interacts with the peptide hormone responsive element located 5 kb upstream of the transcriptional start site.

Glucose starvation leads in rat hepatoma cells to partially N-glycosylated glycoproteins including alpha 1-acid glycoproteins. Identification by endoglycolytic digestions in polyacrylamide gels.

Within minutes of glucose starvation confluent monolayers of rat hepatoma cells synthesize glycoproteins, including alpha 1-acid glycoprotein, which appear on two-dimensional gels as size heterogeneous spot series. The longer the period of glucose starvation the more the production of the glycoproteins is shifted toward smaller molecular weight forms. To compare these forms with the corresponding glycoproteins synthesized either in a cell-free system or by nonstarved cells, a mapping of the N-glycan was done by endo-beta-N-acetylglucosaminidase digestion within a polyacrylamide gel. Glycoproteins from glucose-starved cells contain a reduced number of N-glycans which belong to both the endo H-sensitive and resistant type. The decrease of N-glycosylation may be correlated with the accumulation of truncated lipid-bound oligosaccharides, for the gel chromatography of the oligosaccharides released from the lipid and protein fractions of glucose-starved cells revealed a drastic reduction in their size. Most of the lipid-linked oligosaccharides synthesized during glucose starvation are resistant to endo H digestion. Under conditions of limiting glycosylation we were able to show by glycopeptide analysis, that in the case of alpha 1-acid glycoprotein, N-glycans are added randomly to the 6 possible N-glycosylation sites. Furthermore, non- or partially N-glycosylated proteins do not acquire additional oligosaccharide units after restoration of glucose although the proteins can undergo secondary modification and, in the case of the secretory proteins, can be exported.

Interaction of cytokine- and glucocorticoid-response elements of acute-phase plasma protein genes. Importance of glucocorticoid receptor level and cell type for regulation of the elements from rat alpha 1-acid glycoprotein and beta-fibrinogen genes.

Dexamethasone increased the transscriptional activity of several acute-phase plasma protein genes in cytokine-treated HepG2 cells, suggesting the presence of functional glucocorticoid receptors (GR). The level of GR was, however, insufficient for stimulation of transiently transfected gene constructs containing glucocorticoid-response elements (GRE). By complementation of HepG2 cells with a GR expression vector, a cell system was generated that allowed analysis of the interaction between GRE and cytokine-response elements of the rat alpha 1-acid glycoprotein gene and identification of the principal regulatory elements of the rat beta-fibrinogen gene. Although the expression of plasmid-derived GR mRNA was reduced by dexamethasone treatment, the concentration of GR was sufficient for full, short-term stimulation of GRE-containing vectors. By comparing the pattern of regulation of the cloned GRES in HepG2 and mouse L-cells, an equivalent, cell-type independent dexamethasone response was monitored for the alpha 1-acid glycoprotein element but a response limited to HepG2 cells was found for the beta-fibrinogen element. The data indicate that, although substantial differences exist in the organization and composition of the regulatory elements of the two genes, the overall function is, nevertheless, remarkably similar.

Human hepatocyte-stimulating factor-III and interleukin-6 are structurally and immunologically distinct but regulate the production of the same acute phase plasma proteins.

Human squamous carcinoma (COLO-16) cells synthesize and secrete hepatocyte-stimulating factor-III (HSF-III), a glycoprotein with Mr = 39,000, which stimulates the synthesis of several acute phase plasma proteins in human hepatoma (HepG2) cells. The qualitative response of HepG2 cells to HSF-III is essentially the same as that elicited by human recombinant interleukin-6 (IL-6). Although similar in hepatocyte-stimulating activity, HSF-III and IL-6 are distinct molecules which differ not only in size and charge but also in immunologic properties: no cross-recognition of HSF-III and IL-6 occurs using neutralizing antibodies against IL-6 and HSF-III, respectively. In addition, Northern blot hybridization of IL-6 cDNA to mRNA from COLO-16 cells revealed no detectable IL-6 message. HSF-III does not compete for binding to the IL-6 receptors suggesting that HepG2 cells carry receptors specific for each hormone. Both receptor types may trigger similar intracellular processes explaining the identical regulation of acute phase protein expression.

Distinct sets of acute phase plasma proteins are stimulated by separate human hepatocyte-stimulating factors and monokines in rat hepatoma cells.

A subline of the rat hepatoma (H-35) cells has been identified which responds to hepatocyte-stimulating factors (HSFs) of human squamous carcinoma cells by increased synthesis of all major rat acute phase plasma proteins. The regulation occurs at the level of mRNA. Two HSFs (HSF-I and HSF-II) have been purified from conditioned medium of the squamous carcinoma cells. HSF-I is a protein with an Mr = 18,000 and pI 5.5, and HSF-II is a glycoprotein with an Mr = 34,000 and a broad, neutral to basic charge. In H-35 cells, HSF-I predominantly stimulates the synthesis of complement C3 and haptoglobin and acts synergistically with dexamethasone to stimulate alpha 1-acid glycoprotein. HSF-II stimulates cysteine protease inhibitor, alpha 1-antichymotrypsin, alpha 1-antitrypsin, fibrinogen, and hemopexin, and acts synergistically with dexamethasone to stimulate alpha 2-macroglobulin. Each HSF is between 10 and 100 times less effective in regulating proteins of the other set. Human tumor necrosis factor and interleukin-1 increase complement C3, haptoglobin, and alpha 1-acid glycoprotein, as does HSF-I, but are unable to modulate any of the other acute phase proteins. The monokines differ from HSF-I is their low activity in HepG2 cells and rat hepatocytes.

IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo.

Human rIL-6, produced either in COS cells or Escherichia coli, similarly stimulates the production of acute phase plasma proteins in cultured human and rat hepatoma cells. This anabolic effect in hepatoma cells suggested a potential in vivo role of the cytokine in mediating the hepatic response to inflammation. Injection of IL-6 into adult male rats elicited a cytokine-specific change in the liver expression of acute phase proteins. As predicted from in vitro studies, glucocorticoids were needed to achieve a maximal IL-6 response in vivo. Optimal conditions were found to be two i.p. injections of 35 to 120 micrograms IL-6 and 65 micrograms dexamethasone per kg body weight administered at 12-h intervals. Within 24 h, the plasma concentrations for alpha 2-macroglobulin, fibrinogen, thiostatin, and hemopexin were increased to levels approximating those observed in acute phase animals. These results support the notion that direct interaction of IL-6 with the liver is an essential part in initiating the hepatic acute phase reaction.

Human keratinocytes and monocytes release factors which regulate the synthesis of major acute phase plasma proteins in hepatic cells from man, rat, and mouse.

Human keratinocytes and activated monocytes produces factors which can stimulate the proliferation of thymocytes. The same activity has also been implicated in regulating the expression of plasma proteins in liver cells during the acute phase reaction. To assess whether factors produced by such cells can directly influence liver cells to change the production of acute phase plasma proteins, we studied in tissue culture the response pattern of hepatic cells from three species: human hepatoma cells ( HepG2 cells), and primary cultures of rat and mouse hepatocytes. Conditioned media from the squamous carcinoma COLO-16 cells, normal epidermal cells, and activated peripheral monocytes were able to stimulate the synthesis of specific acute phase plasma proteins: alpha 1-antichymotrypsin in HepG -2 cells, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, alpha 1-acute phase protein, and alpha 2-macroglobulin in rat hepatocytes, and alpha 1-acid glycoprotein, haptoglobin, and hemopexin in mouse hepatocytes. Only in rat cells, dexamethasone was found to have further enhancing effect. The increased production of plasma proteins could be explained by an elevated level of functional mRNA. Comparing thymocyte-stimulating activities with the effects on plasma protein production, we found some difference both between the conditioned media of epidermal cells and monocytes, and between the responses of the three hepatic cell systems. Furthermore, gel chromatography of conditioned media resulted in partial separation of activities regulating liver cells and thymocytes. Since there is no strict correlation between thymocyte- and hepatocyte-stimulating activities, the presence of different sets of specific factors is assumed.

Phorbol ester modulates interleukin 6- and interleukin 1-regulated expression of acute phase plasma proteins in hepatoma cells.

Interleukin 6 (IL 6) and interleukin 1 (IL-1) regulate the expression of acute phase plasma proteins in rat and human hepatoma cells. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), partially mimics the stimulatory effect of IL-6 but reduces that effect of IL-1. TPA and IL-6 act synergistically. These regulatory properties of TPA are also manifested in HepG2 cells transiently transfected with an indicator gene construct carrying the IL-1/IL-6 regulatory enhancer element of the rat alpha 1-acid glycoprotein gene. IL-6 and IL-1 act independently of TPA-inducible kinase C, and of changes in intracellular Ca2+ concentrations. However, prolonged pretreatment of HepG2 cells with TPA results in a drastically reduced cytokine response that is proportional to the loss of cell surface binding activity for the cytokine. These data suggest that hormones activating protein kinase C probably play a contributing role in stimulating the expression of acute phase plasma protein genes but they may be crucial in controlling the responsiveness of liver cells to inflammatory cytokines during subsequent stages of the hepatic acute phase reaction.

Role of CAAT-enhancer binding protein isoforms in the cytokine regulation of acute-phase plasma protein genes.

Rat hepatic cells respond to interleukin (IL) -1, IL-6, and dexamethasone treatment by increasing the transcription rate of acute-phase plasma protein genes. The same conditions lead to changes in the expression of CAAT-enhancer binding protein (C/EBP) isoforms which are specific to the hepatic cell line. To identify the relationship between C/EBP isoforms and acute-phase protein gene activation, the hormone-specific expression of C/EBP alpha, beta, and delta was determined in H-35 and HTC cells and was compared to acute-phase liver. C/EBP beta was found to be the principal isoform in hepatoma cells and to be strongly stimulated by cytokines and dexamethasone in H-35 cells. Transactivating functions were observed for all three C/EBP isoforms by cotransfection of CAT gene reporter constructs containing cytokine and glucocorticoid response elements of acute-phase protein genes and expression plasmids for mouse C/EBP alpha, beta, and delta into rat and human hepatoma cells. The degree of C/EBP-mediated transactivation was, however, extremely variable among the different regulatory elements. Transcription run-on reactions with nuclei from transiently transfected H-35 cells indicated that cotransfected C/EBP beta increases basal expression of reporter gene constructs as well as the dexamethasone-mediated stimulation of constructs containing the glucocorticoid response elements of the rat alpha 1-acid glycoprotein gene, but did not accelerate or enhance hormone-dependent transcription activation of reporter gene plasmids containing the IL-6 regulatory element of the beta-fibrinogen gene. Activation of the reporter gene constructs appeared to be temporally and quantitatively correlated with the amount of nuclear C/EBP as determined by two-dimensional Western and Southwestern blot analyses.

Electroporation-enhanced gene delivery in mammary tumors.

Electroporation was applied to enhance gene transfer into subcutaneous MC2 murine breast tumors. Cultured MC2 cells were also transfected by electroporation or by cationic liposomes in the presence of serum using pSV-luc plasmids. Electroporation parameters and liposome formulation were optimized to achieve the highest relative levels of transfection. An electric field threshold for successful electrotransfection in cultured cells appeared around 800-900 V/cm. The liposomes used contained the cationic lipid dioleoyl-3-trimethylammonium propane (DOTAP). Multilamellar vesicles (MLV) had a 10-fold advantage over small unilamellar vesicles (SUV) in cell culture transfection. For in vivo gene delivery, the plasmids were injected either alone, or in complex with MLV or SUV DOTAP liposomes. A series of six electric pulses 1 ms long were applied across tumors, using caliper electrodes on the skin surface. Electric field strengths ranged from 400-2300 V/cm. Luciferase expression was approximately two orders of magnitude higher than controls in tumors treated with pulses > or =800 V/cm. Differences between enhanced relative levels of transfection using uncomplexed plasmid and lipoplexes were not statistically significant. Distribution of DNA into tumor tissues was monitored by fluorescence in situ PCR. The highest numbers of fluorescent cells were found in tumors electroporated following the injection of plasmid. The significant transfection improvement shows that in vivo electroporation is a powerful tool for local gene delivery to tumors.