Evidence for a shared HLA-A intralocus determinant defined by monoclonal antibody 131.

We describe here a monoclonal antibody, 131, which appears to recognize a determinant shared by HLA-A locus-encoded gene products. Isoelectric focusing analysis demonstrates that 131 reacts with the products of at least seven different HLA-A alleles but none of the five HLA-B allelic products tested. Together with evidence provided by other studies, this finding indicates the existence of A-unique and B-unique determinants, which may have different biological functions. Monoclonal antibody probes, such as the one described here, specific for shared intralocus determinants, may be valuable for assessing these possible functional differences.

Mouse alpha-fetoprotein gene 5' regulatory elements are required for postnatal regulation by raf and Rif.

The mouse alpha-fetoprotein (AFP) gene is expressed at high levels in the yolk sac and fetal liver and at low levels in the fetal gut. AFP synthesis decreases dramatically shortly after birth to low levels that are maintained in the adult liver and gut. AFP expression can be reactivated in the adult liver upon renewed cell proliferation such as during liver regeneration or in hepatocellular carcinomas. Previously, two unlinked genetic loci that modulate postnatal AFP levels were identified. The raf locus controls, at least in part, basal steady-state AFP mRNA levels in adult liver. Rif influences the extent of AFP mRNA induction during liver regeneration. Transgenic mice were used to examine the role of 5' AFP regulatory regions in raf- and Rif-mediated control. A fragment of the AFP 5' region containing enhancer element I, the repressor, and the promoter was linked to the mouse class I H-2Dd structural gene. We demonstrate that this hybrid AFP-Dd transgene is expressed in the appropriate tissues. In addition, it is postnatally repressed and reactivated during liver regeneration in parallel with the endogenous AFP gene. Therefore, proper transcriptional control does not require the AFP structural gene. Furthermore, the AFP 5' control region is sufficient to confer raf and Rif responsiveness to the linked H-2Dd structural gene, suggesting that raf and Rif act at the level of transcriptional initiation.

Role of alpha-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons.

The requirements for activation of the mouse alpha-fetoprotein (AFP) gene in transient heterokaryons were investigated. For this purpose, the 7-kilobases of DNA flanking the 5' end of the AFP gene were linked to a mouse major histocompatibility complex (MHC) class I structural gene. The fusion gene was stably integrated at different sites into mouse L-cells, which do not transcribe the AFP gene. Transient heterokaryon fusions demonstrated that the silent AFP-MHC gene and the endogenous AFP gene were activated by factors present in HepG2 cells, a liver-derived cell line, but not by those in HeLa cells. Activation was detected at the protein level in single heterokaryons by using monoclonal antibodies against the cell surface protein and at the mRNA level in populations of cells. The AFP promoter alone was sufficient for activation could be used for DNA transfer strategies to identify genes which can activate AFP promoter elements in trans.

Individual mouse alpha-fetoprotein enhancer elements exhibit different patterns of tissue-specific and hepatic position-dependent activities.

Transcription of the mouse alpha-fetoprotein (AFP) gene, which is expressed at high levels in the visceral endoderm of the yolk sac and fetal liver and at low levels in the fetal gut, is regulated by three distinct upstream enhancer regions. To investigate the activities of these regions, each enhancer was individually linked to a heterologous human beta-globin promoter fused to the mouse class I H-2Dd structural gene. When tested in transgenic mice, the beta-globin promoter alone has minimal activity. We find that all three enhancers activate the beta-globin promoter in an AFP-like pattern; i.e., activity is detected in the yolk sac, fetal liver, and fetal gut. The enhancers remain active in the livers and guts of adult mice, consistent with previous studies showing that postnatal AFP repression is due not to the loss of enhancer activity but to a dominant repressor region. Enhancer III also functions in the brain. In addition, these studies reveal that the three enhancers exhibit different position-dependent activities in the adult liver. Enhancers I and II are most active in hepatocytes surrounding the central vein, with a gradual decrease in activity along the hepatic plates toward the portal triad. Enhancer III is active exclusively in hepatocytes surrounding the central vein. These data represent the first examples of individual control elements exhibiting positionally regulated activity in adult liver.

A nonimmunoglobulin transgene and the endogenous immunoglobulin mu gene are coordinately regulated by alternative RNA processing during B-cell maturation.

The immunoglobulin (Ig) genes have been extensively studied as model systems for developmentally regulated alternative RNA processing. Transcripts from these genes are alternatively processed at their 3' ends to yield a transcript that is either cleaved and polyadenylated at a site within an intron or spliced to remove the poly(A) site and subsequently cleaved and polyadenylated at a downstream site. Results obtained from expressing modified genes in established tissue culture cell lines that represent different stages of B-lymphocyte maturation have suggested that the only requirement for regulation is that a pre-mRNA contain competing cleavage-polyadenylation and splice reactions whose efficiencies are balanced. Since several non-Ig genes modified to have an Ig gene-like structure are regulated in cell lines, Ig-specific sequences are not essential for this control. This strongly implies that changes in the amounts or activities of general RNA processing components mediate the processing regulation. Despite numerous studies in cell lines, this model of Ig gene regulation has never been tested in vivo during normal lymphocyte maturation. We have now introduced a non-Ig gene with an Ig gene-like structure into the mouse germ line and demonstrate that RNA from the transgene is alternatively processed and regulated in murine splenic B cells. This establishes that the balance and arrangement of competing cleavage-polyadenylation reactions are sufficient for RNA processing regulation during normal B-lymphocyte development. These experiments also validate the use of tissue culture cell lines for studies of Ig processing regulation. This is the first transgenic mouse produced to test a specific model for regulated mRNA processing.

Effect of the peroxisome proliferator ciprofibrate on lipid peroxidation and 8-hydroxydeoxyguanosine formation in transgenic mice with elevated hepatic catalase activity.

Peroxisome proliferators are a group of non-genotoxic hepatic carcinogens which have been proposed to act by increasing oxidative damage in the liver. To test this hypothesis, we have produced a transgenic mouse line that has elevated catalase activity specifically in the liver. In this study, we have examined if catalase overexpression influences the induction of lipid peroxidation or oxidative DNA damage, two mechanisms which have been hypothesized to be important in the carcinogenesis by peroxisome proliferators. Transgenic mice or non-transgenic litter mates were fed either 0.01% ciprofibrate or a control diet for 21 days. The activities of fatty acyl CoA oxidase and lauric acid hydroxylase were not significantly affected by catalase overexpression, although the ratio of fatty acyl CoA oxidase to catalase was significantly decreased in transgenic animals. Hepatic lipid peroxidation was estimated by quantifying the concentrations of malondialdehyde and conjugated dienes. Ciprofibrate treatment did not affect either endpoint, but catalase overexpression increased the concentrations of malondialdehyde (in untreated mice only) and conjugated dienes (in both untreated and ciprofibrate-fed mice). Oxidative DNA damage was estimated by quantifying 8-hydroxydeoxyguanosine (8-OHdG) by high-performance liquid chromatography/electrochemical detection. Ciprofibrate treatment significantly increased hepatic 8-OHdG concentrations, in agreement with several previous studies, but catalase overexpression did not significantly affect them, although 8-OHdG concentrations were decreased 50% in untreated mice. These results imply that the metabolism of hydrogen peroxide by catalase is not an important factor in the development of hepatic lipid peroxidation. The decrease in hepatic 8-OHdG in untreated transgenic mice and the increase seen after ciprofibrate administration imply that hydrogen peroxide is important in the formation of 8-OHdG. While the lack of decreased 8-OHdG levels in ciprofibrate-treated transgenic mice does not support this conclusion, it is possible that catalase levels were not sufficiently high to affect this endpoint. Transgenic mice with higher hepatic catalase activities may be required to resolve this issue.

Activation of nuclear factor-kappaB by the peroxisome proliferator ciprofibrate in H4IIEC3 rat hepatoma cells and its inhibition by the antioxidants N-acetylcysteine and vitamin E.

Peroxisome proliferators are a class of hepatic carcinogens in rodents and are proposed to act in part by increasing reactive oxygen species such as hydrogen peroxide. We previously showed that treatment of rats with ciprofibrate, a peroxisome proliferator, results in increased hepatic nuclear factor-kappaB (NF-kappaB) DNA binding activity. In this study, we have examined the link between peroxisome proliferators and NF-kappaB activation in hepatoma cell lines to test whether increased nuclear NF-kappaB levels activate NF-kappaB-regulated genes and to determine the mechanism of NF-kappaB activation. Electrophoretic mobility shift assays demonstrated NF-kappaB induction by ciprofibrate in peroxisome proliferator-responsive H4IIEC3 rat hepatoma cells but not in peroxisome proliferator-insensitive HepG2 human hepatoma cell lines. In addition, we found that stably transfected NF-kappaB-regulated reporter genes were activated by ciprofibrate in H4IIEC3 cells. This reporter gene activation was blocked by the antioxidants N-acetylcysteine and vitamin E. These studies suggest that hepatocytes are at least partially responsible for peroxisome proliferator-mediated hepatic NF-kappaB activation, and support the possibility that this activation is dependent upon reactive oxygen species.

Increased liver-specific catalase activity in transgenic mice.

Catalase is the major peroxisomal H2O2-detoxifying enzyme and is thought to be critical in maintaining low H2O2 levels within a cell. It has been proposed that increased H2O2 levels may be involved in oxidative DNA damage and tumor promotion induced by peroxisome proliferators and other xenobiotics. To develop a mouse model system to address this issue, we have generated transgenic mice that exhibit a three- to four-fold increase in liver catalase levels. The activities of fatty acyl coenzyme A (CoA) oxidase and lauric acid hydroxylase were unchanged in transgenic mice, demonstrating that elevated catalase levels did not alter the activity of these other peroxisome proliferator-induced enzymes that produce active oxygen. These mice should help elucidate the role of H2O2 in cellular events mediated by peroxisome proliferators and other xenobiotics.

Expression of the hydrogen peroxide-generating enzyme fatty acyl CoA oxidase activates NF-kappaB.

Peroxisome proliferators are a class of hepatic carcinogens in rodents and have been proposed to act in part by increasing oxidative stress. Fatty acyl CoA oxidase (FAO), which is highly induced by peroxisome proliferators, is the hydrogen peroxide-generating enzyme of the peroxisomal beta-oxidation pathway. We previously showed that the treatment of rats and mice with the peroxisome proliferator ciprofibrate resulted in increased hepatic NF-kappaB activity and suggested that this effect may be secondary to the action of H2O-generating enzymes. To test this possibility directly, we have determined whether transient overexpression of FAO, in the absence of peroxisome proliferators, leads to NF-kappaB activation. Here, we show that FAO overexpression in Cos-1 cells, in the presence of an H2O-generating substrate, can activate a NF-kappaB regulated reporter gene. Electrophoretic mobility shift assays further demonstrated that FAO expression increases nuclear NF-kappaB DNA binding activity in a dose-dependent manner. The antioxidants vitamin E and catalase can inhibit this activation. These results indicate that FAO mediates, at least in part, peroxisome proliferator-induced NF-kappaB activation.

A sensitive lacZ-based expression vector for analyzing transcriptional control elements in eukaryotic cells.

We have developed a eukaryotic expression vector that provides a rapid and sensitive measure of transcriptional activity modulated by general and tissue-specific regulatory motifs. The lacZ structural gene has been linked to the minimal promoter of the human liver/bone/kidney alkaline phosphatase gene. In addition, a trimerized cassette of the SV40 polyadenylation region has been placed 5' of this promoter to reduce plasmid-initiated transcripts extending through the lacZ gene that would contribute to background beta-galactosidase (beta-Gal) activity. By combining the weak promoter and the poly(A) cassette, only a very low level of lacZ activity is detected in the absence of additional regulatory sequences. Regulatory domains can be inserted into this vector via a unique Bam HI restriction site and their activity can be rapidly monitored in situ via a colorimetric 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Ga) staining protocol. Also, the activity of linked regulatory domains can be measured quantitatively by assaying beta-Gal levels in cell extracts. We show that derivatives of this vector can be used to monitor the activity of general and tissue-specific control elements and can be transactivated by a single transcription factor in cotransfection experiments.

Differential activation of hepatic NF-kappaB in rats and hamsters by the peroxisome proliferators Wy-14,643, gemfibrozil, and dibutyl phthalate.

Nuclear factor-kappaB (NF-kappaB) is an oxidative stress-activated transcription factor involved in the regulation of cell proliferation and apoptosis. We found previously that the peroxisome proliferator ciprofibrate activates NF-kappaB in the livers of rats and mice. These species are sensitive to the hepatocarcinogenic effects of peroxisome proliferators, whereas other species such as Syrian hamsters are not. In the present study we examined the effects of 3 different peroxisome proliferators on NF-kappaB activation in rats and Syrian hamsters. The peroxisome proliferators Wy-14,643, gemfibrozil, and dibutyl phthalate were administered to animals for 6, 34, or 90 days. NF-kappaB activity was determined using electrophoretic mobility-shift assays and confirmed using supershift assays. Wy-14,643 increased the DNA binding activity of NF-kappaB at all 3 time points in rats and produced the highest activation of the 3 chemicals tested. Gemfibrozil and dibutyl phthalate increased NF-kappaB activation to a lesser extent in rats and not at all times. There were no differences in hepatic NF-kappaB levels between control hamsters and hamsters treated with any of the peroxisome proliferators. This study demonstrates species-specific differences in hepatic NF-kappaB activation by peroxisome proliferators.

Effects of peroxisome proliferators on antioxidant enzymes and antioxidant vitamins in rats and hamsters.

Peroxisome proliferators (PPs) cause hepatomegaly, peroxisome proliferation, and hepatocarcinogenesis in rats and mice, whereas hamsters are less responsive to PPs. PPs increase the activities of enzymes involved in peroxisomal beta-oxidation and omega-hydroxylation of fatty acids, which has been hypothesized to result in oxidative stress. The hypothesis of this study was that differential modulation of antioxidant enzymes and vitamins might account for differences in species susceptibility to PPs. Accordingly, we measured the activities of DT-diaphorase and superoxide dismutase (SOD) and the hepatic content of ascorbic acid and alpha-tocopherol in male Sprague-Dawley rats and Syrian hamsters fed 2 doses of 3 known peroxisome proliferators (dibutyl phthalate [DBP], gemfibrozil, and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) for 6, 34, or 90 days. In untreated animals, the activity of DT-diaphorase was much higher in hamsters than in rats, but the control levels of SOD, ascorbic acid and alpha-tocopherol were similar. In rats and hamsters treated with Wy-14,643, we observed decreases in alpha-tocopherol content and total SOD activity. DT-diaphorase was decreased in activity following Wy-14,643 treatment in rats at all time points and doses, but only sporadically affected in hamsters. Rats and hamsters treated with DBP demonstrated increased SOD activity at 6 days; however, in the rat, DBP decreased SOD activity at 90 days and alpha-tocopherol content was decreased throughout. In gemfibrozil treated rats and hamsters, a decrease in alpha-tocopherol content and an increase in DT-diaphorase activity were observed. In either species, no consistent trend was observed in total ascorbic acid content after treatment with any of the PPs. In conclusion, these data suggest that both rats and hamsters are compromised in antioxidant capabilities following PP treatment and additional hypotheses for species susceptibility should be considered.

Agreement and clinical utility of 2 techniques for measuring cardiac output in patients with low cardiac output.

BACKGROUND: The reliability of cardiac output obtained with the bolus technique is a problem. OBJECTIVES: To compare measurements of cardiac output measured with bolus and continuous techniques in patients with low cardiac output and to determine if measurements obtained with the continuous technique increased the number of subsequent clinical decisions. METHODS: In 60 intensive care patients, a nurse recorded a single continuous cardiac output measurement and then obtained the mean of 3 consecutive bolus determinations. The medical records of these 60 patients (experimental group) for the next 48 hours and of 60 other patients with regular or mixed venous oximetry catheters (control group) were reviewed to assess the occurrence of cardiac output events and the frequency of clinical decisions based on the events. RESULTS: Mean cardiac output was 4.46 L/min by the continuous technique and 5.20 L/min by the bolus technique (P = .011) for the experimental group. Median bias between the 2 types of measurements was -0.10 L/min (P = .79). Twenty-three of the pairs (38%) had an absolute percent difference greater than 15%. Of these, 18 (78%) had a higher bolus reading. Treatment decisions per 48 hours were 9.9 for the experimental group and 8.6 for the control group (P = .014). Median length of stay was 2 days less in the experimental group (P = .02), and mean highest cardiac output was 0.81 L/min higher (P = .009). CONCLUSIONS: Measurements of cardiac output determined with the continuous technique may be more precise than measurements determined with the bolus technique. Continuous cardiac output information increases the number of treatment decisions and actions that may shorten hospital length of stay.

PDGF-A promoter and enhancer elements provide efficient and selective antineoplastic gene therapy in multiple cancer types.

Development of antineoplastic gene therapies is impaired by a paucity of transcription control elements with efficient, cancer cell-specific activity. We investigated the utility of promoter (AChP) and 5'-distal enhancer (ACE66) elements from the platelet-derived growth factor-A (PDGF-A) gene, which are hyperactive in many human cancers. Efficacy of these elements was tested in multiple tumor cell lines, both in cell culture and as tumor explants in athymic nude mice. Plasmid and viral vectors were constructed with the AChP promoter alone or in fusion with three copies of the ACE66 enhancer for expression of the prototype suicide gene, thymidine kinase (TK). ACE/AChP and AChP cassettes elicited ganciclovir (GCV)-induced cytotoxicity in multiple tumor cell lines. The ACE enhancer element also exhibited synergism with placental and liver-specific promoter elements. An adenovirus containing the AChP-TK cassette produced striking increases in GCV sensitivity in cultured tumor cell lines, as well as GCV-induced regression of U87 MG glioblastoma explants in vivo. TK expression was distributed throughout tumors receiving the therapeutic virus, whereas TdT-mediated dUTP nick end labeling (TUNEL) analysis revealed numerous regions undergoing apoptosis. Vascularization and reticulin fiber networks were less pronounced in virus-GCV-treated tumors, suggesting that both primary and stromal cell types may have been targeted. These studies provide proof-of-principle for utility of the PDGF-A promoter and ACE66 enhancer in antineoplastic gene therapy for a diverse group of human cancers.

Transcriptional control in the mammalian liver: liver development, perinatal repression, and zonal gene regulation.

Liver function is crucial for maintaining metabolic homeostasis in mammals. Numerous genes must be properly regulated for the liver to develop and perform a variety of activities. Several recent gene-knockout studies in mice have clarified the roles of GATA6, HNF4alpha, and Foxa1/Foxa2 in early stages of liver formation. After the liver forms, transcriptional changes continue to occur; during the perinatal period, certain genes such as alpha-fetoprotein and H19 are silenced, others are activated, and position-dependent (or zonal) regulation is established. Zhx2 was recently identified as one factor involved in postnatal repression of alpha-fetoprotein and other genes. Furthermore, several studies indicate that negative regulation is involved in the zonal control of glutamine synthetase. Finally, exciting new evidence indicates that signaling through the Wnt/beta-catenin pathway is also involved in zonal regulation in the adult liver.

Alpha-fetoprotein gene regulation: lessons from transgenic mice.

The mouse alpha-fetoprotein (AFP) gene provides an excellent model system to study developmental gene activation and different aspects of liver-specific transcriptional control. AFP is activated early in hepatogenesis, repressed post-natally, and can be reactivated during liver regeneration and in hepatocellular carcinomas. Transgenic studies have also revealed that AFP enhancers, when linked individually to a heterologous promoter, can confer zonal control in the adult liver. Continued transgenic studies, combined with analysis using in vitro and tissue culture systems, will help elucidate mechanisms of transcriptional regulation during liver development and hepatocarcinogenesis.

Endogenous and transfected mouse alpha-fetoprotein genes in undifferentiated F9 cells are activated in transient heterokaryons.

Mouse F9 teratocarcinoma cells provide a system to study developmentally regulated alpha-fetoprotein (AFP) gene expression. AFP is not expressed in undifferentiated F9 cells but is induced when cells differentiate as cell aggregates in the presence of retinoic acid. Previous studies have led to the suggestion that undifferentiated F9 cells contain negative regulators of AFP expression. To test this, we have used transient heterokaryons to ask whether inactive AFP genes in undifferentiated F9 cells are responsive to positively acting trans-acting factors. Our results indicate that silent endogenous and transfected AFP genes are activated when undifferentiated F9 cells are fused to human hepatoma HepG2 cells. This suggests that the lack of AFP expression in undifferentiated F9 cells is due to the absence or insufficient level of positive-acting transcription factors, rather than the presence of dominant negative regulators. We also demonstrate that stably transfected AFP genes, although unmethylated, are properly regulated in F9 cells.

Liver-specific catalase expression in transgenic mice inhibits NF-kappaB activation and DNA synthesis induced by the peroxisome proliferator ciprofibrate.

Peroxisome proliferators are a group of non-genotoxic hepatic carcinogens that have been proposed to act by increasing oxidative damage in the liver. To test this hypothesis, we have examined if hepatic catalase overexpression in peroxisome proliferator-treated mice influences the induction of cell proliferation or the activation of transcription factors involved in cell proliferation. Transgenic mice or non-transgenic littermates were fed either 0.01% ciprofibrate or a control diet for 21 days. Fatty acyl CoA oxidase activity was not significantly affected by catalase overexpression, although the ratio of fatty acyl CoA oxidase to catalase was significantly decreased in transgenic animals. The labeling index in hepatocytes was significantly increased by ciprofibrate in non-transgenic mice, but catalase overexpression significantly inhibited this increase. Ciprofibrate increased the activation of nuclear factor (NF)-kappaB in non-transgenic mice, but this increase was inhibited by catalase overexpression. Ciprofibrate also increased AP-1 activation, but catalase overexpression did not significantly inhibit this increase, although AP-1 activation was 40% lower in transgenic mice. These results support the hypothesis that active oxygen plays a role in the induction of cell proliferation by the peroxisome proliferator ciprofibrate and therefore may be important in the carcinogenicity of these agents.