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.

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.

Effects of peroxisome proliferators on glutathione and glutathione-related enzymes in rats and hamsters.

Peroxisomeproliferators (PPs) cause hepatomegaly, peroxisome proliferation, and hepatocarcinogenesis in rats and mice. Conversely, hamsters are less responsive to these compounds. PPs increase peroxisomal beta-oxidation and P4504A subfamily activity, which has been hypothesized to result in oxidative stress. We hypothesized that differential modulation of glutathione-related defenses could account for the resulting difference in species susceptibility following PP administration. Accordingly, we measured glutathione S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR) activities, and total glutathione (GSH) in male Sprague-Dawley rats and Syrian hamsters fed two doses of three known peroxisome proliferators [dibutylphthalate (DBP), gemfibrozil, and Wy-14,643] for 6, 34, or 90 days. In rats, decreases in GR, GST, and selenium-dependent GPx were observed following PP treatment at various time points. In hamsters, we observed higher basal levels of activities for GR, GST, and selenium-dependent GPx compared to rats. In addition, hamsters showed decreases in GR and GST activities following PP treatment. Interestingly, selenium-dependent GPx activity was increased in hamsters following treatment with Wy-14,643 and DBP. Treatment for 90 days with Wy-14,643 resulted in no change in GPx1 mRNA in rats and increased GPx1 mRNA in hamsters. Sporadic changes in total GSH and selenium-independent GPx were observed in both species. This divergence in the hydrogen peroxide detoxification ability between rats and hamsters could be a contributing factor in the proposed oxidative stress mechanism of PPs observed in responsive and nonresponsive species.

Position-dependent activity of alpha -fetoprotein enhancer element III in the adult liver is due to negative regulation.

alpha-Fetoprotein (AFP) transcription is activated early in hepatogenesis, but is dramatically repressed within several weeks after birth. AFP regulation is governed by multiple elements including three enhancers termed EI, EII, and EIII. All three AFP enhancers continue to be active in the adult liver, where EI and EII exhibit high levels of activity in pericentral hepatocytes with a gradual reduction in activity in a pericentral-periportal direction. In contrast to these two enhancers, EIII activity is highly restricted to a layer of cells surrounding the central veins. To test models that could account for position-dependent EIII activity in the adult liver, we have analyzed transgenes in which AFP enhancers EII and EIII were linked together. Our results indicate that the activity of EIII is dominant over that of EII, indicating that EIII is a potent negative regulatory element in all hepatocytes except those encircling the central veins. We have localized this negative activity to a 340-bp fragment. This suggests that enhancer III may be involved in postnatal AFP repression.

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.

The alpha-fetoprotein promoter is the target of Afr1-mediated postnatal repression.

The alpha-fetoprotein (AFP) gene is transcribed at high levels in the fetal liver and is repressed at birth, leading to low but detectable levels of AFP mRNA in the adult liver. This repression is regulated, in part, by a locus that is unlinked to AFP called Alpha-fetoprotein regulator 1 (Afr1). Previous studies showed that Afr1 regulation is independent of the AFP enhancers but requires the 1-kb AFP promoter/repressor region. Here, we demonstrate that a transgene with the 250-bp AFP promoter region linked to AFP enhancer element EII is expressed in the fetal liver and is postnatally repressed. In addition, this transgene is regulated by Afr1. These data indicate that the promoter is involved in postnatal AFP repression. Furthermore, we provide a high-resolution map of the Afr1 locus on mouse chromosome 15.

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.

Hepatocyte nuclear factor 3 relieves chromatin-mediated repression of the alpha-fetoprotein gene.

The alpha-fetoprotein gene (AFP) is tightly regulated at the tissue-specific level, with expression confined to endoderm-derived cells. We have reconstituted AFP transcription on chromatin-assembled DNA templates in vitro. Our studies show that chromatin assembly is essential for hepatic-specific expression of the AFP gene. While nucleosome-free AFP DNA is robustly transcribed in vitro by both cervical (HeLa) and hepatocellular (HepG2) carcinoma extracts, the general transcription factors and transactivators present in HeLa extract cannot relieve chromatin-mediated repression of AFP. In contrast, preincubation with either HepG2 extract or HeLa extract supplemented with recombinant hepatocyte nuclear factor 3 alpha (HNF3alpha), a hepatic-enriched factor expressed very early during liver development, is sufficient to confer transcriptional activation on a chromatin-repressed AFP template. Transient transfection studies illustrate that HNF3alpha can activate AFP expression in a non-liver cellular environment, confirming a pivotal role for HNF3alpha in establishing hepatic-specific gene expression. Restriction enzyme accessibility assays reveal that HNF3alpha promotes the assembly of an open chromatin structure at the AFP promoter. Combined, these functional and structural data suggest that chromatin assembly establishes a barrier to block inappropriate expression of AFP in non-hepatic tissues and that tissue-specific factors, such as HNF3alpha, are required to alleviate the chromatin-mediated repression.

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.

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.

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.

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.

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.

Activation of hepatic NF-kappaB by phenobarbital in rats.

Nuclear factor kappaB (NF-kappaB) is an important stress-induced transcription factor in many cell types. In this study we examined if the hepatic tumor promoting agent phenobarbital could activate NF-kappaB in the liver. Rats were fed 0.05% phenobarbital in the diet for 3, 6, or 10 days. Benzyloxyresorufin dealkylase activity was greatly increased in rats receiving phenobarbital. The DNA binding activity of NF-kappaB was determined using electrophoretic mobility shift assays. After 3 days, NF-kappaB activity was increased two-fold in rats receiving phenobarbital. NF-kappaB activity continued to increase at 6 and 10 days in rats fed phenobarbital, with a four-fold increase seen at day 10. Cold competition and antibody supershift experiments showed the DNA binding activity to be specific for NF-kappaB. These data show that phenobarbital activates hepatic NF-kappaB in rats.

Treatment of rats with the peroxisome proliferator ciprofibrate results in increased liver NF-kappaB activity.

Nuclear factor kappaB (NF-kappaB) is an important stress-induced transcription factor in many cell types, including hepatocytes. Previous studies have shown that reactive oxygen species, including hydrogen peroxide, are potent activators of NF-kappaB. Peroxisome proliferators are a group of rodent chemical carcinogens that have been proposed to act by increasing reactive oxygen in the liver. These results led us to consider whether peroxisome proliferators would increase NF-kappaB activity in the liver. Here we demonstrate that rats fed diets containing the peroxisome proliferator ciprofibrate exhibit increased hepatic NF-kappaB DNA-binding activity. This observation suggests that NF-kappaB may contribute, in part, to peroxisome proliferator-mediated changes in the liver.

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.