The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

Physiological, pharmacological and toxicological considerations of drug-induced structural cardiac injury.

The incidence of drug-induced structural cardiotoxicity, which may lead to heart failure, has been recognized in association with the use of anthracycline anti-cancer drugs for many years, but has also been shown to occur following treatment with the new generation of targeted anti-cancer agents that inhibit one or more receptor or non-receptor tyrosine kinases, serine/threonine kinases as well as several classes of non-oncology agents. A workshop organized by the Medical Research Council Centre for Drug Safety Science (University of Liverpool) on 5 September 2013 and attended by industry, academia and regulatory representatives, was designed to gain a better understanding of the gaps in the field of structural cardiotoxicity that can be addressed through collaborative efforts. Specific recommendations from the workshop for future collaborative activities included: greater efforts to identify predictive (i) preclinical; and (ii) clinical biomarkers of early cardiovascular injury; (iii) improved understanding of comparative physiology/pathophysiology and the clinical predictivity of current preclinical in vivo models; (iv) the identification and use of a set of cardiotoxic reference compounds for comparative profiling in improved animal and human cellular models; (v) more sharing of data (through publication/consortia arrangements) on target-related toxicities; (vi) strategies to develop cardio-protective agents; and (vii) closer interactions between preclinical scientists and clinicians to help ensure best translational efforts.

The retinoid X receptor enhances the function of the peroxisome proliferator activated receptor.

The peroxisome proliferator activated receptor (PPAR) is a member of the steroid hormone receptor superfamily and is activated by a variety of non-genotoxic rodent hepatocarcinogens termed peroxisome proliferators. A key marker of peroxisome proliferator action is the peroxisomal enzyme acyl-CoA oxidase that is elevated about 10-fold in the liver of treated rodents. We have shown previously that a peroxisome proliferator response element (PPRE) is located 570 bp upstream of the rat acyl-CoA oxidase gene and that PPAR binds to it. We show here that the retinoid X receptor (RXR) is required for PPAR to bind to the PPRE and that the RXR ligand, 9-cis retinoic acid, enhances PPAR action. These results therefore suggest that retinoids may modulate the action of peroxisome proliferators.

Peroxisome proliferator-activated receptor alpha: role in rodent liver cancer and species differences.

Peroxisome proliferators (PPs) are chemicals of industrial and pharmaceutical importance that elicit liver carcinogenesis by a non-genotoxic mechanism. One of the intriguing properties of PPs is that the pleiotropic effects of these compounds (including increased DNA synthesis and peroxisome proliferation) are seen in rats and mice only, but not humans. It is important to determine the risks to humans of environmental and therapeutic exposure to these compounds by understanding the mechanisms of non-genotoxic hepatocarcinogenesis in rodents. To understand this apparent lack of human susceptibility, attention has focused on the peroxisome proliferator-activated receptor alpha (PPARalpha), which appears to mediate the effects of PPs in rodents. It is also known to mediate the hypolipidaemic effects that fibrate drugs exert on humans with elevated plasma cholesterol and triglyceride levels. Human PPARalphas share many functional characteristics with the rodent receptors, in that they can be transcriptionally activated by PPs and regulate specific gene expression. However, one key difference is that PPARalpha is less abundant in human than in rodent liver, which has led to the suggestion that species differences result from quantitative differences in gene expression. In this review we describe the effects of PPs and what is known of the molecular mechanisms of action and species differences with respect to rodents and man. Attention will be given to differences in the amounts of PPARalpha between species as well as the 'qualitative' aspects of PPARalpha-mediated gene regulation which might also explain the activation of some genes and not of others in human liver by PPs.

The peroxisome proliferator-activated receptor:retinoid X receptor heterodimer is activated by fatty acids and fibrate hypolipidaemic drugs.

The peroxisome proliferator-activated receptor (PPAR) is a member of the steroid hormone receptor superfamily and is activated by a variety of fibrate hypolipidaemic drugs and non-genotoxic rodent hepatocarcinogens that are collectively termed peroxisome proliferators. A key marker of peroxisome proliferator action is the peroxisomal enzyme acyl CoA oxidase, which is elevated about tenfold in the livers of treated rodents. We have previously shown that a peroxisome proliferator response element (PPRE) is located 570 bp upstream of the rat peroxisomal acyl CoA oxidase gene and that PPAR binds to it. We show here that the retinoid X receptor (RXR) is required for PPAR to bind to the PPRE, and that the RXR ligand, 9-cis retinoic acid, enhances PPAR action. Retinoids may therefore modulate the action of peroxisome proliferators and PPAR may interfere with retinoid action, perhaps providing one mechanism to explain the toxicity of peroxisome proliferators. We have also shown that a variety of hypolipidaemic drugs and fatty acids can activate PPAR. This supports the suggestion that the physiological role of PPAR is to regulate fatty acid homeostasis, and provides further evidence that PPAR is the target of the fibrate class of hypolipidaemic drugs. Finally, we have demonstrated that a metabolically stabilized fatty acid is a potent PPAR activator, suggesting that fatty acids, or their acyl CoA derivatives, may be the natural ligands of PPAR.

Peroxisome proliferator-activated receptors: structures and function.

We have been attempting to elucidate the molecular mechanisms through which peroxisome proliferators exert their pleiotropic effects, with particular emphasis on understanding why humans appear unresponsive to these compounds. There is a wealth of data to implicate the peroxisome proliferator-activated receptor alpha (PPAR alpha) in mediating these effects in rodent species; PPAR alpha is expressed in tissues that show physiological changes in response to PPs, is transcriptionally activated in vitro by a variety of PPs, and it has been recently demonstrated that mice lacking this receptor are refractory to the effects of clofibrate and Wy-14,643, at least in the short term. It is conceivable that differences in PPAR alpha between responsive rodent and unresponsive human subjects may provide the key to understanding the basis of this species variation in response, and with this in mind we have been studying the biology of PPAR alpha in humans and looking at interindividual variation. There is already published evidence, albeit on only two sequences, for structural and functional polymorphism in human PPAR alphas. We have extended these findings, and shown that: There is considerable variation in hPPAR alpha cDNAs obtained from different individuals, both at the gross structural level (lack of a coding exon) and of a more subtle nature (single base changes leading to amino acid substitutions). One such cDNA, the sequence of which differs at only three amino acids from that published, encodes a receptor that is incapable of transcriptional activation by potent PPs. The degree to which hPPAR alpha transcripts are expressed in human livers can vary by up to an order of magnitude between individuals. The tissue-specific expression profile of PPAR alpha in humans is very different from that in rat and mouse. In particular, the human liver contains generally low levels of PPAR alpha in contrast to the responsive rodents, in which potent PPs cause liver tumors. Taken together, these data suggest first that human and rodent PPAR alphas differ according to a number of molecular and biochemical criteria, and secondly that there is a degree of interindividual variation in PPAR alpha structure and function. Studies are ongoing to clarify this further, but human polymorphism may go some way towards explaining the apparent paradox that active PPAR alpha receptors can be isolated from an "unresponsive" species.

The molecular mechanism of peroxisome proliferator action: a model for species differences and mechanistic risk assessment.

An increasing number of chemicals that produce tumours in rodent bioassays belong to the non-genotoxic class of carcinogens. There are no suitable tests for these carcinogens and our understanding of their mechanism of action is poor. Importantly, assessment of their potential hazard to man is usually difficult without extensive research. Peroxisome proliferators (PP) are a diverse group of rodent non-genotoxic carcinogens that include hypolipidemic drugs, plasticizers and herbicides. We have reported previously the cloning of a member of the nuclear hormone receptor superfamily and, through the use of chimeric receptors, discovered that it could be activated by PPs. The receptor is therefore termed the PP activated receptor (PPAR). The most widely used marker of PP action is the peroxisomal beta-oxidation enzyme acyl CoA oxidase (ACO). Interestingly, it has been speculated that the hydrogen peroxide produced as a result of ACO activity could lead to DNA damage and tumorigenesis. We have now demonstrated that PPAR recognizes a specific PP response element (PPRE) located in the ACO gene promoter and that the response is dependent upon the presence of receptor and the addition of the PP Wy-14,643. These data therefore support a model in which the mechanism of PP action is mediated by PPAR in a manner similar to that of steroid hormone action. Learning more about the function of PPAR offers a unique opportunity to understand the mechanism of action of some non-genotoxic carcinogens. Furthermore, this knowledge when combined with comparison of receptor expression between rodents and man will be important in providing a framework for a new threshold model of risk assessment based upon receptor-mediated carcinogenesis.

Peroxisome proliferator-activated receptor (PPAR) alpha-regulated growth responses and their importance to hepatocarcinogenesis.

Peroxisome proliferators (PPs) are a class of non-genotoxic rodent hepatocarcinogens that act by perturbing liver growth regulation. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGF beta1). More recently, we have demonstrated that PPs can suppress apoptosis induced by more diverse stimuli such as DNA damage or ligation of Fas, a receptor related to the tumour necrosis factor alpha (TNF alpha) family of cell surface receptors. PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha, PPAR alpha, a member of the nuclear hormone receptor superfamily. We investigated whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. hPPAR alpha-6/29 shared the ability of mPPAR alpha to bind to DNA but, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a non-genotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. Recent data have suggested that TNF alpha, perhaps released by liver Kupffer cells in response to PPs, may play a key role in mediating the effects of PPs on hepatocyte growth regulation.

Biology and toxicology of PPARgamma ligands.

The peroxisome proliferator activated receptor-gamma (PPARgamma) is an attractive target for therapeutic intervention, as modulation of PPARgamma-regulated pathways is potentially beneficial in a number of disease areas. This review provides an overview of what is known about the biology of PPARgamma, and an indication of what progress has been made towards drug development in several therapy areas. As well as efficacy, the safety of drugs is of course an important issue, and a substantial volume of preclinical and clinical information has already accumulated for PPARgamma agonists. Here we discuss some of the major toxicology issues with PPARgamma agonists, and give a perspective on likely issues concerning the development of PPARgamma modulators in the future.

Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis.

In this review we have evaluated the relationship between peroxisome proliferation and hepatocarcinogenesis. To do so, we identified all chemicals known to produce peroxisome proliferation and selected those for which there are data (on peroxisome proliferation and hepatocarcinogenesis) which meet certain criteria chosen to facilitate comparison of these phenomena. The summarised data and definition of the methodology used has been collected in appendices. These comparisons enabled us to evaluate the relationship between these phenomena using reliable data. As there is a good correlation between them, we further explored the mechanisms of action that have been proposed (direct genotoxic activity, production of hydrogen peroxide, cell proliferation and receptor activation). The relationship between these events in other species, including humans, was also reviewed and finally an overview of the assessment of human hazard is presented in section IX. Some of the first chemicals which were shown to produce peroxisome proliferation were also hepatocarcinogens whose carcinogenicity could not be readily explained by genotoxic activity. This raised the suggestion that the unusual phenomenon of peroxisome proliferation was intricately linked to the carcinogenic activity of these agents. Three questions have exercised the attention of regulatory, industrial and academic toxicology since then; are chemicals which elicit peroxisome proliferation in the liver actually a coherent class of chemical carcinogens?; does the early biological phenomenon of peroxisome proliferation have real predictive value for and mechanistic association with rodent carcinogenesis?; and what hazard/risk do these agents pose to humans that may be exposed to them? Whether peroxisome proliferators are indeed a discrete class of rodent carcinogens would appear to be the single, most important question. If so, then the assumptions and procedures relevant to human hazard and risk assessment should be applied to the class and should be essentially generic; if not, each chemical should be considered independently. Our critical analysis of the published data for over 70 agents which have been shown to possess intrinsic ability to induce peroxisome proliferation in the livers of rodents has led to the conclusion that there exists a strong correlation between peroxisome proliferation as n early effect in the liver and hepatocarcinogenicity in chronic exposure studies. An almost perfect correlation was observed between the induction of peroxisomes in the rodent liver and the eventual appearance of tumours following chronic exposure The few exceptions to this were largely explainable (section II).(ABSTRACT TRUNCATED AT 400 WORDS)

An epitope-tagging system for studying regulation of the peroxisome proliferator activated receptor alpha (PPAR alpha).

Peroxisome proliferators (PPs) are a group of compounds which cause peroxisome proliferation and hepatocellular carcinomas in rodents, and form a class of non-genotoxic carcinogens. It is thought that PPs act via a receptor similar to members of the nuclear hormone superfamily termed the peroxisome proliferator activated receptor (PPAR). Multiple subtypes (alpha, beta, delta and gamma) of the receptor exist and are differentially expressed between tissues and species. PPAR alpha has been shown to activate transcription by binding to response elements upstream of peroxisome proliferator responsive genes. However, despite the isolation of transcriptionally active human subtypes of the receptor, hPPAR alpha and hNUC1, humans are thought to be non-responsive to PPs. This is possibly due to regulation of PPAR, and it has been recently reported that PPAR alpha is a phosphoprotein in vivo and insulin regulates its phosphorylation. A system employing epitope-tagged receptors has been developed to study this further, with the aim of establishing stably transfected cell lines expressing high levels of epitope-tagged mouse and human PPAR alpha. Our experiments clearly demonstrate that an epitope-tagged mPPAR alpha receptor has an equal ability to modulate transcription as the native receptor in transactivation assays and will be further used to examine the molecular mechanisms of peroxisome proliferation.

The significance of circulating tumour cells in breast cancer: a review.

Haematogenous spread of circulating tumour cells (CTCs) is the principle mechanism for development of metastases. Research into the enumeration and characterisation of CTCs, particularly in the last decade, has allowed the introduction of semi-automated CTC assessment in the clinical setting. In breast cancer, CTC enumeration is being used as a prognostic biomarker, a predictive biomarker of treatment response and is being assessed to guide treatment in both the early and metastatic setting. CTC characterisation has the potential to direct targeted therapies, such as HER2 therapies in HER2 negative primary breast tumour patients. However, CTC assessment has considerable challenges. Capture and identification of these very rare cells is currently largely dependent on a presumed homogeneity of phenotype. In addition, high throughput assays are lacking. The clinical significance of CTCs is incompletely understood. A large proportion of CTC positive patients have no evidence of metastases, raising the issue of either inconsequential tumour dormancy or non-viable CTCs. CTCs may have additional clinical sequelae such as promoting venous thrombosis. However CTCs provide a real-time liquid biopsy of the tumour and represent an exciting, minimally invasive method of assessing disease status and also a novel therapeutic target for malignancy.

Predictive models of hepatotoxicity using gene expression data from primary rat hepatocytes.

With the aim of evaluating the usefulness of an in vitro system for assessing the potential hepatotoxicity of compounds, the paper describes several methods of obtaining mathematical models for the prediction of compound-induced toxicity in vivo. These models are based on data derived from treating rat primary hepatocytes with various compounds, and thereafter using microarrays to obtain gene expression 'profiles' for each compound. Predictive models were constructed so as to reduce the number of 'probesets' (genes) required, and subjected to rigorous cross-validation. Since there are a number of possible approaches to derive predictive models, several distinct modelling strategies were applied to the same data set, and the outcomes were compared and contrasted. While all the strategies tested showed significant predictive capability, it was interesting to note that the different approaches generated models based on widely disparate probesets. This implies that while these models may be useful in ascribing relative potential toxicity to compounds, they are unlikely to provide significant information on underlying toxicity mechanisms. Improved predictivity will be obtained through the generation of more comprehensive gene expression databases, covering more 'toxicity space', and by the development of models that maximize the observation, and combination, of individual differences between compounds.

A human peroxisome-proliferator-activated receptor-gamma is activated by inducers of adipogenesis, including thiazolidinedione drugs.

We have cloned a human cognate of the mouse peroxisome-proliferator-activated receptor-gamma (hPPAR gamma) from a human placenta cDNA library. Sequence analysis reveals a high degree of similarity with the mouse receptor and, like other PPAR, hPPAR gamma forms heterodimers with the retinoid X receptor alpha (RXR alpha) and binds in vitro to DNA elements containing direct repeats of the sequence TGACCT. In common with mouse PPAR gamma, hPPAR gamma is expressed strongly in adipose tissue, but significant levels also are detectable in placenta, lung and ovary. In vitro trans-activation data suggest hPPAR gamma is only poorly activated by xenobiotic peroxisome proliferators, although certain fatty acids and eicosanoids are potent activators of this receptor. Both mouse and human PPAR gamma are capable of being activated by thiazolidinedione drugs, although the two receptors appear to differ in their sensitivity to these compounds. Taken together, these data suggest a high degree of structural and functional similarity between mouse and human PPAR gamma, and provide evidence for variation in human receptor structure which may result in differential sensitivity to activators.

Identification and characterization of DNA elements implicated in the regulation of CYP4A1 transcription.

We have identified a peroxisome proliferator response element (PPRE) approx. 4300 nucleotide upstream of the rat cytochrome P-450 CYP4A1 gene. Two members of the steroid-hormone-receptor superfamily, the peroxisome proliferator-activated receptor-alpha (PPAR alpha) and the retinoid X receptor-alpha (RXR alpha), bind specifically to this element as a heterodimer, and this element confers responsiveness to the peroxisome proliferator Wyeth-14,643 when tested in co-transfection assays. A second element, located 35 nucleotides further upstream, fails to bind PPAR alpha/RXR alpha heterodimers and is unresponsive to Wy-14,643 in co-transfection assays. Both elements are, however, responsive to 9-cis-retinoic acid in the presence of RXR alpha, when tested in the co-transfection assay. As RXR alpha fails to bind to either element as a homodimer, we suggest that RXR alpha interacts with PPAR alpha to regulate transcription via the proximal element, and interacts with some other cellular factor to regulate transcription via the more distal element. This is consistent with previous reports that a number of peroxisome proliferator-regulated genes contain PPRE-like elements as part of their regulatory sequences, which may be recognized by several receptor combinations. This provides further evidence that PPARs and their co-factors are important in mediating the pleiotropic action of peroxisome proliferators.

Two families of sequences in the small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B.

DNA sequence analysis was carried out on the 1-kilobase SacI-EcoRI region of the EcoRI J fragment of four strains of Epstein-Barr virus (EBV) (MABA, P3HR-1, FF41, and NPC-5), and the sequences were compared with the prototype sequence from strain B95-8. Ten single-base changes which grouped the strains into two families (1 and 2) were found. Restriction endonuclease polymorphisms predicted from the sequences were used to classify the EBV DNA from a further 26 EBV-positive cell lines into these two families. The EBNA-2 types (A or B) of the strains were found to correlate with the J region type; EBNA-2 type A DNA regularly contained J region sequence type 1, while EBNA-2 type B DNA generally carried J region sequence type 2. These data are consistent with the notion of there being two distinct families of EBV with discrete, conserved differences in DNA sequence.

Amino acid residues in both the DNA-binding and ligand-binding domains influence transcriptional activity of the human peroxisome proliferator-activated receptor alpha.

We have investigated the basis of the lack of activity of a natural variant human peroxisome proliferator-activated receptor alpha, hPPARalpha6/29. A subcloning approach was used to change the four variant amino acids in the hPPARalpha6/29 sequence, individually and in combination, to those found in an active human PPARalpha. Individual amino acid "back mutations" were unable to confer on hPPARalpha6/29 the ability to be activated by peroxisome proliferators in a transient transfection assay. Although hPPARalpha6/29 was able to bind specifically to DNA in the presence of the retinoid X receptor alpha (RXRalpha), the complete restoration of receptor transcriptional activity required two separate back mutations of the hPPARalpha6/29 sequence, namely amino acid 123 in the DNA binding domain, and amino acid 444 close to the C-terminus. This suggests that sequences in the PPARalpha DNA binding domain influence other receptor functions besides DNA binding.

Evidence for the suppression of apoptosis by the peroxisome proliferator activated receptor alpha (PPAR alpha).

Peroxisome proliferators (PPs) are a class of nongenotoxic rodent hepatocarcinogens. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGFbeta1). PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha (PPAR alpha), a member of the nuclear hormone receptor superfamily. Here, we investigate whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. Electrophoretic mobility shift assays (EMSA) demonstrated that hPPAR alpha-6/29 shared the ability of mPPAR alpha to heterodimerise with the retinoid X receptor (RXR) and bind to DNA. When hPPAR alpha-6/29 was transfected into Hepa1c1c7 cells together with a reporter plasmid containing a PPAR response element (PPRE), hPPAR alpha-6/29, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription since increasing concentrations of hPPAR alpha-6/29 abrogated the activation of co-transfected mPPAR alpha. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a nongenotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. These data indicate that activation of rat liver PPAR alpha provides a survival signal for hepatocytes, preventing their death in response to apoptotic stimuli.