Acute intraoperative heparin-induced thrombocytopenia (HIT) and thrombosis during coronary artery bypass grafting: Two case reports providing evidence for the role of preoperative LMWH in triggering sensitization.

INTRODUCTION: Systemic anticoagulation is necessary during cardiac surgery. To date, the only well established anticoagulation protocol involves the use of heparin. However, heparin can cause heparin-induced thrombocytopenia (HIT) a potentially life threatening immune-mediated thromboembolic syndrome. Until now, devastating consequences of HIT syndrome in patients undergoing heart surgery have been described, but only postoperatively. Here we report the development of HIT syndrome during cardiac revascularization by intra-operative heparin administration in two patients previously exposed to LMWH. PATIENTS/METHODS: We report on two patients who developed rapid and profound intravascular coagulation with severe thrombocytopenia (platelet count decreased from ≥250×109/L to 50×109/L) due to HIT development caused by heparin administration during coronary artery bypass graft surgery. In addition we report that fondaparinux, given intra-operatively in association with antithrombin, may be a suitable alternative anticoagulant for successfully preventing the devastating consequences of intra-operative HIT development. CONCLUSION: To our knowledge, this is the first report describing the development of acute intra-operative HIT, secondary to high-dose UFH administered for coronary revascularization, in which the unexpected presence of platelet-activating anti-PF4/heparin antibodies at surgery was explained by preoperative administration of a one-week course of LMWH but without any preoperative evidence for HIT.

Targeting the endoplasmic reticulum mediates radiation sensitivity in colorectal cancer.

BACKGROUND: Radiotherapy is an established treatment modality for early and locally advanced rectal cancer as part of short course radiotherapy and long course chemoradiotherapy. The unfolded protein response (UPR) is a cellular stress response pathway often activated in human solid tumours which has been implicated in resistance to both chemotherapy and radiotherapy. This research has investigated whether the UPR pathway is upregulated in ex-vivo samples of human colorectal cancer and characterised the interaction between radiotherapy and UPR activation in two human colorectal cancer cell lines in vitro. METHODS: In vitro UPR expression was determined in response to clinical doses of radiotherapy in both the human colorectal adenocarcinoma (HT-29) cell line and a radio-resistant clone (HT-29R) using western blotting and quantitative polymerase chain reaction. The UPR was induced using a glucose deprivation culture technique before irradiation and radiosensitivity assessed using a clonogenic assay. Ex-vivo human colorectal cancer tissue was immuno-histochemically analysed for expression of the UPR marker glucose regulated protein 78 (GRP-78). RESULTS: The UPR was strongly up regulated in ex-vivo human colorectal tumours with 36 of 50 (72.0%) specimens demonstrating moderate to strong staining for the classic UPR marker GRP-78. In vitro, therapeutic doses of radiotherapy did not induce UPR activation in either radiosensitive or radioresistant cell lines. UPR induction caused significant radiosensitisation of the radioresistant cell line (HT-29R SF2Gy=0.90 S.E.M. +/-0.08; HT-29RLG SF2Gy=0.69 S.E.M. +/-0.050). CONCLUSION: This suggests that UPR induction agents may be potentially useful response modifying agents in patients undergoing therapy for colorectal cancer.

An in vivo role for Rho kinase activation in the tumour vascular disrupting activity of combretastatin A-4 3-O-phosphate.

BACKGROUND AND PURPOSE: Combretastatin A-4 3-O-phosphate (CA4P) is in clinical trial as a tumour vascular disrupting agent (VDA) but the cause of blood flow disruption is unclear. We tested the hypothesis that activation of Rho/Rho kinase (ROCK) is fundamental to the effects of this drug in vivo. EXPERIMENTAL APPROACH: Mouse models of human colorectal carcinoma (SW1222 and LS174T) were used. Effects of the ROCK inhibitor, Y27632, alone or in combination with CA4P, on ROCK activity, vascular function, necrosis and immune cell infiltration in solid tumours were determined. Mean arterial BP (MABP) was measured to monitor systemic interactions and the vasodilator, hydralazine, was used to control for the hypotensive effects of Y27632. KEY RESULTS: Y27632 caused a rapid drop in blood flow in SW1222 tumours, with recovery by around 3 h, which was paralleled by MABP changes. Y27632 pretreatment reduced CA4P-induced ROCK activation and partially blocked CA4P-induced tumour vascular effects, in both tumour types. Y27632 also partially inhibited CA4P-induced tumour necrosis and was associated with reduced immune cell infiltration in SW1222 tumours. Hydralazine caused a similar hypotensive effect as Y27632 but had no protective effect against CA4P treatment. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that ROCK activity is critical for full manifestation of the vascular activity of CA4P in vivo, providing the evidence for pharmacological intervention to enhance the anti-tumour efficacy of CA4P and related VDAs.

An automatic algorithm for the segmentation and morphological analysis of microvessels in immunostained histological tumour sections.

A fully automatic segmentation and morphological analysis algorithm for the analysis of microvessels from CD31 immunostained histological tumour sections is presented. Development of the algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, to provide the seeds for a 'region-growing' method for object segmentation in the 3D hue, saturation, value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing objects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels. The automatic segmentation was validated against a hand-segmented set of 44 images from three different SW1222 human colorectal carcinomas xenografted into mice. 96.3 ± 0.9% of pixels were found to be correctly classified. Automated segmentation was carried out on a further 53 images from three histologically distinct mouse fibrosarcomas (MFs) for morphological comparison with the SW1222 tumours. Four morphometric measurements were calculated for each segmented vessel: vascular area (VA), ratio of lumen area to vascular area (lu/VA), eccentricity (e), and roundness (ro). In addition, the total vascular area relative to tumour tissue area (rVA) was calculated. lu/VA, e and ro were found to be significantly smaller in MF tumours than in SW1222 tumours (p < 0.05; unpaired t-test). The algorithm is available through the website http://www.caiman.org.uk where images can be uploaded, processed and sent back to users. The output from CAIMAN consists of the original image with boundaries of segmented vessels overlaid, the calculated parameters and a Matlab file, which contains the segmentation that the user can use to derive further results.

Tumour vascular disrupting agents: combating treatment resistance.

A large group of tubulin-binding microtubule-depolymerizing agents act as tumour vascular disrupting agents (VDAs). Several members of this group are now in clinical trials in combination with conventional anticancer drugs and radiotherapy. Here we briefly update on the development of tubulin-binding combretastatins as VDAs, summarize what is known of their mechanisms of action and address issues relating to treatment resistance, using disodium combretastatin A-4 3-O-phosphate (CA-4-P) as an example. Characteristically, VDAs cause a rapid shutdown of blood flow to tumour tissue with much less effect in normal tissues. However, the tumour rim is relatively resistant to treatment. Hypoxia (or hypoxia reoxygenation) induces upregulation of genes associated with angiogenesis and drug resistance. It may be possible to take advantage of treatment-induced hypoxia by combining with drugs that are activated under hypoxic conditions. In summary, VDAs provide a novel approach to cancer treatment, which should effectively complement standard treatments, if treatment resistance is addressed by judicious combination treatment strategies.

Expression of vascular endothelial growth factor receptors in smooth muscle cells.

Vascular Endothelial Growth Factor (VEGF) has been typically considered to be an endothelial-specific growth factor. However, it was recently demonstrated that VEGF can interact with non endothelial cells. In this study, we tested whether vascular smooth muscles cells (VSMCs) can express VEGF receptors, such as flk-1, flt-1, and neuropilin (NP)-1, and respond to VEGF in vitro. In cultured VSMCs, flk-1 and flt-1 expression was inversely related to cell density. The expression of flk-1 was down-regulated with increasing passage numbers. However, NP-1 levels were not affected by cell density or passage numbers. Flk-1, Flt-1, and NP-1 protein levels were confirmed by Western Blotting. Although the functional mature form of Flk-1 protein is expressed at low levels in VSMCs, phosphorylation of Flk-1 following VEGF(165) stimulation was still observed. SMCs migrated significantly in response to VEGF(165) and VEGF-E, whereas Placenta Growth Factor (PlGF) induced migration only at higher concentrations. Since VEGF-E is a specific activator of flk-1 while PlGF specifically activates only flt-1, SMC migration induced by VEGF(165) is likely to be mediated primarily through the flk-1 receptor. VSMCs did not significantly proliferate in response to VEGF(165), PlGF, and VEGF-E. In conclusion, our studies demonstrate the presence of VEGF receptors on VSMCs that are functional. These studies also indicate that in vivo, VEGF may play a role in modulating the response of VSMCs.

Limitations of the reporter green fluorescent protein under simulated tumor conditions.

This paper reports a detailed analysis of the effect of low oxygen conditions (hypoxia) on the reporter green fluorescent protein (GFP). It questions the feasibility of using GFP for gene expression studies under tumor conditions. Hypoxia is a characteristic of both experimental and clinical tumors. Several important factors are pointed out which need to be considered when using GFP as reporter gene. GFP fluorescence is the final product of a long and complex pathway involving transcription, translation, and posttranslational modifications. All of these steps may be affected by the availability of oxygen. We show specifically that cellular GFP fluorescence decreased with reduced oxygenation, anoxia virtually eliminated fluorescence and protein levels, and fluorescence recovery after anoxia required 5-10 h of reoxygenation. In conclusion, GFP appears to be a good marker gene to study location or movement of proteins or cells but should be used with great caution as a reporter of gene expression under tumor conditions.

Horseradish peroxidase-mediated gene therapy: choice of prodrugs in oxic and anoxic tumor conditions.

We have previously proposed the plant enzyme horseradish peroxidase (HRP) and the plant hormone indole-3-acetic acid (IAA) as an enzyme/prodrug combination for cancer gene therapy. In the current study, we evaluated the potential of HRP/IAA for gene-directed enzyme/prodrug therapy in three human tumor cell lines (T24 bladder carcinoma, MCF-7 breast adenocarcinoma, and FaDu nasopharyngeal squamous carcinoma) and one endothelial cell line (HMEC-1). The action of 10 IAA analogues in combination with HRP was studied in vitro in normoxic conditions as well as in the extreme tumor conditions of anoxia. Compounds characterized by prompt normoxic or anoxic cytotoxic activation and high HRP transfectant killing or selectivity were identified. Some variations were observed in the response of cells of different origin, with IAA, 1-Me-IAA, and 5-Br-IAA representing the most promising candidates for HRP gene therapy. In particular, 5-Br-IAA showed a very prompt and selective activation in anoxia. A strong bystander effect was produced by activated IAA and analogues because 70-90% cell kill was obtained when only 5% of the cells expressed the HRP enzyme. These results indicate that HRP/IAA represents an effective system for enzyme/prodrug-based anticancer approaches, and further improvements could be achieved by the use of novel IAA derivatives.

Cellular effects and signalling pathways activated by the anti-coagulant factor, protein S, in vascular cells protein S cellular effects.

The anticoagulant factor protein S is a secreted vitamin K-dependent gamma-carboxylated protein that is mainly synthesised in the liver but is also made by endothelial cells and megakaryocytes in culture. In previous studies we have shown that protein S acts as a mitogen for cultured human vascular smooth muscle cells. The synthesis and secretion of protein S by endothelial cells suggests that in addition to its role in the coagulation cascade, protein S may be an important autocrine factor implicated in the pathophysiology of the vascular system. The effects of protein S on hVSMC proliferation, migration and survival are discussed. The activation of the components of the MAP kinase pathway, ERK1/2, JNK/SAPK and p38 is also summarised. Binding and chemical cross-linking experiments provided evidence for the existence of a cell surface protein S receptor(s). By virtue of its many cellular effects, it is suggested here that the anticoagulant factor protein S plays an important role in the pathophysiology of the vasculature.

The anticoagulant factor, protein S, is produced by cultured human vascular smooth muscle cells and its expression is up-regulated by thrombin.

The anticoagulant factor protein S is a secreted vitamin K-dependent gamma-carboxylated protein that is mainly made in the liver. Protein S is homologous to the growth arrest specific protein, Gas6, the expression of which is up-regulated in cultured fibroblasts upon serum withdrawal. We report here the synthesis and secretion of protein S by cultured human vascular smooth muscle cells (HVSMCs). Western blot analysis revealed that similar amounts of protein S are secreted by both growing and growth-arrested HVSMCs. HVSMC-derived protein S was found to be gamma-carboxylated as it was precipitated by barium citrate and was shown to possess protein C cofactor activity. Treatment with the vitamin K antagonist warfarin led to the accumulation of intracellular undercarboxylated protein S forms that were rapidly secreted upon the reintroduction of vitamin K. Northern blotting analysis showed that cultured HVSMCs express a protein S transcript. The expression of protein S messenger RNA was unaffected by either warfarin, growth arrest, or various VSMC mitogens, such as platelet-derived growth factor-BB, basic fibroblast growth factor, transforming growth factor-beta, or hepatocyte growth factor. Thrombin, however, induced an up-regulation of protein S expression at both messenger RNA and protein levels. The evidence we provide for protein S secretion by cultured HVSMCs and its up-regulation by thrombin, together with earlier reports showing that protein S acts as a mitogen for these cells, suggests that, in addition to its known role in regulating blood clotting, protein S may also be an important autocrine factor in the pathophysiology of the vasculature. (Blood. 2000;95:2008-2014)

Thrombin activates transcription factors sp1, NF-kappaB, and CREB: importance of the use of phosphatase inhibitors during nuclear protein extraction for the assessment of transcription factor DNA-binding activities.

Thrombin, a serine protease, is an important effector of many cellular processes and has been shown to up-regulate the expression of several genes. The mechanisms underlying thrombin-mediated regulation of gene transcription remain poorly understood. The original aim of this work was to study the effects of thrombin on the activation of transcription factors, Sp1, NF-kappaB, and CREB by means of electrophoretic mobility-shift assays (EMSA). However, an inconsistent pattern of results was observed. We raised the possibility that some EMSA results may have been erroneous by the fact that during the nuclear protein extraction and EMSA procedure, transcription factors are dephosphorylated by cellular phosphatases and hence their DNA-binding capacities are modified. Therefore, we have altered the original nuclear extraction protocol by including a mixture of phosphatase inhibitors during protein extraction and subsequent EMSA steps. We show here that this simple measure led to significant changes in both basal and thrombin-induced levels of activation of Sp1 and CREB, but not of NF-kappaB. In light of the data presented here, it would be important to reexamine the conclusions of many reports in which EMSA was used to assess the basal and agonist-induced levels of transcription factor DNA-binding activities.

Induction of vascular SMC proliferation by urokinase indicates a novel mechanism of action in vasoproliferative disorders.

The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor-independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.

Evidence for cultured human vascular smooth muscle cell heterogeneity: isolation of clonal cells and study of their growth characteristics.

The monoclonal theory of atherosclerosis postulates that the initial vascular smooth muscle cell (VSMC) proliferative event involves the expansion of a single cell or a sub-population of cells thus implying differences in the replicative potential of VSMC. Using the technique of limited dilution, VSMC clones derived from animal tissues have been previously isolated and shown to be morphologically heterogeneous. However, the same technique applied to human VSMC (HVSMC) has been unsuccessful, possibly because HVSMC do not grow when plated at very low densities. In this report, the anchorage-independent growth factor-BB (PDGF-BB) and to lesser extent PDGF-AB and basic fibroblast growth factor (bFGF) induced colony formation. This assay provided a tool for the isolation of HVSMC clones. In terms of their growth characteristics and responsiveness to several growth factors, isolated HVSMC clones and the original parental cell population exhibited marked heterogeneity.

Involvement of pertussis toxin-sensitive and -insensitive G proteins in alpha-thrombin signalling on cultured human vascular smooth muscle cells.

Alpha-thrombin, a key enzyme of the coagulation cascade, is also a potent mitogen for human vascular smooth muscle cells (HVSMC). Here it is demonstrated that the alpha-thrombin-mediated reduction of intracellular cAMP levels is sensitive to pertussis toxin (PTX). cAMP-elevating agents inhibited alpha-thrombin- and serum-induced mitogenesis, thus cAMP confers an anti-mitogenic signal on HVSMC. The PTX-dependent ADP-ribosylation of a 41 kDa Gi alpha protein(s) was significantly inhibited (up to 55%) by thrombin. HVSMC membranes had an intrinsic GTP'ase activity which was significantly increased (up to 36%) by thrombin. PTX treatment did not alter thrombin-induced elevation of GTP'ase activity. Thrombin stimulated phosphatidyl inositol (PI) turnover in a PTX-insensitive manner. This suggested that PTX insensitive G proteins such as Gq are also activated by thrombin. This study on HVSMC provides additional evidence for the involvement of different families of G proteins in thrombin signalling.

Variability in the proliferative responsiveness of cultured human vascular smooth muscle cells to alpha-thrombin.

alpha-Thrombin, a key enzyme of the coagulation cascade, is also a potent mitogen for many cell types. In the present study, the responsiveness to alpha-thrombin of cultured human vascular smooth muscle cells (HVSMC) derived from either vein or normal and atherosclerotic arteries was investigated. All HVSMC populations examined responded mitogenically to alpha-thrombin. However, the extent of this response varied between different cell populations. No significant differences were observed between HVSMC derived from vein versus artery or atherosclerotic versus normal tissues. The responsiveness of a specific HVSMC culture to alpha-thrombin was not affected by cell density and remained constant over several passages. Unlike platelet-derived growth factor BB (PDGF-BB), alpha-thrombin did not exhibit any significant chemotactic effects on HVSMC or induce their anchorage independent growth in semi-solid medium. The hypothesis that the observed variability in HVSMC responsiveness to alpha-thrombin is due to the heterogeneity of cultured HVSMC is raised and discussed.

Prothrombin cleavage by human vascular smooth muscle cells: a potential alternative pathway to the coagulation cascade.

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R155-S156. Cleavage at R271-T272 generated fragment 1.2 and prethrombin 2 whilst cleavage at R284-T285 yielded truncated prothrombin 2 (prethrombin 2'). However, cleavage at R320-I321 which, during prothrombin activation produces two-chain alpha-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1-3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R271-S272 and by alpha-thrombin at R155-S156 and R284-T285. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30-40 kDa.

Thrombin receptor activating peptide (TRAP) stimulates mitogenesis, c-fos and PDGF-A gene expression in human vascular smooth muscle cells.

The synthetic peptide SFLLRNPNDKYEPF, identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage of thrombin, acts a thrombin receptor agonist/activating peptide (TRAP). In this study, Northern blot analysis showed that cultured human vascular smooth muscle cells (HVSMC) express a thrombin receptor transcript. TRAP, in contrast to thrombin was shown to be a weak mitogen for HVSMC. A combination of TRAP and enzymatically-inactivated thrombin (PPACK-thrombin) which provides receptor occupancy, did not potentiate TRAP-induced mitogenesis, indicating that TRAP and PPACK-thrombin do not reproduce the mitogenic effect of enzymatically-active thrombin. Both thrombin and TRAP, induced the expression of c-fos and the PDGF-A gene in a pertussis toxin (PTX)-insensitive manner. Examination of thrombin and TRAP-treated cells by immunofluorescence staining followed by computer assisted image analysis revealed that thrombin and to a lesser extent TRAP induced PDGF-AA protein expression. Antibodies to PDGF-AA partially inhibited thrombin but not TRAP-induced mitogenesis in HVSMC. This study indicates that in addition to the common signalling pathways utilised by thrombin and TRAP, enzymatically-active thrombin activates other signalling pathways and hence TRAP does not mimic fully the biological effect of thrombin on HVSMC.

Structural domains of thrombin involved in the induction of mitogenesis in cultured human vascular smooth muscle cells.

Human alpha-thrombin is a known human vascular smooth muscle cell (HVSMC) mitogen. We have previously reported that gamma-thrombin, in which the anion binding exosite is disrupted, but not catalytically inactivated PPACK-thrombin is mitogenic on HVSMC. Here, the structural requirements for thrombin's mitogenic activity on HVSMC were further investigated. Total inhibition of thrombin-induced DNA synthesis was achieved by AT-III and hirudin. AT-III, added 1 h after exposure of cells to thrombin, failed to alter thrombin-induced mitogenicity. Modification of thrombin's anion binding exosite with peptides derived from the C-terminal sequence of hirudin resulted in a partial loss of thrombin's mitogenic activity. PPACK-inactivated thrombin failed to induce a significant expression of the immediate early gene, c-fos. Unlike PPACK-inactivated thrombin, alpha-thrombin and gamma-thrombin induced a significant increase in the level of the PDGF-A gene expression. A correlation between PDGF-A gene induction and thrombin-induced mitogenicity was suggested by the fact that the mitogenic forms of thrombin also stimulated PDGF-A gene expression. Together, these results indicate that the mitogenic activity of thrombin on HVSMC requires the integrity of the enzyme's active site and is altered by modifications of its anion binding exosite.

Long-term chemotaxis studies on adherent cells: effect of platelet-derived growth factor-BB on human vascular smooth muscle cell migration.

Several chemotaxis methods have been developed which allow the study of different aspects of cell migration. The major limitation of such methods is the lack of a sustained chemotactic signal. Long-term chemotaxis phenomena which are known to take place in vivo have remained largely uninvestigated. Ways to maintain sustained chemotactic signals were sought and the used to investigate the long-term chemotactic effect of platelet-derived growth factor BB (PDGF-BB) on human vascular smooth muscle cells (HVSMC). PDGF-BB was adsorbed onto microcarrier beads and then embedded in agar. PDGF-BB diffusion was slow and a high and sustained local concentration was maintained in the agar. When PDGF-BB-loaded beads embedded in agar were placed at the edge of a tissue culture dish with HVSMC plated in the center, preferential movement was observed in the direction of the PDGF-BB source. This method was subsequently used to study directional movement of HVSMC arising from explants. This report demonstrates that PDGF-BB if present in an anisotropic concentration induces directional cell movement from such explants. By allowing the study of the effect of sustained chemotactic signals upon cultured cells or cells arising from explants, this method may provide a suitable model for investigating in vivo chemotaxis phenomena.

Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents.

The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2'-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.