PFT- has been successfully used in vitro and in vivo to protect normal cells from otherwise lethal doses of chemo and radiotherapy [3,4,10]

PFT- has been successfully used in vitro and in vivo to protect normal cells from otherwise lethal doses of chemo and radiotherapy [3,4,10]. reporter gene assays. In contrast, no inhibition of reporter plasmids comprising Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin- was observed both in vivo and in vitro. Pifithrin- did not inhibit firefly luciferase protein manifestation, but rather suppressed light production/emission, since addition of exogenous pifithrin- to active components inhibited this activity. Furthermore, pifithrin- also inhibited recombinant firefly luciferase protein activity. Conclusions Among its additional biological activities, pifithrin- IL4 is an inhibitor of firefly luciferase activity. Extreme caution must consequently be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene manifestation using transiently transfected reporter plasmids. Furthermore, these results demonstrate Coptisine Sulfate that when using novel compounds, the choice of vectors used in the experimental methods might be of great importance for the correct conclusions to be made. Background The tumour suppressor protein, p53 is one of the most intensively analyzed proteins throughout biomedical study. Due to its central part in genome monitoring, cell cycle arrest Coptisine Sulfate and apoptosis induction, compounds influencing this protein, either re-activating it or inactivating it, are of outstanding interest and use in the field of malignancy, Alzheimer’s disease, Parkinson’s disease, stroke and mind stress [1-3]. In recent years, a chemical inhibitor of p53, Pifithrin-(PFT-), has been recognized and used both in vitro and in vivo to investigate p53 function [4]. PFT- reversibly inhibits p53-transcriptional activity, inhibiting p53-induced apoptosis, cell cycle arrest and DNA-synthesis block [4-9]. PFT- has been successfully used in vitro and in vivo to protect normal cells from normally lethal doses of chemo and radiotherapy [3,4,10]. PFT- therefore provides a useful tool for the recognition of genes under the control of p53 [10]. Despite the fascinating data of these reports, little or nothing is known about the mechanism of action of PFT-, although it is thought to disrupt the nuclear transport of p53 [10]. Recently, the group Coptisine Sulfate that originally found out PFT-, reported that this compound also inhibits the heat shock and glucocorticoid pathways, suggesting that it focuses on a popular protein required for the activity of multiple transcription factors [11]. Reporter gene assays are regularly used to study the control of transcription. This involves the coupling of reporter enzymes such as firefly or Renilla luciferase and Chloramphenicol acetyltransferase to the gene promoter region of interest. Generally, the activity of these enzymes is definitely unaffected by the treatment conditions and this is not regarded as when interpreting the data from these assays. However, it is known that enzymes such as luciferase and -Galactosidase are affected by certain stress conditions such as warmth shock and oxidative stress [12,13]. The fact that these enzymes can be affected by such conditions can give rise to misinterpreted data and compromise the conclusions from these assays. With this report, we have investigated the effect of PFT- on different reporter genes. We Coptisine Sulfate find that PFT- is definitely a specific inhibitor of firefly luciferase. These results indicate that when carrying out practical experiments with this important compound, an appropriate choice of vector should be utilised. These observations also give possible insight into the mechanism of action of PFT- in vivo. Results Effects of PFT- on p53-dependent and self-employed luciferase reporter plasmids To determine the effects of PFT- on p53-dependent and -self-employed transcriptional activity U-2 OS human being osteosarcoma cells, which contain crazy type p53, were transiently transfected with a variety of firefly luciferase reporters. The p53-responsive reporters used were PG13 and p21-luciferase and the unrelated reporters were 3x B and HIV-LTR-luciferase, which are both regulated from the NF-B family of transcription factors. Previously, we have demonstrated the PG13 and 3x B reporters are specifically controlled by p53 and NF-B, actually in unstimulated U-2 OS cells where there is a basal level activity of both transcription factors [14,15]. The p21 and HIV-LTR luciferase reporters are not solely regulated by p53 and NF-B, however, and so effects could result from additional DNA-binding proteins. PFT- was added to the cells at a final concentration of 20 M and cells were harvested 24 hours later. As expected, PFT- strongly downregulated p53-responsive reporters (Number ?(Figure1A)1A) but surprisingly inhibition of the NF-B regulated luciferase reporters was also observed (Figure ?(Figure1B).1B). Further experimentation exposed a dose-dependent inhibition of both p21-luciferase and 3x B luciferase by PFT- (Fig. ?(Fig.1C).1C). Since cross-talk between these transcription factors has been previously observed [16-19], we decided to investigate if this was an effect seen due to PFT-.