Activation Of Luciferase Expression Research Articles

New Cell Biology Assays Using a Stabilized Luciferase Exhibiting Red Luminescence

Bioluminescence is the light produced in certain organisms as a result of luciferase enzyme mediated oxidation of a luciferin substrate. This oxidation is linearly dependent on the presence of adenosine tri‐phosphate (ATP). We have recently developed a codon‐optimized luciferase based upon the gene sequence of the natural luciferase isolated from Luciola cruciata (Japanese firefly). This new gene codes for a modified amino acid sequence which exhibits improved expression levels in mammalian cells, red‐shifted emission wavelengths (560 to 619 nm) as well as improved thermal stability, while still utilizing D‐luciferin and ATP as substrates.This new recombinant enzyme was purified to 98% homogeneity from both transfected E. coli bacteria and HEK 293 mammalian cells using Ni+2‐affinity chromatography as the His6‐tagged protein. Enzymes isolated from either source were found to have equivalent activity in ATP dependent assays. The purified recombinant luciferase was used to develop a number of ultrasensitive biological assays useful in measuring cell number, cell viability as well as the cytotoxicity effects of several chemotherapeutic drugs (Etoposide and Doxorubicin) when added to murine NIH3T3 cells in culture. We also utilized the purified enzyme to sensitively detect bacterial cell number down to 1 × 102 cells and adapted the methods for use in high‐throughput antibiotic screening protocols. By cloning the luciferase downstream of TFkB responsive elements, the activation of TF‐κB (with stimulation by TNFα) in human HEK 293 cells could be quantitated by directly monitoring the increase in luminescence from activation of luciferase expression. Finally, since this new luciferase has a red‐shift in light emission, it was utilized in multiplexed assay conditions with other green (firefly) or blue‐emitting (Renilla) luciferases in a number of coupled biological assays. Moreover, application of this new luciferase for use in in vivo imaging was explored.

Targeting the PAS-A Domain of HIF-1α for Development of Small Molecule Inhibitors of HIF-1

Hypoxia inducible factor-1 (HIF-1) is a master regulator of cellular adaptation to oxygen deprivation and activates transcription of genes involved in tumor metabolism, angiogenesis, invasion and metastasis, all of which are implicated in cancer progression. Several domains of HIF-11α mediate protein-protein interaction, which is essential for the formation of the active heterodimer with HIF-11β. Targeting specific domains of HIF-1α might lead to the identification of more selective inhibitors. HIF-1α and HIF-1β contain two Per-Arnt-Sim (PAS) domains, A and B, both of which appear to be important for heterodimer formation. In an attempt to identify small molecule inhibitors of the PAS-A domain of HIF-1 we expressed proteins containing amino acids 86-165 of HIF-11α and amino acids 159-240 of HIF-1β fused to a His or FLAG tag, respectively. Expressed proteins retained functional activity as indicated by in vitro immunoprecipitation experiments and activation of luciferase expression in a mammalian two-hybrid system. Interestingly, over-expression of HIF-1α-PAS-A domain was sufficient to abrogate hypoxic induction of HIF-1-dependent luciferase expression, supporting its potential role as drug target. An ELISA based on the interaction between FLAG-HIF-1β-PAS-A and HIF-1α-PAS-A-His was developed and used to screen libraries of synthetic compounds. NSC 50352 specifically inhibited PAS-A-dependent interaction between HIF-1α and HIF-1β, but not the interaction mediated by unrelated domains. However, NSC 50352 was devoid of activity in cell-based assays. Our results provide proof-of-principle that the PAS-A domain of HIF-1α is a valid target for development of small molecule inhibitors.

MDM2 interaction with nuclear corepressor KAP1 contributes to p53 inactivation

MDM2 is a RING domain ubiquitin E3 ligase and a major regulator of the p53 tumor suppressor. MDM2 binds to p53, inactivates p53 transcription function, inhibits p53 acetylation, and promotes p53 degradation. Here, we present evidence that MDM2 interacts with the nuclear corepressor KAP1. The binding is mediated by the N-terminal coiled-coil domain of KAP1 and the central acidic domain of MDM2. KAP1 stimulates formation of p53-HDAC1 complex and inhibits p53 acetylation by interacting with MDM2. Expression of KAP1 cooperates with MDM2 to promote p53 ubiquitination and degradation. The tumor suppressor ARF competes with KAP1 in MDM2 binding; oncogene induction of ARF expression reduces MDM2-KAP1 interaction. Depletion of endogenous KAP1 expression by RNAi stimulates p53 transcriptional activity, sensitizes p53 response to DNA damage, and increases apoptosis. Therefore, MDM2 interaction with KAP1 contributes to p53 functional regulation. ARF may regulate p53 acetylation and stability in part by inhibiting KAP1-MDM2 binding.

An expression screen reveals modulators of class II histone deacetylase phosphorylation

Class II histone deacetylases (HDACs) repress transcription by associating with a variety of transcription factors and corepressors. Phosphorylation of a set of conserved serine residues in the N-terminal extensions of class II HDACs creates binding sites for 14-3-3 chaperone proteins, which trigger nuclear export of these HDACs, thereby derepressing specific target genes in a signal-dependent manner. To identify intracellular signaling pathways that control phosphorylation of HDAC5, a class II HDAC, we designed a eukaryotic cDNA expression screen in which a GAL4-dependent luciferase reporter was expressed with the DNA-binding domain of GAL4 fused to the N-terminal extension of HDAC5 and the VP16 transcription activation domain fused to 14-3-3. The transfection of COS cells with cDNA expression libraries results in activation of luciferase expression by cDNAs encoding HDAC5 kinases or modulators of such kinases that enable phosphorylated GAL4-HDAC5 to recruit 14-3-3-VP16 with consequent reconstitution of a functional transcriptional complex. Our results reveal a remarkable variety of signaling pathways that converge on the signal-responsive phosphorylation sites in HDAC5, thereby enabling HDAC5 to connect extracellular signals to the genome.

Electrically stimulated induction of hsp70 gene expression in mouse astroglia and fibroblast cells

Mouse astroglial cells, which were cultured on an electrode, were found responsive to an electric stimulation of sine wave potential in enhancing hsp70 mRNA resulting from an activation of hsp70 gene expression. On the basis of this finding, electrically responsive cells were established by transfecting mouse 3T3-L1 cells with a constructed plasmid encoding hsp70 promoter and the firefly luciferase gene. A stable cell line has been established through selection of heat-stimulated luciferase expression. A 1-h electric stimulation of the cells resulted in activation of luciferase expression, which was confirmed to produce an increase in light emission. The sequential pattern of the electrically stimulated expression of luciferase was found different from that of the heat stimulation. Furthermore, the promoter was activated depending on the potential and duration of the stimulation applied. Consequently, the electric stimulation has proven effective on activating hspP70 promoter. This cell line is feasible in expressing the gene of interest by electrical stimulation, which lead us to construct environment responsive cells in general.

The oestrogen-like effect of 4-hydroxytamoxifen on induction of transforming growth factor alpha mRNA in MDA-MB-231 breast cancer cells stably expressing the oestrogen receptor.

Oestrogens and antioestrogens modulate the synthesis of transforming growth factor alpha (TGF-alpha) in breast cancer cells. The purpose of the present report was to examine regulation of TGF-alpha gene expression by oestradiol (E2) and antioestrogens in MDA-MB-231 breast cancer cells transfected with either the wild-type or mutant oestrogen receptor (ER). We recently reported the concentration-dependent E2 stimulation of TGF-alpha mRNA in MDA-MB-231 ER transfectants (Levenson et al, 1997). We now report that 4-hydroxytamoxifen (4-OHT) shows oestrogen-like effects on the induction of TGF-alpha gene expression in our transfectants. Accumulation of TGF-alpha mRNA in response to both E2 and 4-OHT but not in response to the pure antioestrogen ICI 182,780 suggests that E2-ER and 4-OHT-ER complexes can bind to an oestrogen response element (ERE), located in the promoter region of the TGF-alpha gene and can activate transcription of the gene. Surprisingly, no activation of luciferase expression was observed after transient transfection of the TGF-alpha ERE/luciferase reporter constructs. Possible activation of an alternative ER-mediated pathway responsible for the regulation of TGF-alpha gene expression in the ER transfectants is discussed.