TGF-p receptor mutation is now considered as one of the carcinogenic process of many tumors. To evaluate whether there is an abnormality in TGF-p type II receptor in osteosarcoma cell lines, we performed Northern analysis, cross-linking assay, luciferase activity and TGF-p growth inhibition assay in four osteosarcoma cell lines: G292, U202, HOS and SaOS. We also transfected the tumor cells with normal TGF-p type II receptor sequence to find if there is a possibility of gene therapy in osteosarcoma. In Northern analysis, Type II receptor expressions were decreased at SaOS, U202 and HOS cell lines. In cross-linking assay, all four cell lines didnt show type II receptor at their cell surface. The growth of these tumor cells were not suppressed by TGF-p. From these findings, we concluded that the normal production of TGF-p type II receptor was impaired in osteosarcoma. The transfection of these tumor cells with normal type II receptor sequence restored growth inhibition by TGF-p. This means even though TGF-p type II receptor is abnormal in osteosarcoma, we can restore its function by transfection of normal sequence. We think that the TGF-p type Il receptor gene therapy can be one of the treatment method for osteosarcoma in the future.
Cell Line* ; Genetic Therapy ; Luciferases ; Osteosarcoma* ; Transfection

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection

The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.
Blood Proteins ; Cholesterol* ; Genes, Reporter ; Introns ; Luciferases ; Oligonucleotides, Antisense ; Spermine*

MicroRNAs (miRNAs) are a family of non-coding RNA that are able to adjust the expression of many proteins, including ATP-binding cassette transporter and organic cation transporter. We sought to evaluate the effect of miR-511 on the regulation of OATP1B1 expression by free fatty acids. When using free fatty acids to stimulate Chang liver cells, we found that the expression of miR-511 increased significantly while the expression of OATP1B1 decreased. We also proved that SLCO1B1 is the target gene of miR-511 with a bioinformatics analysis and using the dual luciferase reporter assay. Furthermore, the expressions of SLCO1B1 and OATP1B1 decreased if transfecting Chang liver cells with miR-511, but did not increase when transfecting the inhibitors of miR-511 into steatosis cells. Our study indicates that miR-511 may play an important role in the regulation of OATP1B1 expression by free fatty acids.
Computational Biology ; Fatty Acids, Nonesterified ; Humans ; Liver ; Luciferases ; MicroRNAs ; RNA, Untranslated

PURPOSE: In humans, nineteen types of WNT genes (WNTs) have been hightlighted up to date. The canonical Wnt cascade has recently emerged as a critical regulator of stem cells. To obtain new insights how nineteen WNTs affect mesenchymal stem cells differentiation, we analyzed the transcriptional activity, osteogenic and adipogenic activity of WNTs in mesenchymal stem cells. MATERIALS AND METHODS: Recombinant adenoviruses expressing nineteen WNTs were constructed to infect pluripotent mesenchymal progenitor C3H10T1/2 cells. Transcriptional activity was determined by using the luciferase reporter assay. Osteogenic activity was determined by measuring the induction of alkaline phosphatase upon Wnt stimulation. Adipogenic activity was measured by histochemical Oil red-O staining. RESULTS: WNT1, 2, 3, 3A and 10B significantly induced transcriptional activity in C3H10T1/2 cells. WNT1, 2, 3, 3A and 10B significantly induced alkaline phosphatase activity, but inhibited adipogenic activity in C3H10T1/2 cells. The results of qualitative and quantitative assay of alkaline phosphatase activity were consistent with those of luciferase assay for transcriptional activity and Oil red-O staining for adipogenic activity. CONCLUSION: We could expect that WNT1, 2, 3, 3A and 10B may play a crucial role in inducing osteoblast differentiation of mesenchymal stem cells.
Adenoviridae ; Alkaline Phosphatase ; Cell Line* ; Humans ; Luciferases ; Mesenchymal Stromal Cells ; Osteoblasts ; Stem Cells

BACKGROUND: In mammals, the CLOCK/BMAL1 heterodimer is a key transcription factor complex that drives the cyclic expression of clock-controlled genes involved in various physiological functions and behavioral consequences. Recently, a growing number of studies have reported a molecular link between the circadian clock and metabolism. In the present study, we explored the regulatory effects of SIRTUIN1 (SIRT1), an NAD+-dependent deacetylase, on CLOCK/BMAL1-mediated clock gene expression. METHODS: To investigate the interaction between SIRT1 and CLOCK/BMAL1, we conducted bimolecular fluorescence complementation (BiFC) analyses supplemented with immunocytochemistry assays. BiFC experiments employing deletion-specific mutants of BMAL1 were used to elucidate the specific domains that are necessary for the SIRT1-BMAL1 interaction. Additionally, luciferase reporter assays were used to delineate the effects of SIRT1 on circadian gene expression. RESULTS: BiFC analysis revealed that SIRT1 interacted with both CLOCK and BMAL1 in most cell nuclei. As revealed by BiFC assays using various BMAL1 deletion mutants, the PAS-B domain of BMAL1 was essential for interaction with SIRT1. Activation of SIRT1 with resveratrol did not exert any significant change on the interaction with the CLOCK/BMAL1 complex. However, promoter analysis using Per1-Luc and Ebox-Luc reporters showed that SIRT1 significantly downregulated both promoter activities. This inhibitory effect was intensified by treatment with resveratrol, indicating a role for SIRT1 and its activator in CLOCK/BMAL1-mediated transcription of clock genes. CONCLUSION: These results suggest that SIRT1 may form a regulatory complex with CLOCK/BMAL1 that represses clock gene expression, probably via deacetylase activity.
Cell Nucleus ; Circadian Clocks ; Complement System Proteins ; Fluorescence ; Gene Expression ; Immunohistochemistry ; Luciferases ; Mammals ; Metabolism ; Transcription Factors

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the value of microbubble destruction using low-frequency ultrasound for enhancing gene delivery to skeletal muscles of laboratory animals. MATERIALS AND METHODS: Lac-Z gene was injected into 21 mouse anterior tibialis muscles. Seven muscles received the gene only, and seven each received either 20-kHz ultrasound exposure or ultrasound-PESDA (perfluorocarbon-exposed sonicated albumin) destruction, respectively, following the injection; the extent of Lac-Z expression was then compared. Luciferase gene was injected into the muscles (N=80). The muscles were divided into two groups according to the mixture; in the first group saline was used as the mixture solute, with PESDA used in the second group. The groups were subdivided into two groups, one receiv 10 seconds of ultrasound at the injection site after injection, and the other that received no further intervention. Luciferase activities were measured and compared. RESULTS: The proportions of Lac-Z stained cells were 0, 5.7+/-1.2 and 7.7+/-1.7%, respectively, showing a significant stepwise increase microbubble destruction (p<0.05). Luciferase activities were as follows: Luciferase only (Group 1, N=17), 5727+/-2178 RLU/mg; luciferase plus PESDA (Group 2, N=17), 1170+/-470.7 RLU/mg; luciferase plus ultrasound (Group 3, N=17), 16480+/-5239 RLU/mg; and luciferase plus PESDA destruction (Group 4, N=17), 49910+/-16500 RLU/mg. The activity in group 4 was significantly higher than in group 1 (p<0.01), showing an 8.7-fold increase in gene delivery due to microbubble destruction. CONCLUSION: Microbubble destruction using low-frequency ultrasound is an efficient method for increasing the efficacy of direct gene delivery to skeletal muscles.
Animals ; Animals, Laboratory ; Genetic Therapy ; Luciferases ; Mice* ; Microbubbles* ; Muscle, Skeletal* ; Muscles ; Ultrasonography*

BACKGROUND: The human mic2 gene is a pseudoautosomal gene that encodes a cell surface antigen, CD99. High levels of CD99 constitute a tumor marker in Ewing s sarcoma (ES). We have recently demonstrated that CD99-induced apoptosis occurs only in undifferentiated ES cells, not in differentiated ES cells, raising the possibility of the involvement of CD99 in neural ontogeny. METHODS: To elucidate the relations between the expression of CD99 and the differentiation of neural cells and the mechanism by which the expression of CD99 is regulated, we analyzed the differential patterns of CD99 expression in SH-SY5Y by treatment of 12-O-tetradecanoyl- 13-phorbol acetate (TPA) and retinoic acid. In addition, to explore the transcriptional activity of CD 99 during neural cell differentiation, SH-SY5Y cells were transiently transfected with a CD99 promoter-driven luciferase construct, and treated with the inducers. RESULTS: In immunoblotting and flow cytometry, the expression level of CD99 was increased on differentiated SH-SY5Y cells induced by TPA and retinoic acid. The luciferase activity was elevated by the treatment with TPA, known to mature SH-SY5Y cells toward a sympathetic neuronal lineage, whereas retinoic acid inducing a sympathetic chromaffin lineage displayed little effect. CONCLUSION: The result indicates that CD99 might be expressed only on cells maturing toward a neuronal lineage among differentiating primitive neuronal cells. In addition, the expression of CD99 seems to be regulated at the transcriptional level during the differentiation.
Antigens, Surface ; Apoptosis ; Cell Differentiation ; Cell Line* ; Flow Cytometry ; Humans ; Immunoblotting ; Luciferases ; Neuroblastoma* ; Neurons ; Sarcoma ; Tretinoin

Osteocytes may function as mechanotransducers by regulating local osteoclastogenesis. Reduced availability of oxygen, i.e. hypoxia, could occur during disuse, bone development, and fracture. Receptor activator of nuclear factor-kappaB ligand (RANKL) is an osteoblast/stromal cell derived essential factor for osteoclastogenesis. The hypoxia induced osteoclastogenesis via increased RANKL expression in osteoblasts was demonstrated. Hypoxic regulation of gene expression generally involves activation of the hypoxia-inducible factor (HIF) transcription pathway. In the present study, we investigated whether hypoxia regulates RANKL expression in murine osteocytes and HIF-1alpha mediates hypoxia-induced RANKL expression by transactivating RANKL promoter, to elucidate the role of osteocyte in osteoclastogenesis in the context of hypoxic condition. The expression levels of RANKL mRNA and protein, as well as hypoxia inducible factor-1alpha (HIF-1alpha) protein, were significantly increased in hypoxic condition in MLO-Y4s. Constitutively active HIF-1alpha alone significantly increased the levels of RANKL expression in MLO-Y4s under normoxic conditions, whereas dominant negative HIF-1alpha blocked hypoxia-induced RANKL expression. To further explore to find if HIF-1alpha directly regulates RANKL transcription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity, whereas mutations of putative HIF-1alpha binding elements in RANKL promoter prevented this hypoxia-induced RANKL promoter activity in MLO-Y4s. These results suggest that HIF-1alpha mediates hypoxia-induced up-regulation of RANKL expression, and that in osteocytes of mechanically unloaded bone, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in osteocytes.
Anoxia* ; Bone Development ; Gene Expression Regulation ; Luciferases ; Osteoblasts ; Osteocytes* ; Oxygen ; RANK Ligand* ; RNA, Messenger ; Up-Regulation

BACKGROUND: The histologic characteristics of atopic dermatitis (AD) include perivascular edema and dilated tortuous vessels in the papillary dermis. A single nucleotide polymorphism (SNP) of the fms-related tyrosine kinase 4 (FLT4) gene is associated with AD. OBJECTIVE: To investigate the associations between podoplanin (PDPN) gene SNPs and AD. METHODS: We genotyped 9 SNPs from 5 genes of 1,119 subjects (646 AD patients and 473 controls). We determined the promoter activity of 1 SNP (rs355022) by luciferase assay; this SNP was further investigated using 1,133 independent samples (441 AD patients and 692 controls). RESULTS: The rs355022 and rs425187 SNPs and the C-A haplotype in the PDPN gene were significantly associated with intrinsic AD in the initial experiment. The rs355022 SNP significantly affected promoter activity in the luciferase assay. However, these results were not replicated in the replication study. CONCLUSION: Two SNPs and the C-A haplotype in the PDPN gene are significantly associated with intrinsic AD; although, the results were confirmed by luciferase assay, they could not be replicated with independent samples. Nevertheless, further replication experiments should be performed in future studies.
Dermatitis, Atopic* ; Dermis ; Edema ; Haplotypes ; Humans ; Luciferases ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Protein-Tyrosine Kinases