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*

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

PURPOSE: To investigate the role of STAT5 during osteogenesis differentiation of human mesenchymal stem cells(hMSC). MATERIALS AND METHODS: To assess the expression pattern of STATs during osteogenic differentiation, we performed western blot analysis and RT-PCR. By RNA interference, we have performed effect of STAT5A and STAT5B in osteogenesis. As a result of Luciferase assay, the promoter activity of DLX5 was decreased by STAT5A. RESULTS: To assess the expression pattern of STATs during osteogenic differentiation, we have performed western blot and RT-PCR after 14 day differentiation period. STAT1, 2, 3 and 4 showed no change in expression level during differentiation, while STAT5A and STAT5B displayed steady increase compared to the control. When STAT5A was knock down, the level of osteogenesis was increased. The transcriptional activity of DLX5 was decreased about 70% by STAT5A. CONCLUSION: The expression of STAT5A and STAT5B increased during osteogenesis of hMSC. Also, we shown that STAT5A regulated the transcriptional activity of DLX5. These results indicate that STAT5A acts as a pivotal transcription factor in osteogenesis of hMSC.
Blotting, Western ; Humans ; Luciferases ; Mesenchymal Stromal Cells* ; Osteogenesis* ; RNA Interference ; Transcription Factors

BACKGROUND: S100A8 is differentially expressed in various cell types and is associated with a number of malignant disorders. S100A8 may affect tumor biology. However, its role in cutaneous squamous cell carcinoma (SCC) is not well established. OBJECTIVE: This study aims to investigate the relationship between S100A8 and cutaneous SCC development. METHODS: We performed immunohistochemical staining to detect S100A8 expression in facial skin specimens of premalignant actinic keratosis (AK), malignant SCC, and normal tissues. In addition, we utilized postconfluence and high calcium-induced differentiation in a culture system model. Furthermore, we constructed a recombinant adenovirus expressing GFP-tagged S100A8 to investigate the role of S100A8 in SCC cell differentiation. RESULTS: S100A8 was significantly overexpressed in human cutaneous SCC compared to that in normal and AK tissues. S100A8 was gradually upregulated in SCC cells in a post-confluence-induced differentiation model. Overexpression of S100A8 in SCC cells induced by adenoviral transduction led to increased expression levels of differentiation markers, such as loricrin, involucrin, and filaggrin. S100A8 overexpression also increased loricrin and involucrin luciferase activity. CONCLUSION: S100A8 regulates cutaneous SCC differentiation and induces well-differentiated SCC formation in skin.
Adenoviridae ; Antigens, Differentiation ; Biology ; Carcinoma, Squamous Cell* ; Cell Differentiation ; Humans ; Keratosis, Actinic ; Luciferases ; Skin

Genes, Reporter ; Genetic Vectors ; Luciferases ; genetics ; Transcription, Genetic ; bcl-2-Associated X Protein ; genetics

This study was aimed to establish a high-throughput luciferase reporter system, through which to screen and identify miRNAs directly targeting p21, and to explore the biological function and significance of these miRNAs. Molecular cloning technique was used to construct and identify two lentivirus-expressing vectors-pWPXL-Luc and pWPXL-Luc-P21-3'UTR, virus particles were collected after the pWPXL-Luc or pWPXL-Luc-P21-3'UTR vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pDM2G into HEK-293T cells. Furthermore, two stable cell lines expressing luciferase singly or co-expressed luciferase and P21-3'UTR were established by transducing HEK-293 cells with recombinant lentivirus; the former was used as control in the following experiments. Finally luciferase activity of the latter stable cells was measured by using the luciferase reporter assay system. The results showed that high-titre recombinant lentivirus was produced and two stable cell lines were constructed, also to some certain, the luciferase activity was in direct proportion to the number of cells. In conclusion, the high-throughput luciferase reporter system is established successfully; using this system, the 28 miRNA that directly target P21 Cip1/Waf1 are screened experimentally.
Genes, Reporter ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Luciferases ; genetics ; MicroRNAs ; genetics ; Plasmids ; Transduction, Genetic

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: 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