It is difficult to introduce DNA in non-invasive manner into oral cancer cells as well as primary cells for gene manipulation and expression in vivo. So far, several methods for a gene delivery have been performed to solve this problem. Magnetofection is one of the recent methods for gene transfer, and nanoparticles are applied under a magnetic field for DNA delivery. We investigated whether the magnetofection increases the efficiency of a gene delivery into several oral cell lines. By using a plasmid coding the green fluorescent protein (GFP), the efficiency of gene transfer by magnetofection was compared with those by using the calcium phosphate and the commercial transfection agent. Indeed, the magnetofection increased the green fluorescent signal in cells, suggested that this method apparently enhance the efficiency of gene delivery without any defects in various oral cancer cell lines. Finally, we have shown that magnetofection can be a useful technique for gene delivery to difficult-to-transfect cells to perform a functional study of genes in vivo.

Cellular imrnortali zation is thought to be an ear ly event during tumorigenesis. Telomerase reacLi vaLion by ecLopic hTERT expression is widely used for cellular imrnortali zation. This study was a imed Lo a na lyze establi sh immortalized human ora l epithelial cells(IOEC) and to reconstruct oral precancerous lesion by Lhree dimens iona l cultures. Telomerase activity was analyzed by Telomerase assays and Telomere longLh W:lS dcLccLcd by Termina l restri ction I"ragment analysis. bTERT gene was assayecl by tbe RT-PCR. p16lNK4 a‘ pHb. CDK2. P21CIP1. p27 and p53 were examined by western blotting. Three dimensiona l cu1ture using air - liquicl inLe rl"ace was pe rl"ormed. As results. IOEC was establi shed by ectopic ex pression of catalytic subunit• of telomerase‘ h1'EH1'. which is con tinuously maintained for more tban 120 population doublings(PDs) . IOEC showecl the expression 01" h1'ER1' and h1'H mHNA‘ elongated telomere length and higher telomerase activity. These cel ls showed no ex pression of p16lNK4a with retention 01" pRb and CDK2. Expression of p21CIP1. p27 a nd p53 may have no relation to immorta li ze oraJ epithelial cell s. Three dimensional culture of IOEC showed dysplastic strat ilïed epithelia l cell s. These results may serve as a useful moclel system for the study of oral carcinogenesis.

The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thaliana VrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.

The outer dense fiber 2 (ODF2) protein is an important component of sperm tail outer dense fiber and localizes at the centrosome. It has been reported that the RO072 ES cell derived homozygote knock out of ODF2 results in an embryonic lethal phenotype, and XL169 ES cell derived heterozygote knock out causes severe defects in sperm tail development. The ODF2s splicing variant, Cenexin1, possesses a C-terminal extension, and the phosphorylation of serine 796 residue in an extended C-terminal is responsible for Plk1 binding. Cenexin1 assembles ninein and causes ciliogenesis in early stages of the cell cycle in a Plk1-independent manner. Alternatively, in the late stages of the cell cycle, G2/M phase, Cenexin1 binds to Plk1 and results in proper mitotic progression. In this study, to identify the in vivo function of Plk1 binding to phosphorylated Cenexin1 S796 residue, and to understand the in vivo functional differences between ODF2 and Cenexin1, we generated ODF2/Cenexin1 S796A/Cenexin1 WT expressing transgenic mice in a RO072 ES cell derived knock out background. We observed a severe defect of sperm tail development by ectopic expression of Cenexin1 S796A mutant and no phenotypic differences between the ectopic expression of ODF2/Cenexin1 WT in background and in normal wild type mice.

A low temperature-inducible cDNA designated as VrUBC1 from mungbean (Vigna radiata) was isolated by subtractive hybridization method. By rapid amplification of cDNA end technique, the full-length cDNA of VrUBC1 was obtained. The full-length cDNA of VrUBC1 contains an open reading frame of 444 nucleotides in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.72. VrUBC1 mRNA was induced by NaCl and ABA, but not by wounding and low temperature stress. It was shown that VrUBC1-GFP was localized to the cytoplasm in tobacco cell. To examine the function of VrUBC1, VrUBC1 was expressed in Escherichia coli as His-fusion protein. Purified VrUBC1-His recombinant protein was shown to have ubiquitination activity in vitro. For the in vivo functional analysis of VrUBC1, VrUBC1 was expressed in yeast ubc4/5 double mutant. Stress tolerance was tested in the VrUBC1 overexpressing Arabidopsis transgenic plants. We propose that VrUBC1 play an important role in protein degradation processes during abiotic stress in plants.