Imaging of Viral Thymidine Kinase Gene Expression by Replicating Oncolytic Adenovirus and Prediction of Therapeutic Efficacy

PURPOSE: We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system. MATERIALS AND METHODS: Replication-competent recombinant adenoviral vector (Ad-DeltaE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-DeltaE1A) was generated as a control. Both Ad-DeltaE1B19/55-TK and Ad-DeltaE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment. RESULTS: On gamma counter-analyses, counts/ minute (cpm)/microg of protein showed MOIs dependency with DeltaE1B19/55-TK infection. On MTT assay, Ad-DeltaE1B19/55-TK led to more efficient cell killing than Ad-DeltaE1A-TK. On plate imaging by gamma camera, both Ad-DeltaE1B19/55-TK and Ad-DeltaE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of DeltaE1B19/55-TK infection was remarkably reduced compared to that of Ad-DeltaE1A-TK infection. CONCLUSION: Replicating Ad-DeltaE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-DeltaE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-DeltaE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.ope

Pyruvate Dehydrogenase Kinase 4 Promotes Vascular Calcification via SMAD1/5/8 Phosphorylation

Vascular calcification, a pathologic response to defective calcium and phosphate homeostasis, is strongly associated with cardiovascular mortality and morbidity. In this study, we have observed that pyruvate dehydrogenase kinase 4 (PDK4) is upregulated and pyruvate dehydrogenase complex phosphorylation is increased in calcifying vascular smooth muscle cells (VSMCs) and in calcified vessels of patients with atherosclerosis, suggesting that PDK4 plays an important role in vascular calcification. Both genetic and pharmacological inhibition of PDK4 ameliorated the calcification in phosphate-treated VSMCs and aortic rings and in vitamin D3-treated mice. PDK4 augmented the osteogenic differentiation of VSMCs by phosphorylating SMAD1/5/8 via direct interaction, which enhances BMP2 signaling. Furthermore, increased expression of PDK4 in phosphate-treated VSMCs induced mitochondrial dysfunction followed by apoptosis. Taken together, our results show that upregulation of PDK4 promotes vascular calcification by increasing osteogenic markers with no adverse effect on bone formation, demonstrating that PDK4 is a therapeutic target for vascular calcification

Proteomic analysis of pregnancy-related proteins from pig uterus endometrium during pregnancy

Many important molecular events associated with implantation and development occur within the female reproductive tract, especially within the uterus endometrium, during pregnancy periods. The endometrium includes the mucosal lining of the uterus, which provides a suitable site for implantation and development of a fertilized egg and fetus. To date, the molecular cascades in the uterus endometrium during pregnancy periods in pigs have not been elucidated fully. In this study, we compared the functional regulated proteins in the endometrium during pregnancy periods with those in non-pregnant conditions and investigated changes in expression patterns during pregnancy (days 40, 70, and 93) using two-dimensional gel electrophoresis (2-DE) and western blotting. The functional regulated proteins were identified and discovered from differentially expressed proteins in the uterus endometrium during pregnancy. We discovered 820 protein spots in a proteomic analysis of uterus endometrium tissues with 2-DE gels. We identified 63 of the 98 proteins regulated differentially among non-pregnant and pregnant tissues (matched and unmatched spots). Interestingly, 10 of these 63 proteins are development-, cytoskeleton- and chaperon-related proteins such as transferrin, protein DJ-1, transgelin, galectin-1, septin 2, stathmin 1, cofilin 1, fascin 1, heat shock protein (HSP) 90β and HSP 27. The specific expression patterns of these proteins in the endometrium during pregnancy were confirmed by western blotting. Our results suggest that the expressions of these genes involved in endometrium function and endometrium development from early to late gestation are associated with the regulation of endometrium development for maintaining pregnancy

Expression of Neutrophil Gelatinase-Associated Lipocalin in Calcium-Induced Keratinocyte Differentiation

In a previous search for the differentially expressed genes in keratinocyte differentiation, we identified neutrophil gelatinase-associated lipocalin (NGAL) as a calcium-induced gene. In this study, we further verified the expression of NGAL in cultured keratinocytes as well as in several skin diseases. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and ELISA clearly showed that NGAL expression was markedly increased in calcium-induced keratinocyte differentiation in vitro. However, in our previous report, NGAL expression was not detected in normal skin tissue except for hair follicle by in situ hybridization and immunohistochemistry, indicating the difference of cell status between in vitro and in vitro conditions. Interestingly, NGAL expression was highly increased in psoriasis-like inflammatory disorders (lichen planus and pityriasis rubura pilaris) and skin cancers (keratoacanthoma and squamous cell carcinoma), implying that NGAL may be related with the epidermal hyperplasia. Collectively, these results reveal the potential importance of NGAL in the maintenance of skin homeostasis