STUDY OF ENHANCED IMMUNOGENECITY OF B7-1 GENE TRANSFECTED HUMAN HELA CELL LINE

This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

Withania somnifera Root Extract Enhances Telomerase Activity in the Human HeLa Cell Line

Aging is a decelerating unidirectional process of life. Shortening of telomeric DNA, the (TTAGGG)n hexanucleotide repeats, which form the caps at the chromosome ends, is implicated to determine the aging process, and more importantly the healthy lifespan itself. Telomerase, a ribonucleoprotein having reverse transcriptase activity, arrests telomere loss through addition of the TTAGGG repeats de novo, to the ends of the chromosome. The telomere/telomerase maintenance is an inevitable necessity to delay aging and for a healthy lifespan. Here, we report the potential of full-spectrum, high concentration Ashwagandha (Withania somnifera), an Ayurvedic medicinal herb, root extract to increase telomerase activity. HeLa cells, when treated with various concentrations of Ashwagandha root extract, showed an increase in telomerase activity measured with the established Telomerase Rapid Amplification Protocol (TRAP) assay. Ashwagandha root extract increased telomerase activity with highest enhancement of ~45% at 10 - 50 μg concentration. Thus, Ashwagandha root extract has the anti-aging inducing potential.

In Vitro Biological Activity of Anti-C Ⅱ TA Hammerhead Ribozyme——A Novel Approach for Autoimmune Diseases

This study investigated the feasibility of using an hammerhead ribozyme against C Ⅱ TA, a major regulator of MHC Ⅱ antigens, to repress the expression of MHC Ⅱ molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463-465 GUC triplet of C Ⅱ TA and its target gene were transcribed, then mixed up and incubated in vitro . The cleavage products were analyzed by PAGE and silver staining. Rz464 was then inserted into the pIRES2 EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class Ⅱ MHC induction by recombinant human interferon gamma (IFN γ). mRNA of C Ⅱ TA was measured by RT PCR. Our results showed that Rz464 could exclusively cleave C Ⅱ TA RNA. When induced with IFN γ, the expression of HLA DR, DP, DQ on pRz464 + Hela was induced, and the mRNA content of C Ⅱ TA decreased too. It is concluded that Rz464 could inhibit C Ⅱ TA and thus the family of genes was regulated by C Ⅱ TA:MHC Ⅱ molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto immune diseases.

A DNA-based nanocarrier for efficient cancer therapy

The study aimed to achieve enhanced targeted cytotoxicity and cell-internalization of cisplatin-loaded deoxyribonucleic acid-nanothread(CPT-DNA-NT),mediated by scavenger receptors into HeLa cells.DNA-NT was developed with stiff-topology utilizing circular-scaffold to encapsulate CPT.Atomic force microscopy(AFM)characterization of the DNA-NT showed uniformity in the structure with a diameter of 50-150 nm and length of 300-600 nm.The successful fabrication of the DNA-NT was confirmed through native-polyacrylamide gel electrophoresis analysis,as large the molecular-weight(polymeric)DNA-NT did not split into constituting strands under applied current and voltage.The results of cell viability confirmed that blank DNA-NT had the least cytotoxicity at the highest concentration(512 nM)with a viability of 92%as evidence of its biocompatibility for drug delivery.MTT assay showed superior cytotoxicity of CPT-DNA-NT than that of the free CPT due to the depot release of CPT after DNA-NT internalization.The DNA-NT exhibited targeted cell internalizations with the controlled intracellular release of CPT(from DNA-NT),as illustrated in confocal images.Therefore,in vitro cytotoxicity assessment through flow cytometry showed enhanced apoptosis(72.7%)with CPT-DNA-NT(compared to free CPT;64.4%).CPT-DNA-NT,being poly-anionic,showed enhanced endocytosis via scavenger receptors.

Novel synthesis and characterization of CuO nanomaterials:Biological applications

CuO nanoparticles were synthesized at a relatively low temperature（80 ℃） for 2 h using polyethylene glycol-glycerol mixture which acts as a capping agent.A detailed characterization of the synthesized nanomaterials were performed utilizing X-ray diffraction（XRD）,infra-red spectroscopy（IR）,thermogravimetric analysis（TGA-DTA）,transmission electron microscopy（TEM）,photoluminescence（PL） by studying its crystalline phase,vibrational mode,thermal analysis,morphology and photoluminescence properties.The effect of annealing on the as-prepared nanoparticles were studied and compared with their corresponding bulk counterpart.The synthesized nanoparticles have been screened for in vitro cytotoxicity（1C_（50）） studies against the human cervical adenocarcinoma cell line（HeLa） using WITT assay methods.The as-prepared nanoparticle inhibits the proliferation of this HeLa cell.The standard disc diffusion method has been used to study the antibacterial activity of the samples against the human pathogenic bacteria Escherichia coli（MTCC 729）,Proteus mirabilis（MTCC 425） and Klebsiella pneumoniae subsp.pneumoniae（MTCC 432）.The results have been compared with the positive control antibiotic gentamycin.The synthesized nanoparticles would provide a potential alternative to antibiotics for controlling some of the microorganisms causing urolithiasis.

Microcalorimetric Study of Energy Metabolism of HSV-2 and FMDV Infection Processes

The metabolic thermogenic power data of the HSV-2 infected HeLa cells and the FMDV infected BHK-21 cells were determined by LKB-2277 bioactivity monitor. The aim of the study was to investigate the difference of the cell metabolism under the action of two different viruses and the effects of hyperthermia and drugs on it. The results illustrated that the metabolic thermogenic power of infected cells was larger than the uninfected ones and there was a significant difference between the metabolism heat released by the two types of infected cells. From the maximal thermal power and total metabolism heat, the infection process was observed to be thermosensitive and could be inhibited by interferon. Our experiments also revealed that 6 month storage of FMDV could attenuate its virulence and infectivity. The study shows that microcalorimetry is a potent tool to investigate the metabolism of virus infection process.

Comparative antitumor and anti-proliferative activities of Hippophae rhamnoides L. leaves extracts

Objective:To evaluate the antitumor and anti-proliferative activities of methanol,aqueous,acetone,ethyl acetate,ethanol,chloroform and n-hexane extracts of Hippophae rhamnoides leaves.Methods:Antitumor activities were evaluated by using the antitumor potato disc assay by using inoculums(Agrobacterium tumefaciens)with three different concentrations of test samples(10,100 and 1000 mg/L).Anti-proliferative activity was evaluated by the given method of methyl thiazolyl tetrazolium assay.The concentrations of the extract ranging from 0.039 to 10 mg/mL were tested against HeLa cells.Results:Highest tumors inhibition activity(60.9%and 55.8%)was shown by methanol and ethanol extracts,with EC_(50) values of 424.41 and 434.61 mg/L respectively.At 10 mg/mL,The highest cell inhibition 75.61%was observed in methanol extract and the lowest 36.59%were calculated in n-hexane extract.The difference in tumor and cell inhibition(%)may be due to the different concentration of active compounds responsible for antitumor and anti-proliferative activities.All extracts have considerable level of tumor and cell inhibitiory effect in a dose dependent manner.Conclusions:Our finding showed that Hippophae rhamnoides leaves are a potent natural source of antitumor and antiproliferative agent.