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ABSTRACT: Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases.
In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein.
Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell.
This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.
Iranian Journal of Basic Medical Science 01/2011; 14(6):551-559. · 0.32 Impact Factor
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ABSTRACT: The purpose of the present study is to evaluate the effects of arsenic trioxide (ATO) on human acute promyelocytic leukemia NB-4 cells. Microculture tetrazolium test, bromodeoxyuridine (BrdU) cell proliferation assay, caspase 3 activity assay, cell-based nuclear factor kappa B (NF-kappaB) phosphorylation measurement by ELISA and real-time RT-PCR were employed to appraise the effects of ATO on metabolic activity, DNA synthesis, induction of programmed cell death and NF-kappaB activation. The suppressive effects of ATO on metabolic potential, cell proliferation and NF-kappaB activation were associated with induction of apoptosis in NB-4 cells. In addition, an expressive enhancement in mRNA levels of p73, cyclin-dependent kinase inhibitor 1A (p21), tumor protein 53-induced nuclear protein 1 (TP53INP1), WNK lysine deficient protein kinase 2 (WNK2) and lipocalin 2 coupled with a significant reduction in transcriptional levels of NF-kappaB inhibitor beta (IKK2), Nemo, BCL2-like 1 (BCL-X(L)), inhibitor of apoptosis protein 1 (cIAP2), X-linked inhibitor of apoptosis protein (XIAP), survivin, Bcl-2, TIP60, ataxia telangiectasia (ATM), SHP-2 and sirtuin (SIRT1) were observed. Altogether, these issues show for the first time that ATO treatment could trammel cell growth and proliferation as well as induces apoptosis in NB-4 cells through induction of transcriptional levels of p73, TP53INP1, WNK2, lipocalin 2 as well as suppression of NF-kappaB-mediated induction of BCL-X(L), cIAP2, XIAP and survivin. Furthermore, the inductionary effects of ATO on transcriptional stimulation of p73 might be through cramping the NF-kappaB module (through suppression of p65 phosphorylation as well as transcriptional hindering of IKK2, ATM and Nemo) along with diminishing the mRNA expression of TIP60, SHP-2 and SIRT1.
Medical Oncology 09/2009; 27(3):833-42. · 2.14 Impact Factor
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ABSTRACT: Tamoxifen is the most common antiestrogen used in the treatment of estrogen-positive breast cancer but its adverse effects and also resistance to this drug are serious challenges in the treatment of breast cancer. Characterization of mechanisms responsible for these adverse effects can lead to design of more efficient therapeutic strategies for the treatment of breast cancer. Here, we used a cellular model to evaluate the effects of autocrine expression of human growth hormone on responses of cells to tamoxifen. Our results imply for the first time that autocrine expression of growth hormone in human breast adenocarcinoma cell line, MCF-7, results in increase in cell proliferative capacity of cells even in the presence of tamoxifen. This effect may be due to up-regulation of G-coupled estrogen receptor, GPR30, which is activated by tamoxifen.
Medical Oncology 06/2009; 27(2):474-80. · 2.14 Impact Factor
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ABSTRACT: To investigate the expression of coxsackievirus and adenovirus receptor (CAR) and adenovirus-mediated reporter gene transfer in five human colon cancer cell lines.
Expression of CAR-specific mRNA and protein was analyzed by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Adenovirus-based gene delivery was evaluated by infection of cells with adenoviral vector carrying the green fluorescent protein (GFP) gene.
All the colon cancer cell lines examined (HT29, LS180, SW480, SW948 and SW1116) expressed CAR full-length mRNA and an alternatively-spliced variant that lacks the transmembrane coding exon. All cell lines were detected as CAR-positive by Western blot analysis. Further, all cells we examined were efficiently infected with adenoviral vector-GFP.
The data indicated that the five colon cancer cell lines tested expressed adenovirus primary receptor and could be efficiently infected by adenoviral vectors. Therefore, these cell lines will be useful for adenovirus-based gene transfer and research.
World Journal of Gastroenterology 01/2008; 13(47):6365-9. · 2.47 Impact Factor
