Protein transduction with bacterial cytosine deaminase fused to the TLM intercellular transport motif induces profound chemosensitivity to 5-fluorocytosine in human hepatoma cells

Hillemann, A.; Brandenburg, Boerries; Schmidt, Ulrike; Roos, Martin; Smirnow, Irina; Lemken, Marie-Luise; Lauer, Ulrich M.; Hildt, Eberhard

doi:10.1016/j.jhep.2005.02.031

Cited by 7 publications

(12 citation statements)

References 26 publications

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“…5,13 Moreover, the cargo is transported directly into the cytoplasm: the endosomal compartment is not involved. 5 To investigate whether this holds true as well for the TLM-mediated transfer of nucleocapsids, the endocytotic machinery was blocked by ammonium chloride.…”

Section: Resultsmentioning

confidence: 99%

“…[1][2][3][4][5][6][7][8][9] PTDs have been used to successfully treat preclinical models of human disease such as cancer, psoriasis, and stroke. [10][11][12][13] Transfer of nano-particular structures across cellular membranes is of increasing importance for the development of novel diagnostic and therapeutic tools. The capacity of PTDs to mediate an endocytosis-independent transfer of particles across the plasma membrane into the cytoplasm is still unclear.…”

mentioning

confidence: 99%

“…5 The TLM mediates receptor-and energy-independent transfer of peptides, proteins, and nucleic acids across membranes, when fused to them, without affecting cellular integrity or interfering with intracellular signal transduction cascades. 5,13,23,24 We describe the potential of the TLM to mediate the transport of fully assembled particles into the cytoplasm of target cells in a receptor-and endocytosis-independent manner. Based on this system, we established cell-permeable VLPs harboring marker genes enabling an efficient gene transfer into primary hepatocytes.…”

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confidence: 99%

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A novel system for efficient gene transfer into primary human hepatocytes via cell‐permeable hepatitis B virus–like particle†

et al. 2005

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R ecently, a variety of protein transduction domains (PTDs) have been identified allowing the transfer of peptides, proteins, or nucleic acids across cellular membranes into cells. 1-9 PTDs have been used to successfully treat preclinical models of human disease such as cancer, psoriasis, and stroke. 10-13 Transfer of nano-particular structures across cellular membranes is of increasing importance for the development of novel diagnostic and therapeutic tools. The capacity of PTDs to mediate an endocytosis-independent transfer of particles across the plasma membrane into the cytoplasm is still unclear. To investigate the potential of PTD to mediate the transfer of nano-particles across the cell membrane, virus-like particles (VLPs) harboring a marker gene were instrumental.Hepatitis B virus (HBV) core particles represent a very well-characterized VLP model system. 14,15 The HBV core particle (capsid) is assembled by 180 or 240 core protein monomers (HBcAg), resulting in an icosahedral particle 30 or 34 nm in diameter, respectively. A characteristic of the core protein is a basic arginine-rich C-terminal region, which is responsible for the packaging of DNA 16 and for guidance of the capsid to the nucleus. 17 Based on extensive structural analysis, 18 the hepatitis B capsid was used as a carrier for foreign epitopes to develop new vaccines. 19,20 In the viral context, the core particle harbors the viral genome (nucleocapsid). Efficient in vivo packaging of nucleic acids into capsids requires HBV polymerase and its interaction with a defined secondary structure (termed epsilon) at the 5Ј end of the RNA to be packaged. 21 The ⑀ signal is the primary element of the hepadnaviral packag-

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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A novel system for efficient gene transfer into primary human hepatocytes via cell‐permeable hepatitis B virus–like particle†

et al. 2005

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“…[14][15][16][17][18][19] It is normally sequestered between the inner and outer membranes of the mitochondria and is released with other apoptotic factors during apoptosis. In addition, we have incorporated a TLM 21 into the N-terminus of our chimeric protein to provide a possible bystander capability that can be tested in future applications.…”

Section: Discussionmentioning

confidence: 99%

“…This modification was introduced to tighten the conditional regulation of the toxic gene in hypoxic conditions. A translocation motif (TLM) 21 has also been placed at the N-terminus, to potentially provide bystanderkilling effects in therapeutic settings. Transient expression of TLM-ODD-hEndoG (henceforth referred to as hEndoG) in MDA-MB-435 (henceforth referred to as 435) and PC-3 cells indicated that this protein could conditionally kill cells.…”

Section: Introductionmentioning

confidence: 99%

Hypoxia-induced human endonuclease G expression suppresses tumor growth in a xenograft model

et al. 2008

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We have developed a hypoxia-inducible gene therapy approach for the expression of the mature form of human endonuclease G to facilitate cell death in hypoxic regions of the tumor. The chimeric therapeutic gene is placed under the control of a hypoxia response element based promoter and contains a translocation motif linked in frame to an oxygen-dependent degradation domain and the endonuclease G gene. Transient expression of the chimeric therapeutic gene in breast and prostate cancer cell lines resulted in efficient cell death under hypoxia-mimetic conditions. Stable MDA-MB-435 cells expressing the chimeric therapeutic gene under 1% O 2 showed an increase in stable HIF-1a protein levels and synthesis of the endonuclease G protein in a time-dependent manner. In normoxic conditions, these stable transgenic cells exhibited no change in growth rate, invasion and motility when compared to parental cells. Moreover, xenografts generated using the transgenic cells exhibited highly significant suppression of tumor growth in a preclinical cancer model compared to the parental cell line. Thus, the hypoxia-modulated endonuclease G expression has the potential to be used as a gene-based-therapy system to kill malignant cells within hypoxic regions of tumors.

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The translocation motif of hepatitis B virus improves protein vaccination

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Kammertoens

Hutloff

et al. 2006

Cell. Mol. Life Sci.

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Cell-penetrating peptides (CPPs) have been shown to improve antigen loading of dendritic cell vaccines. Here we asked whether fusion of a CPP to a protein improves its immunogenicity when this fusion protein is directly applied as vaccine. We used the cell-penetrating translocation motif (TLM) derived from the hepatitis B virus, because no size limitation of cargos has been observed. Increased immunogenicity was observed when TLM was fused to ovalbumin (TLM-ova). TLM-ova was found to be superior to ova in inducing proliferation and cytotoxicity of ova-specific CD8+ T cells in vitro and in vivo. Using ovalbumin-expressing thymoma cells (EG7-ova), an improved anti-tumor immune response was observed for TLM-ova vaccination versus vaccination with ova. Moreover, TLM-ova vaccination induced a higher titer of anti-ovalbumin IgG2a antibodies compared to ova. These data demonstrate that CPP-protein vaccines can improve cellular as well as humoral immune responses.

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Protein transduction with bacterial cytosine deaminase fused to the TLM intercellular transport motif induces profound chemosensitivity to 5-fluorocytosine in human hepatoma cells

Cited by 7 publications

References 26 publications

A novel system for efficient gene transfer into primary human hepatocytes via cell‐permeable hepatitis B virus–like particle†

A novel system for efficient gene transfer into primary human hepatocytes via cell‐permeable hepatitis B virus–like particle†

Hypoxia-induced human endonuclease G expression suppresses tumor growth in a xenograft model

The translocation motif of hepatitis B virus improves protein vaccination

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