Kenji Kawamura

Publications (14)48.56 Total impact

• Article: Characterization of gene expression regulation using D-RECS polymer by enzymatic reaction for an effective design of enzyme-responsive gene regulator.

  Yuko T Sato, Kenji Kawamura, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: The development of targeted gene delivery systems has attracted much attention. Nevertheless, target cell-specific gene delivery has not been realized. However, if gene expression can be regulated with a gene carrier, this method could be a powerful tool for target specific gene therapy. We have previously reported some novel artificial gene-regulation systems responding to intracellular signaling enzymes by using graft-type polymers that contain substrate peptide (conjugate) for a target enzyme. The conjugate is able to regulate gene transcription in a cell-free system, in cultivated cells and also in in vivo system. We termed this concept 'D-RECS' which means Drug or gene delivery system responding to cellular signals. In the present study, in order to elucidate the mechanism of this gene expression regulation using the conjugate through enzymatic reactions, we characterized a high order structure of the conjugate/DNA complex and the ability of this conjugate to control gene expression using a Caspase-3 responsive system as a typical example. In addition, we compared these properties with those of a substrate peptide/DNA complex. As a result, we elucidated the importance of the polymer backbone for gene regulation. Because our conjugate contains a large amount of neutral polymer chain, wrapping of the DNA strand with a polymer chain acts as a suppressor for 'sliding' or 'jumping' of sequence-recognizing enzymes such as RNA polymerase or restriction enzymes so that the conjugate can efficiently suppress gene expression or site-specific cleavage. Furthermore, our conjugates formed a loosely packed complex with DNA. This behavior is essential for enzymatic regulation of gene transcription because the complex permits access of essential enzymes such as Caspase-3 but prohibits the access and/or sliding of RNA polymerase.
  Journal of Controlled Release 05/2010; 143(3):344-9. · 5.73 Impact Factor
• Article: Specific transgene expression in HIV-infected cells using protease-cleavable transcription regulator.

  Daisuke Asai, Masanori Kuramoto, Yoko Shoji, Jeong-Hun Kang, Kota Bae Kodama, Kenji Kawamura, Takeshi Mori, Hiroshi Miyoshi, Takuro Niidome, Hideki Nakashima, Yoshiki Katayama
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  ABSTRACT: Gene therapy is a promising strategy for the treatment of HIV infection, but cell specificity remains an issue. Recently we have developed a new concept for a drug or gene delivery system responding to cellular signals (D-RECS) to achieve cell-specific transgene expression using a non-viral polymer-based vehicle. According to this concept, intracellular signaling enzymes, which are activated specifically in target cells, are used to trigger transgene expression. We previously applied this concept to HIV-1 protease and showed that the recombinant protease could act as a suitable signal. Here we further developed this system to achieve highly specific transgene expression in HIV-infected cells. We prepared a polymeric gene regulator grafted with a cationic peptide containing the HIV-Tat peptide via a specific substrate for HIV-1 protease. The regulator formed a stable polyplex with the transgene, suppressing its transcription. HIV-1 protease cleaved the peptide and released the transgene, which was consequently expressed specifically in activated HIV-infected cells, but remained unreleased and inactive in uninfected cells. The validity of this approach was further confirmed by applying it to the CVB1 2A protease of coxsackievirus (Picornaviridae family). This strategy should be widely applicable for specific expression of a variety of therapeutic genes in virus-infected cells.
  Journal of Controlled Release 10/2009; 141(1):52-61. · 5.73 Impact Factor
• Article: Inflammatory cell-specific transgene expression system responding to Ikappa-B kinase beta activation.

  Daisuke Asai, Akira Tsuchiya, Jeong-Hun Kang, Kenji Kawamura, Jun Oishi, Takeshi Mori, Takuro Niidome, Yoko Shoji, Hideki Nakashima, Yoshiki Katayama
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  ABSTRACT: Control of inflammation is essential for the clinical management of many common human diseases. However, there are few generally applicable strategies to convert an abnormal intracellular signal into a gene expression that leads to normalization of the intracellular environment. Recently, we proposed a novel strategy termed D-RECS (i.e. drug or gene delivery system responding to cellular signals) to convert an intracellular signal to transgene expression. In the present study, we applied this concept to inflammatory cells using Ikappa-B kinase as a signal molecule that triggers the gene expression. Candidate cationic substrates of Ikappa-B kinase (IKK)beta were synthesized and their reactivity was investigated. Then, polymers grafted with these peptides were prepared by radical polymerization. Polymer/DNA complexes (polyplexes) were prepared by mixing plasmid DNAs with the polymers. The behaviour of these polyplexes by adding IKKbeta was examined. Furthermore, changes of gene expression were evaluated after the microinjection of polyplex into living cells under conditions of nuclear factor (NF)-kappaB activation. Synthetic peptides with additional lysine residues were well phosphorylated by IKKbeta. Both the polymer and the polyplex were also phosphorylated by IKKbeta. The results of gel shift assay showed that the polyplex was disintegrated and free DNA was released in the presence of IKKbeta. The polyplex comprising-green fluorescent protein plasmid DNA and the polymer expressed the transgene in living cells exposed to a pro-inflammatory stimulus. Our concept of cell-specific gene expression was demonstrated to work in inflammatory cells. This method may provide a unique strategy for gene therapy exclusively in inflammatory cells.
  The Journal of Gene Medicine 05/2009; 11(7):624-32. · 2.48 Impact Factor
• Article: Molecular mechanism of caspase-3-induced gene expression of polyplexes formed from polycations grafted with cationic substrate peptides.

  Kenji Kawamura, Masanori Kuramoto, Takeshi Mori, Riki Toita, Jun Oishi, Yuko Sato, Jeong-Hum Kang, Daisuke Asai, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: We previously reported a novel disease-site-specific gene targeting system that can release plasmid DNA (pDNA) from polymeric carriers responding to abnormally activated signal proteins in disease cells. In this study, the molecular mechanism of the gene targeting system responding to Caspase-3 activity was studied in detail. The polymeric carrier used was composed of a neutral main chain polymer and a grafted oligocationic peptide which contains the substrate sequence of Caspase-3. The polyplex formed from the polymeric carrier and pDNA was stable in physiological saline solution and protected from access of RNA polymerase and the transcriptional factors. These results indicate that the polyplex adopts a core-shell-like structure with a polyion complex core surrounded by neutral main chain polymers. In spite of the inert character of the polyplex to transcription, the polyplex afforded the access of Caspase-3 to the substrate peptide because the electrostatic interaction between each peptide and DNA is essentially weak. After the Caspase-3 reaction, the polyplex was weakened and then became available as a template for transcription.
  Journal of Biomaterials Science Polymer Edition 02/2009; 20(7-8):967-80. · 1.69 Impact Factor
• Article: Drug delivery system based on responses to an HIV infectious signal.

  Daisuke Asai, Kota Bae Kodama, Yoko Shoji, Hideki Nakashima, Kenji Kawamura, Jun Oishi, Masanori Kuramoto, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: Gene therapy is a growing topic in the medical arena. Since the safety system of gene therapy has not been sufficiently established, its clinical use is limited. Recently, we developed a cell-specific gene regulation system based on a new concept, D-RECS, or Drug and Gene Delivery System Responding to Cellular Signals. We hoped here to apply this D-RECS concept to gene therapy for virus infections. In the present study, we report the design, synthesis and characterization of the functional polymers, which are able to discriminate normal and human immunodeficiency virus type 1 (HIV-1) infected cells. In the D-RECS concept, certain intracellular signals, which are extraordinary activated in the target disease cells specifically, are used as a trigger to activate a transgene expression. Thus, we paid attention to HIV protease as a target signal in this case, because HIV protease is essential for the proliferation of HIV. This protease is therefore an indicator of HIV infection. Two types of polymers were designed and synthesized using methacryloyl peptide and acrylamide with radical copolymerization as a functional gene regulator. The grafted peptide possesses a cationic protein transduction domain (PTD) sequence of HIV-Tat protein, GRKKRRQRRRPPQ for cell permeation, which are connected with polyacrylamide backbone via a consensus substrate sequence for HIV protease, SQNY/PIVQ. At first, the polymers were evaluated to see whether they possess DNA binding ability and HIV protease responsibility using gel retardation assay. The results suggested that a polymer could form a stable complex with DNA and release the DNA specifically responding to HIV protease activity. Furthermore, it was shown that this controlled release of DNA by the HIV protease signal-responsive intelligent polymer actually regulated the gene expression in the cell-free system. This system would be a useful tool for gene therapy in HIV infection, and this methodology will be applicable if the cationic peptide is replaced by another virus-specific protease, which is critical for the replication of a corresponding virus.
  Medicinal Chemistry 08/2008; 4(4):386-91. · 1.50 Impact Factor
• Article: Role of plasma as activator and cofactor in phosphorylation catalyzed by protein kinase C.

  Jeong-Hun Kang, Daisuke Asai, Jun Oishi, Kenji Kawamura, Riki Toita, Yuhua Jiang, Takeshi Mori, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: The purpose of this study was to investigate whether plasma can influence the phosphorylation of protein kinase C (PKC). Lysate samples were prepared from normal skin or melanoma tissue and were reacted with a PKC peptide substrate in the presence or absence of plasma. In normal skin tissue lysates, the phosphorylation rates were much lower than those in melanoma tissue lysates. However, the level of phosphorylated peptide was increased in both normal skin and melanoma tissue lysates if plasma was present. Phosphorylation rates in the samples taken from the centre of B16 melanoma tissue were lower than those in samples taken from the edge. Moreover, addition of activator and/or cofactors (diacylglycerol, phosphatidylserine and/or Ca2+) of PKC, or plasma to the lysates contaminated by plasma had no effect on phosphorylation rates for the peptide substrate. These results indicate that plasma can play a role of activator and cofactor for substrate phosphorylation.
  Cell Biochemistry and Function 02/2007; 26(1):70-5. · 1.77 Impact Factor
• Article: Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor.

  Jeong-Hun Kang, Yoshiki Katayama, Aishan Han, Shuhei Shigaki, Jun Oishi, Kenji Kawamura, Riki Toita, Xiao Ming Han, Takeshi Mori, Takuro Niidome
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  ABSTRACT: A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H- and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors.
  Journal of the American Society for Mass Spectrometry 02/2007; 18(1):106-12. · 4.00 Impact Factor
• Article: Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C.

  Jeong-Hun Kang, Yuhua Jiang, Riki Toita, Jun Oishi, Kenji Kawamura, Aishan Han, Takeshi Mori, Takuro Niidome, Masami Ishida, Kenji Tatematsu, Katsuyuki Tanizawa, Yoshiki Katayama
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  ABSTRACT: Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.
  Biochimie 01/2007; 89(1):39-47. · 3.02 Impact Factor
• Article: A protein kinase signal-responsive gene carrier modified RGD peptide.

  Jun Oishi, Moeko Ijuin, Tatsuhiko Sonoda, Jeong-Hun Kang, Kenji Kawamura, Takeshi Mori, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using a cationic polymer. However, this polymer alone cannot deliver any gene into living cells. In the present work, we modified the signal-responsive polymer to the RGD peptide for the introduction of a polymer/DNA complex into living cells and succeeded in regulating the gene expression responding to intracellular PKA activation.
  Bioorganic & Medicinal Chemistry Letters 12/2006; 16(22):5740-3. · 2.55 Impact Factor
• Article: [Gene delivery system responding to the intracellular signal].

  Kenji Kawamura, Yoshiki Katayama
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  ABSTRACT: We designed a peptide-polymer conjugate as a novel gene carrier, which could control gene-expression responding to intracellular cAMP-dependent protein kinase (PKA) or caspase-3 signal. Each polymer containing a substrate peptide (ALRRASLG for PKA or GGGDEVD GGRKKRRQRRRPPQ for caspase-3) could form a tight complex with plasmid DNA via electrostatic interaction, and in this state, the gene expression was totally suppressed. Once the grafted peptides become phosphorylated or hydrolyzed by the appropriate enzyme, the net charge of the peptide changes from cationic to neutral or anionic, respectively, and the polymer then releases the DNA, such that expression of the foreign gene should become activated. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy.
  Nippon rinsho. Japanese journal of clinical medicine 03/2006; 64(2):265-70.
• Article: Intracellular signal-responsive artificial gene regulation.

  Kenji Kawamura, Jun Oishi, Shigeki Sakakihara, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: In gene therapy, in order to avoid serious side effects due to the unexpected expression of the transgene in non-target cells, transgenes have to be delivered only to the target cells. In response to this issue, many researchers have aimed at developing target cell-selective gene carriers using active targeting strategies. However, such methodology does not always work, because an ideal molecular marker, which is specific to the target disease cells, is not always available. In this study, we introduce a new concept regarding target disease cell-selective gene therapy (D-RECS). Here, we use intracellular signals, which are activated to an extraordinary degree only in the target disease cells, as a trigger for transgene expression using polymer-peptide conjugates. This strategy could actually activate gene expression in the target signal-activated cells only. Hyper-activation of certain intracellular signals has been reported in many diseases. Thus, this new strategy is expected to provide a powerful methodology for future gene therapy. In this review, the basic concept, some examples, and the molecular design of D-RECS carriers are introduced.
  Journal of Drug Targeting 02/2006; 14(7):456-64. · 2.70 Impact Factor
• Article: An intracellular kinase signal-responsive gene carrier for disordered cell-specific gene therapy.

  Jun Oishi, Kenji Kawamura, Jeong-Hun Kang, Kota Kodama, Tatsuhiko Sonoda, Masaharu Murata, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using cationic polymer. This cationic polymer (PAK) was a graft-type polymer with an oligopeptide that is a substrate for PKA and could regulate gene-expression in a cell-free system. In the present study, we carried out a detailed characterization of the PAK-DNA complex (AFM observation and DLS measurement) and tried to apply this polymer to living cells. In the unstimulated NIH 3T3 cells, transfection of the PAK-DNA complex showed no expression of the delivered gene. This means that PAK formed a stable complex with DNA in the normal cells to totally suppress gene expression. In contrast, significant expression was seen when the PAK-DNA complex was delivered to forskolin-treated cells. Thus, activated PKA disintegrates the complexes even in living cells, resulting in gene expression. Our results indicate that this type of intracellular signal-responsive polymer will be useful for the cell-specific release of genes.
  Journal of Controlled Release 02/2006; 110(2):431-6. · 5.73 Impact Factor
• Article: Ratiometric direct detection of nitric oxide based on a novel signal-switching mechanism.

  Nobuaki Soh, Toshihiko Imato, Kenji Kawamura, Mizuo Maeda, Yoshiki Katayama
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  ABSTRACT: A novel fluorescent probe, which could be the first example of a ratiometric molecular probe for direct monitoring of NO production, has been developed using a 'spin-exchange' mechanism.
  Chemical Communications 12/2002; · 6.17 Impact Factor
• Article: Intracellular signal-responsive gene carrier for cell-specific gene expression.

  Kenji Kawamura, Jun Oishi, Jeong-Hun Kang, Kota Kodama, Tatsuhiko Sonoda, Masaharu Murata, Takuro Niidome, Yoshiki Katayama
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  ABSTRACT: We designed a peptide-polymer conjugate (CPCCtat) as a novel gene carrier that could control gene expression responding to the intracellular caspase-3 signal. This carrier consists of an uncharged main polymer chain and a cationic peptide side chain, which includes the substrate sequence of caspase-3 and the protein transduction domain sequence of HIV-1 Tat. In the present study, CPCCtat formed a tight complex with DNA through an electrostatic interaction, and in this state the gene expression was totally suppressed. In contrast, the complex disintegrated in the presence of caspase-3 due to cleavage of the cationic portion from CPCCtat. This event led to an activation of gene expression. Our results also indicate that the complex can be delivered into living cells due to the cell-permeable peptide side chain of CPCCtat. This intracellular signal-responsive system with CPCCtat will be useful for the cell-specific gene expression system.
  Biomacromolecules 6(2):908-13. · 5.48 Impact Factor