Jun Oishi

Measurement of homogeneous kinase activity for cell lysates based on the aggregation of gold nanoparticles

Protein kinases are involved in a number of signal transduction pathways and play a crucial role in many cell regulatory processes. Since malfunctions of cellular signaling have been linked with many common diseases, including cancers, considerable efforts have been made to develop analytical techniques for the high-throughput screening (HTS) of kinase activity and kinase-related drugs. Due to the increasing demand for faster screening processes, homogeneous detection methods that can both conduct enzymatic reactions and detect any resulting phosphorylated peptides under homogeneous solution conditions, and that do not require the elaborate washing steps needed for conventional ELISA, are receiving considerable attention. Most of the methods are based on fluorescence readout, and specially designed fluorescence probes are used not only to monitor the activity of purified kinase in real time, but also to detect any kinase activity in cell lysates. Colorimetric assays that make use of the aggregation of gold nanoparticles (GNPs) are simpler but sensitive methods, and can be adapted to HTS systems based on the microwell plate format. Colorimetric assays have been applied to the detection of biomolecules and to the evaluation of enzymatic activity. In most of these colorimetric assays, the GNPs are aggregated by “cross-linking molecules” that have multiple binding sites for molecules immobilized on the GNP surface. Brust’s group successfully detected kinase activity by utilizing this cross-linking-type GNP aggregation. Recently, an alternative GNP-aggregation-based detection system without the use of cross-linking molecules has emerged. In this detection system, biomacromolecules such as DNA and peptides are used as “stabilizers” or inversely as “coagulants” of GNPs. This system, which has been successful in the detection of DNA sequences and phosphatase activity, does not require GNP modifications or complicated procedures. Learning from these previous works, we sought to establish a system for detecting kinase activity, as shown in Figure 1. Here, cationic substrate

Design of Polymeric Carriers for Cancer-Specific Gene Targeting: Utilization of Abnormal Protein Kinase Cα Activation in Cancer Cells

We succeeded in cancer cell specific gene expression by using a polyplex responsive to protein kinase Calpha, which is activated in various types of cancer cells.

An intracellular kinase signal-responsive gene carrier for disordered cell-specific gene therapy

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.

Colorimetric enzymatic activity assay based on noncrosslinking aggregation of gold nanoparticles induced by adsorption of substrate peptides.

The mechanisms of colorimetric assays based on aggregation of gold nanoparticles (GNPs) have been separated into two categories, crosslinking, and noncrosslinking aggregation. The noncrosslinking aggregation has recently been emerging as a simple and rapid mechanism and has been applied to enzymatic activity assays and DNA detection. We report here the detailed study of an enzymatic activity assay for protein kinases based on noncrosslinking aggregation. The principle of the assay is to detect kinase activity by utilizing the difference of coagulating ability of a cationic substrate peptide and its phosphorylated form toward GNPs with anionic surface charge. The critical coagulation concentrations (CCCs) of the peptides were about 10(3) times lower than those of the metal cations with the same cationic charges. The multivalent coordination bonds of the functional groups of the peptides with the GNP surface will strongly support the adsorption of the peptide on the GNP surface. The effect of the GNP size (10, 20, 40, 60 nm) on the dynamic range of OD before and after aggregation was studied. The dynamic range became a maximum for 20 nm GNP among those studied. The difference of CCC between the phosphorylated and nonphosphorylated peptides was governed by (1) the ratio between the peptide concentration and the surface area concentration of GNP and (2) the net charge of the peptides. When the assay system was applied to the activity assessment of protein kinase A, the dynamic range of OD was largest for 20 nm GNPs. However, when the peptide concentration was lowered, the largest 60 nm GNP was advantageous because of its smaller specific surface area.

A short peptide is a protein kinase C (PKC) α-specific substrate

The purpose of this study was to find protein kinase C (PKC) isozyme‐specific peptides. A peptide library containing 1772 sequences was designed using Scansite and screened by MALDI‐TOF MS and kinase activity assays for PKC isozyme‐specificity. A peptide (Alphatomega; H‐FKKQGSFAKKK‐NH2) with high specificity for PKCα relative to other isozymes was identified. The peptide was phosphorylated to a greater extent by tissue lysates from B16 melanoma, HepG2, and human breast cancer, which had higher levels of activated PKCα, when compared to normal skin, liver, and human breast tissue lysates, respectively. Moreover, addition of Ro‐31‐7549, an inhibitor with great specificity for PKCα, to the phosphorylation reaction caused a dose‐dependent reduction in phosphorylation, but no inhibition was identified with the addition of rottlerin and H‐89. These results show that this peptide has great potential as a PKCα‐specific substrate.

Inflammatory cell-specific transgene expression system responding to Iκ-B kinase beta activation

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 Iκ‐B kinase as a signal molecule that triggers the gene expression.

Hepatoma-targeted gene delivery using a tumor cell-specific gene regulation system combined with a human liver cell-specific bionanocapsule

Hepatoma (hepatocellular carcinoma) is the most common type of malignant tumor originating in the liver and has a relatively low 5-year survival rate. The development of hepatoma-targeted therapy is needed to increase treatment efficiency and to reduce the incidence of undesirable side effects. In this study we developed a novel hepatoma-targeted gene delivery system. The gene delivery system was prepared by combining a human liver cell-specific bionanocapsule (BNC) and a tumor cell-specific gene regulation polymer, which responds to hyperactivated protein kinase C alpha in hepatoma cells. The complex of the polymer-DNA with BNCs was delivered into cells and tissues. The developed system showed increased transfection efficiency and resulted in cell-specific gene expression in hepatoma cells and tissues (HuH-7), but no gene expression in normal human hepatocytes or human epidermoid tumor cells (A431). The combination of a tumor cell-specific gene regulation system responding to protein kinase C alpha and BNCs showed excellent potential for the selective treatment of hepatomas. The system could be a useful method with applications in hepatoma-specific gene therapy and molecular imaging. From the clinical editor: Hepatocellular carcinoma is the most common type of malignant tumor in the liver with a low 5-year survival rate. In this study, a novel hepatoma-targeted gene delivery system was prepared by combining a human liver cell-specific bionanocapsule and a tumor cell-specific gene regulation polymer, which responds to hyperactivated protein kinase C (PKC)a in hepatoma cells. The system could be a useful in hepatoma-specific gene therapy and molecular imaging.

A gene-delivery system specific for hepatoma cells and an intracellular kinase signal based on human liver-specific bionanocapsules and signal-responsive artificial polymer

Recently, our group has proposed a novel gene-regulation system responding to cAMP-dependent protein kinase (PKA) that has been applied to living cells. In this study, human liver-specific bionanocapsules (BNCs) are used as a gene-delivery system to increase transfection efficiency and to target specific cell types. BNCs can efficiently deliver a target gene to human hepatocytes and hepatoma cells in vitro or in vivo. The combination of a signal-responsive gene-delivery system with BNCs led to an increase in the transfection efficiency and selectivity for hepatoma cells. Expression from the delivered gene was identified from PKA-activated hepatoma cells (HepG2), but not from colon tumor cells (WiDr). These results show that the combination of a gene-regulation system responding to an intracellular signal with BNC can be used for the selective treatment of human hepatoma cells.

Intracellular signal-responsive artificial gene regulation.

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.

A simple set-and-mix assay for screening of protein kinase inhibitors in cell lysates.

Here we developed a simple set-and-mix assay to perform high-throughput screening of protein kinase A (PKA) inhibitors from the LOPAC 1280 compound library. This assay is based on the color change of gold nanoparticles on aggregation induced by a cationic substrate peptide as coagulant. In spite of the simplicity of this assay system, this assay can be applied to drug screening based on cellular kinases. We successfully found several highly active inhibitors, including compounds that have not been reported before.