Lung Tumor Cell Recruitment Assay.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells.

C1D is not directly involved in the repair of UV-damaged DNA but protects cells from oxidative stress by regulating gene expressions in human cell lines.

A small nuclear protein, C1D, has roles in various cellular processes, transcription regulation, genome stability surveillance, DNA repair and RNA processing, all of which are required to maintain the host life cycles. In the previous report, C1D directly interacts with XPB, a component of the nucleotide excision repair complex, and C1D knockdown reduced cell survival of 27-1 cells, CHO derivative cells, after UV irradiation. To find out the role of C1D in UV-damaged cells, we used human cell lines with siRNA or shRNA to knockdown C1D.

Pleckstrin homology domain of p210 BCR-ABL interacts with cardiolipin to regulate its mitochondrial translocation and subsequent mitophagy.

Chronic myeloid leukemia (CML) is caused by the chimeric protein p210 BCR-ABL encoded by a gene on the Philadelphia chromosome. Although the kinase domain of p210 BCR-ABL is an active driver of CML, the pathological role of its pleckstrin homology (PH) domain remains unclear. Here, we carried out phospholipid vesicle-binding assays to show that cardiolipin (CL), a characteristic mitochondrial phospholipid, is a unique ligand of the PH domain.

Drug Targeting Based on a New Concept-Targeting Against TLR4 as an Example.

TLRs are very important players to regulate innate immune responses. TLR4 controls the host defense by sensing an exotic pathogen, such as lipopolysaccharides. At the same time, some endogenous proteins, including HMGB1 and S100A8, could also function to be a ligand to elicit inflammatory reactions.

Nuclear export of the influenza virus ribonucleoprotein complex: Interaction of Hsc70 with viral proteins M1 and NS2.

The influenza virus replicates in the host cell nucleus, and the progeny viral ribonucleoprotein complex (vRNP) is exported to the cytoplasm prior to maturation. NS2 has a nuclear export signal that mediates the nuclear export of vRNP by the vRNP-M1-NS2 complex. We previously reported that the heat shock cognate 70 (Hsc70) protein binds to M1 protein and mediates vRNP export.

Connexin43 functions as a novel interacting partner of heat shock cognate protein 70.

Regulation of connexin43 (Cx43) expression affects cell proliferation, differentiation and apoptosis in a gap junctional intercellular communication (GJIC)-independent manner. However, the underlying mechanisms of Cx43-mediated cell cycle suppression are still poorly understood. To elucidate the molecular mechanism of Cx43-mediated cell cycle suppression, we searched for Cx43 interacting proteins by using a proteomics approach.

p57(Kip2) and p27(Kip1) cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70.

Cell cycle regulators play critical roles in the balance between hematopoietic stem cell (HSC) dormancy and proliferation. In this study, we report that cell cycle entry proceeded normally in HSCs null for cyclin-dependent kinase (CDK) inhibitor p57 due to compensatory upregulation of p27. HSCs null for both p57 and p27, however, were more proliferative and had reduced capacity to engraft in transplantation.

Bag1 directly routes immature BCR-ABL for proteasomal degradation.

Degradation of BCR-ABL oncoproteins by heat shock protein 90 (Hsp90) inhibitors in chronic myelogenous leukemia is expected to overcome resistance to ABL tyrosine kinase inhibitors. However, the precise mechanisms still remain to be uncovered. We found that while c-Cbl E3 ligase induced ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, another E3 ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) degraded immature BCR-ABL proteins and efficiently suppressed BCR-ABL-dependent leukemic growth.

Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle.

Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70.

N-acetyl-cysteine enhances growth in BCR-ABL-transformed cells.

N-acetyl-cysteine (NAC) has been reported to have anticancer properties such as counteractions against mutagens and prevention of tumor progression by scavenging reactive oxygen species (ROS). However, here we report that NAC can enhance the anchorage-independent growth of cells transformed by activated ABL tyrosine kinases or Ras. This effect was not dependent on loss of focal adhesion kinase activation.

Suppression by 17beta-estradiol of monocyte adhesion to vascular endothelial cells is mediated by estrogen receptors.

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells.

Regulation of nuclear retention of glucocorticoid receptor by nuclear Hsp90.

Heat shock protein 90 (Hsp90) has been demonstrated in both cytoplasm and nucleus, and regulates cytoplasmic retention of glucocorticoid receptor (GR). However, the role of nuclear Hsp90 in GR trafficking is less characterized. The present study examined the role of Hsp90 in nuclear retention of GR after ligand withdrawal.

Hsp90 inhibitors attenuate effect of dexamethasone on activated NF-kappaB and AP-1.

Heat shock protein 90 (Hsp90) regulates the functions of glucocorticoid receptor (GR). Hsp90 inhibitors geldanamycin (GA) and radicicol (Rad) have been studied as anti-inflammatory agents; however, their effects on glucocorticoid-mediated anti-inflammatory mechanism are not known. In the present study, we examined the effects of dexamethasone (Dex) and Hsp90 inhibitors, alone and in combination, on the activation of GR and proinflammatory transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1).

Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70.

Heat shock cognate protein 70 (Hsc70) serves nuclear transport of several proteins as a molecular chaperone. We have recently identified a novel variant of human Hsc70, heat shock cognate protein 54 (Hsc54), that lacks amino acid residues 464-616 in the protein binding and variable domains of Hsc70. In the present study, we examined nucleocytoplasmic localization of Hsc70 and Hsc54 by using green fluorescent protein (GFP) fusions.