Takaki Hiwasa

CD44 signaling through focal adhesion kinase and its anti-apoptotic effect

Adhesion molecules can initiate intracellular signaling. Engagement of CD44 either by its natural ligand hyaluronan or a specific antibody on a cell line induced tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which then associated with phosphatidylinositol 3‐kinase (PI3K) and activated mitogen‐activated protein kinase at its downstream. However, the introduction of dominant negative Rho into the cells inhibited the CD44‐stimulated FAK phosphorylation. Cells expressing CD44 were significantly resistant to etoposide‐induced apoptosis. This anti‐apoptotic effect was cancelled by the inhibition of either Rho, FAK or PI3K. These results may indicate a signaling pathway from CD44 to mediate the resistance against drug‐induced apoptosis in cancer cells.

Facilitation of adenoviral wild-type p53-induced apoptotic cell death by overexpression of p33 ING1 in T.Tn human esophageal carcinoma cells

To investigate the effect of p33ING1 on wild-type p53 gene therapy, T.Tn human esophageal carcinoma cells were stably transfected with p33ING1 cDNA. Infection with Ad-p53 (recombinant adenovirus containing wild-type p53) into p33-transfected cells reduced cell viability, while infection with empty vector had little effect. This reduced viability was shown to be due to apoptotic cell death by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end-labeling) assay. Following infection with Ad-p53, levels of p53 were similar in p33-expressing cells and in the parental line. However, levels of p21 and Mdm2 were elevated in p33-transfected cells. Nonetheless, this enhanced expression of Mdm2 appeared to be ineffective in downregulating p53. Transient transfection with mutant Mdm2 prior to Ad-p53 infection provided a significant protection as compared with cells transfected with wild-type Mdm2. These results imply a synergistic effect between p33 and p53 in the induction of apoptosis of human esophageal carcinoma cells. A role for Mdm2 in this synergism is suggested.

c-Ha-ras gene products are potent inhibitors of cathepsins B and L

c‐Ha‐ras proteins produced by Escherichia coli inhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c‐Ha‐ras proteins and thiol proteinase inhibitors has been found.

Nuclear localization of procathepsin L/MEP in ras-transformed mouse fibroblasts.

It has been well-documented that secretion of procathepsin L is enhanced in ras-oncogene-transformed cells. In the present study, intracellular localization of cathepsin L was investigated by cell fractionation using Nonidet P-40 followed by immunoblot analysis. The results showed that a significant amount of procathepsin L was detectable in the nuclear fraction of Ha-ras, Ki-ras- and erbB2-transformed NIH3T3 mouse fibroblasts while procathepsin L was detected only in the cytoplasmic fraction of NIH3T3 cells and v-mos-transformed cells. These results suggest that the processing and translocation of cathepsin L are seriously impeded in ras- and erbB2-transformed cells.

Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products.

The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.

c-Ha-Ras mutants with point mutations in Gln-Val-Val region have reduced inhibitory activity toward cathepsin B

Protease-inhibitory activity of recombinant Ha-ras gene products (Ras) toward papain and cathepsins B and L was investigated. v-Ha-Ras showed more potent inhibitory activity toward cathepsin B as compared with c-Ha-Ras. We have also investigated protease-inhibitory activity of c-Ha-Ras mutants with point mutations in amino acids between positions 23 and 50. Inhibitory activity of Ras toward papain and cathepsin L was not largely altered among mutants. However, the inhibitory activity toward cathepsin B was significantly impaired by a mutation at position 43, 44, 45 or 48. These results suggest that 43Gln-Val-Val sequence plays an important role at least to inhibit cathepsin B.

Degradation of a cAMP-binding protein is inhibited by human c-Ha-ras gene products

Incubation of the particulate fraction of cell extract prepared from NIH3T3 mouse fibroblasts resulted in preferential proteolytic degradation of a cAMP-binding protein. The proteolysis was inhibited by human c-Ha-ras gene products produced by Escherichia coli. The proteolysis was observed at pH 6 to 7, and inhibited by antipain and leupeptin. These results suggest that cAMP-binding proteins might be cleaved by thiol proteinases. In fact, c-Ha-ras gene products were proved to inhibit the cathepsin B-like activity present in the particulate fraction.

Induction of apoptosis by a calpain stimulator, ONO-3403

The effects of a synthetic serine protease inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 μg/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 μg/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified μ-calpain at a concentration higher than 100 μg/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of μ-calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.

Altered properties of cAMP-dependent protein kinase in H-ras-transformed NIH3T3 cells

cAMP-dependent protein kinase activity in the soluble fraction was decreased in both v-H-ras-transformed and activated-c-H-ras-transformed NIH3T3 cells as compared with that in NIH3T3 cells. Both of the elution profile of type II cAMP-dependent protein kinase from DEAE-cellulose and the electrophoretic behavior of its regulatory subunits in the particulate fraction of H-ras-transformed cells are different from those of control NIH3T3 cells. These results suggest that ras protein causes the alterations of some properties of cAMP-dependent protein kinases.

Enhancement of chemosensitivity toward anticancer drugs by high expression of caspase-1 in NIH 3T3 cells.

It has been well documented that caspase-1 (interleukin-1/Jconverting enzyme, ICE) and its related cysteine proteinases such as caspase-3 (CPP32, apopain) and caspase-2 (ICH-1L) play important roles in apoptosis. In the present study, these genes were inserted into an inducible eukaryotic expression vector, pMSG, and transfected into NIH 3T3 mouse f ibroblasts. The expression of caspases-1 and -3 was effectively induced by treatment with dexamethasone (Dex). The expression of caspase-2 was elevated in the transfected cells without treatment with Dex but was not further stimulated by Dex. High expression of these proteases alone induced neither apoptosis-like cell death nor any morphological change. However, the expression of caspase-1 but not of caspase-2 or -3 enhanced chemosensitivity toward cytotoxic anticancer drugs such as aclarubicin, epirubicin, adriamycin, nimustine and ifosfamide. It is thus concluded that caspase-1 mediates cytotoxic effects of these drugs.