Hiroyuki Iwata

Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells

Mitogen-activated protein (MAP) kinase appears to be one of the key regulators of cell proliferation and differentiation. Very little, however, has been revealed as to how MAP kinase is involved in leukemogenesis. We have studied the activation of the MAP kinase pathway in 100 human primary leukemia cells including 73 acute myelogenous leukemias (AMLs). Forty acute leukemia samples (40% of the total), including 37 AML samples (51% of AML), showed activation of MAP kinase as revealed by the mobility shift of the phosphorylated form of the protein and by in vitro kinase assay. This activation was correlated with MAP kinase kinase activity in these cells. In contrast, none of 14 chronic myelogenous leukemia samples showed the activation of MAP kinase. These results suggest that the MAP kinase pathway is constitutively activated in a subset of primary acute leukemias, and thus indicate the possible role of the constitutively activated MAP kinase in leukemogenesis.

Tyrosine phosphorylation is crucial for growth signaling by tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2)

[3H]Thymidine (TdR) incorporation by human osteosarcoma cell line MG‐63 was significantly stimulated at as early as 3 h after the addition of either TIMP‐1 or TIMP‐2 alone. Maximum stimulation was attained at a concentration of either 20 ng/ml (0.71 nM) TIMP‐1 or 1.0 ng/ml (46 pM) TIMP‐2. Tyrosine kinase inhibitors such as genistein, erbstatin, and herbimycin A almost completely inhibited the [3H]TdR incorporation stimulated by either of the TIMPs. However, essentially no effect was observed with H‐89, H‐7, bisindolylmaleimide and K‐252a. These inhibition studies suggest a crucial role for tyrosine kinase in the signal transduction of TIMPs. Phosphotyrosine‐containing proteins were significantly elevated by the treatment with both TIMPs. We also found that either TIMP stimulated an increase in mitogen‐activated protein (MAP) kinase activity, suggesting that MAP kinase plays a role in TIMP‐dependent growth signaling.

Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A

We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.

Tumor‐specific Activation of Mitogen‐activated Protein Kinase in Human Colorectal and Gastric Carcinoma Tissues

To search for the signaling events in colorectal carcinoma relevant to its tumorigenesis, we investigated the activity of mitogen‐activated protein kinase (MAPK) in human colorectal carcinoma tissues and paired normal tissues. Of 64 cases examined, approximately 75% (48 cases) showed tumor‐specific activation of MAPK by in situ kinase renaturation assay, as well as in vitro kinase assay with immunoprecipitated MAPK. In addition, tumor‐specific activation of MAPK was associated with the activation of MAPK kinase in the cases we examined. However, no clear correlation of MAPK activation with lymph node involvement, metastatic rate, stage, histological classification, age or sex was observed. These results suggest that the MAPK pathway is involved in colorectal tumor development, but its activation alone is not sufficient for malignant conversion. In contrast to colorectal carcinoma, gastric carcinoma tissues showed a lower rate of MAPK activation, suggesting that the signaling pathway activated in colorectal carcinoma tissues may differ in part from that of gastric carcinoma.

Abundant but inactive-state gp140proto-trk is expressed in neuroblastomas of patients with good prognosis

Steady‐state levels of gp140proto‐trk in cell lines and tumor tissues of neurohlastoma were examined by immunoblotting with anti‐gpl40proto‐trk The level of gpl40proto‐trk varied but showed good correlations with the stage of the tumor and the age of the patients at the time of diagnosis. Moreover, patients with higher expression of gpl40proto‐trk clearly had a far better survival rate than those with lower expression, suggesting that suppression of gp140proto‐trkstrongly correlates with the malignant conversion of the tumor. However, we found that neither autophosphorylation of gpl40proto‐trknor tyrosine phosphoryla‐tion of cellular proteins was elevated in tumors of the higher expression group. These results suggest that gpl40proto‐trk does not actively participate in the process of transformation or the suppression of malignant conversion. Rather, the higher level of gpl40proto‐trkmay reflect the greater level of differentiation of tumor cells.

Chemotherapy‐induced expression of αB‐Crystallin in neuroblastoma

Since alpha B-crystallin is known to be expressed in glial tissues of human brain and neuroectodermal tumors, the alpha B-crystallin content of neuroblastomas, may be related to the degree of glial or neuronal differentiation. The alpha B-crystallin content of 73 neuroblastomas, was determined by enzyme immunoassay. The concentration of alpha B-crystallin was examined in light of neuroblastoma prognostic factors. Neuroblastomas from patients who received chemotherapy (n = 23) contained higher concentrations of alpha B-crystallin than those from patients who did not receive chemotherapy (n = 50) (P > 0.05). There was a statistically significant difference in alpha B-crystallin concentrations in advanced stage patients who received preoperative chemotherapy (P < 0.01). Immunohistochemistry demonstrated alpha B-crystallin expression in the nerve-like fibers and a few ganglion-like cells. Staining was not apparent in the less differentiated cells in the tumor cell nest. alpha B-crystallin may play a role in the response to cellular stress in neuroblastoma.

Comparison of Calbindin D-28k and S-100 Protein B in Neuroblastoma as Determined by Enzyme Immunoassay

Levels of two calcium‐binding proteins, calbindin D‐28k (calbindin‐D) and S‐100 protein B (S‐100b), were measured by immunoassay in solid tumors obtained surgically from pediatric patients. Mean concentrations of calbindin‐D and S‐100b in 73 neuroblastomas (23 ganglioneuroblastomas and 50 neuroblastomas) were 10‐ or 25‐fold higher, respectively, than those in other types of solid tumors in pediatric patients (n=15). The mean tumor concentration of calbindin‐D in patients with neuroblastoma (n=73) was 25.1 ng/mg (range 0.20 to 317.0 ng/mg soluble protein, SE=6.26); that of S‐100b was 278.3 ng/mg (range 0.93 to 2521 ng/mg soluble protein, SE=71.7). The mean concentration of calbindin‐D (4.4 ng/mg soluble protein) was significantly (P < 0.05) lower in stage IV, the most advanced stage. The mean concentration of S‐100b (74.0 ng/mg soluble protein) was lower in patients with undifferentiated neuroblastomas (P < 0.01). Tumor levels of the two calcium‐binding proteins were not correlated in patients with neuroblastoma, but each was strongly correlated with outcome in patients with neuroblastoma. The evidence suggests that measurements of the calcium‐binding proteins calbindin‐D and S‐100b would be useful for evaluating the prognosis of patients with neuroblastoma.