GROWTH FACTOR DIVISION

7.GROWTH FACTOR DIVISION

    The main object of our division is to elucidate how cellular signals are modulated by growth factor receptors and other tyrosine kinases during initiation and progression of cancers. One of the goals of own research is to make models of the signal therapy against cancer progression by regulating the phosphotyrosine-dependent interactions between signaling proteins. Our division also promotes studies on the pathophysiology of familial cancer syndromes, new tumor markers, megakaryocyte development and cancer-specific immunotherapy.
Biological Roles of Signal Docking Proteins Involved in Transformation and Migration of Tumor Cells

    In some of the neuroblastoma cell lines, hyperphosphorylation of a docking protein ShcC along with distinct 150-200 kD phosphoproteins associated with ShcC were observed. Anaplastic lymphoma kinase (ALK) was identified as a major component of these phosphoproteins by mass-spectrometry analysis. An in vitro kinase assay revealed that ShcC is a potent substrate of the activated ALK kinase and associated with the PTB domain of ShcC in a phosphorylation dependent manner. The gene amplification of the ALK locus was detected in these cell lines, which will be responsible for the constitutive activation of the ALK kinase ⁽¹¹⁵⁾. Gene amplification of the ALK locus actually exists in some advanced neuroblastoma tissues. Activation of ALK appeared to interfere with signals from other receptor tyrosine kinases including EGFR and TrkA as siRNA, which blocks expression of ALK, caused cell apoptosis in these neuroblastoma cells.
    Cas, a substrate of Src family tyrosine kinases, is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk. Cas-negative fibroblasts obtained from knockout mice have the following phenotypic features: 1) defective in transformation by activated Src(527F Src); 2) lack of actin stress fiber formation; 3) impaired cell migration ⁽¹¹⁶⁾. All of these phenotypes are rescued by the wild-type Cas protein. Cas has a unique structure, with an SH3 domain in the N-terminal region followed by 15 repeats of similar SH2-binding motifs and an Src-binding domain in the C-terminal region. Using 9 different deletion mutants with/without 527F Src, it was found that the central YDxP motifs and the Src-binding domain are important in transformation ⁽¹¹⁷⁾. By generating phospho-specific antibodies against each tyrosine phosphorylation sites of Cas, roles of tyrosine phosphorylation of Cas during cell migration and transformation are being analyzed.
A Peptidomic Approach to Tumor Marker Discovery

    Efforts have been made to screen and identify tumor markers applicable to serum testing for early detection of pancreatic cancer. To achieve this goal, secretory peptides are targeted using mass spectrometric techniques. One approach concerns a serum-free conditioned medium from cultured cells, revealing the secretion of the 29-residue carboxyl-terminal fragment of the putative tumor suppressor protein DMBT1 from pancreatic adenocarcinoma cell lines ^(118,¹¹⁹⁾.
Translational Research of Cancer-specific Immunotherapy

    The translational research bridging the bench to the clinic has been considered to be important for developing a novel immune cell therapy with clinical application. As basic research for developing efficient immunotherapy, in vivo and ex vivo approaches were performed. A tumor lysate-treated dendritic cell (DC) -based vaccine was shown to be effective against an established hamster subcutaneous pancreatic tumor ^(120,¹²¹⁾, and an ex vivo trial in which human IL-12-producing DC derived from CD34+ cells were produced using retroviral-mediated gene transduction technology was successful ⁽¹²²⁾ and Besides tumor antigens, a specific CTL-epitope for cytomegalovirus (CMV) pp65 antigen was identified and demonstrated to be HLA-A24- restricted ⁽¹²³⁾.
Genetic Testing for Familial Cancer Syndrome

    Although sporadic medullary thyroid carcinoma (MTC) tends to be unicentric and confined to one lobe, total thyroidectomy is usually performed because of the risk of a hereditary or bilateral process. Since germline RET mutation analysis can discriminate between hereditary MTC and truly sporadic, non-hereditary MTC, we analyzed 72 patients with MTC to establish the genetic nature and the clinical features of nonhereditary MTC by germline RET mutation analysis. The results of the study suggest that hemithyroidectomy with systemic central and ipsilateral neck dissection is appropriate surgery for non-hereditary MTC ⁽¹²⁴⁾.
Megakaryocyte Development and Platelet Formation

    Platelets derived from the cytoplasm of polyploid megakaryocytes are crucial for human hemostasis. Although megakaryocytes are developed from hematopoietic stem cells just like all other blood cells, they display unique features in that they develop polyploid DNA contents during the normal life cycle of cells and that they release non-nucleated platelets as their progeny. Understanding the mechanisms of megakaryocytic development and platelet formation has been hampered partly by lack of a suitable model system for resolving this stage. We have recently established sublines of an original human megakaryocytic leukemia cell which show distinct phenotypes in respect of megakaryocytic maturation. Candidate genes that regulate the proliferation and differentiation of megakaryocytic progenitor cells are being screened using cDNA microarray analysis and retrovirus-mediated expression cloning.




7.GROWTH FACTOR DIVISION



	The main object of our division is to elucidate how cellular signals are modulated by growth factor receptors and other tyrosine kinases during initiation and progression of cancers. One of the goals of own research is to make models of the signal therapy against cancer progression by regulating the phosphotyrosine-dependent interactions between signaling proteins. Our division also promotes studies on the pathophysiology of familial cancer syndromes, new tumor markers, megakaryocyte development and cancer-specific immunotherapy. Biological Roles of Signal Docking Proteins Involved in Transformation and Migration of Tumor Cells In some of the neuroblastoma cell lines, hyperphosphorylation of a docking protein ShcC along with distinct 150-200 kD phosphoproteins associated with ShcC were observed. Anaplastic lymphoma kinase (ALK) was identified as a major component of these phosphoproteins by mass-spectrometry analysis. An in vitro kinase assay revealed that ShcC is a potent substrate of the activated ALK kinase and associated with the PTB domain of ShcC in a phosphorylation dependent manner. The gene amplification of the ALK locus was detected in these cell lines, which will be responsible for the constitutive activation of the ALK kinase ⁽¹¹⁵⁾. Gene amplification of the ALK locus actually exists in some advanced neuroblastoma tissues. Activation of ALK appeared to interfere with signals from other receptor tyrosine kinases including EGFR and TrkA as siRNA, which blocks expression of ALK, caused cell apoptosis in these neuroblastoma cells. Cas, a substrate of Src family tyrosine kinases, is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk. Cas-negative fibroblasts obtained from knockout mice have the following phenotypic features: 1) defective in transformation by activated Src(527F Src); 2) lack of actin stress fiber formation; 3) impaired cell migration ⁽¹¹⁶⁾. All of these phenotypes are rescued by the wild-type Cas protein. Cas has a unique structure, with an SH3 domain in the N-terminal region followed by 15 repeats of similar SH2-binding motifs and an Src-binding domain in the C-terminal region. Using 9 different deletion mutants with/without 527F Src, it was found that the central YDxP motifs and the Src-binding domain are important in transformation ⁽¹¹⁷⁾. By generating phospho-specific antibodies against each tyrosine phosphorylation sites of Cas, roles of tyrosine phosphorylation of Cas during cell migration and transformation are being analyzed. A Peptidomic Approach to Tumor Marker Discovery Efforts have been made to screen and identify tumor markers applicable to serum testing for early detection of pancreatic cancer. To achieve this goal, secretory peptides are targeted using mass spectrometric techniques. One approach concerns a serum-free conditioned medium from cultured cells, revealing the secretion of the 29-residue carboxyl-terminal fragment of the putative tumor suppressor protein DMBT1 from pancreatic adenocarcinoma cell lines ^(118,¹¹⁹⁾. Translational Research of Cancer-specific Immunotherapy The translational research bridging the bench to the clinic has been considered to be important for developing a novel immune cell therapy with clinical application. As basic research for developing efficient immunotherapy, in vivo and ex vivo approaches were performed. A tumor lysate-treated dendritic cell (DC) -based vaccine was shown to be effective against an established hamster subcutaneous pancreatic tumor ^(120,¹²¹⁾, and an ex vivo trial in which human IL-12-producing DC derived from CD34+ cells were produced using retroviral-mediated gene transduction technology was successful ⁽¹²²⁾ and Besides tumor antigens, a specific CTL-epitope for cytomegalovirus (CMV) pp65 antigen was identified and demonstrated to be HLA-A24- restricted ⁽¹²³⁾. Genetic Testing for Familial Cancer Syndrome Although sporadic medullary thyroid carcinoma (MTC) tends to be unicentric and confined to one lobe, total thyroidectomy is usually performed because of the risk of a hereditary or bilateral process. Since germline RET mutation analysis can discriminate between hereditary MTC and truly sporadic, non-hereditary MTC, we analyzed 72 patients with MTC to establish the genetic nature and the clinical features of nonhereditary MTC by germline RET mutation analysis. The results of the study suggest that hemithyroidectomy with systemic central and ipsilateral neck dissection is appropriate surgery for non-hereditary MTC ⁽¹²⁴⁾. Megakaryocyte Development and Platelet Formation Platelets derived from the cytoplasm of polyploid megakaryocytes are crucial for human hemostasis. Although megakaryocytes are developed from hematopoietic stem cells just like all other blood cells, they display unique features in that they develop polyploid DNA contents during the normal life cycle of cells and that they release non-nucleated platelets as their progeny. Understanding the mechanisms of megakaryocytic development and platelet formation has been hampered partly by lack of a suitable model system for resolving this stage. We have recently established sublines of an original human megakaryocytic leukemia cell which show distinct phenotypes in respect of megakaryocytic maturation. Candidate genes that regulate the proliferation and differentiation of megakaryocytic progenitor cells are being screened using cDNA microarray analysis and retrovirus-mediated expression cloning.