The 2;5 chromosomal translocation is frequently associated with anaplastic large cell lymphomas (ALCLs). The translocation creates a fusion gene consisting of the alk (anaplastic lymphoma kinase) gene and the nucelophosmin (npm) gene: the 3' half of alk derived from chromosome 2 is fused to the 5' portion of npm from chromosome 5. A recent study shows that the product of the npm-alk fusion gene is oncogenic. To help understand how the npm-alk oncogene transform cells, it is important to investigate the normal biological function of the alk gene product, ALK. Here, we show molecular cloning of cDNAs for both the human and mouse ALK proteins. The deduced amino acid sequences reveal that ALK is a novel receptor protein-tyrosine kinase having a putative transmembrane domain and an extracellular domain. These sequences are absent in the product of the transforming npm-alk gene. ALK shows the greatest sequence similarity to LTK (leukocyte tyrosine kinase) whose biological function is presently unknown. RNA blot hybridization analysis of various tissues reveals that the alk mRNA is dominantly detected in the brain and spinal cord. Immunoblotting with anti-ALK antibody shows that ALK is highly expressed in the neonatal brain. Furthermore, RNA in situ hybridization analysis shows that the alk mRNA is dominantly expressed in neurons in speci®c regions of the nervous system such as the thalamus, mid-brain, olfactory bulb, and ganglia of embryonic and neonatal mice. These data suggest that ALK plays an important role(s) in the development of the brain and exerts its e ects on speci®c neurons in the nervous system.

The intracellular protein tyrosine kinase FAK (focal adhesion kinase) was originally identified gy its high level of tyrosine phosphorylation in v-src-transformed cells. FAK is also highly phosphorylated during early development. In cultured cells it is localized to focal adhesion contacts and becomes phosphorylated and activated in response to integrin-mediated binding of cells to the extracellular matrix, suggesting an important role in cell adhesion and/or migration. We have generated FAK-deficient mice by gene targeting to examine the role of FAK during development. Mutant embryos displayed a general defect of mesoderm development, and cells from these embryos had reduced mobility in vitro. Surprisingly, the number of focal adhesions was increased in FAK-deficient cells, suggesting that FAK may be involved in the turnover of focal adhesion contacts during cell migration.

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM).The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lym- (11, 12). We established a cell line of a Ki-1 lymphoma with t(2;5) by maintaining the tumor in severe combined immunodeficiency (SCID) mice, and the cell line was termed AMS3. By examining various signal-transducing molecules and the status of tyrosine phosphorylation of cellular proteins in the AMS3 cells, we found a unique 80-kDa tyrosine-phosphorylated protein and termed it p80. We then purified p80 using anti-phosphotyrosine antibody and determined partial amino acid sequences of some of the tryptic peptides (13). Because the sequences showed similarity to Ltk (leukocyte tyrosine kinase) of the insulin receptor family, we predicted that a novel member of this family was activated and autophosphorylated in the AMS3 cells. Our present study shows the structure of the p80 protein predicted from the cDNA clone, its subcellular localization, and its transforming activity. We also examined the relevance of Shc, IRS-1, and GRB2 to the oncogenic activity of p80. MATERIALS AND METHODSCells and Antibodies. AMS3 cells were established by implanting the tissue of Ki-1 lymphoma in SCID mice as described (13). NIH 3T3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)/5% of calf serum. COS cells were cultured in DMEM/10% of fetal calf serum. Cell culture was performed at 37°C and 5% CO2. Affinity-purified rabbit polyclonal antibody against p80 protein was prepared as described (14). Anti-Shc and anti-IRS-1 polyclonal antibodies were purchased from Upstate Biotechnology. Anti-GRB2 polyclonal antibody was from

The survival probability in patients with resected bile duct cancer was not significantly different between the gemcitabine adjuvant chemotherapy group and the observation group. Registration number: UMIN 000000820 (http://www.umin.ac.jp/).

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Eukaryotic proteins are frequently produced in Escherichia coli as insoluble aggregates. This is one of the barriers to studies of macromolecular structure. We have examined the effect of coproduction of the E. coli thioredoxin (Trx) or E. coli chaperones GroESL on the solubility of various foreign proteins. The solubilities of all eight vertebrate proteins examined including transcription factors and kinases were increased dramatically by coproduction of Trx. Overproduction of E. coli chaperones GroESL increased the solubilities of four out of eight proteins examined. Although the tyrosine kinase Lck that was produced as an insoluble form and solubilized by urea treatment had a very low autophosphorylating activity, Lck produced in soluble form by coproduction of Trx had an efficient activity. These results suggest that the proteins produced in soluble form by coproduction of Trx have the native protein conformation. The mechanism by which coproduction of Trx increases the solubility of the foreign proteins is discussed.

Anaplastic lymphoma kinase (ALK) is a receptor-type protein tyrosine kinase that is expressed preferentially in neurons of the central and peripheral nervous systems at late embryonic stages. To elucidate the role of ALK in neurons, we developed an agonist monoclonal antibody (mAb) against the extracellular domain of ALK. Here we show that mAb16-39 elicits tyrosine phosphorylation of endogenously expressed ALK in human neuroblastoma (SK-N-SH) cells. Stimulation of these cells with mAb16-39 markedly induces the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Shc, and c-Cbl and also their interaction with ALK and activation of ERK1/2. Furthermore, we show that continuous incubation with mAb16-39 induces the cell growth and neurite outgrowth of SK-N-SH cells. These responses are completely blocked by MEK inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, indicating an essential role of the mitogen-activated protein kinase (MAP kinase) signaling cascade in ALK-mediated growth and differentiation of neurons.

IL-8 might act as an angiogenic switch in myometrial invasion in stage I uterine endometrial cancers. Furthermore, IL-8 supplied from infiltrated macrophages within and around the tumor might not be a prognostic indicator of advancement, but may be associated with myometrial invasion in uterine endometrial cancers.