By means of a baculovirus expression system, a large amount of mouse midkine (MK) was produced. The protein was purified to homogeneity by heparin-Sepharose column chromatography. The purified protein was of a mature type; the signal peptide was cleaved at the expected site. To examine the neurite-guiding activity of MK, rat embryonic brain cells (embryonic days 17-18) were cultured on plates coated with purified MK in a grid pattern. The cells attached to and extended their neurites along the substrate pattern. This interaction was strongly inhibited by heparin, but not by other glycosaminoglycans. Treatment of the cells with heparitinase was effective for inhibiting their adhesion to the substrate. These data suggest that the heparin-like domain on cell surface heparan sulfate proteoglycan is the primary site for MK binding upon interaction with nerve cells.

Abstract. Focal adhesion kinase (FAK) was first identified as a viral Src substrate, and substantial experimental data have significantly correlated the elevated FAK expression in human tumor cells with an increased cell adhesion and invasion potential. However, studies investigating the role of FAK in cell proliferation have been limited. Recently, a technique known as RNA interference (RNAi) was successfully adapted to mammalian cells to decrease specifically the expression of targeted cellular genes. In this study, we investigated the role of FAK in cell proliferation, adhesion, and migration by using small interfering RNA (siRNA) technique. Firstly, we constructed a plasmid library expressing short hairpin RNAs (shRNAs) targeting FAK and selected clones substantially suppressing FAK expression in HeLa and HT1080 cells. We then studied the function of FAK in the highly invasive human prostate cancer cell line, PC3M, and mouse breast cancer cell line 4T1, by using selected shRNA clones (#40 and #42) and siRNAs chemically synthesized following the target sequences of #40 and #42. We demonstrated that the decrease of FAK protein expression by treatment with shRNA/siRNA targeting FAK inhibited cell adhesion on a fibronectin/laminin-coated plate, cell migration in a haptotactic migration assay, and cell proliferation in vitro. Furthermore, it suppressed tumor growth in vivo in heterotopic/orthotopic mice models. These results support our hypothesis that FAK plays a crucial role in tumor formation and growth in vivo by regulation of cell adhesion and proliferation by FAK-dependent signals.

TrkB, a member of the tyrosine kinase family of growth factor receptors, is activated by binding of brain-derived neurotrophic factor or neurotrophin 4/5. The intracellular kinase domain of TrkB (ICD-TrkB) was prepared by an insect cell expression system and characterized to identify the mechanism of autophosphorylation. The time course of autophosphorylation, which shows a biphasic progression with a slow nonlinear phase followed by a fast linear phase, indicates the existence of autophosphorylation-induced activation in ICD-TrkB. This is also supported by the finding that phosphorylated ICD-TrkB shows significantly higher activity than control naive ICD-TrkB. Interestingly, the autophosphorylation rate in the linear phase clearly depends on the ICD-TrkB concentration, whereas the rate of initial autophosphorylation is independent of the concentration of ICD-TrkB in the reaction mixture. This observation suggests a two-step autophosphorylation, first an intramolecular activating step and then an intermolecular step. This mechanism is confirmed by the result that only the later phase of autophos-phorylation is inhibited by addition of glycerol which interferes with intermolecular interactions. Therefore, we propose the mechanism of ICD-TrkB autophosphorylation as a sequential cis/trans phosphorylation.

Osteolysis is an important process in the establishment of bone metastasis. The role which cancer cells play in this process is not fully understood. In this study, we first established a reproducible in vivo bone metastasis model using two types of tumor cells, human breast adenocarcinoma (MDA-231) and human lung squamous cell carcinoma (Hara cells), and examined in vitro characteristics of the tumor cells. Tumor cells injected into the left heart ventricle of nude mice preferentially metastasized to bone, 6 weeks after the inoculation. Histological observation of the bone metastatic lesion showed that tumor cells invaded the bone marrow, and osteoclasts adjacent to fibroblasts were actively resorbing the bone matrix. In vitro analysis of the tumor cells showed that MDA-231 cells express cathepsin K, matrix metalloproteinase 9 (MMP-9), and membrane type-1 matrix metalloproteinase (MT1-MMP), all of which are believed to play an important role in osteoclastic bone resorption. In contrast, Hara cells do not express cathepsin K and MT1-MMP. MMP-9 was expressed at a low level. To assess the osteolytic activity of the tumor cells, an in vitro pit assay was performed. The rabbit osteoclasts formed numerous pits on a dentin slice after 18 h of incubation, whereas tumor cells by themselves did not. Taken together, we conclude that MDA-231 and Hara cells, which metastasize to the bone in vivo, do not have enough ability to achieve bone resorption by themselves, but rather achieve it through activation of fibroblast like cells and osteoclasts.

Matrix metalloproteinases (MMP) have been implicated in several steps of tumor metastasis, such as invasion in the extracellular matrix, intravasation, extravasation, and growth in a distant organ site. Various synthetic MMP inhibitors have been reported to suppress tumor metastasis in animal models. However, there are few reports describing which steps in the metastasis process are most critical for inhibition by MMP inhibitors. In the experimental lung colonization model by i.v. injection of mouse B16-F10 melanoma cells, we found that the daily administration of MMI270 for 2 weeks significantly decreased the number of colonies in the lung compared with the control without affecting the size of colony. Micrometastasis was monitored day 7 post-inoculation by measuring the melanin content in the lung as well as by microscopic examination of the lung tissue sections. Even only twice administrations of MMI270 on the first day after tumor injection significantly inhibited micrometastasis in the lung. In the spontaneous metastasis model using B16-BL6 melanoma cells, lung metastasis was not affected by a continuous administration of MMI270 using a mini osmotic-pump. On the contrary, when mice were subjected to popliteal lymphadenectomy on day 7 after the cell inoculation in the footpad subdermis, the continuous administration of MMI270 significantly suppressed the lung metastasis. These results suggest that the tumor cell extravasation in the target organ is the most critical step where MMPs can play their significant role in the experimental metastasis, and that the lymphatic metastasis process is less susceptible to MMI270 than the hematogenic metastasis process in the spontaneous metastasis model.