Importance of Carbohydrates and Platelets in the Early Events of Cancer Metastasis
Hayato Kawakami and Hiroshi Hirano
Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611
Received July 13, 1998; accepted April 9, 1999
Mouse colon carcinoma (colon 26) cells were shown to attach to cultured hepatic sinusoidal endothelial cells only in the presence of PRP (platelet-rich-plasma). Most tumor cells first attached to the endothelial cells, not directly but via aggregated platelets. In addition, almost all of the tumor cells that were attached to the endothelial cells showed positive staining with ECA (Erythrina crystagalli agglutinin), whereas nearly half of the cells before adhesion were negative for ECA binding. ECA-positive cells, when separated from ECA-negative ones, revealed strong potency for metastasis to the liver compared with ECA-negative ones. ECA-positive materials were extracted from the crude membrane fraction of colon 26 cells and were found to suppress the platelet adhesion. These results indicate that the ECA-positive sugar chains on the surface of colon 26 cells should mediate the binding between colon 26 cells and platelets and the attachment of tumor cells to the endothelium. The attachment of colon carcinoma cells to hepatic sinusoidal cells induced as above may cause the successive events in establishing cancer metastasis.
Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582
Received July 11, 1998; accepted April 9, 1999
Employing RMG-1 cells derived from human ovarian adenocarcinoma and COS-1 cells transfected with cDNA for $\beta$ 1,4-galactosyltransferase, we first investigated the intracellular localization of this enzyme and secondly assayed separately the two types of soluble forms of it (designated as normal GalT and GAT) in their culture supernatants. As results, the binding of anti-$\beta$ 1,4-galactosyltransferase monoclonal antibody was strongly positive at the Golgi area surrounding nuclei and the staining pattern was the same between RMG-1 cells and COS-1 transformants. These data demonstrated that the cells were producing $\beta$ 1,4-galactosyltransferase and had the ability to release it into the culture medium. As for the two soluble forms released from the cells, they were simultaneously detected in the culture medium by Western blotting and enzyme immunoassay using monoclonal antibodies, i.e., Mab8628 reactive to both GAT and normal-GalT and Mab8513 specific for GAT, indicating that both were derived from RMG-1 and from the cDNA introduced into COS-1 cells. Since serum-GAT clinically reflects tumor growth more accurately or specifically than normal GalT does, RMG-1 and COS-1 cells having cDNA for $\beta$ 1,4-galactosyltransferase would be promising as sources of these enzymes in order to investigate the differences in the behaviors of normal GalT and GAT associated with ovarian malignant tumors.
Review Differential Distribution of Glycosphingolipid Antigens in the Central Nervous System
Tadashi Tai, Masaharu Kotani and Ikuo Kawashima
Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113-8613
Received October 16, 1997; accepted April 9, 1999
We established an improved method for the generation of mouse monoclonal antibodies (MAbs) to glycosphingolipids especially gangliosides, sialic acid-containing glycosphingolipids, by immunizing mice with purified gangliosides. Using this method, we generated and characterized a large number of the MAbs specific for gangliosides. These MAbs enabled us to examine the distribution of gangliosides in the brain. Immunohistochemical studies revealed that (i) the expression of each ganglioside is restricted to subsets of neuronal cells and their surrounding neuropils in the central nervous system of the adult rat, including cerebrum, cerebellum, hippocampus, and spinal cord, and (ii) the expression of gangliosides changes
during the development. Immunocytochemical studies also revealed a cell type-specific expression of gangliosides in primary cultures of the rat cerebellum. These results suggest that the specific gangliosides may play important roles in various phenomena, involving cell-cell recognition, neurite outgrowth, synaptogenesis, transmembrane signalling, apoptosis, and cell growth and differentiation in the central nervous system.
Key words: Immunohistochemistry, Glycosphingolipid, Monoclonal antibody, Rat brain, Neuron
1 Kanagawa Academy of Science and Technology (KAST), 'Ultimate Mechatronics' Laboratory, KSP Bldg., East 405, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, 2 The University of Tokyo, Department of Precision Machinery Engineering, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656 and 3 Toshiba Machine Co., Ltd. Central Research Laboratory, 2068-3, Ooka, Numazu-shi, Shizuoka 410-8510
Received August 19, 1998; accepted April 9, 1999
The preparation of tissue sections for light microscopy is absolutely necessary in pathology. However, this process is still mainly performed manually. The preparation of many samples from a large number of specimens requires meticulous skills. In addition, it is very difficult to maintain a constant accuracy of the producedure.
To solve these problems we propose two new test machineries for automatic high-speed sample preparations. The first apparatus is conceptually based on attaching pressure-sensitive adhesive tape on the surface of a paraffin block prior to sectioning. This whole process could be repeated automatically. The sectioning is performed by a newly developed high-precision automatic microtome. Using the proposed machine, cut sections could be continuously mounted on the tape and stained automatically.
We also developed the second machine to mount sections on the object glass automatically by using static electricity. The principle of this method is based on electrostatically charging the surface of a paraffin block by which insulation tape was attracted and attached to the surface of the block. After sectioning the block, the section remains adhered on the insulation tape by electrostatic forces. Subsequently, the section is transferred to the object glass by using static electricity as well. In the use of such methods, tissue section on conventional object glass could be made fully automatic.
Review The Practical Side of Image Processing with the Use of a Personal Computer: Automation of the Analyzing Processes Employing NIH Image and Adobe Photoshop{\textregistered} with Power Macintosh Computers
Akihiko Kudo and Hiroshi Hirano
Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611
Received February 15, 1999; accepted May 13, 1999
In histochemistry, image analysis has become quite common and is now regarded as a powerful tool that gives us semi-quantitative and precise interpretation on the obtained image data. However, image analysis itself is a time-consuming and laborious work, and so researchers in the laboratory desire to make this task proceed as automatically as possible. In NIH Image and Adobe Photoshop{\textregistered} software running on Power Macintosh computers, processes by automation are available in both programs, though they are quite different in each software. By use of both of these programs, the analysis processes become easier to set up. To automatically process the data, the suitable preprocessing of digital image data is required. That is, image processing programs are equipped with many processing programs, such as those for filtering, defining the threshold of images, or masking uninteresting areas. Additionally, a method for adequate labeling of the target substances and controlled conditions for the image digitization are always helpful.
Key words: Digital image analysis, NIH Image, Adobe Photoshop{\textregistered}, Automation, Personal computer
Department of Anatomy, Kanazawa Medical University, Uchinada, Ishikawa 920-0293
Received December 5, 1998; accepted May 13, 1999
The generation of reactive oxygen species in the mitochondria was investigated using electron microscopic cytochemistry and energy-filtering transmission electron microscopy in reference to the toxic mediator of paraquat poisoning. When isolated intact mitochondria from rat livers were reacted with paraquat in the presence of NADH, mitochondrial NADH-quinone oxidoreductase generated superoxide anions to cause the destruction of mitochondria which resulted in cell death. Cerium chloride captured hydrogen peroxide and formed cerium perhydroxide precipitates on the outer surface of the mitochondrial outer membrane. This localization was also clearly identified with electron spectroscopic imaging based on electron energy loss spectral analysis.
Key words: Free radicals, Mitochondria, Energy-filtering transmission electron microscope, Electron spectroscopic image
Review Three-dimensional Dynamics of the Golgi Apparatus of Parotid Acinar Cells as Analyzed by Computer Graphics of Cytochemically-marked Serial Sections
Hideaki Tamaki and Shohei Yamashina
Department of Anatomy, Kitasato University School of Medicine, 1-15-1, Kitasato, Sagamihara 228-8555
Received January 5, 1999; accepted March 15, 1999
To correlate the structure and function of the Golgi apparatus, we employed computer-generated three-dimensional reconstruction from electron micrographs of serial sections treated for thiamine pyrophosphatase (TPPase) cytochemistry. Three-dimensional analyses were conducted for rat parotid acinar cells in a resting state, in the course of mitotic division, and during postnatal development.
In resting acinar cells, the TPPase-positive trans elements are the same size as the TPPase-negative middle/cis elements horizontally, and they mutually overlap from side to side. During the mitotic stage, TPPase activity disappears from the disorganized Golgi apparatus and no secretory granule formation is observed. Recovery of the stacked structure and TPPase activity take place in daughter cells at the telophase, which are followed by secretory granule formation. During the differentiation of Golgi apparatus in postnatally developing acinar cells, TPPase activity is expressed following formation of the stacked structure, and then granule formation is initiated. These results clearly demonstrate the close relationship among the stacked structure, TPPase activity and the exocrine function of the Golgi apparatus.
Review In Situ Localization of Gene-Specific Transcription Regulatory Factors by Southwestern Histochemistry
Takehiko Koji
Department of Histology and Cell Biology, Nagasaki University School of Medicine, Sakamoto, Nagasaki 852-8523
Received January 21, 1999; accepted January 22, 1999
To understand better the regulation of gene expression, an analysis of the expression of trans-acting transcription regulatory factors in individual cells is essential. Although cellular analysis of specific mRNA expression is successfully performed by in situ hybridization, the results are not enough to tell whether an elevated level of certain mRNA is due to an increased rate of transcription of the gene or due to a decreased rate of degradation of the mRNA. Recently, we have developed a new method, Southwestern histochemistry, which allows us to localize transcription regulatory factors in situ. In principle, double-stranded (ds) DNA harboring specific response element base sequences is synthesized and labeled with a hapten such as thymine-thymine (T-T) dimer and digoxigenin (Dig). After the reaction of frozen tissue sections with the probe ds DNA, the signal is detected enzyme-immunohistochemically by horseradish peroxidase (HRP)-labeled anti-T-T dimer or anti-Dig, respectively. As a model system, we localized estrogen receptor (ER) in mouse uterine tissue sections by immunohistochemistry with 1D5 monoclonal antibody and Southwestern histochemistry using T-T dimerized estrogen responsive element (ERE) ds DNA of vitellogenin. Consequently, very similar localization of ER was obtained by both methods. This result, together with other examples, further indicates the usefulness of Southwestern histochemistry as a method to localize specific transcription regulatory factors in situ.
Review PCR In Situ Amplification and Catalyzed Signal Amplification: Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization
Yoichi Tani
DAKO LAB, DAKO JAPAN, Kyoto 600-8493
Received December 5, 1998; accepted March 4, 1999
The method of in situ hybridization (ISH) using non-radioactive detection systems has become a very important molecular tool in research and diagnosis. However, nonradioactive ISH techniques generally detect only relatively abundant DNA or RNA molecules. The methods of target amplification, which multiply target molecules, or the methods of signal amplification, which increase the sensitivity of visualization, have been introduced for the detection of rare targets. Polymerase chain reaction (PCR) has been employed in most approaches to target amplification. Although the general principle of PCR in situ amplification is simple, its practical methods have revealed poor amplification efficiency and reproducibility as well as target localization. We have evaluated PCR in situ amplification methods to detect single copies of viral DNA with an experimental model of cervical carcinoma cell lines, and compared them with catalyzed signal amplification methods that we have developed using hapten-conjugated tyramide. Both amplification methods demonstrated the capability of detecting a single copy of DNA. In this overview, the applications and limitations of both target and signal amplification are summarized.
Key words: In situ hybridization, Polymerase chain reaction, Catalyzed signal amplification, Tyramide
For Consistent Detection of Tissue mRNA in Human Surgical Material by Non-Radioactive In Situ Hybridization Technique
Haruo Ohtani
Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ward, Sendai 980-8575
Received December 21, 1998; accepted March 8, 1999
Detection of tissue mRNA in human surgical materials by non-radioactive in situ hybridization technique may be difficult owing to inconsistent retention of tissue mRNA. This inconsistency is generally ascribed to different timing of ligation of feeding arteries during surgical operation. To improve this inconsistency, we adopted fixation in 4% paraformaldehyde/0.5% glutaraldehyde for 12-16 hr with paraffin embedding. This method resulted in improved retention of tissue mRNA: higher in sensitivity and more consistent with cases. Furthermore, this procedure has shortened the color development time of the labeled enzyme, alkaline phosphatase. This helps to reduce non-specific reactivity because prolonged color development time (up to 24) is one of the major causes of non-specific reactions. Cut sections can be stored at 4$\circ$C for more than a year, not resulting in detectable reduction of sensitivity. These are major advantages of this method particularly as human surgical specimens are concerned, because accumulation of cases is important for clinicopathologic analyses. Therefore, this fixation method is, at least, recommendable to improve in situ hybridization technique.
Key words: In situ hybridization, Fixation, Detection sensitivity
Detection of mRNA of Kinesin Superfamily 3A in the Cerebrum and Cerebellum: Biotin-Tyramine-Catalyzed Signal Amplification for In Situ Hybridization
Katsumasa Takahashi 1, Ken Kitamura 1,3 and Toshihiro Takizawa 2,3
1 Departments of Otolaryngology, 2 Anatomy and 3 Center for Molecular Medicine, Jichi Medical School 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498
Received October 26, 1998; accepted April 21, 1999
To improve the sensitivity of in situ hybridization (ISH) carried out using oligonucleotide probes without radionuclides, we employed a new method entailing amplification of ISH signals by catalyzed reporter deposition. This method is based on biotin-tyramine-catalyzed signal amplification (BT-CSA) and was used to detect expression of a relatively scarce polynucleotide, kinesin 3A mRNA, in the central nervous system. Strong signals were detected in the hippocampus and in the granular cell layer of the cerebellum, but expression of kinesin 3A mRNA was not detected in Purkinje cells. ISH performed without the use of radionuclides has the advantage of being comparatively easy to perform, but has the disadvantage of low sensitivity. We showed that the low sensitivity can be overcome by using BT-CSA. Our method also had the advantage of enabling visualization of ISH signals via enzymatic reaction with horseradish peroxidase or by fluorescent staining.
Key words: In situ hybridization, Signal amplification, Tyramine, Oligonucleotide probe, Kinesin 3A
Rapid Communication Osteopontin Expression in Papillary Thyroid Carcinoma
Huagang Zhu 1,3, Yoichiro Kato 2, Reiko Tanaka 3, Takao Obara 3, Kanji Sato 4 and Makio Kobayashi 2
1 Department of General Surgery, First Affiliated Hospital of Anhui Medical University, Jixi Road, Hefei, Anhui, 230022, the People's Republic of China, 2 Department of Pathology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, 3 Department of Endocrine Surgery, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 and 4 Department of Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666
Received May 18, 1999; accepted May 28, 1999
Osteopontin (OPN) is a secreted, adhesive glycophosphoprotein that has been implicated in both normal and pathologic processes. In recent years, it has been suggested that OPN expression also plays an important role in tumorgenesis, tumor progression and tumor metastasis. To investigate OPN expression in papillary thyroid carcinoma, thirty paraffin sections of papillary thyroid carcinoma confirmed pathologically were examined by immunohistochemistry and in situ hybridization. Immunopositive reactions for OPN protein were obtained in the nuclear and perinuclear area in all cases. OPN mRNA was expressed in the carcinoma cells around the microcalcifications. The results demonstrated that OPN mRNA is expressed not only by the infiltrated macrophages but also by the papillary thyroid cancer cells. Hence, OPN expression can be considered a pathological feature of papillary thyroid carcinoma and further study into the relation between OPN and the biological behavior of papillary thyroid carcinoma is warranted.
Note The Use of Alkaline EDTA Solution Improves Heat-induced Epitope Retrieval for Immunohistochemical Localization of MIB-1 Antigen
Young-Sik Kim 1, Sang Ju Lee 1, Insun Kim 1, Gyoung-Mi Kim 2 and Seung R. Paik 2
1 Department of Pathology, College of Medicine, Korea University, Ansan Hospital, Gojan-Dong, Ansan 425-020 and 2 Department of Biochemistry, College of Medicine, Inha University, Nam-Ku, Inchon 402-751, Korea
Received July 30, 1998; accepted March 25, 1999
This study was carried out to show whether the removal of tissue-bound calcium ions during a heat-induced epitope retrieval (HIER) procedure improved immunohistochemical staining of the tissues. In addition, the proteolytic susceptibilities of the fixed tissues before and after various epitope retrieval procedures were also evaluated. Immunohistochemical staining for MIB-1 antigen from paraffin-embedded cases of tonsillitis, gastric adenocarcinomas, and breast carcinomas was performed following heat-treatments for 1, 2, 3, and 5 min in the presence of various buffers such as 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-HCl buffer (pH 8.0), and 1 mM EDTA-NaOH solution (pH 8.0), each of which has different calcium chelating capabilities. In addition, the immunohistochemical staining was performed following 0.05% and 0.1% trypsinizations of the tissues before and after the epitope retrieval. The trypsin-treated tissues were also analyzed with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunohistochemical staining of MIB-1 was most prominent when 1 mM EDTA solution (pH 8.0) was employed during HIER. Although trypsin digestion of the tissues before and after HIER generally increased the staining intensity, the cellular morphology of the background was poorly preserved. In addition, no significant differences were observed with gel electrophoresis of the trypsin digested tissues prepared in the various epitope retrieval solutions. Taken together, it can be suggested that the EDTA solution is more effective than other buffers in exposing epitopes presumably by removing tissue-bound calcium ions. Also, an additional trypsin treatment is not necessary even before and after the HIER as long as the EDTA solution is used.