Heat-shock protein 70 (HSP70) is expressed in response to various stressful stimuli, and generally used as a histological marker of cell damage. While paraquat has previously been shown to cause HSP70 expression as a result of the oxidative cell damage, recent study has shown that HSP70 expression induced by paraquat poisoning is no longer observed at 18hr after the drug administration in the senescence-accelerated mouse liver, thereby raising the question whether the cell damage is always accompanied by elevated HSP70 expression. To affirm the relevance of HSP70 expression as a marker of the cell damage, the expression of HSP70 immunoreactivity and lipid peroxidation in paraquatpoisoned rat liver were examined. The immunostaining of HSP70 was still clearly detected at 24hr after the drug administration. Lipid peroxidation, a biochemical maker of the oxidative cell damage, was also elevated by the same treatment, suggesting that the expression of HSP70 immunoreactivity observed here may reflect the oxidative-damage to liver cells. These results seem to provide evidence supporting the relevance of HSP70 expression as a histological marker of paraquat-induceddamage to rat liver cells.

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Changes in uterine blood vessels were evaluated quantitatively by an immunohistochemical study using an antibody to von Willebrand factor (vWF) during the development of experimentally induced adenomyosis in mice. In all mice treated with pituitary grafting, which is a useful method of inducing adenomyosis, slight adenomyosis was found near the mesometrium 4 weeks after the operation. In the uteri with adenomyosis, the mean surface area and minor axis of blood vessels increased significantly in the endometrium and showed a tendency to increase in the myometrium, while the mean number of blood vessels in both endometrium and myometrium decreased significantly compared with those in the normal uteri. The mean percentage of total surface area of blood vessels to mean total tissue area of either endometrium or myometrium was not different in the two groups. Remarkably dilated venules in the endometrium were frequently found in pituitary-grafted mice. In the myometrium, the mean percentage of surface area and mean number of blood vessels were greater in the mesometrial side than in the antimesometrial side in both the control and pituitary-grafted mice. Thus, pituitary grafting significantly increased the number of dilated venules and decreased the number of microvessels in the uterus, suggesting that vascular changes may be one of the important etiological factors for the development of adenomyosis.

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Osteoclasts secrete protons and lysosomal enzymes through ruffled borders, resulting in demineralization and degradation of bone matrix. Golgi apparatus is thought to play a key role in this process. In order to elucidate the specific functions of Golgi apparatus in osteoclasts, we observed the ultrastructural and cytochemical changes resulting from the administration of brefeldin A (BFA), an inhibitor of Golgi structures, and wortmannin (WT), an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). When treated with BFA, osteoclasts showed a dissociated Golgi apparatus, while the peanut agglutinin (PNA)-positive ruffled borders were poorly developed or disappeared. Moreover, the acid phosphatase (ACPase) activity was somewhat reduced. WT-treated osteoclasts, on the other hand, exhibited no ultrastructural alteration of the Golgi apparatus, but were marked by the disappearance of PNA-positive ruffled borders, and a consequent increase in the number of ACPase-positive vesicles and vacuoles in the cytoplasm. These results indicate that the Golgi apparatus of osteoclasts not only plays an important role in the formation of lysosomal enzymes but also the supply of ruffled border membranes. Furthermore, PI 3-kinase appears to be involved in the transport of lysosomal enzymes and the maintenance of ruffled borders.

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The peroxidase-antiperoxidase (PAP), avidinbiotin-peroxidase complex (ABC) and catalyzed signal amplification (CSA) methods have been used for quantitative or semiquantitative immunohistochemical analysis. It is however unclear whether intensity of immunostaining in sections amplified by using the PAP, ABC or CSA method reflects an antigen content. Thus, relationship between the intensity and the content was examined in antigen-immobilized nitrocellulose filters or serial sections stained by using the PAP, ABC or CSA method. In the present study, we selected cytochromes P-450 2B1/2 (CYP2B1/2), because the immunostaining intensity of the enzymes detected with the indirect immunoperoxidase (IPO) method under saturation conditions is shown to be proportional to the antigen content in sections. For the IPO method, the intensity in filters or sections increased with increasing concentration of anti-CYP2B1/2 antibody, and then saturated. However, a bell-shaped relationship was observed between the intensity and the primary antibody concentration in filters or sections stained by using the PAP, ABC or CSA method. Furthermore, the amplified intensity in filters or sections was disproportionate to the antigen content. Thus, erroneous CYP2B1/2 content was obtained from sections in which the intensity was amplified by the PAP, ABC or CSA method. It is therefore unlikely that the PAP, ABC and CSA methods are suitable for the measurement of antigen content in sections by quantitative immunohistochemistry.

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Extramammary Paget's disease is a slowgrowing malignant disease occurring on the anogenital area and rarely on the axilla. Recent immunohistochemical studies have shown that Paget cells differentiate to secretory cells of sweat glands. However little is known about the expression of glycosaminoglycan in Paget cells and its relation to the differentiation of sweat glands. Therefore we studied the light and electron microscopic localization of anionic sites stained with cationic gold using postembedding method. We also studied the digestibility of anionic sites with enzymes such as neuraminidase, chondroitinase ABC, and heparitinase, because anionic sites in normal eccrine and apocrine sweat glands show different susceptibility to these enzymes. Cationic gold stained 19.6±3.0% of Paget cells at pH 2.0, although most of these anionic sites were not stained at pH 7.4. Anionic sites in Paget cells were completely digested with neuraminidase, however chondroitinase ABC or heparitinase did not digest them. Enzyme susceptibility and paradoxical pH-dependency of anionic sites in Paget cells were the same as those of apocrine secretory cells and completely different from those of eccrine secretory cells, ductal cells of sweat glands or epidermal keratinocytes. Therefore, the expression of glycosaminoglycan labeled with cationic gold indicates that Paget cells differentiate into secretory cells of apocrine sweat gland.

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The in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) method has been used to detect low levels of expression of mRNA in cells and tissues. We applied a newly developed in situ RT-PCR to cells and quantitatively compared the sensitivity of the in situ RT-PCR with that of conventional in situ hybridization (ISH). Human tumor cell lines with either high, moderate, low or no expression of IL-6 mRNA as determined by in vitro competitive RT-PCR were used. In situ RT-PCR was performed using Thermus thermophilus DNA polymerase, special primer sets to form high molecular weight concatemers and an in situ digoxigenin (DIG) direct labeling technique. Conventional ISH was performed using DIG-labeled cRNA probe. Specific signals of IL-6 mRNA were observed in the cytoplasm of positive cells by both in situ RT-PCR and ISH. In situ RT-PCR was more than 100-fold more sensitive than ISH. The specificity of in situ RT-PCR was confirmed by appropriate control tests. The data indicated that the sensitivity of our in situ RT-PCR applied to culture cells was significantly higher than that of ISH, and that the technique retained high specificity.

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The cyclin D1 gene is often amplified in solid tumors. Six human melanoma cell lines derived from tumors at various stages of progression were analyzed by double-target fluorescence in situ hybridization (FISH) and dot blot hybridization to detect cyclin D1 gene amplification and deletion. FISH was performed using a probe for cyclin D1 and an alpha satellite of the chromosome 11 probe, to analyze the cyclin D1 copy number relative to the numbers of chromosome 11 in individual cells. Copies of both the cyclin D1 gene and chromosome 11 were observed in the nuclei of all cell lines. However, the copy numbers of the cyclin D1 gene and of chromosome 11 were closely associated and the copy number ratio of cyclin D1 to chromosome 11 (cyclin D1/chromosome 11) was almost one in all cell lines. This finding indicated that the extra copies of cyclin D1 were always accompanied by extra copies of chromosome 11. At the cellular level, cyclin D1 amplification was detected in fewer than 5% of interphase nuclei in only two cell lines, and the gene was deleted in all cell lines to various degrees. Dot blot hybridization revealed no amplification of cyclin D1 in any of the cell lines. We concluded that amplification of the cyclin D1 gene is rare in these melanoma cell lines.

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To clarify the in vivo effects and the changes in kinetic parameters of soft tissue sarcoma caused by anticancer agents, we performed double-labeling using bromo- (BrdU) and iododeoxyuridine (IUdR). A malignant fibrous histiocytoma cell line was transplanted into nude mice, and treated with 3 anticancer agents: cisplatin, doxorubicin and vincristine. The following parameters were determined: BrdU labeling index (LI), duration of S-phase (Ts), total cell cycle time (Tc), Ki-67 labeling index (KLI), PCNA labeling index (PCNA-LI), and actual tumor volume doubling time (Tv). In addition, the apoptotic cell ratio (apoptotic index; Al) was calculated in the serial specimens using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL).
Vincristine inhibited the tumor growth the most effectively (Tv: -5.8 days). Doxorubicin suppressed the tumor growth better (Tv: 12.7 days) than cisplatin (Tv: 9.1 days). Tc values showed significant differences among the drug groups (ANOVA analysis; p<0.05), and also correlated with Tv (Pearson's analysis; R>0.7, p<0.05) on Day 1. Al peaked on Day 10 and decreased afterward in every drug group.
Anticancer agents effectively suppress tumor growth not only by necrosis and apoptosis, but also by prolonging Tc. Tc values are possibly useful for planning chemotherapy for sarcoma patients.

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