Summary Murine L5178Y-ML cells, when transplanted subcutaneously into the flank of (BALB/c x DBA/ 2)F, mice, grew locally and always formed spontaneous metastases in the liver. Even after surgical removal of the primary tumour mass 5 or 7 days after tumour cell inoculation, all mice died due to liver metastases within 18 days. Using this model of tumour metastasis, we examined whether serine protease or deoxyribonuclease I (DNase I) would affect metastasis. Spontaneous liver metastasis of L5 1 78Y-ML cells was (Sugihara et al., 1990). We also observed that these alterations in frequency of metastasis were direct reflections of changes of sequestration intensity in the lungs of the tumour cells, probably due to aggregation and disaggregation in vivo (Sugihara et al., submitted for publication). Our electron microscopic study showed that this aggregation seemed to be caused by formation of a sleeve-like structure which surrounded the cells and connected them with each other. This structure probably originated from the cell surface glycocalyx in some sort of association with DNA molecules (Sugihara et al., 1991 Recently we examined a spontaneous metastasis model in mice using the L5178Y-ML cell line established by Watanabe and his co-workers (Watanabe et al., 1988). The L5178Y-ML cell line was derived from L5178Y, a murine T-lymphoma cell line isolated from a methylcholanthrene-induced lymphoma in DBA/2 mice. In order to establish a liver-oriented metastatic tumour cell line, cells were propagated further sequential cycles of subcutaneous inoculation of L5178Y cells which were isolated from the metastatic liver masses. L5178Y-ML cells, therefore, metastasised predominantly to the liver after intravenous or subcutaneous injection (Watanabe et al., 1988). In our preliminary study. L5178Y-ML cells were also sensitive to serine proteases and to DNase I in tumour cell aggregation and disaggregation, respectively. The present study was, therefore, undertaken to confirm the effects of protease and nuclease in altering the blood-borne metastatic rate in this model which more closely resembles clinical blood-borne metastasis.
Summary Serine proteases, such as a-chymotrypsin or elastase, caused an aggregation of rat ascites tumour cell lines, AH-130, AH-109A and YS, in a protein free medium which preserved the cell viability. This aggregation, which was monitored spectrophotometrically, was dependent upon the protease activities and was resistant to treatment with either a calcium chelating reagent (EDTA) al., 1973;Liotta et al., 1980;Sloane et al., 1982;Wang et al., 1980). It has also been shown that an important event in blood-borne metastasis is the intercellular adhesion and aggregation of circulating tumour cells (Nicolson & Winkelhake, 1975). The aggregated state of tumour cells in the microcirculation may be favourable to lodgement in distant organs. In this sense, the role of the plasma clotting system (Kinjo, 1978) or platelets (Hara et al., 1980) in blood-borne metastasis has been emphasised with special reference to the formation of floating or plugged masses of tumour cells.In our preliminary experiments, serine proteases such as a-chymotrypsin or elastase caused an aggregation of rat ascites tumour cell lines, AH-130, AH-109A and YS, without any appreciable change in cell viability. In the present study, we have examined a potential augmenting effect of proteaseinduced cell aggregation in vitro and in vivo on blood-borne metastasis using the lung-colonisation model with rat ascites tumour cells. In addition, we also examined the effectiveness of DNase I treatment (which prevented the protease-induced cell aggregation) on blood-borne metastasis in the lung. Materials and methods AnimalsFemale Donryu rats weighing 130-180 g were generally used in this study. These rats received a standard rat pellet diet and tap water ad lib. ReagentsCrystallised bovine pancreatic a-chymotrypsin, elastase, neuraminidase, bovine muscular actin, bovine pancreatic DNase I and calf thymus DNA were purchased from Sigma Chemical Co. (St Louis, MO, USA). Chymostatin and elastatinal were purchased from Peptide Institute Inc. (Osaka, Japan). Hanks' balanced salt solution (HBSS) (without phenol red) was purchased from Nissui Pharm. Co. (Tokyo, Japan), HBSS (without Ca2 , Mg2 + and phenol red) from Gibco (Grand Island, NY, USA). An assay kit for lactate dehydrogenase (LDH) was obtained from Shinotest Laboratories (Kanagawa, Japan).The DNase I was further purified using a high performance liquid chromatography system (LKB) with a TSK gel 3,000 SW gel permeation column (Tosoh). The final preparation was pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate with or without 1% P-mercaptoethanol.
The case of a 59-year-old man with paralytic ileus (pseudo-obstruction) associated with pheochromocytoma is reviewed. Paralytic ileus is believed to have been the result of overstimulation of a and /3 receptors on the intestine by catecholamines. Phentolamine, bunazocin, propranolol, bethanechol and midaglizole in single administrations or in combination were administered. Phentolamine infusion clearly relieved the symptom, but ileus recurred, and the patient died of respiratory failure, liver dysfunction and disseminated intravascular coagulation syndrome. The significant role of catecholamines in causing these symptoms is discussed, and the management of this relatively rare complication is reviewed. Recently we treated a case of severe intestinal distension in a pheochromocytoma patient with multiple metasases. There is, as yet, no definite treatment regimen for paralytic ileus, thus the clinical course and autopsy findings of this case are presented. CASE REPORTA 59-year-old man, brought by ambulance, was admitted to our department because of severe constipation, abdominal distension and one week of jaundice. A diagnosis of malignant pheochromocytoma with multiple metastases had been made previously. The clinical history of the patient from the age of 38 is shown in Table 1. He was immediately transferred to the Intensive Care Unit of our hospital.The patient was drowsy, blood pressure was 130/90 mmHg, and pulse rate was 120/min. Skin and conjunctiva bulbi were icteric. No rale was audible and heart sounds were clear. The abdomen was quite distended, and tympanic. Livedo racemosa was present bilaterally in the upper and lower extremities.Chest X-rays revealed bilateral pleural effusion, enlargement of the heart shadow, and a mass in the right upper field which was thought to be a metastatic lesion of the third rib. Plain films of the abdomen (Fig. 1) demonstrated a distended intestine, but no mechanical obstruction of the colon was found in the endoscopic examination. Echo-
Serine proteases cause aggregation of the rat ascites tumor cell lines AH 130, AH 109A and YS in vitro, and the tumor cell aggregates are dissolved by treatment with DNase I. We previously demonstrated that these events played a critical role in the augmentation or reduction of experimental blood borne metastasis of these cell lines. In the present study, the ultrastructural features of this protease dependent aggregation were analysed. Transmission and scanning electron microscopy revealed that after the protease treatment each tumor cell was surrounded by a thin membranous (sleeve‐like) structure. This sleeve like structure was stained with ruthenium red to an intensity similar to the cell surface of the control. Adjacent cells became attached to each other with microvilli via this fine structure. Immuno‐electron microscopy revealed DNA antigen as dense patches on the sleeve‐like structure or as faint and diffuse deposits on the outer surface of the cells by indirect immunoperoxidase staining using an anti‐DNA monoclonal antibody. Both the sleeve like structure and immunopositive deposits disappeared after treatment with DNase I. Neither cell viability nor the normal ultra‐structure of their organelles was influenced by the enzyme treatment. These results indicate that serine protease induced tumor cell aggregation is due to cellular contact via the sleeve‐like structure, which probably originates from the cell surface glycocalyx in association with DNA molecules of unknown origin.
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