Noritoshi Takeichi

Enhancing Effects of Epidermal Growth Factor on Human Squamous Cell Carcinoma Motility and Matrix Degradation But Not Growth

In order to ascertain the effects of epidermal growth factor (EGF) on human cancer invasion abilities, three cell lines of human oral squamous cell carcinoma were studied using a phagokinetic track assay and zymography. EGF (1-100 ng/ml) was found to inhibit the growth but enhance the random motility of all three cell lines in a concentration-dependent fashion. Exposure to EGF, dose-dependently, led to an increased production of urokinase-type plasminogen activator and M(r) 92 kD matrix metalloproteinase by the same cells. These results strongly suggest that EGF may promote human squamous cell carcinoma invasion and metastasis.

Conversion of Human Colonic Adenoma Cells to Adenocarcinoma Cells Through Inflammation in Nude Mice

The roles of inflammation in the malignant progression of tumors during multistep carcinogenesis have been much discussed but remain to be elucidated. To determine the direct contribution of inflammation to colon carcinogenesis, we established a new model of progression of human colonic adenoma cells using a nude mouse; the progression is accelerated by coimplantation of a plastic plate. The FPCK-1–1 cell line, derived from a colonic polyp in a patient with familial adenomatous polyposis, is nontumorigenic when injected subcutaneously into nude mice in a cell suspension of up to 5 × 106 cells per mouse. However implantation of 1 × 105 FPCK-1–1 cells attached to a plastic plate induced first acute and then chronic inflammation, and formed progressively growing tumors that were histologically determined as moderately differentiated adenocarcinoma in 65% of mice. Moreover cell lines established from the growing tumors were found to be tumorigenic when injected into mice even without a plastic plate. The tumor arising from the adenoma cells implanted attached to a plastic plate was surrounded by highly proliferating fibrous stroma. This fibrous tissue was considered essential for malignant progression, rather than for attachment to the plastic plate substrate, because the tumors were formed after injection of FPCK-1–1 cells into the fibrous tissue from which the plastic plate had been removed before the cell injection. The conditioned medium (CM) obtained from the fibroblasts derived from a plastic plate-associated stromal tissue was found to contain factors that stimulated growth of FPCK-1–1 cells, but not of the derivative progressor cell lines. The factor was stable to heating and neuraminidase treatment, but labile to trypsin treatment. The main growth-potentiating activity was contained in the fraction larger than 100 kDa. In contrast, the activity to promote FPCK-1–1 cell growth was not present in the CM of subcutaneous fibroblasts from untreated nude mice or the fibroblast cell lines C3H10T 1/2 and NIH3T3. These results demonstrated that inflammation-associated stroma promoted the conversion of colonic adenoma cells to adenocarcinoma cells.

SPONTANEOUS HEPATITIS IN LONG-EVANS RATS: A Potential Animal Model for Fulminant Hepatitis in Man

Spontaneous hepatitis associated with severe jaundice occurred in 90% of an inbred strain of Long‐Evans rats. The rapidly progressive syndrome was characterized by abrupt onset, hyperbilirubinemia and increased serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, associated with massive and multifocal necrosis of the liver. This strain should provide a useful animal model for analysis of the pathogenesis of fulminant hepatitis in humans. ACTA PATHOL JPN 38: 1369–1375, 1988.

Natural cytotoxic autoantibody against thymocytes in spontaneously hypertensive rats

Abstract A strain of SHR rats, which spontaneously develops hypertension and periarteritis nodosa, had a decreasing number of rosette-forming T cells in their thymuses and a progressive decline in cellular immune functions by aging. They were found to produce natural thymocytotoxic autoantibody (NTA) detected by a complement-dependent cytotoxicity test. This autoantibody occurred from 1 month of age and throughout life; the incidence was more than 60% of SHR rats at any age. Thymocytes from all six rat strains tested showed similarly high sensitivity to NTA but none of the strains tested produced NTA except the SHR strain. Rosette-forming thymocytes of WKA rats, which were separated by Ficoll gradient, showed much higher cytotoxic sensitivity to NTA than did whole thymocytes and nonrosetting thymocytes. The cytotoxicity of NTA was weak or negative for spleen cells, lymph node cells, bone marrow cells, and blood lymphocytes of WKA rats. However, the cytotoxic activity of NTA was completely absorbed with the thymus, spleen, lymph node cells, and brain homogenates and was partially absorbed with bone marrow cells, but not with liver and kidney homogenates. NTA in SHR rats was an IgM-globulin as determined by sensitivity to 2-ME treatment and by Sephadex G-200 column chromatography. These results suggest that NTA is responsible for the selective suppression of T-cell functions in SHR rats.

Selective removal of copper bound to metallothionein in the liver of LEC rats by tetrathiomolybdate.

LEC rats (Long-Evans with a cinnamon-like coat color) have a genetical defect in Cu metabolism. Male LEC rats aged 10 weeks were injected ip with TTM at a dose of 5 or 10 mg/kg body weight for 8 consecutive days and killed one day after the last injection. Cu that had accumulated in the liver at a concentration of 251 micrograms/g liver was decreased to 82.7 or 74.3 micrograms/g liver respectively, by the treatment. Although most of Cu was bound to MT as a soluble form before TTM treatment, the metal remaining in the liver after the treatment was present almost exclusively in the non-soluble fraction. Zinc (Zn) present, bound to MT before the treatment, was also partly removed from the liver by TTM, and the Zn remaining in the liver after the treatment was revealed to be bound to MT (Zn-MT) by high performance liquid chromatography-atomic absorption spectrophotometry. Iron (Fe) in the liver was not affected by TTM treatment. Cu in the kidneys and spleen increased by TTM treatment, while Zn and iron were not affected. Treatment of LEC rats with severe jaundice effectively cured the animals from otherwise lethal signs by only two ip injections of TTM at a dose of 10 mg/kg body weight.

Correlation between the Presence of Microvilli and the Growth or Metastatic Potential of Tumor Cells

We used an electron microscope to examine microvilli which appear on the surfaces of various tumor cells with high or low growth potential and/or metastatic ability. The results show that a greater number of microvilli appeared on the surfaces of tumor cells (QRpP and ERpP) which possess high growth potential than on tumor cells (QR and ER) with low growth potential. We also observed that microvilli were more abundant on the surface of highly metastatic clone cells, i.e. c‐SST‐2 (cl‐2), mouse B16 melanoma (F‐10) and human colon carcinoma (KM12SM) than on weakly metastatic clone cells, c‐SST‐2 (cl‐4‐2), B16 (F‐1) and (KM12C). At the same time, more microvilli were observed on the surface of B16 BL6 cells, which were obtained from the metastatic site of the B16 F10 cells, than on the surface of the parent B16 F10 cells. Immunoelectron microscopy revealed that the c‐neu oncogene product, which is closely related to an epidermal growth factor receptor, was positively stained in the microvilli of tumor cells (ERpP) with high growth potential and high metastatic ability, whereas the tumor cells (ER) with low growth potential and weak metastatic ability were not stained. These findings suggest that the increased presence of microvilli correlates closely with the growth potential and metastatic ability of tumor cells.

Depression of T Cell-Mediated Immunity and Enhancement of Autoantibody Production by Natural Infection with Microorganisms in Spontaneously Hypertensive Rats (SHR)

We studied the effects of breeding conditions on the development of immunological abnormalities in spontaneously hypertensive rats (SHR) with congenital T cell depression. The depression of T cell functions, the production of natural thymocytotoxic autoantibody (NTA), and the development of polyarteritis nodosa were more evident in SHR reared under a conventional (CV) environment than in specific‐pathogen‐free (SPF) SHR bred in a semi‐barrier system. Enhancement of these immunologic abnormalities was also observed by the conventionalization of SPF‐SHR. A high frequency of antibodies to mouse hepatitis virus (MHV), Sendai virus, and Mycoplasma pulmonis was detected in CV rat sera, whereas no antibodies were detected in SPF‐SHR. The experimental infection of Sendai virus induced the enhancement of T cell depression and of NTA production in SPF‐SHR. We interpret these results to mean that the natural infection of microorganisms causes an acceleration of immunologic abnormalities in SHR reared in a CV environment.

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timms method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving copper as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timms method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with d-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timms method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.

Progression of Weakly Malignant Clone Cells Derived from Rat Mammary Carcinoma by Host Cells Reactive to Plastic Plates

Tumor progression is the process by which tumor cells acquire more malignant properties, such as invasiveness and metastasis, during tumor development. To elucidate mechanisms of tumor progression, we examined the role of interactions between the tumor and its host by using a cloned cell line, ER‐1, which was derived from a rat mammary carcinoma. ER‐1 is weakly tumorigenic and non‐metastatic when s.c. injected into syngeneic hosts in single cell suspension. However, ER‐1 cells show a high incidence of lethal, growth when s.c. implanted (5 × 102 cells), being attached to a 10 × 5 × 1 mm polystyrene plate. Tumor cell lines (PLT) obtained from tumors which had arisen from the plate‐attached ER‐1 cells no longer required plates for their growth in normal hosts, and had acquired metastatic ability to the lungs. The malignant phenotypes of PLT were stable under a usual culture condition for at least 6 months. Furthermore, the incidence of tumor development increased when small numbers of ER‐1 cells were injected onto plates (or at their periphery) which had previously been implanted s.c. without tumor cells. The tumorigenicity of ER‐1 cells increased after they were cocultivated for more than 30 days with host reactive cells obtained from the tissues surrounding the plates. These results suggest that host cells reactive to the foreign body (plastic plate) may not only promote the local growth of ER‐1 cells but also convert them into much more malignant tumors.

Enhancement of Tumorigenicity and Invasion Capacity of Rat Mammary Adenocarcinoma Cells by Epidermal Growth Factor and Transforming Growth Factor‐β

We have studied the effects of growth factors and cytokines on the tumorigenicity and invasion capacity of tumor cells by using regressor and progressor tumor cell lines (ER‐1 and ERpP, respectively) derived from an SHR rat mammary adenocarcinoma. ER‐1 cells regress spontaneously whereas ERpP cells show invasive growth and high metastasis to lung and other organs in syngeneic SHR rats. When ER‐1 cells were pretreated with either epidermal growth factor (EGF) or transforming growth factor‐β (TGF‐β) for 24 h in vitro, and intraperitoneally transplanted into SHR rats, they grew and killed the host, whereas ER‐1 cells pretreated with tumor necrosis factor‐α did not. Tumorigenicity and invasion capacity of ERpP cells were also enhanced by treatment with EGF and TGF‐β. The ER‐1 cells pretreated with EGF, once grown in vivo, had acquired irreversible tumorigenicity and invasion capacity without requiring further EGF treatment, and the enhanced malignancy was irreversible. These findings suggest that growth factors play an important role in acquisition of malignancy of tumor cells.