Takeshi Iwamura

Correlation between spontaneous metastatic potential and type I collagenolytic activity in a human pancreatic cancer cell line (SUIT-2) and sublines

A human pancreatic cancer cell line (SUIT-2) and four sublines cloned in vitro (S2-007, S2-013, S2-020 and S2-028) were inoculated into nude mice for assessment of metastatic potentials. After 16 weeks of subcutaneous injection, the parent SUIT-2 line metastasized to the lungs and lymph nodes in three of six mice. S2-007 cells presented the highest metastatic potential in pulmonary (5/6) and lymph node (2/6) metastases among the four sublines. No metastasis was found in S2-028. The incidence of spontaneous pulmonary metastasis was correlated with that of pulmonary colonization after intravenous (i.v.) injection of cell clusters (r = 0.87, P = 0.056). Pulmonary colonization potential using single cells, however, did not always reflect a spontaneous metastatic ability. Type I collagenolytic activity in serum-free conditioned media of these cells was correlated effectively with the incidence of spontaneous pulmonary metastasis (r = 0.92, P = 0.026) and pulmonary colonization after i.v. injection of cell clusters (r = 0.95, P = 0.013). Thus, type I collagenolytic activity may possibly be essential to spontaneous cancer metastasis.

High collagenolytic activity in spontaneously highly metastatic variants derived from a human pancreatic cancer cell line (SUIT-2) in nude mice

Cell lines with high metastatic capacity to the lung were established by sequential passage of a human pancreatic cancer cell line (SUIT-2) through the lung of a nude mouse, via the lateral tail vein and from a subcutaneous inoculum. Cells of the parental SUIT-2 and sublines S2-VPx (x-cycle selection from SUIT-2 cells, by Vein-Pulmonary metastasis-culture) and S2-CPx (x-cycle selection, by Cutis-Pulmonary metastasis-culture) were injected intravenously or subcutaneously into nude mice to produce experimental or spontaneous lung metastasis. The S2-VP10 cell line produced pulmonary metastases in 100% of the nude mice, when injected intravenously. It failed, however, to produce more lung colonies than its parent cell line, when injected subcutaneously. The S2-CP8 cell line produced extensive pulmonary metastases in 100% of the nude mice, when injected either intravenously or subcutaneously. This study indicates that the nude mouse provided a good model for in vivo selection of metastatic cells from SUIT-2 cells both experimentally and spontaneously, and that the SUIT-2, S2-VPx, and S2-CPx cell lines will be valuable in the study of human cancer metastasis. We previously reported high levels of ezrin expression in the S2-VP10 and S2-CP8 cell lines. Here we show that these cell lines exhibit a greater capacity to invade or attach to various extracellular matrix components than the parent SUIT-2 cells. The S2-CP8 cell lines also exhibit greater level of type-I and type-IV collagen-degrading activity than the parent SUIT-2 cell line and the S2-VP10 cell line, which shows similar collagen-degrading activity to the parent SUIT-2 cells. In RT-PCR studies, SUIT-2, S2-CP8 and S2-VP10 cell lines constitutively expressed many matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP7, MMP-9, MMP-10 and MMP-14). These results suggest that some parameters that enhance adhesion and invasion are important to both experimental and spontaneous metastasis and the collagen degrading enzymes are predicted to play a key-role during spontaneous metastasis.

Heterogeneities of attachment, chemotaxis, and protease production among clones with different metastatic potentials from a human pancreatic cancer cell line

The present study extends our investigations into the metastatic heterogeneity among four clonal cell lines (S2-007:H, S2-013:M1, S2-020:M2, and S2-028:L) from a human pancreatic cancer cell line (SUIT-2), and extends our discussion the positive correlation between metastatic potential and the type I collagenase activity of the cells, focusing on their interaction with extracellular matrix. Ability to attach to the reconstituted basement membrane (Matrigel) was higher for clone H than clone L during an observation period of 30–60 min, whereas clones M1 and M2 were found to be intermediate in ability. In densitometric and radioactive studies, clone L exhibited the lowest collagenolytic activity against mouse and human type IV collagen, while clone H exhibited the highest activity in the densitometric study and clone M1 was the highest in the radioactive study. The production of urinary-type plasminogen activator was highest in clone L and lowest in clone H. On the other hand, tissue-type plasminogen activator was highest in clone M2 and low in both clones H and L. Clone M2 exhibited the highest chemotactic activity toward diluted Matrigel, whereas clone L had the lowest activity. On the whole, these clones showed heterogenous interactions with an extracellular matrix. It is suggested that the attachment activity to basement membrane and the type IV collagenolytic activity of the cells may be positively correlated with their metastatic potential, whereas the production of urinary-type plasminogen activator was negatively correlated, but confirmation of these findings awaits further study.

Tumor Necrosis Factor α Acts on Cultured Human Vascular Endothelial Cells to Increase the Adhesion of Pancreatic Cancer Cells

We studied the effect of tumor necrosis factor a (TNFa), one of the major inflammatory cytokines, on the adhesive reaction of pancreatic cancer cells to human umbilical vein endothelial cells (HUVECs) and on the hepatic metastasis of cancer cells in vivo. After TNFa stimulation, the expression of E-selectin, an adhesion molecule to neutrophils on HUVECs, increased. In addition, the adhesion of pancreatic cancer cells to HUVECs increased after TNFa stimulation, as was observed with neutrophils. The TNFa-induced adhesive response depended on the extent of sialyl Lewis a expression on cancer cells. The hepatic metastasis in vivo was often observed when cancer cells expressing a high amount of sialyl Lewis a were inoculated intrasplenically after increase in plasma TNFa concentration by lipopolysaccharide administration. Because sialyl Lewis a on cancer cells is a ligand for E-selectin on HUVECs, as sialyl Lewis x on neutrophils, TNFa upregulated the adhesive interaction between sialyl Lewis a on cancer cells and E-selectin on HUVECs. These results suggest that production of TNFa after surgical trauma may stimulate the hematogenic metastasis of cancer cells with a high sialyl Lewis a expression.

Newly Established Assay Method for 25‐Hydroxyvitamin D3 24‐Hydroxylase Revealed Much Lower Km for 25‐Hydroxyvitamin D3 than for 1α,25‐Dihydroxyvitamin D3

An accurate assay method of 25‐hydroxyvitamin D3 24‐hydroxylase (24‐hydroxylase) was established. Kidney mitochondria prepared from vitamin D‐replete rats were treated with polyoxyethylenesorbitan monolaurate. The solubilized suspension was ultracentrifuged at 100,000g for 60 minutes and an aliquot of the supernatant was incubated under the saturating concentrations of substrate NADPH and the mitochondrial‐type electron transferring proteins, adrenodoxin and NADPH‐adrenodoxin reductase. Products were analyzed by high‐performance liquid chromatography (HPLC) monitoring effluents at a wavelength of 265 nm. The maximal velocity of the enzyme in vitamin D‐replete rats was 400 pmol/minute per mg of protein, which was considerably higher than those reported by previous authors who used intact kidney mitochondria as the enzyme source. In applying the new assay method, an interesting property was found; Michaelis constant of 24‐hydroxylase for 25‐hydroxyvitamin D3 [25(OH)D3] was 0.6 μM, which was 35‐fold lower than that for 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3] which was 20.9 μM. This fact indicates that affinity of the enzyme to 25(OH)D3 is 35‐fold higher than that to 1α,25(OH)2D3. These data suggest that 25(OH)D3 is the preferred substrate to 1α,25(OH)2D3.

ADENOCARCINOMA IN THE ANAL CANAL ASSOCIATED WITH A FISTULA : REPORT OF A CASE

Adenocarcinoma in the anal canal associated with an anal fistula is extremely rare, and in most cases its origin is difficult to ascertain because the primary sites have already been destroyed before any diagnosis of malignancy is able to be made. We report herein the case of a 62-year-old man found to have papillary adenocarcinoma with partial mucinous carcinoma associated with an anal fistula. The tumor was not exposed to the mucosal surface of the anal canal or rectum and an abdominoperineal resection was carried out. Macroscopic findings suggested that the tumor had developed from the anal fistula; however, the tumor showed a positive result when tested for O-acetylated sialic acids. This test also proved positive in the mucus of normal rectal mucosa, but not in the mucus of the anal glands. We speculated that the results of these tests may indicate that this tumor could have originated from the rectal mucosa, from where it migrated into the anal fistula.

Establishment and Characterization of a Novel Human Pancreatic Cancer Cell Line (SUIT-4) Metastasizing to Lymph Nodes and Lungs in Nude Mice

A new tumor cell line (SUIT-4) derived from ascites of a patient with carcinoma of the pancreas has been established in tissue culture and in nude mice, and maintained for over 7 years. In tissue culture, the cells grew as a confluent monolayer with piling up of cells in some areas. The population doubling time during the exponential phase of the cell growth was 43.9 h in vitro. Chromosome count ranged from 63 to 68 with a modal number of 67. Subcutaneous injection of cultured cells into the flanks of nude mice resulted in tumor formation with a doubling time of 88.8 h. Histopathologically, xenografts in nude mice were moderately differentiated tubular adenocarcinoma, and the tumor cells showed spontaneous metastasis to the regional lymph nodes in 6 of 21 nude mice and to the lung in 4 of 21. Transmission electron microphotographs confirmed the ductal cell origin of the carcinoma and revealed that the cells had abundant mitochondria and lysosomes. SUIT-4 cells released carcinoembryonic antigen (3.08 × 102 ng/1 × 106 cells/24 h) and carbohydrate antigen 19-9 (4.75 × 104 U/1 × 106 cells/24 h) during exponential cell growth in vitro. Reverse transcriptase-polymerase chain reaction studies revealed that SUIT-4 cells expressed matrix metalloproteinases 1, 3, 7, 10 and 14.

A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative determination in human breast cyst and duct fluids

Radioimmunoassay (RIA) is the most prevalent method for measuring small amounts of hormones, peptides, and other compounds in human body fluids. The method, however, has several problems, such as cross reactions or non-specific reactions of the antibody used. In order to establish an improved method for assaying dehydroepiandrosterone sulfate (DHEAS) and cholesterol, which are the largest components of human breast cyst and duct fluids, we describe a simple, accurate, and sensitive method using high-performance liquid chromatography (HPLC). The samples were treated with cholesterol oxidase for quantitation of dehydroepiandrosterone (DHEA) and free cholesterol, and the respective oxidized substances, 4-androstene-3,17-dione and 4-cholesten-3-one, were extracted with n-hexane. The extracts were analyzed by straight phase HPLC. Effluents were monitored by measuring absorption at 240 nm, where a newly introduced chromophoric group, an alpha,beta-unsaturated ketone, showed intense absorption (epsilon = 16,000). When the total amount of DHEA (DHEAS plus DHEA) was measured, the sample had been solvolyzed by sulfatase beforehand. The amounts of DHEAS were quantified by comparing the amounts of DHEA before and after solvolysis. Levels of free cholesterol, DHEAS, and DHEA in human breast cyst fluids (n = 30) were 1.77 +/- 1.12 mmol/dl, 8.27 +/- 10.24 micromol/dl, and 0.02 +/- 0.02 micromol/dl (means +/- SD), respectively. The levels of sterol and steroid measured in breast duct fluids that were turbid, brown, dark green, or milky in color (n = 9) (mean levels, 3.20 +/- 2.97 mmol/dl for free cholesterol and 14.77 +/- 13.75 micromol/dl for DHEAS) were significantly (P < 0.01) higher than the levels in clear or serous breast fluids (n = 21) (mean levels, 0.14 +/- 0.13 mmol/dl for free cholesterol and 0.04 +/- 0.07 micromol/dl for DHEAS).

Primary, solitary, adult T-cell leukemia / lymphoma of the descending colon

We herein report a patient with adult T-cell leukemia/lymphoma (ATLL) of the descending colon. A 64-year-old man was admitted to our hospital complaining of left lower abdominal pain. Endoscopic examination revealed an ulcerative tumor in the descending colon that was diagnosed as T-cell lymphoma by biopsy. Neither distant organ metastasis nor lymph node swelling was observed by radiographic examinations. Curative excision with left hemicolectomy and regional lymph node dissection was performed. Surgical sections contained ulcerative and superficially elevated lesions; these were continuous with each other. Histological examination revealed diffuse proliferation of medium-sized abnormal lymphoid cells. Immunohistochemically, these lymphoid cells were positive for UCHL-1/CD45RO and CD3 and negative for CD79a, indicating that the tumor was a primary malignant T-cell lymphoma of the descending colon. Integration of the proviral DNA of human T-lymphotropic virus type 1 (HTLV-1) was confirmed by Southern blotting analysis.

New technique for enterostomy of extremely low-birth-weight infants—intestinal anchoring with gauze

BACKGROUND In enterostomy for extremely low-birth-weight infants (<1000 g), the technique of anchoring the intestine for a stoma to the abdominal wall is very difficult because of the small size and fragile nature of the intestine. Here we describe a novel technique for intestinal anchoring in such infants. METHODS In our approach to enterostomy, the intestine is anchored only by a strip of gauze packed into the subcutaneous space. No suturing is performed. The efficacy of this technique was evaluated in 21 infants with less than 1000 g of body weight who have intestinal perforation or obstruction. RESULTS Two patients (9.5%) had complications that were related to the enterostomy. The complications were parastomal and intrastomal intestinal prolapse, both of which were treated successfully by reoperation. Eighteen patients (86%) survived to closure of the enterostoma. CONCLUSIONS Intestinal anchoring with gauze is an easy and effective technique for enterostomy in extremely low-birth-weight infants and can be applied in selected cases.