Loss of E-cadherin-dependent cell-cell adhesion due to mutation of the beta-catenin gene in a human cancer cell line, HSC-39.

Detachment of cell-cell adhesion is indispensable for the first step of invasion and metastasis of cancer. This mechanism is frequently associated with the impairment of either E-cadherin expression or function. However, mechanisms of such abnormalities have not been fully elucidated. In this study, we demonstrated that the function of E-cadherin was completely abolished in the human gastric cancer cell line HSC-39, despite the high expression of E-cadherin, because of mutations in one of the E-cadherin-associated cytoplasmic proteins, beta-catenin. Although immunofluorescence staining of HSC-39 cells by using an anti-E-cadherin antibody (HECD-1) revealed the strong and uniform expression of E-cadherin on the cell surface, cell compaction and cell aggregation were not observed in this cell. Western blotting (immunoblotting) using HECD-1 exhibited a 120-kDa band which is equivalent to normal E-cadherin. Northern (RNA) blotting demonstrated a 4.7-kb band, the same as mature E-cadherin mRNA. Immunoprecipitation of metabolically labeled proteins with HECD-1 revealed three bands corresponding to E-cadherin, alpha-catenin, and gamma-catenin and a 79-kDa band which was apparently smaller than that of normal beta-catenin, indicating truncated beta-catenin. The 79-kDa band was immunologically identified as beta-catenin by using immunoblotting with anti-beta-catenin antibodies. Examination of beta-catenin mRNA by the reverse transcriptase-PCR method revealed a transcript which was shorter than that of normal beta-catenin. The sequencing of PCR product for beta-catenin confirmed deletion in 321 bases from nucleotides +82 to +402. Southern blotting of beta-catenin DNA disclosed mutation at the genomic level. Expression vectors of Beta-catenin were introduced into HSC-39 cells by transfection. In the obtained transfectants, E-cadherin-dependent cell-cell adhesiveness was recovered, as revealed by cell compaction, cell aggregation, and immunoflourescence staining. From these results, it was concluded that in HSC-39 cells, impaired cell-cell adhesion is due to mutations in beta-catenin which results in the dysfunction of E-cadherin.

Heparin-induced thrombocytopenia complicated with massive thrombosis of the inferior vena cava after filter placement.

A 45-year-old man presented with deep vein thrombosis of the right leg and bilateral pulmonary embolism. Heparin was administered on the initial one and a half days. On the 3rd day, an inferior vena cava (IVC) filter was placed with a heparin flush, after which massive IVC thrombosis developed. The platelet count was 221000/mm3, decreased 42% from the initial level, but remained within the normal range. Heparin was replaced by argatroban on the 13th day. The platelet count increased to 355000/mm3 on the 15th day. The patient was positive for antibody against complexes of heparin and platelet factor 4, and was diagnosed as heparin-induced thrombocytopenia with thrombosis syndrome (HITTS). When thrombosis develops during heparin treatment, it is important to suspect HITTs and to assay for the associated antibodies, regardless of the actual platelet count.

Concentration of bisphosphonate (incadronate) in callus area and its effects on fracture healing in rats.

The aim of the present study was to investigate effects of incadronate on early stages of fracture healing and to detect its concentration in callus area (Ca.Ar). Rats were injected three times per week with either two doses of incadronate (10 microg/kg and 100 microg/kg) or vehicle for 2 weeks. Femora were then fractured and fixed and animals were divided into pretreatment (P-10 and P-100) and continuous treatment (C-10 and C-100) groups. Incadronate treatment was stopped in P-10 and P-100 groups but continued in C-10 and C-100 groups. Animals were killed at 2 weeks and 4 weeks after fracture. Results showed significantly large callus, compared with the control, only in C-100 group at 4 weeks but not at 2 weeks. Both linear labeled surface (LS) and eroded surface (ES) decreased significantly in C-10 and C-100 groups at 2 weeks and 4 weeks. Osteoclast number (N.Oc) decreased significantly in C-10 and C-100 groups at 2 weeks but increased slightly at 4 weeks. However, there was no significant difference in the above parameters in P-10 and P-100 groups at 4 weeks. Apoptotic osteoclasts were observed only in the C-100 group at 4 weeks. A time-course decrease in incadronate concentration was detected in P-10 and P-100 groups whereas an increase was observed in C-10 and C-100 groups. These findings suggest that larger callus under incadronate treatment may result from the inhibition of bone resorption, histological characteristics of callus may be correlated with incadronate concentration, and metabolism of incadronate in bone may be related to the rate of bone turnover.

Effect of bisphosphonate (incadronate) on fracture healing of long bones in rats.

This study was designed to test whether bisphosphonates disturb the process of fracture healing. Female Sprague-Dawley rats were injected with either two doses of bisphosphonate (incadronate) (10 microg/kg and 100 microg/kg) or vehicle three times a week for 2 weeks. Right femora were then fractured and fixed with intramedullary wires. Incadronate treatment was stopped in pretreatment groups (P-10 and P-100 groups), while the treatment was continued in continuous treatment groups (C-10 and C-100 groups). Animals were sacrificed at 6 and 16 weeks after surgery. Soft X-ray of all fractured femora was taken. After mechanical testing, fractured femora were stained in Villanueva bone stain and embedded in methyl methacrylate. Cross-sections near fracture line were analyzed by microradiography and histomorphometry. Radiographic study showed that bony callus was present in all the fractures and incadronate treatment led to a larger callus, especially in C-100 group at both 6 and 16 weeks. Histologic study showed that the process of fracture healing in pretreatment groups was delayed at 6 weeks, but reached control level thereafter and showed same characteristics as in control at 16 weeks. Woven bony callus could still be seen in continuous treatment groups at 16 weeks. Mechanical study indicated that the ultimate load of C-100 group was slightly higher than the other treatment groups and control. The results suggest that pretreatment with incadronate did not affect fracture healing at 16 weeks after fracture. However, continuous incadronate treatment could lead to larger callus, but it delayed remodeling process during fracture healing, especially with high-dose treatment.

Long-term effect of incadronate disodium (YM-175) on fracture healing of femoral shaft in growing rats.

The aim of this study was to investigate the long-term effect of incadronate on fracture healing of the femoral shaft in rats. Female Sprague-Dawley 8-week-old rats were injected subcutaneously (sc) with either vehicle (V group) or two doses of incadronate (10 microg/kg and 100 microg/kg) three times a week for 2 weeks. Right femoral diaphysis was then fractured and fixed with intramedullary stainless wire. Just after fracture, incadronate treatment was stopped in pretreatment groups (P groups: P-10 and P-100) or continued in continuous treatment groups (C groups: C-10 and C-100). All rats were killed at 25 weeks or 49 weeks after surgery. Fractured femur was evaluated radiologically and mechanically and then stained in Villanueva bone stain and embedded in methyl methacrylate. Undecalcified cross-sections from the fracture area were evaluated microradiologically and histomorphometrically. Radiographic observation showed that the fracture line disappeared in all groups. Cross-sectional area in the C-100 group was the biggest among all groups and in the C-10 group was larger than that in the V group at 25 weeks. Histological and histomorphometric observations showed that the process of fracture healing was delayed under continuous treatment with incadronate as evidenced by the delay of both lamellar cortical shell formation and resolution of original cortex in C groups. Percent linear labeling perimeter, mineral apposition rate (MAR), and bone formation rate (BFR) in C groups significantly decreased compared with the other groups, indicating that the callus remodeling was suppressed under continuous treatment, especially with a high dose. Mechanical study showed that the stiffness and ultimate load of the fractured femur in the C 100 group were the highest among all groups at both 25 weeks and 49 weeks. In conclusion, this study showed that long-term continuous treatment with incadronate delayed the process of fracture healing of femur in rats, especially under high dose but it did not impair the recovery of mechanical integrity of the fracture.

Preadministration of incadronate disodium can prevent bone loss in rat proximal tibial metaphysis when induced by hindlimb immobilization by bandage.

The purpose of this study is to determine whether short-term preadministration of bisphosphonates prevents bone loss in rat proximal tibial metaphysis when induced by hindlimb immobilization by bandage. Six-month-old female Sprague Dawley rats were injected with incadronate disodium (YM-175, 10 micrograms/kg) or vehicle, three times per week for 2 weeks (YM or V groups). Then, the left hindlimb was fixed to the abdomen with a bandage (V-B, YM-B groups), or only the abdomen was bandaged as control (V-SHM, YM-SHM groups), for 4 weeks. The animals were subsequently killed and left proximal tibiae were processed undecalcified for quantitative histomorphometric evaluation. Immobilization-induced cancellous bone loss resulted not only from increased percent eroded surface area but also from decreased percent labeling surface and bone formation rate in V-B compared with V-SHM animals. In contrast, preadministration of YM-175 decreased percent eroded surface significantly and prevented the loss of cancellous bone mass in YM-B compared with V-B animals. Cancellous bone mass was neither increased nor decreased by preadministration of YM-175 in YM-SHM animals. Our results suggest that preadministration of bisphosphonates is effective in prevention of bone loss at the tibial metaphysis when induced by hindlimb immobilization in rats.

Acute exacerbation of hepatitis due to reactivation of hepatitis B virus with mutations in the core region after chemotherapy for malignant lymphoma.

A 43-year-old Japanese man who was positive for hepatitis B surface (HBs) antigen and HB e antibody, underwent chemotherapy for non-Hodgkin's lymphoma. After the chemotherapy he suffered from acute exacerbation of hepatitis because of reactivation of HBV. Recovery was achieved with interferon-alpha, glucagon-insulin therapy, and plasma exchange. Mutations were detected in codons 97, 100, 129, and 131 of the core region of HBV. The peptide encoded from the core region including such mutations possibly had greater antigenicity to induce cytotoxic T cell activity in the host. Core region mutations may be a crucial cause of the acute exacerbation of hepatitis B seen after chemotherapy.

Fibrolamellar carcinoma of the liver.

We report a resected case of fibrolamellar carcinoma (FLC) of the liver that occurred in a 21 year-old Japanese male with a normal liver. Lymph node metastases around the common hepatic artery and the hepatic hilum were revealed by a post-operative histological examination. He was in good health 7 months after the surgery. This case demonstrates typical findings of FLC, radiologically and histologically. The details are described in this report. Moreover, after reviewing 12 Japanese cases, we emphasize the differential diagnosis of focal nodular hyperplasia (FNH) as the following: 1) Calcification is frequently seen in FLC. 2) The central scar of FLC is visualized as an area of low intensity on T2-weighted magnetic resonance imaging (MRI).

Gluteal compartment syndrome after abdominal aortic aneurysm repair.

A 66-year-old man underwent emergency surgery for a ruptured abdominal aortic aneurysm associated with right common and internal iliac aneurysms. Postoperatively, his right buttock was distended and tender to compression. A CT scan revealed an extremely swollen right gluteus maximus with decreased density. Macromyoglobinuria was noted, and creatine kinase and myoglobin were elevated: 87,800 IU/l and 144,300 ng/ml, respectively. Renal function had deteriorated and he was treated with hemodialysis until the 15th postoperative day. The patient recovered without any discomfort to the buttock or intermittent claudication. To our knowledge this is the first documented case of gluteal compartment syndrome after the repair of an abdominal aortic aneurysm.

[Outpatient chemotherapy with continuous infusion of 5-fluorouracil (CI 5-FU) and intravenous bolus leucovorin (IVB LV) in advanced gastrointestinal cancer].

This study was designed to evaluate the efficacy, toxicity, and quality of life (QOL) of outpatient chemotherapy with 5-FU and LV in advanced gastrointestinal cancer. Treatment consisted of CI 5-FU 450-500 mg/body/day, days 1-28, IVB LV 30 mg/body q wk. 5-FU was administered with the Baxter infusor (0.5 ml/hr). LV was not administered if Grade 2 mucositis occurred. Seven patients (pts) with advanced gastrointestinal cancer (esophageal -2, gastric -1, colorectal -4) have been treated to maintain the efficacy of prior inpatient chemotherapy, and two pts with colorectal cancer instead of oral UFT. And to evaluate toxicity and QOL, six pts (bile duct -1, gastric -2, colorectal -3) treated as adjuvant chemotherapy were added. The median duration of response of the nine pts was 3.03 months. Because of mucositis, the administration of LV was restricted. The mean administration of it was 2.1 times (62 mg). Grade 3 or 4 toxicities of mucositis were seen in 40% of the cases. Grade 1 or 2 skin toxicities were seen in all pts. Mean score of QOL in these outpatients chemotherapy was 78.0 +/- 11.5, ranked between inpatient chemotherapy (5-FU+CDDP 59.0 +/- 13.8) and oral UFT (91.8 +/- 6.1). In conclusion, this schedule of 5-FU and LV combination offers a high patient QOL and is suitable for advanced gastrointestinal cancer treatment.

[Outpatient chemotherapy with continuous infusion of 5-fluorouracil (CI 5-FU) and intravenous bolus leucovorin (IVB LV) in advanced gastrointestinal cancer: the second report].

This study was designed to evaluate the efficacy, toxicity, and quality of life (QOL) of outpatient chemotherapy with 5-FU and LV in advanced gastrointestinal cancer. Treatment consisted of CI 5-FU 450 mg/body/day, days 1-28, IVB LV 30 mg/body q wk (schedule, sch. A) and CI 5-FU 200 mg/m2/day, days 1-28, IVB LV 20 mg/m2 q wk (sch. B). 5-FU was administered with the Baxter infusor (0.5 ml/hr). Oral UFT (400-600 mg/day) was administered after these chemotherapy. Sixteen patients (pts) (sch. A 9 pts, sch. B 7 pts) with advanced gastrointestinal cancer have been treated to maintain the efficacy of prior inpatient chemotherapy. Twenty pts treated as adjuvant chemotherapy were added to evaluate toxicity and QOL. The median time to progression in sch. A was 3.0 months and sch. B was 2.4 months. Grade 3 or 4 toxicities of mucositis were seen 40% in sch. A and 0% in sch. B. Grade 1 or 2 skin toxicities were seen 100% in sch. A and 52% in sch. B. Mean score of QOL in sch. A was 78.0 +/- 11.5, sch. B was 89.5 +/- 7.8 ranked between inpatient chemotherapy (5-FU + CDDP 59.0 +/- 13.8) and UFT (91.8 +/- 6.1). In conclusion, these schedule of 5-FU and LV combination offers a high patient QOL and is suitable for advanced gastrointestinal cancer treatment.

[Dysfunction of E-cadherin due to mutation of beta-catenin in a scirrhous gastric cancer cell line].

E-cadherin is a calcium dependent cell-cell adhesion molecule. In cancer tissue, detachment of the adhesion system is indispensable for invasion and metastasis of cancer cells. We have investigated mechanism of the dysfunction of E-cadherin-dependent cell-cell adhesion system in gastric carcinoma cells. Although the high expression of E-cadherin in a scirrhous gastric cancer cell line HSC-39, the function of E-cadherin was completely abolished. Western blotting of beta-catenin in HSC-39 cells demonstrated that a truncated beta-catenin was detected. The truncation was due to partial deletion of beta-catenin DNA. It was concluded that in HSC-39 loss of E-cadherin dependent cell-cell adhesion was due to mutation of beta-catenin gene.

Charcot's joints: a case report. Osteonecrosis may be a predisposing condition for Charcot's joints.

In the case of a patient who had Charcot's joints in her left hip and right shoulders, multiple osteonecrosis in the joints were disclosed by MR imaging. This suggested that osteonecrosis may be a predisposing condition for Charcot's joints.

Altered expression of E-cadherin in gastric cancer tissues and carcinomatous fluid.

Expression of E-cadherin in 21 patients with various histological types of gastric carcinomas was studied by immunoperoxidase staining. Intercellular boundaries of almost all cancer cells in well and moderately differentiated adenocarcinomas stained as deeply for E-cadherin as normal gastric mucosa. However, singly infiltrating cells of those histological types were poorly stained. In poorly differentiated adenocarcinomas, cancer cells forming clusters stained lightly and those infiltrating singly stained even less. In signet ring cell carcinomas, hardly any staining was observed. In each histological type, the staining patterns and intensity at different layers of the gastric wall, were essentially the same. Cancer cells from carcinomatous ascites of gastric adenocarcinomas and pancreatic adenocarcinomas, and those from pleural effusion of lung adenocarcinomas were also studied by immunofluorescence staining. Of 11 specimens, ten were negative and only one from a lung adenocarcinomas was positively stained. By phase-contrast microscopic observations, none of these cancer cells including those from the lung adenocarcinomas, formed obvious cell-cell contacts. Cell aggregation assays confirmed the above results. The molecular weight of E-cadherin of cancer cells of lung adenocarcinomas was less than intact E-cadherin as revealed by Western blot analysis. These results suggest that depressed expression and/or impaired function of E-cadherin in cancer cells, facilitates their liberation from primary sites to infiltrate freely into tissue or fluid.