Tsukasa Ashihara

Photodynamic Inactivation with Acridine Orange on a Multidrug-resistant Mouse Osteosarcoma Cell Line

Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients. In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS/ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line. Cultured cells of MOS and MOS/ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long‐ or short‐term (10 or 1 min) illumination with blue light (466.5 nm) for excitation. Living cells were counted by means of the trypan blue exclusion test. The results showed that AO rapidly bound to DNA, RNA and lysosomes of living MOS and MOS/ADR1 cells and also that most tumor cells in both cell lines died rapidly (viability ratio to untreated cells: 1/1000) within 48 h under conditions of continuous or 15‐min flash exposure to AO at concentrations above 1.0 μg/ ml plus 10‐min illumination with blue light. Even after flash exposure to AO at concentrations above 1.0 μg/ml plus 1‐min illumination, the viability of MOS/ADR1 cells decreased to a viability ratio of less than 1/1000 within 72 h. Based on these results, we concluded that AO with photo excitation has a strong cytocidal effect, not only on chemosensitive mouse osteosarcoma cells, but also on MDR mouse osteosarcoma cells. These results suggested that photodynamic therapy with AO may be a new approach to treating MDR human osteosarcomas.

Development of Bone Canaliculi During Bone Repair

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.

Acridine Orange Excited by Low-Dose Radiation Has a Strong Cytocidal Effect on Mouse Osteosarcoma

The study was conducted to clarify the cytocidal effect of combination therapy consisting of administration of acridine orange (AO), which is a photosensitizer, and radiation therapy using in vitro and in vivo mouse osteosarcoma models. The results revealed that AO combined with low-dose X-ray irradiation of about 1–5 Gy had a strong cytocidal effect on the cultured mouse osteosarcoma cells regardless of their chemosensitivity, and that this combination therapy inhibited growth of the in vivo mouse osteosarcoma by induction of tumor necrosis. This effect was inhibited by L-histidine, but not by mannitol. These findings suggested that AO might be excited by X-rays and kill osteosarcoma cells through the release of singlet oxygen, which is toxic to living cells. This mechanism is similar to that of photodynamic therapy with AO.

Total Tumor Cell Elimination with Minimum Damage to Normal Tissues in Musculoskeletal Sarcomas following Photodynamic Therapy with Acridine Orange

Acridine orange (AO) has unique biological actions enabling tumor visualization (fluorovisualization) and a strong cytocidal effect (photodynamic therapy: AO-PDT) under illumination with blue light. Accordingly, in this study, we attempted to develop a new surgical technique for total tumor cell elimination using these photodynamic reactions with AO in a mouse osteosarcoma model. The results showed that local tumor recurrence was significantly inhibited (23%) in the group treated with curettage under fluorovisualization and AO-PDT, compared to that (80%) in the control group treated with curettage alone under ordinary light. Therefore, we concluded that the combination of curettage under fluorovisualization and AO-PDT may be useful for total tumor cell elimination with minimum damage to normal tissue in musculoskeletal sarcomas.

Actin organization associated with the expression of multidrug resistant phenotype in osteosarcoma cells and the effect of actin depolymerization on drug resistance

We have previously reported that P-glycoprotein (Pgp)-overexpressing multidrug resistant (MDR) osteosarcoma cells were functionally more differentiated than their parent cells. The present study showed that in the parent cells, the actin filaments were sparsely distributed or were diffusely spread throughout the cytoplasm, whereas the MDR osteosarcoma cells exhibited a remarkable increase in well-organized actin stress fibers. Furthermore, dihydrocytochalasin B, a specific inhibitor of actin polymerization, dramatically disrupted this network of stress fibers, increased the intracellular accumulation of doxorubicin (DOX) and modified the resistance against DOX. These results indicate that the organization of actin filaments associated with cellular differentiation may be involved in the expression of Pgp function in the MDR osteosarcoma cells.

Cytogenetic analyses of hepatocellular carcinoma by in situ hybridization with a chromosome-specific DNA probe

Numerical chromosome analysis has been established in solid tumors by using in situ hybridization (ISH) with a chromosome‐specific probe. We analyzed human hepatocellular carcinoma (HCC) by ISH for chromosome 17 and investigated the correlation of its copy number with histologic malignancy, proliferative activity, p53 mutation, and DNA ploidy.

Evaluation of hepatic proliferative activity in chronic liver diseases and hepatocellular carcinomas by proliferating cell nuclear antigen (PCNA) immunohistochemical staining of methanol-fixed tissues.

The proliferative activity of chronic liver diseases and hepatoellular carcinomas (HCCs) was studied by PCNA immunohistochemistry. Human liver tissues were obtained by surgical operation or needle biopsy, and PCNA was detected by immunohistochemistry. PCNA-labelling indices (PCNA-LIs) of methanol-fixed tissues corresponded with the incidence of S-phase cells previously reported, whereas paraformaldehyde-fixed tissues showed extremely high PCNA-LIs in all specimens. Therefore, methanol-fixed tissues were used for evaluation. The PCNA-LIs of the methanol-fixed tissues were: normal liver 0.78 ±0.38%, chronic persistent hepatitis 1.06±0.86%, chronic aggressive hepatitis 2A 1.01±0.50%, chronic aggressive hepatitis 2B 4.20±1.79%, inactive cirrhosis 0.81±0.49%, active cirrhosis 1.96±0.93%, HCC of Edmondsons type I 4.83±1.98%, type II 6.65±1.69%, and type III 38.7±30.6%. PCNA-positive cells showed little specific distribution; in periportal areas in chronic hepatitis, at the margins of pseudolobules in cirrhosis, and throughout the tumor in HCC. These findings indicated that proliferative activity increased during the progression of chronic hepatitis, but that it decreased at the stage of cirrhosis. In chronic liver diseases, the PCNA-LIs reflected hepatitis activity. HCC showed higher proliferative activity than liver cirrhosis, and the histological grade was correlated with the PCNA-LI.

Simultaneous measurement of DNA content and grain count on an autoradiograph of feulgen stained cells

SummaryA microfluorometer is applied for simultaneous measurement of DNA content and grain count on a Feulgen stained cell nucleus in an autoradiograph. The autoradiograph is made on a coverslip and mounted on a slide glass with the emulsion side facing downwards. DNA cytofluorometry by incident light excitation can be carried out without interference from the silver grains. Switch-over from DNA cytofluorometry to grain counting is performed instantaneously by changing excitation filters to a red interference filter. The amount of red light reflected by the silver grains is measured by the microfluorometer. The photometric reading gives a value proportional to the grain count. The measurement of DNA content together with grain counting on a cell is completed within a few seconds.

Disseminated infection of Pneumocystis carinii in a patient with the acquired immunodeficiency syndrome.

This report describes the histopathology of a disseminatedPneumocystis carinii infecton in a 24-year-old Japanese male haemophiliac diagnosed as having the acquired immunodeficiency syndrome. He developed respiratory symptoms, andPneumocystis carinii pneumonia was confirmed by transbronchial lung biopsy. On the 70th day of hospitalization the patient died. Autopsy findings revealedPneumocystis carinii not only in the lungs but also in the stomach, jejunum, ileum, colon, mesoappendix, abdominal lymph nodes, diaphragm, and thyroid gland.

A staining method for bone canaliculi

The modified Bodian method with protargol (silver protein) is ordinarily used to detect nerve fibers. With this technique, applied to decalcified rat bone sections, the bone canaliculi were clearly stained black with good contrast to the bone matrix in both lamellar and woven bone. In addition, the connections between the bone canaliculi and other canaliculi, osteoblasts, osteoclasts, and chondrocytes were easily detectable. We found that the bone canaliculi of woven bone were fewer in number and ran more irregularly than those of lamellar bone. We believe that this staining method for bone canaliculi in decalcified bone is superior to previously reported methods and may be useful in studies on bone pathology.