Adrenergic mechanisms do not contribute to salt-induced vasoconstriction in stroke-prone spontaneously hypertensive rat.
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Adrenergic Fibers↗
Biomedical subjects
T Ashihara
Publications and source records attributed to T Ashihara.
DNA ploidy alterations detected during dedifferentiation of periosteal chondrosarcoma.
DNA ploidy of a case with dedifferentiated periosteal chondrosarcoma was analyzed by DNA cytofluorometry. The diagnosis of primary periosteal chondrosarcoma was made on the basis of the radiographic and histological findings. At 4 years after marginal resection, the tumor recurred locally and metastasized to various organs. The patient died of disease 2 years later. Histologically, there were two components, chondrosarcoma and malignant fibrous histiocytoma, in the recurrent and metastatic tumors. DNA ploidy analysis of multiple samples revealed that the primary lesion was composed of many diploid cells with some tetraploid and octaploid cells, whereas in the dedifferentiated area, there were many aneuploid cells which were not recognized in any area of the primary tumors. This case illustrated that DNA ploidy alteration of euploidy to aneuploidy is closely correlated with the process of dedifferentiation in chondrosarcoma.
Adult↗
Acquisition of multidrug resistance in osteosarcomas, analyzed by doxorubicin binding assay, and histologic response to chemotherapy.
BACKGROUND: The mechanism by which multidrug resistance is acquired in human osteosarcomas remains unclear. In this study, we analyzed the changes in doxorubicin (DOX) binding ability (%DB), showing chemosensitivity before and after chemotherapy with DOX and cisplatin (CDDP), and evaluated the histologic response of human osteosarcomas to chemotherapy. MATERIALS AND METHODS: Eight osteosarcomas were analyzed. %DB by the DOX binding assay was measured in fresh tumor tissues at biopsy and at resection after preoperative chemotherapy. The histologic response to chemotherapy was also evaluated. RESULTS: Changes in %DB before and after chemotherapy were classified into 4 patterns; A: high (> 80%) to low (< 80%), B: high to high, C: low to low, D: low to high. The two tumors, classified as type A and B were responders (> 90% necrosis), whereas the 6 tumors of type C and D were classified as non-responders (< 90% necrosis). Based on these data, we speculated that the fraction of cell populations with different chemosensitivities to DOX and CDDP varied and found that type B tumors have no population of resistant cells, whereas type C tumors have a large fraction of resistant cells. CONCLUSION: We concluded that osteosarcomas are composed of mixed cell populations with different chemosensitivities to anticancer agents and also that survival of multidrug resistant cell populations may be the most important mechanism in acquisition of multidrug resistance of osteosarcomas after chemotherapy.
Adolescent↗
Improving the cytometric detection of doxorubicin resistance in osteosarcoma cells by determining cellular doxorubicin/DNA ratio.
To study the influence of cellular DNA content on the accumulation and efficacy of doxorubicin (DOX), we characterized P-glycoprotein (Pgp)-positive and -negative murine osteosarcoma cell clones that had a different DNA index. Statistical analysis demonstrated that the cytotoxic effects of DOX correlated significantly with the ratio of intracellular DOX accumulation divided by the cellular DNA content (DOX/DNA ratio) (P = 0.001), but not with the intracellular DOX accumulation (P = 0.16). We also tested this relationship for Pgp-negative human osteosarcoma cell lines with a different DNA ploidy, and found that these Pgp-negative cell lines all had similar DOX/DNA ratios. These results indicate that the DOX/DNA ratio is a determinant for the effects of DOX in osteosarcoma cells, regardless of their Pgp status and DNA ploidy. Thus, consideration of the cellular DNA content as well as the intracellular DOX accumulation is important to accurately detect DOX resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Malignant endocrine carcinoma of the stomach.
Small cell carcinoma and small portions of poorly differentiated adenocarcinoma of the stomach sometimes differentiate into secretory cells. We experienced a pure endocrine carcinoma of the stomach. The patient, who had a giant mass in the greater curvature of the body of the stomach and regional lymph node metastasis, underwent total gastrectomy. Immunohistologic staining of the resected specimens revealed that the tumor was an endocrine carcinoma. The tumor cell shape in the present case was polygonal or oval and the cell size was relatively large. The cell nucleus was coarse chromatic and nucleoli were seen, whereas, the nucleus of small cell carcinoma often shows a fine chromatic pattern and nucleoli are not seen. On the basis of these findings, the present case was considered to be a pure endocrine carcinoma which was not classified as a small cell carcinoma.
Biomarkers, Tumor↗
Polyploidization induced by acridine orange in mouse osteosarcoma cells.
This study was undertaken to clarify the in vitro effect of acridine orange (AO) on the cell kinetics of mouse osteosarcoma cells, as well as the mechanism of cell growth inhibition induced by AO. A mouse osteosarcoma cell line (MOS), established from a radiation-induced mouse osteosarcoma, was cultured under exposure to 0.05, 0.5, 5, and 50 micrograms/ml of AO, either continuously or for 10 minutes. The cell kinetic analysis was performed using the following parameters: tumor cell growth by trypan blue exclusion test, mitotic activity, DNA synthetic activity by BrdU labeling and DNA ploidy by cytofluorometry. The results showed that continuous exposure to 5 and 50 micrograms/ml of AO or 10 minute exposure to 50 micrograms/ml of AO quickly killed the tumor cells within 12 hours, whereas continuous exposure to 0.5 microgram/ml of AO or 10 minute exposure to 5 micrograms/ml of AO gradually inhibited tumor cell growth. Under the latter conditions, mitotic activity was rapidly and completely inhibited within 48 hours but DNA synthetic activity was not completely inhibited even after 96 hours. DNA ploidy analysis demonstrated that most of the tumor cells arrested at the S-G2 phase after 12 hours, followed by G2 phase arrest after 24 hours and progressive DNA synthesis to a higher DNA ploidy class after 48 to 96 hours. We therefore concluded that a high concentration of AO has a strong cytocidal effect due to cytotoxicity whilst a moderate concentration of AO induces progressive and synchronous polyploidization by mitotic inhibition without DNA damage in MOS cells. We presume that this in vitro effect on MOS cells may be caused by protein synthetic inhibition after transfer RNA inactivation caused by AO binding.
Acridine Orange↗
Intracellular binding sites of acridine orange in living osteosarcoma cells.
There have been many reports concerning the intracellular binding sites of acridine orange (AO), although the actual localization of AO in living cells remains controversial. This study was undertaken to clarify the intracellular localization of AO in living mouse osteosarcoma cells by cytochemical staining. A mouse osteosarcoma cell line (MOS) was cultured and continuously exposed to 0.5 microgram/ml of AO. The intracellular localization and stainability of AO the living tumor cells was morphologically detected by a high resolution fluorescence microscope. To detect the intracellular microstructure, cytochemical staining with rhodamin 123 for mitochondria, acid phosphatase for lysosome, Sudan-black for fat vesicle and toluidine blue for glucosaminoglycan were performed using fixed cells. The results showed that both the nucleus and cytoplasm of tumor cells at 10 minutes after exposure to 0.5 microgram/ml of AO emitted green fluorescence, which was especially intense in the nucleolus, but not brilliant in the nucleus and was granular orange to red fluorescence in the perinuclear particles. This stainability of AO was different from that of rhodamin 123, Sudan-black or toluidine blue, but similar to that of acid phosphatase. Based on these results, we conclude that the green fluorescence may have derived from AO binding to double stranded RNA, not to DNA, and that orange fluorescence may have derived from aggregated AO binding to lysosome.
Acid Phosphatase↗
Relationship between binuclear and multinuclear cells in giant cell tumor of bone.
Giant cell tumor of bone (GCT) consists of stromal and multinuclear type tumor cells. Although most people believe that the stromal cells are mononuclear, we recently found the existence of many binuclear cells among stromal cells using DNA cytofluorometric examination. This study, using 18 tumors of GCT was conducted to elucidate the cell biological significance of the binuclear cell, especially its relationship to multinuclear cell formation or tumor cell proliferation. The investigation was carried out by means of DNA-RNA cytofluorometry with acridine orange (AO) and histological method. Using fluorescence microscopic observation, we counted the numbers of both mononuclear and binuclear cells and calculated the index of % BNC, which expresses the frequency (percentage) of binuclear cells in a population of mononuclear and binuclear cells. The index of % S-G2 obtained by DNA-RNA cytofluorometry showed the frequency (percentage) of mononuclear cells in the S and G2 phases of the cell cycle. In the histological study, we counted the numbers of multinuclear giant cells with more than 3 nuclei in the cytoplasm and stromal cells including mononuclear and binuclear cells and calculated MNS/SC, which showed the percentage of multinuclear cells in the stromal cells in the microscopic field. Eight tumors showed a value of % BNC greater than 10% and 2 had a value of 40%. The index of % BNC significantly correlated with the average value of MNC/SC in all tumors. There was no significant correlation between % BNC and the average value of % S-G2, in 18 tumors although 4 tumors having a % BNC value greater than 20% showed a % S-G2 value greater than 12% in 18 tumors. These results revealed the presence of many binuclear cells among stromal cells of GCT and suggested that these binuclear cells might be formed in association with the active proliferation of mononuclear cells and closely relate to the formation of multinuclear giant cells.
Adolescent↗
Binuclear cells induced by acridine orange in giant cell tumor of bone.
We have recently found the presence of many binuclear cells among isolated and smeared cells in giant cell tumor of the bone (GCT). These binuclear cells are possibly associated with the formation of multinuclear cells. Therefore, this study was undertaken to clarify the mechanism of binucleation in GCT, using primary culture cells exposed to acridine orange (AO) which is a fluorescent vital staining dye for the cytoplasm and nucleus and which inhibits mitosis. The cells were isolated from explants of fresh tumor materials obtained from two GCT patients (GCT1 and GCT2). These cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% Fetal calf serum (FCS). After exposure to 0.5 microgram/ml AO, for 0, 6, 24, 48, 96 and 144 hours the following parameters were investigated: 1) cell growth rate (GR); 2) frequency of hyperdiploid cells (%HDC) by DNA cytofluorometry; 3) mitotic index (MI); 4) BrdU labeling index (LI); 5) frequency of binuclear cells (%BNC). Compared to the control cells which were cultured in AO-free medium, the GR of both GCT cells exposed to AO was remarkably inhibited. The MI was 0 from 24 to 144 hours. The %HDC was increased at 24 hours and was maintained high until 144 hours. The LI was temporarily increased at 6 hours, but was decreased at 48 hours. The %BNC was gradually increased. AO inhibited DNA synthesis and cell mitotic activity in cultured GCT cells and it finally caused inhibition of cell growth. However, the frequencies of G2 arrest cells and binuclear cells were increased. These results suggested that the binuclear cells in GCT may be formed from G2 arrest cells by amitotic nuclear division, but not by mitosis without cytoplasmic division, or by cell fusion.
Acid Phosphatase↗
Fluorovisualization effect of acridine orange on mouse osteosarcoma.
If the localization of musculoskeletal sarcomas could be visualized during surgery, it would be possible to completely resect the tumor with minimum damage to normal tissues and the patients could retain a functional limb. Therefore, we conducted the present study to clarify the usefulness of acridine orange (AO) for fluorovisualization of tumors using a mouse osteosarcoma model. At 2 hours after injection of 10 mg/kg AO to mice inoculated with MOS mouse osteosarcoma cells, fluorovisualization of mouse osteosarcoma reached the maximum level. Even a 1-mm-diameter lesion of pulmonary metastasis was visualized. The results suggested that AO may be useful for specific fluorovisualization of human osteosarcomas during surgery.
Acridine Orange↗
DNA cytofluorometric analysis of benign and malignant nerve sheath tumors.
The present study was undertaken to examine the ploidy patterns of benign and malignant nerve sheath tumors using PI-DNA cytofluorometry and to clarify the relationship between the ploidy patterns and pathological findings for these lesions. The benign nerve sheath tumors (schwannomas and neurofibromas) were found to be dipolid or to be euploid-polyploidization, with a few DNA synthetic cells. The number of polyploid cells increased with increase in the frequency of cells with large and atypical nuclei in histology, regardless of the natures of cellular arrangements. Malignant schwannomas were composed of many polyploid and aneuploid cells with DNA synthetic cells, indicating their active cell proliferation. Thus, benign nerve sheath tumors exhibited low-proliferative ploidy patterns that were clearly different from those of malignant nerve sheath tumors. As Takeshita has already stated, it is difficult to ascertain why some of the neurogenic tumors show euploid-polyploidization (19). Further investigations will be needed in this direction. Finally, it is clear from the results of this study that the cytofluorometric analysis mentioned above is useful for assessing malignancy and that it may be useful for predicting prognosis of nerve sheath tumors.
Adolescent↗
Relationship between doxorubicin binding ability and tumor volume decrease after chemotherapy in adult malignant soft tissue tumors in the extremities.
We have previously reported that the doxorubicin (DOX) binding ability detected by the DOX (or adriamycin) binding assay closely correlated with the chemosensitivity of human osteosarcomas (1). We performed the present study to clarify the relationship between the DOX binding ability (%DB) and the histologic response, rate of decrease in tumor volume of malignant soft tissue tumors after preoperative chemotherapy and prognosis. Nine malignant soft tissue tumors (4 liposarcomas, 3 synovial sarcomas, one malignant fibrous histiocytoma (MFH) and one extraskeletal osteosarcoma (EOS)) which arose at the extremities of adult patients were analyzed by the DOX binding assay using freshly biopsied specimens. After preoperative chemotherapy including DOX or pirarubicin (THP), the rate of decrease in tumor volume was measured using magnetic resonance imaging, and the histologic response expressed as tumor necrosis to chemotherapy was also investigated. All the patients, apart for one, were continuously disease-free after treatment. One patient with EOS died of metastatic disease before surgery. The histologic response in 8 tumors without EOS was poor. The %DB of 5 tumors was greater than 80% (average: 95.90%), whereas that of 4 tumors was less than 80% (average: 38.33%). Although there was no correlation between the %DB and the histologic response, or prognosis, a significantly positive correlation was found between the %DB and the rate of decrease in tumor volume (r = 0.7455, p < 0.05). These results suggest that in malignant soft tissue tumors, the rate of decrease in tumor volume after chemotherapy might be a better indicator for chemosensitivity than the histologic response and also that the DOX binding ability might be a good predictor for chemosensitivity before chemotherapy.
Adult↗
Assessment of chemosensitivity in patients with malignant bone and soft tissue tumors using thallium-201 scintigraphy and doxorubicin binding assay.
This study was performed to compare the accumulation of thallium (Tl)-201 which is correlated with malignancy and the doxorubicin binding ability, which is correlated with chemosensitivity, in nine patients who received preoperative chemotherapy with doxorubicin and cisplatin. Tl-201 scintigraphy was performed at 15 minutes (early image) and 3 hours (delayed image) after injection of 111 MBq of Tl-201. The change of degree of the radionuclide uptake between the early and delayed images was evaluated before and after preoperative chemotherapy. The doxorubicin binding ability (%DB) to nuclear DNA in living tumor cells isolated from biopsy materials was assessed by doxorubicin binding assay. The histologic response to preoperative chemotherapy was evaluated by the percentage of tumor necrosis. Before preoperative chemotherapy no changes of Tl-201 uptake between the early and delayed images was detected in any tumors. Five patients, who had no change of Tl-201 uptake after preoperative chemotherapy, showed a poor histologic response and had a %DB ranging from 10% to 70% (mean: 36.0%). The other four patients, who had a %DB greater than 90%, showed a good histologic response. All of these four patients had decreased Tl-201 uptake after preoperative chemotherapy. This study demonstrated that doxorubicin binding assay and midcourse Tl-201 scintigraphy are useful methods to assess the response to chemotherapy early in malignant bone and soft tissue tumors.
Adolescent↗
Drug resistance modification using pulsing electromagnetic field stimulation for multidrug resistant mouse osteosarcoma cell line.
Multidrug resistance (MDR) is one of the major problems in osteosarcoma chemotherapy. Therefore, methods of overcoming MDR are urgently needed. In this study, we investigated the effects of pulsing electromagnetic field stimulation (PEMFs) on a MDR murine osteosarcoma cell line which strongly expresses P-glycoprotein (P-gp). To assess the reversal effects of PEMFs on doxorubicin (DOX) resistance, MTT assay was applied. Viable cells were assessed by the trypan blue exclusion test. Fluorescence intensity of DOX binding to nuclear DNA of each cell was measured using a cytofluorometer. Changes in P-gp expression in each cell were detected by the indirect immunofluorescence method using an antibody to Pgp. PEMFs increased DOX binding ability to nuclear DNA and inhibited cell growth, although it had no significant effect on P-gp expression. These findings indicated that PEMFs reversed the DOX resistance of the MOS/ADR1 cells by inhibiting P-gp function. The results suggested that PEMFs may be useful as a local treatment for MDR osteosarcoma.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Oral administration of 1 alpha hydroxyvitamin D3 inhibits tumor growth and metastasis of a murine osteosarcoma model.
We studied the effect of oral administration of 1 alpha hydroxyvitamin D3 (1-D3) on the growth and metastatic ability of Dunn murine osteosarcoma model. A solution of 1-D3 or vehicle alone was administered daily for 2 weeks to tumor-bearing mice using an esophageal tube and tumor size was serially monitored. In 1-D3-treated mice, the growth of Dunn osteosarcoma was significantly suppressed in a dose-dependent manner. Histologically, tumor cells in the control mice proliferated in marginal regions of the tumor with wide central necrosis, whereas in the 1-D3-treated mice, tumor cells were distributed as scattered islands among extensive necrotic tissue. The mean tumor necrosis area was 55.7% in the control tumors and 94.6% in 1-D3-treated tumors (p < 0.001). There were no substantial differences in the cytofluorometric cell cycle distribution or the histological mitotic index between control and 1-D3-treated tumors. When 1-D3 was administered to mice from 2 days before to 2 weeks after transplantation of the tumor, there were significantly fewer metastatic foci in the lungs in 1-D3-treated mice than in control mice. We also tested the effect of coadministration of 1-D3 and doxorubicin on the growth of Dunn osteosarcoma and found that these two drugs act additively to suppress tumor growth. These results indicated that 1-D3 given orally inhibits tumor growth and metastases in a Dunn osteosarcoma model. Although the mechanism remains unknown, oral administration of 1-D3 might be promising as a new method of treating human osteosarcoma.
Administration, Oral↗
Relationship between histologic grade and cytofluorometric cellular DNA and RNA content in primary bone tumors.
The diagnosis and grading of bone tumors remains a challenging problem. We studied the relationship between histologic grade and cytofluorometric cellular DNA and RNA content in 108 primary bone tumors. The data included DNA ploidy, mean DNA content (MDC), S-phase fraction (SPF), mean RNA content (MRC) and RNA/DNA ratio (RDR; MRC/MDC) which represents the RNA content normalized for the DNA content. Benign tumors had a diploid stem line with low MDC (mean; 1.04), low SPF (0.9), high MRC (2.41) and high RDR (2.31). Giant cell tumors of bone, which are locally aggressive benign tumors, showed diploidy with relatively higher MDC (1.07, p < 0.01) and SPF (2.6, p < 0.01) and lower MRC (1.81, p < 0.01) and RDR (1.69, p < 0.01). Similar results were obtained in low-grade sarcomas. In high-grade sarcomas, the data depended on the histologic findings. Pleomorphic sarcomas such as osteosarcomas revealed aneuploidy with remarkably higher MDC (1.70 in osteosarcomas, p < 0.01) and SPF (6.5, p < 0.01), but lower RDR (1.70, p < 0.01). In contrast, small cell sarcomas, such as Ewing's sarcomas, showed diploidy with low MDC (1.11 in Ewing's sarcomas, N.S.) and SPF (2.5, p < 0.01) and extremely low RDR (1.34, p < 0.01). The RDR value was higher in well-differentiated tumors than in primitive tumors, rendering it useful in grading bone tumors with a diploid stem line. By combining the RDR value with the MDC value, 96% of diploid sarcomas could be distinguished from benign tumors. These results indicate that cellular DNA and RNA content analysis may be of value in assessing the malignant potential of diploid as well as aneuploid bone sarcomas.
Bone Neoplasms↗
Avoidance of doxorubicin resistance in osteosarcoma cells using a new quinoline derivative, MS-209.
P-glycoprotein (Pgp), a membrane drug efflux pump, is thought to be responsible for the observed drug resistance in osteosarcoma. We have recently developed Pgp-positive, multidrug resistant (MDR) murine osteosarcoma cell lines, which may be suitable models for the study of drug resistance in osteosarcoma. In this study, we investigated the effect of a newly synthesized quinoline compound, MS-209, on the reversal of doxorubicin (DOX) resistance in these cell lines. Three different types of resistance modifying agents (RMAs) as well as MS-209 were studied. These included the calcium channel blocker verapamil, and the immunosuppressive agents cyclosporin A and FK506. The reversal effects of the RMAs on DOX resistance were assessed by the MTT assay. In the absence of RMAs, the MDR osteosarcoma cells were 20-fold more resistant to DOX than the parental cells. When MS-209 was added at a final concentration of 0.1 to 3 microM to the MDR cells, 3-to 74-fold sensitization was observed. A complete reversal (37-fold sensitization) of the resistance was obtained at 1 microM MS-209. This concentration of MS-209 was 3-, 8- and 28-fold more effective than the same concentration of FK506, verapamil and cyclosporin A, respectively. These results indicate that MS-209 may be a more effective RMA, and that DOX resistance in osteosarcoma cells could be reversed by comparatively low doses of MS-209.
Antineoplastic Agents↗