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Biomedical subjects

K Kusuzaki

Publications and source records attributed to K Kusuzaki.

At least 19 recordsLinked to original sources
PubMed1996

Experimental models for the study of drug resistance in osteosarcoma: P-glycoprotein-positive, murine osteosarcoma cell lines.

P-glycoprotein is an adenosine triphosphate-dependent drug-efflux pump that extrudes drugs from cells and causes drug-resistance. P-glycoprotein is believed to mediate drug-resistance in a wide variety of tumors. In this study, we developed two P-glycoprotein-positive, murine osteosarcoma cell lines that were resistant to Adriamycin (doxorubicin) (MOS/ADR1 and MOS/ADR2). We created the cell lines by short-term pulse exposures of the parent cell line to Adriamycin followed by single-cell cloning. The MOS/ADR1 and MOS/ADR2 cells were sevenfold and eighteenfold more resistant to Adriamycin than the cells from the parent line. Expression of P-glycoprotein, as examined with an immunofluorescence method, was detected in most of the MOS/ADR1 and MOS/ADR2 cells but not in the parent cells. After the cells had been incubated with Adriamycin for one hour, there was less accumulation of the drug in the resistant cell lines than in the parent cell line. The reduced accumulation was due to the increased efflux of Adriamycin. The Adriamycin-resistant cell lines demonstrated greater alkaline phosphatase activity than the parent cell line and produced more differentiated osteoblastic sarcomas in mice. Dose survival studies with use of a tetrazolium colorimetric assay showed that the MOS/ADR1 cells were cross-resistant to vincristine, vinblastine, etoposide, bleomycin, mitomycin C, and actinomycin D but not to dacarbazine, cisplatin, carboplatin, cytosine arabinoside, carmustine, cyclophosphamide, ifosfamide, methotrexate, and 5-fluorouracil. Although the MOS/ADR2 cells exhibited a similar spectrum of cross-resistance, they were more resistant than the MOS/ADR1 cells. We also tested the effect of three different resistance-modifying agents on the reversal of resistance to Adriamycin. We found that verapamil and trifluoperazine substantially reversed resistance to Adriamycin in the P-glycoprotein positive cell lines, whereas cyclosporin A was relatively ineffective. Because these cell lines retain the histological and biochemical features of bone-producing sarcomas and display the multidrug-resistant phenotype, they may be useful models for additional investigations of drug resistance in osteosarcoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Cell proliferation and differentiation of cultured chondrocytes isolated from growth plate cartilage of rat rib.

The present study was undertaken to investigate the relationship among cell morphology, proliferation, and maturation of chondrocytes in primary cultures. Chondrocytes were isolated from the growth cartilages of the rat ribs and cultured for 6 days. In situ DNA cytofluorometry using an inverted epi-illumination cytofluorometer (Nikon P1-I) and 3H-thymidine autoradiography were carried out for the correlated analysis of cell morphology and proliferation. Cytoskeletal staining with fluorescent phalloidin and 35S-sulphate autoradiography were also performed. In addition, in situ hybridization to c-myc mRNA was carried out using DNA probe. According to the results obtained, the cultured chondrocytes were composed of mixed populations of large, polygonal cells and of small, round cells. The round cells showed a significantly higher 35S uptake than the polygonal cells. The cytoskeletal staining clearly revealed stress fibers in the cytoplasm of the polygonal cells, whereas only a fine filamentous structure was shown in the cytoplasm of the round cells. In situ DNA cytofluorometry clearly demonstrated that cell proliferative activity was high in the polygonal cells and low in the round cells. In addition, 3H-thymidine autoradiography with cumulative labeling method revealed that the polygonal cells were changing into the small, round cells. C-myc mRNA signals were detected in the cytoplasm of over a half of the round cells, whereas no evidence of c-myc expression were found in the polygonal cells. From these results, it appears that as the shape of the cultured chondrocytes shifts from polygonal to round, the cell proliferative activity decreases in association with cell differentiation. It was also suggested that c-myc mRNA is amplified in the well differentiated round chondrocytes, and not in the proliferative polygonal cells.

Animals↗
PubMed1995

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.

Animals↗
PubMed1994

An assay to measure adriamycin binding in osteosarcoma cells.

Adjuvant chemotherapy is currently employed in the treatment of patients with osteosarcoma, but the drug regimens, although effective in improving disease-free survival, are unsuccessful in 20-40% of patients and very toxic. It would be useful to know whether tumor cells are sensitive to a given drug prior to its use. To this end, we developed a method of assessing Adriamycin (doxorubicin) binding to tumor nuclei as a possible means of detecting sensitivity to the drug. Adriamycin-sensitive murine osteosarcoma cells were used to develop the assay. The in vitro conditions (drug concentration, duration of incubation, and temperature) were optimized with use of the murine osteosarcoma cells in culture. After the cells had been incubated with Adriamycin, cell viability was determined and Adriamycin fluorescence intensity was measured with a cytofluorometer. The optimal parameters for Adriamycin binding were found to be a 30-minute incubation in a 10 micrograms/ml concentration of Adriamycin at 37 degrees C; the frequency of cells that emitted Adriamycin fluorescence from the nucleus compared with the total number of living cells reached 100% under these conditions. In a murine leukemia cell line with known sensitivity to Adriamycin, the cells emitted red fluorescence from the nucleus and cytoplasm, whereas in a resistant line the cells emitted Adriamycin fluorescence from only the cytoplasm. We demonstrated that it is possible to differentiate nuclear from cytoplasmic concentration of Adriamycin in a tumor cell with use of a fluorescent microscope and that resistant cell lines can be distinguished from sensitive cell lines by this method.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Adriamycin binding assay: a valuable chemosensitivity test in human osteosarcoma.

The reliability of a simple method evaluating the pattern of subcellular binding of Adriamycin (Adriamycin binding assay, ABA) as an index of sensitivity was demonstrated in different primary cultures and in sensitive and resistant cell lines of human osteosarcoma. After exposure to Adriamycin (10 micrograms/ml for 30 min at 37 degrees C), living sensitive cells showed selective intranuclear uptake of the drug, whereas in resistant cells no distinct subcellular distribution was observed. The binding pattern of Adriamycin in sensitive and in highly resistant cells was inversely related to the expression of P-glycoprotein. However, low levels of resistance in vitro, not detectable by increased levels of expression of P-glycoprotein, were revealed by ABA. The use of ABA in combination with the estimate of P-glycoprotein expression is recommended in clinical practice as an accurate means for predicting the sensitivity of osteosarcoma to Adriamycin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Cloning and expression of the rfe-rff gene cluster of Escherichia coli.

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.

Amino Acid Sequence↗
PubMed1991

[Staining of bone canaliculi using decalcified bone tissues].

Recently we were able to stain bone canaliculi using the decalcified bone tissues, according to a modified method of the Bodian staining method, which is commonly used for staining the nerve fibers with silver impregnation of protargol. The bone tissues from normal rabbits, rats, and humans were decalcified with buffered EDTA solution after 10% formalin fixation. The tissues were embedded in paraffin and were sliced at 4 microns then the sections were attached to the gelatin-coated glass slides. These sections were treated first with potassium dichromate followed by 2% protargol solution containing copper. After gold chrolide treatment, they were immersed in oxalic acid. Both the lacunae and canaliculi of osteocytes were stained black in sharp contrast to bone matrix of red purple. Using the present staining method, the bone canaliculi were clearly detectable in fine details without producing apparent artificial damage of soft tissues. The canaliculi showed also markedly complex fine structures. By combining the present staining method with eosin or toluidine blue staining, we were able to recognize even immature osteocytes in enchondral ossification at growth cartilage with fine bone canaliculi. From these results, we conclude that the present method is very useful in histopathological studies of the lacuna-canalicular system of osteocytes in the decalcified bone tissues.

Animals↗
PubMed1991

[The distribution of S-100 protein positive chondrocytes in the human articular cartilages under aging or diseased conditions].

There have been few reports on the localization of S-100 protein positive chondrocytes in the human articular cartilages. We studied 59 articular cartilages of the aged subjects, 65 osteoarthritic (OA) and 39 rheumatoid arthritic (RA) articular cartilages, to detect the histological localization of S-100 protein using immunoperoxidase method (ABC). The results obtained from normal cartilages demonstrated strongly positive cells representing hypertrophic chondrocytes in the perivascular areas of the neonatal articular cartilage and in the deep zone of the infant articular cartilage. The moderately positive cells were found in the intermediate zone of infant and adult articular cartilages. In mild OA, there were many positive chondrocytes in the intermediate zone with erosion of the surface layer, while in moderate or severe OA many strongly positive cells were found in clusters. The hypertrophic cells in the metaplastic cartilage arising from bone marrow in subjects with severe OA, or from pannus after RA were also positive. It is therefore, suggested that S-100 protein may be correlated with the metabolic activity of the cartilage matrix such as collagen and proteoglycan, as reported in the literature. S-100 protein further, appears to be useful for evaluating histologically the activity of cartilage repair in the pathologic human articular cartilages.

Adolescent↗
PubMed1991

In vitro assay of nuclear uptake of doxorubicin hydrochloride in osteosarcoma cells of dogs.

A rapid, simple chemosensitivity assay, assessing tumor cell nuclear uptake of doxorubicin hydrochloride, was evaluated in 16 dogs with appendicular osteosarcoma. Doxorubicin was administered to dogs in 5 biweekly treatments, and surgical resection was performed after the second or third treatment. The chemosensitivity assay was performed on biopsy specimens from all dogs before chemotherapy. It was repeated on tissue from resected tumors, and tumors were evaluated histologically to determine the degree of necrosis resulting from chemotherapy. Disease-free and total survival time correlated significantly (P less than 0.05 in both cases) with the degree of postchemotherapy necrosis of the primary tumors. Significant correlation was not apparent between the percentage of tumor cells with nuclear uptake of doxorubicin (in either biopsy or resection samples) and disease-free or total survival time. The percentage of cells with nuclear uptake of doxorubicin in surgically resected tumors correlated significantly (P less than 0.05) with percentage of necrosis.

Animals↗
PubMed1989

Immunohistochemical localization of interleukin 1 in human growth cartilage.

Although it has been reported that interleukin 1 (IL-1) stimulate chondrocytes to produce collagenase and proteoglycanase in vitro, IL-1 producing cells and the function of IL-1 have not been demonstrated in osteocartilaginous tissue in vivo. Immunohistochemical studies of human cartilaginous epiphysis and growth cartilage demonstrated that IL-1 was detected in: (1) chondrocytes surrounding cartilage canal, (2) hypertrophic chondrocytes in cartilaginous epiphysis, (3) chondrocytes at the hypertrophic and calcified zones in the growth cartilage of actively growing bone. In contrast, few hypertrophic chondrocytes showed positive reactions to IL-1 in growth plates nearing physiologic closure. Furthermore, IL-1 was detected in chondrocytes cultured from human growth cartilage. These results show that IL-1 is produced by matured chondrocytes of human growth cartilage in vivo. Chondrocyte-derived IL-1 might play a key role in the hypertrophy of chondrocytes, in the vascularization of cartilage and in the formation of bone.

Adolescent↗
PubMed1986

[The relationship between cell kinetics and histological features of giant cell tumor of the bone].

This study was undertaken to clarify the relationship between the proliferative activity and histological findings of the giant cell tumor (GCT) of bone by means of an epi-illumination cytofluorometer (NIKON SPM-RF1-D). Fresh tissues of GCT were surgically obtained from two cases. In both cases, small pieces of tumor tissues were obtained from several different regions based on the macroscopic characteristics of the cut surface, and processed for single cell preparation using enzymatic method. These isolated cells were smeared and stained with acridine orange, and then analyzed cytofluorometrically to determine simultaneously DNA and RNA contents of the individual cells. The results showed that the proliferative activity of tumor cells was much higher in the regions composed of both many histiocytic stromal cells having polygonal or ovoid shape and many multinucleated giant cells, than either in the regions composed of fibrocytic stromal cells accompanying abundant collagen fibers or in the regions composed of foamy cells.

Adult↗
PubMed1985

[DNA-RNA cytofluorometric analysis of a case of osteosarcoma in relation to histological characteristics].

We applied DNA-RNA cytofluorometry with AO stain to cell kinetic analysis of osteosarcoma in a 12-year-old girl in relation to its histological characteristics. Histological findings obtained for 9 macroscopically different lesions were grouped into 4 main structural characteristics, but their cytofluorometric results were classified into 2 main patterns of DNA-RNA distribution. One showed remarkable polyploidization with many DNA synthetic cells in the invasive lesions, which were composed of pleomorphic cells forming osteoid or occasionally cartilaginous matrix. The other showed marked accumulation of tetraploid cells almost without DNA synthetic cells, being composed of relatively uniform fibroblastic or stellate-like cells in the cartilaginous matrix. These results indicated a close relationship between cell proliferative activity and the tissue environment.

Bone Neoplasms↗
PubMed1985

[DNA cytofluorometric analysis of nerve sheath tumors].

The present study was undertaken to clarify the ploidy patterns of peripheral nerve sheath tumors by determining nuclear DNA content of the individual tumor cells using PI-DNA cytofluorometry (NIKON SPM-RF1-D), and to investigate the relation between the ploidy patterns and histopathological findings. Most of the solitary neurilemmomas and neurofibromas studied were found to be associated with euploid-polyploidization, almost without DNA synthetic cells. It was also shown that the number of polyploid cells increased in accordance with an increase in the frequency of cells having large, atypical nuclei in histological picture, regardless of the natures of cellular arrangements. Malignant schwannomas, however, were composed of many polyploid and aneuploid cells with an increase of DNA synthetic cells, indicating their active cell proliferation. Thus, the ploidy patterns of nerve sheath tumors were remarkably different between benign and malignant ones. Furthermore, a case of multiple schwannoma suspected to be a variant of von Recklinghausen's disease, showed euploid-polyploidization with many DNA synthetic cells, indicating a ploidy pattern intermediate between benign and malignant tumors. This tumor thus appears to be a premalignant tumor or in the early stages of malignancy.

Adolescent↗
PubMed1984

[DNA cytofluorometry of soft tissue tumors].

We carried out DNA cytofluorometry with propidium iodide stain on the 17 cases of soft tissue tumors including giant cell tumor of the tendon sheath, pigmented villonodular synovitis, 2 hemangiomas, 3 lipomas, 5 schwannomas, 3 neurofibromas, liposarcoma and synovial sarcoma. The benign tumors were characterized by regular polyploidization with very few S-phase cells, indicating slow tumor growth. Most of the malignant soft tissue tumors were associated with remarkable polyploidization with an increase in S-phase cells. However, some malignant tumors did not show polyploidization. We concluded, therefore, that an increase in S-phase cells is an important, cytofluorometric criterion for malignancy of soft tissue tumors.

Adult↗
PubMed1984

[Multiparametric cytofluorometry of the process of cellular proliferation, differentiation and maturation of the chondrocytes in the growing rat].

The present study was undertaken to analyze quantitatively the process of cellular proliferation, differentiation and maturation of the chondrocytes in both the epiphyseal cartilage plates and the articular cartilages of the growing rats, using the multiparametric cytofluorometry (NIKON SPM-RFl-D). The analyses involve the simultaneous determinations by cytofluorometry of nuclear DNA vs. cellular RNA contents, nuclear DNA vs. cellular protein contents, and nuclear DNA vs. silver grain amount of the 3H-leucine, 3H-glycine, 3H-thymidine and the 35SO4 autoradiography. In addition, for the detailed analysis of cell kinetics, DNA-RNA cytofluorometry was carried out in combination with 3H-thymidine autoradiography. According to the results obtained, the chondrocytes were quantitatively divided into subpopulations of G0-G1, S and G2 in the cell cycle by their nuclear DNA and cellular RNA contents, and the maturative chondrocytes had higher cellular RNA content than the proliferative ones. It was suggested that there were, among the chondrocytes in the epiphyseal cartilage plate, two other subpopulations of binuclear cells and mononuclear tetraploid cells, both having higher cellular RNA content than the mononuclear G2 cells. It is reasonable to assume that these two subpopulations are the polyploid cell groups accompanying highly active cell proliferation.

Animals↗
PubMed1984

[DNA-RNA cytofluorometry for a primary cancer of the stomach and choledochus].

We standardized DNA-RNA cytofluorometry with AO stain using a NIKON SPM-RFI-D instrument for cell smears. We applied this technique in a 72-year-old woman with primary double cancer of the stomach and choledochus. We first suspected malignancy of the choledochus and examined the desquamated cells in the bile cytofluorometrically to obtain by X-Y plotting the irregularly increased polyploid cells with a high percentage of cells in S phase. These polyploid cells and S phase cells were found to have an increased RNA content. Before operation, the presence of another malignancy of the stomach was also ascertained. The surgical specimens were diagnosed histopathologically as well differentiated, tubular adenocarcinoma of the stomach and moderately differentiated, tubular adenocarcinoma of the choledochus. By cytofluorometric analysis similar results were obtained, i.e., polyploidization with increase of S phase cells. Based on these results, we concluded that our method is useful for the diagnosis of alimentary tract malignancy, and that the criteria of malignancy appear to be unusual polyploidization with increased fractions of S phase cells.

Adenocarcinoma↗
PubMed1983

[Quantitative analysis of nuclear DNA of rat chondrocytes during the course of growth and aging--Feulgen-DNA cytofluorometry method].

There have been no reported studies on the Feulgen-DNA cytofluorometry of the cartilage cells. We have attempted to devise a method of cell separation from the epiphyseal and articular cartilages of the rats, and to analyze by cytofluorometry the changes in the ploidy patterns of these chondrocytes during growth and ageing of the animals. Chondrocytes were isolated from the proximal cartilage of tibia by dual enzymatic digestions of the cartilage matrix with papain and collagenase, followed by mechanical cell separation with scissors and a micro-homogenizer, and were smeared onto the object glass with PBS. These procedures were found to be suitable for the Feulgen-DNA cytofluorometry of the chondrocytes from our repeated studies. We also carried out Feulgen-DNA cytofluorometry combined with 3H-thymidine autoradiography to determine cellular DNA content of the DNA synthetic chondrocytes in the epiphyseal cartilage. It has been clarified that during the growth course of the rats, the chondrocytes of the epiphyseal cartilage consist of many mononuclear diploid cells, a few mononuclear tetraploid cells and of some fraction of the cells having intermediate DNA values between the diploid and tetraploid levels. Those cells with intermediate DNA values, after autoradiographic studies, were found to correspond to DNA synthetic cells, indicating cell proliferative activity. It has been shown that during ageing of the rats, most of the chondrocytes from the articular cartilage are mononuclear diploid cells. The distribution of each cellular DNA content at the diploid level as determined by Feulgen-DNA cytofluorometry was shown to become gradually broader.

Aging↗