Biomedical subjects

K Scotlandi

Publications and source records attributed to K Scotlandi.

At least 55 records · Page 3Linked to original sources

PubMed1995

High incidence of BK virus large-T-antigen-coding sequences in normal human tissues and tumors of different histotypes.

T-antigen (TAg) coding sequences specific for BK virus (BKV) were detected, by PCR amplification followed by Southern-blot hybridization, in a high percentage of human tumors and tumor-cell lines, as well as in normal tissues, by analysis of 189 specimens. Specifically, the BKV early region was detected in 85% of brain tumors and in all normal brain tissues, in 78% of osteosarcomas, in 38% of Ewing's tumors, in 40% of normal bone specimens and in 71% of normal peripheral blood cell samples. Wilms' tumor tissues used as a control were all negative for BKV sequences. RT-PCR analysis indicated that TAg coding sequences were expressed in specimens carrying BKV early region, ranging from 64% of the osteosarcomas to 100% of glioblastomas, Ewing's tumors, peripheral blood cells and normal bone. Moreover, DNA sequencing performed in 12 different positive samples revealed that the amplified PCR products are identical to the early-region sequence of wild-type BKV. The role of BKV TAg and its possible mechanism of action in human tumorigenesis are discussed.

Antigens, Viral, Tumor↗

PubMed1995

The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit.

The c-MET oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor (HGF), a cytokine that stimulates the invasive growth of normal and neoplastic cells. The Met/HGF receptor is expressed by epithelial cells and its ligand by cells of mesenchymal origin. Receptor-ligand interaction occurs via a paracrine circuit. We studied the expression of the Met/HGF receptor and of its ligand in mesenchymal human tumours by examining 39 clinical samples of bone tumours. The Met/HGF receptor was not detectable in the majority of bone tumours, as expected from their mesenchymal origin. Notably, the receptor was overexpressed in 60% of the osteosarcomas examined. In 12 osteosarcoma cell lines the Met/HGF receptor was overexpressed, phosphorylated by HGF stimulation and fully functional. HGF was detected in two out of seven clinical specimens of osteosarcoma. The ligand and the receptor are co-expressed in two clonal osteosarcoma cell lines. In these lines the Met/HGF receptor was constitutively phosphorylated; phosphorylation was suppressed by suramin treatment, a known blocker of autocrine loops. These data suggest that activation of the Met/HGF receptor by a paracrine or an autocrine mechanism might play a role in the particularly aggressive behaviour of osteosarcomas.

Bone Neoplasms↗

PubMed1995

Clinical relevance of Ki-67 expression in bone tumors.

BACKGROUND: The availability of Ki-67 monoclonal antibody has opened new possibilities for an extensive analysis of cell kinetics in human neoplasms. Ki-67 antibody reveals a nuclear antigen that is expressed in proliferating but not in quiescent cells. Although the reliability of Ki-67 immunostaining has been evaluated in different tumor types, little information has been reported on bone neoplasms. METHODS: Cell proliferation, as determined by Ki-67 expression, was measured by immunofluorescence on representative cytospins obtained from 205 patients with bone tumors. In each sample, the percentage of Ki-67-positive cells was quantified on at least 500 cells and expressed as Ki-67 labeling index (LI). RESULTS: Ki-67 LI was lower in benign and low grade lesions as compared with high grade malignant lesions. A correlation between Ki-67 LI and histologic grade was observed in osteosarcoma and chondrosarcoma. In osteosarcoma, among the 43 primary lesions included in this study, 30 patients, all treated with the same regimen of chemotherapy and limb-salvage surgery, were selected to establish the prognostic significance of cell proliferation. The Ki-67 labeling was higher in patients with a good histologic response to chemotherapy. However, at a 24-month follow-up, a worse prognosis was associated with a higher proliferative activity, whereas no correspondence was found between the histologic response to preoperative chemotherapy and the disease free survival, suggesting that in high grade osteosarcoma the biologic aggressiveness expressed by high levels of Ki-67 LI may be clinically more relevant than the responsiveness to antineoplastic agents. CONCLUSIONS: In bone tumors, the level of Ki-67 expression correlates with the level of malignancy and is diagnostically and prognostically useful.

Adolescent↗

PubMed1995

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses.

Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Analysis of P-glycoprotein expression in osteosarcoma.

Current treatment of high-grade osteosarcoma combines surgical removal of the lesion with chemotherapy. In this study we evaluated whether the expression of P-glycoprotein, a protein closely associated with multidrug resistance, may be helpful in identifying the patients whose tumours will be further resistant to specific agents. By using multidrug-resistant osteosarcoma cell lines as standards, the expression of P-glycoprotein was evaluated in 105 cases of primary and metastatic osteosarcoma by semiquantitative immunofluorescence. Overexpression of the protein was shown in 23% of primary and in 50% of metastatic lesions. In 38 cases, homogeneously treated and followed-up for at least 24 months, overexpression of P-glycoprotein appeared to be associated with a higher relapse rate and with a trend toward a worse outcome. These data support the role of P-glycoprotein in the response to chemotherapy and its involvement in the determination of the outcome of osteosarcoma patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism.

Calcitonin (CT) is a peptide hormone that interacts with the cAMP-and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 micrograms/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+]i, consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of [Ca2+]i. However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu2)cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 micrograms/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+]i in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [35S]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha 3-, -alpha v-, -beta 1-, and -beta 3-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha v beta 3 and the alpha 3 beta 1 receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-IIA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.

Animals↗

PubMed1995

Nuclear immunolocalization of P-glycoprotein in multidrug-resistant cell lines showing similar mechanisms of doxorubicin distribution.

The MDR1 gene product P-glycoprotein is a plasma membrane efflux pump which is responsible for multiple drug resistance of cancer cells. Although the ability of multidrug-resistant cells to exclude drugs from the nucleus is a distinctive and possibly the main mechanism for resistance against a number of drugs, including doxorubicin, this phenomenon is not entirely understood. In this paper, the relationship between doxorubicin subcellular distribution and P-glycoprotein activity at different cell sites has been investigated by different techniques. Cytofluorometry and confocal microscopy were used to study doxorubicin subcellular distribution in U-2 OS human osteosarcoma cells and in the multidrug-resistant variant U-2 OS/DX580. Stable levels of doxorubicin accumulation were found in the nuclei of sensitive cells, whereas the absence of detectable levels of drug in the nuclei of resistant cells could be attributed to an energy-dependent mechanism. Moreover, in resistant cells, inhibition of P-glycoprotein activity was able to induce drug accumulation in the nuclei of resistant cells and to achieve cytotoxic effects comparable to those observed in sensitive cells. Similar results were also found in isolated nuclei from U-2 OS/DX580 cells. The expression of P-glycoprotein in U-2 OS/DX580 and in two other multidrug-resistant cell lines (SW948-R-300 and LoVo-R-100) was investigated by confocal microscopy and immunoelectron microscopy, by using a panel of monoclonal antibodies directed against this protein. Higher levels of P-glycoprotein expression, not only in the plasma membrane and inside the cytoplasm, but also in the nucleus, were found in U-2 OS/DX580 and in LoVo-R-100 multidrug-resistant cells compared to their corresponding sensitive cells. SW948-R-300 cells, featuring increased amounts of MDR1 mRNA but lacking P-glycoprotein expression at the cell surface, showed a higher P-glycoprotein immunolabeling only in the nucleus and in the cytoplasm. The localization of P-glycoprotein in the nucleus of multidrug-resistant cells was confirmed also by studies on isolated nuclei and nuclear matrices, and by Western blot analysis on total cell and isolated nuclear extracts. These findings, suggesting the possible involvement of nuclear P-glycoprotein in the regulation of subcellular doxorubicin distribution in multidrug-resistant cells, open new insights on the mechanisms of P-glycoprotein-mediated resistance to anticancer drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Pre-treatment of human osteosarcoma cells with N-methylformamide enhances P-glycoprotein expression and resistance to doxorubicin.

N-methylformamide (NMF), a powerful differentiating agent, has been extensively used in experimental and preclinical cancer chemotherapy studies, alone or in association with conventional anti-cancer drugs. To evaluate the use of this molecule in the treatment of osteosarcoma (OS), we have analyzed the effects of NMF and doxorubicin (DXR) on DXR-sensitive and -resistant human OS cell lines. Our study shows that NMF exerts remarkable effects on cell proliferation and, in Saos-2 and SARG cells, also induces differentiation, as shown by increasing alkaline phosphatase activity. Moreover, NMF increases the cytotoxic activity of DXR when administered after the drug, in both DXR-sensitive and -resistant cells. However, when this agent is given before DXR, it enhances P-glycoprotein expression in U-2 OS cell lines. This over-expression is associated with reduced DXR accumulation within cells and with significant enhancement of resistance to DXR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

New model for bone resorption study in vitro: human osteoclast-like cells from giant cell tumors of bone.

Cells harvested from 12 human giant cell tumors of bone and kept in culture for several passages were characterized for bone-resorbing capability, total and tartrate-resistant acid phosphatase activity, response to the calciotropic hormone calcitonin, cell proliferation, multinucleation after passages, and presence of calcium sensing. Cells obtained from three tumors presented a complete panel of osteoclast characteristics and maintained their multinuclearity after several passages. Cells from four other tumors increased their cAMP levels after treatment with calcitonin, and the other five apparently consisted of cells of stromal origin. These human cell populations with osteoclast characteristics may provide valid in vitro models for the investigation of osteoclastic differentiation and activity.

Acid Phosphatase↗

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↗

PubMed1992

Evaluation of osteonectin as a diagnostic marker of osteogenic bone tumors.

Osteonectin (ON), a 32,000-kd glycoprotein involved in the early steps of mineralization of skeletal tissue, is a recognized differentiation marker of normal osteogenic cells. The expression of ON was evaluated in vitro and in tissue sections by the polyclonal antibody bON II. In different cell cultures immunocytochemistry and molecular biology displayed a nonspecific reaction for the antibody, which showed itself to be useless for the in vitro identification of cells of the osteoblastic lineage. The diagnostic use of bON II antibody was investigated by immunohistochemistry on a series of osteogenic and nonosteogenic bone tumors. A strongly positive stain of the entire neoplastic component of osteosarcoma and osteoblastoma and a weaker stain of the mononuclear component of giant cell tumor and chondroblastoma were observed. On the other hand, stains for chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, and brown tumor from hyperparathyroidism were entirely negative. Our results indicate that ON may be helpful in the histologic diagnosis of bone tumors, particularly in differentiating small cell osteosarcoma from other small round cell tumors.

Biomarkers, Tumor↗

PubMed1992

SARG: a new human osteosarcoma cell line. Expression of bone markers and of major histocompatibility antigens.

A new cell line (SARG) was established from a human radiation-induced osteosarcoma (OSA). It showed an epithelial-like morphology with polymorphous and sometimes bizarre nuclei. SARG had an osteoblastic differentiation pattern: almost 100% of the cells were positive for alkaline phosphatase, type I and III collagens and osteonectin. The expression of class I HLA antigens was detectable even after 40 in vitro passages. The expression of MHC antigens was greatly increased after in vitro treatment with interferon gamma (IFN-gamma), whereas interferon alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) increased the expression of class I antigens, but not of class II antigens. SARG was tumorigenic after subcutaneous injection in nude mice. Experimental metastases were never detected.

Adult↗

PubMed1992

Interphase cytogenetics for the detection of the t(11;22)(q24;q12) in small round cell tumors.

Among the small round cell tumors differential diagnosis is particularly difficult for their undifferentiated or primitive character. In this mixed group of tumors, only the primitive neuroectodermal tumors, which include Ewing's sarcoma (ES), show the unique and consistent feature of the (11;22)(q24;q12) translocation, which can therefore be considered a hallmark of these neoplasias. We analyzed four primitive neuroectodermal tumor cell lines, one osteosarcoma cell line, and 11 patients by fluorescent in situ hybridization with cosmid clones 23.2 and 5.8, bracketing the t(11;22) at 11q24. Metaphase spreads from tumor cell lines, and from biopsy specimens of three patients with ES were analyzed. In the remaining eight patients comprising five ES, two small cell osteosarcomas and one chronic osteomyelitis, only nuclei preparations were available for analysis. We detected the t(11;22) in interphase nuclei of the four primitive neuroectodermal tumor cell lines, of three patients in which the karyotype demonstrated the translocation and in five cases of ES in which cytogenetic analysis had not been possible. Two cases of small cell osteosarcoma and one chronic osteomyelitis were also analyzed and were both normal with respect to the t(11;22). By analyzing cell lines and small round cell tumor samples by fluorescent in situ hybridization, we established that interphase cytogenetics is a rapid alternative to chromosomal analysis for the detection of the t(11;22) and represents an invaluable tool for the differential diagnosis of small round cell tumors.

Adolescent↗

PubMed1992

Functional and biochemical characterization of osteoclast-like cells derived from giant cell tumours of bone.

Cells harvested from human giant cell tumours of bone were characterized on the basis of morphological features, proliferative capacity, total(AP) and tartrate resistant acid phosphatase (TRAP) activity, and hormonal response. Culture were formed by mononucleated and multinucleated cells. Mononucleated cells showed fibroblastic morphology, whereas multinucleated cells showed osteoclastic phenotype. We conclude that in these cultures mature osteoclasts and their mononuclear precursors are present.

Bone Neoplasms↗

PubMed1992

Integrin expression and adhesion property of osteoclast-like cells from giant cell tumours of bone.

Cells cultured from human giant cell tumours of bone were used to study interactions with different extracellular matrix proteins as Collagen, Fibronectin, Osteocalcin, Thrombospondin and Bone Sialoprotein II. Cells were capable of recognizing these substrata; beta 3 integrin subunit was distributed in focal adhesions, together with beta 1 on BSPII, FN, and in presence of serum, whereas and presented a diffuse organization onto the other substrate. beta 1 alone was expressed over collagen coated coverslips.

Bone Neoplasms↗

PubMed1991

Immunological and non-immunological influence of H-2Kb gene transfection on the metastatic ability of B16 melanoma cells.

The H-2b-negative B78HI clone (derived from B16 melanoma) was transfected with the H-2Kb gene; 4 cell clones expressing membrane H-2Kb antigens and 2 control clones (transfected with pSV2neo alone) were used for studies of metastatic ability, immunogenicity, NK sensitivity and homotypic adhesion. The experimental metastatic capacity of H-2Kb transfectants in syngenic mice was greatly diminished in comparison with control and parent cells. Both immune-mediated and intrinsic properties of transfectants correlated with their lower metastatic ability. A cell-mediated cytotoxic response was induced by repeated in vivo immunizations of syngeneic mice followed by in vitro restimulation of effectors when transfectants (but not controls) were used as immunizers and as targets. Moreover, homotypic adhesion of H-2Kb transfectants was significantly lower than that of controls. Sensitivity to NK cells of transfectants was not decreased in comparison to H-2-negative controls. It is known that in vitro treatment with IFN-gamma of H-2-positive B16 melanoma cells induces a simultaneous increase in H-2 expression and in experimental metastasis; treatment of H-2Kb transfectants with IFN-gamma induced a higher Kb expression, but no increase in metastatic ability, thus suggesting that the IFN-sensitive component that mediates enhancement of metastasis is not H-2Kb.

Animals↗

PubMed1991

In vitro differentiation of rhabdomyosarcomas induced by nickel or by Moloney murine sarcoma virus.

In vitro cultures and clonal derivatives have been established from rat rhabdomyosarcomas induced by Moloney-Murine Sarcoma Virus (MSV) or by nickel sulfide; differentiation ability has been studied as expression of desmin, embryonic and adult myosin isoforms, alpha-actin isoforms and cellular fusion. The two rhabdomyosarcoma models showed different levels of myogenic differentiation. Multinucleated myotube-like structures were frequently observed in cultures derived from nickel-induced tumours. Desmin was present in 50-80% of cells and embryonic myosin in up to 10%. In MSV-tumour-derived cultures and in their metastases or clonal derivatives two cell types are present in different ratios: spindle-shaped cells, adherent to plastic surfaces, and rounded cells, loosely attached or floating free in the medium. These cultures showed features of myogenic differentiation (10-80% desmin-positive cells), but embryonic myosin expression and production of multinucleated myotube-like structures were very rare events. Cultures from autochthonous lymph node and lung metastatic cells showed similar patterns of differentiation. Retinoic acid increased differentiated features (myotube formation and embryonic myosin expression) only in nickel-induced rhabdomyosarcoma cells. The two models described here mimic the heterogeneity in differentiation pattern found among human rhabdomyosarcomas. Myogenic differentiation ability was retained at a good level by nickel-induced tumours, whereas it was strongly impaired in MSV-induced tumours.

Animals↗

PubMed1991

Reduced metastatic ability of in vitro differentiated human rhabdomyosarcoma cells.

We studied the human embryonal rhabdomyosarcoma cell line RD and 8 derivatives obtained in our laboratory either by cell cloning or by culturing in vitro cells from tumors or secondaries grown in nude mice. The expression of desmin and of the embryonic isoform of myosin and the formation of multinuclear myotube-like structures were studied as specific markers of myogenic differentiation. During continuous growth, each derivative contained a proportion (ranging from 5 to 80% among derivatives) of desmin-positive cells and a small number of myosin-positive or multinuclear elements. Cells from continuous cultures were injected intravenously in nude mice, producing lung and kidney/adrenal nodules. No correlation was found between the proportion of cells expressing desmin and metastatic capacity. When cultures were grown in differentiation medium (Dulbecco's minimal essential medium + 2% horse serum) some derivatives (designated type A) showed a strong increase in the proportion of myosin-positive cells, while others (type B) showed no increase. In vitro differentiation significantly reduced the metastatic ability of type A cells, while no modification was observed in type B after growth in differentiation medium. The proliferative ability of type A and type B cells grown in differentiation medium did not correlate with the proportion of myosin-positive cells, and extensive formation of multinuclear myotubes was never observed. It was concluded that reduction of experimental metastatic ability was mediated by events related to late, though not necessarily terminal, differentiation of rhabdomyosarcoma cells.

Animals↗