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Background: To evaluate the impact of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes on methotrexate (MTX) resistance in osteosarcoma cells in relation to retinoblastoma (RB1) gene status., Materials and Methods: A series of human osteosarcoma cell lines-either sensitive or resistant to MTX-and 16 osteosarcoma tumour samples were used in this study., Results: In U-2OS MTX-resistant variants, and in other RB1-positive cell lines, MTX resistance was associated with increased levels of DHFR and with a slight decrease of RFC gene expression. In Saos-2 MTX-resistant variants, and in another RB1-negative cell line, development of MTX resistance was associated with a decrease in expression of RFC, without any significant involvement of DHFR. In osteosarcoma clinical samples, amplification of the DHFR gene at clinical onset appeared to be more frequent in RB1-positive compared with RB1-negative tumours., Conclusions: Amplification of the DHFR gene may occur more frequently in the presence of RB1-mediated negative regulation of its activity and can be present at clinical onset in osteosarcoma patients. Simultaneous evaluation of RFC, DHFR and RB1 gene status at the time of diagnosis may become the basis for the identification of potentially MTX-unresponsive osteosarcoma patients, who could benefit from treatment protocols with alternative antifolate drugs.

Relatively little is known about the biologic relevance of numerical chromosomal changes in relation to DNA content in osteosarcoma. In this study, by using a series of human osteosarcoma cell lines, we standardized a method for the assessment, on the same nuclei specimen, of both specific chromosome copy numbers by fluorescence in situ hybridization (FISH) and the DNA content by static cytofluorometry or image cytometry. On the same cell lines, we also evaluated the DNA content by using flow cytometry and the chromosome number distribution by metaphase analysis. Comparison between these different methods showed that DNA ploidy level as determined by FISH or metaphase analysis is frequently lower than the ploidy pattern as defined by cytometric methods. By using comparative genomic hybridization, we were able to demonstrate that these discrepancies were due to the presence of several unbalanced chromosome aberrations, specifically gains and high-level amplifications, which affect the total DNA content with less effect on the total chromosome number. Thus, evaluation of DNA ploidy in osteosarcoma cells is needed for a correct interpretation of FISH or cytogenetic data concerning numerical chromosomal changes. Evaluation of tumor ploidy in a series of clinical samples demonstrated that in high-grade osteosarcoma, flow cytometry sometimes may give false results because of the presence of high proportions of contaminating, nonneoplastic cells that cannot be excluded from the flow cytometric assessment but that do not interfere with the evaluation of DNA ploidy by static cytofluorometry or image cytometry, in which only tumor cells are selected for the analysis. The possibility of using this method to evaluate, on the same nuclei sample, both specific chromosomal aberrations and DNA ploidy may allow a better determination of numerical chromosomal changes that may be relevant for the biologic behavior of osteosarcoma.

Alkaline phosphatases are a family of glycoproteins that are able to hydrolize various monophosphate esters at a high pH optimum. Liver/bone/kidney (L/B/K) alkaline phosphatase (ALP) is one of the four major isoenzymes that belong to this family. Apart from its role in normal bone mineralization, other functions of L/B/K ALP remain obscure, both in physiological and in neoplastic conditions, including the bone-forming tumor osteosarcoma. In this study, we transfected the U-2 OS osteosarcoma cell line, which does not show any basal expression of this enzyme, with the full-length gene of L/B/K ALP, and analyzed the in vitro and in vivo features of four transfectants showing different expression of L/B/K ALP. A reduced in vitro ability to invade Matrigel and to grow in a semi-solid medium, together with a lower tumorigenic and metastatic ability in athymic mice, was found to be associated with a high level of cell surface L/B/K ALP activity. Moreover, L/B/K ALP transfectants showed a reduced secretion of matrix metalloproteinase-9 enzyme. These findings indicate a loss of aggressiveness of osteosarcoma cells after the expression of L/B/K ALP on their surface and suggest a new role for this enzyme.

The transcription of MDR1 gene may be increased by mutation or loss of function of p53 gene. In this study, we investigated whether in osteosarcoma, the p53 status is correlated with overexpression of the MDR1 gene product P-glycoprotein. The relationship between P-glycoprotein expression and p53 status was analyzed by immunohistochemistry in 64 primary and 11 metastatic high-grade osteosarcomas. In the same series, we also assessed the nuclear accumulation of MDM2 protein, whose binding to p53 protein provides an alternative mechanism of p53 inactivation. No association was found between mutant-p53 and MDM2 nuclear accumulation either with P-glycoprotein expression or with clinical course. Only increased expression of P-glycoprotein in tumor cells was significantly associated with a poor outcome, further supporting the adverse prognostic value of this marker in osteosarcoma.

The relationship between P-glycoprotein expression and malignancy is controversial. We have recently found that, in osteosarcoma, multidrug resistance (MDR) is associated with a less aggressive behavior, both in vitro and in clinical settings. In this study, we evaluated whether P-glycoprotein overexpression has a cause-effect relationship with the reduced metastatic potential of MDR cells, or rather reflects a more complex phenotype. MDR1 gene-transfected osteosarcoma cell clones, showing different levels of P-glycoprotein expression, were analysed for their in vitro characteristics and their tumorigenic and metastatic ability in athymic mice. Apart from the different levels of P-glycoprotein, no significant change in the expression of surface antigens or in the differentiative features were observed in the MDR1 gene transfectants compared to the parental cell lines or control clones, obtained by transfection with neo gene alone. In contrast to controls, however, MDR1 transfectants showed a significantly lower ability to grow in semi-solid medium and were completely unable to grow and give lung metastases in athymic mice. These findings indicate that P-glycoprotein overexpression is causally associated with a low malignant potential of osteosarcoma cells, and open new insights on the role and functions of P-glycoprotein activity.

Background: Increased levels of P-glycoprotein occur in some osteosarcomas. In this study we determined the relation between P-glycoprotein status and outcome in patients with high-grade osteosarcoma., Methods: P-glycoprotein status was determined immunohistochemically in specimens of osteosarcoma of the extremities (stage II) from 92 patients who were treated with surgery and chemotherapy. The P-glycoprotein status was analyzed in relation to the length of event-free survival., Results: The presence of increased levels of P-glycoprotein in the osteosarcoma was significantly associated with a decreased probability of remaining event-free after diagnosis (P = 0.002). In a multivariate analysis, P-glycoprotein status (P = 0.001) and the extent of tumor necrosis after preoperative chemotherapy (P = 0.04) were independent predictors of clinical outcome. The risk of adverse events was increased substantially (rate ratio, 3.37; 95 percent confidence interval, 1.60 to 7.10) among patients with increased levels of P-glycoprotein in tumor cells, as compared with patients who did not have increased levels of P-glycoprotein in tumor cells., Conclusions: In patients with high-grade osteosarcoma treated with surgery and chemotherapy, the presence of increased levels of P-glycoprotein in tumor cells is associated with a significantly increased risk of adverse events and is independent of the extent of necrosis after preoperative chemotherapy.

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.

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.

Multidrug resistant variants of two human osteosarcoma cell lines (U-2 OS and Saos-2) were selected by continuous exposure to doxorubicin. The in vitro and in vivo growth characteristics of these sublines as well as the expression of osteoblastic markers and of some surface antigens were analyzed. Resistant variants showed a higher doubling time and a lower cloning efficiency, and a lower metastatic ability after i.v. injection than corresponding parental cell lines. All the sublines showed overexpression of p-glycoprotein (referred to as p170). The level of expression of this protein in the different cell lines was directly related to the degree of resistance as shown by the in vitro sensitivity to doxorubicin and other multidrug-related drugs. In sublines showing the highest levels of resistance (over 300-fold), p170 overexpression was associated with mdr 1 gene amplification. These are the first multidrug resistant human osteosarcoma cell lines ever reported. They may be used as a model for further investigations into the mechanisms of drug resistance in osteosarcoma and as a standard for the assessment of chemosensitivity in clinical samples.

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.