A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression

Abstract

Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.

Glycosaminoglycan composition changes with MG‐63 osteosarcoma osteogenesis in vitro and induces human mesenchymal stem cell aggregation

“…Cell culture and dose-response assays MG-63 osteosarcoma cells (ATCC, Manassas, VA) and human mesenchymal stem cells (hMSCs, Cambrex Corporation, East Rutherford, NJ) were used as previously described (Kumarasuriyar et al, 2007). Cultures were seeded at 3,000 cells/cm 2 in 6-well plates under differentiating conditions until assay endpoint.…”

“…DNA was extracted by lysing cells in dH 2 O and freeze-thawing four times before centrifugation. Alkaline phosphatase (ALP) activity was assessed as previously described (Kumarasuriyar et al, 2007). To measure calcium content, cell layers were lysed with 1% trichloroacetic acid and the supernatant mixed 1:10 with calcium arsenazo reagent (Pointe Scientific, Canton, MI).…”

“…GAGs were harvested from MG-63 cells as previously described (Kumarasuriyar et al, 2007) and characterized by size exclusion chromatography. Prepacked Superdex 75 HR columns (10 mm  300 mm, GE Healthcare, Uppsala, Sweden) were equilibrated with AKTA Explorer System (Amersham Pharmacia Biotech, Uppsala, Sweden) at 0.4 ml/min in 150 mM NaCl, 10 mM HEPES buffer (pH 7.2) and the column void (V o ) and total (V t ) volumes determined using blue dextran 2000 and sodium dichromate, respectively.…”

Modulation of Glycosaminoglycans Affects PrPScMetabolism but Does Not Block PrPScUptake

“…Sulfate moieties of GAG mimetics are required for the antiprion activity (40). Sodium chlorate, a competitive inhibitor of the cellular 3=-phosphoadenosine 5=-phosphosulfate, prevents both HS and CS sulfation (41)(42)(43) and also decreases PrP Sc accumulation in persistently infected cells (31,44). GAG sulfation also affects PrP Sc formation in in vitro assays and thus directly acts on PrP Sc amplification (45).…”

Control of cell fate decisions

“…Perhaps the use of a more naïve source, or more homogenous population of stem cells, such as those found in amniotic fluid (Kolambkar et al 2007), or following immuno-selection ), may prove more successful. One approach to overcome this is the transplantation of an acellular bioactive construct containing heparan (Kumarasuriyar et al 2007;Woodruff et al 2007) or chondroitin sulfate (Hui et al 2007), molecules that are thought to stimulate autologous stem cells within the host.…”