Figure 3

Heparanase overexpression induces Akt/PKB phosphorylation and facilitates xenograft growth. (a, b) Heparanase activity. Heparanase cDNA was stably transfected into HT29 cells. Control Vo (♦) and heparanase-transfected (▴) cells (1 × 10⁶) were plated intact on ³⁵S-labeled extracellular matrix (b) or subjected to three cycles of freeze/thaw before incubation (18 h, 37°C, pH 6.0) with the labeled extracellular matrix. (a). Sulfate-labeled degradation fragments released into the incubation medium were subjected to gel filtration on Sepharose 6B, as described in ‘Materials and methods’. (Insets) Heparanase expression and secretion. Cell lysates (a) and conditioned medium (b) of control Vo and heparanase-transfected (Hepa) cells were immunoblotted with anti-heparanase 1453 antibodies to reveal heparanase expression (a) and secretion into the culture medium (b). (c) Akt/PKB phosphorylation. Lysates of control (Vo) and heparanase-transfected (Hepa) HT29 cells were immunoblotted with anti-phospho (left, upper panel) and total (left, lower panel) Akt/PKB antibodies. HT29 cells were left untreated (0) or incubated with recombinant heparanase (1 μg/ml) for the time indicated (min) and cell lysates were immunoblotted with antibody directed against phosphorylated (right, upper panel) and total (right, lower panel) Akt/PKB. (d, e) Xenograft development. (d) Tumor volume. Control Vo and heparanase-overexpressing HT29 cells (5 × 10⁶) were implanted subcutaneously at the flank of SCID mice (n=5). Xenograft size was measured twice a week with a caliper and tumor volume was calculated. Tumor weight (g) measured at the end of the experiment on day 62 is shown in (e). A 2.5-fold increase in tumor weight is noted in tumors produced by heparanase-overexpressing cells.