PEGylation did not alter the inhibitory properties of D2A-Ala. Human tumour xenografts in the immunodeficient nude mice using HT 1080 and HT 29 cell lines showed that PEG-D2A-Ala significantly prevents tumour growth decreasing size, weight and density of tumours.
MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls.
To test the radiosensitivity of acutely and chronically hypoxic cells in tumors, we isolated high-EGFP ("chronically hypoxic") and low-EGFP cells (containing both acutely hypoxic and aerobic cells) from HT 1080 xenograft tumors by fluorescence-activated cell sorting (FACS), immediately after in situ treatment with 20 Gy (ambient or clamped), and plated the cells to determine clonogenic survival in vitro.
In the light of this, the applicability of a hypoxia-responsive enhanced green fluorescent protein (EGFP)-based system to the measurement of tumor hypoxia was tested in human HT 1080 fibrosarcoma cells stably transfected with a destabilized EGFP vector containing the hypoxia-responsive 5HRE-hCMVmp promoter or, as a positive control, the strong constitutive CMV promoter.