CCN3/nephroblastoma overexpressed matricellular protein regulates integrin expression, adhesion, and dissemination in melanoma (vol 68, pg 715, 2008)
Authors: V. Vallacchi, M. Daniotti, F. Ratti, D. Di-Stasi, P. Dcho, A. De-Filippo, G. Tragni, A. Balsari, A. Carbone, L. Rivoltini, G. Parmiani, N. Lazar, B. Perbal, M. Rodolfo
Journal: Cancer Research (2007)

2051  Introduction: The efficacy of many chemotherapeutic agents and of radiotherapy is directly linked with adequate oxygen supply. As hypoxic regions are frequently found in solid tumors, this implies that tumors often contain viable cells that are more resistant to cytotoxic drugs and irradiation. In this study, we investigated the impact of moderate hypoxia (1% O2) and extreme hypoxia (anoxia, 0% O2) on the characteristics of 3 tumor cell lines. In addition, the cytotoxicity of gemcitabine (dFdC) was studied under normoxic versus hypoxic or anoxic conditions.  Materials & methods: The human tumor cell lines investigated are A549 (lung carcinoma cell line, wt p53), A549-E6 (transfected cell line with abrogated p53) and A549-LXSN (vector transfected control). During hypoxic exposure, cells were kept in an anaerobic chamber, continuously flushed with a premixed gas containing 1% or 0% oxygen. At several time points (1h, 24h and 48h hypoxia; 24h and 96h reoxygenation), cells were sampled. Growth curve studies determined the influence on cell proliferation. The effect of hypoxia on cell cycle and apoptosis was flow cytometrically examined after Vindelov staining and Annexin V - PI labeling. To validate the hypoxic conditions, expression of VEGF was measured by quantitative real time RT-PCR. For the cytotoxicity experiments, cells were treated with dFdC (0 - 100 nM) for 24h and hypoxic incubation was varied from 4 till 24 hours. The chemosensitivity was assessed by the sulforhodamine B assay.  Results: Hypoxic or anoxic incubation for 24h or 48h inhibited cell proliferation in all cell lines examined, but only significantly at some time points. We observed a small non-significant increase in G1/0 phase and a small decrease in the number of cells in S phase after 48h hypoxia in A549 and A549-LXSN. No significant induction of apoptosis was found in any cell line, at any time point. The expression of VEGF increased after 24h hypoxia, which was even more pronounced after 48h hypoxia. Upregulation of VEGF after anoxic incubation was much higher than after hypoxic incubation.  A549 cells were treated with dFdC for 24 hours and subsequently placed under 1% O2. After 24 hours, cells were reoxygenated. Hypoxic exposure induced a small decrease in chemosensitivity. Using anoxic conditions, a gradual increase in chemoresistance was seen when cells were exposed to anoxia for a longer time prior to treatment with dFdC.  Conclusion: An in vitro model was established and could be used to examine the effects of cytotoxic drugs under low oxygen conditions. Preliminary results suggest that reduced oxygen conditions could be involved in mediating the sensitivity to dFdC. However, these experiments have to be repeated before final conclusions can be drawn. Experiments, investigating the impact of oxygen deficiency on the radiosensitizing effect of dFdC are currently ongoing.