Visit our Expo - Redox and Inflammation signaling 2012

Session XV : Reactive oxygen species and cell signaling Poster XV, 5
Functional Genetic Screens to Identify Genes in Adaptative Responses to Hypoxia
Celine Rofel1, Iris Partouns1, Raymond Hessing1, Elena Bardina1, Marianne
Koritzinsky2, Brad Wouters2, Jan Willem Voncken1
1) Department of Molecular Genetics
2) Department of Radiation Oncology; Research Institute Growth & Development,
Maastricht University, Universiteitssingel 50 6229 ER; Maastricht, The Netherlands
Tumors need oxygen and nutrient to grow, but these two factors are rapidly depleted, due to a
rapid growth, leading to hypoxic tissue in the tumor. Hypoxia remains a constant feature of
these tumors and contributes to tumor progression by triggering adaptative mechanisms.
HIF-1 is one of most important transcription factor up-regulated during hypoxia.
HIF-1 alpha appear to be important for tumor vascularization and metabolic adaptation to
hypoxia, which are essential for tumor progression.
To further investigate genes involved in mechanism of adaptation to hypoxia we screened a
placenta retroviral cDNA library for genes capable to bypass a growth arrest induced by
hypoxic conditions.
The HCT116 colon carcinoma cell line were selected to screen the library based on their
capacity to form colonies, to be genetically stable and sensitive to hypoxia.
The HCT116 were infected by the cDNA retroviral library 2 days before applying the hypoxic
treatment of 0% during 5 days, and then placed under normoxia (21%) for 10 days. The DNA
from the cells was isolated and a PCR was performed to amplify the integrated cDNA. Two
intense bands of 1kb and 4kb were observed. The 1kb band was isolated, subcloned into a
PCR4 topo plasmid and sequenced. 2 clones were identified as placental lactogen cDNA. The
cDNA of placental lactogen was cloned into a retroviral plasmid to validate the gene function
and elucidate the mechanism. To confirm that placental lactogen increase the survival of the
cells submitted to hypoxia reoxygenation we will analyze HCT116 expressing placental
lactogen survival by colony formation assay. To evaluate the capacity of placental lactogen to
give a growth advantage under hypoxia we will submit the cells to 0% during 2 and 3 days to
achieve a growth curve.
Based on the literature this gene plays a role in suppression of apoptosis pathway, thus it
could be interesting to investigate whether it also plays a role in hypoxia-induced apotosis.
Therefore we will assess the number of apoptotic cells submitted to hypoxic condition
expressing or not placental lactogen.
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