Research Interests
Identification of oncogenic events collaborating with loss of function of the Retinoblastoma gene family in tumorigenesis
 Tinke Vormer |
 Camiel Wielders |
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We have previously shown that ablation of Rb and p107 or Rb and p130 in primary mouse embryonic fibroblasts (MEFs) was sufficient to cause immortality and to reverse the growth inhibitory effect of RASV12 expression into a proliferative stimulus. Yet, Rb-/-p107-/-, Rb-/-p130-/- (DKO) and even Rb-/-p107-/-p130-/- (TKO) MEFs were not transformed by RASV12 as they were unable to grow anchorage independently and did not form tumors in nude mice (Peeper et al., 2001). Apparently, in pocket-protein-defective cells, a cell cycle mechanism still operates to restrict proliferation under non-adherent conditions. To uncover this mechanism, we performed genetic screens to identify events that permit anchorage-independent growth of RASV12-expressing DKO and TKO MEFs.
GAIN-OF-FUNCTION SCREENS (Vormer et al., 2009).
Loss of adhesion induces G1 and G2 arrest in RASV12-expressing DKO and TKO cells (dependent on the level of pocket protein activity).
By screening a cDNA library, we found over-expression of the oncogene TBX2 to alleviate cell cycle arrest and to promote anchorage-independent growth.
TBX2-induced transformation only occurred in the absence of pocket proteins and could be attributed to down regulation of the p53 pathway.
Indeed, RNAi-mediated knockdown of p19ARF, p53 or p21CIP1 induced anchorage-independent growth, but again, only in the absence of pocket proteins.
These results suggest that a balance between pocket protein and p53 activity determines the level of transformation in MEFs.
Since transformation of human fibroblasts also requires ablation of both pathways, the mechanism of transformation of human and mouse cells are not as different as previously claimed.
LOSS OF FUNCTION SCREENS USING CLONED RNAI LIBRARIES
RNA interference (RNAi) has emerged as a powerful tool for gene silencing.
The knock-down of genes is achieved by introducing small double-stranded RNA molecules in cells, which are complementary to the target gene to be silenced and mediate sequence-specific mRNA destruction.
Such RNA duplexes can be transiently transfected into cells or be stably expressed as short hairpins (shRNA) from pSuper plasmids.
Based on the pSuper RNAi technology, synthetic shRNA libraries have been constructed that can be used for genome-wide screening, e.g.,
for tumor suppressor genes whose shRNA-mediated knockdown confers a growth advantage to cells.
As an alternative to synthetic shRNA libraries we have developed new technique to produce shRNA libraries enzymatically from cDNAs.
By this approach, we have identified several novel suppressors of anchorage-independent growth.