C87
The RhoA kinase (ROCK) inhibitor Y27632 and specific novel structural analogues of this compound cause irreversible elimination of transformed NIH3T3 cells from cultures
Lynne Hampson1, Xiaotong He1, Anthony Oliver1, John Hadfield2, Timothy Kemp2, John Butler2, Alan McGown2, Henry Kitchener1, Ian Hampson1
1University of Manchester, Manchester, UK, 2University of Salford, Manchester, UK
Intoduction
NIH3T3 cells will readily transform in vitro with accompanying loss of cell polarity and contact inhibited growth. As a consequence, these cells have been used extensively as a model system for the study of cellular transformation.
Method
Stable transfection of NIH3T3 cells with guanidine exchange factor (GEF) 16 caused the formation of multilayered non-contact inhibited transformed colonies. GEF16 transfected cells were treated with either the ROCK inhibitor Y27632 or one of 64 novel structural analogues. Their ability to effect cell growth and transformed colony formation and persistence following drug withdrawal was evaluated. In vitro kinase inhibitory activity assays were carried out with selected compounds against X40 recombinant kinases (SelectScreenTM Invitrogen).
Results
At 10μM concentration, Y27632and three other structural analogues prevented the formation of and irreversibly eliminated transformed colony forming cells from GEF16 transfected NIH3T3 cells. None of these compounds showed any adverse toxicity against sub-confluent cells in logarithmic growth. Interestingly the three novel Y27632 analogues had minimal ROCK inhibitory activity- showing instead maximum activity against the kinases Aurora A, p38 (MAPK14) and Hgk (MAP4K4).
Conclusion
Our data are the first to indicate that blockade of different yet specific kinases can cause irreversible elimination of cells with the ability to undergo loss of contact inhibition and polarity whilst having no discernible effect against non transformed cells. These observations imply that chemical blockade of selected kinases may form the basis of novel strategies for cancer chemoprevention and NIH3T3 cells may provide a convenient means of identifying compounds with this activity.
