LNZ308 and LN229 glioma cell lines stably expressing p14ARF under a tet-on inducible system
To study the biological effects of ARF, we have restored ARF gene expression in ARF-deficient human glioblastoma tumor cells. We engineered two ARF-null cell lines to stably express a cDNA for conditional expression of p14ARF using the tet-on system. We generated Tet-on p14ARF clones in wt-p53 cell line LN229-L16 (clones A5, A18) and p53-null cell line LNZ308-C16 (clone C19). We found that ARF expression is tightly induced by doxycycline (dox) and is associated with an increase in the mRNA levels of the p53-target gene, p21 as a result of p53 stabilization by ARF in A5 and A18 but not in C19 cells as they are p53-null (Fig A). Western blot analyses confirmed tightly regulated p14ARF induction by dox with concomitant stabilization of p53 and downstream p21 activation (Fig B). This data shows that we successfully generated ARF inducible-cells that can be used in a variety of studies.
A: Northern blots showing increased ARF mRNA levels upon dox treatment (2 ug/ml for 48hrs) in clones A5 and A18 derived from tet-on LN229-L16 cells (upper panel) and in clone C19 derived from tet-on LNZ308-C16 cells (lower panel) and its consequence on p21 mRNA levels.
B: Western blots showing the induction of p14ARF in A5, A18 (upper panel) and C19 clones (lower panel) upon treatment with dox (2ug/ml for 48hrs) and its effect on p53 stabilization and p21 expression in A5 and A18 cells positive for p53. Because C19 are p53 null, the effect of ARF on p53 was not tested.