In order to establish a causal relationship between p53 deficiency and increased Pancreatic cancer sensitivity of SCCHN cells to ATO we assessed whether reconstitution of wt p53 in p53-deficient SCC9 cells would decrease their sensitivity to ATO treatment. SCC9 cells were stably transfected with the tetracycline-inducible expression vector pRTS1 [26], either encoding for wt p53 (SCC9-wtp53) or as an empty vector (SCC9-vc). In the absence of doxycycline (Dox), expression of p53 and p21 was not detectable in SCC9-wtp53 cells. Addition of Dox led to a significant induction of wt p53 expression in a dose- (Figure 2 A) and time-dependent manner (Figure 2 B) which preceded upregulation of its target gene p21 (Figure 2 C). No induction of p53 and p21 was observed after Dox treatment of SCC9-vc cells (data not shown).

We then evaluated the effect of ATO on the clonogenic survival of SCC9-wtp53 cells in the absence (-Dox) or presence of wt p53 (+Dox). Reconstitution of wt p53 decreased clonogenic survival of SCC9-wtp53 cells (Figure 2 D left panel). After correcting for this growth-inhibitory effect of wt p53 reconstitution itself we observed a significantly reduced sensitivity of these cells to treatment with ATO at low doses (Figure 2 D right panel). Figure 2 Reconstitution of p53-deficient SCC9 cells with wt p53 renders them less sensitive to ATO treatment. Combined treatment of SCCHN cells with ATO and IR inhibits their clonogenic survival in an additive manner Based on previous reports of a significant radiosensitizing activity of ATO in a xenograft model of oral squamous cell carcinoma [9] we next asked whether we could observe such effect in our SCCHN cell lines as well and whether the radiosensitizing activity of ATO would also depend on the p53 status.

When cells were treated with ATO 2 hs before IR their clonogenic survival was inhibited more effectively than by either treatment alone. After correction for the cytotoxic activity of ATO itself no significant radiosensitizing activity, neither in p53-deficient nor p53-proficient SCCHN cell lines with the exception of the UM-SCC-25 cell line, could be observed (Figure 3). Drug_discovery The calculated combinatory indices (Table 1) indeed suggested an additive but not synergistic effect of the combination regimen in 9 of 10 cell lines. Since there is evidence from previous studies that the interaction between ATO and IR could depend on the sequence of their combination we also treated cells with ATO 2 hs after IR. Again, only additive effects of the combined treatment were observed (data not shown).