Incompatible effects of p53 and HDAC inhibition on p21 expression and cell cycle progression

Nutlin-3 selectively activates p53 by inhibiting the interaction of this tumor suppressor with its negative regulator murine double minute 2 (mdm2), while trichostatin A (TSA) is one of the most potent histone deacetylase (HDAC) inhibitors currently available. As both Nutlin-3 and TSA increase the levels of the cell cycle inhibitor p21(cip1/waf1) in cells, we investigated whether a combination of these compounds would further augment p21 levels. Contrary to expectations, we found that short-term exposure to Nutlin-3 and TSA in combination did not have an additive effect on p21 expression. Instead, we observed that activation of p53 prevented the ability of TSA to increase p21 levels. Furthermore, TSA inhibited Nutlin-3-induced expression of p53-dependent mRNAs including P21. This negative effect of TSA on Nutlin-3 was significantly less pronounced in the case of hdm2, another p53 downstream target. Aside from suggesting a model to explain these incompatible effects of Nutlin-3 and TSA, we discuss the implications of our findings in cancer therapy and cell reprogramming.

mock-treated sample of each experiment was set at 1.

Supplementary Figure S3
Various anti-p53 antibodies indicate that p53 protein levels and its subcellular localization do not change upon short-term treatment with TSA. MCF7 cells plated on 10 cm dishes were treated with TSA [0.1 µM] for 6 h; 1 h after TSA treatment, Nutlin-3 [5 µM] was added.
Afterwards, cells were washed twice with PBS and scraped off the culture dish in PBS. Cells were spun down at 7000 rpm for 3 min and supernatants were discarded. Pellets were resuspended in 200 µl Buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl 2 ; 340 mM sucrose; 10% glycerol; 1 mM dithiothreitol (DTT); protease inhibitor cocktail (PIC, # 11836170001, Roche)) and incubated on ice for 5 min. Triton X-100 was added to a final concentration of 0.1%, followed by an incubation on ice for 8 min. Samples were centrifuged at 1300 x g at 4°C for 5 min. Supernatants (fraction S1) were further centrifuged at 20000 x g at 4°C for 5 min. The resulting supernatants (fraction S2) and pellets (fraction P2) were kept on ice until further processed. The pellets of the previous step (fraction P1) were washed once with 100 µl Buffer A (without DTT and PIC) and spun down at maximum velocity for 1 min. Supernatants were discarded and pellets lysed in 100 µl Buffer B (3 mM EDTA pH 8.0, 0.2 mM EGTA pH 8.0, 1 mM DTT and PIC) for 30 min on ice, followed by centrifugation at 1700 x g at 4°C for 5 min. Resulting supernatants (fraction S3) were kept on ice until further processed. Resulting pellets (fraction P3) was washed once with Buffer B (without DTT and PIC) and spun down at maximum velocity for 1 min. Supernatants were discarded and pellets resuspended in 100 µl RIPA buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate and PIC), followed by incubation on ice for 10 min and subsequent centrifugation at maximum velocity for 10 min.
Lithium dodecyl sulphate (LDS) containing 100 mM DTT was added to the resulting supernatants (fraction S4) and pellets (fraction P4) as well as all other fractions from previous steps. Afterwards, samples were heated at 70°C for 10 min and fractions P2 and P4 sonicated for 2 x 10 sec. Subsequently, Western blots were performed as described in the Materials and Methods section. To detect total p53 levels, antibodies PAb421 (1) and CM-1 (2) were used (both were a kind gift of David P. Lane). Antibody # 07-329 (Upstate, Lake Placid, NY, UK) was used to detect acetylated histone H4 (K16).

Supplementary Figure S4
The subcellular localization of p53 does not change upon short-term treatment with TSA.
MCF7 cells seeded in 2-well glass object slides (Nunc) were treated as in shown in Figure   2A and stained as described previously (3). Pictures were taken with a Zeiss AxioVert 40C microscope equipped with a high-resolution AxioCam MRc5 camera. Brightness of images was adjusted with Adobe Photoshop CS4 Extended in accordance with instructions given by this journal.