In this study, we searched for the cause of varying cellular responses elicited by two human uterine sarcoma cell lines upon treating them with the HDACi SAHA. By screening several important pro-apoptotic key molecules, we found that expression of the tumor suppressor protein p53 was completely undetectable in ESS-1 cells when compared to the easily detectable and abundant expression in MES-SA cells. Furthermore, only basal PUMA levels were present in relation to MES-SA cells. Irrespective of absent p53 expression, the promising anti-cancer compound SAHA increased expression of the cell cycle kinase inhibitor p21
WAF1 which downregulates tumor cell proliferation via cell cycle arrest in the G1/S phase. This observation confirms a previous study which reported, in contrast to normally tightly p53-controlled expression, the possibility of p53-independent activation of the p21
WAF1 promoter by direct SAHA-mediated activation of SP1 sites [
32]. Overall, this finding was in consistency with prevailing induction of autophagic cell death in ESS-1 cells [
10] and the predominant apoptotic response in MES-SA cells provoked by SAHA as previously elaborated [
20,
21]. Therefore, we searched for the molecular mechanism in which the absence of p53 expression originated. We detected a homozygous 637C>T transition in exon 6 of the
TP53 gene in the ESS-1 cell line which results in the formation of a premature stop codon. Since codon 213 consists of a CpG dinucleotide, which is a target for cytosine methylation, the nonsense mutation at codon 213 could have occurred in vitro in this cell line as the result of endogenous deamination of 5-methylcytosine to thymine. Upon database screening, we were also able to confirm this mutation by an entry in the COSMIC (catalogue of somatic mutations in cancer) database for human immortal cancer cell lines (
http://www.cancer.sanger.ac.uk/cancergenome/projects/cell_lines/). Moreover, this mutation has been verified and annotated for endometrial carcinoma [
33]. The R213X non-sense mutation, which is not present in MES-SA cells, is located at the N-terminus of the DNA binding domain (DBD) of p53 and leads to a truncated protein that is missing the last ~180 of 393 amino acids. Although we used a monoclonal human-specific p53 antibody (clone DO-7) that recognizes an epitope between amino acids 1–45 of wildtype as well as mutant forms of p53, we were not able to detect the presumptive truncated p53 protein in our experiments. In support of our finding, also ovarian cancer patients possessing the R213X mutation exhibited null type mutations that could not be detected by immunohistochemical staining for unknown reasons [
34].
The apoptosis-inducing functions of p53 are exerted in two ways: one is through the transcriptional activation of pro-apoptotic proteins such as Puma, Noxa and Bax, the other is via direct physical and functional interaction with members of the Bcl-2 protein family that cause mitochondrial membrane permeabilization. The identified homozygous p53 mutation thus clearly explains apoptosis resistance in ESS-1 cells as in the case of complete p53-deficiency, no physical interactions or transcriptional activities can take place at all. Alternatively, in case of undetectable p53 protein, it was previously reported that the R213X mutation disrupted the efficiency of p53 transactivation by its inability to bind consensus binding sequences of p53 in the regulatory region of the p21WAF gene [
35]. In addition, this mutation was also found to be a dominant negative p53 variant which suppresses the endogenous wild-type function [
36]. Beyond that, we hypothesized that p53 could be the missing link leading to either SAHA-induced autophagy in the absence of the p53 protein or preferential SAHA-stimulated apoptosis when a functional wild type molecule is present in the cell. In support of this, in addition to its many other tumor suppressing activities, p53 has been identified as a major regulator of autophagy in recent time [
37‐
40]. Autophagy induced by p53 may facilitate p53's cell cycle arrest activities, such that autophagy mediates the selective degradation of damaged molecules and organelles in order to provide an energy source for the damage repair process and promote ‘cell healing’. Alternatively, when the extent of damage is beyond repair, autophagy may act to synergize with accelerated cell death in response to p53 activation. While the autophagy-promoting activity of p53 requires the presence of p53 in the nucleus, associated with transcriptional activity, the autophagy-suppressing function of p53 was found to completely depend on its cytoplasmic presence without transcriptional dependence. Both, autophagy-promoting as well as -inhibiting activities of p53, engage the mTOR signaling pathway which in response to genotoxic or metabolic stress cross-talks with p53 in a coordinated fashion [
41]; thus, both signaling machineries regulate cell growth, proliferation, and death together. This mechanism of p53-induced autophagy involves activation of 5′ AMP-activated protein kinase (AMPK) as well as the tuberus sclerosis complex kinases, TSC1 and TSC2, which finally inhibit mTOR kinase. Indeed, after rescue of ESS-1 cells with wildtype
TP53 we found in our experiments that the balance between predominant autophagy and apoptosis was directed towards prevailing apoptosis and basic autophagy; this observation was supported by upregulation of PUMA, as well as by activation of the apoptosis initiator and executioner caspases 9,-3 and -7, and finally by PARP-1 cleavage. In contrast, downregulation of autophagy was supported by mTOR/phosphor-mTor immunoblotting, staining with the autophagic marker LC3 as well as specific autophagosome staining with MDC [
25]. These results could identify p53 as a molecular switch that directly mediates the response of SAHA by either executing pro-apoptotic signalling; mechanistically this could possibly be accomplished by direct acetylation of the protein or by promoting the formation of autophagy upon the absence of p53 in the tumor cell. Accordingly, a study demonstrated that complexes constituted by acetylated p53 as well as acetylated histones and coactivators were held responsible for HDACi-induced apoptosis in HepG2 cells [
42]. If proven true, these findings furthermore might have immediate implications in the choice of cancer therapeutics and make our experimental system interesting for further molecular analysis regarding SAHA-provoked cell death regulation.