The current study shows two major findings. One is represented by the significant changes in nuclear size and chromatin organization in UC and its two preinvasive lesions compared with NU. The other is represented by an altered expression of the chromatin remodeler DAXX in UC as well as in CIS and HGPCa, compared with NU. The nuclear size depends on the DNA content as well as on the status of chromatin organization. The latter depends on epigenetic events, such as DNA methylation and histone modifications, and is regulated by the chromatin remodeler DAXX [10–13].
Compared to normal cells, DNA of cancer cells is generally hypomethylated, while promoters of certain genes are hypermethylated, in the context of CpG islands. Such promoter-specific increase in methylation leads to silencing of the affected gene that might have functioned as, for instance, a tumor suppressor. Transcriptional repression by DNA methylation is mediated by a class of methyl DNA binding proteins which, by virtue of recognizing specifically methylated DNA sequences, recruit repressive protein complexes including histone deacetylases to gene promoters (see below) [8, 9, 19]. The combination of CpG island methylation, proteins that binds to them, and repressive histone modifications generates localized regions of specialized chromatin, which can inhibit transcription. Despite a growing list of genes including tumor suppressors and DNA repair genes that are aberrantly hypermethylated in different cancers, only a limited number of the identified hypermethylated genes have demonstrated any potential utility in clinical decision making. As opposed to single-gene analysis, the integrated information on methylation patterns of multiple genes may reflect the functional status of several cellular pathways [19, 20].
To investigate DNA methylation in situ an immunohistochemical approach was adopted by our group using a monoclonal antibody that recognize the presence of a methyl group on the carbon 5 of cytosine. This allowed the analysis of global methylation to be performed on interphase nuclei, on a cell by cell basis by microscopy . Negative nuclei and those with weak-to-moderate intensity were considered unmethylated and hypomethylated, respectively, whereas those strongly stained as hypermethylated. That study showed an altered global DNA methylation pattern in UC compared with NU, the proportion of nuclei with weak-to-moderate intensity in the former being greater than that of strongly stained. This was interpreted as an increased global DNA unmethylation and hypomethylation in UC compared with NU [8, 9].
Histones are subject to a variety of post-translational modifications, including acetylation of lysines. Such modifications play fundamental roles in gene regulation and other chromatin-based processes. Histone-modifying enzymes affect histones either locally, through targeted recruitment by sequence specific transcription factors , or globally throughout the genome in an untargeted manner, affecting virtually all nucleosomes . Such widespread functions that occur independently of apparent sequence-specific DNA binding proteins are referred to as global histone modifications. Like their targeted effects, the global activity of the histone modifying enzymes can modulate gene activity . Therefore, histones are modified locally and globally through multiple histone-modifying enzymes with different substrate specificities, generating hierarchical patterns of modifications from single promoters to large regions of chromosomes and even single cells.
Since histone modifications occur throughout the genome, any potential change in the activity of the histone modifying enzymes results in changes in specific histone patterns detectable at the level of individual nuclei by immunohistochemistry. While the immunohistochemical approach provides information on global histone acetylation, it does not give information on the genomic, gene–gene differences in distribution of histone modifications. To investigate global histone acetylation in UC an immunohistochemical approach was previously adopted by our group using a polyclonal antibody raised against acetylated H3K9 (i.e., lysine 9 (K9) of histone 3) [8, 9]. The study showed an altered pattern of global histone acetylation in UC, the percentages of positive nuclei being higher than in NU.
These two previous investigations pointed out that the chromatin pattern in UC is associated with an increased global DNA unmethylation and hypomethylation and with an altered pattern of global histone acetylation compared with NU [8, 9]. The current study, performed on the same series of cases, indicates that such changes in UC are associated with an increased number of DAXX positive nuclei whose intensity of nuclear immunostaining is lower than in NU by a factor of 0.94. Since DAXX is a repressor of gene expression that binds DNA methyl transferases, histone deacetyl transferases and chromatin-modifying proteins, the findings in the current study are of paramount importance to explain the changes in DNA methylation and histone acetylation in UC.
There is only one previous study by Segersten et al.  on DAXX immunohistochemistry in bladder neoplasms. The investigation was performed on tissue microarray (TMA) material comprising a range of noninvasive and invasive bladder cancer. The aim was to screen a publicly available immunohistochemistry based web-atlas to identify key proteins that might serve as potential biomarkers. The study did not focus on DAXX, as in our study, but involved a series of proteins. The expression of ALCAM, CN130, DAXX, GAL1, PHF6 and XPA were significantly correlated with tumor grade and stage. ALCAM and GAL1 expression were increased with advanced stage, whilst the others were decreased. In particular, GAL1 expression was higher in poorly differentiated tumors, whilst DSG3 and DAXX protein expression was decreased. Our current study confirms Segersten et al’s findings of a decreased DAXX protein expression in UC and expands our knowledge in this field because it shows that its altered expression correlates with previously observed changes in DNA methylation and histone acetylation.
In conclusion, the quantitative immunohistochemical analysis shows an altered expression of chromatin remodeler DAXX in UC and in its preinvasive phases, when compared with NU. In particular, the evaluation of the DAXX protein expression, if associated with other markers related to global DNA methylation and histone acetylation, could be used in clinical practice as a marker of aggressiveness.