New method to detect histone acetylation levels by flow cytometry.
BACKGROUND
Reversible histone acetylation affects chromatin structural organization, thus regulating gene expression and other nuclear events. Levels of histone acetylation are tightly modulated in normal cells, and alterations of their regulating mechanisms have been shown to be involved in tumorigenesis.


METHODS
We developed a new flow cytometric technique for detection of histone acetylation, based on a specific monoclonal antibody that recognizes acetylated histone tails. Bivariate analysis for histone acetylation levels and DNA were performed to study modulation of chromatin organization during the cell cycle and after induction of histone hyperacetylation by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Histone acetylation and transcription levels were monitored during differentiation induced by retinoic acid alone or in combination with TSA. Blood samples from patients were analyzed with the described protocol to monitor the effects of HDAC inhibitors in vivo and validate the developed protocol for clinical usage.


RESULTS
Flow cytometric detection of acetylation status can successfully detect modifications induced by HDAC inhibitor treatment in vivo as demonstrated by analysis of various blood samples from patients treated with valproic acid. Changes in acetylation levels during the cell cycle demonstrated a reproducible increase in histone acetylation during the replication phase that was subsequently decreased at the G2M entrance, thus paralleling the behavior of DNA replication and transcriptional activity.


CONCLUSIONS
Multiparameter analysis of histone acetylation and expression of molecular markers, DNA ploidy, and/or cell cycle kinetics can provide a quick and statistically reliable tool for the diagnosis and evaluation of treatment efficacy in clinical trials using HDAC inhibitors.
Ronzoni
Simona
S
European Institute of Oncology, Milan, Italy.
Faretta
Mario
M
Ballarini
Marco
M
Pelicci
Piergiuseppe
P
Minucci
Saverio
S
D000107:Acetylation; D000208:Acute Disease; D000704:Analysis of Variance; D014408:Biomarkers, Tumor / Q000378:metabolism; D001773:Blood Cells / Q000378:metabolism*; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000097:blood
D014408:Biomarkers, Tumor; D056572:Histone Deacetylase Inhibitors; D006657:Histones