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Alterations of histone H1 phosphorylation during bladder carcinogenesis.

ABSTRACT: There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. Epigenetic alterations, such as histone modifications, are implicated in the genetic dysregulation that is fundamental to carcinogenesis. Here we focus on profiling the histone modifications during the progression of bladder cancer. Histones were extracted from normal human bladder epithelial cells, an immortalized human bladder epithelial cell line (hTERT), and four human bladder cancer cell lines (RT4, J82, T24, and UMUC3) ranging from superficial low-grade to invasive high-grade cancers. Liquid chromatography-mass spectrometry (LC-MS) profiling revealed a statistically significant increase in phosphorylation of H1 linker histones from normal human bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer cells. This finding was further validated by immunohistochemical staining of the normal epithelium and transitional cell cancer from human bladders. Cell cycle analysis of histone H1 phosphorylation by Western blotting showed an increase of phosphorylation from G0/G1 phase to M phase, again supporting this as a proliferative marker. Changes in histone H1 phosphorylation status may further clarify epigenetic changes during bladder carcinogenesis and provide diagnostic and prognostic biomarkers or targets for future therapeutic interventions.

SUBMITTER: Telu KH

PROVIDER: S-EPMC3743963 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Alterations of histone H1 phosphorylation during bladder carcinogenesis.

Telu Kelly H KH Abbaoui Besma B Thomas-Ahner Jennifer M JM Zynger Debra L DL Clinton Steven K SK Freitas Michael A MA Mortazavi Amir A

Journal of proteome research 20130617 7

There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. Epigenetic alterations, such as histone modifications, are implicated in the genetic dysregulation that is fundamental to carcinogenesis. Here we focus on profiling the histone modifications during the progression of bladder cancer. Histones were extracted from normal human bladder epithelial cells, an ...[more]

PMID: 23675690

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Project description:Structural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes. Changes in the expression and genomic distributions of H1 variants during cell differentiation appear to contribute to phenotypic differences between cell types, but few details are known about the roles of individual H1 variants and the significance of their disparate capacities for phosphorylation. In this study, we investigated the dynamics of interphase phosphorylation at specific sites in individual H1 variants during the differentiation of pluripotent NT2 and mouse embryonic stem cells and characterized the kinases involved in regulating specific H1 variant phosphorylations in NT2 and HeLa cells.Here, we show that the global levels of phosphorylation at H1.5-Ser18 (pS18-H1.5), H1.2/H1.5-Ser173 (pS173-H1.2/5) and H1.4-Ser187 (pS187-H1.4) are regulated differentially during pluripotent cell differentiation. Enrichment of pS187-H1.4 near the transcription start site of pluripotency factor genes in pluripotent cells is markedly reduced upon differentiation, whereas pS187-H1.4 levels at housekeeping genes are largely unaltered. Selective inhibition of CDK7 or CDK9 rapidly diminishes pS187-H1.4 levels globally and its enrichment at housekeeping genes, and similar responses were observed following depletion of CDK9. These data suggest that H1.4-S187 is a bona fide substrate for CDK9, a notion that is further supported by the significant colocalization of CDK9 and pS187-H1.4 to gene promoters in reciprocal re-ChIP analyses. Moreover, treating cells with actinomycin D to inhibit transcription and trigger the release of active CDK9/P-TEFb from 7SK snRNA complexes induces the accumulation of pS187-H1.4 at promoters and gene bodies. Notably, the levels of pS187-H1.4 enrichment after actinomycin D treatment or cell differentiation reflect the extent of CDK9 recruitment at the same loci. Remarkably, the global levels of H1.5-S18 and H1.2/H1.5-S173 phosphorylation are not affected by these transcription inhibitor treatments, and selective inhibition of CDK2 does not affect the global levels of phosphorylation at H1.4-S187 or H1.5-S18.Our data provide strong evidence that H1 variant interphase phosphorylation is dynamically regulated in a site-specific and gene-specific fashion during pluripotent cell differentiation, and that enrichment of pS187-H1.4 at genes is positively related to their transcription. H1.4-S187 is likely to be a direct target of CDK9 during interphase, suggesting the possibility that this particular phosphorylation may contribute to the release of paused RNA pol II. In contrast, the other H1 variant phosphorylations we investigated appear to be mediated by distinct kinases and further analyses are needed to determine their functional significance.

Project description:Cruciferous vegetable intake is associated with reduced risk of bladder cancer, yet mechanisms remain unclear. Cruciferous vegetable isothiocyanates (ITCs), namely sulforaphane (SFN) and erucin (ECN), significantly inhibit histone deacetylase (HDAC) activity in human bladder cancer cells representing superficial to invasive biology (59-83% inhibition with 20μM, 48h treatment), and in bladder cancer xenografts (59±3% ECN inhibition). Individual HDACs inhibited by SFN and ECN include HDACs 1, 2, 4 and 6. Interestingly, global acetylation status of histones H3 or H4 remain unaltered. The interplay between HDAC inhibition and modest modulation of AcH3 and AcH4 status is partially explained by decreased histone acetyl transferase activity (48.8±5.3%). In contrast, a significant decrease in phosphorylation status of all isoforms of histone H1 was observed, concomitant with increased phosphatase PP1β and PP2A activity. Together, these findings suggest that ITCs modulate histone status via HDAC inhibition and phosphatase enhancement. This allows for reduced levels of histone H1 phosphorylation, a marker correlated with human bladder cancer progression. Therefore, ITC-mediated inhibition of histone H1 phosphorylation presents a novel direction of research in elucidating epidemiological relationships and supports future food-based prevention strategies.SignificanceCollectively, our findings suggest that the cruciferous vegetable isothiocyanates: sulforaphane (SFN) and erucin (ECN), impact histones status in bladder cancer cells by modulating specific HDACs and HATs, and enhancing phosphatase activity, resulting in reduction of histone H1 phosphorylation. These findings are significant due to the fact that our previous work positively correlated histone H1 phosphorylation with bladder cancer carcinogenesis and progression. Therefore, we propose that SFN and ECN may inhibit bladder carcinogenesis via epigenetic modulation of gene expression associated with histone H1 phosphorylation. These efforts may elucidate biomarkers useful in epidemiologic studies related to cruciferous vegetable intake and cancer risk or provide intermediate biomarkers for food-based clinical intervention studies in high-risk cohorts.

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Alterations of histone H1 phosphorylation during bladder carcinogenesis.

Publications

Alterations of histone H1 phosphorylation during bladder carcinogenesis.

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