Elevated urine histone 4 levels in women with ovarian endometriosis revealed by discovery and parallel reaction monitoring proteomics.

Endometriosis is a common gynecologic disorder and due to a lack of non-invasive detection methods, it can take up to 12 years before an affected woman obtains a diagnosis and receives appropriate treatment. Therefore, the identification of a specific biomarker that can be detected quickly and non-invasively is urgently needed. In this study, the urine proteome, a potentially rich source of biomarkers, is examined in patients with or without endometriosis in an attempt to identify novel protein biomarkers that can be used to diagnose endometriosis. This study is the first to combine tandem mass tags and parallel reaction monitoring approaches to aid in identifying and validating urine biomarkers for endometriosis. The findings presented herein support previous conclusions that endometriosis is a chronic inflammatory disease. Additionally, Histone 4 was identified as a potential biomarker and/or therapeutic target for endometriosis. At a cutoff of 14.2, the area under the curve for H4 was 0.848, with a sensitivity of 70% and specificity of 80%. Moreover, to our knowledge, this is the first study to observe an elevated histone level in body fluids obtained from endometriosis patients. While this study provides a good foundation, further studies are required to further validate the results presented. SIGNIFICANCE: Endometriosis is a common gynecologic disorder and due to a lack of non-invasive detection methods, it can take up to 12 years before an affected woman obtains a diagnosis and receives appropriate treatment. Therefore, the identification of a specific biomarker that can be detected quickly and non-invasively is urgently needed. We believe our results have an important impact on detection and treatment of endometriosis. Firstly, this study is the first to combine tandem mass tags and parallel reaction monitoring approaches to aid in identifying and validating urine biomarkers for endometriosis, which has established the methodology required for subsequent studies. Secondly, this is also the first study to observe an elevated histone level in body fluids obtained from endometriosis patients. Compared with other urine biomarkers reported in literature, histone 4 has a potential to serve as a biomarker of endometriosis and a therapeutic target. Thirdly, our study supports previous conclusions that endometriosis is a chronic inflammatory disease. These findings can warrant further investigation of the pathophysiology of endometriosis.

IMAC fractionation in combination with LC-MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers.

Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.

Associations between arsenic exposure and global posttranslational histone modifications among adults in Bangladesh.

BACKGROUND: Exposure to arsenic (As) is associated with an increased risk of several cancers as well as cardiovascular disease, and childhood neuro-developmental deficits. Arsenic compounds are weakly mutagenic, alter gene expression and posttranslational histone modifications (PTHMs) in vitro. METHODS: Water and urinary As concentrations as well as global levels of histone 3 lysine 9 di-methylation and acetylation (H3K9me2 and H3K9ac), histone 3 lysine 27 tri-methylation and acetylation (H3K27me3 and H3K27ac), histone 3 lysine 18 acetylation (H3K18ac), and histone 3 lysine 4 trimethylation (H3K4me3) were measured in peripheral blood mononuclear cells (PBMC) from a subset of participants (N = 40) of a folate clinical trial in Bangladesh (FACT study). RESULTS: Total urinary As (uAs) was positively correlated with H3K9me2 (r = 0.36, P = 0.02) and inversely with H3K9ac (r = -0.47, P = 0.002). The associations between As and other PTHMs differed in a gender-dependent manner. Water As (wAs) was positively correlated with H3K4me3 (r = 0.45, P = 0.05) and H3K27me3 (r = 0.50, P = 0.03) among females and negatively correlated among males (H3K4me3: r = -0.44, P = 0.05; H3K27me3: r = -0.34, P = 0.14). Conversely, wAs was inversely associated with H3K27ac among females (r = -0.44, P = 0.05) and positively associated among males (r = 0.29, P = 0.21). A similar pattern was observed for H3K18ac (females: r = -0.22, P = 0.36; males: r = 0.27, P = 0.24). CONCLUSION: Exposure to As is associated with alterations of global PTHMs; gender-specific patterns of association were observed between As exposure and several histone marks. IMPACT: These findings contribute to the growing body of evidence linking As exposure to epigenetic dysregulation, which may play a role in the pathogenesis of As toxicity.

DNA methylation biomarkers of prostate cancer: confirmation of candidates and evidence urine is the most sensitive body fluid for non-invasive detection.

BACKGROUND: A prostate cancer (PCa) biomarker with improved specificity relative to PSA is a public health priority. Hypermethylated DNA can be detected in body fluids from PCa patients and may be a useful biomarker, although clinical performance varies between studies. We investigated the performance of candidate PCa DNA methylation biomarkers identified through a genome-wide search. METHODS: Real-time PCR was used to measure four DNA methylation biomarkers: GSTP1 and three previously unreported candidates associated with the genes RASSF2, HIST1H4K, and TFAP2E in sodium bisulfite-modified DNA. Matched plasma and urine collected prospectively from 142 patients referred for prostate biopsy and 50 young asymptomatic males were analyzed. RESULTS: Analysis of all biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers discriminated PCa from biopsy negative patients with AUCs ranging from 0.64 for HIST1H4K (95% CI 0.55-0.72, P < 0.00001) to 0.69 for GSTP1 (95% CI 0.60-0.77, P < 0.00001). All biomarkers showed minimal correlation with PSA. Multivariate analysis did not yield a panel that significantly improved performance over that of single biomarkers. All biomarkers showed greater sensitivity for PCa in urine than in plasma DNA. CONCLUSIONS: Analysis of the biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers provided information independent of PSA and may warrant inclusion in nomograms for predicting prostate biopsy outcome. The biomarkers' PCa sensitivity was greater for urine than plasma DNA. The biomarker performances in urine DNA should next be validated in formal training and test studies.

The 17 kDa band identified by multiple anti-aquaporin 2 antisera in rat kidney medulla is a histone.

The osmotic water permeability of epithelial cells of the inner medullary collecting duct of the kidney is regulated by antidiuretic hormone (ADH). ADH causes the insertion and removal of cytoplasmic vesicles containing the aquaporin (AQP-2) water channel protein which is recognized by multiple rabbit antipeptide antisera raised against amino acid sequences comprising its cytoplasmic carboxyl terminal. Immunoblots of rat kidney membrane fractions as well as human urine have all shown that AQP-2 is expressed exclusively by collecting duct cells and have identified a 29 kDa band (corresponding to the nonglycosylated AQP-2 protein), a broad 35-45 kDa band (corresponding to the mature glycosylated form of AQP-2 protein) and an additional immunoreactive 17 kDa band of unknown origin. We now report that the 17 kDa band identified by these anti-AQP-2 antisera is not an AQP-2 component but rather a denatured histone protein type H2A1. This binding of anti-AQP-2 antisera to denatured H2A1 present in protein samples derived from both kidney inner medulla and human urine is blocked specifically by preincubation of immunoblots with solutions containing the acidic protein gelatin.