A Conformational Variant of p53 (U-p53AZ) as Blood-Based Biomarker for the Prediction of the Onset of Symptomatic Alzheimer's Disease.

BACKGROUND: Ongoing research seeks to identify blood-based biomarkers able to predict onset and progression of Alzheimer's disease (AD). OBJECTIVE: The unfolded conformational variant of p53 (U-p53AZ), previously observed in AD individuals, was evaluated in plasma samples from individuals participating in the Australian Imaging, Biomarkers and Lifestyle (AIBL) cohort for diagnostic and prognostic assessment, validated on a neuropsychological-based diagnosis, over the course of six years. DESIGN: Retrospective Longitudinal Prognostic biomarker study. SETTING: Single-center study based on the AIBL cohort. PARTICIPANTS: 482 participants of the AIBL cohort, aged 60-85 years, without uncontrolled diabetes, vascular disease, severe depression or psychiatric illnesses. MEASUREMENTS: The AlzoSure® Predict test, consisting of immunoprecipitation (IP) followed by liquid chromatography (LC) tandem mass spectrometry (MS/MS), was performed to quantify the AZ 284® peptide as readout of U-p53AZ and compared with an independent neuropsychological diagnosis. The amyloid load via amyloid ß-positron emission tomography (Aß-PET) and supporting clinical information were included where possible. RESULTS: U-p53AZ diagnostic and prognostic performance was assessed in both time-independent and time-dependent (36, 72 and 90 months following initial sampling) analyses. Prognostic performance of Aß-PET and survival analyses with different risk factors (gender, Aß-PET and APOE ε4 allele status) were also performed. U-p53AZ differentiated neuropsychologically graded AD from non-AD samples, and its detection at intermediate/high levels precisely identified present and future symptomatic AD. In both time-independent and time-dependent prognostic analyses U-p53AZ achieved area under the curve (AUC) >98%, significantly higher than Aß-PET AUCs (between 84% and 93%, P respectively <0.0001 and <0.001). As single factor, U-p53AZ could clearly determine the risk of AD neuropsychological diagnosis over time (low versus intermediate/high U-p53AZ hazard ratio=2.99). Proportional hazards regression analysis identified U-p53AZ levels as a major independent predictor of AD onset. CONCLUSIONS: These findings support use of U-p53AZ as blood-based biomarker predicting whether individuals would reach neuropsychologically-defined AD within six years prior to AD diagnosis. Integration of U-p53AZ in screening processes could support refined participant stratification for interventional studies.

Development of a prognostic risk model for clear cell renal cell carcinoma by systematic evaluation of DNA methylation markers.

BACKGROUND: Current risk models for renal cell carcinoma (RCC) based on clinicopathological factors are sub-optimal in accurately identifying high-risk patients. Here, we perform a head-to-head comparison of previously published DNA methylation markers and propose a potential prognostic model for clear cell RCC (ccRCC). PATIENTS AND METHODS: Promoter methylation of PCDH8, BNC1, SCUBE3, GREM1, LAD1, NEFH, RASSF1A, GATA5, SFRP1, CDO1, and NEURL was determined by nested methylation-specific PCR. To identify clinically relevant methylated regions, The Cancer Genome Atlas (TCGA) was used to guide primer design. Formalin-fixed paraffin-embedded (FFPE) tissue samples from 336 non-metastatic ccRCC patients from the prospective Netherlands Cohort Study (NLCS) were used to develop a Cox proportional hazards model using stepwise backward elimination and bootstrapping to correct for optimism. For validation purposes, FFPE ccRCC tissue of 64 patients from the University Hospitals Leuven and a series of 232 cases from The Cancer Genome Atlas (TCGA) were used. RESULTS: Methylation of GREM1, GATA5, LAD1, NEFH, NEURL, and SFRP1 was associated with poor ccRCC-specific survival, independent of age, sex, tumor size, TNM stage or tumor grade. Moreover, the association between GREM1, NEFH, and NEURL methylation and outcome was shown to be dependent on the genomic region. A prognostic biomarker model containing GREM1, GATA5, LAD1, NEFH and NEURL methylation in combination with clinicopathological characteristics, performed better compared to the model with clinicopathological characteristics only (clinical model), in both the NLCS and the validation population with a c-statistic of 0.71 versus 0.65 and a c-statistic of 0.95 versus 0.86 consecutively. However, the biomarker model had limited added prognostic value in the TCGA series with a c-statistic of 0.76 versus 0.75 for the clinical model. CONCLUSION: In this study we performed a head-to-head comparison of potential prognostic methylation markers for ccRCC using a novel approach to guide primers design which utilizes the optimal location for measuring DNA methylation. Using this approach, we identified five methylation markers that potentially show prognostic value in addition to currently known clinicopathological factors.

High content image cytometry in the context of subnuclear organization.

The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fast-growing need for image-based cytometry. Simultaneously, the massive amount of data that is generated by image-cytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA damage repair patterns in response to genotoxic stress and radiation, and we show the potential of data mining in pinpointing specific phenotypes after transient transfection. The presented approach allowed for systematic analysis of subnuclear features in large image data sets and accurate classification of phenotypes at the level of the single cell. Consequently, this type of nuclear fingerprinting shows potential for high-throughput applications, such as functional protein assays or drug compound screening.

Relation between telomerase activity, hTERT and telomere length for intracranial tumours.

Human linear chromosomes are capped by specialized DNA-protein structures called telomeres. The present study analysed the telomerase activity, hTERT protein and telomere length in meningiomas and gliomas in relation to their WHO grading. Fifty-three freshly dissected tumour biopsies were analysed for telomerase activity, hTERT protein expression and telomere length. Telomerase activity was examined in 41 of the 53 biopsies. Telomerase activity was detected in 3 of 35 (8.6%) screened meningiomas (1 benign, 1 atypical and 1 malignant meningioma). For hTERT expression, 56.4% of meningiomas were positive with a mean labelling index (hTERT LI) of 31.3% (SD=26.5) for the hTERT positive meningiomas. The mean telomere length for meningiomas was 6.983 kb (SD=1.969). For gliomas, no active telomerase was detected in 2 low-grade astrocytomas, whereas three of the four screened glioblastomas were positive for telomerase activity. The only hTERT protein positive astrocytoma had a mean labelling index of 9.0%. On the other hand, the hTERT LI for glioblastomas was 53.6% (SD=28.0). The two low-grade astrocytomas had a telomere length of 14.310 and 9.236 kb. The anaplastic astrocytoma had a telomere length of 4.903 kb and the glioblastomas 5.767 kb (SD=2.042). The normal meningeal and neuronal tissue is negative for telomerase activity and hTERT. The length was +/-10.000 kb. These results indicate that telomere shortening may be a critical step in pathogenesis of atypical and malignant meningiomas and gliomas. Critical telomere shortening in vitro was shown to activate telomerase.

Cellular distribution of somatogenic receptors and insulin-like growth factor-I mRNA in the rat liver.

Parenchymal and non-parenchymal cell fractions isolated from rat liver were analysed for the distribution of somatogenic receptors and for insulin-like growth factor-I (IGF-I) mRNA. The effect of hypophysectomy either alone or in combination with a single injection of human GH (hGH) on the levels of IGF-I mRNA was also studied in the two cell fractions. The contamination of parenchymal cells in the non-parenchymal cell fraction was in the range of 2-3%. Somatogenic receptors were found only in the parenchymal cell fraction. IGF-I mRNA was detected in both cell fractions, although the level of this mRNA was about fivefold higher in parenchymal cells. When RNA was studied by Northern gel analysis no major differences were observed in the size distribution of IGF-I transcripts in the two cell fractions. In hypophysectomized animals, the IGF-I mRNA level was decreased to 10 and 30% of control values in the parenchymal and non-parenchymal cell fractions respectively. Treatment of hypophysectomized animals with a single dose of hGH restored IGF-I mRNA in parenchymal and in non-parenchymal cells to the extent found in intact animals. In conclusion, our data indicate that somatogenic receptors are exclusively, and IGF-I mRNA predominantly, expressed in liver parenchymal cells compared with a total non-parenchymal cell fraction. The most marked effect of GH, correlating with the presence of somatogenic receptors, was seen in the parenchymal cells. The minor non-GH dependent expression of IGF-I mRNA suggests that this peptide may elicit effects in addition to the well-established GH-dependent activity.

Heparin-protamine complexes in the production of heparin rebound and other complications of extracorporeal bypass procedures.

Neutralization by protamine of the heparin activation of antithrombin III in plasma, in vitro, represents an equilibrium reaction in which by mass action heparin remains complexed only in the presence of an excess of protamine. Loss of this excess through enzymatic breakdown of the free protamine leads to instability of the complexes with liberation of the heparin, reestablishing antithrombin activity. This "heparin rebound" can also be produced by an increase in heparin levels or increased amounts of antithrombin III. These phenomena could occur after extracorporeal bypass procedures by an increase in heparin from whatever source or by added antithrombin III, through transfusions of fresh frozen plasma. A larger excess of protamine does not itself act as an anticoagulant but produces large heparin-protamine complexes that can still activate antithrombin III. Such large complexes, formed in vivo, could possibly block the pulmonary microcirculation and cause the acute pulmonary hypertension that has been reported after protamine infusions.

Transcriptional regulation of Wnt inhibitory factor-1 by Miz-1/c-Myc.

The Wnt signaling pathway is capable of self-regulation through positive and negative feedback mechanisms. For example, the oncoprotein c-Myc, which is upregulated by Wnt signaling activity, participates in a positive feedback loop of canonical Wnt signaling through repression of Wnt antagonists DKK1 and SFRP1. In this study, we investigated the mechanism of Wnt inhibitory factor-1 (WIF-1) silencing. Mapping of CpG island methylation of the WIF-1 promoter reveals regional methylation (-295 to -95 bp from the transcription start site) that correlates with transcriptional silencing. We identified Miz-1 as a direct activator of WIF-1 transcriptional activity, which is found at WIF-1 promoter. In addition, we show that c-Myc contributes to WIF-1 transcriptional repression in a Miz-1-dependent manner. Although the transient repression mediated by Miz-1/c-Myc is independent of de novo methylation, the stable repression by this complex is associated with CpG island methylation of the critical -295 to -95-bp region of the WIF-1 promoter. Importantly, Miz-1 and c-Myc are found at WIF-1 promoter in WIF-1 non-expressing cell lines DLD-1 and 209myc. Transient knockdown or somatic knockout of c-Myc in DLD-1 failed to restore WIF-1 expression suggesting that c-Myc is involved in initiating rather than maintaining WIF-1 epigenetic silencing. In a genome-wide screen, DNAJA4, TGFß-induced and TRIM59 were repressed by c-Myc overexpression and DNA promoter hypermethylation. Our data reveal novel insights into c-Myc-mediated DNA methylation-dependent transcriptional silencing, a mechanism that might contribute to the dysregulation of Wnt signaling in cancer.

Re-expression of CXCL14, a common target for epigenetic silencing in lung cancer, induces tumor necrosis.

Chemokines are important regulators of directional cell migration and tumor metastasis. A genome-wide transcriptome array designed to uncover novel genes silenced by methylation in lung cancer identified the CXC-subfamily of chemokines. Expression of 11 of the 16 known human CXC-chemokines was increased in lung adenocarcinoma cell lines after treatment with 5-aza-2'-deoxycytidine (DAC). Tumor-specific methylation leading to silencing of CXCL5, 12 and 14 was found in over 75% of primary lung adenocarcinomas and DAC treatment restored the expression of each of the silenced gene. Forced expression of CXCL14 in H23 cells, where this gene is silenced by methylation, increased cell death in vitro and dramatically reduced the in vivo growth of lung tumor xenografts through necrosis of up to 90% of the tumor mass. CXCL14 re-expression had a profound effect on the genome altering the transcription of over 1000 genes, including increased expression of 30 cell-cycle inhibitor and pro-apoptosis genes. In addition, CXCL14 methylation in sputum from asymptomatic early-stage lung cancer cases was associated with a 2.9-fold elevated risk for this disease compared with controls, substantiating its potential as a biomarker for early detection of lung cancer. Together, these findings identify CXCL14 as an important tumor suppressor gene epigenetically silenced during lung carcinogenesis.