The dynamics of DNA methylation covariation patterns in carcinogenesis.

Recently it has been observed that cancer tissue is characterised by an increased variability in DNA methylation patterns. However, how the correlative patterns in genome-wide DNA methylation change during the carcinogenic progress has not yet been explored. Here we study genome-wide inter-CpG correlations in DNA methylation, in addition to single site variability, during cervical carcinogenesis. We demonstrate how the study of changes in DNA methylation covariation patterns across normal, intra-epithelial neoplasia and invasive cancer allows the identification of CpG sites that indicate the risk of neoplastic transformation in stages prior to neoplasia. Importantly, we show that the covariation in DNA methylation at these risk CpG loci is maximal immediately prior to the onset of cancer, supporting the view that high epigenetic diversity in normal cells increases the risk of cancer. Consistent with this, we observe that invasive cancers exhibit increased covariation in DNA methylation at the risk CpG sites relative to normal tissue, but lower levels relative to pre-cancerous lesions. We further show that the identified risk CpG sites undergo preferential DNA methylation changes in relation to human papilloma virus infection and age. Results are validated in independent data including prospectively collected samples prior to neoplastic transformation. Our data are consistent with a phase transition model of carcinogenesis, in which epigenetic diversity is maximal prior to the onset of cancer. The model and algorithm proposed here may allow, in future, network biomarkers predicting the risk of neoplastic transformation to be identified.

CD133 (Prominin) negative human neural stem cells are clonogenic and tripotent.

BACKGROUND: CD133 (Prominin) is widely used as a marker for the identification and isolation of neural precursor cells from normal brain or tumor tissue. However, the assumption that CD133 is expressed constitutively in neural precursor cells has not been examined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrate that CD133 and a second marker CD15 are expressed heterogeneously in uniformly undifferentiated human neural stem (NS) cell cultures. After fractionation by flow cytometry, clonogenic tripotent cells are found in populations negative or positive for either marker. We further show that CD133 is down-regulated at the mRNA level in cells lacking CD133 immunoreactivity. Cell cycle profiling reveals that CD133 negative cells largely reside in G1/G0, while CD133 positive cells are predominantly in S, G2, or M phase. A similar pattern is apparent in mouse NS cell lines. Compared to mouse NS cells, however, human NS cell cultures harbour an increased proportion of CD133 negative cells and display a longer doubling time. This may in part reflect a sub-population of slow- or non-cycling cells amongst human NS cells because we find that around 5% of cells do not take up BrdU over a 14-day labelling period. Non-proliferating NS cells remain undifferentiated and at least some of them are capable of re-entry into the cell cycle and subsequent continuous expansion. CONCLUSIONS: The finding that a significant fraction of clonogenic neural stem cells lack the established markers CD133 and CD15, and that some of these cells may be dormant or slow-cycling, has implications for approaches to identify and isolate neural stem cells and brain cancer stem cells. Our data also suggest the possibility that CD133 may be specifically down-regulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells.

Cyclosporine pharmacokinetics and blood pressure responses after conversion to once-daily dosing in maintenance liver transplant patients.

In this six-month randomized multicenter trial, we characterized cyclosporine pharmacokinetics and blood pressure profiles in maintenance liver transplant patients converting from twice-daily to once-daily cyclosporine dosing. A total of 60 patients were randomized as follows: group A (n = 14) maintained twice-daily dosing; group B (n = 24) converted to once-daily dosing at the same total daily dose as pre-conversion; and group C (n = 22) was treated the same as group B but with a 25% reduction in dose and C2 at two to three wk post-conversion. After conversion to once-daily dosing in groups B and C, trough blood levels (C0) did not change; whereas, C2 nearly doubled. The total daily area under the concentration-time curve AUC(0-24) increased by 29%. After the dose reduction in group C, the AUC(0-24) was similar to the pre-conversion value. Hence, a 25-30% dose reduction can be considered after conversion to once-daily dosing. In the study observation period in weeks 4-15, the median (25-75 percentile) C2 was 568 (469-750) ng/mL for group A; 1055 (840-1224) ng/mL for group B; and 764 (575-959) ng/mL for group C. Conversion to once-daily dosing was associated with a decrease in nighttime mean arterial blood pressure.