33 research outputs found
Telomere length after recovery from <i>WRN siRNA</i> transfection.
<p>After 54 PD of continuous <i>WRN</i> knockdown (after telomeres had shortened by almost 50% of the original length), <i>WRN siRNA</i> transfections of a subset of HeLa and VA-13 cells were ceased and cells allowed to recover in culture for an additional 54 PD. Average relative telomere repeat lengths were then measured by qRT-PCR. Results are graphically represented as the difference between a telomere-specific PCR reaction in comparison to the single copy gene albumin PCR reaction as delta C<sub>t</sub>.</p
Immunofluorescence of WRN and BRCA1 in telomerase-positive and ALT cells.
<p><b>A</b>. Representative micrograph images of cells from the telomerase-positive cell line HeLa and three ALT cell lines, VA-13, Saos-2 and U-2 OS. Cells were fixed and immunofluorescently labeled with antibodies to BRCA1 (red) and WRN (green), and the nucleus is stained with DAPI (blue). <b>B</b>. A graphical representation of at least three independent experiments averaged to yield the percentage of each cell type with co-localized BRCA1-WRN foci.</p
<i>WRN siRNA</i> knockdown and telomere length of U-2 OS cells.
<p><b>A</b>. U-2 OS cells were mock transfected or transfected with SC or <i>WRN siRNAs</i>. Whole cell extracts were collected, separated by SDS-PAGE and western blotted with antibodies to WRN to confirm the knockdown (top panel), cleaved PARP1 to examine apoptosis induction (middle panel) and lamin B as a loading control (bottom panel). Treatment with camptothecin is a positive control for the induction of apoptosis. <b>B</b>. To inhibit apoptosis, U-2 OS cells were transfected with <i>WRN</i> and <i>p53 siRNAs</i>. Whole cell extracts were separated by SDS-PAGE and western blotted with antibodies specific to WRN (top panel), p53 (middle panel) and lamin B (loading control; bottom panel). <b>C</b>. DNA from transfected U-2 OS cells was collected at 77 PD and telomere length was measured by qRT-PCR. Average relative telomere repeat lengths are graphically represented as the difference between a telomere-specific PCR reaction in comparison to the single copy gene <i>ALB</i> PCR reaction, or delta C<sub>t</sub>. (ALT = alternative lengthening of telomeres; PD = population doubling)</p
<i>WRN siRNA</i> knockdown and telomere length of HeLa, MCF7, VA-13, and Saos-2 cells.
<p><b>A</b>. Pooled <i>WRN siRNAs</i> or scrambled control (SC) <i>siRNAs</i> were transfected into immortalized human cell lines and whole cell extracts were collected 48 hours after transfection. Each lysate was separated by SDS-PAGE and western blotted with antibodies to WRN (top), BLM (to ensure specificity of the <i>siRNAs,</i> middle), and lamin B (as a loading control, bottom). <b>B</b>. Relative telomere length measurement by qRT-PCR. The difference in the cycle threshold (C<sub>t</sub>) between a telomere-specific PCR reaction and a single copy gene (<i>ALB</i>) PCR reaction is calculated for each sample as the ΔC<sub>t</sub>, which represents the average relative telomere repeat length. Telomere length in Saos-2 ALT cells decreases after stable <i>BLM</i> knockdown as measured by TRF Southern blot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093991#pone.0093991-Bhattacharyya1" target="_blank">[23]</a>. Here, we confirm detection of these length changes by qRT-PCR (far right). Telomere lengths of telomerase-positive cells, HeLa and MCF7, and ALT cells, VA-13 and Saos2, were measured following transfection of either SC or <i>WRN siRNAs</i>. Relative lengths depicted represent the final measurement for each cell type, which was taken at the population doubling number (PD) indicated. (ALT = alternative lengthening of telomeres; TRF =  telomere restriction fragment; PD = population doubling).</p
WRN Loss Induces Switching of Telomerase-Independent Mechanisms of Telomere Elongation
<div><p>Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates <i>in vitro</i>. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term <i>siRNA</i> knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50–70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.</p></div
Telomere recombination measured by T-SCE is variably affected by <i>WRN</i> and/or <i>BRCA1</i> knockdown.
<p>ALT cell lines VA-13, Saos-2, and U-2 OS were transfected with <i>WRN</i>, <i>BRCA1</i>, or <i>WRN/BRCA1 siRNAs</i> and metaphase spreads were processed for T-SCE after 72 hours. Telomeres were fluorescently labeled and analyzed. <b>A</b>. A representative image of a metaphase spread from VA-13 cells shows DAPI-labeled chromosomes in blue and Cy-3 labeled telomeres in red. <b>B</b>. Results depict the frequency of T-SCE per chromosome analyzed from at least 30 metaphase spreads per treatment group. Western blots depict the ability of <i>BRCA1 siRNAs</i> to knock down BRCA1 in each cell line.</p
WRN immunoprecipitation from telomerase-positive and ALT cells.
<p><b>A</b>. Nuclear extracts were immunoprecipitated with antibodies to BRCA1 and the immunoprecipitated proteins subjected to western blotting with an antibody to WRN (right panels). Immunoprecipitation with anti-BLM serves as a positive control for the interaction with WRN in Saos-2 cell extracts, while an anti-BRCA1 immunoprecipitation from AG11395 WS cell extracts and an anti-IgG immunoprecipitation from Saos-2 cell extracts serve as negative controls. Immunoprecipitation input lanes (10% of the total extract) of WRN and BRCA1 are shown in the left panels. <b>B</b>. VA-13 cells were synchronized with aphidicolin and collected at various time points post-release from aphidicolin arrest. Cell cycle distribution was analyzed by flow cytometry at 2, 6 or 12 hours post-release as depicted in the histograms. <b>C</b>. Synchronized nuclear extracts were immunoprecipitated with antibodies to WRN and the immunoprecipitated proteins subjected to western blotting with antibodies to WRN (top) or BRCA1 (bottom). Immunoprecipitation with anti-BLM serves as a positive control for the interaction with WRN and BRCA1 in VA-13 cell extracts, while an anti-WRN immunoprecipitation from AG11395 WS cell extracts serves as a negative control.</p
ALT-associated PML body (APB) formation following <i>WRN</i> knockdown.
<p><b>A</b>. Representative confocal micrograph images of cells from two telomerase-positive cell lines, HeLa and MCF7, and three ALT cell lines, VA-13, Saos-2 and U-2 OS. Cells were fixed 48 hours after transfection with scrambled control (SC) <i>siRNAs</i>, <i>BLM siRNAs</i> or <i>WRN siRNAs</i> and immunofluorescently labeled with antibodies to PML and TRF2. We have previously shown a reduction in APBs following <i>BLM</i> knockdown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093991#pone.0093991-Bhattacharyya1" target="_blank">[23]</a>, so cells transfected with BLM <i>siRNAs</i> served as a positive control. PML is labeled in red, TRF2 in green, and nuclei stained with DAPI (blue). <b>B</b>. A graphical representation of at least 3 independent experiments depicts the percentage of cells displaying co-localization of TRF2 and PML, indicative of APBs. Co-localization of PML/TRF2 foci was scored as a positive indication of APBs; at least three independent experiments were averaged to yield the percentage of each cell type. Western blots confirm the ability of <i>BLM siRNAs</i> to reduce BLM expression.</p
Identification of endometrial cancer methylation features using combined methylation analysis methods
<div><p>Background</p><p>DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP) can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed.</p><p>Methods</p><p>Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA) were compared to MethylCap-seq data.</p><p>Results</p><p>Analysis of methylation in promoter CpG islands (CGIs) identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a “hypermethylator state.” High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA.</p><p>Conclusion</p><p>We identified a methylation signature for a “hypermethylator phenotype” in endometrial cancer and developed methods that may prove useful for identifying extreme methylation phenotypes in other cancers.</p></div
Promoter CGIs validated by Infinium beadchip analysis.
<p>Promoter CGIs validated by Infinium beadchip analysis.</p