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Depletion of Ku70/80 reduces the levels of extrachromosomal telomeric circles and inhibits proliferation of ALT cells.

Li B, Reddy S, Comai L - Aging (Albany NY) (2011)

Bottom Line: In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence.Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length.These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

ABSTRACT
In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence. In contrast, cancer cells counteract the loss of telomeric DNA either by inducing the expression of telomerase or by activating the alternative lengthening of telomeres (ALT) pathway. ALT cells are characterized by heterogeneous telomeres and the presence of extrachromosomal circular double-stranded DNA molecules containing telomeric repeat sequences. These telomeric circles (t-circles) are though to be generated through a recombination process and utilized as templates for telomere elongation by rolling-circle-replication, although their precise mechanism of formation and role in telomere maintenance and cell proliferation is largely unknown. Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length. These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.

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Depletion of WRN does not influence the levels of t-circles in CCL75.1 cells(A) CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs for WRN or GFP and analyzed 3 days (shWRN) and 7 days (shWRN and shGFP) after the addition of 1.0 mg/ml DOX to the media. Protein levels were determined by Western blotting with WRN antibodies. We estimated that WRN shRNAs reduce its cognate gene expression ~75% compared to GFP shRNAs control after 7 days of DOX induction. Antibody against GAPDH was used as a loading control. (B) Genomic DNA isolated from CCL75.1 cells expressing shRNAs targeting WRN or GFP for 7 days was digested with the restriction endonucleases HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA)4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate levels of t-circles present in each sample (expressed as a percentage of the total telomeric DNA) were estimated as in [14] and is shown in the upper right corner.
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Figure 5: Depletion of WRN does not influence the levels of t-circles in CCL75.1 cells(A) CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs for WRN or GFP and analyzed 3 days (shWRN) and 7 days (shWRN and shGFP) after the addition of 1.0 mg/ml DOX to the media. Protein levels were determined by Western blotting with WRN antibodies. We estimated that WRN shRNAs reduce its cognate gene expression ~75% compared to GFP shRNAs control after 7 days of DOX induction. Antibody against GAPDH was used as a loading control. (B) Genomic DNA isolated from CCL75.1 cells expressing shRNAs targeting WRN or GFP for 7 days was digested with the restriction endonucleases HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA)4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate levels of t-circles present in each sample (expressed as a percentage of the total telomeric DNA) were estimated as in [14] and is shown in the upper right corner.

Mentions: Genetic knock-out of the Ku80 gene in telomerase-positive human cells results in reduced cell proliferation and is accompanied by dramatic shortening and loss of telomeres [16, 17]. Moreover, 50% reduction in the expression of Ku80 either by small interfering RNA (siRNA) or by functional inactivation of one allele in two telomerase-positive carcinoma cell lines, HeLa and HCT116, is sufficient to alter telomere homeostasis, as demonstrated by rapid telomere shortening and, at least in one of the studies, stronger telomere overhang signal [16, 18]. To determine whether depletion of Ku70/80 influences telomere homeostasis in ALT cells, we measured telomere length, single-stranded telomere overhang signal and t-circles in ALT cells transduced with lentiviruses for the conditional expression of shRNAs targeting Ku70 and Ku80. Cells were subjected to selections and DNA was isolated before induction and at 7 and 10 days after DOX addition. DNA was digested with HinfI and RsaI, resolved on agarose gel and hybridized to a radiolabeled telomeric probe under either denaturing or native conditions. This analysis show that within the time frame of this experiment, depletion of Ku70/80 does not significantly influence overall telomere length or the single-stranded telomeric overhang (Figure 3). In parallel, DNA isolated from the transduced cells was separated by 2-dimensional gel electrophoresis (2-DGE) and probed with a radiolabeled telomeric probe. This analysis shows that depletion of Ku70/80 is accompanied by a significant decrease in the intensity of the arc representing extrachromosomal t-circles (Figure 4). To corroborate this finding, we analyzed telomere length, overhang signal and t-circles in another ALT cells line, Saos2, and show that, as observed in CCL75.1, depletion of Ku70/80 in Saos2 does not affect overall telomere length nor 3′ overhang but results in a significant decrease in the levels of t-circles (Figure 4 and Supplementary Figure S3). These results indicate that Ku70/80 downregulation negatively influences t-circle formation in ALT cells, thus implicating non-homology-based processes in t-circles homeostasis in these cells. In contrast, shRNA-mediated silencing of the Werner syndrome protein (WRN), a factor that binds to Ku70/80 and has been implicated in the repression of t-circles in telomerase-positive cells [14], does not alter the levels of t-circles in ALT cells (Figure 5). As a control we show that downregulation of MRE11/NBS1 in both CCL75.1 and Saos2 cells also results in a significant reduction in the levels of t-circles (Supplementary Figure S4), which is in agreement with published results [2].


Depletion of Ku70/80 reduces the levels of extrachromosomal telomeric circles and inhibits proliferation of ALT cells.

Li B, Reddy S, Comai L - Aging (Albany NY) (2011)

Depletion of WRN does not influence the levels of t-circles in CCL75.1 cells(A) CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs for WRN or GFP and analyzed 3 days (shWRN) and 7 days (shWRN and shGFP) after the addition of 1.0 mg/ml DOX to the media. Protein levels were determined by Western blotting with WRN antibodies. We estimated that WRN shRNAs reduce its cognate gene expression ~75% compared to GFP shRNAs control after 7 days of DOX induction. Antibody against GAPDH was used as a loading control. (B) Genomic DNA isolated from CCL75.1 cells expressing shRNAs targeting WRN or GFP for 7 days was digested with the restriction endonucleases HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA)4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate levels of t-circles present in each sample (expressed as a percentage of the total telomeric DNA) were estimated as in [14] and is shown in the upper right corner.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117455&req=5

Figure 5: Depletion of WRN does not influence the levels of t-circles in CCL75.1 cells(A) CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs for WRN or GFP and analyzed 3 days (shWRN) and 7 days (shWRN and shGFP) after the addition of 1.0 mg/ml DOX to the media. Protein levels were determined by Western blotting with WRN antibodies. We estimated that WRN shRNAs reduce its cognate gene expression ~75% compared to GFP shRNAs control after 7 days of DOX induction. Antibody against GAPDH was used as a loading control. (B) Genomic DNA isolated from CCL75.1 cells expressing shRNAs targeting WRN or GFP for 7 days was digested with the restriction endonucleases HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA)4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate levels of t-circles present in each sample (expressed as a percentage of the total telomeric DNA) were estimated as in [14] and is shown in the upper right corner.
Mentions: Genetic knock-out of the Ku80 gene in telomerase-positive human cells results in reduced cell proliferation and is accompanied by dramatic shortening and loss of telomeres [16, 17]. Moreover, 50% reduction in the expression of Ku80 either by small interfering RNA (siRNA) or by functional inactivation of one allele in two telomerase-positive carcinoma cell lines, HeLa and HCT116, is sufficient to alter telomere homeostasis, as demonstrated by rapid telomere shortening and, at least in one of the studies, stronger telomere overhang signal [16, 18]. To determine whether depletion of Ku70/80 influences telomere homeostasis in ALT cells, we measured telomere length, single-stranded telomere overhang signal and t-circles in ALT cells transduced with lentiviruses for the conditional expression of shRNAs targeting Ku70 and Ku80. Cells were subjected to selections and DNA was isolated before induction and at 7 and 10 days after DOX addition. DNA was digested with HinfI and RsaI, resolved on agarose gel and hybridized to a radiolabeled telomeric probe under either denaturing or native conditions. This analysis show that within the time frame of this experiment, depletion of Ku70/80 does not significantly influence overall telomere length or the single-stranded telomeric overhang (Figure 3). In parallel, DNA isolated from the transduced cells was separated by 2-dimensional gel electrophoresis (2-DGE) and probed with a radiolabeled telomeric probe. This analysis shows that depletion of Ku70/80 is accompanied by a significant decrease in the intensity of the arc representing extrachromosomal t-circles (Figure 4). To corroborate this finding, we analyzed telomere length, overhang signal and t-circles in another ALT cells line, Saos2, and show that, as observed in CCL75.1, depletion of Ku70/80 in Saos2 does not affect overall telomere length nor 3′ overhang but results in a significant decrease in the levels of t-circles (Figure 4 and Supplementary Figure S3). These results indicate that Ku70/80 downregulation negatively influences t-circle formation in ALT cells, thus implicating non-homology-based processes in t-circles homeostasis in these cells. In contrast, shRNA-mediated silencing of the Werner syndrome protein (WRN), a factor that binds to Ku70/80 and has been implicated in the repression of t-circles in telomerase-positive cells [14], does not alter the levels of t-circles in ALT cells (Figure 5). As a control we show that downregulation of MRE11/NBS1 in both CCL75.1 and Saos2 cells also results in a significant reduction in the levels of t-circles (Supplementary Figure S4), which is in agreement with published results [2].

Bottom Line: In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence.Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length.These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

ABSTRACT
In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence. In contrast, cancer cells counteract the loss of telomeric DNA either by inducing the expression of telomerase or by activating the alternative lengthening of telomeres (ALT) pathway. ALT cells are characterized by heterogeneous telomeres and the presence of extrachromosomal circular double-stranded DNA molecules containing telomeric repeat sequences. These telomeric circles (t-circles) are though to be generated through a recombination process and utilized as templates for telomere elongation by rolling-circle-replication, although their precise mechanism of formation and role in telomere maintenance and cell proliferation is largely unknown. Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length. These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.

Show MeSH
Related in: MedlinePlus