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

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.

Figure 1.. Short hairpin RNAs (shRNAs) for Ku70/80 inhibit the proliferation of ALT cells

(A) shRNAs for Ku70/80 reduce the levels of Ku70/80 in CCL75.1 cells. CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs targeting Ku70 and Ku80 or GFP and cultured for 3, 7 and10 days in the presence of 1.0 mg/ml Doxycycline (DOX). The levels of Ku70 and Ku80 were analyzed by immunoblotting with antibodies against Ku70 and Ku80. Antibodies against tubulin were used as a loading control. We estimated that 7 to 10 days after induction, Ku70/80 shRNAs-expressing cells display an 80 to 85% decrease in Ku70/80 expression compared to GFP shRNAs control. (B) Growth curves of CCL75.1 expressing shRNAs for Ku70/80 or GFP. After 5 days in Geneticin and hygromycin selection, 1.0ug/ml DOX was added to the media and the growth rate of each CCL75.1 cell line was measured by counting viable cells every 2 days. Cells were seeded at a low density, and the medium was changed every 2 days. Values represent the mean ± the standard deviation of three experiments (n = 3). (C) Detection of SA-β-gal activity. CCL75.1 cells transduced with lentiviruses for the conditional expression of shRNAs targeting K70/80 or GFP were grown for 3, 6, 9, or 12 days after addition of DOX and were stained for SA-βgal activity as previously described [14]. Values are the mean ± the standard deviation of three independent experiments (n = 3) carried out in duplicates in which 500 cells were scored for SA-β galactosidase. Student's t test was used to evaluate differences in means between two groups, and P < 0.05 was considered statistically significant. ALT cells (CCL75.1 and Saos2) were purchased from ATCC.

Figure 2.. Depletion of Ku70/80 in CCL75.1 cells induces transient p53 activation

The levels of p21, p53, and p53 phosphorylated at serine 15 were assessed by Western blotting in extracts prepared from CCL75.1 cells collected either before, or 3 to 6 days after addition of DOX. Tubulin serves as a loading control. Western blot analyses were performed with antibodies against p53, p53-phosphoserine15, p21, and tubulin.

Figure 3.. Depletion of Ku70/80 in CCL75.1 cells does not affect telomere length nor overhangs signal

(A) Terminal Restriction Fragment (TRF) analysis. CCL75.1 cells transduced with lentiviruses for the conditional expression of shRNAs targeting Ku70 and Ku80 or GFP were harvested at 7 (shKu70/80) and 10 days (shKu70/80 and shGFP) after induction with DOX. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (CCCTAA)₄probe. The molecular mass standards shown on the right lane were generated by digestion of lambda DNA with the restriction endonuclease HindIII. (B) Telomere 3′ overhang signal analysis. CCL75.1 cells transduced with lentiviruses for the conditional expression of shRNAs targeting either Ku70/80 or GFP were harvested at 7 (shKu70/80) and 10 days (shKu70/80 and shGFP) after induction with DOX. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel. The gel was dried under native conditions and then hybridized to a radiolabeled (CCCTAA)₄probe.

Figure 4.. Depletion of Ku70/80 reduces the levels of t-circles in CCL75.1 and Saos2 cells

(A) Genomic DNA isolated from CCL75.1 cells expressing shRNAs targeting Ku70/80 or GFP for 7 days was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA)₄probe. 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. (B) (top panel) Saos2 cells were infected with lentiviruses for the conditional expression of shRNAs for Ku70and Ku80 or GFP and analyzed either before or 3, 7 or 10 days after the addition of 1.0 mg/ml DOX to the media. The levels of Ku70 and Ku80 were determined by Western blotting with antibodies against Ku70 and Ku80. We estimated that Ku70/80 shRNAs reduce Ku70/80 expression ~80% compared to GFP shRNAs control. Antibody against tubulin was used as a loading control. (middle and bottom panels) DNA isolated from Saos2 cells 7 days after DOX addition was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA)₄probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions and the approximate levels of t-circles present in each sample (expressed as a percentage of the total telomeric DNA) is shown in the upper right corner.

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.

Figure 6.. Telomere FISH analysis of ALT cells expressing shRNAs for Ku70/80 or GFP

CCL75.1 cells expressing shRNAs for Ku70/80 or GFP were treated with 0.5 mg/ml of colcemid for 1.0 hour, harvested by trypsinization and swollen in 0.075M KCl for 20 min. The cells were fixed and washed with methanol:acetic acid (3:1) for three times and dropped into slides. Images shown are representative of more than 50 metaphases analyzed for each cell line.