Volume 19, Issue 12, Pages 2544-2556 (June 2017) Extensive Proliferation of Human Cancer Cells with Ever-Shorter Telomeres  Rebecca A. Dagg, Hilda A. Pickett, Axel A. Neumann, Christine E. Napier, Jeremy D. Henson, Erdahl T. Teber, Jonathan W. Arthur, C. Patrick Reynolds, Jayne Murray, Michelle Haber, Alexander P. Sobinoff, Loretta M.S. Lau, Roger R. Reddel  Cell Reports  Volume 19, Issue 12, Pages 2544-2556 (June 2017) DOI: 10.1016/j.celrep.2017.05.087 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 19, 2544-2556DOI: (10.1016/j.celrep.2017.05.087) Copyright © 2017 The Authors Terms and Conditions

Figure 1 A Unique Subgroup of High-Risk MYCN Non-amplified Neuroblastomas with Long Telomeres Are ALT-Negative and Have Poor Survival (A) Relative telomere content (TC) in arbitrary units (AU), measured by telomere qPCR for individual patient tumors (n = 149), are plotted according to ALT status. ALT+ was defined by a relative C-circle (CC) level ≥ 7.5 compared to the ALT+ IIICF/c cell line with an arbitrary value of 100. Horizontal red bar indicates median. ∗p < 0.0001. (B) Relative TC of MYCN non-amplified tumors (n = 94) is plotted according to ALT status. A subgroup of ALT− tumors (n = 17) had very long telomeres (TC ≥15, indicated by the bracket). (C) Terminal restriction fragment (TRF) analysis of 13 selected tumor samples with differing TC and CC profile. TC and CC status for each sample measured by telomere qPCR is indicated at the bottom together with mean TRF lengths. Black boxes represent the four ALT− samples that have long, heterogeneous telomeres indistinguishable from those of the ALT+ samples (green boxes). ALT− samples with low TC are indicated by white boxes. (D and E) Event-free (D) and overall survival (E) of long telomere/ALT− (TC ≥15/ALT−) (n = 17), ALT+ (n = 36), and MYCN amplified (amp) (n = 55) tumors. Fifty-one percent of patients with long telomere/ALT− tumors died because of their disease. See also Figure S1 and Table S3. Cell Reports 2017 19, 2544-2556DOI: (10.1016/j.celrep.2017.05.087) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Two Neuroblastoma Cell Lines, LA-N-6 and COG-N-291, Are Phenotypically Similar to the Long Telomere/ALT-Negative Tumor Group (A) Terminal restriction fragment (TRF) analysis of a panel of cell lines. LA-N-6 and COG-N-291 both have extremely long telomeres and a unique banding pattern that is not present in the SK-N-FI, CHLA-90, and U-2 OS ALT+ cell lines. SH-SY5Y and SK-N-BE2c are telomerase-positive (TEL+) NB cell lines with homogeneously short telomeres. (B) Telomere fluorescent in situ hybridization (FISH) in a panel of cell lines. Telomeres are represented by green signals at the end of the chromosomes. Quantitation of relative telomere fluorescent signals in telomere FISH shows LA-N-6 and COG-N-291 have the highest mean telomere fluorescence (red horizontal bar). HT1080 hTR cells are HT1080 cells overexpressing hTR with a corresponding increase in telomerase activity and telomere length. Dots represent individual telomere signals and 15 metaphases were analyzed per cell line. (C) Telomere repeat amplification protocol (TRAP) to detect telomerase activity (indicated by the presence of a six base-pair ladder) in a panel of cell lines. Cell lysates were immunopurified (IP) with anti-hTERT antibody to remove potential inhibitors. The ALT+ samples (U-2 OS, CHLA-90, and SK-N-FI) LA-N-6 and COG-N-291 are negative for telomerase. (D) APBs (co-localization of telomeric foci (green) and PML nuclear body (red), indicated by arrows) are frequently found in ALT+ cell lines (U-2 OS, GM847, SK-N-FI, and CHLA-90) and infrequently in telomerase (TEL+) cell lines (GM639, HT1080, SH-SY5Y, and SK-N-BE2c). Like TEL+ cell lines, LA-N-6 and COG-N-291 have infrequent APBs. Data represent mean + SD, n = 3 independent experiments. Scale bar, 3 μM. (E) Representative C-circle (CC) assay with and without Φ29 polymerase, followed by detection using a 32P-labeled telomeric probe in slot-blot analysis. CC level was calculated relative to that of the ALT+ U-2 OS cell line (designated to be 100 AU). TEL+ cell lines are CC− and ALT+ cell lines are CC+. LA-N-6 and COG-N-291 have very low levels of CC. See also Figures S2 and S3 and Tables S1, S2, and S3. Cell Reports 2017 19, 2544-2556DOI: (10.1016/j.celrep.2017.05.087) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Two Neuroblastoma Cell Lines, LA-N-6 and COG-N-291, Are Capable of Long-Term Proliferation Despite the Ever-Shorter Telomeres Phenotype (A) Terminal restriction fragment (TRF) analysis of COG-N-291 and LA-N-6 shows telomere shortening at progressive population doublings (PDs). (B) The average rate of telomere shortening was calculated by plotting the average TRF length of four individual TRF bands (indicated by asterisks in A) against the number of PDs. The telomeres of COG-N-291 and LA-N-6 shortened at a rate of 80 and 55 bases/PD, respectively. (C) Growth curves of COG-N-291 and LA-N-6 demonstrate long-term proliferation. (D) Telomere content, measured by telomere qPCR, confirms telomere shortening in LA-N-6 and COG-N-291. Data represent mean + SD, n = 3. (E) Frequency of APBs remained unchanged after extensive culturing of LA-N-6 and COG-N-291. Data represent mean + SD of three independent experiments. (F) C-circle levels (detected using 32P-labeled telomeric DNA probe on slot blot) remained low during long-term proliferation. Levels were calculated relative to those of the ALT+ MeT-4A cell line (designated to be 10 AU). SK-N-BE2c (telomerase-positive) was included as a negative control. See also Figure S4 and Tables S2 and S3. Cell Reports 2017 19, 2544-2556DOI: (10.1016/j.celrep.2017.05.087) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Ectopic Expression of Telomerase Rescues the EST Phenotype Telomerase activity was induced in LA-N-6 cells by ectopic expression of the telomerase catalytic subunit (hTERT) or hTERT plus the telomerase RNA component (hTR). (A) mRNA expression of the three telomerase components (hTR, hTERT, and dyskerin; measured by RT-qPCR) in LA-N-6 infected with plasmids for either hTR (clones, hTR C3 and C4), hTERT (clones, hTERT C4 and C23), or hTR/hTERT (clones, hTR/hTERT C19 and C23). (B) Telomerase activity was detected by TRAP in clones shown in (A). Expression of hTERT or hTERT plus hTR, but not hTR alone, resulted in telomerase activity in corresponding LA-N-6 clones. U-2 OS and SH-SY5Y cells are negative and positive controls, respectively, for telomerase activity. (C) Growth curves of LA-N-6 derivatives demonstrate telomerase activity resulted in a significant increase in growth rate. ∗p < 0.0003. (D and E) Telomerase activity resulted in telomere length maintenance or lengthening in corresponding LA-N-6 clones, as shown by TRF analysis (D) and telomere content by qPCR (E) at the indicated population doublings (PDs). Clones without telomerase expression had continued telomere shortening. qPCR data represent mean of triplicates + SD. (F) Relative telomere fluorescent signals measured from telomere FISH show telomerase activity increased the mean telomere length (15 metaphases analyzed per cell line). Dots represent individual telomere signals and red horizontal bar indicates mean. (G) Telomerase lengthened the shortest telomeres as indicated by a reduction in the percentage of chromosome ends without telomere signal in telomere FISH. See also Figure S5. Cell Reports 2017 19, 2544-2556DOI: (10.1016/j.celrep.2017.05.087) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Rescue of the EST Phenotype with Activation of the Alternative Lengthening of Telomeres (A) p53 immunoblot of LA-N-6 cells with wild-type and mutant p53. LA-N-6 MUT p53 represents a culture of LA-N-6 that spontaneously acquired a stop codon (Y236∗) at PD90, resulting in the loss of protein expression. GAPDH was used as loading control. (B) TRF analysis of the LA-N-6 culture from acquisition of the p53 mutation (PD104) at progressive PDs. Despite loss of wild-type p53, telomeres continued to shorten until PD263, after which telomere lengthening was observed. (C) Percentage of chromosome ends free of telomere signal (obtained from telomere FISH) in LA-N-6 parental (wild-type p53) and LA-N-6 MUT p53 cells. Reduction in signal-free chromosome ends in MUT p53 cells indicate alternative lengthening of telomeres (ALT) lengthened the shortest telomeres. See also Figure S6. (D) Growth curves of LA-N-6 parental (wild-type p53) and LA-N-6 MUT p53 culture demonstrate ALT activation did not result in increased proliferation. (E) Telomerase activity was monitored by TRAP at the specified PDs in LA-N-6 MUT p53 cells. The culture remained telomerase-negative. Lysates were immunopurified (IP) with anti-hTERT antibody to remove potential inhibitors. SH-SY5Y and SK-N-BE2c were used as positive controls. Cell Reports 2017 19, 2544-2556DOI: (10.1016/j.celrep.2017.05.087) Copyright © 2017 The Authors Terms and Conditions

Figure 6 EST Cells Do Not Form T-Circles with Activation of ALT or Extreme Telomere Lengthening by Telomerase Activity (A) Two-dimensional gel electrophoresis of restriction-digested genomic DNA. Denatured gels were hybridized with a 32P-labeled C-rich telomeric DNA probe. T-circle arcs (indicated by arrow) are present in ALT+ cells but are absent from telomerase-positive and EST cells. (B) hTR and hTERT expressing LA-N-6 clones do not have t-circles at early or later population doublings (PD). (C) T-circles (indicated by arrow) are present in the ALT+ control but not in the LA-N-6 MUT p53 clone. Cell Reports 2017 19, 2544-2556DOI: (10.1016/j.celrep.2017.05.087) Copyright © 2017 The Authors Terms and Conditions