1. Volume 19, Issue 12, Pages 2529-2543 (June 2017)
Highly Aggressive Metastatic Melanoma Cells Unable to Maintain Telomere Length 
Nikenza Viceconte, Marie-Sophie Dheur, Eva Majerova, Christophe E. Pierreux, Jean-François Baurain, Nicolas van Baren, Anabelle Decottignies 
Cell Reports 
Volume 19, Issue 12, Pages (June 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 2529-2543DOI: (10.1016/j.celrep.2017.05.046)
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3. Figure 1
Most, but Not All, Melanoma Metastases Express the Telomerase Subunits
(A) Measurement of hTERT expression in melanoma cell lines (blue) and tumors (black). hTERT cDNA values were normalized first to ACTB cDNA and then to relative hTERT expression in SW39/TEL+ cell line used as positive control. IMRB/ALT+ cells were used as negative control. SW39/TEL+ and IMRB/ALT+ are post-crisis SV40T-immortalized fibroblasts; NHEM are normal human epithelial melanocytes.
(B) hTERT promoter mutations in melanoma cell lines. All the three double mutations occurred in the same allele. Lower panel: Sanger sequencing of the cloned PCR-amplified hTERT promoter fragment in LB3129-MELB. Arrows indicate C228T and C250T mutations.
(C) Telomerase activity measured by qTRAP in the indicated cell lines. Values were normalized to SW39/TEL+ cells. The gray rectangle delineates background based on values obtained from the IMRB/ALT+ negative control. Arrows indicate cell lines with no detectable telomerase activity.
(D) Measurement of hTR expression by qRT-PCR.
(E) H3K9me3 and H3K4me3 densities at hTR promoter in LB2870-MEL hTRpos and LB3129-MELB hTRneg cell lines, normalized to H3. The following control loci with low/no transcriptional activity were included: promoter of TSH2B testis-specific histone 2B variant and subtelomeric 4qHOX locus (Boros et al., 2014). Data are presented as mean ± SEM.
(F) Telomerase activity measured by qTRAP in metastatic tumor tissue extracts. Values were normalized to SW39/TEL+ cell line.
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4. Figure 2
Telomerase-Deficient Melanoma Cell Lines Do Not Show Any Sign of ALT Mechanism
(A) TRF analysis in melanoma cell lines using HinfI/RsaI restriction enzymes. SW39/TEL+ and IMRB/ALT+ control cell lines are included. Note the characteristic heterogeneous TRF profile in IMRB/ALT+ cells. TRAPneg cell lines/tumors are indicated in red.
(B) ALT-associated PML body (APB) analysis by immunofluorescence (IF) and fluorescent in situ hybridization (FISH) in the indicated cell lines using IMRB/ALT+ cells as positive control. Green, PML by IF; red, telomere DNA by FISH. Nuclei are stained in blue with DAPI. Scale bar, 5 μm. TRAPneg cell lines/tumors are indicated in red.
(C) Staining for telomeres by FISH (green) and for PML by IF (red) in LB2901-tum, LB3129-tumA, LB3129-tumB tissues, and in LB2687-tum/TEL+ control tumor. Scale bar, 10 μm. TRAPneg cell lines/tumors are indicated in red.
(D) Detection of ATRX by IF in the same cell lines as in (B). Scale bar, 5 μm. TRAPneg cell lines/tumors are indicated in red.
(E) C-circle analysis in the indicated cell lines and tumors. IMRB/ALT+ and U2OS/ALT+ cell lines were used as positive controls for C-circles and SW39/TEL+ and MG63/TEL+ as negative controls. TRAPneg cell lines/tumors are indicated in red.
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5. Figure 3
Telomerase-Deficient Melanoma Cell Lines Are Not Immortal and Display Ever-Shorter Telomeres upon Cell Divisions
(A) Cumulative population doublings (PDs) for the indicated cultured cell lines. The passage number at which growth curves were initiated is indicated. TRAPneg/ALTneg cell lines/tumors are indicated in red.
(B) TRF analyses in the indicated melanoma cell lines upon passages. TRF is also shown for LB3129-tumA and LB3129-tumB tissues. Genomic DNA was digested with HinfI/RsaI restriction enzymes and telomere length (kb), estimated with Telometric software, is indicated below. TRAPneg/ALTneg cell lines/tumors are indicated in red.
(C) Representative pictures of telomere-induced foci (TIF) analysis in LB2870-MEL/TEL+ (p21), LB2687-MEL/TEL+ (p25), and in LB2901-MEL (p16) and LB3129-MELB (p21) cell lines with no TMM. Telomeres are detected by FISH (red) and DNA damage is detected with an antibody against 53BP1 (green). Nuclei are stained in blue with DAPI. Scale bar, 5 μm. TRAPneg/ALTneg cell lines/tumors are indicated in red.
(D) Quantification of TIF in the indicated cell lines. Frequencies of nuclei with no (black), 1–3 (green), 4–6 (blue), or above 6 (orange) TIF are shown. TRAPneg/ALTneg cell lines/tumors are indicated in red.
(E) Representative pictures of meta-FISH analyses in LB2901-MEL (p16), LB3129-MELA (p16), and LB3129-MELB (p21) cell lines showing chromosome fusions (arrowheads). Telomeres are shown in green. Scale bar, 5 μm. TRAPneg/ALTneg cell lines/tumors are indicated in red.
(F) Quantification of chromosome fusions based on meta-FISH analyses in the indicated cell lines. Average numbers (±SEM) of chromosome fusions/metaphase are indicated (20–25 metaphases were analyzed for each condition). TRAPneg/ALTneg cell lines/tumors are indicated in red.
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6. Figure 4
Telomerase Overexpression Rescues Ever-Shorter Telomeres of TMM-Negative Melanoma Cells and Is Sufficient for Immortalization
(A) Measurement of hTERT and hTR expression in LB3129-MELB and hTR-complemented LB3129-MELB cells. cDNA values were normalized first to ACTB cDNA and then to relative expression in SW39/TEL+ cell line used as hTRpos control. TRAPneg cell lines/tumors are indicated in red.
(B) Telomerase activity in LB3129-MELB and hTR-complemented LB3129-MELB cells measured by qTRAP. Values were normalized to background measured in IMRB/ALT+ cell line and shown with a gray rectangle. TRAPneg cell lines/tumors are indicated in red.
(C) TRF analysis in LB3129-MELB cells at p25 and after complementation with hTR. px+1 refers to the first passage after selection of transfected cells with antibiotic. Genomic DNA was digested with HinfI/RsaI restriction enzymes and telomere length (kb) was estimated using Telometric software (below). TRAPneg cell lines/tumors are indicated in red.
(D) LB3129-MELB cells were transfected with hTR plasmid at p20. Cumulative population doublings (PDs) with time in culture (days) are shown for both cell lines: red, LB3129-MELB; black: LB3129-MELB + hTR. TRAPneg cell lines/tumors are indicated in red.
(E) Xenograft experiments. NSG immunocompromised mice (three to four animals/condition) were injected with 106 cells of the indicated cell lines and tumor volume (mm3) was evaluated each week using a digital caliper (mean ± SEM). Inset: representative picture of mouse injected with LB3129-MELB + hTR cell line and the corresponding tumor after excision. Ruler graduation is in cm. TRAPneg cell lines/tumors are indicated in red.
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7. Figure 5 Melanoma Tumors Generally Display Long Telomeres
(A) Telomeres are detected by FISH (red) and nuclei are stained in blue with DAPI. Scale bar, 5 μm. For each human tissue sample, a piece of mouse heart tissue was added on the slide and processed simultaneously. To account for the presence of much longer telomeres in mouse cells, laser power was set to 10% to image mouse nuclei and to 25% to image human nuclei. TMMneg tumors and cell lines are indicated in red.
(B) Quantification of (A) using VIS software. qFISH values for human tissues were normalized first to mouse heart and then to nevus. TMMneg tumors and cell lines are indicated in red.
(C) Quantification of telo-FISH intensity in the two sub-populations of LB2805 tumor cells indicated by either an arrow (long) or an asterisk (short) in (A). TMMneg tumors and cell lines are indicated in red.
(D) TRF analysis in the indicated melanoma tumor samples using HinfI/RsaI restriction enzymes. TMMneg tumors and cell lines are indicated in red.
(E) Measurement of POT1 expression in melanoma cell lines. cDNA values were normalized first to ACTB cDNA and then to relative expression in LB3080-MELA cell line used as reference. TMMneg tumors and cell lines are indicated in red.
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8. Figure 6
Clinical History of Patients with Tumors Lacking TMM and Proposed Crisis-Independent Model of Melanoma Formation
(A) Clinical history of patients LB2901 and LB3129. Timescale (years) is indicated above. Tumor cell lines were established from the indicated metastases and are shown with gray rectangles. Treatments are indicated below and blue rectangles indicate their respective duration. See legend inside for more details.
(B) Proposed history of disease evolution in patient LB2901. Dotted arrows indicate possible links between tumors.
(C) TRF analysis in nevus, normal skin, LB3129-tumA, and LB3129-tumB tissues using HinfI/RsaI restriction enzymes.
(D) Possible disease evolution in patient LB3129. Radiotherapy is shown in yellow. Dotted arrows indicate possible links between tumors.
(E) The current model of cellular immortalization postulates that TMM acquisition during tumorigenesis involves a passage through short telomere-driven crisis (M2) after cells escaped from the first senescence barrier (M1) by inactivating cell-cycle checkpoints. In this exit-from-crisis model, reactivation of telomerase does not restore initial telomere length (gray arrow) (Shay and Wright, 2011). We propose that telomerase activation occurs in the context of still very long telomeres during melanocyte transformation but that the resulting telomerase activity levels are not high enough to maintain the length of long telomeres that progressively shorten until they likely stabilize at reduced length (dotted black arrow). We further found that TMM activation is not necessary for melanoma metastasis formation.
(F) Proportion of cancer patients with short telomeres in leukocytes as defined by Zhu et al. (2016). Control individuals are shown in black, patients with skin cancers are shown in white, and other cancer patients are shown in blue. Student’s t tests were applied to evaluate the significance of differences with control cohort (∗p < 0.1, ∗∗p < 0.01; n.s., not significant).
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