Division of Hematology/Oncology, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA; Texas Children's Cancer Center, Texas Children's Hospital, Houston, Texas, USA.
Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA.
J Biol Chem. 2023 May;299(5):104665. doi: 10.1016/j.jbc.2023.104665. Epub 2023 Mar 30.
Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect circular DNA with extrachromosomal telomere repeats. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (∼23 kb telomeres) and HeLaE1 (∼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.
端粒长度的维持对于癌细胞的永生至关重要。多达 15%的癌症利用一种非端粒酶依赖的、基于重组的机制，称为端粒的替代性延长（ALT）。目前，主要的 ALT 生物标志物是 C 环，一种具有染色体外端粒重复（cECTR）的环状 DNA 类型。C 环的形成方式尚未得到很好的描述。我们研究了人 cen3tel 细胞系中的 C 环形成，该细胞系是一种具有长端粒、端粒酶+（LTT+）的细胞系，其端粒逐渐超伸长（长达约 100 kb）。在 2D 凝胶和 C 环测定中观察到 cECTR 信号，但在 t 环测定中没有观察到，后者也检测到具有染色体外端粒重复的环状 DNA。在克隆群体的分析中，端粒酶活性和 C 环信号不可分离，这与 C 环的产生发生在端粒酶+细胞内一致。我们在另外两个 LTT+细胞系 HeLa1.3（约 23 kb 端粒）和 HeLaE1（约 50 kb 端粒）中观察到类似的 cECTR 结果。在 LTT+细胞中，端粒酶活性不会直接影响 C 环信号；相反，C 环信号与端粒长度相关。与 ALT+细胞系相比，LTT+细胞系对羟基脲的敏感性较低，这表明 ALT 状态对复制应激水平的贡献强于端粒长度。此外，与复制应激相关的蛋白 FANCM 并没有像在 ALT+细胞中那样抑制 LTT+细胞中的 C 环。因此，C 环的形成可能由端粒长度驱动，独立于端粒酶和复制应激，突出了 C 环作为独立的 ALT 生物标志物的局限性。
