Morphogenesis of giant telomeric nuclear bodies in cancer cells with alternative lengthening of telomeres

Some cancer cells lengthen their telomeres by alternative lengthening of telomeres (ALT); these are referred to as ALT cancer cells and do not express telomerase. The ALT mechanism involves the elongation of telomeric DNA repeats by homologous recombination. In interphase nuclei of ALT cancer cells, giant telomeres can be specifically observed by fluorescence in situ hybridization (FISH) to detect telomeric repeats, and they are co-localized with promyelocytic-leukemia nuclear-bodies (PML-NBs). However, it how large telomeres specific to ALT and how they form a structural relation with PML-NBs. We refer to giant telomeres specific to interphase ALT cancer cells giant-telomeric nuclear-bodies (GT-NBs). To quantitatively define GT-NBs, we performed telomeric FISH of both interphase nuclei and metaphase chromosomes and analyzed telomere sizes by integrated FISH signals. The distributions of telomere sizes in telomerase-positive cells were similar in interphase nuclei and chromosomes. However, the distribution of telomere sizes in ALT cancer cells differed between interphase nuclei and chromosomes. Giant telomeres that were larger than those at chromosomal ends were only observed in interphase nuclei of ALT cancer cells. Accordingly, GT-NBs could be quantitatively defined as larger than the maximum size of telomeres at chromosomal ends. Furthermore, ALT cancer cells demonstrated fewer telomeric signals in interphase nuclei than in chromosomes. These findings indicate that GT-NBs could be formed by the aggregation of two or more telomeres at chromosomal ends. Furthermore, super-resolution microscopy showed that GT-NBs contain one or two PML-NBs. GT-NBs are considered aggregates of telomeres and could contain multiple sites of homologous recombination accompanied by PML-NBs. These findings may contribute to the development of therapeutic approaches for ALT cancer. (251 words)

mechanism involves the elongation of telomeric DNA repeats by homologous 23 recombination. In interphase nuclei of ALT cancer cells, giant telomeres can be 24 specifically observed by fluorescence in situ hybridization (FISH) to detect telomeric 25 repeats, and they are co-localized with promyelocytic-leukemia nuclear-bodies (PML- 26 NBs). However, it how large telomeres specific to ALT and how they form a structural 27 relation with PML-NBs. We refer to giant telomeres specific to interphase ALT cancer 28 cells giant-telomeric nuclear-bodies (GT-NBs). To quantitatively define GT-NBs, we 29 performed telomeric FISH of both interphase nuclei and metaphase chromosomes and  Most cancer cells express telomerase to lengthen telomeric DNA, and these are referred 48 to as telomerase-positive cancer cells (Tel (+) cancer cells) [4][5][6]. Nevertheless, 10%-49 15% of human cancers exhibit little telomerase activity; in these cells, telomeric DNA is 50 elongated by an alternative mechanism [6][7][8][9], termed alternative lengthening of telomeres 51 (ALT), involving homologous recombination between telomeric DNAs [5][6][7][8][9][10][11]. Cancer 52 cells that lengthen telomeres by the ALT mechanism are referred to as ALT cancer cells.

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Telomerase inhibitors are not expected to be effective anticancer therapies in ALT cancer.

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Telomeres of ALT cancer cells vary from 3 to 20 kb but are generally longer than those 4 55 of Tel (+) cancer cells [3][4]. Telomeres can be detected by fluorescence in situ 56 hybridization (FISH) and quantified by integrated brightness fluorescence intensities for 57 telomere signals, reflecting the amount of telomeric DNA. The signals of giant telomeres 58 are observed by FISH in interphase nuclei of ALT cancer cells [4,[12][13][14][15]. However, it is 59 not clear how large telomeres are specific to ALT cancer cells and how they form in their 60 interphase nuclei. In the present study, we refer to ALT-specific giant telomeres as "giant-61 telomeric nuclear-bodies (GT-NBs)" and attempted to define GT-NBs in a quantitative 62 manner and to clarify their morphogenesis.

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Preparation of chromosome specimens 99 To analyze telomeric signals during metaphase, chromosomes were prepared. After    were statistically significant by Welch's t-test (P < 0.05) for the three ALT cancer cells.

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Distributions of telomeric FITC IVs in chromosomal specimens (c) and interphase nuclei   Telomere elongation by the ALT mechanism is suggested by telomeric homologous 271 recombination. This homologous recombination (HR) mechanism involves two process.  In has recently been reported that ALT cancer cells possess telomeric sequences 286 containing the intranuclear receptor-related TCAGGG sequence throughout their 287 genomes. According to one such report, giant telomeric signals disappear by knocking 17 288 down an intracellular receptor, NuRD-ZNF827, in ALT cancer cells. Therefore, ALT-289 specific GT-NBs suggested that the TCAGGG sequence is involved in nuclear receptor 290 activity. Further analyses of GT-NB formation will elucidate the mechanism underlying 291 ALT.