Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform

Abstract

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.

Keywords: BIBR1532; Hutchinson-Gilford progeria syndrome; M-FISH; Q-FISH; SUN1; SUN1 isoform 9; chromosome territories; genomic instability; hTERT; telomeres.

Figure 1

Analysis of chromosomes in the immortalized cell lines. Representative images of metaphase chromosome spreads of NB1T (A), T06 (B) and T08 (C) cells. Scale bar: 5 μm. The graph in panel D reveals the number of chromosomes plotted against frequency (%) for each cell line, binned for chromosome number (D). Representative M‐FISH karyotype of T08 cell line which is cell 21 displaying genomic instability 83,XXX,t(X;1),ins(1;14),der(1)t(14;1;17),del(2p),del(3p),der(4)t(4;19),der(5)t(5;10), del(5p),‐6,der(7)t(7;21),‐13,‐14,der(14)t(10;14),‐15,der(15)t(8;15),‐16,del(17q) (E) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Telomere distribution in the immortalized cell lines. Representative digital images of NB1, NB1T, T06 and T08 cells in interphase after hybridization with cy3‐conjugated telomeric peptide nucleic acid (PNA) oligonucleotides (A, B, C, D, respectively) in red and nuclear DNA in blue. Corrected calibrated fluorescence (CcFL) telomere signal intensity for NB1T, T06, and T08 cells relative to the control NB1 cell line (E). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent SE of the mean (SEM). F displays the nuclear position of the telomeres in NB1, NB1, T06, and T08 cells after erosion analysis33, 77 measuring the percentage of the cy3 telomere signal (%), normalized by the percentage of DAPI signal, over five concentric shells of equal area from the nuclear periphery to interior. The x‐axis displays the shells from 1 to 5 (left to right), with 1 being the most peripheral shell and 5 being the most internal shell. The y‐axis shows the normalized signal (%)/DAPI (%), error bars representing the SE of mean (SEM). Significant differences are denoted by stars (*P ≤ 0.05; ** P ≤ 0.01) (B). Scale bar = 5 μm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Nuclear locations of chromosome territories. Representative images displaying examples of peripheral, intermediate, and internal positioned chromosome territories in proliferating NB1, NB1T, T06, and T08 cell lines for chromosome 18 (A‐D) and chromosome X (I‐L). Fibroblasts were subjected to 2D‐FISH using whole chromosome painting probes specific to chromosomes 18 and X. The probes were labeled with biotin by degenerate oligonucleotide primed‐polymerase chain reaction (DOP‐PCR) and detected using streptavidin conjugated to cyanine 3 (colored green) and the nuclei were counterstained with DAPI (blue). Scale bar: 5 μm. The bar charts in panels E‐H (chromosome 18) and M‐P (chromosome X) display the distribution of the chromosome signal in 50‐55 nuclei for each chromosome for as analyzed by erosion analysis for NB1, T06, and T08 cells. The x‐axis displays the shells from 1 to 5 (left to right) with 1 being the most peripheral shell and 5 being the most internal shell. The y‐axis shows chromosome signal (%)/DAPI (%) signal. Bars represent the mean normalized proportion (%) of chromosome signal for each human chromosome. Error bars represent SEM [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Alterations of telomere length and proliferating cells in hTERT‐immortalized cells treated with BIBR1532. Corrected calibrated fluorescence (CcFl) before and after treatment with BIBR1532 NB1T and T08 cell lines relative to the control NB1 cell line. Every 2 weeks from 4 weeks onwards treated and untreated NB1T and T08 cells were measured for telomere fluorescence intensity by performing IQ‐FISH (A, B). *P ≤ 0.05; **P < 0.01; ***P < 0.001. Error bars represent SEM. Ki67 in hTERT‐immortalized cells treated with BIBR1532. Panel E represents Ki67 nuclei with Ki67 in red and the nuclear DNA stained by DAPI in blue. The fraction of cells displaying positive Ki67 staining was scored with and without the BIBR1532 drug over the culture period of 0‐8 weeks and is presented by the graphs (B, D). *P ≤ 0.05; **P < 0.01; ***P < 0.001. Error bars represent SEM [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Chromosome repositioning after BIBR1532 treatment. Representative images of the position of chromosome 18 and X within NB1, NB1T, and T08 fibroblasts nuclei before and after drug treatment (A‐E, K‐O, U‐W and A’‐C’). Fibroblasts were subjected to 2D‐FISH using probes specific to chromosomes 18 and X. Whole chromosome painting probes were labeled with biotin and detected using streptavidin conjugated to cy3 (green) and the nuclei were counterstained with DAPI (blue). Ki‐67 staining is not shown in the images. Histograms displaying the nuclear positions of chromosomes 18 and X territories in Ki‐67 positive NB1 and NB1T cells before and after drug treatments (F‐J) and (P‐T). Erosion analyses were performed by ascertaining the distribution of the mean proportion of hybridization signal per chromosome (%), normalized by the percentage of DAPI signal, over five concentric shells of equal area from the nuclear periphery to center. The x‐axis displays the shells from 1 to 5 (left to right), with 1 being the most peripheral shell and 5 being the most internal shell. The y‐axis shows signal (%)/DAPI (%). Error bars representing SEM were plotted for each shell for each graph (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). Scale bar = 5 μm. Bar charts displaying the position of human chromosomes 18 and X territories with Ki‐67 positive in T08 cells before and after drug treatments (X‐Z and D’‐F’) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

Expression differences of nuclear envelope proteins. Representative images of lamin B receptor (red), SUN1 (red) in NB1T (A, C, respectively) and T08 cells (B, D, respectively). Nuclear DNA is counter‐stained with DAPI (blue). Scale bar = 5 μm. Samples of NB1T control and atypical HGPS (T08) cell lines in 3X SDS sample buffer were resolved on 10% SDS‐PAGE gels, and anti‐SUN1 and anti‐LBR antibodies were used to identify SUN1 and LBR in western blots. All samples were loaded equally, with 2 × 10⁵ cells per lane. α‐tubulin was visualized to normalize the level of proteins [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7

Cloning of the LBR and SUN1 fragments generated from cDNA of HGPS T08 cells for sequencing analysis. Schematics of the in silico designed DNA fragments generated using primers designated by the arrows (A). The expected sizes of DNA fragments are shown in numbers of base‐pairs. (B) Gel electrophoresis analysis of LBR fragments (lanes 2, 3, 4) and the SUN1 fragments (lanes 5, 6, 7) amplified from cDNA of T08 cells using the pairs of primers indicated above the lanes. Lanes 1 and 8 are the DNA molecular weight markers, BIOLINE Hyperladder II and I, respectively. The sizes of the DNA markers in base‐pairs are shown on the left and the right side

Figure 8

Schematic presentation of the human SUN1 isoforms annotated under O94901 at UniProt database as well as other relevant sequences deposited into GenBank. A novel isoform identified in this work is shown at the bottom of the schematic. The exon numbers are annotated in Ensembl database for SUN1‐001 transcript (ENSG00000164828). The exons are shown as boxes with the corresponding number. The exons 10‐19 are presented as a dash line as they are identical for all the isoforms containing the C‐terminal half. A vertical bar in exon 6 represents the 10‐aa peptide missing in the canonical isoform‐1 that was identified during phosphoproteomics analysis by93 [Color figure can be viewed at wileyonlinelibrary.com]