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.

Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen

“…To-date, many immortalized genes have been discovered, amongst which SV40 large T antigen is the most widely studied. SV40-LT is known to inactivate Rb through p53 to promote cell immortalization by preventing cell senescence and apoptosis [7,8,25]. In most cases, the T antigen enters the cell cycle by inducing the inactivation of Rb proteins (pRB, p130, and p107), thereby activating E2F-dependent transcription and promoting S phase progression, proliferation, and immortalization [26].…”

“…MC growth can be stimulated by external factors including viral genes that stimulate M1 progression, leading to immortalization and indefinite cell division in vitro. Exogenous genes commonly used to establish immortalized cell lines include Epstein–Barr virus, human papilloma virus (HPV), simian virus 40 Large T (SV40-LT), and telomerase reverse transcriptase [8].…”

Conditional reprogramming: next generation cell culture

“…Overexpression of hTERT has long been used as a tool to promote cell immortalization 44 . Although there are many successful cases 45 , 46 , 47 , this method is confronted with similar problems to the use of viral oncogenes 10 , 48 .…”

The Nuclear Lamina: Protein Accumulation and Disease

“…There are 10 predicted splice variants of SUN1 (not all of which have been detected), with all variation occurring in the N-terminal nucleoplasmic DNA binding domain [53,87,88]. There is limited information on the specific functions of these variants and it has been shown that HGPS cells express a previously uncharacterized isoform of SUN1 which may be responsible for the observed genomic instability and disruption of chromosome positioning within the nuclear volume of HGPS cells [89,90]. Although the mechanisms controlling the expression of these variants are still unknown, the accumulation of different isoforms may have functional consequences.…”