Super-telomeres in transformed human fibroblasts

Abstract

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis.

The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation

“…While depleting DAXX or ATRX had little effect on wildtype J1 cells, TKO cells exhibited increased fragile telomeres, with smeared or double telomere signals on metaphase chromosomes (Figure 5C). Dysregulated telomere length control can result in abnormally long telomeres with average lengths of >1MB (super telomeres) (Chiodi et al, 2013). Again, we could observe super telomeres in TKO but not wildtype cells (Figure 5C).…”

Cellular response to glutamine and/or glucose deprivation in in�vitro transformed human fibroblasts

“…Cells and cell culture. The cen3tel cells used in this study belong to the cen3tel cellular system, which was developed from telomerase-immortalized fibroblasts that gradually underwent transformation during propagation in culture (13)(14)(15)(16)(18)(19)(20). Cen3tel cells underwent approximately 1,000 population doublings after telomerase immortalization.…”

Cells with stemness features are generated from in vitro transformed human fibroblasts

“…The cen3tel cellular system comprises cells at different stages along the way to transformation, well characterized at the cellular and molecular level 9 – 14 . Gene and miRNA expression profiles of cells at the different stages are available 13 .…”

“…In our laboratory, an in vitro model system for human fibroblast transformation was developed from hTERT immortalized fibroblasts 9 – 14 . Immortalized cells, named cen3tel, spontaneously and gradually underwent neoplastic transformation during culture propagation, becoming able to induce tumors when injected into immunocompromised mice.…”