Abstract
Telomeres, the natural ends of chromosomes, hide the linear telomeric DNA from constitutive exposure to the DNA damage response with a lariat structure or t-loop. Progressive telomere shortening associated with DNA replication in the absence of a compensatory mechanism culminates in t-loop collapse and unmasked telomeres. Dysfunctional telomeres can suppress cancer development by engaging replicative senescence or apoptosis, but they can also promote tumour initiation when cell cycle checkpoints are disabled. In this setting, telomere dysfunction promotes increasing chromosome instability (CIN) through breakage-fusion-bridge cycles. Excessive instability may hamper cell proliferation but might allow for the appearance of some rare advantageous mutations that could be selected and ultimately favour neoplastic progression. With the aim of generating pre-malignant immortalised cells, we ectopically expressed telomerase in telomere-compromised variant human mammary epithelial cells (vHMECs), proficient and deficient for p53, and analysed structural and numerical chromosomal aberrations as well as abnormal nuclear morphologies. Importantly, this study provides evidence that while immortalisation of vHMECs at early stages results in an almost stable karyotype, a transient telomere-dependent CIN period—aggravated by p53 deficiency—and followed by hTERT overexpression serves as a mechanism for the generation of immortal unstable cells which, due to their evolving karyotype, could attain additional promoting properties permissive to malignancy.
Highlights
Telomerase reactivation is a hallmark of carcinogenesis, and the vast majority of human tumours have telomerase activity by upregulating expression of telomerase’s catalytic subunit [1]
We show that genomic alterations acquired in immortalised genome-unstable variant human mammary epithelial cells (vHMECs) are a mixture of random and fixed chromosomal rearrangements that could be potential sources of oncogenic changes and malignant evolution
Genetic modifications consisted of the generation of an immortalised vHMEC cell line by ectopical expression of the catalytic subunit of telomerase (Figure 1). hTERT immortalisation was performed at an early population doubling (PD) before vHMECs developed chromosome instability (CIN) associated with extensive telomere shortening
Summary
Telomerase reactivation is a hallmark of carcinogenesis, and the vast majority of human tumours have telomerase activity by upregulating expression of telomerase’s catalytic subunit (hTERT) [1]. Previous telomere and cytogenetic studies have documented CIN, reduced telomere length and telomere end fusions in early-stage human breast cancers [20,21,22,23], supporting telomere dysfunction as a driver of CIN and an inducer of intratumour diversity in this emerging malignancy [24] These findings, along with the detection of telomerase activity in some breast carcinomas in situ [25,26,27,28], suggest that immortalisation of telomere unstable cells through the activation of telomerase could be an early event in the progression of breast carcinogenesis. We show that genomic alterations acquired in immortalised genome-unstable vHMECs are a mixture of random and fixed chromosomal rearrangements that could be potential sources of oncogenic changes and malignant evolution
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