Background: Telomere shortening is linked to a range of different human diseases, hence reliable measurement methods are needed to uncover such associations. Among the plethora of telomere length measurement methods, qPCR is reported as easy to conduct and a cost-effective approach to study samples with low DNA amounts. Methods: Cancer cells' telomere length was evaluated by relative and absolute qPCR methods. Results: Robust and reproducible telomere length measurements were optimized taking into account a careful reference gene selection and by knowing the cancer cells ploidy. qPCR data were compared to "gold standard" measurement from terminal restriction fragment (TRF). Conclusions:Our study provides guidance and recommendations for accurate telomere length measurement by qPCR in cancer cells, taking advantage of our expertise in telomere homeostasis investigation in primary cutaneous T-cell lymphomas.Furthermore, our data emphasize the requirement of samples with both, high DNA quality and high tumor cells representation. K E Y W O R D S cancer, qPCR, southern blot, telomere length, tumor 3154 | ROPIO et al. (ERDF), COMPETE 2020 -Operacional Programme for Competitiveness and Internationalization (POCI) and by Portuguese funds through FCT -Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior.
Telomerase expression and telomere maintenance are critical for cell proliferation and survival, and they play important roles in development and cancer, including hematological malignancies. Transcriptional regulation of the rate-limiting subunit of human telomerase reverse transcriptase gen (hTERT) is a complex process, and unveiling the mechanisms behind its reactivation is an important step for the development of diagnostic and therapeutic applications. Here, we review the main mechanisms of telomerase activation and the associated hematologic malignancies.
Cutaneous T-cell lymphomas (CTCL) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its reexpression. We analyzed hTERT promoter methylation status in CTCL cells compared to healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from-650bp to-150bp and a hypomethylated proximal region from-150bp to +150bp. Interestingly, the hypermethylated region matches with the recently named TERT Hypermethylated Oncogenic Region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect on THOR of two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment as well as a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.
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