Cannabidiol (CBD) and cannabidivarin (CBDV) are natural cannabinoids which are consumed in increasing amounts worldwide in cannabis extracts, as they prevent epilepsy, anxiety, and seizures. It was claimed that they may be useful in cancer therapy and have anti-inflammatory properties. Adverse long-term effects of these drugs (induction of cancer and infertility) which are related to damage of the genetic material have not been investigated. Therefore, we studied their DNA-damaging properties in human-derived cell lines under conditions which reflect the exposure of consumers. Both compounds induced DNA damage in single cell gel electrophoresis (SCGE) experiments in a human liver cell line (HepG2) and in buccal-derived cells (TR146) at low levels (≥ 0.2 µM). Results of micronucleus (MN) cytome assays showed that the damage leads to formation of MNi which reflect chromosomal aberrations and leads to nuclear buds and bridges which are a consequence of gene amplifications and dicentric chromosomes. Additional experiments indicate that these effects are caused by oxidative base damage and that liver enzymes (S9) increase the genotoxic activity of both compounds. Our findings show that low concentrations of CBD and CBDV cause damage of the genetic material in human-derived cells. Furthermore, earlier studies showed that they cause chromosomal aberrations and MN in bone marrow of mice. Fixation of damage of the DNA in the form of chromosomal damage is generally considered to be essential in the multistep process of malignancy, therefore the currently available data are indicative for potential carcinogenic properties of the cannabinoids.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2322-9) contains supplementary material, which is available to authorized users.

Long non-coding transcripts from telomeres, called telomeric repeat-containing RNA (TERRA), were identified as blocking telomerase activity (TA), a telomere maintenance mechanism (TMM), in tumors. We expressed recombinant TERRA transcripts in tumor cell lines with TA and with alternative lengthening of telomeres (ALT) to study effects on TMM and cell growth. Adeno- and lentivirus constructs (AV and LV) were established for transient and stable expression of approximately 130 units of telomere hexanucleotide repeats under control of cytomegalovirus (CMV) and human RNase P RNA H1 (hH1) promoters with and without polyadenylation, respectively. Six human tumor cell lines either using telomerase or ALT were infected and analyzed for TA levels. Pre-infection cells using telomerase had 1%–3% of the TERRA expression levels of ALT cells. AV and LV expression of recombinant TERRA in telomerase positive cells showed a 1.3–2.6 fold increase in TERRA levels, and a decrease in TA of 25%–58%. Dominant-negative or small hairpin RNA (shRNA) viral expression against human telomerase reverse transcriptase (hTERT) results in senescence, not induced by TERRA expression. Population doubling time, cell viability and TL (telomere length) were not impacted by ectopic TERRA expression. Clonal growth was reduced by TERRA expression in TA but not ALT cell lines. ALT cells were not affected by treatments applied. Established cell models and tools may be used to better understand the role of TERRA in the cell, especially for targeting telomerase.

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Most tumors express telomerase for infinite cell growth capacity. Telomerase was recognized recently as central regulator of all of the hallmarks of cancer and synthetic inhibitors of telomerase, such as Imetelstat blocking the RNA subunit of telomerase TERC, are in clinical trials. RNA transcripts from telomeres called TERRA were identified to block telomerase activity (TA) potentially via direct binding to TERC. We established recombinant expression systems to modulate TERRA transcripts in cell culture cells and study in vitro cell growth and viability directly and in combination with telomerase inhibitors. Adeno- and lentivirus constructs (AV and LV) were established for transient and stable recombinant TERRA expression in TA tumor cell lines (N=3) and mortal fibroblast cells as control. AV and LV express TERRA under control of hH1 promoter with around 120 units of UUAGGG repeats in sense (S) or anti-sense (AS) orientation. Similarly, AV and LV express polyadenylated form of TERRA under control of CMV promoter and polyadenylation signaling sites. Telomere length (TL), endogenous and recombinant TERRA expression was analyzed by qPCR, TA by TRAP assay. Population doubling (PD) times were calculated from cell growth numbers of LV clones. Small molecule telomerase inhibitors and AVs expressing dominant-negative telomerase and shRNA against hTERT were used in MTT cell viability and clonogenicity assays. TERRA expression was modulated transiently and stably by recombinant AVs and LVs as compared to virus controls expressing eGFP. Moderate 1.5-1.7 fold elevation of recombinant TERRA-S transcripts caused reduction of TA to 23-38%. In contrast, AS expression reduced TERRA 3-8 fold without reduction of TA. TL, PD time, cell viability and clonogenicity were not affected by TERRA-S and -AS expression up to 3 weeks in culture. Single cell clones were isolated from cells infected by LVs. Significant improve of telomerase targeting approaches was identified as IC-50 values decreased 2.0-2.6 fold in LV cell clones expressing moderate recombinant TERRA-S compared to TERRA-AS and eGFP. Similarly, clone formation capacity decreased 1.3-1.7 fold. Human fibroblasts showed 15 fold increased TERRA expression compared to tumor cells and were not affected by treatments applied. Preliminary data with modified RNA oligonucleotides containing TERRA sequences indicate that up-take by cells was efficient. Furthermore, IC-50 values of TA inhibitor MST-312 in combination with 3 nM TERRA oligonucleotides decreased 1.9-3.9 fold compared to mismatch controls. Effective concentrations were 1000 fold lower compared to DNA oligonucleotide telomerase inhibitors currently in clinical trials. We demonstrated that recombinant expression of TERRA significantly improved telomerase targeting approaches in tumor cells but not in mortal fibroblasts. TERRA oligonucleotides are candidates for in vivo application in combination with telomerase targeting approaches or other tumor therapies. Citation Format: Sandra Sampl, Doris Mejri, Christian Stern, Hui Wang, Klaus Holzmann. Telomere transcripts improve synthetic inhibitors of telomerase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2743. doi:10.1158/1538-7445.AM2014-2743